Document [original]

Ethnopharmacology

of medicinal plants traditionally used in the

Greater Mpigi region, Uganda

vorgelegt von

M.Sc.

Fabien Schultz

ORCID: 0000-0003-1904-2430

an der Fakultät III – Prozesswissenschaften

der Technischen Universität Berlin

zur Erlangung des akademischen Grades

Doktor der Naturwissenschaften

– Dr. rer. nat. –

genehmigte Dissertation

Promotionsausschuss:

Vorsitzender: Prof. Dr. Lorenz Adrian

Gutachterin: Prof. Dr. Vera Meyer

Gutachter: Prof. Dr. Juri Rappsilber

Gutachter: Prof. Dr. Leif-Alexander Garbe

Gutachter: Prof. Dr. Michael Heinrich

Tag der wissenschaftlichen Aussprache: 11. Juni 2021

Berlin 2021

Ethnopharmacology [constitutes] a respectful marriage between modern

science and ancient wisdom with much to be gained in both directions.

Dr. Graham Jones (Heinrich and Jäger, 2015)

Abstract

Relevance: This PhD thesis provides an ethnopharmacological assessment of 16 medicinal

plant species used by traditional healers for the treatment of various infectious and

inflammatory diseases in the Greater Mpigi region of Uganda. It includes the first detailed

report on selected medicinal plant use in the local traditional medicine system. The

ethnobotanical data obtained supports the conservation of local traditional knowledge and will

enable future drug discovery research. As conventional drug therapies are becoming

increasingly limited and inefficacious, some of the greatest threats to global health today are

addressed in this work, including antibiotic resistance, malaria, cancer, inflammation, and

related symptoms such as pain and fever.

Aims of the PhD thesis: The overarching objectives were 1) the documentation of the medicinal

use of (often understudied) plant species; 2) the development of a bibliographic assessment

tool for selection of medicinal plants for lab studies; 3) the creation of a plant extract library;

4) the pharmacological in vitro assessment of these species regarding their respective

traditional uses; 5) the contribution to early drug discovery stages and thereby the provision of

a scientific basis for future drug lead identification endeavors; and 6) the integration of aspects

of community work while fostering a bidirectional communication with indigenous

communities.

Methods: The results of diverse ethnopharmacological field research methods and

pharmacological lab assays are reported in this dissertation, including a) an ethnobotanical

survey using structured interviews with 39 traditional healers from 29 villages; b) the novel

Degrees of Publication (DoP) method; c) antibacterial screenings against a panel of clinical

isolates of multidrug-resistant human pathogens; d) antivirulence experiments, e.g., monitoring

of quorum sensing inhibition and direct protein output (į-toxin) in Staphylococcus aureus;

e) antiinflammatory investigations (e.g., cyclooxygenase-2 inhibition); f) antimalarial studies

against chloroquine-resistant Plasmodium falciparum K1; g) cytotoxicity counterscreens

(e.g., against human MRC-5SV2 lung fibroblasts and HaCaT keratinocytes); h) a genotoxicity

assessment model with simulation of human liver metabolism; and i) a method for transferring

research results back to traditional healers in rural indigenous communities.

Results: The results of the survey indicated a high level of local traditional use, as the 16 plant

species are frequently used to treat a total of 75 different medical disorders. Many of these use

reports were documented for the first time. The development and application of the

DoP method allowed for assessment of what was already known about a species, while also

estimating the quality of the evidence. In total, 634 peer-reviewed publications covering the

period of 1960-2019 were reviewed. Six of the 16 species were then classified as being highly

understudied, and three species as being understudied, which provided further justification for

the subsequent lab investigations. The results of the pharmacological experiments generally

showed a high correlation between the pharmacological activity and the respective traditional

use. In the following, some examples of the strongest bioactive extracts are provided for

a) growth inhibition activity: extracts of Zanthoxylum chalybeum and Harungana

madagascariensis stem bark against S. aureus (MIC: 16 and 32 ȝg/mL) and Enterococcus

faecium (MIC: 32 ȝg/mL); b) quorum sensing inhibition activity against four S. aureus

accessory gene regulator (agr) alleles: extracts of Solanum aculeastrum root bark and Sesamum

calycinum subsp. angustifolium leaves (IC50: 1–64 ȝg/mL); c) selective COX-2 inhibition

activity: an extract of Leucas calostachys leaves (IC50: 0.66 ȝg/mL); and d) antiplasmodial

activity against P. falciparum K1: extracts of W. ugandensis stem bark and L. calostachys

leaves (IC50: 0.5 and 5.7 ȝg/mL). The transfer of lab results back to the traditional healers

culminated in a video article showing a two-day workshop.

Conclusions: The conservation of traditional knowledge continues to be vital for future

generations, especially when facing the need for novel and more effective drugs, which have

often been discovered from natural products in the past. The newly introduced DoP method is

a useful tool for selecting traditionally used species for future lab studies, like costly

pharmacological experiments and time-consuming isolation procedures. The results of the

pharmacological studies provided scientific support for the potential therapeutic effects of the

medicinal plants used in the Greater Mpigi region.

III

Zusammenfassung

Relevanz: Diese Doktorarbeit beschäftigt sich mit der ethnopharmakologischen Untersuchung

von 16 Medizinpflanzen, die von traditionellen Heilerinnen und Heilern zur Behandlung von

diversen Infektions- und Entzündungskrankheiten in der Großregion Mpigi in Uganda genutzt

werden. Sie beinhaltet erstmalig einen detaillierten Bericht zur selektiven

Medizinpflanzennutzung im örtlichen traditionellen Gesundheitssystem. Die erhobenen

ethnobotanischen Daten tragen zum Erhalt des lokalen traditionellen Wissens bei und

ermöglichen zukünftige Medikamentenwirkstoffentdeckungen und -entwicklungen. Da

herkömmliche medikamentöse Therapien zunehmend in ihrer Anwendung beschränkt und

wirkungslos sind, werden in dieser Arbeit einige der größten Bedrohungen für die globale

Gesundheit thematisiert, darunter Antibiotikaresistenzen, Malaria, Krebs und Entzündungen

sowie damit in Zusammenhang stehende Symptome wie Schmerzen und Fieber.

Ziele der Doktorarbeit: Die übergeordneten Ziele sind 1) die Dokumentation der medizinischen

Anwendung von (oftmals bislang wenig erforschten) Pflanzenspezies; 2) die Entwicklung einer

bibliographischen Bewertungsmethode für die Auswahl von Medizinpflanzen für

Laborstudien; 3) der Aufbau einer Pflanzenextrakt-Datenbank; 4) die pharmakologische

in vitro Untersuchung dieser Spezies bezüglich ihrer traditionellen Anwendungen; 5) die

Mitwirkung an frühen Wirkstofffindungs- und -entwicklungsphasen durch die Bereitstellung

der wissenschaftlichen Basis für die zukünftige Identifikation von Arzneimittel-Leitstrukturen;

und 6) die Integration von Aspekten der lokalen Community-Arbeit in die Forschung mit dem

Ziel der Förderung einer bidirektionalen Kommunikation mit indigenen Gemeinschaften.

Methoden: In dieser Dissertation werden Resultate von diversen ethnopharmakologischen

Feldforschungsmethoden und pharmakologischen Labor-Assays präsentiert, unter anderem

von a) einer ethnobotanischen Umfrage bei 39 traditionellen Heilerinnen und Heilern aus

29 Dörfern mittels strukturierten Interviews; b) der neuartigen „Degrees of Publication“-

Methode (DoP-Methode); c) antibakteriellen Screenings gegen ein Panel bestehend aus

klinischen Isolaten multiresistenter Humanpathogene; d) Antivirulenz-Experimenten, z.B. der

Überwachung der Quorum Sensing Inhibition und der direkten Protein-Sekretion (į-Toxin) bei

Staphylococcus aureus; e) antiinflammatorischen Untersuchungen (z.B. der Cyclooxygenase-

2-Inhibition); f) Antimalaria-Studien gegen den chloroquin-resistenten Plasmodium

falciparum K1-Stamm; g) Cytotoxizitäts-Counterscreenings (z.B. gegen menschliche

MRC-5SV2-Lungenfibroblasten und HaCaT-Keratinozyten); h) einem Gentoxizitäts-

Beurteilungs-Modell mit Simulierung des menschlichen Lebermetabolismus; und von i) einer

Methode zum Transfer von Forschungsergebnissen zurück zu den traditionellen Heilerinnen

und Heilern in ländlichen indigenen Gemeinschaften.

Ergebnisse: Die lokalen Umfrageresultate zeigten, dass die 16 Pflanzenarten häufig bei der

traditionellen Behandlung von insgesamt 75 Erkrankungen angewendet werden. Viele dieser

Anwendungsberichte wurden erstmalig dokumentiert. Die Entwicklung und Anwendung der

DoP-Methode ermöglichte eine Bewertung darüber, inwiefern eine Spezies bereits erforscht

wurde. Zudem wurde die Qualität der wissenschaftlichen Evidenz eingeschätzt. Insgesamt

wurden 634 von Experten begutachtete wissenschaftliche Publikationen aus einem Zeitraum

von 1960 bis 2019 geprüft. Sechs der 16 Spezies wurden dabei als hochgradig untererforscht

und drei Spezies als untererforscht eingestuft, was die darauffolgenden Laboruntersuchungen

zusätzlich legitimierte. Die Ergebnisse der pharmakologischen Experimente zeigten im

Allgemeinen eine hohe Korrelation zwischen der pharmakologischen Aktivität und der

jeweiligen traditionellen Anwendung. Im Folgenden sind einige Beispiele für die in den

Versuchen am stärksten wirkenden Extrakte aufgeführt: a) Wachstumshemmungsaktivität:

Extrakte aus Zanthoxylum chalybeum- und Harungana madagascariensis-Stammrinde gegen

S. aureus (MIC: 16 und 32 ȝg/mL) und Enterococcus faecium (MIC: 32 ȝg/mL); b) Quorum

Sensing-Hemmaktivität gegen vier S. aureus-Accessory-Gene-Regulator-Allele: Extrakte aus

Solanum aculeastrum-Wurzelrinde und Sesamum calycinum subsp. angustifolium-Blättern

(IC50: 1–64 ȝg/mL); c) selektive COX-2-Hemmaktivität: ein Extrakt aus Leucas calostachys-

Blättern (IC50: 0,66 ȝg/mL); und d) antiplasmodiale Aktivität gegen P. falciparum K1: Extrakte

aus W. ugandensis-Stammrinde und L. calostachys-Blättern (IC50: 0,5 und 5,7 ȝg/mL). Die

Rückführung der Laborergebnisse zu den traditionellen Heilerinnen und Heilern resultierte in

einem Videoartikel, der einen zweitägigen Workshop veranschaulicht.

Fazit: Die Konservierung des traditionellen Wissens der lokalen Heilerinnen und Heiler ist

fortwährend unerlässlich für zukünftige Generationen. Dieses ist vor allem bezogen auf die

Nachfrage nach neuen, effektiveren Medikamenten der Fall, die in der Vergangenheit bereits

häufig in Naturstoffen entdeckt und daraus entwickelt wurden. Die neu vorgestellte

DoP-Methode erwies sich als ein leistungsfähiges Instrument für die Auswahl von traditionell

genutzten Spezies für zukünftige Laborstudien, wie z.B. kostenintensive pharmakologische

Experimente und zeitaufwendige Naturstoffisolationen. Die Ergebnisse der

pharmakologischen Studien unterstützen die potenziellen therapeutischen Wirkungen der in

der Großregion Mpigi genutzten Medizinpflanzen.

Table of contents

Abstract I

Zusammenfassung III

Acknowledgements VI

Abbreviations VII

Thesis structure IIX

Introduction 1

Publication I:

"Ethnobotanical study of selected medicinal plants traditionally used in the rural

Greater Mpigi region of Uganda" 13

Publication II:

"A bibliographic assessment using the Degrees of Publication method: Medicinal

plants from the rural Greater Mpigi region (Uganda)" 32

Publication III:

"Targeting ESKAPE pathogens with anti-infective medicinal plants from the

Greater Mpigi region in Uganda" 51

Publication IV:

"Antiinflammatory medicinal plants from the Ugandan Greater Mpigi region act

as potent inhibitors in the COX-2 / PGH2 pathway" 86

Manuscript V:

"Pharmacological assessment of the antiprotozoal activity, cytotoxicity and

genotoxicity of medicinal plants used in the treatment of malaria in the Greater

Mpigi Region in Uganda" 126

Manuscript VI – Video article with accompanying short written article:

"Transferring ethnopharmacological results back to traditional healers in rural

indigenous communities – The Ugandan Greater Mpigi region example" 169

Discussion and conclusions 181

Awards, achievements, plenary talks, conference and poster presentations,

honorary positions, and press features linked to the PhD studies 193

References 200

Acknowledgements

My eternal gratitude to my supervisor, supporter, mentor, and friend Professor Leif-Alexander

Garbe, who always put his trust in me, let me independently define my own research approach,

and unconditionally facilitated the opportunity for me to follow my dreams, while turning my

passion into an occupation. Thank you, Leif.

Mein größter Dank gebührt meiner Familie, insbesondere meiner Mutter, die immer an mich

glaubte. Durch ihre unerschöpfliche Liebe wurde ich zu der Person, die ich heute bin.

My heartfelt thanks to the traditional healers in the Greater Mpigi region and neighboring

regions who provided the ethnobotanical information that formed the foundation of this thesis.

I would also like to thank my supervisors, Professor Vera Meyer and Professor Juri Rappsilber,

for their valuable guidance and for being available as reviewers for my dissertation. Thanks to

my mentors, Professor Michael Heinrich and Professor Luc Pieters, for providing important

feedback on my studies, and for always being available.

Thanks to Professor Cassandra Quave, who has been an outstanding, supportive, and inspiring

Fulbright host and mentor to me. Dr. Quave has always been nothing but kind, while pushing

me towards greater productivity with her professionalism and expertise.

Thanks to my Ugandan colleague Godwin Anywar and our student assistants for the fruitful

collaboration.

Thanks to Fulbright Germany, the BMBF, the DFG, and the DAAD for funding my research.

Special thanks to the HSNB for letting me conduct research on rather out-of-the-ordinary

topics, and to Mrs. Gaschler, who has been the best university third-party-funded project

coordinator I could ever imagine.

Thanks to my buddy Logan Penniket for the (most likely tiresome) proofreading of most of our

publications.

And lastly, but most importantly, my deepest gratitude to my beautiful wife and best friend,

Inken, who always has my back and supports me unconditionally. Thank you for putting all

your efforts into the various fieldwork stays in Uganda, which we always experienced together.

I am beyond lucky and so grateful to have had you always by my side, for better or for worse,

for more than 12 years now. And it makes me even happier that our partnership will now be

crowned by the birth of our wonderful daughter. I love you, Inken.

VII

Abbreviations

AA: arachidonic acid

AChE: acetylcholinesterase

ACTs: artemisinin combination therapies

2-AF: 2-aminofluorene

agr: accessory gene regulator

AMR: antimicrobial resistance

ATCC: American Type Culture Collection

BHI: brain heart infusion

BUTHGA: Buyijja Traditional Healers Association

CAMHB: cation-adjusted Mueller Hinton Broth

CBD: Convention on Biological Diversity

CC50: 50% cytotoxic concentration

CDC: Centers for Disease Control and Prevention

CFU: colony forming units

CHA: chlorogenic acid

COPE: Committee on Publication Ethics

COX: cyclooxygenase

COX-1: cyclooxygenase-1

COX-2: cyclooxygenase-2

DHA: docosahexaenoic acid

DMSO: dimethyl sulfoxide

DoP: Degrees of Publication

DPPH: 1,1-diphenyl-2-picrylhydrazyl radical

DSMZ: German Collection of Microorganisms and Cell Cultures

ELISA: enzyme-linked immunosorbent assay

EPA: eicosapentaenoic acid

ESI: electrospray ionization

ESKAPE: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,

Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter

species

FC: frequency of citation

GEO: Emory University Herbarium

GI: growth inhibition

GMR: Greater Mpigi region

12(S)-HpETE: 12-S-hydroxyeicosatetraenoic acid

15(S)-HpETE: 15-S-hydroxyeicosatetraenoic acid

IC50: half maximal inhibitory concentration

IL-1: Interleukin-1

IL-4: Interleukin-4

LC-FTMS: liquid chromatography-Fourier transform mass spectrometry

LDH: lactate dehydrogenase

LOX: lipoxygenase

12/15-LOX: 12/15-lipoxygenase

LXA4: lipoxin A4

m.a.s.l.: meters above sea level

MHB: Mueller Hinton Broth

MI: mutagenicity index

MIC: minimum inhibitory concentration

VIII

MMS: methyl methanesulfonate

MRSA: methicillin-resistant Staphylococcus aureus

MS: mass spectrometry

MUH: Makerere University Herbarium

NADP: ß-nicotinamide adenine dinucleotide phosphate disodium salt

2-NF: 2 nitrofluorene

NGO: non-governmental organization

NSAIDs: nonsteroidal antiinflammatory drugs

OD: optical density

PG: prostaglandin

PGD2: prostaglandin D2

PGE2: prostaglandin E2

PGF2Į: prostaglandin F2Į

PGG2: prostaglandin G2

PGH2: prostaglandin H2

PGI2: prostaglandin I2

QSI: quorum sensing inhibition

RFCs: relative frequencies of citation

RvE3: resolvin E3

SDS: sodium dodecyl sulfate

SERNEC: SouthEast Regional Network of Expertise and Collections

SI: selectivity index

sox. Soxhlet extraction

sox. succ.: successive Soxhlet extraction.

TI: therapeutic index

TNF: tumor necrosis factor

TNFĮ: tumor necrosis factor alpha

TSA: tryptic soy agar

TSB: tryptic soy broth

TPC: total phenolic content

TX: thromboxane

WHO: World Health Organization

YFP: yellow fluorescent protein

Thesis structure

This doctoral thesis encompasses six combined manuscripts, which in the following sections

are referred to by their corresponding Roman numerals (I-VI). The dissertation is therefore

presented in cumulative form. All manuscripts were drafted with first authorship and

subsequently submitted to international peer-reviewed journals. Each manuscript, including a

prefaced statement regarding my personal contribution, information about its publication, and

a subsequent supplementary material document (where applicable), represents a separate

chapter of the thesis. Four of these manuscripts have recently been published and are inserted

herein as postprint versions. The remaining two manuscripts are currently undergoing peer-

review processes and are included as preprint versions. The last manuscript is presented in an

innovative new format: a 26-minute video article, which has been submitted to the Video

Journal of Education and Pedagogy. An access link to the video is provided in this thesis, using

an online hosting platform, along with a brief written manuscript, which was also part of the

submission to the journal. In addition, the print version of this thesis includes an enclosed USB

flash drive containing the video article file.

The six manuscripts are listed below:

Publication I:

"Ethnobotanical study of selected medicinal plants traditionally used in the rural Greater Mpigi

region of Uganda"

Schultz, F.; Anywar, G.; Wack, B.; Quave, C.L.; Garbe, L.-A.

published in the Journal of Ethnopharmacology (publisher: Elsevier)

Volume 256, 28 June 2020, https://doi.org/10.1016/j.jep.2020.112742

Permission by Elsevier to reprint the material at no charge in my thesis was obtained, and the

reproduction of the article is confined to the purpose for which permission was given.

Publication II:

"A bibliographic assessment using the Degrees of Publication method: Medicinal plants from

the rural Greater Mpigi region (Uganda)"

Schultz, F.; Anywar, G.; Quave, C.L.; Garbe, L.-A.

published in Evidence-Based Complementary and Alternative Medicine

(publisher: Wiley/Hindawi)

6661565, 2021, https://doi.org/10.1155/2021/6661565

This is an open access article distributed under the Creative Commons Attribution 4.0

International License (CC BY 4.0).

Publication III:

"Targeting ESKAPE pathogens with anti-infective medicinal plants from the Greater Mpigi

region in Uganda"

Schultz, F.; Anywar, G.; Tang, H.; Chassange, F.; Lyles, J.T.; Garbe, L.-A.; Quave, C.L.

published in Scientific Reports (publisher: Nature Research)

volume 10, Article number: 11935, 2020, https://doi.org/10.1038/s41598-020-67572-8

This is an open access article distributed under the Creative Commons Attribution 4.0

International License (CC BY 4.0).

Publication IV:

"Antiinflammatory medicinal plants from the Ugandan Greater Mpigi region act as potent

inhibitors in the COX-2 / PGH2 pathway"

Schultz, F.; Osuji, F. O.; Wack, B.; Anywar, G.; Garbe, L.-A.

published in Plants (publisher: MDPI)

10(2), 351, 2021; https://doi.org/10.3390/plants10020351

This is an open access article distributed under the Creative Commons Attribution 4.0

International License (CC BY 4.0).

Manuscript V:

"Pharmacological assessment of the antiprotozoal activity, cytotoxicity and genotoxicity of

medicinal plants used in the treatment of malaria in the Greater Mpigi Region in Uganda"

Schultz, F.; Osuji, O. F.; Nguyen, A.; Anywar, G.; Scheel, J. R.; Caljon, G.; Pieters, L.;

Garbe, L.-A.

submitted to Frontiers in Pharmacology on March 9, 2021

(in review, publisher: Frontiers Research Foundation)

If accepted, this will be an open access article distributed under the Creative Commons

Attribution 4.0 International License (CC BY 4.0).

Manuscript VI – Video article and accompanying written short article:

"Transferring ethnopharmacological results back to traditional healers in rural indigenous

communities – The Ugandan Greater Mpigi region example"

Schultz, F.; Dworak-Schultz, I.; Olengo, A.; Anywar, G.; Garbe, L.-A.

submitted to the Video Journal of Education and Pedagogy on February 18, 2021

(in review, publisher: Brill)

If accepted, this will be an open access article distributed under the Creative Commons

Attribution 4.0 International License (CC BY 4.0).

Introduction

Ethnopharmacology

The science of ethnopharmacology seeks to investigate the medicinal use of natural materials,

such as plants, macrofungi, microorganisms, animals, and minerals, by humans, while applying

pharmacological, anthropological, and socio-cultural research methods. Various other

branches of science may also be involved in this highly interdisciplinary field (Heinrich, 2014;

Heinrich and Jäger, 2015). For example, some ethnopharmacologists study how indigenous

peoples use plants for the treatment of diseases and healing, and pharmacologically investigate

the recorded and collected species in a laboratory or clinical setting regarding their potential

medicinal effects. Here, the initial fieldwork stages of research are often closely linked to the

scientific discipline of ethnobotany, the study of relationships between plants, culture, and

humans (Alexiades and Sheldon, 1996; Martin, 2004; Balick and Cox, 2020). Ethnobotanical

information, such as the identification of plants used and the traditional methods of harvesting,

preparation, and administration, are often vital for the successful replication and assessment of

pharmacological effects in in vitro and in vivo models (Harrison et al., 2015; Balick and Cox,

2020). The results can be the starting point for the discovery of novel drug candidates and

botanical formulations for therapies, as well as evidence-based safety assessments (Mukherjee

et al., 2010; Heinrich and Jäger, 2015; Balick and Cox, 2020). In today's drug discovery led by

the modern Western pharmaceutical industry, the key role of traditional medicine is evident,

as up to 50% of all FDA-approved drugs on the market are currently derived directly or

indirectly from natural sources (Veeresham, 2012; Atanasov et al., 2015; Li et al., 2019). The

most famous example of an ethnopharmacology-driven success story with worldwide

significance for human health was the discovery and isolation of artemisinin (Qinghaosu) from

Artemisia annua (sweet wormwood), a Chinese medicinal herb called Qinghao (Qinghaosu

Research Group, 1977; Tu, 2004; Tu, 2011). The research leading to this discovery was

conducted by Dr. Youyou Tu and her team, who found a diethyl ether extract of the plant's

leaves that strongly inhibited malaria (Czechowski et al., 2020). Initially, Youyou Tu had

obtained the ethnobotanical information on the traditional use against malaria fevers from an

ancient Chinese herbal book compiled by Ge Hong in China's Eastern Jin Dynasty around

317–420 A.D. (Kong and Tan, 2015; Czechowski et al., 2020). Today, A. annua remains the

sole global source of the drug, and derived artemisinin combination therapies (ACTs) are

considered first-line drugs to combat malaria, saving millions of lives, especially among

children in developing countries (Miller and Su, 2011; Su and Miller, 2015; Czechowski et al.,

2020; Ma et al., 2020). In honor of her contribution to the discovery of artemisinin, the Chinese

ethnopharmacologist Youyou Tu was awarded the Nobel Prize in Physiology or Medicine in

2015 (Croft and Ward, 2015; Efferth et al., 2015). Other well-known examples of outstanding

ethnopharmacological research in the past have been the discovery of 1) paclitaxel (Taxol),

first isolated from the Pacific yew (Taxus brevifolia) and widely used in the chemotherapy of

various types of cancers today; 2) morphine, an analgesic isolated from the opium poppy

(Papaver somniferum); and 3) quinine, derived from the bark of Cinchona spp., which was

originally used medicinally by the indigenous peoples of the Andes and most likely first used

for the treatment of malaria by a European in the 1630s (Klockgether-Radke, 2002; Heinrich

et al., 2015; Permin et al., 2016; Walker and Nesbitt, 2019). An example of a traditionally used

botanical drug that provides the basis for modern phytomedicines is the herb St. John's wort

(Hypericum perforatum), which is prescribed globally to treat mild and moderate cases of

depression (Heinrich et al., 2015; Volz, 2020; Moragrega and Ríos, 2021).

In my personal interpretation of the science of ethnopharmacology, research activities

involve a) field studies (such as ethnobotanical studies in local communities, surveys,

interviews, first-time documentation of medicinal use, ritual use, or religious aspects); b) the

pharmacological assessment of species regarding their respective traditional uses in a

laboratory setting ("bioactivity studies"); and c) the discovery of pharmacologically active

natural products via pharmacognostic approaches, e.g. bioassay-guided fractionation and

isolation of bioactive secondary metabolites (Schultz et al., 2021b). However, these activities

may be further expanded to include community work because I believe that

ethnopharmacologists have a responsibility for the respective indigenous communities they

collaborate with and should therefore also act as their advocates.

Ugandan traditional medicine and its significance

My field research was conducted in the tropical country Uganda, which is located on the

northern shores of Lake Victoria, bordered by Rwanda and Tanzania to the south, the

Democratic Republic of Congo to the west, South Sudan to the north, and Kenya to the east.

Uganda is characterized by a very rich biological diversity due to its unique bio-geographical

location (Kalema, 2005). Seven of Africa's 18 phytogeographical regions occur in Uganda

(Davenport and Matthews, 1995), making it the most phytogeographically diverse country on

the African continent (White, 1983). In total, there are more than 5,000 species of higher plants

present in the indigenous Ugandan flora (Hamilton et al., 2016). From an anthropological

viewpoint, Uganda is also highly diverse in terms of ethnic groups (>50) and languages

(Brockerhoff and Hewett, 2000).

Humans around the world have used plants as medicine since time immemorial, and

traditional medicine continues to be of highest importance for all human beings (Yuan et al.,

2016; Bussmann et al., 2018; Kigen et al., 2019). Africa is more reliant on traditional medicinal

healthcare than any other continent, as up to 90% of its population seeks traditional treatment

provided by healers (WHO, 2013; Ekor, 2014). According to the World Health Organization

(WHO, 2016), Uganda has one physician for every 10,752 patients, which is the lowest

proportion of healthcare professionals with modern medical training per capita in East Africa

(a ratio of modern physicians to inhabitants of 1:1,000 is considered an indicator of a good

health system by the WHO). At the same time, Ugandan traditional healers provide significant

basic medical treatment that has been passed down by previous generations for centuries

(estimated healer to patient ratio in Uganda: 1:100) (Green, 1997; Nyamukuru et al., 2017;

Schultz et al., 2020b). It is important to mention that, in most cases, traditional information on

the use of local herbal drugs has never been pharmacologically investigated or even

documented. These traditional practices and applications of medicinal plants in Uganda

significantly vary between different ethnic groups, cultures, and even neighboring villages. It

is an unfortunate fact that much of this traditional knowledge has already been lost due to

economic drift to the cities (rural-urban migration, Western influence, and deforestation)

(Bussmann et al., 2018). Thus, the work on the documentation of medicinal applications of

understudied plant species that is presented in this PhD thesis further contributes to the

preservation of traditional knowledge, which will be vital for future generations.

The Greater Mpigi region

My field research site in West-Central Uganda covered an area of 715 km2 in the Mpigi,

Butambala, and Gomba districts, which I refer to in the following as the "Greater Mpigi

region". The area of the research site was calculated by drawing a perimeter line around the

locations of 29 villages and 39 traditional healers (study participants). At this field site, the

local vegetation is described as a tropical, moist evergreen forest / savanna mosaic (Barbour,

1987; Howard, 1991). The Greater Mpigi region, including geographical, anthropological, and

socio-economic data, is further described in "Publication I" as part of this PhD thesis (Schultz

et al., 2020b). There is a high dependency on local traditional healers and medicinal plants for

covering primary healthcare needs in this remote area. Nevertheless, reports on medicinal plant

use and the documentation of traditional methods of preparation and administration in the

treatment of individual medical disorders were still scarce at the start of this study (Adia et al.,

2014; Kibuuka and Anywar, 2015; Nyamukuru et al., 2017).

PhD research

In my PhD studies, I investigated 16 selected Ugandan medicinal plants that had been

identified in preliminary studies during my previous field research in Uganda. The approach

applied in this dissertation is highly interdisciplinary and based on ethnopharmacological,

ethnobotanical, ethnomedicinal, pharmacological, and anthropological best-practice methods.

For example, procedures for plant collection, species identification, and the assignment of

scientific names followed the current standards for conducting and reporting

ethnopharmacological field studies at all times (Alexiades and Sheldon, 1996; Martin, 2004;

Heinrich et al., 2018; Weckerle et al., 2018).

The overarching objectives of this thesis were a) the first-time documentation of the

medicinal use of (in many cases understudied) plant species; b) the development of a

bibliographic assessment tool for selection of medicinal plants for lab studies; c) the creation

of a plant extract library; d) the pharmacological in vitro assessment of the 16 species regarding

their respective traditional uses; e) the contribution to early drug discovery stages and thereby

the provision of a scientific basis for future drug lead identification endeavors; and f) the

integration of aspects of community work while fostering a bidirectional communication with

indigenous communities. More detailed objectives for the individual studies summarized in

this thesis are specified in each of the six manuscripts.

The research structure, including individual study stages and the resulting

publications/manuscripts, is illustrated in Figure 1. Each of the six presented manuscripts

contains a thorough introduction, leading to a more profound understanding of the individual

study objectives and their background. Therefore, the various research topics addressed in this

thesis are only introduced briefly in the following paragraphs and subsequently presented in

detail in the manuscripts' introductions (including the corresponding literature reviews).

The first stage of field research was the collection of plant material, guided by traditional

healers. This work included the preparation of herbarium voucher specimens for taxonomic

identification or confirmation, which was carried out subsequently at the Makerere University

herbarium. The collected plant material was processed (e.g., bark was cut into small pieces)

and dried in the shade. An ethnobotanical and anthropological survey among 39 traditional

healers followed, resulting in "Publication I" (Schultz et al., 2020b).

Figure 1: Overview of the structure of my PhD research, including individual study stages and

the resulting publications/manuscripts. GMR: Greater Mpigi region; ESKAPE: Enterococcus

faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,

Pseudomonas aeruginosa and Enterobacter species; agr: accessory gene regulator; MRSA:

methicillin-resistant S. aureus; LC-FTMS: liquid chromatography-Fourier transform mass

spectrometry; COX-1: cyclooxygenase-1; COX-2: cyclooxygenase-2; 12/15-LOX: 12/15-

lipoxygenase; TPC: total phenolic content

In addition, the survey assessed the healers' general knowledge of Western medicine,

including their understanding of infectious diseases, the concept of microbial pathogens, and

cancer. Another topic addressed was the identification of the traditional healers' needs and

future expectations regarding our collaboration.

Next, I planned to develop an urgently needed novel tool for literature assessment in

ethnopharmacological research that would determine which species merit the costly lab studies,

e.g., pharmacological assays and subsequent isolation of active natural product compounds.

This novel method should therefore assess the degree to which a species has already been

studied in the past and, additionally, allow to estimate the quality of the journals in which the

studies have been published. This development work led to the introduction of the "Degrees of

Publication" (DoP) method in a standalone publication ("Publication II"), which also included

a thorough literature review for the 16 selected medicinal plant species from the Greater Mpigi

region (Schultz et al., 2021a).

The plant samples collected during fieldwork on multiple research stays between 2013

and 2017 were regularly taken to the lab, where a total of 86 different extracts were produced.

The extraction procedures are further described in "Publications III and IV" (Schultz et al.,

2020a; Schultz et al., 2021c) and in "Manuscript V." One objective of this PhD research was

the creation of a unique extract library of medicinally used species, which I intend to

continuously expand over the course of my academic career. Once this library had been

established, diverse pharmacological investigations, screening the extract library, were

performed in accordance with the use reports given by the traditional healers (in vitro

bioactivity assessment).

In the context of the ethnobotanical survey in the Greater Mpigi region, the 16

medicinal plant species were found to be critical to anti-infective traditional medicine practices

(in particular, wound infections, skin infections, and symptoms associated with bacterial

infections). Therefore, one study involved screening for growth inhibitory activity against a

panel of life-threatening ESKAPE pathogens. In addition, the extract library was screened for

antivirulence activity against quorum sensing processes in methicillin-resistant Staphylococcus

aureus (MRSA), which represents a promising alternative therapeutic, yet non-antibiotic,

strategy. The study also involved a library counterscreen for cytotoxicity against human

keratinocytes (HaCaT cells), further confirmation of quorum quenching activity via direct

protein output assessment (į-toxin), and the elucidation of putative matches of compounds

present in the best-performing extracts via LC-FTMS. In order to realistically assess the results

of this study for future advances in drug discovery, multidrug-resistant clinical isolates were

used in the experiments. Antimicrobial resistance (AMR) is considered "one of the biggest

threats to global health, food security, and development today," potentially affecting anyone of

any age around the globe (WHO, 2018). For example, in 2017, MRSA alone was associated

with 19,832 deaths and 119,247 severe blood-stream infections in the United States (Kourtis

AP, 2019). It is estimated that, by 2050, more than 10 million people will die annually due to

AMR and associated multidrug-resistant bacterial pathogens worldwide. This figure is even

higher than the current number of annual cancer-related fatalities (O’Neill, 2016). Bacteria can

develop and accumulate adaptations that enable them to survive the drug exposure originally

designed to eradicate them. AMR develops through the long-term accumulation of these

adaptations (Lomazzi et al., 2019), accelerated by the broad deployment of antibiotics (Llor

and Bjerrum, 2014). The worldwide consumption of antibiotics increased by 65% between

2000 and 2015, substantially driven by low- and middle-income countries, where the use of

antibiotic drugs doubled (Klein et al., 2018). Factors leading to the overuse and misuse of

antibiotics in humans, animals, and plants included complacency about the consequences, a

fear of bacteria, a lack of knowledge, and the passing of responsibility between physicians,

patients, pharmacies, and the pharmaceutical industry (Vazquez-Lago et al., 2012). As a

consequence, current antibiotic drug therapies are becoming increasingly inefficient and

limited. This is where traditionally used Ugandan plants and the development of novel

therapeutic strategies come into play. Part of my research therefore sought to tackle the global

health threat posed by AMR in an initial drug discovery study, investigating crude extracts of

species medicinally used by Ugandan traditional healers for the treatment of bacterial

infections. This study subsequently resulted in "Publication II" (Schultz et al., 2020a).

In a second "bioactivity" study, the 16 medicinal plant species were investigated via

in vitro assessment of inhibition of proinflammatory enzymes such as cyclooxygenases (COX)

in the human PGH2 pathway. The ethnopharmacological justification for this study was the fact

that the Ugandan traditional healers had been cited to use the plants in the treatment of

inflammation and related disorders, including pain, redness, heat, fever, wounds, general

infections, and even types of cancer (Schultz et al., 2020b). Inflammation is regarded as one of

the most important human host defense mechanisms, as it represents the immune system's

reaction to invading pathogens and injury (Chaplin, 2010; Ricciotti and FitzGerald, 2011;

George et al., 2014). Diverse medical disorders and their pathogeneses are implicated with

(over- or chronic) inflammation, potentially leading to tissue damage, failure of vital organs

and death (Michaëlsson et al., 1995; Stuhlmüller et al., 2000; George et al., 2014). Mediators

of inflammation are involved in various biochemical signaling pathways. A key role is

attributed to the COX-2 and COX-1 pathways that describe the biosynthesis of prostaglandin

signaling molecules via the precursor prostaglandin H2 (PGH2) (Williams et al., 1999; Ricciotti

and FitzGerald, 2011). However, the main human cyclooxygenase isoforms, COX-2 and

COX-1, do not both play a major role in inflammatory response. COX-1, which is

constitutively present in human cells, catalyzes the biosynthesis of signaling molecules

involved in normal, hemostatic functions, e.g., macrophage differentiation, cytoprotection of

gastric mucosa, renal blood flow, hemostasis, and regulation of cells (Kurumbail et al., 2001;

Botting, 2006). By contrast, the isoform COX-2 is underexpressed in cells under normal

conditions and is rapidly upregulated to elevated levels during inflammation (Botting, 2006;

Cao et al., 2010). The resulting prostanoids include potent proinflammatory mediators that

contribute to or induce medical disorders such as pain, swelling, and fever, and are even

implicated with allergies, arthritis, stroke, types of cancer, asthma, and Alzheimer's disease

(Konturek et al., 2005; Young et al., 2008; Saba et al., 2009; Cao et al., 2010; Ricciotti and

FitzGerald, 2011; Schneider and Pozzi, 2011; Bitto et al., 2017; AlFadly et al., 2019; Hashemi

Goradel et al., 2019; KuĨbicki and BroĪyna, 2020; Sheng et al., 2020). Commercial painkillers

of the group of nonsteroidal antiinflammatory drugs (NSAIDs) are sold and administered

globally on a large scale. Examples of NSAIDs are Paracetamol, Aspirin, and ibuprofen, which

share the capacity for COX inhibition, thus causing a reduction of pain, fever, and inflammation

in the patient. However, these NSAIDs exhibit low selectivity to COX-2, thereby generating

various side effects due to unwanted COX-1 inhibition (Pirlamarla and Bond, 2016; Ho et al.,

2018; Wongrakpanich et al., 2018; Varrassi et al., 2020). Therefore, both human recombinant

isoforms, COX-2 and COX-1, were included in the pharmacological experiments of this study

to make the assessment of the selectivity of the strongest COX-2 inhibitors among the plant

extracts in the library possible. There is a vital need for discovery of novel antiinflammatory

drug leads, and medicinal plants and ethnopharmacological approaches have regained

momentum for the treatment of inflammatory disorders (Ghasemian et al., 2016; Shaikh et al.,

2016). In addition to screening for potential in vitro antiinflammatory effects of extracts, the

study also screened for 12/15-lipoxygenase (12/15-LOX) inhibition activity in the

15-S-hydroxyeicosatetraenoic acid (15(S)-HpETE) pathway and for further antibacterial

activity against multidrug-resistant strains of Listeria innocua, Listeria monocytogenes,

Escherichia coli K12, and S. aureus MRSA, applying a resazurin-based bioassay. In order to

help rule out a potential mechanism of action for the COX-2/1 and 15-LOX inhibition activity

due to an increased presence of polyphenols and free radical scavengers in the complex

extracts, an in vitro antioxidant activity screening model and the determination of total phenolic

contents (TPCs) of individual extracts were also included in the experimental design of this

study. The results were reported in "Publication IV" (Schultz et al., 2021c).

The next study to investigate the pharmacological effects of the 16 medicinal plants

claimed by the traditional healers focused on their antimalarial properties and safety in terms

of cytotoxicity and genotoxicity. During the ethnobotanical survey in the Greater Mpigi region,

the plant species were additionally found to be frequently used in the treatment of malaria,

fever, and related disorders (Schultz et al., 2020b). Although the prevalence of malaria

infection and incidence of related clinical treatment have significantly decreased in sub-

Saharan Africa in recent years (Bhatt et al., 2015; Snow et al., 2017), malaria remains one of

the most severe public health problems in the world (CDC, 2021). In 2020, 5% of the world's

malaria cases were reported in Uganda, while the whole continent of Africa accounted for 94%

of the cases (215 million) and 384,000 deaths (WHO, 2020). Human malaria is transmitted

through the bites of female Anopheles mosquitoes that are infected with protozoan parasites of

the genus Plasmodium (Kotepui et al., 2020). Here, the greatest malaria threat globally is posed

by the species Plasmodium falciparum, which is also responsible for 94% of the malaria cases

in Africa (WHO, 2020; CDC, 2021). Children below five years of age continue to be the most

vulnerable patients, accounting for 274,000 of the malaria deaths worldwide in 2019 (WHO,

2020). The objectives of this study were, therefore, a) the pharmacological investigation of the

16 species regarding their traditional use in treatment of malaria and related fevers by

identifying promising candidates from the extract library via the application of a hemozoin

formation inhibition screening model. Active hits were subsequently followed up through the

assessment of their antiprotozoal effects against chloroquine-resistant Plasmodium falciparum

K1. A cytotoxicity counterscreen against human MRC-5SV2 lung fibroblasts was conducted,

and selectivity indices were calculated. In addition to the traditional use of the plants against

malaria, traditional healers at another of our study sites near the village Nakawuka in Wakiso

District indicated that one of the plants, Plectranthus hadiensis, is frequently used as a ritual

plant to "prepare young women and teenagers for marriage" and to boost their fertility. Details

on the method of preparation and administration are given in "Manuscript V". However, in

these Ugandan communities and according to anecdotal reports, health providers observed a

high incidence of undiagnosed, rapidly growing large breast masses in young female patients

(not necessarily breast cancer). At the study site, breast cancer detection and treatment are

scarce due to inefficiencies in the healthcare system and are often unaffordable for the rural

Ugandan population (Foerster et al., 2019; Nakaganda et al., 2021). During puberty and shortly

thereafter, the development of mammary glands in lifecycle windows represents a window of

susceptibility for breast cancer in teenagers and young women due to DNA damage caused by

mutagens/carcinogens during the increased proliferation of cells (Davis and Lin, 2011; Macias

and Hinck, 2012; Martinson et al., 2013; Natarajan et al., 2020). The frequent ritual use of

P. hadiensis in the Nakawuka area could potentially cause a first incident of cell growth

perturbation. In the long term, this could facilitate more incidents, leading to tumor growth.

Few studies report plants that are suspected of promoting the formation of breast tumors

through growth stimulation by phytoestrogens or mutagenesis (Stopper et al., 2005; Bilal et al.,

2014). Therefore, in the study, the potential genotoxic effects derived from the 16 medicinal

plants, including P. hadiensis, were preliminarily investigated for the first time (objective b)).

This was accomplished by a genotoxicity assessment of the extract library using a Salmonella

reverse mutation bioassay, both without and with metabolic bioactivation after pre-treatment

of extracts with human S9 liver fraction (simulation of human liver metabolism). A manuscript,

resulting from this study, is currently under review with the journal Frontiers in Pharmacology.

The research was conducted in accordance with the international, national, and

institutional recommendations considering the Convention on Biodiversity and the Nagoya

Protocol (at the beginning of the project, no active implementation was in place based on the

information obtained from our Ugandan collaborators). Collaboration agreements between the

universities were signed and are available upon request. Export permits for transfer of plant

samples were obtained from the Ugandan Ministry of Agriculture, Animal Industry and

Fisheries / Plant Health and Inspection Services. Researchers and academic institutions

involved in no way sought to benefit financially from the traditional knowledge shared. Written

informed consent was obtained from all study participants. All results from the lab work on

plants collected and the survey data was transferred back to traditional healers and local

participants. The sixth manuscript therefore addresses an often-neglected issue in

ethnopharmacological research and is of particular importance for this PhD thesis. In the past,

the intellectual property rights of indigenous peoples have often not been recognized. The

Convention on Biological Diversity (CBD) and the Nagoya Protocol finally provided

international agreements for financial benefit sharing and a nation's sovereignty over its

biodiversity (Alexiades and Sheldon, 1996; Heinrich and Jäger, 2015; Heinrich et al., 2018;

Balick and Cox, 2020). However, non-financial benefits, such as the transfer of knowledge in

both directions, are poorly defined in these agreements. Unfortunately, ethnopharmacologists

still rarely return to the local communities once a study has been completed and published

(Maregesi et al., 2007; Schultz et al., 2020b). As a consequence, the extraction of

ethnomedicinal information from indigenous communities and subsequent collection of

samples under the traditional healer's guidance often marks the end point of a one-sided

collaboration. However, the research continues, as samples and data are analyzed, interpreted,

and published, potentially leading to unique, significant discoveries, and the results of these

studies would certainly be of high interest to the local study participants. This issue has been

addressed in the past, e.g., in the book Giving Back - Research and Reciprocity in Indigenous

Settings (Herman et al., 2018), and remains a major problem in the field of ethnopharmacology

today. During the ethnobotanical survey ("Publication I"), one question was added to the

questionnaires, asking the traditional healers about their motivation to collaborate with me, a

Western scientist, and with my Ugandan colleague from Makerere University. Moreover, we

specifically asked them what they expected from us researchers in this study. Their responses

were quite interesting. Although the traditional healers live in rather low socio-economic

circumstances, only 5% stated that they would like to benefit financially from the collaboration.

In fact, the three most frequent responses were, in descending order, that they would like 1) to

receive scientific support through information on whether the investigated plants actually

possessed the claimed medicinal properties; 2) to get feedback on the actual findings of the

pharmacological lab studies that followed the fieldwork; and 3) to continue and improve our

collaboration (Schultz et al., 2020b). These findings indicate that there is a vital need for

feedback and a strong interest in continued collaboration after the study is completed. The

transfer of research results back to local study participants usually fosters an equal

co-partnership and might even empower them locally (Cordell, 1995; Unander et al., 1995;

Vandebroek et al., 2011). I believe that ethnopharmacologists have the important responsibility

to ensure that their collaboration and communication with local study participants is

bidirectional. This can be accomplished, for example, by sharing information, knowledge, and

scientific results, thereby generating a benefit for both the local informant and the scientist. In

this sixth study, a transfer of research results back to the traditional healers who initially

participated in the ethnobotanical survey was accomplished in the form of a two-day workshop.

The description of this method, providing an example for transferring fieldwork and lab results

back to indigenous communities, is presented as a 26-minute video article in this PhD thesis.

The video article uses the same structure as a written scientific article (introduction, methods,

results and discussion, conclusions). It is currently under review with the Video Journal of

Education and Pedagogy, along with a brief accompanying written manuscript. Once accepted,

it will be freely available to the traditional healers and the public via online video platforms.

Publication I:

"Ethnobotanical study of selected medicinal plants traditionally used

in the rural Greater Mpigi region of Uganda"

Pages: 14-31

Personal contribution

In the following, my personal contribution to the presented study and manuscript is briefly

described: I designed the overall strategy of the study and the questionnaires for the

ethnobotanical survey. I conducted part of the fieldwork. I contributed to the processing of the

survey data and the collection of plant material for future lab analysis. Moreover, I interpreted

the majority of the data and wrote most parts of the manuscript. A more detailed author-

contribution statement is given in the published article.

Information on publication

This study was published in the Journal of Ethnopharmacology in June 2020 and is available

at https://www.sciencedirect.com/science/article/abs/pii/S0378874119332313. Permission by

Elsevier to reprint the material at no charge in my thesis was obtained, and reproduction of the

article is confined to the purpose for which permission was given.

Schultz, F.; Anywar, G.; Wack, B.; Quave, C.L.; Garbe, L.-A.: Ethnobotanical study of selected

medicinal plants traditionally used in the rural Greater Mpigi region of Uganda. Journal of

Ethnopharmacology, Volume 256, June 28, 2020

https://doi.org/10.1016/j.jep.2020.112742

Graphical abstract

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journal homepage: www.elsevier.com/locate/jethpharm

Ethnobotanical study of selected medicinal plants traditionally used in the

rural Greater Mpigi region of Uganda

Fabien Schultz

a,c,d,∗

, Godwin Anywar

, Barbara Wack

, Cassandra Leah Quave

d,f

Leif-Alexander Garbe

a,c,e

Institute of Biotechnology, Faculty III - Process Sciences, Technical University of Berlin, Gustav-Meyer-Allee 25, Berlin, 13355, Germany

Department of Plant Sciences, Microbiology and Biotechnology, Makerere University, P.O Box 7062, Kampala, Uganda

Department of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences, Brodaer Str. 2, Neubrandenburg, 17033, Germany

Department of Dermatology, Emory University School of Medicine, 615 Michael St., Atlanta, 30322, Georgia, USA

ZELT - Neubrandenburg Center for Nutrition and Food Technology gGmbH, Seestraße 7A, Neubrandenburg, 17033, Germany

Center for Study of Human Health, Emory University College of Arts and Sciences, 615 Michael St., Atlanta, 30322, Georgia, USA

ABSTRACT

Ethnopharmacological relevance: This study provides the first report on selected traditional medicinal plant use, including parts used and methods of preparation, in

the Greater Mpigi region of Uganda. This data supports the conservation of local traditional ecological knowledge and will facilitate future drug discovery research.

Aim of the study: Our study aimed to conserve culturally and scientifically-valuable medical knowledge of 16 plant species traditionally used in the Greater Mpigi

region in Uganda, namely Albizia coriaria,Cassine buchananii,Combretum molle,Erythrina abyssinica,Ficus saussureana,Harungana madagascariensis,Leucas calostachys,

Microgramma lycopodioides,Morella kandtiana,Plectranthus hadiensis,Securidaca longipedunculata,Sesamum calycinum subsp. angustifolium,Solanum aculeastrum,

Toddalia asiatica,Warburgia ugandensis and Zanthoxylum chalybeum. An additional objective of the study was an ethnological investigation of the socio-cultural

background and medical understanding of diseases treated by traditional healers in the study area.

Materials and methods: A pilot survey in the study area revealed that 16 plant species were frequently used in treatment of a variety of medical disorders. In order to

obtain more complete information, we conducted a broader ethnobotanical survey using structured interviews with 39 traditional healers from 29 villages, speci-

fically asking about the traditional uses of these 16 medicinal species.

Results: Results of the survey confirmed a high level of traditional use of these species in the Greater Mpigi region. In addition, various other traditional uses and

methods of preparation were recorded, most of them for the first time. In total, 75 different medical disorders treated with the plants were documented.

Conclusions: Conservation of traditional knowledge for future generations is vital, as loss has already been recorded due to multiple causes. The need for novel and

more effective drugs derived from natural products is more important than ever, making future studies on herbal remedies both justified and urgently required. The

traditional healers surveyed in this project also have expectations of the research – they would like to be updated about any resulting studies into the pharmacological

efficacy of medicinal plants so that the research findings can inform their confidence in each herbal remedy.

1. Introduction

Plants have been used traditionally as a source of medicine and

natural remedies throughout history by humans across the globe and

medicinal plant use is still the predominant form of healthcare services

in East and Central Africa (Bussmann et al., 2018;Kigen et al., 2019). In

Uganda, four out of five people primarily seek care from traditional

healers and previous studies report that there is at least one traditional

healer per village (Abbo, 2011;THETA, 2001). Subsequently, Uganda

has been reported to have many more indigenous traditional healers

than Western-trained doctors. The traditional healer-to-population ratio

in Uganda is 1:200 compared to 1:20,000 for Western-trained physi-

cians (Abbo, 2011;King, 2002;Tuck and Green, 1997), thus resulting in

100 times as many traditional healers as Western-trained physicians.

Especially in rural areas, these Western-trained physicians are absent,

although the WHO recommends a ratio of at least 1 physician to 1000

people (WHO, 2016). On the other hand, traditional healers meet the

healthcare needs of most Ugandans in a culturally appropriate manner.

Uganda has a very rich biological diversity deriving from its unique

bio-geographical location despite its small size (Kalema and Bukenya-

Ziraba, 2005). The East African country boasts seven of Africa's 18

phytogeographical regions (Davenport and Matthews, 1995). This tally

is higher than that of any other African country (White, 1983). Its

biodiversity includes more than 5000 species of higher plants in the

indigenous flora (Hamilton et al., 2016).

Roughly 86% of Uganda's population are predominantly farmers

who rely on subsistence agriculture and live in rural areas (Turyahabwe

et al., 2013). These farmers are generally poor and 40% live on less than

https://doi.org/10.1016/j.jep.2020.112742

Received 16 August 2019; Received in revised form 2 March 2020; Accepted 3 March 2020

∗

Corresponding author. Institute of Biotechnology, Faculty III - Process Sciences, Technical University of Berlin, Gustav-Meyer-Allee 25, Berlin, 13355, Germany.

E-mail address: [email protected] (F. Schultz).

Journal of Ethnopharmacology 256 (2020) 112742

Available online 26 March 2020

a US dollar per day, which is below the global poverty line

(Turyahabwe et al., 2013). Previous studies amongst the traditional

medicine practitioners in our study region indicate they generally have

very low levels of literacy and schooling (Adia et al., 2014;Nyamukuru

et al., 2017).

Practices of African traditional medicine and application of medic-

inal plants vary between different cultures, geographic and climatic

regions, and even between neighboring villages. In most parts of Africa,

including Uganda, traditional knowledge of healing using plants is

transferred orally from one generation to the other and is often never

documented (Adia et al., 2014;Nyamukuru et al., 2017). Un-

fortunately, much of this traditional knowledge on the medicinal ap-

plication of plants has already been lost due to deforestation, economic

drift to the cities (rural-urban migration), or Western influence in

general (Bussmann et al., 2018). Thus, this first documentation of tra-

ditional knowledge concerning the use of selected medicinal plants in

the Greater Mpigi region is vital for the conservation of traditional

knowledge for future generations.

The aims of this study were two-fold: 1) To document the traditional

use of selected medicinal plant species from tropical Uganda, specifi-

cally in the Greater Mpigi region; and 2) To undertake an ethnological

assessment of the socio-cultural background and medical understanding

of diseases treated by traditional healers in the study area.

2. Materials and methods

2.1. Study area

The study was conducted with traditional healers in the Greater

Mpigi region of central Uganda–a tropical country located on the

northern shores of Lake Victoria, bordered by Kenya to the east, South

Sudan to the north, the Democratic Republic of Congo to the west, and

Rwanda and Tanzania in the south. Apart from the high biodiversity,

Uganda is also characterized by its diversity in terms of ethnic groups

(> 50) and languages (Brockerhoff and Hewett, 2000).

The Greater Mpigi region consists of Mpigi, Butambala and Gomba

districts. Butambala and Gomba were originally counties within Mpigi

district, but were elevated to district status in 2010 (Mpigi-Local-

Government, 2019;UBOS, 2012). Mpigi District is located 0.2274° N,

32.3249° E, whereas Butambala is located at 0° 8′ 16 N and 32° 13′ 5 E

and Gomba is 0° 12′ 0 N and 31° 45′ 0 E. The population of Mpigi,

Butambala and Gomba districts is 273,900, 105,400 and 169,000 re-

spectively, according to the Uganda Bureau of Statistics (UBOS, 2018).

The study area lies between 1182–1341 m above sea level and receives

heavy rainfall in the range of 1513 mm per annum. The main ethnic

group here are the Buganda and the main language spoken is Luganda

(UBOS, 2012). The local vegetation is characterized as a tropical, moist

evergreen forest/savanna mosaic (Barbour et al., 1987;Howard, 1991).

A total of 39 traditional healers from 29 different villages were in-

terviewed. The locations of the villages are shown in Fig. 1. The peri-

meter in which the 29 villages are located adds up to an estimated total

study area of 715 km

. People living in this remote area are highly

dependent on medicinal plants and local traditional healers to cover

their primary healthcare needs.

Most of the traditional healers visited and interviewed belonged to

the Buyijja Traditional Healers Association (BUTHGA), which is af-

filiated with an international NGO called PROMETRA that has a branch

in Uganda (www.prometraug.com). The PROMETRA Uganda head-

quarters is located within the study area in Buwama-Buyija, 67 km from

Kampala along Masaka road. The institution is situated on more than

100 acres of forested land. Through the platform created by PROME-

TRA, traditional healers meet once a week to share ideas, remedies and

learn from each other during workshops.

2.2. Collection of ethnopharmacological data

During preceding pilot study field research expeditions in 2015 and

2016, 16 different medicinal plant species were collected, identified

and the traditional use was recorded through three informants, ac-

companied by a thorough literature review. All of these 16 species were

verbally reported to be highly used medicinally in the rural Greater

Mpigi region. In 2018, ethnobotanical interviews were conducted in the

study area with 39 informants. The three traditional healers from the

pilot study were not among these 39 informants, but all informants

were local practicing traditional healers in their respective home vil-

lages. The ethnopharmacological survey questionnaires were specifi-

cally designed to collect in-depth data on each species (use, parts used,

methods of preparation) because for many of these species, this would

be the first report of their medicinal use in this particular region of

Uganda.

Our research group specializes in ethnopharmacological fieldwork

in Africa and subsequent evaluation of traditional use through phar-

macological bioassays. Therefore, the questionnaires were designed to:

a) collect specific information on the traditional use in treatment of

those diseases where our in vitro model expertise lies, namely ma-

laria, bacterial infections, antiinflammatory disorders and cancer.

This strategy will guide towards further selection and prioritization

of medicinal plants for future pharmacological studies through the

ethnobotanical approach;

b) gather data on the totality of ethnopharmacological uses of the

studied plants within the study area;

c) assess the general knowledge regarding Western Medicine of the

traditional healers in the study area, including understanding of

infectious diseases, the concept of microbial pathogens, and cancer.

Listing known infectious diseases contributed to the identification of

the culturally most-important diseases;

d) identify the traditional healers' needs and future expectations of our

research endeavor and collaboration.

The methodological standards of the survey and the questionnaires

were evaluated prior to the field research according to established re-

commendations (Heinrich et al., 2009;Weckerle et al., 2018). Before

undertaking the survey, written prior informed consent was obtained

from all traditional healers participating in the study after explaining to

them the study aims and what would be involved in obtaining the data.

The questionnaires were in the English and Luganda language

(Appendix A and B Supplementary data). Interviews were conducted in

the Luganda language by GA.

2.3. Collection of plant material and identification of specimens

Linking local plant names with collected plant samples is one of the

major challenges of ethnopharmacological field research (Bennett and

Balick, 2014;Rivera et al., 2014). The 16 selected plant specimens were

collected with representative morphological features under guidance of

the traditional healers. The collection was conducted following the

standard collection procedures (Martin, 2004).

The methodology for plant identification and assignment of scien-

tific names was adapted from Weckerle et al. (2018) in terms of col-

lecting specimens for herbarium vouchers, linking plant names given

during interviews to plants collected for herbarium voucher prepara-

tion, as well as exhaustive collection of plant material and application

of visual aids for identification (KEW database). Scientific names were

cross-checked with http://www.theplantlist.org on August 11th

2019

and family assignments follow The Angiosperm Phylogeny Group IV

guidance (The Angiosperm Phylogeny, 2016). Voucher specimens of all

species collected were deposited at Makerere University Herbarium in

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

Kampala, Uganda and select specimens were also deposited at Emory

University Herbarium (GEO) in Atlanta, GA, USA and digitized speci-

mens are available for viewing through the SERNEC portal at http://

sernecportal.org/portal/.

2.4. Data analysis

Following completion of fieldwork, all questionnaires were pro-

cessed for data analysis, e.g. scanned and data transferred to Excel

sheets. All parameters, such as methods of preparation, information on

herbal drug administration, number of citations of a species for a spe-

cific traditional use or demographic data, were analyzed through de-

scriptive statistics, described below.

2.4.1. FC

Transparency of this study is mainly conferred through information

on the Frequency of Citation (FC), making the study comparable with

other studies in the region and resulting in a reliable assessment of the

cultural use of a plant species (Heinrich et al., 2009). The FC expresses

the absolute number of informants interviewed that use a certain plant

species for a specific event (non-specified medicinal use/specified

medicinal condition or disease). For a single event, it varies from 0

(none of the informants uses this plant species in a specific event) to 39

(maximum number of informants use this plant species in a specific

event; n = 39). The ethnobotanical index FC

event

represents the number

of use reports of a medicinal plant mentioned in each previously de-

fined event (“malaria”, “inflammatory disorders”, “treatment of symp-

toms of general infections”, “skin infections”, “tuberculosis”, “cancer”).

If set into relation with the total number of informants, this absolute

value serves as a parameter for prioritizing the selected plant species for

our future evaluation of pharmacological effects through in vitro

models. The event “total” and the FC

total

describe the total sum of all

use reports for treatment of all medical conditions combined.

3. Results and discussion

3.1. Demographic data

Thirty-nine informants participated in the study; all were rural

traditional healers. The demographic data of these informants is re-

ported in Table 1. The majority were female (24, 61.5%), whereas 15

male traditional healers (38.5%) were interviewed. The age of the in-

formants ranged from 23 to 74 years. The age groups 36–45 (28.2%),

46–55 (23.1%) and 56–65 years (23.1%) were more represented than

younger informants from the age groups 25–35 years (10.3%) or

younger (2.6%). The distribution of traditional knowledge among elder

generations, without passage of knowledge to younger generations has

been exacerbated by economic drift to the cities and Western influence.

This gap in traditional knowledge between the older and younger

generations is more pronounced in neighboring districts, because tra-

ditional medicine is already being taught in organized sessions by the

BUTHGA and the PROMETRA network in the study area. Informants

older than 65 years (12.8%) were also less represented, reflecting that

Fig. 1. Map of the study area in the Greater Mpigi region (715 km

), illustrating the 29 villages where the ethnobotanical survey was conducted (Satellite Data:

Copernicus Sentinel, 2019; processed by the European Space Agency).

Table 1

Demographic data of the informants (n = 39).

Group n%

Gender Female 24 61.5

Male 15 38.5

Age Younger than 25 years 1 2.6

25–35 years 4 10.3

36–45 years 11 28.2

46–55 years 9 23.1

56–65 years 9 23.1

Older than 65 years 5 12.8

Tribe Baganda 33 84.6

Bakiga 2 5.1

Banyankole 2 5.1

Bagwere 1 2.6

Banyarwanda 1 2.6

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

depth of traditional knowledge is vanishing due to the death of rural

elderly people. Most of the informants were members of the Baganda

tribe (33, 84.6%). Other ethnic groups stated include two informants

each from the Bakiga (2) and the Banyankole (5.1%) tribes, as well as

one informant each from the Bagwere (2.6%) and Banyarwanda tribes

(2.6%).

The informants live and practice traditional medicine in a total of 29

different villages, small towns or communities within the study area.

The names of all locations and their approximate coordinates are given

in Table 2. The highest proportion of informants (10.3%) reside in

Buwama, one of the smaller towns along Kampala-Masaka Road, which

is the major road connecting the capital Kampala with the South-

Western parts of the country. Apart from Mpigi town, most of the other

communities are very small, sometimes remote villages and commu-

nities that cannot be reached during heavy rain due to muddy dirt

roads. One community, Bunjako Island, is interestingly located on a

small island in the swamps of Lake Victoria.

3.2. Background of traditional healers

Interviews began with collection of background information on each

healer. This included questioning the traditional healers about their

experience in practicing their profession, about the number of patients

each healer treats per month and about the source of their botanical and

medical knowledge. Results are listed in Table 3.

The level of experience of the survey participants ranged from 2 to

40 years of practicing. This discrepancy is largely due to the variation of

age of the traditional healers, described in subsection 3.1. The largest

part of the informants (41.0%) had practiced medicinal plant use and

healing for 11–20 years in their individual local communities at the

time of the survey. Table 3 also shows the monthly numbers of patients

treated, which ranged from 1 to 40 patients per month.

The source of traditional knowledge varied between the informants:

12.8% of the respondents stated that they acquired their medicinal

knowledge from their parents and 15.4% from their grandparents, re-

sulting in a total of 28.2% of the traditional practitioners where tradi-

tional knowledge was solely transferred from one generation to another

within the family. Ten percent of the respondents mentioned their

knowledge of botanical medicine was obtained via apprenticeship

under other traditional healers. However, with 51.3% the majority of

respondents claimed to have acquired their traditional knowledge by

participating in the BUTHGA platform and the associated PROMETRA

network, reflecting the important work and gain in conservation of

traditional ethnobotanical knowledge performed by these associations

in the region. Of the survey participants, 10.3% reported that their

source of traditional knowledge can be described by a combination of

the traditional knowledge transfer within the family and the modern

BUTHGA/PROMETRA approach.

Another aspect of documenting the background of the traditional

healers was learning more about their awareness and conception of

infectious diseases, as well as their understanding of cancer. During

interviews, healers were asked about the general concept of infectious

diseases and were asked to state three infectious diseases they know

about. According to their own statements, this general concept, such as

that diseases are caused by very tiny organisms invisible to the naked

eye, was clear to them (100% confirmation). However, results indicate

that the Western concept of pathogenic microbiology generally or at

least the differentiation between cancer and infectious diseases is not

fully understood (Table 4). Nearly one third of participants (30.8%)

listed “cancer” as an infectious disease. Other non-infectious diseases

stated were sickle cell disease (5.1%), diabetes (2.6%) and asthma

(2.6%). Malaria (41.0%), HIV/AIDS (33.3%), syphilis (28.2%) and tu-

berculosis (15.4%) were the infectious diseases most prominently

mentioned. Of note, 25.6% of the traditional healers failed to name

more than two infectious diseases/conditions and one healer (2.6%)

was only able to state one such disorder. All participants could name at

least one infectious disease/condition.

All of the 39 informants were aware of diseases summarized under the

general term “cancer”. Following this, they were asked to define ‘cancer’.

Results are shown in Table 5. The greatest proportion of the traditional

healers defined cancer as “wounds that are not healing” (28.2%) or “in-

creased, uncontrolled or abnormal growth of cells” (20.5%). Cancer was

generally referred to as “Kookolo”, which is the condition of cancer

within the body in Luganda, the most common local language. Some of

the answers of informants were related to individual experiences and

hearsay, e.g. “Cancer is caused by eating food sprayed with pesticides”,

“Cancer can be caused by having teeth removed” or “Some menstruating

women develop cervical cancer because of using dirty sanitary pads”.

Table 2

Locations of practice of interviewed traditional healers in rural Central Uganda

(n = 39).

Village n% Approx. coordinates

latitude longitude

Buwama 4 10.3 0.06424 32.10788

Buwere 2 5.1 0.08945 32.10866

Buyijja 2 5.1 0.10659 32.14712

Gombe 2 5.1 0.25657 32.27275

Kamengo 2 5.1 0.1433 32.22426

Kibanga 2 5.1 0.23616 32.2276

Kibibi 2 5.1 0.23859 32.16125

Mpigi 2 5.1 0.22735 32.32492

Nsangwa 2 5.1 0.1 32.11666

Bunjako Island 1 2.6 0.00454 32.13912

Busolo 1 2.6 0.26527 32.14555

Butambala 1 2.6 0.17425 32.10646

Kabira 1 2.6 0.17263 32.26706

Kalamba 1 2.6 0.27824 32.19894

Kibissi-Mawokota 1 2.6 0.04958 32.10692

Kisubi 1 2.6 0.01368 32.09051

Kyabadaza 1 2.6 0.12299 32.1903

Lungala 1 2.6 0.23308 32.35043

Magejjo 1 2.6 0.17883 32.23543

Magya 1 2.6 0.06617 32.12849

Mbizzi Nnya 1 2.6 0.04126 32.08208

Mitala Maria 1 2.6 0.0756 32.11034

Mpenja 1 2.6 0.21894 32.05835

Ntolomwe 1 2.6 0.14685 32.11476

Nvule 1 2.6 0.00179 32.09894

Ssango 1 2.6 0.0351 32.1044

Ssenero 1 2.6 0.11527 32.22715

Ssenge 1 2.6 0.20705 32.19522

Table 3

Information on the background of the interviewed traditional healers (n = 39).

Group n%

Experience as traditional

healer

Less than 5 years 7 17.9

5–10 years 11 28.2

11–20 years 16 41.0

More than 20 years 5 12.8

No. of patients per month Less than 4 3 7.7

5–9 14 35.9

10–14 9 23.1

15–19 2 5.1

20–24 8 20.5

25–29 0 0.0

30–34 2 5.1

35–39 0 0.0

More than 40 1 2.6

Origin of traditional

knowledge

Parents 5 12.8

Grandparents 6 15.4

Other traditional healer 4 10.3

Buyijja Traditional Healers

Association (BUTHGA)

20 51.3

Combination of above 4 10.3

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

3.3. Collection of plant species

In total, 16 medicinal plants were collected in the Greater Mpigi

region. Table 6 lists these selected plant species, along with their plant

families, life forms, voucher specimen details and local names in Lu-

ganda. Some of these species are highly understudied, whereas others

are known to be incorporated in the African traditional medicine

system inter-regionally.

Voucher specimens of all 16 selected species were deposited at the

Makerere University Herbarium in Kampala, Uganda. Additional vou-

cher specimens of Leucas calostachys, Sesamum calycinum subsp. angu-

stifolium,Morella kandtiana,Harungana madagascariensis and Warburgia

ugandensis were deposited at the Emory University Herbarium in

Atlanta, GA, USA and made available as part of the digitized collection

in the SERNEC portal (SERNEC, 2019). Herbarium voucher numbers

are provided in Table 6.

3.4. Relative importance of medicinal plants

The majority of the 16 plant species focused on in this study have

recently been reported to be used medicinally in Uganda, the East

African region or different parts of the continent, demonstrating their

relative significance in African traditional medicine (Alebie et al., 2017;

Bunalema et al., 2014;Jima and Megersa, 2018;Katumba et al., 2004;

Kibuuka and Anywar, 2015;Lamorde et al., 2010;Lukhoba et al., 2006;

Malan et al., 2015;Maroyi, 2013;Mongalo and Makhafola, 2018;Moshi

et al., 2012;Muazu and Kaita, 2008;Mukungu et al., 2016;Nyamukuru

et al., 2017;Ochwang'i et al., 2014;Orwa et al., 2008;Shaheen et al.,

2017;Ssegawa and Kasenene, 2007;Tabuti, 2008;Tabuti et al., 2003;

Tariq et al., 2017;Tuasha et al., 2018;Tugume et al., 2016;Vanga

et al., 2018;Wambugu et al., 2011).

Taking the Greater Mpigi region into account, the selection of

medicinal plant species as study objects was done prior to the survey

and based on pilot study interviews with three traditional healers from

the study area. Plant species were chosen because they were cited as

playing a significant role in the local traditional medicine system, al-

though the majority of these species are still understudied in a la-

boratory setting. Results of the survey show that this pre-assessment

was accurate, as all of the 16 selected medicinal plant species were

Table 4

Knowledge of traditional healers regarding the connection between diseases

and contagiousness through microorganisms (n = 39).

Group n%

Knowledge about infectious diseases

and microorganisms

Yes 39 100.0

No 0 0

If yes, which three infectious diseases

were named

Malaria 16 41.0

HIV/AIDS 13 33.3

Syphilis 11 28.2

Tuberculosis 6 15.4

Typhoid fever 5 12.8

STDs 5 12.8

Candidiasis 4 10.3

Gonorrhea 4 10.3

Ringworms 2 5.1

Measles 2 5.1

Skin rashes 2 5.1

Flu 2 5.1

Mumps 1 2.6

Skin infections 1 2.6

Fungal infections 1 2.6

Bacterial infections 1 2.6

Potentially infectious

conditions stated instead:

Stomachache/vomitting 3 7.7

Fever 2 5.1

Cough 2 5.1

Non-infectious diseases

stated:

Cancer 12 30.8

Sickle Cell Disease 2 5.1

Diabetes 1 2.6

Asthma 1 2.6

Inability to name three

diseases:

Just two diseases/

conditions stated

10 25.6

Just one disease/condition

stated

1 2.6

No diseases/condition

stated

0 0.0

Table 5

View of interviewed traditional healers on cancer (n = 39).

Group n%

Knowledge about cancer Yes 39 100.0

No 0 0.0

If yes, how would you define of cancer External or internal wounds that are not healing result in cancer. 11 28.2

Cancer is a malignant disease caused by overproliferation/increased, uncontrolled or abnormal growth of body cells. 8 20.5

When cells and body parts die and refuse to leave the body, they form “Kookolo” known as cancer. 4 10.3

Cancer is a disease resulting in spoilt/destroyed cells in the body. 3 7.7

Cancer is a disease that is incurable. 3 7.7

Cancer is a prolonged body disorder/sickness. 3 7.7

Cancer is an internal wound that does not heal and is infected by bacteria. 2 5.1

Cancer is a disease caused by heavy metals. 2 5.1

Cancer is an abnormal functioning of the body. 2 5.1

Wounds/conditions that are not treated results in cancerous cells. 2 5.1

Cancer is a non-contagious disease. 1 2.6

Cancer are wounds that affect the uterus, lungs and liver. 1 2.6

Cancer are wounds that are internal and destroy the bones. 1 2.6

Cancer is a disease that cannot easily be treated. 1 2.6

Cancer attacks the breast. 1 2.6

Cancer needs Western diagnosis unless it is skin cancer. 1 2.6

Cancer is caused by eating food sprayed with pesticides. 1 2.6

Cancer is when white blood cells begin functioning abnormally and rebel against the body system. 1 2.6

Cancer is an infection for example of the breast, uterus, or wounds (Candida causes uterus cancer). 1 2.6

Some menstruating women develop cervical cancer because of using dirty sanitary pads. 1 2.6

Cancer is a disease caused by diet or change of behavior. 1 2.6

Cancer can be caused by having teeth removed. 1 2.6

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

confirmed to be frequently used as medicines in the study area. Table 6

reports the number of survey participants (FCs) that claim to use a

particular species at all, verifying the general knowledge of the selected

medicinal plant species and their traditional use.

Albizia coriaria and Erythrina abyssinica are the two species that are

used medicinally by all participants. In total, 10 out of 16 of the se-

lected plant species were mentioned by at least 87.2% or more of the

survey participants. Only Zanthoxylum chalybeum,Leucas calostachys,

Microgramma lycopodioides and Securidaca longipedunculata were re-

corded by less than half of the traditional healers interviewed. Still,

these plant species are also regarded as being widely used in the local

traditional health system as 18 informants, 17, 17 and 15 respectively,

claimed to utilize these species regularly.

By determining the information whether a specific plant is used in

this particular study area in the Greater Mpigi region (FC), our research

sought to establish a comparable relationship between local traditional

use and traditional use within the East African region. Our following

assessment on the total use reports not only takes the five predefined

medical conditions into account (“malaria”, “inflammatory disorders”,

“skin infections”, “tuberculosis”, “cancer”), but also every single in-

dividual medicinal use stated in the questionnaire category “other”

(FC

total

= sum of all use reports in the survey). Calculated FC

total

values

are shown in Fig. 2.

A. coriaria, Warburgia ugandensis and E. abyssinica were the pre-

dominant species that exhibited the highest number of use reports in

the study area, followed by Plectranthus hadiensis,Ficus saussureana and

Toddalia asiatica. These plants reflect a high use in the treatment of a

large variety of diseases and medical disorders. The lowest numbers of

use reports were calculated for M. lycopodioides,L. calostachys and S.

longipedunculata. The low number of use reports was as expected for L.

calostachys and M. lycopodioides, as these are understudied species.

However, in case of S. longipedunculata, the low number of use reports

was surprising as it grows and is used medicinally all over the African

continent (Mongalo et al., 2015;Okoli et al., 2005).

3.5. Specification of traditional use

Selection of medicinal plants was based on previous pilot ethno-

botanical studies within the study area. Particular emphasis was placed

on questioning the survey participants about their traditional use of

plants in treatment of malaria, inflammatory disorders, treatment of

symptoms of general infections, skin infections, tuberculosis and

cancer. Apart from plants described in the treatment of a certain disease

or medical condition, information about the methods of preparation,

administration and the plant parts used were also recorded. Plant parts

used medicinally and therefore mentioned in the survey were bark (B),

leaves (L), roots (R), root bark (RB), seeds (S), stem (ST), stem bark

(STB). fruits (FR), flowers (F) and the whole plant (WP).

The following subsections provide summaries of the results for each

of these medical conditions and their treatment with the selected

plants.

3.5.1. Malaria

Forty-one percent of the traditional healers interviewed mentioned

malaria among the three infectious diseases that came first to their

mind (see Table 4). In Uganda, malaria still kills more than 200 chil-

dren daily (Sub-Saharan Africa > 1200), making research on botanical

antimalarial treatments and the discovery of “novel” natural remedies a

high priority (Chinsembu, 2015). In the African traditional medicine

system, medicinal plants for treatment of malaria are well established,

yet many species remain unknown to scientists, undocumented, or at

least have never been investigated for antimalarial activity and efficacy

in the lab (Onguéné et al., 2013;Titanji et al., 2008).

The traditional use of plants in treatment of malaria, plant parts

used and methods of preparation and administration are summarized in

Table 7. As indicated in our initial pilot study, all 16 plant species were

confirmed to be used against malaria in traditional medicine within the

Greater Mpigi region. W. ugandensis (27), A. coriaria (26), T. asiatica

(25) and E. abyssinica (25) were the species most often mentioned and

reached the highest numbers of malaria-specific use reports. Species

with low FCs

malaria

within the study area are M. lycopodioides (4), L.

calostachys (7) and S. longipedunculata (7). Eight out of the nine plant

species with high FC

malaria

(> 14) were previously described in the

ethnobotanical literature for their traditional use in malaria treatment

in Mpigi District, Uganda, which is a part of the Greater Mpigi region

(Adia et al., 2014). Our study therefore confirms and extends the list of

plants used against malaria in the study area.

For the vast majority of plant species, the stem bark, the leaves, or

both are used in traditional medicine within the study area. Often, the

whole plant, meaning multiple plant parts at once, are also boiled and

Table 6

Medicinal plants selected for the ethnobotanical survey and related FCs, confirming general knowledge and high traditional use of the selected species in the study

area (n = 39).

Botanical name Family Local name (Luganda) Life form Voucher specimen no. FC

Albizia coriaria Oliv. Fabaceae Mugavu Tree AG203

Cassine buchananii Loes. Celastraceae Mbaluka Shrub/small tree AG198

Combretum molle R.Br. ex G.Don Combretaceae Ndagi Shrub/small tree AG191

Erythrina abyssinica DC. Fabaceae Jjirikiti Shrub/small tree AG199

Ficus saussureana DC. Moraceae Omuwo Strangler/tree AG219

Harungana madagascariensis Lam. ex Poir. Hypericaceae Mukabiiransiko Shrub/small tree AG230

23180

Leucas calostachys Oliv. Lamiaceae Kakuba musulo Herb AG195

23175

Microgramma lycopodioides (L.) Copel. Polypodiaceae Kukumba Fern AG639

Morella kandtiana (Engl.) Verdc. & Polhill Myricaceae Mukikimbo Shrub AG201

23174

Plectranthus hadiensis (Forssk.) Schweinf. ex Sprenger Lamiaceae Kibwankulata Herb AG210

Securidaca longipedunculata Fresen. Polygalaceae Omukondwe Tree AG196

Sesamum calycinum subsp. angustifolium (Oliv.) Ihlenf. & Seidenst. Pedaliaceae Lutungotungo Herb AG205

23173

Solanum aculeastrum Dunal Solanaceae Ekitengo Shrub/small tree AG193

Toddalia asiatica (L.) Lam. Rutaceae Kawule Shrub AG190

Warburgia ugandensis Sprague Canellaceae Abasi Tree AG220

23181

Zanthoxylum chalybeum Engl. Rutaceae Ntaleyaddungu Shrub/tree AG204

deposited at Makerere University herbarium

deposited at Emory University herbarium

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

Fig. 2. Total number of use reports (FC

total

) of the selected medicinal plant species in the study area.

Table 7

Results of analysis of traditional use of selected plant species in treatment of malaria (n = 39); FCs for total specific use and for the sum of FCs for mode of

preparations/administrations per species might differ due to participants mentioning multiple parts used and/or methods.

Plant species FC

malaria

Parts used Mode of preparation and administration (FC)

Albizia coriaria 26 STB boiled (20); powdered into water (5); pounded and boiled (1), taken orally

Cassine buchananii 11 B boiled (7), taken orally

L boiled (3); powdered into tea (2), taken orally

STB mixed with other herbs, boiled (1), taken orally

Combretum molle 19 STB boiled (12); powdered into water (2); mixed with other herbs, boiled (1), taken orally

L boiled (7), taken orally

Erythrina abyssinica 25 STB boiled (18); powdered into water (6), taken orally

L boiled (2), taken orally

F boiled (1), taken orally

Ficus saussureana 19 STB boiled (13); powdered into water (6), taken orally

Harungana madagascariensis 14 STB boiled (12); powdered into water (5), taken orally

L boiled (3); powdered into tea (1), taken orally

S boiled (1), taken orally

WP boiled (1), taken orally

Leucas calostachys 7 L boiled (5); powdered into tea (1), taken orally

WP boiled (1), taken orally

Microgramma lycopodioides 4 L boiled (3); powdered into tea (2), taken orally

R boiled (1), taken orally

Morella kandtiana 16 RB boiled (2); powdered into cold water (1), taken orally

L boiled (2); fresh leaves chewed (1); powdered, then licked (1), taken orally

R boiled (6); powdered, boiled (4), taken orally

WP boiled (2); steamed (1), taken orally

Plectranthus hadiensis 23 L boiled (19); powdered into tea (1); cold pressed, boiled (1); mixed with other herbs, boiled (1), taken orally

R boiled (1), taken orally

ST boiled (1), taken orally

WP boiled (1), taken orally

Securidaca longipedunculata 7 B boiled (5), taken orally

L boiled (1), taken orally

R powdered, boiled (1), taken orally

Sesamum calycinum subsp. angustifolium 11 L boiled (8); powdered into tea (1), taken orally

WP boiled (2), taken orally

Solanum aculeastrum 11 STB boiled (1), taken orally

L boiled (2), taken orally

R boiled (8); powdered, boiled (1), taken orally

Toddalia asiatica 25 STB boiled (1); powdered into cold water (2), taken orally

L boiled (7), taken orally

R boiled (11); powdered, boiled (9), taken orally

Warburgia ugandensis 27 STB boiled (15); powdered into tea (5); fresh bark chewed (1); taken orally

L boiled (8); powdered into tea (2), taken orally

Zanthoxylum chalybeum 12 STB boiled (3), taken orally

L boiled (4); powdered into tea (1); mixed with other herbs, boiled (1), taken orally

R boiled (3), taken orally

key: B = bark, L = leaves, R = roots, RB= root bark, S = seeds, ST = stem, STB = stem bark, FR = fruits, F = flowers, WP = whole plant

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

consumed for treatment. In addition, other plant parts prepared and

administered, but less often cited, include stems, seeds, flowers and

roots. The predominant mode of preparation is as aqueous decoction.

Some plant parts are also combined with tea leaves when preparing tea

(e.g. Cassine buchananii leaves, W. ugandensis stem bark and leaves, M.

lycopodioides leaves), chewed (M. kandtiana leaves, W. ugandensis stem

bark) or licked (M. kandtiana leaves). The method of drug delivery for

treatment of malaria with the plants of interest is always oral admin-

istration.

3.5.2. Inflammatory disorders

Inflammation is one of the most important human host defense

mechanisms, as it is the immune system's reaction to injury and in-

vading pathogens. Although these mechanisms are essential for life, in

some cases acute or chronic inflammation may lead to tissue damage

and lethal failure of vital organs (George et al., 2014;Ricciotti and

FitzGerald, 2011). In recent years, more studies have focused on dis-

covery of novel antiinflammatory herbal remedies, following ethno-

botanical leads. Prior to these endeavors, the traditional use of medic-

inal plants to treat inflammatory disorders in different cultures needed

to be thoroughly preserved and documented. In our survey, we sought

to investigate treatment of inflammation in the study area and the

cardinal signs of acute inflammation, i.e. pain, swelling, heat, redness

and wound infection/healing were combined in the category “in-

flammatory disorders”. Results are reported in Table 8.

W. ugandensis (19), A. coriaria (16) and T. asiatica (13), H. mada-

gascariensis (12), and E. abyssinica (12) showed the highest numbers of

use reports (FC

inflammation

). Subsequently, these species were most often

associated with treatment of inflammatory disorders among the selec-

tion of medicinal plants investigated in this study. Plant species less

used to treat inflammatory disorders in the study region are L. calos-

tachys (2), S. longipedunculata (2) and M. lycopodioides (5). Generally,

plant parts mostly used are stem bark, roots, root bark and leaves. Some

exceptions are S. aculeastrum fruits and seeds, and A. coriaria seeds.

The modes of administration differed depending on the area of in-

flammatory disorder and the type of symptoms. In most instances, plant

parts, powders or aqueous decoctions are taken orally as an analgesic.

Another common method is applying processed plant parts, decoctions

or powders incorporated in petroleum jelly directly on wounds or

swollen body parts. Wounds are sometimes treated by pressing leaves

directly against the wounds, e.g. C. buchananii (fresh leaves), Sesamum

calycinum subsp. angustifolium (boiled leaves) and Leucas calostachys

(boiled leaves). Some more rare modes of preparation and adminis-

tration include sniffing M. kandtiana root powder, warming P. hadiensis

leaves over the fire and application on the inflamed body part, and

burning dry fruits from S. aculeastrum prior to the ashes being added to

petroleum jelly and subsequent smearing onto the inflamed body area.

In some cases, traditional uses in pain management of certain body

areas and corresponding FCs were additionally recorded: a) headache,

b) joint pain, c) back pain, d) chest pain, e) bone pain, and f) labor pain.

In our study, we also requested information on the herbal treatment of

toothache. Four of the 16 plant species investigated are traditionally

used for toothache in the study area. Here, aqueous decoctions of A.

coriaria stem bark, P. hadiensis leaves or Z. chalybeum roots are used to

rinse the mouth without swallowing. Fresh P. hadiensis leaves are also

chewed on the site of the toothache. One informant stated that he

prescribes C. buchananii root powder as a painkiller, which is then

sniffed in the case of toothache.

3.5.3. Treatment of symptoms of general infections

In traditional medicine practices, identification of pathogenesis is

also often based on holism, including spiritual, philosophical and socio-

cultural conceptions, as well as the character and emotions of the pa-

tient. Unlike Western medicine and the pharmaceutical industry, where

accurate extractions of plants or selective synthesis of pure active mo-

lecules are performed according to established protocols, in traditional

medicine on the community level, plants or plant parts are mostly used

with individually varying methods of preparations, e.g. as decoctions,

pills, juices or fresh (Firenzuoli and Gori, 2007). As reported in other

studies, indigenous peoples often do not clinically diagnose a particular

disease, but rather prescribe herbal drug mixtures to treat the totality of

symptoms that are mentioned by the patient (Vandebroek et al., 2008;

Wachtel-Galor and Benzie, 2011). As all of the 16 selected medicinal

plants are widely used against an array of diseases and medical con-

ditions in the Greater Mpigi region, this subsection elaborates on their

use for treatment of symptoms that indicate contagion with an in-

fectious disease. This disease might be of more serious nature, such as

tuberculosis, malaria, typhus or cholera, but also of less harmful nature,

e.g. the common cold.

Symptoms categorized in this subsection were sore throat, fever,

stomachache/gastrointestinal tract (GI) disorder, nausea and cough.

The FCs for the traditional use of these symptoms are reported in Fig. 3.

F. saussureana,T. asiatica, P. hadiensis and S. longipedunculata are the

species most often cited in treatment of sore throat. Considering our

previous results on the general knowledge and traditional use of the

selected plant species among the participants (Table 6), it is surprising

that S. longipedunculata was mentioned amongst the highly used spe-

cies. This is because 61.5% of the informants claimed that they do not

use S. longipedunculata at all, making it the least used and least known

plant species among the 16 species investigated. Thus, this species re-

veals a high specialization in treatment of sore throats within the study

area. In the literature, mainly the roots and bark are cited for treatment

of different conditions in African traditional medicine (Borokini et al.,

2013;Okoli et al., 2005;Semenya et al., 2013;Sobiecki, 2008). In our

study area, an aqueous decoction of the leaves of S. longipedunculata,

combined with a few drops of lemon juice, is used for treatment of sore

throat. To the best of our knowledge, this specific use of the leaves

against sore throats is reported for the first time. Only four ethnobo-

tanical studies report use of the roots for flu symptoms, such as cough in

Nigeria (Motlhanka and Nthoiwa, 2013;Mustapha, 2013), influenza in

the Ugandan Bulamogi county (Tabuti et al., 2003) or cough and oral

sores for treatment of opportunistic infections among people living with

HIV/AIDS in Uganda (Anywar et al., 2020). One paper from 1962

mentions the use of the leaves in treatment of skin wounds and sores in

Eastern Tanzania (Watt and Breyer-Brandwijk, 1962). When it comes to

results of pharmacological assays published, most studies also in-

vestigate the roots. However, one study reported certain antibacterial

and antifungal properties of a crude extract made from S. long-

ipedunculata leaves, supporting the traditional application of the leaves

in the Greater Mpigi region (Karou et al., 2012). Interestingly, T. asia-

tica is used by the Keiyo Community in the Kenyan Elgeyo Marakwet

County to treat common colds (chewed leaves and bark) and a disease

called “Koroitab mokto” that translates to “Disease of the Throat”

(Kigen et al., 2014). After documenting a description of the symptoms

by local herbalists, the research team concluded that the symptoms

were similar to those of throat cancer. Here, an aqueous concoction of

the roots along with a combination of other plant roots is prepared and

administered orally.

Plants most often referred to for treatment of fever were T. asiatica,

P. hadiensis,W. ugandensis and E. abyssinica. These four species were

also among those most often used for treatment of inflammatory dis-

orders. This overlap may be explained by the generalized molecular

mechanism of action of non-steroidal antiinflammatory drug molecules

(NSAIDs). NSAIDs achieve their pharmacological activity by inhibition

of cyclooxygenase-2 (COX-2) in the prostaglandin H2 (PGH2) signaling

pathway, therefore relieving pain and reducing fever non-selectively

(Ho et al., 2018;Simmons et al., 2000;Steinmeyer, 2000). P. hadiensis is

used in Ayurvedic formulations to treat any type of carcinoma and

chronic inflammation, and showed promising antiinflammatory and

cytotoxic properties in two pharmacological studies by an Indian re-

search group (Menon et al., 2011,2014). Moreover, a total of 15 species

of the genus Plectranthus are known to be used for treatment of fever

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

and infections (Lukhoba et al., 2006). However, according to data ob-

tained from our literature assessment, the use of P. hadiensis against

fever is now reported for the first time. W. ugandensis has previously

been reported as an anti-fever remedy in Kenya (Jeruto et al., 2008). In

other Ugandan regions, e.g. in Butebo County in the Eastern part of the

country, E. abyssinica is used against fever, among other traditional uses

(Anywar et al., 2020;Philip et al., 2017).

In terms of treatment of stomachache or GI tract related disorders,

T. asiatica, A. coriaria and F. saussureana were most often cited.

Generally, most of the selected plants were used to treat symptoms of

this category, as 13 of the 16 plant species were cited by 5 informants or

more.

E. abyssinica was most often named when participants were ques-

tioned about herbal medication against nausea, followed by P. ha-

diensis,T. asiatica, W. ugandensis and M. kandtiana. The sap of E. abys-

sinica was previously described to be used to prevent vomiting (Chitopo

et al., 2019).

Almost half of the traditional healers questioned stated that they

Table 8

Medicinal use of the study species in treatment of inflammatory disorders, such as pain, redness, heat, swelling and wound treatment in the Greater Mpigi region

(n = 39); FCs for total specific use and for the sum of FCs for mode of preparations/administrations per species might differ due to participants mentioning multiple

parts used and/or methods.

Plant species FC

inflammation

Parts used Mode of preparation and administration (FC)

Albizia coriaria 16 STB boiled (9), taken orally; powdered in petroleum jelly, then smeared on body part (5); powdered on wound

(1), applied topically

S broken and then smeared on wound/swollen body part (1), applied topically

Cassine buchananii 7 B boiled (5), taken orally; powdered in petroleum jelly, then smeared on body part/wound (1), applied

topically

L boiled (1), taken orally; boiled, then pressed on wound/swollen body part (1), applied topically

R powdered and sniffed (1), nasal administration

Combretum molle 10 STB boiled (7), taken orally; powdered in petroleum jelly, then smeared on body part/wound (1), applied

topically

L boiled (3), taken orally; boiled, then pressed on wound/swollen body part/wound (1), applied topically

Erythrina abyssinica 12 STB boiled (7); mixed with other herbs, boiled and decoction is drunk (2), taken orally; powdered in petroleum

jelly, then smeared on body part/wound (2); mixed with other herbs, boiled in petroleum jelly and then

pressed against wound/swollen body part (1), applied topically

F boiled (1), taken orally; boiled, pressed around the pain area (1), applied topically

Ficus saussureana 11 STB boiled (6); powdered into tea (2), taken orally; boiled and used for cleaning wound (1), applied topically

L boiled (1), taken orally; juice extracted from fresh leaves and smeared on the affected area (1); fresh leaves

directly put on swollen wound (1); boiled and used for cleaning wound (1), applied topically

Harungana madagascariensis 12 STB boiled (8), taken orally; powdered in petroleum jelly, then smeared on body part/wound (1); mixed with

other herbs, boiled in petroleum jelly and then pressed against wound/swollen body part (2), applied

topically

L boiled (1), taken orally

RB boiled (1), taken orally

Leucas calostachys 2 L boiled (1), taken orally; boiled, then pressed on wound/swollen body part (1); juice extracted from fresh

leaves and smeared on the affected area (1), applied topically

Microgramma lycopodioides 5 L boiled (2); powdered into water (1); squeezed into cold water and drunk (1), taken orally; boiled, then

pressed on wound/swollen body part (1); juice extracted from fresh leaves and smeared on the affected area

(1), applied topically

R boiled (1), taken orally

Morella kandtiana 8 RB mixed with other herbs, boiled and decoction is drunk (1), taken orally; powdered in petroleum jelly, then

smeared on body part/wound (1), applied topically

L powdered, then licked (1), taken orally

R boiled (3); crushed, boiled (1), taken orally; powdered, sniffed (1), nasal administration

Plectranthus hadiensis 11 L boiled (6); fresh leaves chewed near location of toothache (3), taken orally; boiled, then pressed on wound/

swollen body part (2); powdered in petroleum jelly, then smeared on body part/wound (1); juice extracted

from fresh leaves and smeared on the affected area (2); warmed over fire, applied on inflamed part (2),

applied topically

Securidaca longipedunculata 2 B boiled (2), taken orally

Sesamum calycinum subsp. angustifolium 10 L boiled (4); powdered into cold water (1), taken orally; juice extracted from fresh leaves and smeared on the

affected area (1); boiled, then pressed on wound/swollen body part (3), applied topically

WP mixed (fresh) with other herbs, then put on a swollen wound (1), applied topically

Solanum aculeastrum 9 L powdered in petroleum jelly, then smeared on body part/wound (1), applied topically

R boiled (3); crushed, boiled (1), taken orally

FR mixed with petroleum jelly, rubbed on swollen body part (1); dry fruits burned to ash, ash added to

petroleum jelly, then smeared onto inflamed area (1), applied topically

S crushed, then smeared on swollen body part/wound (2), applied topically

Toddalia asiatica 13 STB powdered in petroleum jelly, then smeared on body part/wound (2), applied topically

L boiled (3), taken orally

R boiled (6); crushed, boiled (3), taken orally

RB mixed with other herbs, boiled and decoction is drunk (1), taken orally

Warburgia ugandensis 19 STB boiled (11); powdered into tea (1), taken orally; powdered in petroleum jelly, then smeared on body part/

wound (3), applied topically

L boiled (5), taken orally; powdered in petroleum jelly, then smeared on body part (3); juice extracted from

fresh leaves and smeared on the affected area (1), applied topically

R boiled (1), taken orally

Zanthoxylum chalybeum 7 STB boiled (1), taken orally; powdered in petroleum jelly, then smeared on body part/wound (1), applied

topically

L boiled (2), taken orally; boiled, then pressed on wound/swollen body part (1), applied topically

R boiled (3), taken orally

key: B = bark, L = leaves, R = roots, RB= root bark, S = seeds, ST = stem, STB = stem bark, FR = fruits, F = flowers, WP = whole plant

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

prescribe C. molle for treatment of cough. Other often-cited plants in

this category were A. coriaria,W. ugandensis and M. kandtiana.

According to a book published in 1976, C. molle is taken to treat chest

conditions in Kenya (Kokwaro, 1976).

The calculated FCs

symptoms

combine the total use of the five in-

dividual, symptom-specific FCs per species and are shown in Table 9,

along with the plant parts used, and modes of preparation and ad-

ministration.

3.5.4. Skin infections

Skin and soft tissue infections (SSTIs) are frequently encountered by

medical staff and traditional healers in Uganda and worldwide. The

human skin is characterized by a notable ecological diversity of mi-

croorganisms that could cause infection, if unbalanced or disturbed.

SSTIs encompass a broad set of conditions, and pathogenesis may range

from simple infections, e.g. subcutaneous abscesses or pyoderma, to

life-threatening infections, e.g. necrotizing fasciitis (Dryden, 2009;Ki

and Rotstein, 2008). Bacterial strains involved in pathogenesis are of

limited number, but possess unique virulence and transmissibility fac-

tors that account for the majority of SSTIs (Gorwitz, 2008). Amongst

these are Staphylococcus aureus, a leading cause of SSTIs that is also

implicated in atopic dermatitis, Acinetobacter baumannii,Klebsiella

pneumoniae,Pseudomonas aeruginosa,Streptococcus pyogenes and Cuti-

bacterium acnes. As antibiotic resistance has been reported in these

strains in recent years, herbal remedies used by traditional healers and

herbalists are increasingly frequently being investigated for their

pharmacological efficacy and could be leveraged as starting points for

future anti-infective drug development (Salam and Quave, 2018).

Part of our survey aimed to investigate traditional plant use against

skin infections. Results are shown in Table 10. Generally, “skin infec-

tions” was the category with the lowest number of use reports com-

pared to the other categories. According to our informants, 11 of the 16

plant species are used in treatment of skin infections in the Greater

Mpigi region. C. buchananii,M. kandtiana,S. longipedunculata,S. acu-

leastrum and Z. chalybeum are not utilized medicinally at all, while M.

lycopodioides roots, C. molle stem bark and L. calostachys leaves/whole

plant were only cited by one or two informants. The species and plant

parts most often stated to be used to treat skin infections were H. ma-

dagascariensis stem bark, leaves and fruits, A. coriaria stem bark and

leaves, and S. calycinum subsp. angustifolium leaves. In Mawokota

county, which is part of the Greater Mpigi region in Central Uganda, A.

coriaria bark has previously been described by Adia et al. (2014) as a

natural remedy against skin disorders. Another plant cited is P. ha-

diensis, which has previously only been reported to be used in treatment

of wounds in the Malabar region of Kerala in India where leaves are

rubbed onto the wound (Deepthy and Ramashree, 2014). The modes of

preparation and administration differed considerably compared to the

other categories of traditional use. This was mainly due to the need for

topical application in most cases. The predominant method of pre-

paration was bathing the affected skin or soft tissue body part in an

herbal aqueous decoction. Another way of preparation regularly cited

was boiling plant parts in petroleum jelly, which is then smeared onto

the infected skin.

3.5.5. Tuberculosis

This category of traditional use focuses on the application of the

selected species in treatment of tuberculosis (TB). TB has co-evolved

with humans over thousands of years, and it is one of the most ancient

diseases of humanity (Sandhu, 2011). Molecular evidence of the disease

was found in human remains from the Stone Age, recovered 9000 years

later from Neolithic settlement in the Eastern Mediterranean

(Hershkovitz et al., 2008). Along with HIV/AIDS, TB remains the top

cause of death from a single infectious agent worldwide, accounting for

more than 10 million infections and 1.6 million deaths in 2017 (WHO,

Fig. 3. FCs of plants used in treatment of individual symptoms of general infections (n = 39).

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

2018). Patients cured from the disease can still suffer substantial re-

duction in quality of life due to lifetime sequelae, such as parenchymal,

airway, vascular, mediastinal, pleural and chest wall lesions (Glaziou

et al., 2015;Kim et al., 2001;Miller et al., 2009). TB is mainly caused

by Mycobacterium tuberculosis and it is most commonly transmitted from

people with infectious pulmonary TB to others via droplet infection,

e.g. through sneezing, coughing and speaking (Glaziou et al., 2015;

Nardell, 2016). A previous study by Bunalema et al. (2014) investigated

the tuberculosis health situation in the Greater Mpigi region. This study

showed that the majority of the local traditional healers closely as-

sociate TB with HIV/AIDS, substantiated by the fact that 50% of TB

patients are infected with HIV and that 30% of all AIDS-related deaths

are attributed to TB.

Africa harbors the Cradle of Humanity (Dirks and Berger, 2013).

Since TB co-evolved with humans, traditional cultural knowledge of

medicinal plants used to counteract TB also co-evolved with the ancient

African traditional medicine system. This is also true of local herbal

medicine use in the Greater Mpigi region, where, according to our

ethnobotanical survey, all of the 16 plant species are used alone or in

herbal mixtures to treat TB (Table 11). Plant species displaying the

highest TB-specific numbers of use reports are A. coriaria (25), W.

ugandensis (24), C. molle (21) and E. abyssinica (21). There was high

consensus regarding these species, as more than half of the traditional

healers interviewed stated they use these plants in treatment of TB. The

lowest TB-specific numbers of use reports were recorded for L. calos-

tachys leaves and the whole plant and M. lycopodioides roots and leaves

which were named by only two and three of the informants respec-

tively. Plant parts used and methods of preparation/administration are

generally similar to those already described in the traditional use ca-

tegory “malaria” (subsection 3.5.1). For the majority of interviews and

plants, these are aqueous decoctions that are drunk as medication.

Another regularly mentioned mode of preparation is mixing leaves into

tea. Other methods recorded include licking powdered stem bark or

powdered roots, and chewing raw stem bark. Two of the sources

mentioned that they steam fresh P. hadiensis leaves over the fire, which

they then give to their patients to chew. According to our literature

review, use of P. hadiensis in treatment of tuberculosis is reported for

the first time. One study of another species of the genus Plectranthus, P.

amboinicus, that grows in Puerto Rico, showed low antibacterial prop-

erties against growth of M. tuberculosis (Frame et al., 1998). One

Table 9

FCs

symptoms

, plant parts used and modes of preparation and administration in treatment of symptoms of general infections (n = 39); FCs

symptoms

combine the total use

of the five individual, symptom-specific FCs per species.

Plant species FC

symptoms

Parts used Mode of preparation and administration

Albizia coriaria 37 STB aqueous decoction; powdered into tea, taken orally

Cassine buchananii 16 B aqueous decoction, taken orally

L aqueous decoction, taken orally

Combretum molle 36 STB aqueous decoction; powdered into tea; powdered and licked, taken orally

L aqueous decoction; fresh leaves chewed; dried leaves powdered and licked, taken orally

R aqueous decoction, taken orally

Erythrina abyssinica 38 STB aqueous decoction; powdered into tea; fresh bark powdered in cold water; powdered bark licked; sliced bark

chewed, taken orally

L aqueous decoction, taken orally

F aqueous decoction, taken orally

Ficus saussureana 38 STB aqueous decoction; powdered into tea, taken orally

L aqueous decoction; dried leaves powdered and licked, taken orally

Harungana madagascariensis 25 STB aqueous decoction; powdered into tea, taken orally

L aqueous decoction; powdered into tea, taken orally

R aqueous decoction, taken orally

P aqueous decoction; dried, powdered and licked, taken orally

WP aqueous decoction, taken orally

Leucas calostachys 13 L aqueous decoction; fresh leaves chewed; burned to ashes, then licked, taken orally

WP aqueous decoction, taken orally

Microgramma lycopodioides 11 R aqueous decoction, taken orally

L aqueous decoction; crushed into fruit juice, taken orally

WP aqueous decoction, taken orally

Morella kandtiana 31 RB aqueous decoction; powdered into tea, taken orally

L aqueous decoction; powdered and licked, taken orally

R aqueous decoction; powdered into tea, powdered and licked, taken orally; powdered and sniffed; steam bath,

nasal administration

STB aqueous decoction, taken orally

Plectranthus hadiensis 36 L aqueous decoction; fresh leaves chewed; powdered into tea; powdered, mixed into milk; crushed into fruit

juice, taken orally

Securidaca longipedunculata 15 L aqueous decoction; lemon juice added to decoction, taken orally

Sesamum calycinum subsp. angustifolium 16 L aqueous decoction; powdered into tea; fresh leaves chewed; crushed into fruit juice, taken orally; decoction

mixed with salt to rinse the mouth

WP aqueous decoction, taken orally

Solanum aculeastrum 15 L aqueous decoction, taken orally

FR fruits squeezed, juice then taken orally

R burned, then ashes mixed with salt; roots chewed, taken orally

S burned, then ashes mixed with salt, licked, taken orally

Toddalia asiatica 41 STB aqueous decoction; powdered and licked; bark sliced and chewed, taken orally

L aqueous decoction; fresh leaves chewed, taken orally

R aqueous decoction, powdered into tea; roots chewed, taken orally

P aqueous decoction, taken orally

Warburgia ugandensis 37 STB aqueous decoction; powdered into tea; powdered and licked, taken orally

L aqueous decoction; powdered and licked, taken orally

Zanthoxylum chalybeum 12 STB aqueous decoction; powdered into tea, taken orally

L aqueous decoction; powdered into tea, taken orally

R aqueous decoction, taken orally

key: B = bark, L = leaves, R = roots, RB = root bark, S = seeds, ST = stem, STB = stem bark, FR = fruits, F = flowers, WP = whole plant.

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

traditional healer stated that dried, powdered fruits of S. aculeastrum

are added to cold water and then consumed orally. The use of S. acu-

leastrum in treatment of tuberculosis has also not been reported before.

3.5.6. Cancer

Although nowadays cancer is not a rare disease in tropical Africa, it

continues to be a low priority for health care services. The reason is

undoubtedly the overwhelming burden of communicable diseases, as

reflected by the high number of deaths suffered from malaria, tu-

berculosis, HIV/AIDS, amongst others (Jemal et al., 2012;Sitas et al.,

2006). Epidemiological data on cancer in Uganda is still insufficient and

scarce (Jemal et al., 2012). This is mostly due to absence of modern

Western clinical facilities for cancer diagnosis in many regions, re-

sulting in a high estimated number of unknown/undetected cases. This

lack of modern facilities and technologies means that Ugandan patients

being treated in hospitals using Western medicine, experience much

lower survival rates than patients in other, non-African developing

countries (Gondos et al., 2005). In Uganda, according to the authors’

knowledge, there is insufficient data on survival rates of patients

treated by traditional healers with medicinal plants and/or witchcraft,

making assessment of efficacy of these treatments extremely difficult.

The selected 16 medicinal plant species are also widely used for

treatment of a broad array of types of cancer in the study area.

Regarding the types of cancer mentioned by the traditional healers, the

calculated FCs

cancer

show that some species are used more regularly

than others. Beginning with the highest number of use reports against

cancer, in general: A. coriaria (31), F. saussureana (30), P. hadiensis (26),

H. madagascariensis (22), T. asiatica (22), E. abyssinica (0.51), W.

ugandensis (18), C. molle (16), M. kandtiana (12), S. calycinum subsp.

angustifolium (12), S. longipedunculata (10), S. aculeastrum (9), L. calos-

tachys (8), C. buchananii (7), M. lycopodioides (6) and Z. chalybeum (6).

Table 12 shows the types of cancer named by the informants in the

survey, as well as those plant species used for treatment and cure of

these specific cancer variants.

In our study, we also wanted to investigate how particular types of

cancer can be specifically treated with medicinal plants in the absence

of clinical diagnosis available in Ugandan traditional medicine, espe-

cially in the remote parts of the Greater Mpigi region. How do our in-

formants know about these different types of cancer while living in

remote villages, far away from hospitals which use Western medical

techniques? Another question was how this relatively new knowledge

can be described as “traditional and transferred orally over genera-

tions”. The answer to these questions is that, as when HIV/AIDS was a

new disease in the early 1980's, traditional healers treat cancers based

on the similarity of symptoms. A small number of patients may have the

possibility to travel and can afford a Western-medicine hospital diag-

nosis. After receiving a cancer diagnosis, these patients report to their

traditional healer in the village. The traditional healers treat their pa-

tients for the symptoms they have, such as pain, swelling, visible tumor

growth, etc. Consequently, they connect the symptoms and medicinal

plants used with the previously obtained specific cancer diagnosis from

the hospital. Cases of patients reporting to the traditional healers from

the hospital may be very rare in the Greater Mpigi region, but new

information about a patient with a certain type of cancer being treated

with a certain plant, resulting in a positive change in pathogenesis, can

spread fast at the community level among traditional medicine collea-

gues.

3.5.7. Other traditional medicinal uses recorded

With our questionnaires, we also sought to document all other tra-

ditional uses that do not fall in one of the six categories above. These

“other” traditional uses, corresponding plant parts used and the re-

spective FCs are shown in Table 13. Results of the full range of tradi-

tional uses of the selected medicinal plant species were diverse and

numerous. A total of 141 “other” traditional uses for the 16 selected

species were recorded, describing their use in the treatment of 44 dif-

ferent diseases and (medical) conditions. The informants responded in

many different ways, e.g. they referred to Z. chalybeum and M.

Table 10

Results of analysis of traditional use of selected plant species in treatment of SSTIs (n = 39); FCs for total specific use and for the sum of FCs for mode of preparations/

administrations per species might differ due to participants mentioning multiple parts used and/or methods.

Plant species FC

skin_infections

Parts used Mode of preparation and administration (FC)

Albizia coriaria 8 STB boiled, herbal bath (3); boiled in petroleum jelly (1) or powdered and mixed in petroleum jelly (3), then

smeared on skin, applied topically

L boiled (1), herbal bath

Cassine buchananii – – –

Combretum molle 1 STB pounded and mixed with petroleum jelly (1), then smeared on skin, applied topically

Erythrina abyssinica 3 STB boiled in petroleum jelly (3), then smeared on skin, applied topically; boiled, filtered and then the infected

skin part is bathed in (1), herbal bath

Ficus saussureana 5 STB boiled in petroleum jelly (4) or powdered and mixed in petroleum jelly (1), then smeared on skin, applied

topically

Harungana madagascariensis 12 STB boiled, herbal bath (2); boiled in petroleum jelly (2) or powdered and mixed in petroleum jelly (6), then

smeared on skin, applied topically

L powdered and mixed in petroleum jelly (1), then smeared on skin, applied topically

FR powdered and mixed in petroleum jelly (1), then smeared on skin, applied topically

Leucas calostachys 2 L powdered and mixed in petroleum jelly (1), then smeared on skin, applied topically

WP boiled (1), herbal bath

Microgramma lycopodioides 1 R boiled in petroleum jelly (1), then smeared on skin, applied topically

Morella kandtiana – – –

Plectranthus hadiensis 5 ST boiled in petroleum jelly (1) or powdered and mixed in petroleum jelly (1), then smeared on skin, applied

topically

L boiled (3), herbal bath

Securidaca longipedunculata – – –

Sesamum calycinum subsp. angustifolium 6 L boiled (2), herbal bath; powdered and mixed in petroleum jelly (4), then smeared on skin, applied topically

Solanum aculeastrum – – –

Toddalia asiatica 3 STB boiled in petroleum jelly (1), then smeared on skin, applied topically

R boiled (1), herbal bath; chewing of roots (1), taken orally

Warburgia ugandensis 4 STB boiled in petroleum jelly (1) or powdered and mixed in petroleum jelly (2), then smeared on skin, applied

topically

L boiled (1), herbal bath

Zanthoxylum chalybeum – – –

key: B = bark, L = leaves, R = roots, RB= root bark, S = seeds, ST = stem, STB = stem bark, FR = fruits, F = flowers, WP = whole plant

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

kandtiana use with the Luganda word “Bulalu”. Literally translated this

means “madness”, which is why we classified it as traditional use in

treatment of psychosis. The term “madness” as a disease/medical con-

dition has previously been described and literally translated from local

languages in other East African countries and ethnic groups, such as the

Jo-Luo and the Kakwa in South Sudan, the Sebei in southeast Uganda,

the Pokot and Kamba in Kenya, the Hehe in Tanzania, the Tutsi and

Hutu in Burundi and the Wanande in the Democratic Republic of the

Congo (Edgerton, 1966;Ventevogel et al., 2013). The local conception

of mental illnesses and the generalized East African definition of

“madness” relate to all conditions of severe behavioral disturbances,

ranging from chaotic behavior (collecting rubbish, walking aimlessly or

naked), talking nonsense, conversing with oneself, doing things con-

sidered foolish, and types of interpersonal violence to posttraumatic

stress disorders and major depression (Ventevogel et al., 2013).

Some traditional uses cited deal with sexual performance and re-

production, e.g. plants as aphrodisiacs, for vaginal dryness, as a birth

control agent, or against infertility and importance in men. Moreover,

some plants are highly used in treatment of erectile dysfunction within

the study area, e.g. C. buchananii,S. aculeastrum and S. calycinum subsp.

angustifolium.

As mentioned before, traditional medicine in Uganda is holistic,

taking both the physical and the psycho-spiritual condition of a patient

into account. Therefore, medicinal plants may be prescribed to appease

the spirits, or to remove charms or spells of witchcraft. This treatment

might be more expensive than others, e.g. treatment of physical ill-

nesses, and sacrifice of animals and mob justice might also be involved

(Allen and Reid, 2015;Tabuti et al., 2003). M. lycopodioides and Z.

chalybeum leaves were both cited to be used to cure bewitchment and

chase away evil spirits. Here, the mention of the polypodiaceous fern M.

lycopodioides is of high interest because, this plant has only been

documented for its traditional use as a medicinal plant for treatment of

diseases in two publications so far. Just as in our study in the Greater

Mpigi region, it has previously been reported to be used to treat anemia

in Tanzania, and additionally for removal of lice in South Africa

(Maroyi, 2017). More interestingly, in 1988, Dr. Philip A. Dennis

published a paper on herbal medicine among the Zambo-Miskito of

Eastern Nicaragua, in which he also described the use of M. lycopo-

dioides against witchcraft attacks (Dennis, 1988). Surprisingly, although

the territory of the Miskito Indians in Nicaragua and the Greater Mpigi

region are separated by more than 12,800 km, both ethnic groups seem

to utilize the same plant spiritually against witchcraft and sorcery.

M. lycopodioides naturally occurs in South and Central America, Sub-

Saharan Africa and in the Caribbean (Mucunguzi, 2007;Pereira et al.,

2013;Steege and Cornelissen, 1989;Walker, 1973). It is likely that a

shipwrecked African slave ship on the shallow-water coast of Nicaragua

Table 11

Traditional use of selected plant species in treatment of tuberculosis (n = 39); FCs for total specific use and for the sum of FCs for mode of preparations/admin-

istrations per species might differ due to participants mentioning multiple parts used and/or methods.

Plant species FC

tuberculosis

Parts used Mode of preparation and administration (FC)

Albizia coriaria 25 STB boiled (19) or powdered, then boiled (5); powdered and licked (1), taken orally

Cassine buchananii 11 B boiled (8) or powdered, then boiled (1); powdered in tea, (1), taken orally

L powdered into tea (1), taken orally

Combretum molle 21 STB boiled (17) or powdered, then boiled (3); chewed raw (1), taken orally

L boiled (4), taken orally

R boiled (1), taken orally

Erythrina abyssinica 21 STB boiled (17) or powdered, then boiled (4), taken orally

L boiled (1), taken orally

F boiled (1), taken orally

Ficus saussureana 15 STB boiled (9) or powdered, then boiled (6), taken orally

Harungana madagascariensis 10 STB boiled (8), taken orally

L boiled (1); powdered into tea (1), taken orally

WP boiled (1), taken orally

Leucas calostachys 2 L boiled (2), taken orally

WP boiled (1), taken orally

Microgramma lycopodioides 3 R boiled (1), taken orally

L boiled (1); powdered into tea (1), taken orally

Morella kandtiana 12 RB powdered, then boiled (1); powdered and licked (1), taken orally

L powdered into tea (1); powdered and licked (1), taken orally

R boiled (4) or powdered, then boiled (4); powdered and licked (1), taken orally

WP boiled (1), taken orally

Plectranthus hadiensis 16 L boiled (8); dried leaves powdered into tea (2); fresh leaves cold pressed, boiled (2); steamed over fire, then

chewed (2), taken orally

ST boiled (1), taken orally

WP boiled (2), taken orally

Securidaca longipedunculata 6 B boiled (3), taken orally

L boiled (1), taken orally

R boiled (2), taken orally

FR boiled (1), taken orally

Sesamum calycinum subsp. angustifolium 9 L boiled (5); powdered into tea (1), taken orally

WP boiled (3), taken orally

Solanum aculeastrum 10 L boiled (1), taken orally

R boiled (7) or powdered, then boiled (1), taken orally

FR dried, powdered and mixed with cold water (1), taken orally

Toddalia asiatica 18 STB boiled, taken orally (2); powdered and licked (1), taken orally rally

L boiled (2), taken orally

R boiled (6) or powdered, then boiled (7), taken orally

Warburgia ugandensis 24 STB boiled (14) or powdered, then boiled (2); powdered and licked (2); chewed raw (1), taken orally

L boiled (7); powdered into tea (2), taken orally

Zanthoxylum chalybeum 9 STB boiled (3), taken orally

L boiled (3); powdered into tea (1); powdered and licked (1), taken orally

R boiled (2), taken orally

key: B = bark, L = leaves, R = roots, RB= root bark, S = seeds, ST = stem, STB = stem bark, FR = fruits, F = flowers, WP = whole plant

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

in 1641 facilitated the intercontinental transfer of spiritual knowledge

about M. lycopodioides from Africa to America. In 1711, the bishop of

Nicaragua, Benito Garret y Arlovi, wrote a letter to the King of Spain, in

which he described the events of 1641, when about a third of the

Africans who survived the shipwreck escaped and hid in the forest, then

violently fought and defeated the local Amerindian groups, reproducing

with the Amerindian women by intermarriage, ultimately followed by

peaceful communal life (Arlovi, 1711;Cwik, 2019). These events,

especially the intermarriage, are considered to be the birth and fun-

dament of the Zambo-Miskito culture, which is therefore an ethnic

group originally composed of Amerindians and former African slaves

(Arlovi, 1711;Dennis and Olien, 1984;Iglesias, 2012). Similar tradi-

tional use of M. lycopodioides against witchcraft in both regions can

therefore be logically explained by this historical context and the power

Table 12

Types of cancer mentioned in the survey and plant species used for individual treatment (n = 39).

Type of cancer Plant species used and their specific FCs

abdominal cancer Albizia coriaria (4), Combretum molle (1), Erythrina abyssinica (2), Ficus saussureana (1), Plectranthus hadiensis (1), Sesamum calycinum subsp. angustifolium

(1)

blood cancer/leukemia Albizia coriaria (1), Cassine buchananii (2), Combretum molle (2), Erythrina abyssinica (2), Ficus saussureana (2), Harungana madagascariensis (2), Leucas

calostachys (1), Microgramma lycopodioides (1), Plectranthus hadiensis (1), Securidaca longipedunculata (1), Solanum aculeastrum (1), Toddalia asiatica (3),

Warburgia ugandensis (3)

bone cancer Combretum molle (1), Ficus saussureana (2), Leucas calostachys (2)

bone marrow cancer Albizia coriaria (1), Securidaca longipedunculata (1)

brain cancer Ficus saussureana (1), Morella kandtiana (1), Plectranthus hadiensis (1)

breast cancer Microgramma lycopodioides (1), Morella kandtiana (1), Plectranthus hadiensis (1), Securidaca longipedunculata (1), Sesamum calycinum subsp. angustifolium

(2), Toddalia asiatica (3), Warburgia ugandensis (1)

cervical cancer Albizia coriaria (5.1), Cassine buchananii (1), Combretum molle (1), Erythrina abyssinica (1), Ficus saussureana (3), Harungana madagascariensis (1), Morella

kandtiana (2), Securidaca longipedunculata (1), Toddalia asiatica (1), Warburgia ugandensis (1)

intestinal cancer Albizia coriaria (6), Cassine buchananii (3), Combretum molle (5), Erythrina abyssinica (3), Ficus saussureana (4), Harungana madagascariensis (4), Leucas

calostachys (3), Morella kandtiana (3), Plectranthus hadiensis (4), Sesamum calycinum subsp. angustifolium (3), Solanum aculeastrum (1), Toddalia asiatica

(7), Warburgia ugandensis (6), Zanthoxylum chalybeum (1)

liver cancer Combretum molle (2), Ficus saussureana (2), Harungana madagascariensis (1), Microgramma lycopodioides (1), Morella kandtiana (1), Securidaca

longipedunculata (2), Solanum aculeastrum (1), Zanthoxylum chalybeum (1)

lung cancer Cassine buchananii (1), Erythrina abyssinica (1), Morella kandtiana (2), Plectranthus hadiensis (1), Solanum aculeastrum (1)

prostate cancer Albizia coriaria (2), Erythrina abyssinica (1), Ficus saussureana (1), Plectranthus hadiensis (1), Securidaca longipedunculata (1), Toddalia asiatica (1),

Warburgia ugandensis (1)

skin cancer Albizia coriaria (14), Combretum molle (3), Erythrina abyssinica (10), Ficus saussureana (13), Harungana madagascariensis (14), Leucas calostachys (2),

Microgramma lycopodioides (2), Morella kandtiana (1), Plectranthus hadiensis (14), Securidaca longipedunculata (3), Sesamum calycinum subsp.

angustifolium (5), Solanum aculeastrum (4), Toddalia asiatica (5), Warburgia ugandensis (5), Zanthoxylum chalybeum (4)

stomach cancer Morella kandtiana (1)

throat cancer Albizia coriaria (1), Combretum molle (1), Ficus saussureana (1), Microgramma lycopodioides (1), Plectranthus hadiensis (1), Sesamum calycinum subsp.

angustifolium (1), Solanum aculeastrum (1), Toddalia asiatica (2), Warburgia ugandensis (1),

uterine cancer Plectranthus hadiensis (1)

Table 13

Other traditional uses recorded for the 16 selected medicinal plant species (n = 39).

Plant species Other traditional uses recorded (parts used, FCs)

Albizia coriaria aphrodisiac (L, 1), fibroids (STB, 3), heart diseases (STB, 1), hernia (STB, 2), HIV/AIDS (STB, 3), STIs (STB, 1), syphilis (STB, 4),

typhoid fever (STB, 1), ulcers (STB, 1), vaginal dryness (STB, 3)

Combretum molle aphrodisiac (STB, 1), diarrhea (STB, 1), hemorrhoids (L, 1), HIV/AIDS (STB, 1), kidney failure (STB, 1), syphilis (STB, 2; L, 1; R, 1),

ulcers (P, 1)

Cassine buchananii erectile dysfunction (STB, 8; R, 3), flu (R, 1), HIV/AIDS (B, 3), kidney failure (B, 1), liver disease (1), sinusitis (S, 1), syphilis (B, 2),

yellow fever (B, 1)

Erythrina abyssinica anemia (STB, 3), birth control (F, 1), brain disorders (L, 1), dehydration (STB, 2), diabetes (STB, 1), diarrhea (STB, 1), eye dryness (L,

1), fallopian tube blockage (STB, 1), fibroids (1), HIV/AIDS (STB, 3), STIs (STB, 1), syphilis (STB, 10)

Ficus saussureana diabetes (STB, 1; L, 1), fallopian tube blockage (STB, 2), HIV/AIDS (STB, 2), infertility in men (STB, 1), STIs (STB, 1), syphilis (STB, 7;

L, 1; R, 1), typhoid fever (STB, 1), ulcers (L, 1; R, 2)

Harungana madagascariensis anemia (L, 1), diarrhea (STB, 1), hernia (STSB, 1), syphilis (STB, 4; L, 2), typhoid fever (S, 1), ulcers (STB, 1; L, 1), worms (STB, 1)

Leucas calostachys anemia (L, 1), bed-wetting (1), candidiasis (L, 1), hemorrhoids (L, 1), hernia (R, 1), HIV/AIDS (L, 1), vaginal dryness (L, 2)

Microgramma lycopodioides anemia (L, 1; R, 1), chasing away evil spirits (L, 1), flu (L, 1), heart diseases (WP, 1), HIV/AIDS (R, 1)

Morella kandtiana anemia (R, 1), diarrhea (WP, 1), epilepsy (RB, 1), fallopian tube blockage (R, 1), influenza (L, 3; R, 3), fungal infections (L, 1), HIV/

AIDS (R, 2), psychosis (RB, 1), sinusitis (RB, 3; R, 2), typhoid fever (R, 1)

Plectranthus hadiensis diarrhea (L, 2), eye diseases (L, 3), fallopian tube blockage (L, 2), flu (L, 1), fibroids (L, 1), gonorrhea (ST, 1), HIV/AIDS (L, 1),

infertility in men (ST, 1), kidney failure (ST, 1), measles (L, 2), psychosis (ST, 1), syphilis (L, 3), typhoid fever (1), ulcers (L, 5),

Securidaca longipedunculata diarrhea (B, 1; L, 1), liver disease (1), syphilis (B, 1), ulcers (B, 1)

Sesamum calycinum subsp. angustifolium anemia (L, 1), erectile dysfunction (L, 3; WP, 2), bed-wetting (L, 1), dehydration (WP, 1), diarrhea (L, 1), vaginal dryness (WP, 1),

impotence/infertility (WP, 2), prolapsed rectum (L, 1), syphilis (L, 1), typhoid fever (L, 2), vaginal dryness (L, 4)

Solanum aculeastrum erectile dysfunction (STB, 1; L, 2; R, 7), fallopian tube blockage (R, 1), fibroids (R, 1), impotence (R, 1), infertility in men (FR, 1),

kidney failure (R, 1), snake bite (1), worms (R, 1)

Toddalia asiatica anemia (R, 1), aphrodisiac (L, 1; R, 2), brain disorders (L, 1), diabetes (R, 3), diarrhea (STB, 1; R, 1), HIV/AIDS (STB, 1; R, 1),

hypertension (R, 1), menstrual cramps (R, 1), typhoid fever (STB, 1; R, 1), snake bite (STB, 1; R, 1), ulcers (L, 1; R, 2), worms (STB, 1; R,

Warburgia ugandensis anemia (L, 1), aphrodisiac (STB, 1; L, 2), candidiasis (L, 1), fallopian tube blockage (STB, 1), influenza (L, 1), kidney failure (STB, 1),

HIV/AIDS (STB, 4; L, 1; R, 1), hypertension (STB, 2), measles (L, 1), miscarriage (L, 1), nose bleeding (L, 1), nasal congestion (L, 1),

syphilis (STB, 1), ulcers (STB, 4)

Zanthoxylum chalybeum chasing away evil spirits (L, 1), epilepsy (1), fallopian tube blockage (L, 1), psychosis (1), syphilis (L, 1; R, 1)

key: B = bark, L = leaves, R = roots, RB = root bark, S = seeds, ST = stem, STB = stem bark, FR = fruits, F = flowers, WP = whole plant.

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

of oral transmission of traditional knowledge from 1641 to 1988 is once

more remarkable.

3.6. Combinations of medicinal plants

In Western medicine and pharmaceutical industries, purified sub-

stances from extracts or (semi-) synthesized active compounds are sold

and prescribed for treatment of medical conditions and diseases. Plant

extracts prescribed and marketed as “botanicals” on the Western mar-

kets are still rather rare and are highly standardized regarding their

chemical composition, as well as their individual pharmacological ac-

tivity and efficacy. This general approach is different in traditional

medicine, as here mixtures of many plants are often used, bringing

together synergistic effects and supporting a holistic treatment strategy

(Chan et al., 2010;Firenzuoli and Gori, 2007;Fu et al., 2014). Fig. 4

shows the response of informants in percentage regarding single plant

use only, use in mixtures, or both, for each plant species when pre-

scribing herbal medicines. The majority of the plants are often used

both in mixtures with other medicinal plants and singly (> 20%), ex-

cept for S. aculeastrum (17.9%), M. lycopodioides (17.6%), H. mada-

gasciesis (15.8%) and Z. chalybeum (11.1%). Eleven of 16 plant species

are predominantly used in herbal mixtures (> 40%). The plant species

most often prescribed alone (> 40%) are M. kandtiana and S. aculeas-

trum.

3.7. Motivation of traditional healers to participate in study

The researchers adhered to the ethical principles of the

International Society of Ethnobiology, and in addition to acquiring

appropriate prior informed consent from study participants, efforts

were made to engage in access and benefit sharing – specifically with

the return of knowledge collected and laboratory results. Bidirectional

communication with healers was a priority and the study also aimed to

learn more about the healers’ motivation for participating in the study

and define their future expectations from the authors.

Fig. 5 displays the responses of the traditional healers and their

percentage ratios. Multiple responses per participant were welcome.

Only 2% of the traditional healers stated that they are interested in

future Western drug development and only 5% revealed that they want

to benefit financially through the scientific information gained. Some

traditional healers prioritized the conservation of their ethnomedicinal

knowledge (5%) and the protection of their medicinal plant resources

(5%). It is of high interest to 9% of the traditional healers to collaborate

for improvement in treatment of patients and 11% want to reinforce

their collaboration with the authors, and researchers in health and

medicine, in general. The second most common expectation, with 18%,

was to receive feedback on the actual findings of the pharmacological

studies after the fieldwork and lab experiments are completed. Finally,

more than a quarter of the informants stated that getting evidence of

whether the plants investigated really do have the claimed medicinal

properties is of highest interest to them, as it will boost their confidence

in using the plants for individual treatment. Their responses show that

there is a high interest in collaboration and also the vital need for

feedback.

Unfortunately, very few scientific findings are transferred back to

the indigenous peoples and traditional healers that originally set the

foundation for advanced ethnopharmacological research endeavors

(Maregesi et al., 2007). One of the reasons is that scientific articles are

incomprehensible and inaccessible to them (Jaeger, 2005). Screening

random plants for bioactivity yields significantly fewer hits than drug

development research based on ethnomedicinal uses, making it ur-

gently important to transfer useful findings of lab studies back to in-

formation providers in a way that is appropriate to the level of their

understanding (Cordell, 1995;Maregesi et al., 2007;Unander et al.,

Fig. 4. Percentages of informants using the plant species singly, in mixtures only or both.

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

1995). Some plants may even pose a threat to local communities, be-

cause they might turn out to be toxic and harmful to patients as a direct

outcome of treatment. It is the responsibility of ethnopharmacologists

to contribute to improving local medicinal plant use, help reduce health

hazards derived from herbal drugs, and create a good relationship for

future collaborations. Consequently, we commit to transferring all re-

sults of our pharmacological evaluation of bioactivity back to our in-

formants in the Greater Mpigi region, e.g. through medicinal plant

workshops and continued collaboration.

4. Conclusion

Recording information about traditional medicinal plant use, plant

parts used and methods of preparation and administration results in its

conservation and facilitates future drug discovery research endeavors,

based on ethnobotanical hints. Concerns about the loss of traditional

knowledge have been equally expressed by both researchers and pol-

icymakers (Bussmann et al., 2018). This study contributes to the con-

servation of culturally and scientifically valuable medical knowledge of

the 16 selected Ugandan plant species.

The species were selected because preliminary studies in the study

area suggested a high level of medicinal use in the treatment of malaria,

inflammatory disorders, symptoms of general infections, tuberculosis

and cancer for all of these species. Results of the ethnobotanical survey

fully confirmed these claims. Additionally, various other traditional

uses were documented; many for the first time. Moreover, the tradi-

tional healers who participated in our survey signified their expecta-

tions of the team of researchers: they collectively asked to receive

feedback on the findings of any resulting pharmacological study

investigating the efficacy of medicinal plants that might lower or boost

their confidence in individual herbal remedies.

Author contributions

FS designed the overall strategy of the study and the questionnaires

for the ethnobotanical survey. GA and FS conducted fieldwork and

collected plant material for future lab analysis. GA collected plant

specimens for the Makerere University and Emory University herbaria,

prepared the herbarium vouchers and performed plant identifications.

FS and BW processed the survey data. FS and CLQ interpreted the data.

FS, GA and CLQ wrote the manuscript. LAG directed the study. All

authors read, revised and approved the final manuscript.

Funding

This work was supported by a grant from the German Federal

Ministry of Education and Research (13FH026IX5, PI: LAG and Co-I:

FS). The content is solely the responsibility of the authors and does not

necessarily reflect the official views of the funding agency. The funding

agency had no role in study design, data collection and analysis, deci-

sion to publish, or preparation of the manuscript.

Declaration of competing interest

This study was performed according to the international, national

and institutional rules considering the Convention on Biodiversity and

the Nagoya Protocol. Informed consent was requested and obtained

Fig. 5. Future expectations concerning our scientific findings.

F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

from all participants of the ethnobotanical survey. All results from fu-

ture lab work on mentioned plants will be transferred back to tradi-

tional healers and survey participants through workshops. The authors

declare that the research was conducted in the absence of any com-

mercial or financial relationships that could be construed as a potential

conflict of interest.

Acknowledgements

Greatest thanks to the 39 traditional healers who participated in the

survey and to PROMETRA Uganda for providing a network of contacts

within surveyed communities. Thanks to research assistants Alex

Olengo, Kibuuka Sserwano Moses and Kasozi Dauda for assisting during

data collection. Thanks to Inken Dworak-Schultz for photography

during fieldwork (e.g. figures in graphical abstract). Thanks to Tina

Seehafer for her assistance in transferring the handwritten survey re-

sponses into digital versions. Thanks to Vanessa Rabus for her assis-

tance with processing the Copernicus Sentinel satellite data and the GIS.

Thanks to Logan Penniket for proof-reading the manuscript. Special

thanks to the Neubrandenburg University of Applied Sciences for sup-

porting FS's fieldwork activities in Uganda in terms of working hours.

Appendix A and B Supplementary data. Supplementary data

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.jep.2020.112742.

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F. Schultz, et al. Journal of Ethnopharmacology 256 (2020) 112742

Publication II:

"A bibliographic assessment using the Degrees of Publication method:

Medicinal plants from the rural Greater Mpigi region (Uganda)"

Pages: 33-50

Personal contribution

In the following, my personal contribution to the presented study and manuscript is briefly

described: I designed the overall strategy of the study and the novel DoP concept. I conducted

the literature review and processed the data. I interpreted the majority of the data. I wrote most

of the manuscript. A more detailed author-contribution statement is given in the published

article.

Information on publication

This study was published in Evidence-Based Complementary and Alternative Medicine in

January 2021 and is available at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822678. It

is an open access article distributed under the Creative Commons Attribution 4.0 International

License (CC BY 4.0).

Schultz, F.; Anywar, G.; Quave, C.L.; Garbe, L.-A.: A bibliographic assessment using the

Degrees of Publication method: Medicinal plants from the rural Greater Mpigi region

(Uganda). Evidence-Based Complementary and Alternative Medicine, 6661565, 2021.

https://doi.org/10.1155/2021/6661565

Research Article

A Bibliographic Assessment Using the Degrees of Publication

Method: Medicinal Plants from the Rural Greater Mpigi

Region (Uganda)

Fabien Schultz

Godwin Anywar,

Cassandra Leah Quave,

and Leif-Alexander Garbe

Institute of Biotechnology, Faculty III - Process Sciences, Technical University of Berlin, Gustav-Meyer-Allee 25,

Berlin 13355, Germany

Department of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences, Brodaer Str. 2,

Neubrandenburg 17033, Germany

Department of Dermatology, Emory University School of Medicine, 615 Michael St., Atlanta, 30322 GA, USA

Department of Plant Sciences, Microbiology and Biotechnology, Makerere University, P.O Box 7062, Kampala, Uganda

Center for Study of Human Health, Emory University College of Arts and Sciences, 615 Michael St., Atlanta, 30322 GA, USA

ZELT - Neubrandenburg Center for Nutrition and Food Technology gGmbH, Seestraße 7A, Neubrandenburg 17033, Germany

Correspondence should be addressed to Fabien Schultz; [email protected]

Received 6 November 2020; Accepted 30 November 2020; Published 15 January 2021

Academic Editor: Luiz Felipe Domingues Passero

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In ethnopharmacological research, many ﬁeld assessment tools exist. Yet, these miss that critical point of how to really determine

which species merit the costly lab studies, e.g., evaluation of traditional use via pharmacological assays and isolation of bioactive

secondary metabolites. This gap can be ﬁlled with the introduction of a new tool for literature assessment: the Degrees of

Publication (DoPs). In this study, its application is illustrated through an extensive bibliographic assessment of 16 medicinal plant

species that were recently identiﬁed in the Greater Mpigi region of Uganda as being frequently used by local traditional healers in

the treatment of medical disorders (namely, Albizia coriaria,Cassine buchananii,Combretum molle,Erythrina abyssinica,Ficus

saussureana,Harungana madagascariensis,Leucas calostachys,Microgramma lycopodioides,Morella kandtiana,Plectranthus

hadiensis,Securidaca longipedunculata,Sesamum calycinum subsp. angustifolium,Solanum aculeastrum,Toddalia asiatica,

Warburgia ugandensis, and Zanthoxylum chalybeum). These species are suspected to be understudied, and a thorough bib-

liographic assessment has not been previously performed. Thus, the objectives of our study were to undertake a comparative

assessment of the degree to which each of these plant species has been studied in the past, including evaluation of the quality of the

journals where results were published in. The determination of the DoPs enabled successful assessment of the degrees to which

each individual plant species has been studied so far, while also taking into account the methodological “research chain of

ethnopharmacology” from ethnobotanical studies (“traditional use”) to pharmacological assays (“bioactivity”) and ﬁnally to

pharmacognostic research (“structure elucidation”). The signiﬁcance of a research paper was assessed by determining whether its

journal and publishing house were members of the Committee on Publication Ethics (COPE). In total, 634 peer-reviewed

publications were reviewed covering the period of 1960–2019, 53.3% of which were published in journals and by publishing houses

aﬃliated with COPE (338 publications). The literature assessment resulted in the identiﬁcation of understudied plants among the

selected species. The majority of plants reviewed have not been suﬃciently studied; six species were classiﬁed as being highly

understudied and three more as being understudied: C. buchananii,F. saussureana,L. calostachys,M. lycopodioides,M. kandtiana,

and S. calycinum subsp. angustifolium and A. coriaria,P. hadiensis, and S. aculeastrum, respectively. The newly introduced DoPs

are a useful tool for the selection of traditionally used species for future laboratory studies, especially for pharmacological

bioassays, isolation procedures, and drug discovery strategies.

Hindawi

Evidence-Based Complementary and Alternative Medicine

Volume 2021, Article ID 6661565, 18 pages

https://doi.org/10.1155/2021/6661565

1. Introduction

Throughout human history and across the globe, plants were

regarded as the major source of medicine and natural

remedies. Traditional medicine is deﬁned by the World

Health Organization (WHO) as “the knowledge, skills, and

practices based on the theories, beliefs, and experiences

indigenous to diﬀerent cultures, used in the maintenance of

health and in the prevention, diagnosis, and improvement or

treatment of physical and mental illness” [1]. In the de-

veloping world, over 80% of the population still rely on

traditional herbal medicines for their day-to-day healthcare

needs [2–4]. This is largely attributed to their ease of access,

aﬀordability, perceived fewer side eﬀects, and cultural ap-

propriateness, among other reasons [5]. Despite the general

loss of cultural practices worldwide [6, 7], traditional

medicine practices and medicinal plant use are still the

predominant form of healthcare services in East and Central

Africa today [8, 9]. The global importance of plants as a

source of medicine is also often emphasized by scientists

worldwide [10–14]. Around 25% of the Western drugs

prescribed contain active ingredients that were initially

isolated as natural products from plants [10]. Still, the

majority of Earth’s plant species has never been screened for

pharmacological eﬀects in a research facility [10, 15].

In consideration of this global importance, there are

many assessment tools applied when reporting ﬁeld studies

in the science of ethnopharmacology. These include ﬁeld

assessment indices for medicinally used species, such as the

frequency of citation, use value, informant consensus factor,

and ﬁdelity level, among others. However, none of these take

into account how to really determine which species merit the

costly lab studies. This is why we introduce the Degrees of

Publication (DoPs), providing a standardized way to ex-

amine how well studied individual species are (or are not) in

an ethnopharmacological context. In this study, 16 medic-

inal plant species from the Greater Mpigi region were se-

lected to illustrate how the new tool works.

Situated in West-Central Uganda, the tropical Greater

Mpigi region displays a high abundance of traditional

medicine practitioners and diverse use of a vast amount of

medicinal plant species [14, 16, 17]. Consequently, local

people are still highly dependent on these traditional healers

and their medicinal plants in order to secure their primary

health care.

A recently published ethnobotanical survey from the

Greater Mpigi region [14] and an ethnopharmacological

study [18] identiﬁed 16 medicinal plant species that are often

used in the treatment of medical disorders in the local

traditional medicine system while displaying high phar-

macological activity in our ongoing in vitro evaluation in a

lab setting. A preliminary literature review resulted in a few

results. Therefore, these 16 plants are suspected to be

understudied species, and a thorough literature review using

the new DoP method for bibliographic assessment enables

the selection of traditionally used species for pharmaco-

logical bioassays and drug discovery strategies. Our study,

therefore, aims to undertake a comparative literature as-

sessment, applying the DoP method, regarding (a) other

reports of these species, (b) the quality of the journals where

results were published in (assessment of international

standards and best practice in scholarly publication ethics),

and (c) the degree to which each plant species has been

studied thus far.

2. Materials and Methods

2.1. Study Objects. Our study objects are 16 tropical plant

species identiﬁed to be frequently used by Ugandan tradi-

tional healers in treatment of diverse medical disorders in the

Greater Mpigi region. This choice of species can be considered

taxonomically diverse, representing 13 diﬀerent plant fami-

lies. Table 1 lists these species, stating their scientiﬁc names,

local names at the study site (Luganda language), their plant

families, and their Relative Frequencies of Citation (RFC),

calculated from absolute values of the ethnobotanical survey

(n�39) previously published by Schultz et al. [14].

2.2. Literature Review. Our research strategy included pri-

oritization of some of the collected plant species for future

pharmacological bioassays. Here, the results of the ethno-

botanical survey on traditional use were the major indicator

[14]. However, another parameter for this assessment was

conducting a literature survey, identifying those medicinal

plant species that are currently understudied, hereby lim-

iting duplication of research eﬀorts.

A literature search of electronic databases included

GoogleScholar and the Web of Science Core Collection,

using the scientiﬁc name of each plant as keywords (syn-

onyms included). As suggested by Heinrich et al. [19],

NAPRALERT

, a comprehensive natural-product database

containing ethnomedical and pharmacological information

of extracts and isolated compounds, was consulted as an

additional tool (http://www.napralert.org). Membership in

the Committee on Publication Ethics (COPE) was assessed

by searching for an individual journal and the corresponding

publishing house on the COPE website (http://www.

publicationethics.org/members). Digitalized herbarium

voucher specimens were obtained from the JSTOR Global

Plants Database (https://plants.jstor.org).

2.3. Data Analysis: Degrees of Publication. The results of the

literature survey were analyzed by categorizing published

studies on the 16 medicinal plant species. A new indicator

was introduced: the Degrees of Publication (DoPs). DoPs

were deﬁned as “Traditional Use,” “Bioactivity,” “Structure

Elucidation,” “Other,” and “Total (without <other>).”

“Traditional use” are sources stating that a plant species iis

used traditionally in an ethnopharmacological context. The

2Evidence-Based Complementary and Alternative Medicine

DoP “Bioactivity” describes the number of published studies

investigating a potential pharmacological activity of an

extract from a plant i. “Structure elucidation” includes

studies that resulted in isolation of (bioactive) secondary

metabolites and their structure elucidation. These three

DoPs are consecutive steps in the bioassay-guided discovery

of novel bioactive natural products based on the ethno-

pharmacological approach of investigating traditional use

reports. This classiﬁcation, therefore, may lead to an as-

sessment of the degree to which each plant species has been

studied so far. The DoP “Other” classiﬁes all publications

mentioning a plant species iin a non-ethnopharmacological

context (e.g., studies on the morphology of the species, on

the distribution of species, or on non-medicinal traditional

use of a species). The DoP “Total (without <other>)”

summarizes the three ethnopharmacologically relevant

DoPs and is deﬁned as the sum of “Traditional Use,”

“Bioactivity,” and “Structure Elucidation.” For each DoP,

absolute numbers were given as value N

all

and N

COPE

whereas N

all

describes all publications discovered in the

literature survey, and N

COPE

lists all publications in scientiﬁc

journals whose publishing houses are members of the

Committee on Publication Ethics (COPE, http://www.

publicationethics.org). Members of the COPE must accept

the international standards and best practice in the ethics of

scholarly publishing, meaning that membership of the

COPE is an appropriate indicator for high-quality research.

A DoP “Total (without <other>)” of N

all

ranging from 0 to

14 classiﬁes a plant species as being “highly understudied,”

while 15–29 is “understudied,” 30–44 is “moderately stud-

ied,” and 45-∞is “highly studied.”

3. Results and Discussion

3.1. Species Information. Figure 1 is a compilation of digitized

herbarium voucher specimens to give an overview of the

appearance of each of the 16 plant species. Sections 3.1.1–3.1.16

provide information on synonyms, geographical distribution

(in Uganda, in particular), life forms, ecological growth con-

ditions and climate zones, local names in East Africa, and some

basic characteristics for each of the selected plants.

3.1.1. Albizia coriaria. A. coriaria is a pioneer tree that is

found throughout Uganda on forest edges, wooded grass-

lands, woodland, and thickets. The tree is large and de-

ciduous. Although it can reach a height of up to 18 m, it is

frequently smaller with a ﬂat, spreading crown [20, 21]. It is

an indigenous plant that is also known as the “giant albizia”

[20, 22]. A. coriaria can generally be found from Sudan to

southern Angola [20]. It grows on various soil types at an

altitude of 850–1,680 m above sea level (m.a.s.l.) [23].

A. coriaria can be propagated from seeds, and wild plants

can be collected and planted. The seeds have a good ger-

mination rate [23]. The stem bark was formerly utilized as a

ﬁsh poison in the Madi and West Nile areas of Uganda [24].

Local names in diﬀerent Ugandan languages are as follows:

Luganda: mugavu [14, 20–23], Lusoga: musita [20, 21, 23],

Ateso: etek and etekwa [20, 23], Kwamba: musisiya [23],

Lugishu: chesovio and kumoluko [23], Swahili: mugavu

[20, 22], Lugwe: mubere [23], Luo (Acholi): latoligo and

ayekayek [20, 23], Luo (Jopadhola): omogi and ober

[20, 23], Luo (Langi): itek and bata [23], Madi: oyo [23],

Rukiga: muyenzayenze [23], Runyankore: musisa and

murongo [20, 23], Runyoro: musisa [20, 23], Rutoro:

musisa [20, 23], and Ik: kiluku [21]. A local name in other

East African countries is as follows: Luhya: omubele [20].

3.1.2. Cassine buchananii. Some synonyms include: Elaeo-

dendron buchananii (Loes.) Loes., E. keniense Loes., E. stolzii

Loes., E. warneckei Loes., E. afzelii Loes., and E. friesianum

Loes. This indigenous species is better known as the “moth

tree” or the “leathery-leaved saﬀron” [20]. It is a small shrub

to large tree up to 24 m high with a round compact crown

that commonly occurs in grasslands in parts of Uganda

[24, 25], but can also be found in dry upland evergreen

Table 1: Overview of medicinal plant species investigated in this study, indicating high traditional use in treatment of medical disorders in

the Greater Mpigi region (n�39).

Botanical name Local name

(Luganda language) Family RFC (%)

Albizia coriaria Oliv. Mugavu Fabaceae 100.0

Cassine buchananii Loes. Mbaluka Celastraceae 61.5

Combretum molle R.Br. ex G.Don Ndagi Combretaceae 89.7

Erythrina abyssinica DC. Jjirikiti Fabaceae 100.0

Ficus saussureana DC. Muwo Moraceae 94.9

Harungana madagascariensis Lam. ex Poir. Mukabiiransiko Hypericaceae 97.4

Leucas calostachys Oliv. Kakuba musulo Lamiaceae 43.6

Microgramma lycopodioides (L.) Copel. Kukumba Polypodiaceae 43.6

Morella kandtiana (Engl.) Verdc. & Polhill Mukikimbo Myricaceae 87.2

Plectranthus hadiensis (Forssk.) Schweinf. ex Sprenger Kibwankulata Lamiaceae 97.4

Securidaca longipedunculata Fresen. Mukondwe Polygalaceae 38.5

Sesamum calycinum subsp. angustifolium (Oliv.) Ihlenf. & Seidenst. Lutungotungo Pedaliaceae 87.2

Solanum aculeastrum Dunal Kitengo Solanaceae 71.8

Toddalia asiatica (L.) Lam. Kawule Rutaceae 97.4

Warburgia ugandensis Sprague Abasi Canellaceae 92.3

Zanthoxylum chalybeum Engl. Ntaleyaddungu Rutaceae 46.2

Evidence-Based Complementary and Alternative Medicine 3

forests, forest remnants, and riverine woodland (growing at

an altitude of 1,200–2,100 m.a.s.l.) [20]. Its ripe fruits are

green-orange and ovoid (up to 2.5 cm). Parts of the tree are

known to be extremely toxic to livestock, especially when the

leaves are ingested. Death occurs suddenly. Interestingly,

giraﬀes eat the leaves of C. buchananii without notable

Harungana

madagascariensis

Zanthoxylum

chalybeum

Solanum

aculeastrum

Sesamum calycinum

subsp. angustifolium

Albizia

coriaria

Combretum

molle

Cassine

buchananii

Erythrina

abyssinica

Ficus

saussureana

Leucas

calostachys

Microgramma

lycopodioides

Morella

kandtiana

Plectranthus

hadiensis

Securidaca

longipedunculata

Toddalia

asiatica

Warburgia

ugandensis

Figure 1: Digitized herbarium voucher specimens showing the 16 selected medicinal plant species used in the Greater Mpigi region, Uganda

(source: JSTOR Global Plants Database).

4Evidence-Based Complementary and Alternative Medicine

adverse eﬀects [24]. The local name in Uganda is as follows:

Luganda: mbaluka [14, 21, 26]. Local names in other East

African countries are as follows: Kisii: enkanda [24], Meru:

mutimweru [24]; Kikamba: mutanga and mutanya [24],

Kipsigis/Lumbwa: sawanet [24], Sebei: sunwa [24], and

Kinyaramba: mtuwilang’holo [24].

3.1.3. Combretum molle. Some of the synonyms are

C. welwitschii Engl. & Diels, C. arbuscula Engl. & Gilg, C. nyikae

Engl., C. boehmii Engl., C. holtzii Diels, C. schelei Engl., and

C. ankolense Bagsh. C. molle is a slow-growing tree widespread

in wooded grasslands and bushlands in Uganda and the rest of

the African continent. It also commonly grows on stony hills up

to an altitude of 2,300 m.a.s.l. [20]. The seeds germinate easily if

fresh [23]. It is usually 5–7 m in height and branching near its

base [20]. The names in Ugandan local languages are as follows:

Ateso: ekworo and eworo [21, 23], English: velvet-leaved

Combretum and velvet bushwollow [20, 23], Luganda: ndagi

[14, 20, 21, 23], Lugbara: geleo [23], Lugishu: shikimira [23],

Lugwe: muchuta [23], Lugwere: kinakworo [23], Luo (Acholo):

okechu and oduk [20, 23], Luo (Jopadhola): deda [23], Luo

(Langi): iworo and iyoro [23], Lusoga: ndawa, daha, and

nfodwa [23], Madi: otubi and lebilebi [23], Runyoro: murama

[23], Sebei: kembei [23], and Ik : ngulara [21]. Local names in

other regions of East Africa are as follows: Luhya: mukhungula

[20], Maasai: ol-mororoi [20], Swahili: mgurure [20], Sukuma:

kagua [20], Kamba: muama [20], Kikuyu/Meru: murema and

murama [20], Taita: mwama [20], and Haya/Nyamwezi:

mlama [20].

3.1.4. Erythrina abyssinica. E. abyssinica is a deciduous tree,

reaching a height of 6–12 m. It has a short trunk and thick

spreading branches. It has a rounded crown and occurs in

savannah woodland, grassland, and scrubland throughout

Uganda [20, 22, 23]. It propagates through seeds and cuttings,

but the seeds have a low germination rate. E. abyssinica is an

indigenous species that is also known as the “red-hot poker

tree,” the “ﬂame tree,” the “Uganda coral tree,” or the “lucky

bean tree” [20, 22]. The tree is called “ﬂame tree” because of its

orange-red ﬂowers. Common synonyms are E. bequaerti De

Wild., E. kassneri Baker f., E. tomentosa R. Br., Chirocalyx

abyssinicus (Lam.) Hochst., C. tomentosus Hochst., and Cor-

allodendron suberifera (Welw. ex Baker) Kuntze. The local

names in diﬀerent languages in Uganda are as follows: Lu-

ganda: muyirikiti and jirikiti [14, 20–23], Lugbara: oluo and

olugo [21, 22], Runyankore: muko, kiko, and murinzi [14, 23],

Lugishu: cheroguru and muragolo [20, 22, 23], Lugwe:

mutembetembe [22, 23], Lunyuli: mudongodongo and mukobe

[22, 23], Swahili: mwamba-ngoma [20, 22], Luo (Acholi):

lochoro, kisoro, oding, and loting [22, 23], Luo (Jophadhola):

koli [23], Luo (Langi): ewilakot [23], Madi: olawu [22, 23],

Rukiga: bwiko [23], Runyoro: mudoti, muko, and kiko [23],

Rutoro: muko and kiko, Sebei: kaborte [23], Ateso: engosorot

[23], Kwamba: kikiri [23], and Lusoga: muyirikiti [21]. Local

names in other East African countries include the following:

Chagga: mriri [20, 22], Kamba: muvuti [20], Taita: mulungu

[20, 22], Kisii: omotembe [20, 22], Hehe: muhemi [20, 22],

Pare: muungu [20, 22], and Ateso: engosorot [20, 22].

3.1.5. Ficus saussureana. Some synonyms include

F. eriobotryoides Kunth & C.D. Bouch´

e, F. afeelii Kunth and

C.D.Bouch´

e, F.dawei Hutch, F. murrayana Miq., F.mon-

buttuensis Warb., F. dawei Hutch, or Galoglychia saussur-

eana Gasp. It is a large, mostly epiphytic, hemi-epiphytic, or

terrestrial tree [27, 28]. The base of the trunk consists of a

mass of fused aerial roots. It produces large amounts of white

latex. The slash typically discolours, but the latex does not

[28]. It is a widely distributed tree in West Africa and the

eastern and western margins of the Congo Basin [29]. In

Uganda, it mainly occurs in the northern, western, and

south-central parts [28]. F. saussureana prefers riverine,

groundwater, and lowland forest areas [27]. The local name

in Luganda language is as follows: muwo [14, 21].

3.1.6. Harungana madagascariensis. Some common syno-

nyms include: Haronga madagascariensis (Lam. ex Poir.),

Haronga paniculata Lodd. ex Steud., Haronga pubescens Steud.,

and Arungana paniculata Pers. The vernacular name is “or-

ange-milk tree” [20]. It is a pioneer, evergreen shrub or tree,

reaching 3–18 m in height, whose bark, leaves, and stem

produce a brilliant orange sap that turns blood-red on expo-

sure. The outer layers of the wood and the innermost layer of

the bark yield a yellow sap. This sap is traditionally used as a dye

[20, 23, 30]. The bark mixed with the highly poisonous

Mansonia altissima is used as B´

et´

earrow poison in the Daloa

region of the Ivory Coast [30]. H. madagascariensis occurs

throughout tropical Africa, from Senegal to East Africa. It is a

common and widely distributed pioneer tree species in

Uganda, where it grows along forest edges, in areas where

forests have been cleared, in secondary scrubland, around

termite mounds, and in riverine areas at medium to low al-

titudes [20, 23, 30]. The local names in diﬀerent languages in

Uganda are as follows: Luganda: mulirira and mukabiiransiko

[14, 20, 23, 30], Madi: asonbere and serubele [14, 30], Rukiga:

mungolero, munianga, and muliamanga [23], Runyankore:

mutaha [20, 23], Rutoro: murinda, murunda, and musoga

[20, 23, 30], Luo: aremo [20], Kirundi: umushayishyi [30],

Nyankole: omutaha [30], Kiga: omungolero mniananga and

muliamanga, and Swahili: mkekundu, mdamudamu, mpula-

pula, nkekundu, nrimba, ngoningoni, kunamaji, funa maji,

mdura, and mgondogado [30]. Local names in other East

African languages and countries are as follows: Luhya-Bukusu:

namalasile [20], Luhya-Kisa: omwinyala amatsai [20], Nandi:

chepsebil [20], Meru: munyanwe [20], Embu: munyanwe [20],

Sambaa: mkuntu [20], Ngindo: muhekara [30], Mbunga:

mtelekajugo [30], Ruﬁji: mulungamo [30], Pogoro: mson-

goliko [30], Hehe: mtunu [30], and Digo: marindazia [30].

3.1.7. Leucas calostachys. Synonyms are Leucas calostachys

var. calostachys and Leucas calostachys var. fasciculata (Baker)

Sebald. L. calostachys is an aromatic herb that occurs in some

parts of Uganda, including the Greater Mpigi region [14].

However, there is limited literature on this species. The local

name in Luganda language is as follows: kakuba musulo [14].

3.1.8. Microgramma lycopodioides. Known synonyms for

this species are Pleopeltis lycopodioides (L.) C. Presl,

Evidence-Based Complementary and Alternative Medicine 5

Polypodium lycopodioides L., Niphobolus lycopodioides (L.)

Keyserl., and Phymatodes lycopodioides (L.) Millsp. It is an

epiphytic or terrestrial fern that has been reported in tropical

America, especially Brazil and Mexico, in sub-Saharan

Africa, and in the Caribbean [31–38]. In Uganda,

M. lycopodioides has been recorded in Masaka district, Lake

Nabugabo, Mengo, Entebbe, Kibale forest, and in the

Greater Mpigi region [14, 39, 40]. The local name in Uganda

is as follows: Luganda: kukumba [14].

3.1.9. Morella kandtiana. There is one synonym: Myrica

kandtiana Engl. M. kandtiana is an herb, shrub, or short

multibranched tree that spreads. The ﬂowers are on the

inﬂorescences, which are greenish yellow. The inﬂorescences

occur on the older rather than on the younger branches. It

grows in grasslands, in seasonal swamps, or swampy areas,

but is very rare nowadays [41, 42]. Local names in diﬀerent

languages in Uganda are as follows: Luganda: mukikimbo,

bowolola omusajja, and enkikimbo [14, 41, 43] and Run-

yankore/Runyoro: omujeje [41].

3.1.10. Plectranthus hadiensis. Common synonyms are

P. cyaneus G¨

urke, P. forsskaolii Vahl, Coleus personatus

Lem., and C. forsskaolii Briq. It is a widespread, semi-suc-

culent, herbaceous perennial herb in East and Central Africa.

It has also been reported in South Africa. P. hadiensis can

grow 10–150 cm high [41, 44, 45]. Local names in Uganda

are as follows: Luganda: mukikimbo [14] and Lusoga: kiraga

and kigalama [41].

3.1.11. Securidaca longipedunculata. Some of the common

synonyms include Elsota longipedunculata (Fresen.) Kuntze

and S. longipedunculata var. longipedunculata. It is a semi-

deciduous shrub or small tree that can reach a height of

2–6 m. S. longipedunculata is widespread throughout

tropical Africa from Kenya and Uganda to South Africa. It

occurs in wooded and savannah grassland and woodland,

preferring dry areas, and it is associated with Hymenocardia

acida and Combretum spp. The plant easily propagates

through seedlings, but seeds germinate with diﬃculty if not

pretreated. The roots are yellow, and if cut, this species

radiates an intense aromatic smell. The ﬂowers are sweet

scented, in numerous racemes, and magenta, purple, or

violet in color [23, 24, 30]. According to Neuwinger [30],

S. longipedunculata is “one of the most beautiful African

ﬂowering shrubs or trees.” Interestingly, the plant is highly

toxic to humans, which is why it has been used as a hunting

poison in Africa, but much more often as a trial-by-ordeal

and murder poison. For example, the plant has been de-

scribed as the most often used ordeal poison among the

Gbaya people in the Central African Republic. Sadly, the

Lunda women of the Democratic Republic of Congo,

Zambia, and Angola consider the root pulp or the peeled

root the “best known and most eﬀective of all the intra-

vaginal poisons” used for suicide [30]. Local names in

diﬀerent languages in Uganda are as follows: Ateso: elilyoi

and elilie [23], Lugbara: oiyofe [23], Lugishu: wadambasima

[23], Lugwe: mwiabala and amwiabala [23, 30], Lugwere:

loloyi [23, 30], Luo (Acholi): aliya, lalia, and lalon [23, 30],

Luo (Jophadhola): lilyo [23, 30], Luo (Langi): elila [23, 30],

Madi: lio [23, 30], Runyankore: mweya and omweya

[23, 30], Runyoro: nkondwe and nkungwe [23, 30], Lu-

ganda: lilo and mukondwe [14, 21, 23, 30], Swahili: Nzigi,

muteya, matungunungu, and mzigi [30], Lusoga:

mukondwa [21, 23], Teso: elilyoi and elilie [30], and Soga:

mukondwa [30]. Local names in other East African countries

and languages are as follows: Nyarwanda: umunyagazozi

and umukuyu [30], Kirundi: umunyagazozi [30], Hehe:

muhulatangu and mukenegatangu [30], Zigua: mkola and

mkala [30], Zinza: mweyo [30], Sukuma: hengo-hengo,

nengo-nengo, and mbaso [30], Yao: chiguluka [30], Ngindo:

kiguraka [30], Mwera: mtikwi [30], Shambaa: mbazo [30],

Kamba: ithithi [30], Kikuyu: muguraka [30], and Digo:

muteya, mzidvi, mzidyi, and mzisi [30].

3.1.12. Sesamum calycinum Subsp. angustifolium. There are

two synonyms: Sesamum angustifolium (Oliv.) Engl. and

Sesamum indicum var. angustifolium Oliv. S. calycinum

subsp. angustifolium is an erect, annual to perennial herb

with or without side branches. It can reach a height of

0.4–2.0 m. The ﬂowers appear pink or purple and often have

spots within. Its distribution encompasses eastern tropical

Africa, including Uganda, Tanzania, Democratic Republic of

Congo, and Kenya and south to Malawi, Zambia, and

Mozambique. It is occasionally cultivated as a vegetable and

prefers sandy soil. It frequently grows by roadsides, in

grasslands, and open woodlands [46]. Local names in dif-

ferent languages in Uganda are as follows: Luganda:

lutungotungo [14, 21] and Lusoga: lutungotungo [21].

3.1.13. Solanum aculeastrum. This plant species is a large

shrub or small tree, and it was reported to be cultivated in

Rugazi, Bynyaruguru, and Ankole in western Uganda [41]. It

can reach up to 6 m in height [20]. S. aculeastrum is a native

African plant that occurs from the South African Cape to the

Imatong mountains in Sudan and westwards to Cameroon

[47]. Its branchlets are densely covered in woolly hairs and

possess sharp, curved thorns [48]. It ﬂowers from September

to July, peaking in November and March, and fruits from

April to January, peaking in June and November [49]. The

fruits are extremely bitter and highly toxic due to the

presence of the poisonous alkaloid solanine [24, 50]. The

species is regionally known as “bitter apple” [20]. Local

names in Uganda are as follows: Luganda: ekitengo, entego

eddene, and entengo lyabalalo [14, 20, 41]. Local names in

other East African countries and languages are as follows:

Kikuyu: mutura [20], Kipsigis: siganet [20], and Maasai:

osigawai [20].

3.1.14. Toddalia asiatica. Synonyms include Aralia labordei

H.L´

ev., Cranzia aculeata (Sm.) Oken., Paullinia asiatica L.,

Toddalia aculeata (Sm.) Pers., and Toddalia ﬂoribunda Wall.

T. asiatica is a woody liana or shrub widely distributed in

Southeast Asia, South Africa, and tropical Africa [51]. In

6Evidence-Based Complementary and Alternative Medicine

Uganda, it is cultivated by traditional healers and was

designated as a multipurpose slow-growing shrub with

important therapeutic values [52]. It commonly grows in

tropical forests, especially near anthills, near rivers or

streams, and it grows fairly well in clay soils [53]. In East

Africa, this indigenous species commonly grows in riverine

and forest edge habitats from where it is harvested. Local

herbalists in Uganda exclusively harvest it from the wild

[54]. Local names in Uganda are as follows: Luganda:

kawule [14, 55] and Luo: ajua [22]. Local names in other East

African countries and languages are as follows: Maasai: ole-

barmonyo [22], Digo: chikombe za chui [22], Kikuyu:

mwikunya [22], Kamba: maluia [22], Luhya: luabare [22],

Marakwet: kipkeres [22], Meru: mukonguru [22], Tugen:

ketemwe [22], Nandi: usuet [22], Turkana: etokebengu [22],

and Samburu: llaramunyo [22].

3.1.15. Warburgia ugandensis. Some common synonyms are

Dawea ugandensis Sprague ex Dawe and Warburgia ugan-

densis subsp. ugandensis. W. ugandensis is an evergreen tree

with a dense leafy rounded canopy that is widely distributed

in lower rainforest and drier highland forest areas of East

Africa. It is also known as the “East African greenheart” and

the “pepper-bark tree.” The species can grow up to 25 m

high. It occurs between 1,000 and 2,000 m.a.s.l. In Uganda, it

grows in colonizing forests, forest edges, and thickets, as well

as often on dry sites [20, 22, 23]. W. ugandensis is one of the

most commonly used multipurpose medicinal plant species

in Uganda [14, 21]. It is a fairly slow-growing tree whose

seeds quickly lose viability. The wood has high oil content

[23]. Local names in Uganda are as follows: Luganda: abasi,

muya, and mukazanume [14, 20, 21], Mukuzanume, dialect

Buddu: muwiya [23], Lusoga: balwegiira [21, 23], Lugishu:

balwegira and abasi [21], Luo (Langi): abac [21], Runyoro:

musizambuzi and mwiha [20, 22, 23], Rutoro: muharami

[20, 22, 23], and Lugwere: muwiya [22]. Local names in

other East African countries and languages include the

following: Kikuyu: muthiga [20], Maasai: osogonoi and

msokonoi [20], Rangi: osogonoi and msokonoi [20], Kisii:

omenyakige [20], Luhya: apacha [20], Meru: musunui [20],

Nandi: soget and sorget [20], Tugen: soget and sorget [20],

Kipsigis: sogoet [20], Goro: sagonai [20], Haya: muhiya

[20], and Sambaa: mdee and mlifu [20].

3.1.16. Zanthoxylum chalybeum. Synonyms include Zan-

thoxylum chalybeum var. chalybeum and Fagara chalybea.

Z. chalybeum, also known as the “lemon-scented knob-

wood,” is a spiny deciduous shrub or tree that can reach up

to 8 m in height [20]. Its crown is open rounded. It grows in

medium to low altitudes up to 1,500 m.a.s.l., mainly in dry

woodlands, bushlands, or grasslands and often on termite

mounds and in rocky places. The bole has characteristic

large, conical woody knobs with sharp prickles. Twigs and

branches have single recurved spines that are up to 2 cm long

and dark red. It can be propagated through seeds and

cuttings obtained from wild or cultivated plants. The seeds

lose viability quickly [20, 23, 30]. The leaves have a strong

lemon smell if crushed [30]. Local names in diﬀerent

languages in Uganda are as follows: Ateso: eusuk and

musuku [20, 23, 30], Luo (Acholi/Alur): kichuk and roki

[23, 30], Luganda: ntaleyedungu, ntaleyaddungu, and nta-

liyedongu [14, 21, 23, 30], Lugwere: musuku [20, 30],

Lusoga: ddungu lya ntale [23], Ik: rukuts [23], Lugbara:

outiku [23], Swahili: mjafari, mkununungu, and mtata

[20, 30], and So (Tepes): wangok and ongokat [30]. Local

names in other East African countries and languages are as

follows: Maasai: ol-oisugi and ol oissugu [20, 30], Zaramo:

mnungu [20], Nandi: sagawaita [20], Kipsigis: sagawaita

[20], Digo: mdungu, mdhungu, and mundungu [20, 30],

Chonyi: mdungu and mdhungu [20], Giriama: mdungu and

mdhungu [20, 30], Kamba: mukenea [20], Mbeere:

mugucua [20], Meru: mugucua [20], Tharaka: muguuchwa

[20], Marakwet: sangoja and songuruwa [30], Luguru:

mhunungu [20], Nywarwanda: intare y’irungu [30], Teita:

genika [30], Samburu: l’oisug-i and l’oisuk-i [30], Boran:

adda [30], Boni: arer and arere [30], Shambaa: mfuakumbi

[30], Hehe: mulungulungu [30], Mbunga: muhuluhumbi

and mulunguhumbi [30], Nyamwezi: mnugunugu [30],

Zigua: mhombo and mkunungu [30], and Sukuma: nun-

gunungu [30].

3.2. Literature Review. The determination of the DoPs en-

abled successful assessment of the degrees to which each

individual plant species has been studied so far, while also

taking into account the methodological “research chain of

ethnopharmacology” from ethnobotanical studies (“tradi-

tional use”) to pharmacological assays (“bioactivity”) and

ﬁnally to pharmacognostic research (“structure elucida-

tion”). The signiﬁcance of a research paper was also assessed

by determining whether its journal and publishing house

were members of the COPE.

The literature survey was completed on 31 July 2019 and

covered the period of 1960–2019. In total, 634 peer-reviewed

publications were reviewed, 53.3% of which were published

in journals, and by publishing houses aﬃliated with the

COPE (338 publications). These articles were published in

304 diﬀerent academic journals, of which 114 are COPE

members. A cloud-based literature library was successfully

created, ﬁrst categorizing publications according to the

selected plant species mentioned in the paper and subse-

quently, according to their individual DoPs (“Traditional

use,” “Bioactivity,” “Structure elucidation,” and “Other”).

Excluding the DoP “Other,” there were a total of 441 ﬁeld-

related original research papers, of which 245 were pub-

lished by journals with COPE membership (55.6%).

3.3. DoP Analysis on the Totality of Selected Plant Species.

Figure 2 shows the distribution of papers by DoPs. A total of

191 publications (30.1%) were allocated to the DoP “Other,”

as these were mostly non-ﬁeld-related publications and a few

review papers. Reference to the plant species of interest was

often in the form of documentation of traditional knowledge

and medicinal application, and those papers were allocated

to the DoP “Traditional use” (139 papers). This represents

21.9% of all original research publications mentioning one of

the 16 plant species or about a third of all ﬁeld-related

Evidence-Based Complementary and Alternative Medicine 7

publications (31.5%). With 186 articles (42.2%), the largest

share of ﬁeld-related publications were classiﬁed to the DoP

“Bioactivity,” depicting the importance of in vitro and in vivo

evaluation of traditional use and pharmacology activity in

the ﬁeld of ethnopharmacology. DoP “Bioactivity” catego-

rized articles made up 29.3% of all published papers (in-

cluding DoP “Other”). As this is the ﬁnal stage of the

bioassay-guided fractionation methodology in drug dis-

covery, original research dealing with structure elucidation

of bioactive compounds made up the smallest share (DoP

“Structure elucidation” �116), representing 18.3% of all

recorded publications and 26.3% of the ﬁeld-related pub-

lications (without DoP “Other”).

3.4. Journal Analysis and COPE Assessment.

Subsequently, the frequency of each DoP term’s publication

in individual peer-reviewed journals (“abundance of pub-

lication”) was assessed. The results are shown in Figure 3.

The signiﬁcance of COPE member Journal of Ethno-

pharmacology (JEP) to the ﬁeld can be aﬃrmed, as by far the

greatest proportion of related articles describing “Traditional

use” and “Bioactivity” were published in the JEP, as well as

the fourth-highest number of papers recorded for the DoP

“Structure elucidation.” Overall, 36.7 % of all publications

categorized under the DoP “Traditional use” (51 articles)

were printed in the JEP, 15.1% in the case of the DoP

“Bioactivity” (28 articles), and 4.3% for the DoP “Structure

elucidation” (5 articles).

Other journals that published the greatest proportion of

“Traditional use”-related papers on the 16 selected medicinal

plants are the Journal of Ethnobiology and Ethnomedicine

(7.9%, 11 articles, COPE member), the Journal of Herbal

Medicine (4.3%, 6 articles, COPE member), the African

Journal of Traditional, Complementary and Alternative

Medicines (2.9%, 4 articles), Ethnobotany Research &

Applications (2.9%, 4 articles), the European Journal of

Medicinal Plants (2.9%, 4 articles), the Journal of Medicinal

Plants Research (2.9%, 4 articles), the Journal of Medicinal

Plant Studies (2.9%, 4 articles), and the South African Journal

of Botany (2.9%, 4 articles, COPE member). The rest of the

“Traditional use”-related publications (33.8%, 47 articles)

were printed in 37 other journals, of which the majority fail

to be COPE members (26 journals).

In terms of the DoP “Bioactivity,” the second-largest

proportion of papers was published in the African Journal of

Traditional, Complementary and Alternative Medicines

(6.5%, 12 articles), followed by Phytotherapy Research (3.5%,

6 articles, COPE member) and African Health Sciences

(2.7%, 5 articles). Ninety-seven other journals with minor

article distribution (<2.5%) were identiﬁed and summarized

under “Other” (72.6%, 135 articles). The majority of these

journals are not COPE members (64 journals).

The most dominant journals for the DoP “Structure

elucidation” were the COPE members Phytotherapy (11.2%,

13 articles) and Journal of Natural Products (9.5%, 11 ar-

ticles). These journals are also followed by COPE members

to the biggest part: Planta Medica (5.2, 6 articles), the JEP

(4.3%, 5 articles, COPE member), Phytotherapy Research

(4.3%, 5 articles, COPE member), Phytochemistry Letters

(3.4%, 4 articles, COPE member), Phytomedicine (3.4%, 4

articles, COPE member), Bioorganic & Medicinal Chemistry

Letters (2.6%, 3 articles, COPE member), the Bulletin of the

Chemical Society of Ethiopia (2.6%, 3 articles), and Phar-

maceutical Biology (2.6%, 3 articles, COPE member). A total

of 59 papers were published in 50 other journals (50.9%), of

which 24 are COPE members.

Statistical analysis of the DoP “Other” resulted in the

identiﬁcation of three journals that were most abundant:

the African Journal of Ecology (4.2%, 8 articles, COPE

member), the African Journal of Biotechnology (2.6%, 5

articles), and the Uganda Journal of Agricultural Sciences

(2.6%, 5 articles). Moreover, the majority of papers, in-

cluding their corresponding journals, were summarized as

“Other” (<2.5% of articles in DoP “Other” published in this

journal), consisting of a total of 173 articles (90.6%) printed

in 138 diﬀerent journals, whereas only 58 of these are

COPE members. Research categorized under the DoP

“Other” and its individual journals was diverse, ranging

from journals on botany (e.g., Planta,Systematic Botany,

and American Fern Journal), nature conservation (e.g.,

Biological Conservation and Journal of Threatened Taxa),

geography (e.g., Applied Geography and Journal of Bioge-

ography), ecology (e.g., Journal of Chemical Ecology,Plant

Ecology,Oecologia, and Advanced Journal of Ecology and

Ecosystems), animal sciences (e.g., Journal of Advanced

Veterinary and Animal Research and Livestock Science),

and insect studies (e.g., Journal of Applied Entomology,

Journal of Insect Physiology,Applied Entomology and Zo-

ology, and Entomologia Experimentalis et Applicata) to

more abstract journals (e.g., Polymers,International

Journal of Creative Research Thoughts,International

Journal of Cosmetic Science,Journal of Archaeological

Science, and Digest Journal of Nanomaterials and Bio-

structures, Biomass and Bioenergy), and others.

DoPs

“Traditional use”

“Bioactivity”

“Structure elucidation”

“Other”

21.9%

29.3%

18.3%

30.1%

Figure 2: Distribution of peer-reviewed articles within the diﬀerent

DoPs (total number of articles: 634; number of species of interest:

16).

8Evidence-Based Complementary and Alternative Medicine

3.5. Assessment of Study Progress for Each Species. The DoPs

were used as a tool for assessment of the degree to which a

species has been studied so far. Results on individual plants

are shown in Figure 4 (accumulated DoPs, excluding the

DoP “Other”) and Table 2 (absolute values of individual DoP

categories). Values in red or in square brackets state the total

number of articles published in journals with COPE

membership that committed themselves to reaching highest

standards and best practice in scholarly publication ethics.

3.5.1. Highly Understudied Species. Plant species identiﬁed as

being highly understudied are M. lycopodioides (DoP

total

= 2

(1)), M. kandtiana (DoP

total

= 2 (2)), F. saussureana (DoP

total

3 (3)), S. calycinum subsp. angustifolium (DoP

total

= 5 (4)),

C. buchananii (DoP

total

= 6 (5)), and L. calostachys (DoP

total

12 (4)). Numbers in brackets correspond to absolute numbers

of journal articles from publishing houses with COPE

membership.

According to results of our literature review,

M. lycopodioides has been studied and mentioned in a total

of 63 journal articles over the past 60 years. However, the

vast majority of these papers deal with non-medicinal use

and are not part of drug discovery-related research. The

papers mainly describe the occurrence of the fern species in

the American tropical forests and its extraordinary mor-

phology/biology; traditional medicinal use has only been

mentioned in two publications so far. Here, it has been

reported to be used for removal of lice and to treat anemia in

South Africa and Tanzania [56]. The second publication

European Journal of

Medicinal Plants

2.9%

Journal of Medicinal

Plants Research

2.9%

Journal of Medicinal

Plants Studies

2.9% South African

Journal of Botany

2.9%

Ethnobotany Research

and Applications

2.9%

African Journal Traditional

Complementary and

Alternative Medicines

2.9%

Journal of Ethnobiology

and Ethnomedicine

7.9%

Journal of Herbal

Medicine

4.3%

Other (<2.5% of articles)

33.8%

Journal of

Ethnopharmacology

36.7%

Abundance of journals for DoP ‘Traditional use’

(a)

African Journal

Traditional,

Complementary

& Alternative

Medicines

6.5%

African Health Sciences

2.7%

Other (<2.5%/articles)

72.6%

Journal of

Ethnopharmacology

15.1%

Phytotheraphy

Research

3.2%

Abundance of journals for DoP ‘Bioactivity’

(b)

Phytochemistry

11.2%

Other (<2.5% of articles)

50.9%

Pharmaceutical

Biology

2.6%

Bulletin of the Chemical

Society of Ethiopia

2.6%

Bioorganic & Medicinal

Chemistry Letters

2.6%

Phytochemistry

Letters

3.4%

Phytotheraphy

Resarch

4.3%

Journal of

Ethnopharmacology

4.3%

Planta Medica

5.2%

Journal of Natural

Products

9.5%

Phytomedicine

3.4%

Abundance of journals for DoP ‘Structure elucidation’

(c)

Other (<2.5% of articles)

90.6%

African Journal

of Ecology

4.2%

African Journal of

Biotechnology

2.6%

Uganda Journal of

Agricultural Sciences

2.6%

Abundance of journals for DoP ‘Other’

(d)

Figure 3: Distribution of scientiﬁc articles within peer-reviewed journals (total number of articles: 634; number of species of interest:16). (a)

Abundance of journals for DoP “traditional use.” (b) Abundance of journals for DoP “bioactivity.” (c) Abundance of journals for DoP

“structure elucidation.” (d) Abundance of journals for DoP “other.”

Evidence-Based Complementary and Alternative Medicine 9

reports traditional use by the Zambo-Miskito ethnic group

of Eastern Nicaragua to cure bewitchment and chase away

evil spirits [57].

Traditional use of the shrub M. kandtiana has also

only been previously described in two publications fol-

lowing ethnobotanical surveys. Just as in our ethnobo-

tanical study in the Greater Mpigi region [14], its roots

were cited to be used in treatment of HIV/AIDS in one of

the four surveyed Ugandan districts [58]. The second

paper names M. kandtiana bark, leaves, and fruits as a

natural remedy in treatment of tuberculosis (fruits,

leaves, roots, and bark used) in the Butambala and Mpigi

Districts, which constitute a major part of the Greater

Mpigi region [43]. In our ethnobotanical survey [14], we

were able to conﬁrm these citations of traditional use in

the study area (root bark, leaves, and roots). There were

no other publications from any other ﬁeld identiﬁed,

mentioning M. kandtiana.

Table 2: Literature survey overview: DoPs and individual categories; number �total number of journal articles published for a certain DoP

category, (COPE) �number of journal articles from publishing houses with COPE membership.

Plant species

Degree of publication (DOP)

Traditional use Bioactivity Structure

elucidation Other

Total

(without

“other”)

Number

(COPE)

Number

(COPE)

Number

(COPE)

Number

(COPE)

Number

(COPE)

Albizia coriaria 11 (9) 4 (4) 2 (2) 9 (5) 17 (15)

Cassine buchananii 2 (1) 1 (1) 3 (3) 1 (1) 6 (5)

Combretum molle 8 (7) 30 (15) 11 (6) 21 (8) 49 (28)

Erythrina abyssinica 18 (9) 7 (2) 16 (11) 7 (4) 41 (22)

Ficus saussureana 1 (1) 1 (1) 1 (1) 2 (2) 3 (3)

Harungana madagascariensis 11 (5) 38 (12) 13 (10) 11 (2) 62 (27)

Leucas calostachys 9 (3) 3 (1) — 1 (-) 12 (4)

Microgramma lycopodioides 2 (1) — — 61 (25) 2 (1)

Morella kandtiana 2 (2) — — — 2 (2)

Plectranthus hadiensis 3 (2) 13 (2) 6 (3) 24 (13) 22 (7)

Securidaca longipedunculata 28 (16) 26 (8) 12 (8) 13 (1) 66 (32)

Sesamum calycinum subsp. angustifolium 4 (3) — 1 (1) 9 (8) 5 (4)

Solanum aculeastrum 6 (5) 10 (7) 6 (2) 3 (2) 22 (14)

Toddalia asiatica 15 (6) 21 (12) 28 (21) 9 (8) 64 (39)

Warburgia ugandensis 8 (7) 15 (5) 14 (11) 16 (11) 37 (23)

Zanthoxylum chalybeum 11 (9) 17 (8) 2 (2) 6 (3) 30 (19)

0 10 20 30 40 50 60 70

DoPs

Microgramma lycopodioides

Morella kandtiana

Ficus saussureana

Sesamum calycinum subsp. angustifolium

Cassine buchananii

Leucas calostachys

Albizia coriaria

Plectranthus hadiensis

Solanum aculeastrum

Zanthoxylum chalybeum

Warburgia ugandensis

Erythrina abyssinica

Combretum molle

Harungana madagascariensis

Toddalia asiatica

Securidaca longipedunculata

Figure 4: Summarized “total” DoPs (without “Other”) for assessment of degree to which the species have been studied so far; blue �total

number of journal articles; red �number of journal articles from publishing houses with COPE membership.

10 Evidence-Based Complementary and Alternative Medicine

As far as M. lycopodioides and M. kandtiana are con-

cerned, no studies on bioactivity or isolation and elucidation

of active secondary metabolites have been published so far.

In the case of L. calostachys, research has shifted slightly

towards the investigation of pharmacological activity. The

majority of papers mention its traditional medicinal use

(DoP

traditional use

= 9 (3)), while three publications investigated

the antiplasmodial activity of the plant (DoP

bioactivity

= 3 (1)),

reporting moderate to low antiplasmodial activity of crude

extracts [59–61]. Traditional uses were recorded in Kenya only

and encompass treatment of malaria [62, 63], gastrointestinal

disorders [64–68], ulcers [65, 67], tructure elucidation of

compounds from P. pneumonia [69], colic pain in infants [65],

stomach ache [68, 69], heartburn [65, 67], cough [70], amoe-

biasis [65], headache [65], heart diseases [65], renal disorders

[65], ﬂu [68, 70], arthritis [65], skin diseases [65], and cancer

[65]. There were no articles found describing isolation and

identiﬁcation of bioactive natural products from L. calostachys.

F. saussureana, S. calycinum subsp. angustifolium, and

C. buchananii are still highly understudied, but research has

progressed to the bioanalytical stage of structure identiﬁcation.

Regarding F. saussureana, the intra- and interspeciﬁc

variations in vacuolar ﬂavonoids among Ficus species from

the Budongo Forest, Uganda, were described [71]. Another

paper, which undertook a pharmacological evaluation of

bioactivity, investigated the vasodilating eﬀect of the root

bark extract of F. saussureana on the guinea pig aorta [72]. In

addition, F. saussureana has also recently been mentioned in

Ugandan traditional medicine for the use of its leaves and

stem bark in the treatment of HIV/AIDS in parts of the

Greater Mpigi region [17].

Only four publications mentioned the traditional use of

S. calycinum subsp. angustifolium; however, there was one

article on bioactive natural product structures isolated from

this species.The ﬁrst paper classiﬁes S. calycinum subsp.

angustifolium as a weed that is used medicinally as an emetic

and contraceptive, as well as for treatment of eye diseases,

diarrhea, burns, and wounds by the Haya people in the

Kagera region, Tanzania [73]. In the second paper, Kibuuka

and Anywar describe the traditional use of S. calycinum

subsp. angustifolium against hernias in Central Uganda [16].

The third paper mentions the use of the fresh leaves in

treatment of hypertension in Bulamogi county, Uganda

(eaten together with Arachis hypogaea or Sesamum indicum)

[74]. The fourth ethnobotanical study from the DoP

traditional

use

was conducted in rural and urban areas across Central

Uganda (including part of the Greater Mpigi region). The

article mentions the use of the root powder (1 tablespoon) in

200 mL water, which is boiled for 5 minutes and then drunk

twice a day to induce vomiting [75]. In terms of the

DoP

structure elucidation

, Chidewe et al. isolated six compounds

from S. calycinum subsp. angustifolium [76]. Two were al-

ready known (the hydrocarbon nonacosane and the glu-

cosinolate glucoiberverin), while the other four remained

unidentiﬁed and were not structure elucidated.

C. buchananii was mentioned twice as being used in other

African countries and cultures, namely, as a natural remedy

against fungal infections in the southern highlands of Tanzania

[77] and against back pain, hernia, and erectile disfunction in

the Kagera region, northwest Tanzania [78]. At our study site in

the Greater Mpigi region [14], we also documented the

common practice of using this plant medicinally in the

treatment of erectile dysfunction. The Kagera region is rela-

tively close to the Greater Mpigi region and also situated in the

Lake Victoria basin. Among other Tanzanian medicinal plants

traditionally used to treat fungal infections, C. buchananii has

been investigated for cytotoxic, genotoxic, and CYP450 en-

zymatic competition eﬀects [79]. In addition, in the 1990s,

three compounds were isolated from C. buchananii: (1) ela-

bunin, a novel dammarane triterpene from the root bark with

moderate cytotoxic activity against L-1210 leukemic cells [80];

(2) mutangin, a novel sesquiterpene from the plant’s fruit, with

moderate antifeedant activity [81]; and (3) buchaninoside, a

steroidal glycoside from the plant’s fruit, with antifeedant

activity against Spodoptera exempta larvae [82].

3.5.2. Understudied Species. Plant species identiﬁed as still

being understudied are A. coriaria (DoP

total

�17 (15)),

P. hadiensis (DoP

total

�22 (7)), and S. aculeastrum (DoP

total

�22

(14)).

In contrast to M. lycopodioides,M. kandtiana, and

L. calostachys, studies on A. coriaria (DoP

structure_elucidation

�2

(2)), P. hadiensis (DoP

structure_elucidation

�6 (3)), and

S. aculeastrum (DoP

structure_elucidation

�6 (2)) already resulted in

isolation of secondary metabolites and structure elucidation

[83–96].

A. coriaria was reported to be used traditionally in 11

publications that show results of surveys conducted in

Uganda, except for the one in Kenya. Forty traditional

healers from the Greater Mpigi region named A. coriaria as

one of their priority plants for the treatment of tuberculosis

[43]. Two more studies from Mpigi district identiﬁed the

stem bark and the leaves for treatment of HIV/AIDS and

related medical disorders [17, 58]. At another study site in

Uganda, Katabi subcounty in Wakiso district, the leaves and

the bark were used in treatment of wounds and skin rashes

[97]. In Kakamega county, Kenya, the bark and the leaves

were reported to be used in treatment of breast, uterine, and

skin cancer [98]. Another publication assessed the ecological

status and ethnobotany of A. coriaria in Budondo sub-

county, eastern Uganda, highlighting its use, local harvesting

patterns, and local attitudes towards its conservation.

Community members consider the species as being rare, and

abundance is declining in the region. Among many non-

medicinal uses, the root and the stem bark were used in the

treatment of syphilis, skin diseases, jaundice, eye diseases,

cough, sore throat, and to concentrate breast milk in humans

[99]. Another study assessed the possibility of setting up

multipurpose tree gardens to provide traditional healers

with species used for medicine [100]. In the county of

Bulamogi in Uganda, the bark is used by traditional healers

in the treatment of diarrhea, “lameness” (butenge), syphilis,

snake bites, and amoebiasis. The roots are used for the

treatment of pyomyositis and amoebiasis. The leaves were

also reported to be used against snake bites [74]. In Kibale

rainforest, an aqueous decoction of the fresh stem bark is

drunk to treat cough [101]. One Ugandan study assessed the

Evidence-Based Complementary and Alternative Medicine 11

domestication of medicinal tree species in the Victoria

lakeshore region, including A. coriaria, and their distribu-

tion by vendors on local markets [102]. With regard to the

DoP

bioactivity

, four publications were recorded. One article

reported low antigiardial activity of a mixture of roots and

bark against Giardia lambia at 500 µg/mL [64]. Another

study investigated the stem bark and reported moderate in

vitro antiplasmodial activity against Plasmodium falciparum

D6 (IC

: 37.83 µg/mL) and low antileishmanial activity

[103]. A dichloromethane extract of the stem bark displayed

moderate antiplasmodial activity against P. falciparum D6

(IC

: 10.68 µg/mL) and the chloroquine-resistant

P. falciparum W2 strain (IC

: 6.80 µg/mL) [104]. Three stem

bark extracts with varying solvents displayed moderate to

low growth inhibitory activity against ﬁve African livestock

pathogens of the genus Mycoplasma [105]. Publications

categorized under the DoP

structure elucidation

include reporting

of the isolation of two new oleanane-type saponins, cor-

iariosides A and B, along with a known saponin, gummi-

feraoside C, from the roots of A. coriaria. As part of this

study, coriarioside A and gummiferaoside C displayed cy-

totoxic activity against the colorectal human HCT116 and

HT29 cancer cells [83]. The same group of researchers

published the isolation and structure elucidation of cor-

iarioside C, D, and E from the roots shortly after [84].

P. hadiensis has been only mentioned to be traditionally

used (DoP

traditional use

) in three ethnobotanical studies so far.

One paper described the use of leaves for wound healing in

the Malabar Region of Kerala, India [106]. Another article

reported the use of seeds and stem bark as a ﬁshing poison in

South Africa [107]. The third article mentioned P. hadiensis

and related species in the context of medicinal plant use to

treat respiratory infections, digestive disorders, and skin in-

fections [108]. Following these ethnobotanical data, the

plant’s leaves have recently been reported in a preliminary

study to display antibacterial activity against S. aureus, iso-

lated from wounds in Bushenyi district, Uganda [109]. A

study investigated the antibacterial eﬀect of the essential oil

from the aerial parts against P. aeruginosa,S. aureus,E. coli,

and S. mutans, which showed no signiﬁcant activity [110].

Another study published by the same group investigated the

antioxidant activity of the aerial parts [111]. A leaf extract

displayed low larvicidal activity against the fourth instar

larvae of Aedes aegypti, a dengue fever vector (LC

489.278 µg/mL) [112]. A terpene-rich methanolic extract of

the shoot part and a methanolic extract of the stem was

investigated for cytotoxicity using a shrimp brine lethality

assay (LC

: 145 µg/mL) and against HeLa cells (141.3 μg/mL)

[113, 114]. Other studies include (a) antibacterial suscepti-

bility single dose and antioxidant activity studies [115–117];

(b) two studies investigating the antioxidant, antiproliferative,

and antiinﬂammatory properties of the shoot parts [118, 119];

and (c) a study screening several Plectranthus species, in-

cluding P. hadiensis, for antiinﬂammatory eﬀects [120].

Isolation and structure elucidation of compounds from

P. hadiensis (DoP

structure elucidation

) resulted in ﬁve new

abietane-type diterpenoids (7β-acetoxy-6β-hydroxyr-

oyleanone, 7β, 6β-dihydroxyroyleanone, 11,20-dihydrox-

ysugiol, 11-hydroxysugiol, 1,11-epoxy-6,12-dihydroxy-20-

norabieta-1(10), and 5,8,11,13-pentaen-7-one) [91, 92], a

known stereoisomer (7α-acetoxy-6β-hydroxyroyleanone)

[91], carnosolon [92], and 25 known compounds detected in

the essential oil extracted from the seeds [95]. A terpenoid

fraction of P. hadiensis, containing 1-octern-ol, linalool, nerol,

Z-citral, geraniol, neryl acetate, α-copaene, geranyl acetate,

δ-cadinene, β-cubebene, α-cadinol, and valencene induced

apoptosis in human colon cancer HCT15 cells [96].

Concerning S. aculeastrum and the DoP

traditional use

multiple medicinal uses were recorded. These include tra-

ditional use of (a) the berries and leaves in treatment of

lymphatic ﬁlariasis in the KwaZulu-Natal and Mpumalanga

regions of South Africa [121]; (b) the berries, leaves, roots,

and bark against cancer in the Kakamega county of Kenya

and Eastern Cape Province of South Africa [98, 122]; (c) the

roots to treat stomachache in Limpopo Province of South

Africa [123]; and (d) the berry juice against ditlapedi (a facial

skin condition) in the Central Sekhukhuneland of South

Africa [124]. Although the practice is regarded by Rwandan

women as “a positive force in their lives,” S. aculeastrum has

been described as being used as medicine applied during

stretching sessions for elongation of the labia minora, which

is classiﬁed as Type IV female genital mutilation by the

World Health Organization [125]. Publications, categorized

under the DoP

bioactivity

, reported low antioxidant activity of

the berries and low antimicrobial activity of the berries and

leaves against ten bacterial and ﬁve fungal strains [126–129].

A methanolic extract from the berries displayed low activity in

inhibiting the growth of promastigotes in Leishmania major

infection in BALB/c mice (IC

: 78.62 μg/ml) [130]. Methanolic

extracts of the berries showed antiproliferative activity against

human HeLa, MCF7, and HT29 tumour cell lines, while the

leaf extracts displayed no cytotoxic activity [131]. In another

study, the methanolic extracts of the leaves and the berries

showed moderate activity against host snails of schistosomiasis

[132]. MeOH-CH2Cl2 (1 :1, v/v) extracts from the stem bark

and the berries showed growth inhibitory activity against ﬁve

African livestock pathogens of the genus Mycoplasma, dis-

playing a mean MIC value of 20 µg/mL [105]. One acute

toxicity study of an extract of the unripe berries in Wistar rats

resulted in toxicity symptoms such as respiratory distress,

epistaxis, and hypoactivity that disappeared 72 h after treat-

ment. Above 125 mg/kg body weight, the extract produced

mortality in the Wistar rats, and the latency was inversely

proportional to the doses [133]. Another study investigated the

toxicological eﬀect of the aqueous extract of fresh, dried, and

boiled berries in male Wistar rats at 1, 10, and 25 mg/kg body

weight for 28 days. The rats gained weight, but showed no signs

of clinical toxicity at the doses tested [134]. Concerning the

DoP

structure elucidation

, two new steroidal alkaloids were isolated

from the root bark, along with known compounds such as

solamargine and ß-solamargine [89, 90]. Steroid alkaloids,

namely, solasodine and tomatidine, isolated from the berries,

displayed cytotoxic eﬀects on the growth of HeLa, MCF7, and

HT29 cancer cell lines and antioxidant properties [87, 88].

Solamargine, isolated from the berries, induced nonselective

cytotoxicity and P-glycoprotein inhibition [85]. Volatile oil

fractions from the leaves and berries were investigated via GC-

MS analysis and contained mainly alkanes and alkenes [86].

12 Evidence-Based Complementary and Alternative Medicine

3.5.3. Moderately Studied Species. Z. chalybeum

(DoP

total

�30 (19)), W. ugandensis (DoP

total

�37 (23)), and

E. abyssinica (DoP

total

�41 (22)) were classiﬁed as having

been moderately studied in the past. A discussion of all the

published literature was not conducted at this point because

this would merit its own standalone review article for each of

the three species. Review articles of the genera Warburgia

[135, 136], Zanthoxylum [137], and Erythrina [138] have

been published.

3.5.4. Highly Studied Species. Plant species identiﬁed as

having been highly studied in the past are C. molle

(DoP

total

�49 (28)), H. madagascariensis (DoP

total

�62 (27)),

T. asiatica (DoP

total

�64 (39)), and S. longipedunculata

(DoP

total

�66 (32)). A discussion of all the published liter-

ature was not conducted at this point because this would

merit its own standalone review article for each of the four

species. A review article of the genus Combretum [139], a

minireview article of H. madagascariensis [140], and a review

article of S. longipedunculata [141] have been published.

4. Conclusions

An extensive literature survey successfully assessed the degree to

which a plant species has been studied so far, introducing a new

indicator: the Degrees of Publication (DoPs). This literature

assessment resulted in the identiﬁcation of understudied plants

among the selected 16 species. Three plant species were iden-

tiﬁed as being moderately studied (E. abyssinica,W. ugandensis,

and Z. chalybeum), while four have already been highly studied

over the past decades (C. molle,H. madagascariensis,

S. longipedunculata, and T. asiatica). More importantly, the

majority of plant species surveyed have not yet been investigated

suﬃciently. Six species were classiﬁed as being highly under-

studied (C. buchananii,F. saussureana,L. calostachys,

M. lycopodioides,M. kandtiana, and S. calycinum subsp.

angustifolium) and three more species as being understudied

(A. coriaria,P. hadiensis, and S. aculeastrum). Due to the ab-

sence of any bioactivity-related publications for S. calycinum

subsp. angustifolium,M. lycopodioides, and M. kandtiana,

pharmacological evaluation of these species should be priori-

tized. The need for research and development of novel natural

products is more vital than ever, making future studies on

traditional herbal remedies justiﬁed and urgently required.

Generally, there was no signiﬁcant correlation between

the DoPs (Figure 4) and the RFCs (Table 1) of individual

plant species, previously published by Schultz et al. [14]. This

is most likely due to the fact that the species are highly used

within the study area (Greater Mpigi region, Uganda) but

occur interregionally on the African continent, and some are

even native to other continents (e.g., M. lycopodioides or

T. asiatica). From the literature survey, it was noted that

other natural plant habitats and ethnic groups than the

people of the Greater Mpigi region were more often sur-

veyed in the past, followed by ethnopharmacological lab

studies of some of the plant species. In conclusion, the

discrepancy between high regional medicinal use and low

DoPs conﬁrms the demand for studying the diverse cultures

and ethnomedical practices of the Greater Mpigi region, in

particular, and the 16 selected species, in general.

The new DoPs indicator proved to be a valuable tool that

ﬁlls a gap compared to other ethnopharmacological tools. Other

than existing ﬁeld assessment tools, e.g., the relative frequency of

citation, the ﬁdelity level, the use value, and the informant

consensus factor, the DoPs tool can be leveraged to better

identify those species that are understudied and merit deeper

investigation. This includes using the tool for selection of species

for costly lab studies, thereby avoiding reproduction of results,

while facilitating a more time-eﬃcient approach to ethno-

pharmacological research. When applied by other researchers in

the future, another value of the new tool will be that gaps in the

literature could be ﬁlled more strategically, making ethno-

pharmacological research more targeted and eﬃcient.

It should be noted that focusing on a logic approach to the

ﬁeld of ethnopharmacology, such as (a) starting with eth-

nobotanical/ethnopharmacological ﬁeld studies; (b) con-

tinuing with validation of traditional medicine via in vitro and

in vivo pharmacological assays in the lab, and (c) progressing

to bioassay-guided fractionation, natural product isolation

and structure elucidation studies, cannot always, though in

most cases, be applied within the ﬁeld. One such example

would be ritual use or where pharmacological eﬀects cannot

be “measured,” which would still be of value to the ﬁeld, e.g.,

with symbolic signiﬁcance to an indigenous community.

Here, it would be categorized under the DoP

traditional use

, thus

recognized, and lab studies may not follow.

Data Availability

The data supporting this bibliographic assessment are from

previously published studies, which have been cited. Indi-

vidual information on publications categorized under dif-

ferent Degrees of Publication is available from the

corresponding author upon request.

Disclosure

The content is solely the responsibility of the authors and

does not necessarily reﬂect the oﬃcial views of the funding

agency. The funding agency had no role in study design, data

collection and analysis, decision to publish, or preparation of

the manuscript.

Conflicts of Interest

The authors declare that the research was conducted in the

absence of any commercial or ﬁnancial relationships that

could be construed as potential conﬂicts of interest.

Authors’ Contributions

FS designed the overall strategy of the study and the DoP

concept, conducted the literature review, and processed the

data. FS and CLQ interpreted the data. FS, GA, and CLQ

wrote the manuscript. LAG directed the study. All authors

read, revised, and approved the ﬁnal manuscript.

Evidence-Based Complementary and Alternative Medicine 13

Acknowledgments

This work was supported by a Fulbright Fellowship (FS) and

a grant from the German Federal Ministry of Education and

Research (13FH026IX5, PI : LAG, and Co-I : FS). The authors

acknowledge support for the Article Processing Charge from

the Open Access Publication Fund of Neubrandenburg

University of Applied Sciences (HSNB). The authors thank

the Ugandan traditional healers for the collaboration and

sharing of ethnobotanical information. The authors thank

Ogechi Favour Osuji and Kristine Kossol for their assistance

in collecting relevant literature on the medicinal plant

species. They thank Logan Penniket for proof-reading the

manuscript. They also thank Dr. Michael Heinrich for advice

on the quality assessment of individual papers.

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18 Evidence-Based Complementary and Alternative Medicine

Publication III:

"Targeting ESKAPE pathogens with anti-infective medicinal plants from

the Greater Mpigi region in Uganda"

Pages: 52-85

Personal contribution

I performed the lab work leading to this publication at the Quave Lab for Medical Ethnobotany

and Drug Discovery as a visiting researcher at the Emory University School of Medicine in

Atlanta, Georgia, USA (Fulbright scholarship 2018-2019). In the following, my personal

contribution to the presented study and manuscript is briefly described: I contributed to the

collection and processing of the plant material in Uganda. I prepared the extracts and performed

the antibacterial, quorum sensing inhibition and į-toxin experiments. I conducted most of the

cell culture experiments, and I contributed to the HPLC analyses. I contributed to the data

analysis. I wrote the majority of the manuscript. A more detailed author-contribution statement

is given in the published article.

Information on publication

This study was published in Scientific Reports in July 2020 and is available at

https://www.nature.com/articles/s41598-020-67572-8. It is an open access article distributed

under the Creative Commons Attribution 4.0 International License (CC BY 4.0).

Schultz, F.; Anywar, G.; Tang, H.; Chassange, F.; Lyles, J.T.; Garbe, L.-A.; Quave, C.L.:

Targeting ESKAPE pathogens with anti-infective medicinal plants from the Greater Mpigi

region in Uganda. Nature Scientific Reports, volume 10, Article number: 11935, 2020

https://doi.org/10.1038/s41598-020-67572-8

Vol.:(0123456789)

Scientific RepoRtS | (2020) 10:11935 | https://doi.org/10.1038/s41598-020-67572-8

www.nature.com/scientificreports

targeting eSKApe pathogens

with anti‑infective medicinal plants

from the Greater Mpigi region

in Uganda

fabien Schultz1,2,3, Godwin Anywar4, Huaqiao Tang6, François Chassagne6, James T. Lyles6,

Leif‑Alexander Garbe2,3,5 & Cassandra L. Quave1,6,7*

Antibiotic resistance poses one of the greatest threats to global health today; conventional drug

therapies are becoming increasingly inefficacious and limited. We identified 16 medicinal plant species

used by traditional healers for the treatment of infectious and inflammatory diseases in the Greater

Mpigi region of Uganda. Extracts were evaluated for their ability to inhibit growth of clinical isolates

of multidrug‑resistant ESKAPE pathogens. Extracts were also screened for quorum quenching activity

against S. aureus, including direct protein output assessment (δ‑toxin), and cytotoxicity against

human keratinocytes (HaCaT). Putative matches of compounds were elucidated via LC–FTMS for the

best‑performing extracts. These were extracts of Zanthoxylum chalybeum (Staphylococcus aureus:

MIC: 16 μg/mL; Enterococcus faecium: MIC: 32 μg/mL) and Harungana madagascariensis (S. aureus:

MIC: 32 μg/mL; E. faecium: MIC: 32 μg/mL) stem bark. Extracts of Solanum aculeastrum root bark and

Sesamum calycinum subsp. angustifolium leaves exhibited strong quorum sensing inhibition activity

against all S. aureus accessory gene regulator (agr) alleles in absence of growth inhibition (IC50 values:

1–64 μg/mL). The study provided scientific evidence for the potential therapeutic efficacy of these

medicinal plants in the Greater Mpigi region used for infections and wounds, with 13 out of 16 species

tested being validated with in vitro studies.

The rise of antimicrobial resistance (AMR) requires mobilization of political, financial and research investment

due to its emergence as a global health hazard that threatens the ability to treat infectious

diseases1. According

to the World Health Organization, AMR poses “one of the biggest threats to global health, food security, and

development today” and can affect anyone in any country and of any

age2. Today, AMR already accounts for

700,000 deaths annually. By 2050, this figure is estimated to reach more than 10 million deaths per year, which is

more people than currently die from cancer3. Because effective antibiotics are critical for treatment of bacterial

infections and for procedures where there is a high risk of infection, e.g. surgery, new anti-infectives are needed

to overcome this global threat4. The issue of resistance is not uniformly spread across all bacteria5. Six species

have been identified by the Infectious Disease Society of America (IDSA) as being especially dangerous due to

their potential multidrug resistance mechanisms and virulence. They are referred to as ‘ESKAPE’ pathogens,

which is an acronym for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter

baumannii, Pseudomonas aeruginosa and Enterobacter species. This group of pathogenic bacteria encompasses

both Gram-negative and Gram-positive species that are capable of ‘escaping’ bactericidal action of conventional

open

1Department of Dermatology, Emory University School of Medicine, 615 Michael St., Atlanta, GA 30322,

USA. 2Institute of Biotechnology, Faculty III - Process Sciences, Technical University of Berlin, Gustav-Meyer-Allee

25, 13355 Berlin, Germany. 3Department of Agriculture and Food Sciences, Neubrandenburg University of

Applied Sciences, Brodaer Str. 2, 17033 Neubrandenburg, Germany. 4Department of Plant Sciences, Microbiology

and Biotechnology, Makerere University, P.O. Box 7062, Kampala, Uganda. 5ZELT - Neubrandenburg Center for

Nutrition and Food Technology gGmbH, Seestraße 7A, 17033 Neubrandenburg, Germany. 6Center for Study of

Human Health, Emory University College of Arts and Sciences, 615 Michael St., Atlanta, GA 30322, USA. 7Emory

Antibiotic Resistance Center, Emory University, 615 Michael St., Atlanta, GA 30322, USA. *email: cquave@

emory.edu

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www.nature.com/scientificreports/

antibiotics6,7. ESKAPE pathogens are common causes of deadly or life-threatening infections, especially among

children, immunocompromised, and critically-ill people8.

Antibiotics are not the only anti-infectives that could provide an effective weapon against these pathogens.

Another therapeutic, yet non-antibiotic, strategy is targeting bacterial virulence controlled by quorum sensing

processes. The quorum-sensing mechanism mediated by signal molecules regulates the expression of virulence

genes in the majority of pathogenic bacteria, meaning that quorum-sensing inhibitors are expected to be one of

the best alternatives to antibiotics9,10. Autoinducers, self-secreted signal molecules, are regulated by a density-

dependent synchronized gene expression system during quorum sensing11. Biofilm formation, toxin production

and other virulence factors are controlled by quorum sensing and the production of virulence factors can weaken

the balance of host defense mechanisms9. Initiation of toxin production occurs when extracellular signaling and

communication indicates that a threshold population of bacteria has been achieved12. Inhibition of quorum

sensing induced by secondary plant metabolites can significantly attenuate bacterial virulence and substantially

enhance vulnerability to conventional antibiotics and to the immune system9,12–14.

It is estimated that more than 25% of the Western drugs prescribed contain plant-derived natural products as

active ingredients15. Yet, only a small proportion of plant species has ever been investigated for pharmacological

activity in a laboratory setting16,17. In East and Central Africa, medicinal plant use and traditional medicine prac-

tices are still the predominant form of healthcare18,19. In Uganda, four out of five patients primarily seek medical

treatment from traditional healers instead of Western-trained physicians and there is at least one traditional

healer per village practicing traditional use of medicinal plants20,21. Despite its small size, Uganda is characterized

by its very rich biological diversity of 5,000 species of higher plants in the indigenous flora22, resulting from its

unique bio-geographical location23. Documentation of traditional use and ethnopharmacological evaluation of

this wealth of plant species can still be considered an understudied field.

A recent ethnobotanical study by Schultz etal. identified 16 medicinal plant species that play a significant

role in the local traditional medicine of the Greater Mpigi region located in West-Central Uganda24. The local

vegetation at the study site is characterized as a tropical, moist evergreen forest/savanna mosaic25,26. Here, people

are highly dependent on medicinal plants and local traditional healers for primary health care. Apart from many

other traditional uses documented, 16 medicinal plants were found to be critical to anti-infective traditional

medicine practices in the Greater Mpigi region (in particular, skin and wound infections, and symptoms associ-

ated with bacterial infections). The majority of the plant species have not been studied for potential bioactivity

yet24. As the ethnopharmacological basis for this study, these species, their traditional use in treatment of infec-

tions and the relative frequency of citation in % (n = 39) are illustrated in Fig.1.

Figure1. Ethnopharmacological information on the medicinal use of plant candidates from the Greater Mpigi

region in Uganda (with emphasis on infections and symptoms of infections). The stacked histogram figure

shows the relative frequencies of citation (RFC) in % in treatment of relevant medical disorders, calculated from

data obtained through an ethnobotanical survey of 39 traditional healers. Here, the RFC assesses the importance

of a plant species used for a specific medical condition relative to the total number of informants interviewed

in the study. It varies from 0% (none of the informants uses this plant species in treatment of a specific medical

condition) to 100% (maximum number of informants use this plant species in treatment of a specific medical

condition)24. Consequently, the higher the value of cumulated RFCs (x-axis), the higher the traditional use of a

plant species in treatment of medical conditions relevant to this study.

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We screened 86 plant extracts derived from these 16 medicinal species for antibacterial activity against a

panel of multidrug resistant ESKAPE pathogens associated with the medical disorders stated in Fig.1, and for

antivirulence activity in S. aureus (Fig.2). The extracts were produced from plant material collected in the Greater

Mpigi region during fieldwork in 2015, 2016 and 2017. The overarching aims of the study were to contribute

to drug discovery and pharmacological evaluation of traditional use. Specifically, the study objectives were to

investigate the potential (1) growth inhibitory impact of the medicinal plants on a panel of ESKAPE pathogens;

(2) quorum-quenching activity targeting the agr system of S. aureus; (3) mammalian cytotoxicity against the

HaCaT keratinocyte cell line from adult human skin; (4) inhibition of δ-toxin production in S. aureus; and (5)

to conduct a chemical characterization for putative natural product matches of the four most bioactive extracts.

Results

Extraction and information on plant species. Extractions were achieved by means of (a) maceration

in either methanol, ethanol, ethyl acetate or diethyl ether; (b) Soxhlet extraction using n-hexane and successively

methanol; and (c) aqueous decoction. These procedures yielded a total of 86 different plant crude extracts from

16 medicinal plant species. Details on the medicinal plants investigated, herbarium voucher specimen numbers,

local plant names in Luganda, plant parts investigated, extract identification numbers (extract IDs) and solvents

used for extraction are reported in Supplementary TableS1.

Growth inhibition library screen and dose–response study against multi‑resistant ESKAPE

panel. Extracts were initially screened for growth inhibition of one clinical isolate of each ESKAPE pathogen

at a concentration of 256μg/mL. Extracts displaying an inhibition percentage above 40 for an individual strain

were further investigated by dose–response experiments in order to obtain the

IC50 and MIC (IC90) values. In

this initial library screen, none of the extracts from Ficus saussureana, Microgramma lycopodioides, Plectranthus

hadiensis and Securidaca longipedunculata displayed significant activity at this initial screening concentration

and were therefore eliminated from further experiments. However, 26 of the 86 extracts were investigated fur-

ther. In the second experimental stage, a total of 10 extracts from seven plant species inhibited the growth of

E. faecium (EU-44). While growth of S. aureus (UAMS-1) was significantly inhibited by 14 extracts from nine

plant species at 256μg/mL, only six extracts from three species were active against K. pneumoniae (CDC-004).

Fifteen extracts from nine plant species were introduced to dose–response studies against A. baumannii (CDC-

0033), and eight extracts from six plant species against P. aeruginosa (AH-71) respectively. Only the ethanolic

and diethyl ether extracts from Harungana madagascariensis stem bark(etE011-18, dietE011) showed growth

Figure2. Research methodology for the study—16 plant species were identified in close collaboration with the

traditional healers of the Greater Mpigi region based on the species’ traditional use in treatment of infections.

After collecting specimens and producing a medicinal plant extract library, our invitro study commenced,

targeting bacterial virulence and growth of multidrug-resistant ESKAPE pathogens. After initial growth

inhibition, quorum quenching and cytotoxicity library screenings, hits were followed up via dose–response

studies, a δ-toxin production inhibition assay and chemical characterization. Results of this study will ultimately

be transferred back to the traditional healers through field workshops.

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inhibition above 40% against E. cloacae (CDC-0032) at the initial screening concentration of 256μg/mL. The

individual plant extracts selected for the dose-response study and their results are shown in Table1.

The diethyl ether extract of Zanthoxylum chalybeum stem bark(dietE017a) displayed the highest inhibitory

activities in the study: S. aureus (IC50: 4μg/mL; MIC: 16μg/mL) and E. faecium (IC50: 8μg/mL; MIC: 32μg/

mL). Ethanolic (etE011-18), diethyl ether (dietE011) and hexane extracts (hE011-18) of H. madagascariensis

stem barkwere the second most active extracts against growth of S. aureus (IC50: 8μg/mL; MIC: 32μg/mL) and

the ethanolic stem barkextract displayed considerable antibiotic properties against E. faecium, resulting in the

same IC50 (8μg/mL) and MIC (32μg/mL) values as Z. chalybeum. None of the extracts yielded an MIC at the

concentration range tested (≤ 256μg/mL) in the experiments with K. pneumoniae, A. baumannii and E. cloa-

cae. Furthermore, 50% growth inhibition of E. cloacae was not achieved by the two H. madagascariensis stem

barkextracts (etE011-18, dietE011) that were tested, meaning that none of the 86 extracts were active against the

Scientific name Extract ID

Enterococcus

faecium

EU-44

Staphylococcus

aureus

UAMS-1

Klebsiella

pneumoniae

CDC-004

Acinetobacter

baumannii

CDC-0033

Pseudomonas

aeruginosa

AH-71

Enterobacter

cloacae

CDC-0032

IC50 MIC IC50 MIC IC50 MIC IC50 MIC IC50 MIC IC50 MIC

Sesamum calycinum subsp.

angustifolium eE004-18 – 256 > 256 ––––

Leucas calostachys eE005-18 – – – > 256 > 256 – –

hE005-18 128 256 256 > 256 > 256 > 256 – 256 > 256 –

Solanum aculeastrum hE006 256 256 32 128 – – – –

Albizia coriaria etE007 – – – > 256 > 256 32 > 256 –

Erythrina abyssinica etE008 64 > 256 32 64 – > 256 > 256 – –

Toddalia asiatica

etE010 – – > 256 > 256 –––

etE010a – – > 256 > 256 –––

eE010 – – > 256 > 256 –––

dietE010 128 256 256 > 256 256 > 256 > 256 – – –

Harungana madagascariensis

etE011-18 8 32 8 32 – 256 > 256 – > 256 > 256

eE011 128 128 – – – – –

dietE011 – 8 32 – > 256 > 256 – > 256 > 256

dietE011-18 – – – > 256 > 256 – –

hE011-18 – 8 32 – – – –

Morella kandtiana

etE012 – – – 256 > 256 32 > 256 –

etE012a – – – 256 > 256 32 > 256 –

etE012-18a – 128 > 256 – 128 > 256 – –

wE012-18 – – – 128 > 256 32 256 –

Cassine buchananii etE013 – 64 > 256 ––––

Warburgia ugandensis

dietE014-18 128 > 256 32 64 – – – –

eE014-18 128 > 256 –––––

hE014-18 – 64 64 – > 256 > 256 – –

etE014-18 256 256 128 128 256 > 256 128 – 64 > 256 –

Combretum molle etE015 – – – 32 > 256 16 128 –

Zanthoxylum chalybeum dietE017a 8 32 4 16 – > 256 > 256 – > 256 –

Gentamicin – – 4 4 – < 1 < 1 1,024 > 1,024

Meropenem – – – – – 16 16

Vancomycin – 4 4 – – – –

Ampicillin – – – – > 256 > 256 –

Tetracycline – – – 2 4 – –

Chloramphenicol 4 32 – – – – –

Table 1. Results of growth inhibition of selected ESKAPE pathogens by medicinal plant samples from the

Greater Mpigi region in Uganda. Only extracts that showed growth inhibition above 40% in the initial screen

are listed. Crude extracts obtained during maceration were labeled according to their extraction solvent: (a)

methanol (mEXXX); (b) ethanol (etEXXX); (c) ethyl acetate (eEXXX); (d) diethyl ether (dietEXXX), where

‘XXX’ stands for the sample number assigned to a given plant species. Crude extracts produced via Soxhlet

extraction were labeled: (e) n-hexane (hEXXX); (f) methanol, successive extraction (smEXXX). In most cases,

we recorded that the traditional healers prepare herbal drugs by boiling the plant material in water. Therefore,

the original method of preparation was simulated by an aqueous decoction (wEXXX). Results are reported

as the minimum concentration of extract that achieved 50% inhibition (IC50) and 90% inhibition (MIC) of

growth as detected by optical density measures. IC50 and MIC values are expressed as concentration (μg/mL).

The maximum concentration at which extracts were tested was 256μg/mL. Dashes indicate that a sample was

not tested.

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multidrug-resistant CDC-0032 strain. The ethanolic extract of Combretum molle stembark(etE015) exhibited

modest activity against A. baumannii (IC50: 32μg/mL; MIC: > 256μg/mL). Growth of P. aeruginosa was mod-

erately inhibited by the ethanolic extract of C. molle stem bark(etE015; IC50: 16μg/mL; MIC: 128μg/mL) and

Morella kandtiana roots(etE012-18a; IC50: 32μg/mL; MIC: 256μg/mL).

Quorum sensing inhibition in Staphylococcus aureus. In S. aureus, a number of quorum-sensing

component pathways are encoded by the accessory gene regulator (agr) system, which plays a key role in the

species’ pathogenesis14. There are four allelic groups on the agr gene locus: agr I–IV27. The importance of the agr

system to abscess formation has previously been confirmed by means of genetic and agr-inhibiting tools28–32.

During an initial screening, all 86 extracts were tested for inhibition of quorum sensing against the strain

S. aureus agr I reporter strain AH-1677 at 16μg/mL (sub-IC50 concentrations for growth were used to avoid

potential growth inhibition effects). A total of 11 extracts from seven plant species revealed quorum-sensing

inhibition activity above 40% and were selected for dose-response experiments with four reporter strains of S.

aureus agr subtypes (agr I: AH-1677, agr II: AH-430, agr III: AH-1747, agr IV: AH-1872). These plant species,

which were significantly active in the initial screen, were Sesamum calycinum subsp. angustifolium (both hexane

leaveextracts; hE004 and hE004-18), Leucas calostachys (hexane leaveextract; hE005), Solanum aculeastrum

(hexane rootextract; hE006, and ethyl acetateroot extract; eE006), Z. chalybeum (ethyl acetate stembarkextract;

eE009), M. kandtiana (both diethyl etherroot extracts; dietE012 and dietE012-18), Warburgia ugandensis (diethyl

ether stembarkextract; dietE014) and P. hadiensis (hexane leaveextract; hE016, and diethyl etherleave extract;

dietE016) (Table2). None of the extracts from S. longipedunculata, M. lycopodioides, F. saussureana, Albizia

coriaria, Erythrina abyssinica, T. asiatica, H. madagascariensis and C. molle inhibited quorum sensing above

40% at 16μg/mL.

Ugandan medicinal plant species exhibit dose‑dependent quorum‑sensing inhibition

in vitro. The transcription of each of the four known agr allelic groups was inhibited by all of the selected 11

crude extracts from seven plant species. Strains were additionally monitored for potential growth inhibition by

optical density (600nm). Dose-response curves, indicating the percent growth inhibition and quorum sensing

inhibition (QSI) activity of the vehicle control (dimethyl sulfoxide [DMSO]), were calculated to evaluate the

antivirulence activity (Fig.3). Agr subtype-specific IC50 values are reported in Table3. The two hexane extracts

of S. calycinum subsp. angustifoliumleaves, hE004 and hE004-18, were identified as the most active quorum-

sensing inhibitors. The IC50 against agr I–IV were 2, 2, 16 and 32μg/mL (hE004), 4, 2, 16 and 32μg/mL (hE004-

18) respectively. Another plant extract highly active in tackling bacterial virulence was the ethyl acetate extract

of S. aculeastrum roots(eE006), which scored agr subtype-dependent IC50 values of 4, 1, 16 and 64μg/mL. Two

Table 2. Results of quorum-sensing inhibition plant extract library screen on S. aureus agr I reporter strain at

16μg/mL. –, Quorum-sensing inhibition below 40 percent; + , quorum-sensing inhibition above 40 percent.

Plant species Extract ID %I ≥ 40 Plant species Extract ID %I ≥ 40 Plant species Extract ID %I ≥ 40 Plant species Extract ID %I ≥ 40

Securidaca

longipedunculata

eE001 –

Leucas

calostachys

eE005 –

Toddalia asiatica

etE010 –Cassine

buchananii

etE013 –

smE001 –eE005-18 –etE010a –etE013a –

wE001 –smE005 –eE010 –eE013 –

mE001 –smE005-18 –dietE010 –

Warburgia

ugandensis

dietE014 +

hE001 –wE005 –

Harungana

madagascariensis

etE011 –dietE014-18 –

Microgramma

lycopodioides

hE002 –mE005-18 –etE011a –eE014-18 –

mE002 –hE005 + etE011-18 –wE014-18 –

wE002 –hE005-18 –eE011 –hE014-18 –

smE002 –

Solanum

aculeastrum

eE006 + eE011-18 –smE014-18 –

eE002 –hE006 + dietE011 –etE014a –

Ficus saussureana

smE003 –wE006 –dietE011-18 –etE014-18 –

wE003 –smE006 –wE011-18 –Combretum

molle

etE015 –

eE003 –Albizia coriaria etE007 –hE011-18 –eE015 –

mE003 –eE007 –smE011-18 –Plectranthus

hadiensis

hE016 +

hE003 –Erythrina

abyssinica

etE008 –

Morella

kandtiana

etE012 –dietE016 +

Sesamum

calycinum subsp.

angustifolium

smE004 –eE008 –etE012a –

smE004-18 –

Zanthoxylum

chalybeum

etE009 –etE012-18a –

mE004 –eE009 + etE012-18b –

hE004 + etE017 –eE012-18 –

hE004-18 + etE017a –wE012-18 –

eE004 –dietE017 –dietE012 +

eE004-18 –dietE017a –dietE012-18 +

wE004 –

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Figure3. Results of the quorum-sensing inhibition invitro dose-response studies: Data shown as serial

dilution and percent agr activity or growth of the vehicle control (DMSO) at 22h; FLD: fluorescence detector

(measuring quorum sensing activity), represented by solid lines; OD: optical density at 600nm (measuring

bacterial growth), represented by dashed lines.

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more promising quorum-sensing inhibitors were the hexane extract of S. aculeastrum roots(hE006, agr I–III

IC50: 12, 2 and 16μg/mL), which only displayed moderate activity against agr IV (IC50: 64μg/mL), and the hex-

ane extract of L. calostachys leaves(hE005, agr I–II: 4μg/mL), which was moderately active against agr III and

IV (IC50: 64μg/mL). Extracts of W. ugandensis and Z. chalybeum stem barkshowed low IC50 values ranging from

8–32μg/mL, but were eliminated from the anti-agr assessment due to their strong growth inhibitory activity

on our reporter strains. No MIC values were detected (either > 64μg/mL or growth inhibition), except for the

hexane extract of S. calycinum subsp. angustifolium leaves(hE004-18, MIC: 64μg/mL).

δ‑Toxin production and quantification assay. The phenol-soluble modulin peptide δ-toxin (also

known as δ-hemolysin) is responsible for various pathophysiological effects caused by S. aureus as it seeks to

evade host defense mechanisms33–36. These effects include cytolysis of red and white blood cells, followed by

cell death, as well as triggering of inflammatory responses33,36. Extracts hE004, hE004-18, hE005, eE006, hE006,

which displayed strong quorum sensing inhibitory activity (Table3), were selected for further confirmation

of antivirulence effects on the translational products of agr in S. aureus. These invitro experiments aimed to

measure δ-toxin levels during extract treatment at sub-growth inhibitory concentrations through examination

of the bacterial supernatant using hydrophobic interaction chromatography (HIC)37. The experiments were con-

ducted with two high-toxin-producing strains of S. aureus: AH1263 and NRS243. All tested extracts were effec-

tive in significantly reducing δ-toxin in AH1263, confirming their antivirulence activity. The hexane extracts

of S. calycinum subsp. angustifolium leaves(hE004, hE004-18) and the ethyl acetate extract of S. aculeastrum

roots(eE006) displayed the highest inhibition activity against NRS243. Extracts hE005 and hE006 showed mod-

erate activity against NRS243 (Fig.4).

Medicinal plants from the Greater Mpigi region exhibit low toxicity to human keratino‑

cytes. In an effort to assess the cytotoxicity of the plant extracts, all 86 extracts were screened in a human

keratinocyte toxicity assay at 64μg/mL, using HaCaT cells. In this library screen, only one of the 86 extracts from

16 plant species exhibited a cytotoxicity above 50%. The only extract displaying cytotoxic activity in the initial

screen was the methanolic extract of S. aculeastrum rootssmE006 (I%: 51.8 ± 1.5). Results of the cytotoxicity

library screen are shown in Supplementary TableS3. Subsequently, dose-response experiments to assess cytotox-

icity were conducted on extract smE006, the 26 active hits from the growth inhibition library screen (see Table1)

and the five most active quorum-sensing inhibitors that were introduced to the δ-toxin production inhibition

assay (see Figs.3 and 4, Table3). Results of this counterscreen are shown in Table4, along with the calculated

therapeutic indices for growth inhibition (TIgrowth inhibition) for individual strains tested and quorum-sensing inhi-

bition (TIquorum quenching) for each reporter gene targeted. The therapeutic index is used as an important parameter

in drug discovery to assess an appropriately balanced safety-efficacy profile for a given indication, as it ena-

bles for characterization and optimization of efficacy and safety of drug candidates38. The majority of extracts

tested in our dose-response study displayed no toxicity to the HaCaT cells (20 extracts, 64.5%). However, some

extracts did show low toxicity with IC50 values ranging from 512 to 256μg/mL: (1) the ethyl acetate extract of

S. calycinum subsp. angustifolium leaves(eE004-18); (2) three extracts of L. calostachys leaves(eE005-18, hE005,

hE005-18); (3) two extracts of S. aculeastrum roots(hE006, smE006); (4) the diethyl ether extract of Toddalia asi-

atica leaves/stem bark(dietE010); and (5) four extracts of H. madagascariensis stem bark(etE011-18, dietE011,

dietE011-18, hE011-18). As expected, extract smE006 remained the most cytotoxic sample in the extract library,

displaying an IC50 of 64μg/mL.

Table 3. Results of the quorum-sensing inhibition invitro dose-response studies: IC50 and MIC values.

The calculated

IC50 and MIC values of plant extracts, represented in μg/mL, are displayed. The most active

extracts were selected for confirmation of antivirulence activity via a δ-toxin production and quantification

assay; > 16/32 GI describes undetectable

IC50 and MIC values due to growth inhibition at 16 or 32μg/mL. GI,

growth inhibition.

Plant species Extract ID

agr 1

AH 1677 agr 2

AH 430 agr 3

AH 1747 agr 4

AH 1872

IC50 MIC IC50 MIC IC50 MIC IC50 MIC

Sesamum calycinum subsp. angustifolium hE004 2 > 64 2 > 64 16 > 64 32 > 64

hE004-18 4 64 2 > 64 16 > 64 32 > 64

Leucas calostachys hE005 4 > 64 4 > 64 64 > 64 64 > 64

Solanum aculeastrum eE006 4 > 32 GI 1 > 32 GI 16 > 32 GI 64 > 64

hE006 16 > 32 GI 2 > 64 16 > 32 GI 64 > 64

Zanthoxylum chalybeum eE009 16 > 32 GI 8 > 64 16 > 32 GI 16 > 16 GI

Morella kandtiana dietE012 64 > 64 4 > 64 16 > 64 64 > 64

dietE012-18 64 > 64 4 > 64 64 > 64 64 > 64

Warburgia ugandensis dietE014 32 > 32 GI 32 > 32 GI > 32 GI > 32 GI > 16 GI > 16 GI

Plectranthus hadiensis hE016 64 > 64 16 > 64 64 > 64 64 > 64

dietE016 64 > 64 16 > 64 64 > 64 64 > 64

224CF2c (positive control) 16 > 32 GI 16 > 32 GI 16 > 32 GI 32 > 32 GI

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LC–MS analysis of plant extracts for putative matches. The two best performing extracts of the

growth inhibition experiments (dietE017a and etE011-18), as well as of the quorum-sensing and δ-toxin pro-

duction assays (hE004-18 and eE006) were further investigated by chemical characterization via liquid chro-

matography–mass spectrometry (LC–MS) analysis and searched for putative matches. The base peak negative

mode electrospray ionization (ESI) LC–MS chromatograms for the four extracts are shown in Fig.5. A total of

60 peaks were identified and screened for putative matches (Fig.5 and Table5). This resulted in 10 peaks having

putative matches for etE011-018, 9 peaks for hE004-18, 9 peaks for eE006, and 2 peaks for dietE017a. Most of the

Figure4. Five extracts from three Ugandan medicinal plant species exhibited strong δ-toxin production

inhibition activity against S. aureus AH1262 (A) and moderate activity against S. aureus NRS243 (B); extracts

were tested at 32, 16 and 8μg/mL (sub-growth inhibition concentrations) and compared to the untreated

control (UT). The positive control 224CF2c was additionally tested at 64μg/mL. All samples were normalized

for growth

(OD600nm) during supernatant harvest. Results are reported as the total peak area and peaks are

identified as deformylated (blue) and formylated (red) δ-toxin peak areas. Statistical significance is denoted as

*P value < 0.05, ‡P value < 0.01, †P value < 0.001.

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ions yielded several putative matches which are isomers of the experimentally determined empirical formula.

The only putative match for dietE017a, peak 54 and 55, was cyclozanthoxylane A, which has a mass difference

of over 13ppm from the experimentally determined mass. While this is a low probability match, it was the only

putative match for the Z. chalybeum sample from 9,463 published compounds in the genus. Chemical structures

for the putative matches from the four extracts are provided in Supplementary FiguresS3, S4, S5 and S6.

Discussion

The study provides scientific evidence for the therapeutic use of medicinal plants in the Ugandan Greater Mpigi

region. Traditional use in treatment of infections and wounds was successfully validated in 13 out of 16 medici-

nal plant species investigated using invitro studies. Extracts of species displaying no pharmacological activity

in these experiments were S. longipedunculata, M. lycopodioides and F. saussureana. On the contrary, different

extracts of S. calycinum subsp. angustifolium, L. calostachys, S. aculeastrum, M. kandtiana, W. ugandensis and

Z. chalybeum simultaneously displayed both growth inhibition and quorum-sensing inhibition effects on the

strains investigated. Extracts from the same species distinguished themselves in terms of polarity of extraction

solvent used (“pre-fractionation”). Except for the hexane extract of S. aculeastrum rootshE006, there was no

extract that was simultaneously active in inhibiting bacterial growth and quorum quenching, which highlights

the need for bioassay-guided fractionation and isolation of active compounds from these species in the future.

In general, extracts produced as aqueous decoction, which is consistent with the majority of the traditional

preparations in the Greater Mpigi region24, failed to display bioactive effects in our invitro models. The excep-

tion was one aqueous extract of M. kandtianaroots, which exhibited lowinhibitory effects on the growth of

Table 4. The 30 most active Ugandan plant extracts are either non-toxic or show low toxicity to human

HaCaT cells (Table showing results of cytotoxicity dose–response experiments and calculated therapeutic

indices). IC50 values are given in µg/mL. † E. faecium EU-44; *S. aureus UAMS-1; §K. pneumoniae CDC-004; °A.

baumannii CDC-033; ǂP. aeruginosa AH-71; ±E. cloacae CDC-0032; --, cannot be calculated; –, not tested.

Plant species Extract ID

Cytotoxicity

TIgrowth inhibition

TIquorum quenching

IC50 agr I agr II agr III agr IV

Sesamum calycinum subsp. angustifolium

hE004 > 512 – > 256 > 256 > 32 > 16

hE004-18 > 512 – > 128 > 256 > 32 > 16

eE004-18 512 2* – – – –

Leucas calostachys

eE005-18 256 <1° – – – –

hE005 256 – 64 64 4 4

hE005-18 512 4†; 2*; < 2§; 2ǂ– – – –

Solanum aculeastrum

eE006 > 512 – > 128 > 512 > 32 > 8

hE006 512 2†; 16* 32 256 32 8

smE006 64 – – – – –

Albizia coriaria etE007 > 512 --°; > 16ǂ– – – –

Erythrina abyssinica etE008 > 512 > 8†; > 16*; --° – – – –

Toddalia asiatica

etE010 > 512 --§– – – –

etE010a > 512 --§– – – –

eE010 > 512 --§– – – –

dietE010 256 2†; 1*; 1§; < 1° – – – –

Harungana madagascariensis

etE011-18 256 32†; 32*; 1°; < 1±– – – –

eE011 > 512 > 4†; – – – –

dietE011 256 32*; < 1°; < 1±– – – –

dietE011-18 256 < 1° – – – –

hE011-18 256 32* – – – –

Morella kandtiana

etE012 > 512 > 2°; > 16ǂ– – – –

etE012a > 512 > 2°; > 16ǂ– – – –

etE012-18a > 512 > 4*; > 4° – – – –

wE012-18 > 512 > 4°; > 16ǂ– – – –

Cassine buchananii etE013 > 512 > 8* – – – –

Warburgia ugandensis

dietE014-18 > 512 > 4†; > 16* – – – –

eE014-18 > 512 > 4†– – – –

hE014-18 > 512 > 8*; --° – – – –

etE014-18 > 512 > 2†; > 4*; > 2§; > 4°; > 8ǂ– – – –

Combretum molle etE015 > 512 > 16°; > 32ǂ– – – –

Zanthoxylum chalybeum dietE017a > 512 > 64†; > 128*;

--°; --ǂ– – – –

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A. baumannii CDC-0033 (IC50: 128μg/mL; MIC: > 256μg/mL) and moderate effects on P. aeruginosa AH-71

(IC50: 32μg/mL; MIC: 256μg/mL). One possible factor that could contribute to this phenomenon is the fact

that extracts are standardized in the lab (filtered before solvent evaporation), unlike during traditional treatment

where solids are swallowed along with the infused water. In this way, apolar pharmacologically active secondary

plant metabolites bound to the solids remain in the decoction and could potentially yield a pharmacological

effect in the patient.

Extract dietE017a, a diethyl ether extract of Z. chalybeum stem bark, displayed the highest growth inhibitory

activity of all extracts against growth of S. aureus (IC50: 4μg/mL; MIC: 16μg/mL) and E. faecium (IC50: 8μg/mL;

MIC: 32μg/mL). Although representing a mixture of hundreds of secondary plant metabolites, dietE017a sur-

prisingly reached a similar level of antibiotic activity exhibited invitro by the single compound positive controls,

namely chloramphenicol (S. aureus, IC50: 4μg/mL; MIC: 32μg/mL) and vancomycin (E. faecium, IC50: 4μg/mL;

MIC: 4μg/mL). Extract dietE017a exhibited no cytotoxic effects in the human keratinocyte cell line (Cytotoxicity

IC50: > 515μg/mL). The calculated therapeutic index (TI) demonstrated that cytotoxicity to human cells was at

concentrations > 128 (S. aureus) and > 64 (E. faecium) times higher than that required for growth inhibition of

these pathogenic bacteria. In the Greater Mpigi region, none of the informants stated that Z. chalybeum is used as

an herbal drug for skin infections. Instead, 18% of the traditional healers interviewed stated that this deciduous

shrub or tree is used for wound disinfection and treatment. It was also reported that itis used medicinally for

treatment of stomach/GI tract disorders (13%), nausea (8%) and sore throat (8%)24. These results support the

traditional use of Z. chalybeum stem bark as an anti-infective therapy. Z. chalybeum has been moderately studied

Figure5. The ESI negative mode base peak LC-FTMS chromatogram for (A) etE011-18, H. madagascariensis,

(B) hE004-18, S. calycinum subsp. angustifolium, (C) eE006, S. aculeastrum, and (D) dietE017a, Z. chalybeum.

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Peak no. RT (min) % Area m/z* MS–MS Empirical formula Putative match (CAS no.)

etE011-18, Harugana madagascariensis

1 6.9 1.6 465.1050, 931.2171 345.1, 375.1 C21H21O12 (2.4) no matches

2 18.0 8.4 589.1005, 1,179.2081 463.1 C30H21O13 (2.9) no matches

3 18.7 3.6 573.1053, 589.1004, 1,147.2178 463.1 C30H21O13 (2.9) no matches

4 19.1 1.5 447.0734, 573.1050, 589.1006 463.1 C30H21O13 (3.0) no matches

5 24.8 1.7 424.1896, 493.2603 381.2 C25H28O6 (1.1) calycinigin A (1384180-74-8)

6 25.4 3.5 475.2498, 493.2606 406.2 C30H35O5 (1.8) bazouanthrone (942983-94-0), Kenganthranol B

(879208-71-6)

7 25.8 1.3 475.2498, 895.5402 406.2 C30H35O5 (1.8) see peak 7

8 28.2 5.9 322.1213, 391.1919 322.1 C25H27O4 (1.1) no matches

9 30.8 1.0 406.1793, 455.3537 363.2 C25 H26 O5 (1.8) mammeisin (18483-64-2), 3-geranylemodin

(87605-71-8), 2-geranylemodin (97399-77-4),

kengaquinone (879208-69-2)

10 31.2 1.8 491.2447 423.2, 473.3 C30H35O6 (1.6) no matches

11 32.7 1.3 475.2498, 797.3711 292.1, 347.2 C30H35O5 (1.7) see peak 7

12 34.1 22.3 390.1841, 459.2547 390.2 C30H35O4 (1.4) harunganin (3736-60-5), harungin anthrone

(59204-72-70), ferruginin B (73210-80-7), ferrug-

inin A (73210-81-8), harunganol B (84393-25-9)

13 36.0 1.0 781.3761 322.1, 712.2 C50H53O8 (2.0) bianthrone A1 (97399-74-1)

14 37.2 1.7 475.2501, 489.2292, 933.4983 457.3 C30H35O5 (2.2) see peak 7

15 37.9 13.4 390.1841, 459.2548 390.2 C30H35O4 (1.5) see peak 12

16 40.7 2.5 601.3555, 917.5029 409.2, 465.4 C38H49O6 (3.3)

xanthochymol (52617-32-0), cambogin (71117-97-

0), garcinol (78824-30-3), guttiferone F (219538-

86-0), coccinone F (1141870-97-4), coccinone G

(1141870-99-6), coccinone H (1141871-01-3),

coccinone A (1141871-31-9)

17 42.0 1.9 865.4342 407.2, 796.3 C55H61O9 (2.5) no matches

18 44.8 2.7 849.4391 322.1, 780.3 C55H61O8 (2.1) no matches

19 45.9 7.7 527.3179, 865.4345 407.3, 796.2 C55H61O9 (2.7) no matches

20 46.3 1.8 527.3179, 865.4345, 949.4933 422.2, 880.3 C53H73O15 (-2.5) no matches

hE004-18, Sesamum calycinum subsp. angustifolium

21 24.1 12.4 293.2123, 309.2073, 609.4149, 844.6181 209.1, 291.2 C18H29O4 (0.6) tetrahydrotrisporic acid C (35996-92-0)

22 24.6 12.7 295.2283, 564.4143, 860.6514 ND C55H88O7 (-2.5) no matches

23 25.0 5.3 295.2281 171.0, 195.1, 277.2 C18H31O3 (0.68) vernolic acid (503-07-1)

24 25.5 1.8 293.2124, 471.3490, 564.4149 270.3, 547.2 C37H56O4 (5.2) no matches

25 26.3 3.9 295.2281 171.1, 195.1, 251.2, 277.2 C18H31O3 (0.68) see peak 23

26 29.0 9.0 277.2175, 933.4965 233.2, 259.2 C18H29O2 (0.6)

alpha-linolenic acid (463-40-1), eleostearic acid

(506-23-0), gamma-linolenic acid (506-26-3),

trichosanic acid (544-72-9), beta-eleostearic acid

(544-73-0), 9, 12, 15- octadecatrienoic acid (1955-

33-5), 5, 9, 12- octadecatrienoic acid (13237-97-3),

elaeostearic acid (13296-76-9), linolenelaidic acid

(28290-79-1)

27 31.5 5.8 279.2332, 455.3539, 933.4983 407.4 C30H47O3 (1.8) betulinic acid (472-15-1), oleanic acid (508-02-1),

boswellic acid (631-69-6)

28 31.7 16.7 279.2331, 455.3536, 933.4961 407.4 C30H47O3 (1.1) see peak 27

29 34.2 10.9 933.4965, 949.4937 ND C53H73O15 (-1.9) no matches

30 35.5 6.7 255.2330, 281.2488 237.2, 255.3 C16H31O2 (0.1) palmitic acid (57-10-3), ethyl myristate (124-06-1),

methyl pentadecanoate (7132-64-1)

31 36.5 10.9 281.2487, 865.4341 263.3, 281.3 C18H33O2 (2.4)

elaidic acid (112-79-8), oleic acid (112-80-1),

11Z-octadecenoic acid (506-17-2), (6Z)-6-octa-

decenoic acid (593-39-5), (11E)-11-octadecenoic

acid (693-72-1), methyl 9,10-methylenehexade-

canoate (10152-61-1), (7Z)-7-octadecenoic acid

(13126-31-3), ethyl 9-hexadecenoate (54546-22-4)

32 39.2 3.8 455.3544, 865.4335 393.3, 407.4, 409.4, 437.4 C30H47O3 (4.1) see peak 27

eE006, Solanum aculeastrum

33 16.3 14.5 766.4394, 912.4975 866.4 C43H76O20 (4.4) no matches

34 17.1 16.3 720.4350, 766.4398 246.8, 574.1 C39H62NO11 (2.8)

γ2-Solamarine (11034-34-7), γ1-solamarine

(15299-06-6), β2-solamargine (32449-98-2), β2-

solanine (61877-94-9), β1-solasonine (73069-18-8),

β1-solamargine (73069-20-2), β-d-glucopyranoside

derivative of solanidane (81920-14-1), β-d-

tomatid- 5- en- 3β- ol 4- o- α- l- rhamnopyranosyl-

glucopyranoside (906342-97-0)

Continued

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Peak no. RT (min) % Area m/z* MS–MS Empirical formula Putative match (CAS no.)

35 20.3 1.6 299.0563, 599.1220 284 C16H11O6 (0.7)

3′-Methoxyapigenin (491-71-4), diosmetin (520-

34-3), 7-methylkaempferol (569-92-6), 6-meth-

oxyapigenin (1447-88-7), 3-methylkaempferol

(1592-70-7), 8-hydroxyacacetin (51876-19-8),

5,8,4′-trihydroxy-7-methoxyflavone (56595-23-4)

36 24.9 4.8 295.228 171.1, 195.1, 277.2 C18H31O3 (0.4)

trans-3- oxo- 2- pentyl- cyclopentaneoctanoic

acid (91403-58-6), (1R, 5R) - rel-2- oxo- 5- pentyl-

cyclopentaneoctanoic acid (282091-22-9),

vernoleic acid (503-07-1), coronaric acid

(16833-56-0), α-artemisolic acid (18104-45-5),

(±)-α-dimorphecolic acid (98524-19-7), (Z,E)-

9-hydroxy-10,12-octadecadienoic acid (109281-

79-0)

37 25.1 14.0 293.2123, 311.2230 293.2 C18H31O4 (0.8)

Octadecenedioic acid (4494-16-0), (9Z)

-13-

hydroxy- 12- oxo-9- octadecenoic acid (5502-89-6),

(E,Z)-9-hydroperoxy-10,12-octadecadienoic acid

(5502-91-0), 9- hydroperoxy-10, 12- octadecadi-

enoic acid (7324-20-1), 13-hydroperoxylinoleic

acid (7324-21-2), 9, 11- 13- (9Z, 11E) -hydroper-

oxy-

octadecadienoic acid (23017-93-8), (11E)

-13- hydroxy- 10- oxo-11- octadecenoic acid

(28979-44-4), (9S, 10E, 12Z) -9- hydroperoxy-10,

12-

octadecadienoic acid (29774-12-7), (9Z,

11E,

13S) -13- hydroperoxy-9, 11- octadecadienoic acid

(33964-75-9), 9-D-hydroperoxy-10,12-octadeca-

dienoic acid (39692-45-0), (10E, 12E) -9- hydrop-

eroxy- 10, 12- octadecadienoic acid (63121-49-3),

(9R, 12Z) -9- hydroxy- 10- oxo- 12- octadecenoic acid

(70144-92-2), (θS, 2S, 3S) -3- (1Z) - 1- hepten- 1- yl- θ-

hydroxy-2-

oxiranenonanoic acid (282091-26-3),

3- [(1R, 2Z) - 1- hydroxy- 2- octenyl] - (2S, 3R) -oxirane-

octanoic acid (166735-97-3)

38 26.1 1.7 295.2281, 313.2388 295.2 C18H33O4 (1.2)

10-

dibutyl decanedioic acid ester (109-43-3), 1,

6- dihexyl-hexanedioic acid ester (110-33-8), 16-

hydroxy- 9- oxo-octadecanoic acid (132796-50-0),

9- hydroxy- 16- oxo-octadecanoic acid (132828-

40-1)

39 26.3 2.1 295.2282, 313.2388 295.2 C18H33O4 (1.3) see peak 38

40 31.3 26.1 279.233 261.2 C18H31O2 (0.1) stereo isomers of 9,

12-octadecadienoic acid

(2197-37-7)

41 35.1 2.1 255.2331, 458.2477, 915.4877 403.3, 415.2 C30H34O4 (3.6) no matches

42 36.1 10.2 281.2487 263.3, 281.3 C18H33O2 (0.4) stereo isomers of 9-octadecenoic acid (112-79-8)

43 45.1 1.3 283.2645, 933.4976 846.3 C46H77O19 (-9.5)

β-d-glucopyranoside, (3β, 5α, 6α,

22α,

25S) - 26-

(β-d- glucopyranosyloxy) - 3- hydroxy- 22- methoxy-

furostan- 6- yl 3- O- (6- deoxy- α- L- mannopyranosyl)

(1418008-88-4)

44 45.5 1.1 283.2647, 607.4019 589.3 C38H55O6 (2.5) no matches

dietE017a, Zanthoxylum chalybeum

45 21.3 3.8 465.2293, 931.4689 406.3, 421.3 C28H33O6 (2.3) no matches

46 22.1 3.3 421.2399, 451.2500, 903.5092 407.3, 436.3 C28H35O5 (2.2) no matches

47 23.0 3.1 435.2552, 451.2507, 453.2667, 871.5184 ND C28H37O5 (4.5) no matches

48 23.2 2.1 435.2552, 465.2665, 871.5193, 901.5299 ND C29H37O5 (4.0) no matches

49 24.0 2.5 437.2709, 453.2655 315.3, 425.3, 435.3 C28H37O5 (2.0) no matches

50 24.3 2.2 437.2709, 453.2655 315.3, 425.3, 435.3 C28H37O5 (2.0) no matches

51 24.5 3.3 449.2344, 899.4781 406.3, 434.2 C28H33O5 (2.4) no matches

52 25.2 1.7 451.2499 436.3 C28H35O5 (2.0) no matches

53 30.7 2.0 933.4972, 949.4942 ND C53H73O15 (-1.4) no matches

54 33.2 9.7 390.1845, 459.2550 390.2 C23H33N5O5 (13.6) cyclozanthoxylane A (1384258-42-7)

55 34.0 14.1 459.2551, 933.4975 403.2 C23H33N5O5 (13.8) see peak 54

56 35.1 16.0 458.2473, 527.3181, 933.4977 458.2 C35H43O4 (2.6) no matches

57 37.9 11.1 849.4393, 933.4981 390.2, 780.2 C55H61O8 (2.5) no matches

58 40.8 5.7 933.4972 407.3, 864.3 C60H69O9 (2.6) no matches

59 43.8 2.2 503.2812, 933.4977 459.2 C32H39O5 (1.8) no matches

60 44.3 10.4 933.4974 407.3, 864.3 C60H69O9 (2.9) no matches

Table 5. LC–MS data and putative matches for extracts etE011-18, hE004-18, eE006, and dietE017a. ND, not

detected. *When multiple base ions were detected, the m/z in bold fond indicates the ion used to predict the

empirical formula and which underwent MS2 fragmentation.

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in the past. The majority of publications documented its traditional use in Uganda24,39–42, Kenya43, Tanzania44

and Ethiopia45. Fagaramide, an antiplasmodial natural product, was previously isolated from Z. chalybeum stem

bark46. In contrast to our results, diverse extracts of stem bark did not show any antibacterial activity against S.

aureus up to a concentration of 100mg/mL in two other studies published in 2001 and

201147,48. The present

work offers the first report of antibiotic properties of Z. chalybeum stem bark against growth of multiresistant

S. aureus and E. faecium.

Another highly active extract in terms of growth inhibition was an ethanolic extract of H. madagascariensis

stem bark (etE011-18), displaying the same

IC50 (8μg/mL) and MIC (32μg/mL) values against E. faecium EU-44

as Z. chalybeum (dietE017a). Extract etE011-18 also highly inhibited growth of S. aureus UAMS-1

(IC50: 8μg/

mL; MIC: 32μg/mL). Although there was low cytotoxicity against human keratinocytes recorded (IC50: 256μg/

mL), the TI still reached an excellent value of 32 for both strains. H. madagascariensis has been extensively stud-

ied in the past and reports on traditional medicine describe medicinal use all over the African

continent49–54.

One study sought to evaluate the antibacterial activity of stem bark from H. madagascariensis against bacterial

species also tested in our

study55. In this study, a hydro-ethanolic extract displayed low inhibitory effects on

two P. aeruginosa strains (MIC: 500μg/mL) and moderate effects on two S. aureus strains (MIC: 62.5μg/mL

and 125μg/mL). Although much work has been done on this evergreen shrub or tree, including isolation of

compounds56–59, our study is the first to report on the strong growth inhibitory activity of the stem bark of this

species, targeting multiresistant ESKAPE pathogens, especially E. faecium and S. aureus. This finding strongly

supports the traditional medicinal use of H. madagascariensis in the Greater Mpigi region, where it was highly

cited to be efficient in treatment of skin infections (relative frequency of citation: 31%, n = 39); wound treatment

(31%); stomach/gastrointestinal (GI) tract disorders (21%); nausea (13%), sore throat (13%); and fever (18%)24.

As stated above, the stem barks of Z. chalybeum and H. madagascariensis are particularly often used in

plant-based antibiotic treatment of stomach/GI tract disorders and wounds in the Greater Mpigi region. Our

study identified certain extracts of these stem barks as being highly effective in inhibiting bacterial growth of

multidrug-resistant strains of E. faecium and S. aureus. Enterococci are mostly commensal non-pathogenic

bacteria, present in the GI tract without causing human infections60. However, in past decades, E. faecium

strains have emerged as one of the most pervasive nosocomial pathogens worldwide that caused numerous

outbreaks of serious

infections61,62. E. faecium managed to circumvent conventional antibiotics, such as vanco-

mycin, and successfully adapted to hospital environments, making it difficult to target pharmacologically63,64.

With regards to its prevalence in Africa, regional pathogenic strains of E. faecium were identified to possess the

lowest vancomycin resistance rates worldwide, but at the same time the highest resistance to ampicillin (data

provide by WHO regional offices)65. This might be due to regional scarcity and high prices for wide-spectrum

antibiotics and higher prescription of narrow-spectrum antibiotics66. The traditional use of Z. chalybeum and H.

madagascariensis is still widely practiced for treatment of stomach/GI disorders in our study region and specific

use against E. faecium was therefore validated in this study. S. aureus is a ubiquitous colonizer of the human

epithelia, e.g. the skin, the upper respiratory tract and the GI

tract67. Methicillin-resistant S. aureus (MRSA) can

cause serious, sometimes fatal, infections upon invading the blood-stream or internal tissues, whereas wounds

are often the source of

infection68,69. According to the results of our study, the traditional use of Z. chalybeum

and H. madagascariensis stem barks in wound treatment and disinfection therefore seems justified in order to

prevent and combat a S. aureus infection, among others.

None of the extracts reached an MIC in the growth inhibition experiments with K. pneumoniae, A. baumannii

and E. cloacae (maximum concentration tested at 256µg/mL) and the lowest IC50 values reported were 256μg/mL

for K. pneumoniae (dietE010, T. asiatica) and 32μg/mL for A. baumannii (etE015, C. molle). Moreover, none of

the 86 extracts were showed antibacterial effects on the growth of the multiresistant E. cloacae CDC-0032 strain.

Another set of extracts displayed antivirulence activity and was highly effective in the quorum sensing inhi-

bition and the δ-toxin production screen. Selectively inhibiting quorum sensing pathways could prove to be an

efficient alternative to antibiotics that simply try to kill the pathogen. One advantage of targeting the agr system

is disruption of a wide variety of virulence factors, instead of targeting each virulence factor individually70,71. Use

of botanical formulations or small molecule quorum sensing inhibitors isolated from medicinal plants might

offer some additional benefits, e.g. protection of commensal bacteria that induce protective responses to prevent

invasion and colonization by pathogens as part of the human host defense70,72,73. Many virulence factors are not

of relevance to the overall survival of the pathogen. QSI therefore provides a less selective pressure towards resist-

ance, facilitating a promising alternative therapy when combatting pathogens that are likely to develop resistance

mechanisms during strong selective pressure of conventional treatment with antibiotics73–75.

The ethyl acetate root extract of S. aculeastrum (eE006) was among the two most QSI-active extracts, showing

reporter gene subtype-dependent IC50 values of 4, 1, 16 and 64μg/mL (agr I-IV). Its antivirulence activity was

successfully confirmed in the δ-toxin production screen, where it significantly attenuated δ-toxin biosynthesis

in our high-toxin-producing model strains. Extract eE006 exhibited no cytotoxicity in our model at the highest

tested concentration of 512μg/mL and calculated TIs were as high as > 128 (agr I)., > 512 (agr II)., > 32 (agr III).

and > 8 (agr IV). S. aculeastrum is regarded an understudied species and our study accomplished to identify the

roots of S. aculeastrum as strong quorum sensing inhibitor for the first time. Previous publications encompass

use in African folklore medicine24,76–79. Pharmacological studies published on this species investigated the fruits

or leaves, but not the roots80–84. For instance, fruits and leaves were investigated for antimicrobial activity against

food-borne pathogens, but MIC values were only in the mg/mL range85. Steroidal alkaloids have previously been

isolated from the root bark and fruits, such as solaculine A86 which induced induces non-selective cytotoxicity

and P-glycoprotein inhibition87. At our field study location, the Greater Mpigi region, this poisonous nightshade

species, whose berries contain α-solanine88, is often used in disinfection and treatment of wounds (23%) and

fever (15%)24. The pharmacological effects of the roots, claimed by the traditional healers, might be explained by

its now reported antivirulence activity, but should be further investigated through additional studies.

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The hexane extract of S. calycinum subsp. angustifolium leaves (hE004-18) also exhibited strong antiviru-

lence effects. Quorum sensing inhibition

IC50 values against agr I–IV were as low as 4, 2, 16 and 32μg/mL. No

cytotoxicity was found, suggesting that use of this plant extract and species is safe to human cells. Calculated

TIs are reported as > 128 (agr I), > 256 (agr II), > 32 (agr III) and > 16 (agr IV). S. calycinum subsp. angustifolium

isstill a highly understudied species with only five studies previously being published on its traditional medici-

nal use24,89–92 and one study that reported the presence of the hydrocarbon nonacosane and the glucosinolate,

glucoiberverin93 in its leaves. Traditional healers in the Greater Mpigi region claimed that the leaves of this

medicinal herb are often used to treat skin infections (15%), wounds (26%), disorders of the stomach/GI tract

(13%), sore throats (10%) and fever (10%)24. Our study provides the first report of antivirulence activity, targeting

quorum sensing and δ-toxin production in S. aureus, validating S. calycinum subsp. angustifolium application

as anti-infective herbal drug.

Furthermore, our quorum sensing inhibition experiments showed that eE006 and hE004-18 displayed lower

IC50 and MIC values than the positive control (224CF2c). This is particularly interesting because 224CF2c is not

a crude extract, but a refined fraction of the European chestnut (Castanea sativa) that was previously identified to

be highly active against agr I–IV14. Future bioassay-guided fractionation of the crude extracts eE006 and hE004-

18 from the Ugandan rainforests could result in promising novel natural products aiming towards discovery of

antivirulence drugs. The traditional use of S. aculeastrum roots(eE006) and S. calycinum subsp. angustifolium

leaves (hE004-18) in wound

treatment24 indicates that these extracts might demonstrate a significant reduc-

tion in dermonecrosis after infection with a virulent strain of MRSA, as shown with QSI-active fractions from

Schinus terebinthifolia (Brazilian Peppertree) and Castanea sativa (European Chestnut)

before13,14. Moreover, it

will be essential to further investigate the ability of eE006 and hE004-18 in limiting the severity of disease and

in increasing efficacy of conventional antibiotics. This includes potential activation of other virulence pathways,

such as biofilm formation and secretion systems. Further experiments are also needed in order to assess the

actual decrease of S. aureus virulence invivo.

Methods

Ethnobotanical data. Information on traditional use for medical treatment among 39 traditional healers

in the Greater Mpigi region in Uganda was obtained by means of an ethnobotanical survey. Results of this study

were previously

published24 and serve as a basis for the antibacterial and antivirulence experiments.

Collection and identification of plant material. Plant specimens were collected under guidance of the

traditional healers during fieldwork in 2015, 2016 and 2017, while following standard collection procedures94.

The approach for plant identification and assignment of scientific names was adapted from Weckerle etal. 95. Sci-

entific names were cross-checked with https ://www.thepl antli st.org. Plant family assignments follow The Angio-

sperm Phylogeny Group IV

guidance96. Voucher specimens of all species collected were deposited at Makerere

University Herbarium in Kampala, Uganda and select specimens were also deposited at the Emory University

Herbarium (GEO) in Atlanta, GA, USA and made digitally available on the SERNEC

portal97 (Supplementary

TableS1).

Extraction. Plant samples were shade dried and ground prior to extraction (Supplementary FigureS1).

Extractions were performed as described in the flow sheet (Supplementary FigureS2). Briefly, plant material was

either extracted by maceration, Soxhlet extraction or aqueous decoction. In order to selectively extract different

compounds from the samples, extraction procedures were conducted using solvents of different polarities. Some

plant species were collected for a second time in order to facilitate for productionof higher amounts of extract.

These upscaled extractions were performed in 2018 and resulting extracts received the additional information

“-18” in their extract ID.

Bacterial strains. Multidrug-resistant clinical isolates were used in all growth-inhibition experiments in

order to realistically assess the results of this study for future drug discovery advances for AMR threats. This

study used 12 strains from six bacterial species recognized as ESKAPE pathogens, including Gram-negative

[Klebsiella pneumoniae (CDC-004), Acinetobacter baumannii (CDC-0033), Pseudomonas aeruginosa (AH-71)

and Enterobacter cloacae (CDC-0032)] and Gram-positive [Enterococcus faecium (EU-44) and Staphylococcus

aureus (UAMS-1, AH-1677, AH-430, AH-1747, AH-1872, AH-1872, NRS243)] species. Strain characteristics,

antibiotic resistance profiles and sources are reported in Supplementary TableS2. After streaking from freezer

stock and overnight incubation at 37°C, all strains were maintained on tryptic soy agar (TSA). Overnight liquid

cultures were achieved in tryptic soy broth (TSB) at 37°C and with constant shaking at 230rpm. Appropriate

positive controls (antibiotics or quorum quenchers) and negative controls (vehicle control, sterile media control)

were always incorporated into the assays. All bacterial experiments were conducted in triplicate and repeated at

least once on a separate day.

Growth inhibition assay. All growth inhibition experiments were conducted following the guidelines

set by the Clinical and Laboratory Standards Institute for broth microdilution testing98. Standardized work-

ing cultures were calculated and diluted from TSB overnight cultures in cation-adjusted Müller-Hinton broth

(CAMHB). This was achieved using a BioTek Cytation3 and based on the cultures’ optical density (OD590nm)

to a confluence of 5 × 105CFU/mL. The working culture was pipetted into 96-well microtiter plates (Greiner

Bio-One International, CELLSTAR 655–185) and extracts and controls were added. Vehicle controls, sterility

controls and antibiotic controls (1–64µg/mL) were included on the plate setup. After initial optical density read-

ingsat 600nm to account for extract absorbance, plates were incubated at 37°C for 18h (E. faecium, S. aureus,

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P. aeruginosa, E. cloacae) or for 22h (A. baumannii, K. pneumoniae). A final optical density measurement was

performed, and the percent inhibition was calculated as previously

described99. Growth inhibition is reported as

the IC50 (the lowest concentration at which a sample displayed ≥ 50% inhibition) and MIC (the lowest concentra-

tion at which a sample displayed ≥ 90% inhibition).

All extracts were tested at a concentration of 256µg/mL during an initial screen. Extracts that displayed a

percent inhibition above 40% for an individual strain were further examined by dose-response experiments to

obtain the

IC50 and MIC values. Using two-fold serial dilution, extracts and vehicle were tested at concentrations

ranging from 2 to 256µg/mL.

agr reporter assay. An initial library screen was first conducted against the agr I reporter strain of S. aureus

at 16μg/mL (sub-MIC concentrations). After identification of extracts that displayed > 40% inhibition, candi-

dates were further examined by dose-response studies (0.5–64μg/mL) against all four accessory gene regulator

(agr) subtypes of S. aureus. Crude extracts were tested as previously

described14,100. Briefly, the agr reporter

strains were grown and maintained in TSB and TSA, supplemented with chloramphenicol (10μg/mL). All agr

inhibition assays were conducted in 96-well, tissue culture-treated, black-sided microtiter plates (Costar 3,603,

final well volume: 200 μL). Microtiter plates were incubated in a humidified chamber at 37°C, while shaking at

1,200rpm (Stuart SI505). At initial (0h) and final (22h) time points,

OD600nm and fluorescence (493nm excita-

tion, 535nm emission) were measured using a plate reader (BioTek Cytation3). Controls included a vehicle con-

trol (DMSO) and a positive control (224CF2c). 224CF2c is an QSI-active fraction extracted from the European

chestnut (Castanea sativa), as reported in a previous study by the

authors14. The quorum-quenching activity

was reported as percent vehicle of the signal of the individual reporter train’s yellow fluorescent protein (YFP).

Dose–response curves were generated using the GraphPad Prism 8 software (GraphPad Software, La Jolla, CA,

USA). IC50 and MIC values were calculated as in the growth inhibition experiments described above.

Production of δ‑toxin and quantification by HIC‑HPLC. To confirm antivirulence activity on the

translational products of agr in S. aureus (decline in δ-toxin biosynthesis), the most active extracts of the agr

reporter assay were tested at 8, 16, and 32μg/mL and compared to an untreated control in a δ-toxin production

assay using high-toxin-producing strains of S. aureus (AH1262 and NRS243) by hydrophobic interaction chro-

matography as previously described37. The positive control 224C-F2c was additionally tested at 64μg/mL. Data

integration was normalized for growth (OD600nm) during supernatant harvest and reported as formylated and

deformylated δ-toxin, visualized by a stacked histogram chart. The data was analyzed using a Student’s t-test and

statistical significance was denoted as *P value < 0.05, ‡P value < 0.01, †P value< 0.001.

Human keratinocyte toxicity assay. Potential cytotoxicity of extracts was assessed using immortalized

human keratinocytes (HaCaTs cells) combined with a lactate dehydrogenase (LDH) test kit (G-Biosciences, St.

Louis, MO, USA) as previously described by Quave etal.14. All extracts were initially tested at a concentration

of 64μg/mL. In effort to calculate the therapeutic index (TI), samples selected for dose-response cytotoxicity

testing were extracts that (a) displayed cytotoxic activity above 50% inhibition in the library screen at 64μg/mL,

(b) were introduced as library-screen-active candidates to the growth inhibition dose-response studies and (c)

were identified as being most active in the quorum sensing-dose-response studies and were investigated further

in the δ-toxin production and quantification assay. Dose-response cytotoxicity experiments were conducted at

a concentration range of 2–256μg/mL. Percent of the vehicle (DMSO, v/v) in the well was < 2% for all experi-

ments. All human keratinocyte toxicity experiments were conducted in triplicate and repeated at least once on a

separate day. The TI for growth inhibition and the TI for quorum-sensing inhibition (agr I-IV) were calculated

by dividing the IC50 for extract cytotoxicity by the IC50 for its respective antibacterial activity.

LC–MS characterization of extracts. Extracts displaying the highest anti-growth and antivirulence

activity in the invitro assays, as well as the highest TI, were selected for chemical characterization. These

extracts were examined by negative ESI mode Liquid chromatography-Fourier transform mass spectrometry

(LC-FTMS) using a Thermo Scientific LTQ-FT Ultra mass spectrometer equipped with a Shimadzu SIL-ACHT

auto sampler and Dionex 3600SD HPLC pump. A 20 μL injection of the extract at 10mg/mL dissolved in ethyl

acetate, 1:1 ethyl acetate:methanol, or DMSO was made onto a PhenomenexKinetexC18 150 × 2.1mm, 2.6µm

with compatible guard column at room temperature. The mobile phase consisted of (A) 0.1% formic acid in

water and (B) 0.1% formic acid in Optima LC/MS acetonitrile (Fisher Scientific) at a flow rate of 0.2mL/min.

The gradient program began with initial conditions of 98:2 A:B,which were held for 3min, then changed to

100% B over 15min using a linear gradient, 100% B was held for 25min, before returning to initial conditions

to equilibrate the column. The capillary temperature and voltage were 275.0°C and − 48.00, the sheath gas flow

40, source voltage and current − 5.0kV and 100.0 μA. All MS data was collected in negative MS1 mode scanning

from m/z 150–1,500 with data dependent MS2 collected on the top 4 most abundant ions. The data was collected

and processed using Thermo Scientific Xcalibur 2.2 SP1.48 software.

Putative compounds for each extract were determined by searching The Dictionary of Natural Products

(CRC Press) and Scifinder (Chemical Abstracts Service) for compounds consistent with each MS1 peak’s parent

ion m/z (± 1Da). For The Dictionary of Natural Products ions were searched against all compound records for

theextract’s genus. Duringsearches in Scifinder, ions werescreened against compounds published from the same

genus as the extract in books, clinical trials, commentaries, conference proceedings, dissertations, editorials,

journals, letters, reports, and review articles; entries frompatents and preprints were not included in thesearch.

Any matches from these databases were compared to the empirical formulas derived from the experimental MS

data. Compounds which matched the empirical formula with a calculated mass error < 10ppm were investigated

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further in the literature and reported. When no matches in the literature were found, the hydrocarbon with the

lowest mass error was reported. Due to search limitations in Scifinder, only compounds published prior to 2005

were searched for the genus Solanum. All searches were performed in Feb. 2020.

Received: 23 March 2020; Accepted: 22 May 2020

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Acknowledgements

This work was supported by a Fulbright Fellowship (FS), a Grant from the BMBF—German Federal Ministry

of Education and Research (13FH026IX5, PI: LAG and Co-I: FS), and a Grant from the National Institutes of

Health,National Institute of Allergy and Infectious Disease (R21 AI13656s3, PI: CQ). We acknowledge support

for the Article Processing Charge from the German Research Foundation (DFG, 414051096) and the Open

Access Publication Fund of Neubrandenburg University of Applied Sciences (HSNB).The content is solely the

responsibility of the authors and does not necessarily reflect the official view of NIAID, NIH, Fulbright, DFG,

HSNB or BMBF. The funding agencies had no role in the study design, data collection and analysis, decision to

publish, or preparation of the manuscript. We thank Alex Horswill (UC Denver) for providing the Staphylococ-

cus aureus agr reporter isolates: AH1677, AH430, AH1747, and AH1872. Thanks to research assistants Kristine

Kossol, Tidjani Cisse and Tina Seehafer for assisting during the extractions of plant material. Thanks to Micah

Dettweiler for assisting in strain maintenance.Thanks to Logan Penniket for proof-reading the manuscript.

Author contributions

F.S. and G.A. collected and processed the plant material. G.A. prepared herbarium voucher specimens and

identified the plant species. F.S. prepared extracts and performed the antibacterial, quorum sensing inhibition

and δ-toxin experiments. F.S. and F.C. conducted the cell culture experiments. F.S., J.T.L. and H.T. performed the

HPLC analyses. J.T.L. performed and analyzed the LC–MS experiments. F.S., J.T.L. and C.L.Q. analyzed the data

Vol.:(0123456789)

Scientific RepoRtS | (2020) 10:11935 | https://doi.org/10.1038/s41598-020-67572-8

www.nature.com/scientificreports/

and wrote the manuscript. L.A.G. provided oversight of extraction procedures and fieldwork. C.L.Q. directed

the study. All authors read, revised and approved the final manuscript.

competing interests

The authors declare no competing interests.

Additional information

Supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-67572 -8.

Correspondence and requests for materials should be addressed to C.L.Q.

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Targeting ESKAPE pathogens with anti-infective medicinal

plants from the Greater Mpigi region in Uganda

Fabien Schultza,b,c, Godwin Anyward, Huaqiao Tangf, François Chassagnef,

James T. Lylesf, Leif-Alexander Garbeb,c,e, Cassandra L. Quavea,f,g剷

aDepartment of Dermatology, Emory University School of Medicine, 615 Michael St., Atlanta,

30322, Georgia, United States of America

bInstitute of Biotechnology, Faculty III - Process Sciences, Technical University of

Berlin, Gustav-Meyer-Allee 25, Berlin, 13355, Germany

cDepartment of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences,

Brodaer Str. 2, Neubrandenburg, 17033, Germany

dDepartment of Plant Sciences, Microbiology and Biotechnology, Makerere University, P.O Box

7062,Kampala, Uganda

eZELT - Neubrandenburg Center for Nutrition and Food Technology gGmbH, Seestraße 7A,

Neubrandenburg, 17033, Germany

fCenter for Study of Human Health, Emory University College of Arts and Sciences, 615 Michael

St., Atlanta, 30322, Georgia, United States of America

gEmory Antibiotic Resistance Center, Emory University, 615 Michael St., Atlanta, 30322,

Georgia, United States of America

剷Corresponding author (

Email address: cquave@emory.edu)

Supplementary information

Table of contents

Supplementary Figure S1:

Photos of dried plant material page 3

Supplementary Figure S2:

Workflow of extractions page 4

Supplementary Table S1:

Description of collected plant species page 5

Supplementary Table S2:

Description of bacterial strains used in the study page 7

Supplementary Table S3:

Results of HaCaT cytotoxicity library screen at 64 μg/mL page 9

Supplementary Figure S3:

Chemical structures for the putative matches from the extract of

H. madagascariensis, etE011-18 page 12

Supplementary Figure S4: Chemical structures for the putative

matches from the extract of S. calycinum subsp. angustifolium,

hE004-18 page 13

Supplementary Figure S5:

Chemical structures for the putative matches from the extract of

S. aculeastrum, eE006 page 14

Supplementary Figure S6: Chemical structures for the putative

matches from the extract Z. chalybeum, dietE017a page 15

References cited in supplementary files page 15

Supplementary Figure S1: Photos of dried plant material

Supplementary Figure S2: Workflow of extractions

Supplementary Table S1: Description of collected plant species. All specimens deposited

at Emory University have been digitized and are available for viewing at

http://sernecportal.org/portal/.

Extract ID

Type of

extract

Scientific name

Local name in

Luganda

Plant

part

Voucher

specimen #

eE001

ethyl acetate

Securidaca

longipedunculata

Fresen., Polygalaceae

ukondwe

stem

AG196°

wE001

water

hE001

hexane (sox.)

mE001

methanol

smE001

methanol

(sox. succ.)

eE002

ethyl acetate

Microgramma

lycopodioides

(L.) Copel.

Polypodiaceae

Kukumba

root

(rhizomes)

AG639°

wE002

water

hE002

hexane (sox.)

mE002

methanol

smE002

methanol

(sox. succ.)

eE003

ethyl acetate

Ficus saussureana

DC.,

Moraceae

Muwo

stem

AG219°

wE003

water

hE003

hexane (sox.)

mE003

methanol

smE003

methanol

(sox. succ.)

eE004

ethyl acetate

Sesamum calycinum

subsp. angustifolium

(Oliv.) Ihlenf. &

Seidenst., Pedaliaceae

Lutungotungo

leaves

AG205°

23173*

wE004

water

hE004

hexane (sox.)

mE004

methanol

smE004

methanol

(sox. succ.)

eE004-18

ethyl acetate

hE004-18

hexane (sox.)

smE004-18

methanol

(sox. succ.)

eE005

ethyl acetate

Leucas calostachys

Oliv.

, Lamiaceae

akuba

musulo

leaves

AG195°

23175*

wE005

water

hE005

hexane (sox.)

smE005

methanol

(sox. succ.)

eE005-18

ethyl acetate

hE005-18

hexane (sox.)

mE005-18

methanol

smE005-18

methanol

(sox. succ.)

eE006

ethyl acetate

Solanum aculeastrum

Dunal

, Solanaceae

Kitengo

root

AG193°

wE006

water

hE006

hexane (sox.)

smE006

methanol

(sox. succ.)

eE007

ethyl acetate

Albizia coriaria Oliv.,

Fabaceae

Mugavu

bark

AG203°

etE007

ethanol

eE008

ethyl acetate

Erythrina abyssinica

DC., Fabaceae

Jjirikiti

bark

AG199°

etE008

ethanol

eE009

ethyl acetate

Zanthoxylum chalybeum

Engl., Rutaceae

Ntaleyaddungu

bark

AG204°

etE009

ethanol

eE010

ethyl acetate

Toddalia asiatica

(L.) Lam.

, Rutaceae

Kawule

leaves,

bark

AG190°

etE010

ethanol

dietE010

diethyl ether

etE010a

ethanol

eE011

ethyl acetate

Harungana

madagascariensis

Lam. ex Poir.

Hypericaceae

Mukabiiransiko

bark

AG230°

23174*

etE011

ethanol

dietE011

diethyl ether

etE011a

ethanol

eE011-18

ethyl acetate

wE011-18

water

etE011-18

ethanol

dietE011-18

diethyl ether

hE011-18

hexane (sox.)

smE011-18

methanol

(sox. succ.)

eE012

ethyl acetate

Morella kandtiana

(Engl.) Verdc. & Polhill,

Myricaeae

ukikimbo

root

AG201°

23174*

etE012

ethanol

dietE012

diethyl ether

etE012a

ethanol

eE012-18

ethyl acetate

wE012-18

water

etE012-18a

ethanol

etE012-18b

ethanol

dietE012-18

diethyl ether

eE013

ethyl acetate

Cassine buchananii

Loes.

, Celastraceae

Mbaluka

bark

AG198°

etE013

ethanol

etE013a

ethanol

eE014

ethyl acetate

Warburgia ugandensis

Sprague, Canellaceae

Abasi

bark

AG220°

23181*

etE014

ethanol

dietE014

diethyl ether

etE014a

ethanol

eE014-18

ethyl acetate

wE014-18

water

etE014-18

ethanol

dietE014-18

diethyl ether

hE014-18

hexane (sox.)

smE014-18

methanol

(sox. succ.)

eE015

ethyl acetate

Combretum molle

R.Br. ex G.Don

Combretaceae

Ndagi

bark

AG191°

etE015

ethanol

dietE016

diethyl ether

Plectranthus hadiensis

(Forssk.) Schweinf. ex

Sprenger, Lamiaceae

Kibwankulata

leaves

AG210°

hE016

hexane

etE017

ethanol

Zanthoxylum chalybeum

Engl.

, Rutaceae

Ntaleyaddungu

bark

AG204°

dietE017

diethyl ether

etE017a

ethanol

dietE017a

diethyl ether

° deposited at Makerere University herbarium * deposited at Emory University herbarium

Abbreviations: sox.: Soxhlet extraction; succ.: successive extraction

Supplementary Table S2: Description of bacterial strains used in the study

Species

Strain ID

Characteristics*

Ref.

Enterococcus

faecium

EU-44

HM-959; Strain 513

Resistance: AMC, RIF, SXT, TET, TZP

Human clinical sample, source: BEI Resources

Staphylococcus

ureus

UAMS-1

ATCC49230

linical MSSA isolate from osteomyelitis

Source: Dr. Mark Smeltzer, University of

Arkansas for Medical Sciences

AH-1677

AH845 + pDB59 cmR; Resistance: OXA

agr

type I YFP reporter

Source: Dr. Alex Horsewill, UC Denver

AH-430

SA502a + pDB59 cmR;

agr

type II YFP reporter

Source: Dr. Alex Horsewill, UC Denver

AH-1747

MW2 + pDB59 cmR; Resistance: OXA

agr

type III YFP reporter

Source: Dr. Alex Horsewill, UC Denver

AH-1872

MN EV(AH407) + pDB59 cmR

agr

type IV YFP reporter

Source: Dr. Alex Horsewill, UC Denver

NRS243

HT20020252

Resistance: ERY, PEN

Intermediate re

sistance: CIP

high delta toxin producing strain

associated with

pneumonia

Source: NARSA Library

AH1263

LAC CA-MRSA USA300 clinical isolate

Resistance: OXA

high delta toxin producing strain

Source: Source: Dr. Alex Horsewill, UC Denver

Klebsiella

pneumoniae

CDC-004

AR-BANK#0004

Resistance: AMC, AMP, ATM, CAZ, CFZ, CIP,

CRO, CTX, DOR, ETP, FEP, FOX, IPM, LVX,

MEM, SAM, SXT, TET, TOB, TZP

Clinical isolate, source: CDC Antimicrobial

Resistance Bank

Acinetobacter

baumannii

CDC-0033

AR-BANK #0033

esistance: CAZ, CIP, CLI,

CRO, CTX. DOR,

FEP, GEN, IPM, LVX, MEM, SAM, SXT, TOB,

TZP

Clinical isolate, source:

CDC Antimicrobial

Resistance Bank

Pseudomonas

aeruginosa

AH-71

PAO1

Laboratory strain

, source: Dr. Alex Horswill,

University of Colorado, Denver

Enterobacter

loacae

CDC-0032

AR-BANK #0032

Resistance:

AMC, AMP, ATM, CAZ, CFZ,

CRO,

CTX

, ETP, FEP, FOX, GEN, IPM, MEM,

SAM,

SXT

, TZP

Intermediate resistance: DOR, TOP

Clinical isolate, source:

CDC Antimicrobial

Resistance Bank

*abbreviations:

agr: accessory gene regulator; AMC: amoxicillin-clavulanic acid; AMP: ampicillin; ATM:

aztreonam; CAZ: ceftazidime; CFZ: cefazolin; CIP: ciprofloxacin; CLI: clindamycin; CRO:

ceftriaxone; cmR: Chloramphenicol resistance protein; CTX: cefotaxime; DOR: doripenem;

ERY: erythromycin; ETP: ertapenem; FEP: cefoxitin; FOX: cefoxitin; GEN: gentamicin; IPM:

imipenem; LVX: levofloxacin; MEM: meropenem; MSSA: methicillin sensitive

Staphylococcus aureus; NARSA: network on antibiotic resistant Staphylococcus aureus; OXA:

oxacillin; PEN: penicillin; RIF: rifampicin; SAM: ampicillin-sulbactam; SXT: trimethoprim-

sulfamethoxazole; TET: tetracycline; TOB: tobramycin; TZP: piperacillin-tazobactam; YFP:

yellow fluorescent protein

Supplementary Table S3: Results of HaCaT cytotoxicity library screen at 64 μg/mL

scientific name

extract ID

HaCaT

cytoxoxicity

≥ 50

Securidaca longipedunculata

eE001

negative

smE001

negative

wE001

negative

mE001

negative

hE001

negative

Microgramma lycopodioides

hE002

negative

mE002

negative

wE002

negative

smE002

negative

eE002

negative

Ficus saussureana

smE003

negative

wE003

negative

eE003

negative

mE003

negative

hE003

negative

Sesamum calycinum subsp.

angustifolium

smE004

negative

smE004-18

negative

mE004

negative

hE004

negative

hE004-18

negative

eE004

negative

eE004-18

negative

wE004

negative

Leucas calostachys

eE005

negative

eE005-18

negative

smE005

negative

smE005-18

negative

wE005

negative

mE005-18

negative

hE005

negative

hE005-18

negative

Solanum aculeastrum

eE006

negative

hE006

negative

wE006

negative

smE006

positive

51.8

1.5

Albizia coriaria

etE007

negative

eE007

negative

Erythrina abyssinica

etE008

negative

eE008

negative

Zanthoxylum chalybeum

etE009

negative

eE009

negative

etE017

negative

etE017a

negative

dietE017

negative

dietE017a

negative

Toddalia asiatica

etE010

negative

etE010a

negative

eE010

negative

dietE010

negative

Harungana madagascariensis

etE011

negative

etE011a

negative

etE011-18

negative

eE011

negative

eE011-18

negative

dietE011

negative

dietE011-18

negative

wE011-18

negative

hE011-18

negative

smE011-18

negative

Morella kantiana

etE012

negative

etE012a

negative

etE012-18a

negative

etE012-18b

negative

eE012-18

negative

wE012-18

negative

dietE012

negative

dietE012-18

negative

Cassine buchananii

etE013

negative

etE013a

negative

eE013

negative

Warburgia ugandensis

dietE014

negative

dietE014-18

negative

eE014-18

negative

wE014-18

negative

hE014-18

negative

smE014-18

negative

etE014a

negative

etE014-18

negative

Combretum molle

etE015

negative

eE015

negative

Plectranthus hadiensis

hE016

negative

dietE016

negative

Supplementary Figure S3: Chemical structures for the putative matches from the

extract of H. madagascariensis, etE011-18

Supplementary Figure S4: Chemical structures for the putative matches from the

extract of S. calycinum subsp. angustifolium, hE004-18

Supplementary Figure S5: Chemical structures for the putative matches from the

extract of S. aculeastrum, eE006

Supplementary Figure S6: Chemical structures for the putative matches from the

extract Z. chalybeum, dietE017a

References cited in supplementary files

1 Gillaspy, A. F. et al. Role of the accessory gene regulator (agr) in pathogenesis of

staphylococcal osteomyelitis. Infect. Immun. 63, 3373-3380 (1995).

2 Kirchdoerfer, R. N. et al. Structural basis for ligand recognition and discrimination of a

quorum-quenching antibody. J. Biol. Chem. 286, 17351-17358,

doi:10.1074/jbc.M111.231258 (2011).

Publication IV:

"Antiinflammatory medicinal plants from the Ugandan Greater Mpigi region

act as potent inhibitors in the COX-2 / PGH2 pathway"

Pages: 87-125

Personal contribution

In the following, my personal contribution to the presented study and manuscript is briefly

described: I contributed to the collection and processing of the plant material in Uganda. I

contributed to the extraction procedures and the preparation of the extract solutions for all

assays. I created the extract library. I contributed to the work involved in the COX-2 and

COX-1 inhibition experiments, the 15-LOX inhibition assay, the DPPH assay, the TPC

determination experiments, and the antibacterial experiments. I analyzed the majority of the

data. I wrote most of the manuscript. A more detailed author-contribution statement is given in

the published article.

Information on publication

This study was published in Plants in February 2021 and is available at

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918315. It is an open access article

distributed under the Creative Commons Attribution 4.0 International License (CC BY 4.0).

Schultz, F.; Osuji, F. O.; Wack, B.; Anywar, G.; Garbe, L. A.: Antiinflammatory Medicinal

Plants from the Ugandan Greater Mpigi Region Act as Potent Inhibitors in the COX-2 / PGH2

Pathway. Plants, 10(2), 351, 2021; https://doi.org/10.3390/plants10020351

Plants 2021, 10, 351. https://doi.org/10.3390/plants10020351 www.mdpi.com/journal/plants

Article

Antiinflammatory Medicinal Plants from the Ugandan Greater

Mpigi Region Act as Potent Inhibitors in the COX-2 / PGH

Pathway

Fabien Schultz

1,2,

*, Ogechi Favour Osuji

, Barbara Wack

, Godwin Anywar

and Leif-Alexander Garbe

2,4

Institute of Biotechnology, Faculty III - Process Sciences, Technical University of Berlin, Gustav-Meyer-Allee

25, 13355 Berlin, Germany

Department of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences, Brodaer

Str. 2, 17033 Neubrandenburg, Germany; [email protected] (O.F.O.);

[email protected] (B.W.); [email protected] (L.-A.G.)

Department of Plant Sciences, Microbiology and Biotechnology, Makerere University, P.O. Box 7062,

Kampala, Uganda; [email protected]

ZELT - Neubrandenburg Center for Nutrition and Food Technology gGmbH, Seestraße 7A,

17033 Neubrandenburg, Germany

* Correspondence: Fabien.Schult[email protected]berlin.de; Tel.: +49-395-5693-2704

Abstract: Our study investigates 16 medicinal plants via assessment of inhibition of proinflamma-

tory enzymes such as cyclooxygenases (COX). The plants are used by traditional healers in the

Greater Mpigi region in Uganda to treat inflammation and related disorders. We present results of

diverse in vitro experiments performed with 76 different plant extracts, namely, (1) selective COX-2

and COX-1 inhibitor screening; (2) 15-LOX inhibition screening; (3) antibacterial resazurin assay

against multidrug-resistant Staphylococcus aureus, Listeria innocua, Listeria monocytogenes, and Esche-

richia coli K12; (4) DPPH assay for antioxidant activity; and (5) determination of the total phenolic

content (TPC). Results showed a high correlation between traditional use and pharmacological ac-

tivity, e.g., extracts of 15 out of the 16 plant species displayed significant selective COX-2 inhibition

activity in the PGH2 pathway. The most active COX-2 inhibitors (IC50 < 20 μg/mL) were nine extracts

from Leucas calostachys, Solanum aculeastrum, Sesamum calycinum subsp. angustifolium, Plectranthus

hadiensis, Morella kandtiana, Zanthoxylum chalybeum, and Warburgia ugandensis. There was no coun-

teractivity between COX-2 and 15-LOX inhibition in these nine extracts. The ethyl acetate extract of

Leucas calostachys showed the lowest IC50 value with 0.66 μg/mL (COX-2), as well as the most prom-

ising selectivity ratio with 0.1 (COX-2/COX-1). The TPCs and the EC50 values for DPPH radical scav-

enging activity showed no correlation with COX-2 inhibitory activity. This led to the assumption

that the mechanisms of action are most likely not based on scavenging of reactive oxygen species

and antioxidant activities. The diethyl ether extract of Harungana madagascariensis stem bark dis-

played the highest growth inhibition activity against S. aureus (MIC value: 13 μg/mL), L. innocua

(MIC value: 40 μg/mL), and L. monocytogenes (MIC value: 150 μg/mL). This study provides further

evidence for the therapeutic use of the previously identified plants used medicinally in the Greater

Mpigi region.

Keywords: inflammation; antibiotics; ethnopharmacology; traditional medicine; pain; fever; cy-

clooxygenase; lipoxygenase; Albizia coriaria; Cassine buchananii; Combretum molle; Erythrina abys-

sinica; Ficus saussureana; Harungana madagascariensis; Leucas calostachys; Microgramma lycopodioides;

Morella kandtiana; Plectranthus hadiensis; Securidaca longipedunculata; Sesamum calycinum subsp. an-

gustifolium; Solanum aculeastrum; Toddalia asiatica; Warburgia ugandensis; Zanthoxylum chalybeum

Citation: Schultz, F.; Osuji, O.F.;

Wack, B.; Anywar, G.; Garbe, L.-A.

Antiinflammatory Medicinal Plants

from the Ugandan Greater Mpigi Re-

gion Act as Potent Inhibitors in the

COX-2 / PGH2 Pathway. Plants 2021,

10, 351. https://doi.org/

10.3390/plants10020351

Academic Editor: Sebastian Granica,

and Francesca Pintus

Received: 31 December 2020

Accepted: 09 February 2021

Published: 12 February 2021

Publisher’s Note: MDPI stays neu-

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Licensee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license

(http://creativecommons.org/licenses

/by/4.0/).

Plants 2021, 10, 351 2 of 30

1. Introduction

Approximately 80% of Africa’s population relies almost entirely on plants for medi-

cation [1–3]. The knowledge of plants in Uganda and their medicinal uses is mainly trans-

ferred orally from one generation to the next by traditional healers, leading to the potential

for loss of vital information due to lack of records [4,5]. A previous ethnopharmacological

study from the Greater Mpigi region documented the traditional use of 39 healers [5]. In

this study, Schultz et al. described the medicinal uses of 16 plant species used in treatment

of diverse medical disorders. The 16 Ugandan medicinal plant species were Albizia cori-

aria, Cassine buchananii, Combretum molle, Erythrina abyssinica, Ficus saussureana, Harungana

madagascariensis, Leucas calostachys, Microgramma lycopodioides, Morella kandtiana, Plectran-

thus hadiensis, Securidaca longipedunculata, Sesamum calycinum subsp. angustifolium, Solanum

aculeastrum, Toddalia asiatica, Warburgia ugandensis, and Zanthoxylum chalybeum. Another

study applying the Degrees of Publication (DoP) method as a tool for literature assessment

in ethnopharmacological research classified six of these 16 plant species as being “highly

understudied” and three species as “understudied” [6]. This DoP analysis further

strengthened the justification for conducting pharmacological lab studies, investigating

these select medicinal plant species from the Greater Mpigi region. The ethnobotanical

survey specifically sought to investigate the treatment of cardinal signs of acute inflam-

mation, which is relevant to the present study. Uses documented for each species include

the treatment of pain, fever, redness, heat, wounds, cancer, and general infections [5]. Fig-

ure 1 depicts the relative frequencies of citation (RFCs, n = 39) for these use reports.

Inflammation is the reaction of the immune system to injury and invading pathogens

and can be considered one of the most important human host defense mechanisms [5,7,8].

The scientific pursuit of novel antiinflammatory therapeutics and drug leads, e.g., for

treatment of pain, is complex and challenging [9,10]. Inflammation has also been impli-

cated in the pathogeneses of diverse medical disorders, and over- or persistent inflamma-

tion can cause tissue damage, failure of vital organs, and death [8,11,12]. Its mediators are

involved in diverse biochemical signaling pathways. One of these pathways is the cy-

clooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) pathway, which plays a key role

in the production of eicosanoids (Figure 2). It is also known as the prostaglandin H2

(PGH2) pathway, named after the resulting prostaglandin precursor of the COX-catalyzed

reaction of arachidonic acid in the human body [7,13]. The main human cyclooxygenases,

COX-1 and COX-2, are prostaglandin (PG) endoperoxide synthases (E.C.1.14.99.1) that

catalyze the metabolic biosynthesis of arachidonic acid to prostanoids, encompassing po-

tent proinflammatory signaling molecules such as prostaglandin F2΅ and prostaglandin E2

[14–17]. Each of these COX isoforms catalyzes the reaction of individual prostanoids,

whereas products of COX-1 catalysis are involved in normal, homeostatic functions, such

as cytoprotection of gastric mucosa, renal blood flow, macrophage differentiation, and

hemostasis. These prostaglandins are also involved in regulating normal cells in general,

which is why COX-1 is constitutively present in human cells. The concentration in the

body generally remains stable [15,18–20]. The isoform COX-2, however, plays a major role

in inflammatory response. While underexpressed in cells under normal conditions, COX-

2 expression is upregulated during inflammation as part of the immune response, rapidly

displaying elevated levels. Stimuli that induce COX-2 expression in cells can include pro-

inflammatory cytokines (TNF΅, IL-1) or growth factors [8,15,17–20]. Proinflammatory

prostaglandins produced through the COX-2 pathway contribute to or induce pain, fever,

and swelling, and are even implicated with types of cancer, allergy, asthma, arthritis,

stroke, and Alzheimer’s disease [7,13,19,21–32].

Plants 2021, 10, 351 3 of 30

Figure 1. Ethnopharmacological information, describing the traditional use of 16 medicinal plants from the Greater Mpigi

region in Uganda (with emphasis on the treatment of cardinal signs of acute inflammation, cancer, and stomach and gas-

trointestinal (GI) tract infections). The histogram shows the relative frequencies of citation (RFCs) in %, a field assessment

index that was calculated from an ethnobotanical survey of 39 traditional healers. The RFC describes the use of plants to

treat a specific medical condition relative to the total number of interviewees in the study, assessing the significance of a

plant species in the local traditional medicine system (y-axis). This ethnobotanical index can vary from 0% (no survey

participant uses this plant in treatment of a specific medical condition) to 100% (all survey participants use this plant in

treatment of a specific medical condition) [5].

Large-scale applied nonsteroidal antiinflammatory drugs (NSAIDs), such as ibu-

profen, Paracetamol, or Aspirin, share the capacity for COX / PGH2 inhibition, thereby re-

ducing pain, fever, and inflammation. Yet the vast majority of the NSAIDs on the market

exhibit no selectivity to COX-1 and COX-2, leading to various side effects caused by inhi-

bition of COX-1 regulated “housekeeping” functions in the body (such as ulceration and

gastrointestinal bleeding) [7,20,33–38]. In the past, a few selective COX-2 inhibitors were

discovered and marketed, e.g., celecoxib and rofecoxib. These drug molecules selectively

inhibited COX-2 catalyzed biosynthesis of proinflammatory prostaglandins and were tre-

mendously more potent at inhibiting COX-2 than COX-1 [38–41]. However, these selective

COX-2 inhibitors (members of the diaryl heterocycle group of drug molecules) exhibited

severe skin-related and cardiovascular toxicities, including myocardial infarction, leading

to partial withdrawal of this class of compounds from the market [8,38,42,43]. As a result,

Plants 2021, 10, 351 4 of 30

medicinal plants have regained momentum for treatment of inflammatory diseases, mak-

ing research in this particular field a hot topic [8,17,44–46].

Figure 2. Cyclooxygenase-1/2 pathway and its physiological functions; COX, cyclooxygenase; NSAIDs, nonsteroidal anti-

inflammatory drugs; IL-1, interleukin 1; IL-4, interleukin 4; TNF, tumor necrosis factor; PGH

, prostaglandin H2; PGE

prostaglandin E

; PGF

2΅

, prostaglandin F

2΅

; PGD

, prostaglandin D

; PGI

, prostaglandin I

; TX, thromboxane.

Another pathway of inflammatory response in the human body is the lipoxygenase

(LOX) pathway. Here, long-chain polyunsaturated fatty acids, such as arachidonic acid,

are enzymatically peroxidized by lipoxygenases to the corresponding hydroperoxyl deriv-

atives to produce eicosanoid signaling metabolites (Figure 3) [47].

Plants 2021, 10, 351 5 of 30

Figure 3. Overview of the 12/15-lipoxygenase (12/15-LOX) pathway, highlighting biosynthesis of the antiinflammatory

mediator lipoxin via a 15-S-hydroxyeicosatetraenoic acid (15(S)-HpETE) precursor with relevance to the 15-LOX inhibition

assay presented in this study [48].

While COX-2- and 5-LOX-mediated reactions generally produce prostaglandins and

leukotrienes that act as proinflammatory mediators involved in pathogenesis, 12/15-LOX

generates protectins and resolvins derived from n-3 polyunsaturated fatty acids, as well

as lipoxins derived from arachidonic acid (n-6) [48]. A hydroperoxy cis-trans-1,3-conju-

gated pentadienyl moiety within the unsaturated fatty acid is the initial, unstable product

of the LOX reaction [49–51]. Protectins, resolvins, and lipoxins are considered antiinflam-

matory mediators involved in the regulation of inflammatory responses and resolution of

acute inflammation. Therefore, they are required in order to maintain homeostasis

[48,52,53]. With regard to lipoxin, there is typically an inverse concentration between anti-

inflammatory lipoxin and proinflammatory leukotriene present at the site of inflamma-

tion [54]. 15-LOX is also involved in the development and progression of cancer, yet its

role is complex and still controversial [52]. Due to the presence of two 15-LOX isoforms in

human tumor biopsies and its implication in carcinogenesis of some cancers, the literature

suggests procarcinogenic as well as anticarcinogenic roles [52,55–59]. Thus, discovery of

15-LOX inhibitors derived from medicinal plants may provide promising, novel, and se-

lective therapies for certain cancers [48,56,57].

We screened up to 76 different extracts derived from these 16 medicinal plants from

the Greater Mpigi region in Uganda for antiinflammatory, antioxidant, and antibacterial

activity associated with the traditional use of medical disorders described in Figure 1. The

main objectives of the study were (a) the pharmacological evaluation of traditional use

and (b) contributing to drug discovery. Specifically, the study aims were to investigate the

potential in vitro (1) human recombinant COX-2 inhibition activity; (2) human recombi-

nant COX-1 inhibition activity; (3) 15-LOX inhibition activity; (4) free radical scavenging

activity; (5) growth inhibitory activity against multidrug-resistant Listeria innocua, Listeria

monocytogenes, Escherichia coli K12 and Staphylococcus aureus; and (6) to determine the total

phenolic content (TPC) of the plant extracts.

Plants 2021, 10, 351 6 of 30

2. Results

2.1. Information on Plant Species and Extractions

Table 1 shows taxonomic information on the 16 medicinal plant species studied, ex-

tract identification numbers (extract IDs), extraction solvents used, local names in the Lu-

ganda language, plant parts selected for investigation, and herbarium voucher specimen

numbers and locations. Extracts were produced through different methods: (a) macera-

tion in either methanol, ethanol, ethyl acetate or diethyl ether, (b) Soxhlet extraction using

n-hexane and successively methanol, and (c) aqueous decoction, which simulated the

original methods of traditional preparation [5].

Table 1. Description of collected plant species and different extracts investigated in this study.

Scientific Name Family Local Name in

Luganda Plant Part Voucher Specimen

Number and Location

Extraction

Solvent Extract ID

Securidaca

longipedunculata Fresen. Polygalaceae Mukondwe stem

AG196

(Makerere University

herbarium, Uganda)

ethyl acetate 1 eE001

water 1 wE001

n-hexane

(sox.) 1

hE001

methanol 1 mE001

methanol

(sox. succ.) 1

smE001

Microgramma

lycopodioides

(L.) Copel.

Polypodiaceae Kukumba root (rhizomes)

AG639

(Makerere University

herbarium, Uganda)

ethyl acetate 1 eE002

aqueous 1 wE002

n-hexane

(sox.) 1

hE002

methanol 1 mE002

methanol 1

(sox. succ.)

smE002

Ficus saussureana DC. Moraceae Muwo stem

AG219

(Makerere University

herbarium, Uganda)

ethyl acetate 1 eE003

aqueous 1 wE003

n-hexane

(sox.) 1

hE003

methanol 1 mE003

methanol

(sox. succ.) 1

smE003

Sesamum calycinum

subsp. angustifolium

(Oliv.) Ihlenf. &

Seidenst.

Pedaliaceae Lutungotungo leaves

AG205

(Makerere University

herbarium, Uganda)

23173 *

(Emory University

herbarium, USA)

ethyl acetate1 eE004

water 1 wE004

n-hexane

(sox.) 1

hE004

methanol 1 mE004

methanol

(sox. succ.) 1

smE004

ethyl acetate5 eE004-18

n-hexane

(sox.) 5

hE004-18

Leucas calostachys Oliv. Lamiaceae Kakuba musulo leaves

AG195

(Makerere University

herbarium, Uganda)

23175 *

(Emory University

herbarium, USA)

ethyl acetate 1 eE005

water 1 wE005

n-hexane

(sox.)1

hE005

methanol

(sox. succ.) 1

smE005

ethyl acetate 5 eE005-18

n-hexane

(sox.) 5

hE005-18

methanol 5 mE005-18

methanol

(sox. succ.) 5

smE005-18

Solanum aculeastrum

Dunal Solanaceae Kitengo root

AG193

(Makerere University

herbarium, Uganda)

ethyl acetate 1 eE006

water 1 wE006

n-hexane

(sox.) 1

hE006

methanol smE006

Plants 2021, 10, 351 7 of 30

(sox. succ.) 1

Albizia coriaria Oliv. Fabaceae Mugavu stem bark

AG203

(Makerere University

herbarium, Uganda)

ethyl acetate 2 eE007

ethanol 2 etE007

Erythrina abyssinica DC. Fabaceae Jjirikiti stem bark

AG199

(Makerere University

herbarium, Uganda)

ethyl acetate 2 eE008

ethanol 2 etE008

Zanthoxylum chalybeum

Engl. Rutaceae Ntaleyaddungu stem bark

AG204

(Makerere University

herbarium, Uganda)

ethyl acetate 2 eE009

ethanol 2 etE009

ethanol 3 etE017

diethyl ether 3 dietE017

ethanol 4 etE017a

diethyl ether 4 dietE017a

Toddalia asiatica

(L.) Lam. Rutaceae Kawule leaves, bark

AG190

(Makerere University

herbarium, Uganda)

ethyl acetate 2 eE010

ethanol2 etE010

diethyl ether 4 dietE010

ethanol 4 etE010a

Harungana

madagascariensis

Lam. ex Poir.

Hypericaceae Mukabiiransiko stem bark

AG230

(Makerere University

herbarium, Uganda)

23180 *

(Emory University

herbarium, USA)

ethyl acetate 2 eE011

ethanol 2 etE011

diethyl ether 4 dietE011

ethanol 4 etE011a

ethyl acetate 5 eE011-18

n-hexane 5 hE011-18

ethanol 5 etE011-18

diethyl ether 5 dietE011-18

Morella kandtiana (Engl.)

Verdc. & Polhill Myricaeae Mukikimbo root

AG201

(Makerere University

herbarium, Uganda)

23174 *

(Emory University

herbarium, USA)

ethyl acetate 2 eE012

ethanol 2 etE012

ethanol 4 etE012a

diethyl ether 4 dietE012

ethyl acetate 5 eE012-18

diethyl ether 5 dietE012-18

Cassine buchananii Loes. Celastraceae Mbaluka stem bark

AG198

(Makerere University

herbarium, Uganda)

ethyl acetate 2 eE013

ethanol 2 etE013

ethanol 4 etE013a

Warburgia ugandensis

Sprague Canellaceae Abasi stem bark

AG220

(Makerere University

herbarium, Uganda)

23181 *

(Emory University

herbarium, USA)

ethanol 4 etE014a

diethyl ether 4 dietE014

ethyl acetate 5 eE014-18

water wE014-18

diethyl ether 5 dietE014-18

n-hexane

(sox.) 5

hE014-18

ethanol etE014-18

Combretum molle

R.Br. ex G.Don Combretaceae Ndagi stem bark

AG191

(Makerere University

herbarium, Uganda)

ethyl acetate 2 eE015

ethanol 2 etE015

Plectranthus hadiensis

(Forssk.) Schweinf. ex

Sprenger

Lamiaceae Kibwankulata leaves

AG210

(Makerere University

herbarium, Uganda)

diethyl ether 4 dietE016

n-hexane 4 hE016

* Specimens have been digitized and are available for viewing at http://sernecportal.org/portal/; 1 collected in Apr. 2016;

2 collected in Oct. 2015; 3 collected in Sep. 2013; 4 collected in Sep. 2016; 5 collected in Dec. 2017; sox. = Soxhlet extraction;

sox. succ. = successive Soxhlet extraction.

2.2. Selective COX-2 Inhibition Library Screen

The plant extract library was initially screened for COX-2 inhibition activity at a con-

centration of 50 μg/mL. Extracts displaying a COX-2 inhibition percentage above 80 were

further investigated by dose-response experiments in order to obtain IC50 values. The COX

(human) inhibition assay has two steps involving a COX reaction and a PG-acetylcholin-

esterase (AChE) competitive ELISA for direct spectrophotometric quantification of PGF2΅

by Tin(II) chloride reduction of the PGH2 output produced in the COX reaction (Figure 4).

The two distinct COX isoforms are bifunctional enzymes, displaying both COX and pe-

Plants 2021, 10, 351 8 of 30

roxidase activity [60]. Thus, arachidonic acid is first converted by the prostaglandin syn-

thase active site to a hydroperoxyl endoperoxide (PGG2), and then further reduced by the

peroxidase synthase active site to the corresponding alcohol (PGH2), which is the precur-

sor for PG mediator molecules. The ELISA utilizes a broadly specific antiserum capable

of binding to all major PG compounds. It is based on a PG tracer (PG-AChE conjugate)

and PGs present in the sample that compete for a limited amount of PG antiserum. Since

the PG concentration varies depending on the COX inhibitory activity of plant extracts

tested, while the concentration of PG-AChE conjugate is constant, the concentration of PG

in the sample is inversely proportional to the amount of PG-AChE conjugate that can bind

to the PG antiserum.

Figure 4. Scheme describing the COX inhibition assay used for screening plant extracts for antiinflammatory activity.

In this initial library screen, extracts of 15 out of 16 species inhibited COX-2 at

50 μg/mL. The only exception was extracts of C. molle, which did not display any inhibi-

tory activity on COX-2 (I = 0%). Details of the results of the prescreen are given in Supple-

mentary Data Table S1. In total, out of 58 extracts screened, 19 extracts from nine species

did not show COX-2 inhibition activity, 15 extracts from 10 species resulted in percentage

inhibition of 0–40, and 15 extracts from 10 species exhibited percentage inhibition values

between 40 and 80. Nine extracts from seven species were identified as particularly prom-

ising due to their high percent inhibition values (%I > 80). These were the ethyl acetate

and the n-hexane extract of S. calycinum subsp. angustifolium leaves (eE004, hE004), the

ethyl acetate and the n-hexane extract of S. aculeastrum root (eE006, hE006), the diethyl

ether extract of W. ugandensis stem bark (dietE014), the ethyl acetate extract of L. calostachys

leaves (eE005), the diethyl ether extract of Morella kandtiana root (dietE012), the diethyl

ether extract of P. hadiensis leaves (dietE016), and the ethanolic extract of Z. chalybeum stem

bark (etE009). These nine extracts were selected for the next stage of COX experiments

and subsequently introduced to the dose-response COX-2 and COX-1 inhibition studies.

Plants 2021, 10, 351 9 of 30

2.3. Dose-Response COX-2 Inhibition Experiments

The results of the dose-response COX-2 inhibition experiments, further investigating

the most promising nine extracts from seven species identified in the library screen, are

reported in Table 2.

Table 2. Results of COX-2 and COX-1 inhibition by medicinal plant samples from the Greater Mpigi region in Uganda;

extracts are sorted from highest to lowest COX-2 sensitivity; IC50 values are given in μg/mL (positive control: ng/mL); SEM

= standard error of the mean.

Extract ID Plant Species Type of Extract

IC50 ± SEM Ratio

COX-2

COX-1

COX-2 COX-1

eE005 Leucas calostachys ethyl acetate 0.66 ± 0.66 7.76 ± 1.58 0.1

eE006 Solanum aculeastrum ethyl acetate 1.74 ± 0.28 9.72 ± 0.28 0.2

hE006 Solanum aculeastrum n-hexane 3.19 ± 0.43 3.99 ± 3.92 0.8

hE004 Sesamum calycinum subsp.

angustifolium n-hexane 3.65 ± 0.56 8.57 ± 2.03 0.4

dietE016 Plectranthus hadiensis diethyl ether 4.55 ± 0.76 5.83 ± 3.79 0.8

eE004 Sesamum calycinum subsp.

angustifolium ethyl acetate 6.05 ± 0.20 11.47 ± 2.89 0.5

dietE014 Warburgia ugandensis diethyl ether 13.33 ± 4.36 11.05 ± 1.43 1.2

etE009 Zanthoxylum chalybeum ethnanol 16.07 ± 2.29 24.89 ± 4.16 0.7

dietE012 Morella kandtiana diethyl ether 17.24 ± 2.79 15.01 ± 1.14 1.2

positive control DuP-769 - (pure compound) 0.93 ± 0.20 >100.0 >0.001

Calculated IC50 values for these nine extracts ranged from 0.66 to 17.24 μg/mL. The

ethyl acetate extract of L. calostachys leaves (eE005) displayed the highest inhibitory activ-

ity against human recombinant COX-2 in the study (IC50: 0.66 μg/mL). The second most

active extract in inhibiting COX-2 was the ethyl acetate extract of S. aculeastrum root

(eE006), reaching an IC50 value of 1.74 μg/mL. Further, high COX-2 inhibition activity can

be reported for the n-hexane extract of S. aculeastrum root (hE006; IC50: 3.19 μg/mL) and

the n-hexane extract of S. calycinum subsp. angustifolium leaves (hE004; IC50: 3.65 μg/mL).

There was only one extract among the most active nine extracts that was produced using

a polar extraction solvent (ethanol, etE009, Z. chalybeum stem bark), meaning that most of

the extracts were apolar (extraction solvent: n-hexane) or somewhat apolar extracts (ex-

traction solvents: diethyl ether, ethyl acetate).

2.4. COX-1 Inhibition Analysis and Selectivity Ratio Determination

The nine most active plant extracts, selected in the initial COX-2 inhibition library

screen and followed up on via dose-response COX-2 inhibition studies, were further as-

sayed to assess their potential inhibition activity against human recombinant COX-1. The

calculation of the COX-2/COX-1 selectivity ratio for balance of inhibition can be used for

the assessment of side effects and efficacy [61,62]. Results are given in Table 2.

All nine extracts inhibited COX-1 enzyme activity and their IC50 values ranged from

3.99 to 24.89 μg/mL. Extract eE005, which was previously identified as the strongest COX-

2 inhibitor in the extract library, showed a COX-1 inhibition IC50 value of 7.76 μg/mL, lead-

ing to a calculated COX-2/COX-1 selectivity ratio of 0.1. The second most active COX-2

inhibitor, eE006, displayed moderate COX-1 inhibition activity (IC50: 7.76 μg/mL) and a

COX-2/COX-1 selectivity ratio of 0.2. The analysis of extract hE004, which was previously

highly active against COX-2, resulted in an IC50 value of 8.57 μg/mL and a selectivity ratio

of 0.4. The most active COX-1 inhibitors among the nine extracts were hE006 (n-hexane

extract of S. aculeastrum root; IC50: 3.99 μg/mL; selectivity ratio: 0.8) and dietE016 (diethyl

ether extract of P. hadiensis leaves; IC50: 5.83 μg/mL; selectivity ratio: 0.8). Two extracts

exhibited stronger COX-1 than COX-2 inhibitory effects. These were the diethyl ether ex-

tracts of W. ugandensis stem bark (dietE014; selectivity ratio: 1.2) and M. kandtiana root

(dietE012; selectivity ratio: 1.2).

Plants 2021, 10, 351 10 of 30

2.5. 15-LOX Inhibition Counterscreen

In an effort to estimate the 15-LOX counteractivity, the extract library, containing 58

plant extracts previously investigated for COX-2 inhibition activity, was screened at a con-

centration of 10 μg/mL. The results of extracts inhibiting 15-LOX enzyme activity are re-

ported in Figure 5.

Figure 5. Results of the 15-LOX inhibition extract library counterscreen at 10 μg/mL; positive control tested at 3.024 μg/mL

and 0.3024 μg/mL.

In total, only nine extracts from six plant species exhibited 15-LOX inhibition activity

at 10 μg/mL, whereas 49 extracts from 16 species did not display inhibitory activity (I =

0%). These nine extracts were wE002 (aqueous extract, M. lycopodioides roots/rhizomes),

hE003 and mE003 (n-hexane and methanolic extracts, F. saussureana stems), wE004 (aque-

ous extract, S. calycinum subsp. angustifolium leaves), wE006 (aqueous extract, S. aculeas-

trum roots), eE008 and etE008 (ethyl acetate and ethanolic extracts, E. abyssinica stem bark),

etE011 (ethanolic extract, H. madagascariensis stem bark), and etE013 (ethanolic extract, C.

buchananii stem bark). Interestingly, except for extracts hE003 and eE008, these active ex-

tracts were all polar extracts (extraction solvents: water, methanol, ethanol). Extracts with

the highest 15-LOX activity at 10 μg/mL were the aqueous root extract from S. aculeastrum

(I: 58.5%) and the n-hexane stem extract from F. saussureana (I: 51.9%).

2.6. DPPH Assay for Antioxidant Activity and TPC Determination

The plant extract library was further screened for free radical scavenging potential

(antioxidant activity) and the total phenolic content (TPC) was determined. Both assays

were conducted to rule out a potential mechanism of action for the COX-2/1 and 15-LOX

inhibition due increased presence of free radical scavenging compounds in highly active

plant extracts. Many phenolic compounds, such as tannins or flavonoids, are considered

to act via their free radical scavenging activities, facilitating the inhibition of proinflam-

matory enzymes, e.g., COX and LOX, during host immune response [63,64]. For example,

reactivity with the radical trap DPPH (1,1-diphenyl-2-picrylhydrazyl radical) in the pres-

ence of each plant extract was evaluated to elucidate the potential of lipid-derived radical

scavenging in the mechanism of the 15-LOX enzyme inhibition previously assessed. Re-

sults are summarized in Figure 6 and absolute values are reported in Supplementary Ta-

ble S2.

Plants 2021, 10, 351 11 of 30

Figure 6. Results of the in vitro investigation of antioxidant activity (free radical scavenging activity) and determination of

TPC for assessment of potential mechanism of action of the COX-2/1 and 15-LOX inhibition activity; plant extracts identi-

fied in the initial library COX-2 screen for COX-2/COX-1 dose-response inhibition experiments are marked with *; n.a. =

not available.

Plants 2021, 10, 351 12 of 30

The results show that there is poor correlation between the TPC and the correspond-

ing EC50 values (antioxidant activity), which is further addressed in the Discussion section.

Extracts containing the highest TPC in the extract library were etE013 (C. buchananii; 32.69

mg chlorogenic acid equivalent/g dry extract), eE009 and etE009 (Z. chalybeum; 32.29 mg

chlorogenic acid equivalent/g dry extract), and etE011a (H. madagascariensis; 32.09 mg

chlorogenic acid equivalent/g dry extract). Analysis of hE006 (S. aculeastrum; 0.61 mg

chlorogenic acid equivalent/g dry extract), hE003 (F. saussureana; 1.01 mg chlorogenic acid

equivalent/g dry extract), and eE010 (T. asiatica; 3.00 mg chlorogenic acid equivalent/g dry

extract) resulted in the lowest TPCs in the library.

Interestingly, the two extracts from C. molle (the only species inactive in the initial

COX-2 inhibition library screen) exhibited the lowest EC50 values for free radical scaveng-

ing activity in the library, resulting in 8.26 μg/mL (eE015) and 8.73 μg/mL (etE015). Other

extracts in a similar EC50 range were etE012a and etE012 (M. kandtiana; EC50: 8.97 μg/mL

and 9.03 μg/mL). The highest EC50 value for free radical scavenging potential in the extract

library was recorded for COX-2 inhibitor dietE016 (P. hadiensis; EC50: 181.00 μg/mL). A

total of 15 extracts did not reach an EC50 value in the tested concentration range, which

included the top three performing COX-2 inhibitors eE005 (L. calostachys), eE006 and

hE006 (S. aculeastrum), identified above. Except for etE009 (relatively high TPC), none of

the nine extracts that exhibited high COX-2 inhibition activity showed an increased anti-

oxidant activity or TPC compared to the other extracts in the library.

2.7. Antibacterial Resazurin Bioassay

The plant extract library was further screened for growth inhibition activity against

multidrug-resistant strains of S. aureus and E. coli K12, and L. innocua (no resistances re-

ported) to further evaluate their potential for treatment of wounds and infections of the

stomach/GI tract. Extracts that were active against the non-pathogenic L. innocua were

subsequently tested against multidrug-resistant pathogenic L. monocytogenes. Minimal in-

hibition concentration (MIC) values are reported in Table 3.

Table 3. Resazurin bioassay growth inhibition results of medicinal plants from the Greater Mpigi region; MIC values with

standard deviations are expressed as concentration (μg/mL). The maximum concentration at which extracts were tested

was 500 μg/mL. Dashes indicate that a sample was not tested.

Scientific Name Extract ID S. aureus

ATCC 25923

E. coli K12

ATCC 23716

L. innocua

ATCC 33090

L. monocytogenes

ATCC 15313

Securidaca

longipedunculata

eE001 104.17

± 29.46 >500 500.00 ± 0 -

wE001 >500 >500 >500 -

hE001 125.00

± 0 >500 250.00 ± 0 250.00 ± 0

mE001 83.33

± 29.46 >500 >500 -

smE001 104.17

± 29.46 >500 >500 -

Microgramma

lycopodioides

eE002 500.00

± 0 >500 >500 -

wE002 >500 >500 >500 -

hE002 - >500 >500 -

mE002 250.00

± 0 500.00 ± 0 500.00 ± 0 250.00 ± 0

smE002 26.04

± 7.37 >500 >500 -

Ficus saussureana

eE003 500

± 0 500.00 ± 0 >500 -

wE003 >500 >500 >500 -

hE003 500.00

± 0 500.00 ± 0 >500 -

mE003 20.83

± 7.37 500.00 ± 0 >500 -

smE003 500

± 0 500.00 ± 0 >500 -

Sesamum calycinum

subsp. angustifolium

eE004 125.00

± 0 500.00 ± 0 >500 -

wE004 500 ± 0 >500 >500 -

hE004 125.00

± 0 500.00 ± 0 >500 -

Plants 2021, 10, 351 13 of 30

mE004 250.00

± 0 500.00 ± 0 >500 -

smE004 250.00

± 0 >500 >500 -

eE004-18 250.00

± 0 - - -

hE004-18 31.25

± 0 - - -

Leucas calostachys

eE005 500.00

± 0 >500 >500 -

wE005 - >500 >500 -

hE005 62.50

± 0 500.00 ± 0 >500 -

smE005 500

± 0 >500 >500 -

eE005-18 500.00

± 0 - - -

hE005-18 104.17

± 29.46 - - -

mE005-18 500

± 0 - - -

smE005-18 104.17

± 29.46 - - -

Solanum aculeastrum

eE006 500.00

± 0 500.00 ± 0 >500 -

wE006 500.00 ± 0 >500 >500 -

hE006 125.00

± 0 >500 >500 -

smE006 11.72

± 5.52 >500 >500 -

Albizia coriaria eE007 250.00

± 0 250.00 ± 0 >500 -

etE007 500.00 ± 0 500.00 ± 0 >500 -

Erythrina abyssinica eE008 83.33

± 29.46 500.00 ± 0 >500 -

etE008 62.50 ± 0 >500 >500 -

Zanthoxylum chalybeum

eE009 31.25

± 0 - >500 -

etE009 500.00 ± 0 >500 >500 -

etE017 500.00

± 0 >500 >500 -

dietE017 250.00

± 0 >500 >500 -

etE017a >500 250.00

± 0 - -

dietE017a 13.02

± 3.62 >500 - -

Toddalia asiatica

eE010 31.25

± 0 >500 >500 -

etE010 31.25 ± 0 >500 >500 -

dietE010 20.83

± 7.37 >500 >500 -

etE010a 83.33

± 29.46 >500 >500 -

Harungana

madagascariensis

eE011 125.00

± 0 250.00 ± 0 500.00 ± 0 >500

etE011 57.29 ± 7.37 500.00 ± 0 500.00 ± 0 >500

dietE011 13.02

± 3.68 500.00 ± 0 41.67 ± 14.73 125 ± 0

etE011a 125.00

± 0 250.00 ± 0 500.00 ± 0 >500

eE011-18 52.08

± 14.73 - - -

hE011-18 31.25

± 0 - - -

etE011-18 31.25

± 0 - - -

dietE011-18 52.08

± 14.73 - - -

Morella kandtiana

eE012 250.00

± 0 - - -

etE012 500.00 ± 0 500.00 ± 0 >500 -

etE012a 500.00

± 0 250.00 ± 0 >500 -

dietE012 250.00

± 0 500.00 ± 0 >500 -

eE012-18 500.00

± 0 - - -

dietE012-18 500.00

± 0 - - -

Cassine buchananii

eE013 500.00

± 0 500.00 ± 0 >500 -

etE013 500.00 ± 0 500.00 ± 0 >500 -

etE013a 500.00

± 0 500.00 ± 0 >500 -

Warburgia ugandensis

etE014a 500.00

± 0 500.00 ± 0 >500 -

dietE014 31.25 ± 0 500.00 ± 0 500.00 ± 0 125 ± 0

eE014-18 31.25

± 0 - - -

wE014-18 >500 - - -

Plants 2021, 10, 351 14 of 30

dietE014-18 31.25

± 0 - - -

hE014-18 31.25

± 0 - - -

etE014-18 41.67

± 14.73 - - -

Combretum molle

eE015 500.00

± 0 250.00 ± 0 >500 -

etE015 500.00 ± 0 500.00 ± 0 >500 -

Plectranthus hadiensis dietE016 104.17

± 29.46 >500 >500 -

hE016 62.50 ± 0 >500 >500 -

ciprofloxacin - 0.19

± 0.06 7.81 ± 0 0.12 ± 0.00 0.12 ± 0.00

Compared to the other bacteria tested, the extracts were significantly more active

against the Gram-positive S. aureus (ATCC 25923). Here, the majority of the plant extracts

showed at least a minor inhibitory impact on S. aureus growth at the highest concentration

tested (500 μg/mL). Thirty-one of 75 plant extracts (41.3%) displayed MIC values below

125 μg/mL. The highest growth inhibitory activity (<50 μg/mL) was recorded for 11 ex-

tracts from seven plant species: (1) the methanolic Soxhlet extract of S. aculeastrum roots

(smE006, MIC: 11.72 μg/mL); (2) the diethyl ether, n-hexane, and ethanolic extracts of H.

madagascariensis stem bark (dietE011, MIC: 13.02 μg/mL; hE011-18 and etE011-18, MIC:

31.25 μg/mL); (3) the methanolic extract of F. saussureana stems (mE003, MIC: 20.83

μg/mL); (4) the methanolic Soxhlet extract of M. lycopodioides roots/rhizomes (smE002,

MIC: 26.04 μg/mL); (5) the n-hexane extract of S. calycinum subsp. angustifolium leaves

(hE004-18, MIC: 31.25 μg/mL); (6) ethyl acetate and ethanolic extracts of T. asiatica leaves

and stem bark (eE010 and etE010, MIC: 31.25 μg/mL); and (7) the diethyl ether and etha-

nolic extracts of W. ugandensis stem bark (dietE014 and eE014-18: 31.25 μg/mL).

The experiments screening the extract library against multidrug-resistant E. coli K12

(ATCC 23716) generally resulted in low growth inhibitory activity. None of the extracts

reached a MIC below 250 μg/mL. The antibiotic screen against L. innocua (ATCC 33090)

resulted in low growth inhibition activities of tested extracts. The exception was extract

dietE011 (MIC: 41.67 μg/mL), which is the diethyl ether extract of H. madagascariensis stem

bark and which was also the second most active extract in the S. aureus inhibition bioas-

says reported above. Seven extracts from four plant species were further investigated for

growth inhibition activity against L. monocytogenes. The results indicate that extract di-

etE011 is less effective against this pathogenic strain of Listeria than against L. innocua

(MIC: 125 μg/mL). Extract dietE14 (W. ugandensis stem bark extract; also one of the high-

performing S. aureus growth inhibitors) showed similar activity on L. monocytogenes,

reaching a MIC of 125 μg/mL.

3. Discussion and Conclusion

The results of this study provide scientific evidence for the therapeutic use of medic-

inal plants from the Ugandan Greater Mpigi region in treatment of inflammatory disor-

ders and infections. Antiinflammatory (COX-2 inhibition) and antibacterial (growth inhi-

bition of S. aureus) effects were recorded for most plant species, successfully validating

traditional use in 15 out of 16 medicinal plant species investigated in the in vitro studies.

The only species exhibiting no COX-2 inhibition activity in the experiments was C. molle.

All 16 species displayed at least low inhibitory effects on S. aureus growth. The determi-

nation of the TPC and the assessment of the DPPH radical scavenging activity of the

strongest COX-2 inhibitors led to the assumption that a high concentration of phenols and

free radical scavenging seem not to play the crucial role in the mechanism of action of the

most active plant extracts.

Extracts of the same species distinguished themselves in terms of method of extrac-

tion and polarity of extraction solvent used (“pre-fractionation strategy”). The most active

COX-2 inhibitors in the extract library were extracts from L. calostachys (eE005), S. aculeas-

trum (eE006, hE006), S. calycinum subsp. angustifolium (eE004, hE004), P. hadiensis (di-

etE016), M. kandtiana (dietE012), Z. chalybeum (etE009), and W. ugandensis (dietE014). There

Plants 2021, 10, 351 15 of 30

was no counteractivity between COX-2 and 15-LOX inhibition in these nine extracts from

seven plant species. Except for the ethanolic extract of Z. chalybeum stem bark, all of these

highly active extracts were produced using an apolar or somewhat apolar extraction sol-

vent, namely n-hexane, ethyl acetate, or diethyl ether. In general, aqueous extracts, whose

lab preparation simulated the traditional methods of preparation in the Greater Mpigi

region [5], often failed to exhibit bioactive effects in our antiinflammatory and antibacte-

rial in vitro models. The present result is similar to our previous findings that investigated

the extract library with other pharmacological test methods [65]. This phenomenon might

be explained by the fact that the lab-produced extracts are filtered prior to solvent evapo-

ration as part of extract standardization procedures. This led to the removal of tiny solids

present in the traditional preparations, which are normally swallowed by patients along

with the infused water. This way, apolar pharmacologically active secondary plant me-

tabolites may remain in the traditional herbal remedy, but only occur in the apolar extracts

in our plant extract library.

The ethyl acetate extract of L. calostachys leaves (eE005) showed the lowest IC50 value

for COX-2 inhibition (0.66 μg/mL). With 0.1, this extract also displayed the most promis-

ing selectivity ratio (COX-2/COX-1). The IC50 value for COX-1 inhibition was 7.76 μg/mL.

As a comparison, the IC50 values of Aspirin and ibuprofen in the literature are 210 μg/mL

and 46 μg/mL (COX-2), and 5 μg/mL and 1 μg/mL (COX-1) [39,40], respectively. This

leads to a poor selectivity ratio of 42 (Aspirin) and 46 (ibuprofen), which is characteristic

for most commercial NSAIDs [7,36,39–41]. Thus, extract eE005 seems to be a much more

potent COX-2 inhibitor than Aspirin or ibuprofen, while also displaying much higher se-

lectivity for COX-2 in contrast to COX-1, thereby potentially generating fewer side effects

due to decreased COX-1 and increased COX-2 inhibition. The in vitro performance of

eE005 can be further highlighted by the fact that it is a crude extract containing a complex

mixture of hundreds or thousands of compounds, whereas Aspirin and ibuprofen are

pure substances. The TPC content of extract eE005 was not significantly higher than those

of the inactive extracts in the library and no EC50 value was reached in the DPPH assay at

the highest test concentration, indicating low antioxidant activity and eliminating free

radical scavenging as a potential mechanism of action. Moreover, extract eE005 only ex-

hibited low growth inhibition activity against S. aureus (MIC: 500 μg/mL) and no antibac-

terial effects on L. innocua and E. coli (MICs: >500 μg/mL). L. calostachys is an aromatic herb

occurring in some parts of Uganda. It was recently identified by the DoP method as a

“highly understudied” species [6]. In fact, not much research has been done on this species

so far. Three studies reported moderate to low antiplasmodial activity of L. calostachys

crude extracts [66–68]. The n-hexane extract of the leaves (hE005) displayed significant

quorum sensing inhibition activity against the accessory gene regulator (agr) system in S.

aureus [65]. Thus, our study provides the first report of strong in vitro antiinflammatory

activity of L. calostachys. Other publications in the literature describe the traditional uses

of L. calostachys in Kenya, which include use for the treatment of ulcers [69–71], colic pain

in infants, cancer, skin diseases, headache, arthritis, heart diseases [69], malaria [72,73],

gastrointestinal disorders [69,71,74–76], flu [76,77], and stomach ache [70,76]. Our data

provides further evidence for some of these traditional therapeutic uses. According to the

authors’ knowledge, there have been no articles published so far reporting isolation and

identification of bioactive natural products from L. calostachys.

Other strong COX-2 inhibitors identified were the ethyl acetate extract of S. aculeas-

trum root (eE006; COX-2 IC50: 1.74 μg/mL; COX-2 IC50: 9.72 μg/mL; selectivity ratio: 0.2),

the n-hexane extract of S. aculeastrum root (hE006; COX-2 IC50: 3.19 μg/mL; COX-2 IC50:

3.99 μg/mL; selectivity ratio: 0.8), and the n-hexane extract of S. calycinum subsp. angusti-

folium leaves (hE004; COX-2 IC50: 3.65 μg/mL; COX-2 IC50: 8.57 μg/mL; selectivity ratio: 0.4).

S. aculeastrum is a small tree or large shrub with branchlets covered in dense woolly

hairs and sharp, curved thorns [6,78]. In our assessment of antioxidant activity, the ex-

tracts eE006 and hE006 displayed significantly lower TPCs than other extracts of the same

extraction solvent in the extract library (5.06 and 0.61 mg chlorogenic acid equivalent/g

Plants 2021, 10, 351 16 of 30

extract), as well as no EC50 value reached in the DPPH assay. We therefore hypothesize

that the mechanism of action for the COX-2 inhibition is not due to free radical scavenging

and high phenol content, as often proposed for antiinflammatory medicinal plants [79–

82]. Extract hE006 exhibited moderate antibacterial activity against multidrug-resistant S.

aureus (MIC: 125 μg/mL) and no inhibitory activity against L. innocua (MIC: >500 μg/mL),

whereas extract eE006 showed low antibacterial activity against S. aureus and L. innocua

(MIC: 500 μg/mL). Interestingly, a previous study by Schultz et al. [65] identified extract

eE006 as one of the two most active extracts in the library for quorum sensing inhibition

(agr system in S. aureus), exhibiting reporter gene subtype-dependent IC50 values of 4, 1,

16, and 64 μg/mL (agr I-IV). This antivirulence activity was successfully confirmed via a

direct protein output assessment (Έ-toxin). S. aculeastrum is one of the Ugandan species

that were recently classified via the DoP method as being “understudied” [6]. Published

studies focus on documentation of traditional use, and pharmacological and phytochem-

ical investigation of the berries and the leaves [6], not the roots that were investigated in

this study. For instance, these include reports of low antioxidant and antimicrobial activ-

ity of the berries and leaves [83–85]; antiproliferative activity against human HeLa, MCF7,

and HT29 tumor cell lines of methanolic berry extracts [86]; and toxicity studies of berry

extracts in Wistar rats [87,88], mainly published by the Afolayan research group at Fort

Hare University, South Africa. The new steroidal alkaloids solamargine, Ά-solamargine,

solasodine and tomatidine were isolated from S. aculeastrum root bark and berries [89–92],

and solamargine induced P-glycoprotein inhibition and non-selective cytotoxicity [93].

Apart from traditional uses reported from the Greater Mpigi region [5], few other publi-

cations also mentioned traditional uses of S. aculeastrum, e.g., use of the roots, berries,

leaves, and bark to treat cancer in Kenya and South Africa [94,95]; use of the roots to treat

stomach ache in South Africa [96]; use of the berry juice to treat ditlapedi (a facial skin

condition) in South Africa [97]; and use of the berries and leaves to treat lymphatic filari-

asis in South Africa [98].

S. calycinum subsp. angustifolium leaves displayed high COX-2 inhibition activity in

this study. This species is an erect, annual to perennial herb with spotted pink or purple

flowers, reaching a height of 0.4–2.0 m. It can often be seen in Uganda along the roadside

[6,99]. Yet it has also been classified as a “highly understudied” species with regard to

ethnopharmacological research [6]. This is because only four publications mention the tra-

ditional use of the herb (excluding the study from the Greater Mpigi region [5]) [6]. To

briefly summarize, S. calycinum subsp. angustifolium is used in the treatment of burns,

wounds, eye infections, and diarrhea, and as a contraceptive and emetic in Tanzania [100].

In Uganda, it was reported to be used to treat hernias [101], to induce vomiting [102], and

to treat hypertension in combination with other herbs [103]. There have been no pharma-

cological investigations published on this species so far, except for the antibiotic, cytotox-

icity, and antivirulence study investigating the same extract library [65]. Here, extract

hE004, just as extract eE006, was among the two most active agr system quorum sensing

inhibitors (IC50 values: 2, 2, 16, and 32 μg/mL (agr I-IV)). The antiinflammatory activity of

the n-hexane extract of S. calycinum subsp. angustifolium leaves (hE004) described in this

study is the first report in the literature to date, as well as the first scientific evidence for

its therapeutic use in the Greater Mpigi region of Uganda.

Phenolic compounds are often thought to possess antiinflammatory properties. The

mechanisms of action of many phenolic compounds are most likely associated with their

inhibition of proinflammatory enzymes in the arachidonic acid pathway (e.g., COX-2 and

5-LOX) or with their free radical scavenging activity [79–82]. Except for extract etE009

(relatively high TPC), none of the nine extracts that exhibited high COX-2 inhibition ac-

tivity showed an increased antioxidant activity or calculated TPC compared to the other

extracts in the library. Interestingly, the lowest EC50 value for free radical scavenging/an-

tioxidant activity was recorded for the two extracts of C. molle (eE015, EC50: 8.26 μg/mL;

etE015, EC50: 8.73 μg/mL), the only species that did not display any inhibitory activity on

COX-2 in the initial library screen. These findings suggest that free radical scavenging and

Plants 2021, 10, 351 17 of 30

a high concentration of phenols in general seem not to be involved in the mechanism of

action of the most active COX-2 inhibiting plant extracts. In theory, there is a direct rela-

tionship between the TPC and the free radical scavenging activity because phenols signif-

icantly contribute to the antiradical activity [104]. The poor correlation between high TPC

and low EC50 values reported for some samples may be attributed to the different quality

of phenols present in the samples, resulting in varying antioxidant activities. Constituents

other than phenols, such as carbohydrates, fatty acids, phospholipids, etc., may also play

a role in the antioxidant activity of the sample. Correlations between the phytochemical

composition of the plant species and their bioactive properties should be further investi-

gated by more advanced methods of analytical chemistry, such as LC-MS-MS profiling,

enabling identification of specific sets of molecules present in the extract.

In the antibiotic resazurin bioassay, the diethyl ether extract of H. madagascariensis

stem bark (dietE11) displayed high growth inhibition activity against S. aureus (MIC

value: 13 μg/mL), L. innocua (MIC value: 42 μg/mL), and L. monocytogenes (MIC value: 125

μg/mL). H. madagascariensis, the “orange-milk tree,” is an evergreen shrub or tree whose

sap is orange and turns blood-red upon exposure [105–107]. It is not considered an under-

studied species because it has been extensively studied in the past [6]. Traditional use has

been reported in many regions of the African continent [73,108–112].

Another plant extract exhibiting strong growth inhibitory effects against S. aureus

was the diethyl ether extract of Z. chalybeum stem bark (dietE017a; MIC: 13 μg/mL). This

species is a spiny deciduous tree or shrub, reaching heights of about 8 m. It occurs in dry

woodland, bushland, or grassland in medium to low altitudes throughout Uganda (up to

1500 m.a.s.l.) [105–107]. According to the DoP analysis [6], Z. chalybeum is regarded a

“moderately studied species.” None of the traditional healers were cited to use this me-

dicinal plant as a remedy for skin infections. However, 18% of the survey participants

stated that it is used for disinfection of wounds, as well as treatment of sore throat (8%)

and disorders of the stomach/GI tract (13%) (Figure 1 and [5,65]). The present data, there-

fore, supports the traditional use of Z. chalybeum stem bark in the study area.

A polar extract of S. aculeastrum root (smE006; methanolic Soxhlet extraction) dis-

played the highest antibacterial activity against S. aureus with a MIC of 12 μg/mL (species

discussed above). The results of the antibiotic resazurin assay against multidrug-resistant

S. aureus strongly support the traditional medicinal use of S. aculeastrum, H. madagascari-

ensis, and Z. chalybeum in treatment of wounds and infections.

Another bioactive medicinal plant that inhibited the growth of S. aureus, L. innocua,

and L. monocytogenes is S. longipedunculata. This species is mentioned in the literature in

connection with treatment of measles [113]. In Uganda, measles is one of the major dis-

eases responsible for fatalities in children. Pneumonia is the most common severe compli-

cation, leading to the most measles-associated deaths. It can be caused by the measles

virus alone, secondary viral infection, or also secondary bacterial infections. Here, S. au-

reus is the most abundant organism of secondary bacterial infections [114]. In parts of

Uganda, the roots of S. longipedunculata are prescribed by traditional healers to control

secondary bacterial and measles infections [115].

The same plant extract library was previously screened in another study against a

panel of multidrug-resistant ESKAPE pathogens, in which we used a different method to

test antibacterial growth inhibition [65]. The bacterial species and strains tested also var-

ied, however, the S. aureus (UAMS-1 strain) can be compared to the S. aureus strain (ATCC

25923) used in this study. Generally, some MIC values differed. However, many values

were confirmed by the resazurin bioassay in our study, e.g.:

•extract hE005-18 (L. calostachys, MICUAMS-1: >256 μg/mL, MIC25923: 500 μg/mL);

•extract hE006 (S. aculeastrum, MICUAMS-1: 128 μg/mL, MIC25923: 125 μg/mL);

•extract etE008 (E. abyssinica, MICUAMS-1: 64 μg/mL, MIC25923: 63 μg/mL);

•extract etE011-18 (H. madagascariensis, MICUAMS-1: 32 μg/mL, MIC25923: 31 μg/mL);

•extract hE011-18 (H. madagascariensis, MICUAMS-1: 32 μg/mL, MIC25923: 31 μg/mL); and

•extract etE013 (C. buchananii, MICUAMS-1: >256 μg/mL, MIC25923: 500 μg/mL).

Plants 2021, 10, 351 18 of 30

For some extracts, approximately one additional two-fold dilution could be reported

as MIC value against S. aureus:

•extract dietE011 (H. madagascariensis, MICUAMS-1: 32 μg/mL, MIC25923: 13 μg/mL);

•extract dietE017a (Z. chalybeum, MICUAMS-1: 32 μg/mL, MIC25923: 13 μg/mL);

•extract dietE014-18 (W. ugandensis, MICUAMS-1: 64 μg/mL, MIC25923: 31 μg/mL); and

•extract hE014-18 (W. ugandensis, MICUAMS-1: 64 μg/mL, MIC25923: 31 μg/mL).

Extract smE006, which was the strongest S. aureus growth inhibitor in this study, did

not display antibiotic effects against the UAMS-1 strain [65]. A similar result was obtained

for extract dietE10 (T. asiatica). This significant discrepancy can most likely be explained

by the different resistance profiles of the two strains investigated. The results of this study

also showed that the antibacterial potential of medicinal plants depends on the species,

which plant part is used, the time and location of harvest, and on the solvents used for

extraction.

The results of this study, reporting pharmacological effects of medicinal plants on

inflammatory enzyme cascades and growth of bacterial pathogens, could be the starting

point for subsequent studies to investigate potential leads for the development of potent

antiinflammatory drugs or antibiotics. Further work is required to characterize the ex-

tracts phytochemically in order to identify compounds responsible for the antiinflamma-

tory, antioxidant, and antibacterial properties of the plant species. This could be achieved,

for instance, via bioassay-guided fractionation experiments and investigation of the mech-

anisms of action. However, these effects might also be due synergistic relationships of

multiple active ingredients within the plant extracts. To assess the plant species’ potential

for drug discovery endeavors, future research should also include evaluation of toxicity

(e.g., cytotoxicity and genotoxicity) and in vivo studies. Regarding future in vivo studies

and for more accurate validation of traditional use, it will also be essential to continue

including the original preparation cited by the traditional healers in the experimental

setup, as well as their route of administration and dose. Extracts that displayed strong

antibacterial activity need to be further investigated regarding their ability to limit the

severity of disease, as well as their potential of increasing the efficacy of conventional an-

tibiotics (that pathogens may have acquired resistance to already). Future studies should

therefore also focus on the deactivation of other virulence pathways, such as secretion

systems and biofilm formation.

4. Materials and Methods

4.1. Ethnobotanical Data

Ethnobotanical information on the traditional use of the 16 plant species for treatment

of inflammatory disorders was obtained from a previously published survey among 39

traditional healers in the Greater Mpigi region in Uganda [5]. Traditional use reports from

this study served as the basis for antiinflammatory, antioxidant, and antibacterial experi-

ments.

4.2. Collection and Identification of Plant Material

Following standard collection procedures [116–118] and under guidance of the tra-

ditional healers, plant specimens were collected during fieldwork in 2015, 2016, and 2017.

For all collected samples of species, voucher specimens were prepared and deposited at

the Makerere University Herbarium in Kampala, Uganda. Additional select specimens

were deposited at the Emory University Herbarium (GEO) in Atlanta, GA, USA, which

are also digitally available on the SERNEC portal [119]. Voucher specimen numbers are

given in the Table 1. Plant identification and assignment of scientific names (cross-checked

with http://www.theplantlist.org) were conducted following the best practice in the field

of ethnopharmacology [120]. Assignments of plant family correspond to The Angiosperm

Phylogeny Group IV guidance [121].

Plants 2021, 10, 351 19 of 30

4.3. Extractions

Collected plant samples were dried in the shade, taken to the laboratory, and ground.

Extractions were performed as described in detail in the flow sheet of the supplementary

material Figure S2 of a previous publication [65]. Briefly, methods applied to extract plant

samples were either maceration, aqueous decoction, or Soxhlet extraction. Extraction pro-

cedures were conducted using different solvents, aiming to achieve selective extraction of

biomolecules of different polarities from the samples. Individual crude extract samples

were labeled according to their extraction solvent and the collection number (EXXX) as-

signed to a plant species during the field studies, ranging from E001 to E017. Regarding

the maceration procedure, the extraction solvents used were (a) methanol (mEXXX), (b)

ethanol (etEXXX), (c) ethyl acetate (eEXXX), and (d) diethyl ether (dietEXXX). According

to the ethnobotanical survey [5], traditional healers usually prepare their herbal drugs as

aqueous decoctions. In order to simulate this original method of preparation, plant sam-

ples were also boiled in (e) water at 95 °C for 30 min while being stirred (wEXXX). Soxhlet

extraction crude extracts were extracted using (f) n-hexane (hEXXX) or (g) methanol

(smEXXX; successive extraction of corresponding EXXX material). Some of the plant spe-

cies had to be collected again in 2018 due to the requirement to obtain higher amounts of

extract, e.g., for future bioassay-guided fractionation strategies. Their resulting extracts

were additionally labeled with “-18” in their sample ID.

4.4. Sample Preparation

Crude extracts were dissolved in DMSO (Carl Roth) at 10 mg/mL. Sonication and

temperature increase up to 55 °C were applied for some samples with moderate or low

solubility experienced at RT. Extract solutions were stored at ƺ20 °C until assaying.

4.5. COX-1/2 Inhibition Screening Assays

First, an initial COX-2 inhibition library screen was performed at 50 μg/mL. Extracts

exhibiting a COX-2 inhibition value above 80% were introduced to the COX-2 and COX-

1 dose-response studies.

Materials and chemicals for the COX inhibition screening assays were sourced from

Cayman Chemical, Ann Arbor, MI, USA (Cayman Item No. 701070-96 and 701080-96).

This assay for assessment of human recombinant cyclooxygenase inhibitory potential of

plant extracts was divided into two steps (described in the following paragraphs): (1) the

COX reaction step and (2) the ELISA step. The ELISA step was performed to quantify the

prostaglandin product generated in the COX reaction step.

COX reaction step: 5 mL reaction buffer (Cayman Item No. 460104) was mixed with

45 mL ultrapure water (COX buffer). 80 μL human recombinant COX-2 (Cayman Item No.

460121) or COX-1 (Cayman Item No. 460108) solution were diluted with 320 μL of COX

buffer and stored on ice, resulting in a quantity sufficient for 40 COX reactions (COX so-

lution). To obtain a heme solution necessary for the reaction, a commercial heme in DMSO

solution (Cayman Item No. 460102) was further diluted (40 μL with 960 μL of COX buffer;

stable at room temperature for 12 h). 50 μL of arachidonic acid in ethanol (Cayman Item

No. 460103) were mixed with 50 μL of 0.1 M potassium hydroxide, vortexed, and further

diluted with 400 μL of ultrapure water (final concentration of substrate solution: 2 mM;

to be used within 1 hour if kept on ice). 5 mL of 1 M hydrochloric acid were added to a

crystalline stannous chloride vial (Cayman Item No. 460107) and vortexed to obtain a sat-

urated solution, which was stable for 8 hours at RT. The selective COX-2 inhibitor com-

pound DuP-769 was used as a positive control. The COX reaction procedure is described

in Supplementary Table S4.

Briefly, the background tubes were used to generate the background values. These

two tubes contained an inactivated COX solution (10 μL; produced by placing a 500 μL

microfuge tube containing 20 μL COX solution in boiling water for 3 min), 160 μL of COX

buffer, and 10 μL of heme solution. The sample (or positive control) tubes and two COX

Plants 2021, 10, 351 20 of 30

100% initial activity tubes were prepared by adding 160 μL of COX buffer, 10 μL of heme

solution, and 10 μL COX solution. 10 μL of DMSO (sample vehicle) were added to each

background and COX 100% tube. 10 μL of sample or positive control solution were then

transferred to each sample tube. All tubes were incubated for 10 min at 37 °C. Initiation of

the COX reaction was completed by adding 10 μL of arachidonic acid substrate solution

and incubating for exactly 2.00 min at 37 °C (final substrate concentration in the reaction:

100 μM). The enzymatic reaction was stopped through the addition of 30 μL of saturated

stannous chloride solution and the tubes were further incubated for 5 min at RT. The tubes

were then tightly capped and stored at 4 °C for up to 3 days (produced PG F2΅ is stable for

one week). A more detailed description of the assay procedure is available [122].

ELISA step: PG F2΅ was then quantified via Cayman Chemical’s Prostaglandin

Screening AChE ELISA kit (Cayman Item No. 514012). The AChE competitive ELISA pro-

cedure was performed according to the manufacturer’s instructions [122,123]. Briefly, the

COX reaction tubes, containing prostanoids, were diluted in ELISA assay buffer (1:2000

and 1:400 dilutions were run in the ELISA). A prostaglandin standard screening solution

was freshly prepared and diluted (twofold), ranging from 2000.0–15.6 pg/mL. Each plate

setup contained a minimum of two blanks, two non-specific binding wells, two maximum

binding wells, one total activity well, an eight-point standard curve run in duplicate, and

the COX 100% activity and COX reaction samples at 1:2000 and 1:4000 dilution in dupli-

cate, respectively. Samples and controls were transferred to pre-coated mouse monoclonal

anti-rabbit IgG antibodies. After the addition of a PG-AChE tracer to each well (except for

the total activity and the blank wells) and a specific PG antiserum, the plate was incubated

for 18 hours at room temperature on a rotary microtiter plate shaker. After incubation, the

wells were emptied and rinsed five times with wash buffer in order to remove all unbound

reagents. The plate was developed by adding Ellmann’s reagent (AChE substrate) to all

wells, as well as addition of the tracer to the total activity wells, and shaking on a rotary

microtiter plate shaker in the dark for 75 min. The yellow color of the reaction product of

AChE was then measured spectrophotometrically at 412 nm. The intensity is proportional

to the amount of PG tracer bound to the well (determined from the PG standard curve

from the plate), thus inversely proportional to the quantity of free PG present in the well

(Figure 4).

The calculation of % COX inhibition values was performed using the Cayman Chem-

ical’s Workshop Sheets Excel template (Version 11 October, 2011). IC50 values were calcu-

lated using the GraphPad Prism 9.0.0 software and a log(inhibitor) vs. response—variable

slope (four parameters) model with duplicate and triplicate determinations. Error propa-

gation was performed as described by the software manufacturer [124] and in the litera-

ture [125].

4.6. 15-LOX Inhibition Assay

The extract library previously screened for COX-2 inhibition activity was subse-

quently counterscreened for 15-LOX inhibition activity at 10 μg/mL. The 15-LOX inhibi-

tion assay was performed using a lipoxygenase inhibitor screening assay kit, manufac-

tured by Cayman Chemical, Ann Arbor, MI, USA (Cayman Item No. 760700). This 96-well

microtiter plate-based method utilizes the 15-LOX catalyzed enzymatic reaction between

a polyunsaturated free fatty acids with a cis,cis-1,4-pentadiene-type structure and molec-

ular oxygen (Figure 7). In the assay, the hydroperoxides, namely 12(S)-hydroxyeicosatet-

raenoic acid (12(S)-HpETE) and 15(S)-hydroxyeicosatetraenoic acid (15(S)-HpETE), are

detected and measured.

Plants 2021, 10, 351 21 of 30

Figure 7. LOX-15 inhibition assay flow sheet depicting the experimental procedure.

Purified soybean 15-LOX (Cayman Item No. 760714) was used to facilitate lipoxy-

genation (final concentration in each well: 200 U/mL). Arachidonic acid was selected as

the substrate (final concentration in each well: 125 mM). Nordihydroguaiaretic acid

(NDGA), a non-selective LOX inhibitor, was used as a positive control (Cayman Item No.

760717). The assay was performed according to the manufacturer’s instructions [126] and

as previously described [127–130]. Briefly, individual plant extract solutions were incu-

bated with LOX assay buffer containing 15-LOX for 5 min at RT. Three blank control wells,

three 100% initial activity wells, three negative control wells, and NDGA positive control

wells were also included in each plate setup. After the addition of the substrate and initi-

ation of the reaction, the uncovered plate was placed on a shaker at 500 rpm for 20 min.

After incubation, chromogen was rapidly added to stop enzyme catalysis and the plates

were covered and placed on a shaker at 500 rpm for 5 min to develop the reaction. The

absorbance at 495 nm was then read using a plate reader.

All experiments were conducted in triplicate. During data analysis, the average ab-

sorbance of the blank, 100% initial activity, positive control, and sample wells were deter-

mined. After subtraction of the average blank absorbance from the average 100% initial

activity and sample wells, the % inhibition for each sample was calculated using the fol-

lowing equation:

% inhibition = ((100% initial activity ƺ sample)/ 100% initial activity) * 100

4.7. DPPH Assay

A quantitative DPPH assay for free radical scavenging activity (antioxidant poten-

tial) of plant extracts was conducted as previously described [131–133]. The 96-well mi-

crotiter plates were incubated in the dark for 30 min at RT. Absorbance was measured at

517 nm via UV-vis spectrophotometer. All plant extracts were assayed at 500 μg/mL in

their initial well and double-fold diluted down to 0.24 μg/mL. Quercetin was used as a

positive control and DMSO as the vehicle control. All experiments were performed in

triplicate. The % inhibition was calculated with the following equation:

% inhibition = ((absorbanceblank ƺ absorbancesample)/absorbanceblank) * 100

The EC50 values were calculated via linear regression using Microsoft Excel®.

4.8. TPC Determination

Determination of the total phenolic content (TPC) of plant extracts was performed in

96-well microtiter plates as previously described [134]. Briefly, Folin–Ciocalteu reagent

was used and a standard curve with chlorogenic acid (CHA) was prepared (serial dilution

ranging from 100 μg/mL (or 282.24 μM) to 0.049 μg/mL (or 0.1383 μM)). Spectrophoto-

metric measurement was conducted at 765 nm. Quercetin was used as a positive control

and DMSO as a vehicle control. All experiments were performed in triplicate. For data

analysis, the CHA standard curve was plotted and linear regression applied using the

software GraphPad Prism 9.0.0. After subtracting plant extract blank absorbances from

the sample absorbance, the results were interpolated in the standard curve to determine

the equivalent CHA concentration for each extract.

Plants 2021, 10, 351 22 of 30

4.9. Bacterial Strains

In order to realistically evaluate the extracts’ potential for future drug discovery ad-

vances for antimicrobial resistance threats, multidrug-resistant isolates of three bacterial

species, (a) E. coli K12 (ATCC 23716), (b) S. aureus (ATCC 25923), and (c) L. monocytogenes

(ATCC 15313), were selected for experiments. As a prescreen, a non-pathogenic L. innocua

strain (ATCC 33090) was used as a substitute (no resistances reported), and hits were fol-

lowed up with the human pathogen L. monocytogenes. Antibiotic resistance profiles, strain

numbers and characteristics, and sources are reported in Supplementary Table S3. All

strains were streaked from freezer stock and maintained on tryptic soy agar (TSA) via

overnight incubation at 37 °C. Overnight liquid cultures were prepared in Mueller-Hinton

(MHB, S. aureus, E. coli) or brain heart infusion (BHI) broth at 37 °C with constant shaking

at 200 rpm. For S. aureus, a bacterial growth curve was generated, allowing for determi-

nation of the growth phase and CFU/mL for standardization. After incubation of the over-

night culture, 1.0 mL of the bacteria culture was taken and pipetted into a sterile flask

containing 30 mL of broth. After 5–6 h of incubation, the bacteria were in the exponential

phase according to the growth curve. In order to get rid of the preculture broth medium,

20 mL of the bacterial culture were centrifuged at 4000 rpm for 5 min. The supernatant

was discarded, and the bacterial pellet was resuspended in 20 mL of sterile saline. After

vortexing, the suspension was centrifuged again under the same conditions as stated

above. Those steps were repeated three times until the supernatant was clear. The pellet

was once again resuspended in 20 mL sterile saline and the optical density (OD) was

measured at 600 nm. The OD of the culture and the growth curve were used to calculate

the dilution factor to achieve a final concentration of 5 × 106 CFU/mL for the resazurin

bioassay. As a control and to ensure correct bacterial concentration in the assay, the bac-

terial suspension was additionally spread onto TSA plates, incubated overnight at 37 °C,

and colonies were counted. The other three strains were standardized via cell counting in

a Thoma chamber, followed by calculation of the cell concentration, calculation of the cor-

rect dilution factor, and dilution.

4.10. Resazurin Bioassay

An in vitro 96-well microtiter plate-based antibacterial bioassay, incorporating resaz-

urin as a colorimetric indicator of cell growth, was used to assess the growth inhibitory

effects of plant extracts against tested bacterial pathogens. The method was previously

described by Sarker et al. [131,135]. The plates were labelled as shown in Supplementary

Material Figure S1. Sterility and vehicle/growth controls, as well as a positive control

(ciprofloxacin), were incorporated on each microtiter plate of the bioassay. A schematic

description of the bioassay procedure is given in Supplementary Material Figure S2.

Briefly, 50 μL of sterile saline was pipetted in all wells, except for the first row (20 μL of

sterile saline). Next, (a) in column 1 to 4 80 μL of extract solution, (b) in the two X columns

80 μL of sterile saline (sterility control), (c) in column Y 80 μL of DMSO (vehicle control),

and (d) in column Z 80 μL of ciprofloxacin (10 μg/mL) as a positive control were added.

A serial dilution from row 1 to row 12 was performed. After each dilution step, the pipette

tips were discarded. A total of 30 μL of 3.3 x strength MHB and 10 μL of resazurin solution

(0.05 % (w/v) resazurin sodium salt in sterile ultrapure water) were added to all wells.

Except for column X, all wells were inoculated with 10 μL of standardized bacterial sus-

pension, resulting in a final bacterial concentration of 5 × 105 CFU/mL in the wells. An

amount of 10 μL of sterile saline was added to the sterility control (column X). The plate

was sealed with a microtiter plate foil to prevent draining and the plate was shaken on an

orbital shaker at 500 rpm for 5 min. The plate was then incubated in the dark for 18 h at

37 °C. After incubation, the MIC was determined by visual assessment of the color change.

Any color change from blue to pink was recorded as negative, indicating bacterial growth.

Plants 2021, 10, 351 23 of 30

Blue was interpreted as inhibition of growth by the individual plant extract (Supplemen-

tary Material Figure S1). The lowest concentration at which color change occurred was

taken as the MIC value. All bacterial experiments were performed as biological triplicates.

Supplementary Materials: The following are available online at www.mdpi.com/2223-

7747/10/2/351/s1, Table S1: Results of the initial COX-2 extract library screen at 50 μg/mL, Table S2:

Detailed data of the TPC determination and the DPPH assay, Table S3: Information on bacterial

strains used in the study, Table S4: Procedure of the COX reaction step of the COX inhibition library

screening assay, Figure S1: Plate layout and setup during the resazurin bioassay, Figure S2: Sche-

matic description of the resazurin bioassay for growth inhibition, and references cited in the sup-

plementary files.

Author Contributions: F.S. and G.A. collected and processed the plant material. G.A. prepared

herbarium voucher specimens and taxonomically identified the plant species. F.S. and O.F.O. pre-

pared the extracts and built up the extract library. F.S. and B.W. performed the COX-2 and COX-1

inhibition experiments. F.S. and O.F.O. conducted the 15-LOX inhibition assays, the DPPH assays,

and the TPC determination experiments. F.S., O.F.O., and B.W. performed the antibacterial experi-

ments. F.S., O.F.O., and B.W. analyzed the data. F.S. and B.W. wrote the manuscript. L.-A.G. pro-

vided oversight of lab work and directed the study. All authors have read and agreed to the pub-

lished version of the manuscript.

Funding: This work was supported by two grants from the BMBF—German Federal Ministry of

Education and Research (13FH026IX5, 13FH066PX5; PI: L.A.G. and Co-I: F.S.). We acknowledge

support for the article processing charge from the German Research Foundation (DFG, 414051096)

and the Open Access Publication Fund of Neubrandenburg University of Applied Sciences

(HSNB). The content is solely the responsibility of the authors and does not necessarily reflect the

official view of the DFG, HSNB, or BMBF.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The data presented in this study are available in this article and in the

Supplementary Material. Ethnobotanical data are available in Schultz, F.; Anywar, G.; Wack, B.;

Quave, C. L.; Garbe, L.-A., Ethnobotanical study of selected medicinal plants traditionally used in

the rural Greater Mpigi region of Uganda. J. Ethnopharmacol. 2020, 256, 112742.

Acknowledgments: Greatest thanks to the Ugandan traditional healers in the Greater Mpigi re-

gion and neighboring regions who provided ethnobotanical information as a foundation for this

study, and who provided guidance during the collection of plant materials. Thanks to student

assistant Kristine Kossol, and research assistants Tidjani Cisse, and Tina Seehafer for assisting dur-

ing the extractions of plant material. Thanks to student assistant Kristine Kossol and to Inken

Dworak-Schultz for assistance in graphic design. Thanks to Logan Penniket for proofreading the

manuscript.

Conflicts of Interest: The authors declare no conflict of interest. The funding agencies had no role

in the study design, data collection and analysis, decision to publish, or preparation of the manu-

script.

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Antiinflammatory Medicinal Plants from the Ugandan

Greater Mpigi Region act as potent inhibitors in the

COX-2 / PGH2 pathway

Fabien Schultz1,2,*, Ogechi Favour Osuji2, Barbara Wack2, Godwin Anywar3, and Leif-Alexander

Garbe2,4

1 Institute of Biotechnology, Faculty III - Process Sciences, Technical University of Berlin,

Gustav-Meyer-Allee 25, Berlin, 13355, Germany

2 Department of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences, Brodaer Str.

2, Neubrandenburg, 17033, Germany

3 Department of Plant Sciences, Microbiology and Biotechnology, Makerere University, P.O Box 7062,

Kampala, Uganda

4 ZELT - Neubrandenburg Center for Nutrition and Food Technology gGmbH, Seestraße 7A,

Neubrandenburg, 17033, Germany

* Correspondence: Fabien[email protected]erlin.de; Tel.: +49-395-5693-2704

Supplementary information

Table of contents

Supplementary Table S1:

Results of the initial COX-2 extract library screen at 50 µg/mL page 3

Supplementary Table S2:

Detailed data of the TPC determination and the DPPH assay page 5

Supplementary Table S3:

Information on bacterial strains used in the study page 7

Supplementary Table S4:

Procedure of the COX reaction step of the COX inhibition library

screening assay page 8

Supplementary Figure S1:

Plate layout and setup during the resazurin bioassay page 8

Supplementary Figure S2:

Schematic description of the resazurin bioassay for growth inhibition page 9

References cited in supplementary files page 9

Supplementary Table S1:

Results of the initial COX-2 extract library screen at 50 µg/mL

scientific name extract ID COX-2 %I at 50 µg/mL

41-80

>80

Securidaca longipedunculata

eE001

smE001

wE001

mE001

hE001

Microgramma lycopodioides

hE002

mE002

wE002

smE002

eE002

Ficus saussureana

smE003

wE003

eE003

mE003

hE003

Sesamum calycinum subsp.

angustifolium

smE004

mE004

hE004

eE004

wE004

Leucas calostachys

eE005

smE005

wE005

mE005

hE005

Solanum aculeastrum

eE006

hE006

wE006

smE006

Albizia coriaria

etE007

eE007

Erythrina abyssinica

etE008

eE008

Zanthoxylum chalybeum

etE009

eE009

etE017

etE017a

dietE017

dietE017a

Toddalia asiatica

etE010

etE010a

eE010

dietE010

Harungana madagascariensis

etE011

etE011a

etE011

eE011

dietE011

hE011

Morella kandtiana

etE012

etE012a

eE012

wE012

dietE012

Cassine buchananii

etE013

etE013a

eE013

Warburgia ugandensis

dietE014

eE014

wE014

hE014

etE014a

etE014

Combretum molle

etE015

eE015

Plectranthus

hadiensis

hE016

dietE016

“nt” indicates that a sample was not tested.

Supplementary Table S2:

Detailed data of the TPC determination and the DPPH assay

Plant species and extract ID

TPC DPPH scavenging

activity

chlorogenic

acid

equivalent / g

extract

SEM EC50 (µg/ml) SEM

Securidaca longipedunculata (eE001) 26.00 0.25 160.50 30.94

Securidaca longipedunculata (hE001) 24.56 0.06 68.10 26.36

Securidaca longipedunculata (mE001) 24.95 0.50 81.14 12.67

Securidaca longipedunculata (smE001) 24.92 0.40 55.31 9.45

Microgramma lycopodioides (eE002) 26.57 2.84 n.a. / no correlation -

Microgramma lycopodioides (wE002) 5.02 0.53 n.a. / no correlation -

Microgramma lycopodioides (hE002) 23.86 0.32 n.a. / no correlation -

Microgramma lycopodioides (mE002) 23.95 0.63 91.27 23.09

Microgramma lycopodioides (smE002) 23.30 0.37 161.80 22.86

Ficus saussureana (eE003) 25.52 0.21 53.71 14.09

Ficus saussureana (wE003) 27.19 0.74 n.a. / no correlation

Ficus saussureana (hE003) 1.01 0.91 n.a. / no correlation

Ficus saussureana (mE003) 15.52 7.17 33.55 4.01

Ficus saussureana (smE003) 26.36 1.91 15.81 2.00

Sesamum calycinum subsp. angustifolium

(eE004) 26.64 0.29 96.65 18.53

Sesamum calycinum subsp. angustifolium

(wE004) 26.34 0.39 n.a. / no correlation

Sesamum calycinum subsp. angustifolium

(hE004) 8.89 0.70 121.00 38.33

Sesamum calycinum subsp. angustifolium

(mE004) 25.64 0.92 26.99 4.39

Sesamum calycinum subsp. angustifolium

(smE004) 26.64 0.59 25.27 3.25

Leucas calostachys (eE005) 20.49 0.44 n.a. / no correlation -

Leucas calostachys (wE005) 25.77 0.60 n.a. / no correlation -

Leucas calostachys (hE005) 9.51 0.44 n.a. / no correlation -

Leucas calostachys (smE005) 26.50 0.68 19.70 4.63

Solanum aculeastrum (eE006) 5.06 0.26 n.a. / no correlation -

Solanum aculeastrum (wE006) 24.77 0.60 n.a. / no correlation -

Solanum aculeastrum (hE006) 0.61 0.43 n.a. / no correlation

Solanum aculeastrum (smE006) 25.16 0.86 n.a. / no correlation -

Albizia coriaria (eE007) 28.37 0.34 18.39 2.23

Albizia coriaria (etE007) 28.36 0.97 22.98 2.47

Erythrina abyssinica (eE008) 28.37 0.34 45.57 7.21

Erythrina abyssinica (etE008) 28.36 0.97 68.72 9.00

Zanthoxylum chalybeum (eE009) 32.39 0.23 106.00 25.33

Zanthoxylum chalybeum (etE009) 32.39 0.23 52.02 9.11

Zanthoxylum chalybeum (etE017) 26.39 0.24 n.a. / no correlation -

Zanthoxylum chalybeum (etE017a) 28.19 0.13 44.60 7.12

Zanthoxylum chalybeum (dietE017) 23.81 0.82 91.61 31.57

Zanthoxylum chalybeum (dietE017a) 27.47 0.21 45.58 4.36

Toddalia asiatica (eE010) 3.00 0.29 n.a. / no correlation -

Toddalia asiatica (etE010) 25.34 0.50 139.40 31.40

Toddalia asiatica (etE010a) 25.49 0.53 165.40 28.24

Toddalia asiatica (dietE010) 26.89 0.77 60.10 21.78

Harungana madagascariensis (eE011) 25.54 0.77 20.14 3.00

Harungana madagascariensis (etE011) 23.97 0.15 27.64 4.05

Harungana madagascariensis (etE011a) 32.09 0.45 33.19 4.35

Harungana madagascariensis (dietE011) 27.25 0.36 47.87 7.87

Morella kandtiana (etE012) 29.86 0.15 9.03 0.75

Morella kandtiana (etE012a) 29.88 0.48 8.97 8.97

Morella kandtiana (dietE012) 26.16 0.38 28.05 5.70

Cassine buchananii (eE013) 26.44 0.11 26.91 4.91

Cassine buchananii (etE013) 32.69 0.81 50.52 3.71

Cassine buchananii (etE013a) 26.75 0.78 23.78 2.39

Warburgia ugandensis (etE014a) 27.12 0.90 10.33 0.61

Combretum molle (eE015) 28.92 0.72 8.26 0.58

Combretum molle (etE015) 30.40 0.60 8.73 1.10

Plectranthus hadiensis (dietE016) 17.74 0.14 181.00 24.22

Plectranthus hadiensis (hE016) 8.03 0.38 23.63 12.71

quercetin 306.80 9.77 0.41 0.03

DMSO 0.00 0.00 - -

Supplementary Table S3:

Information on bacterial strains used in the study

Species Strain IDs Characteristics* Ref.

Escherichia coli ATCC 23716

DSM # 498

Resistance: BAC, CLI, LIN, LZD,

NYT, OXA, PEN-G, Q-D, TEC,

VAN

K12 strain, mesophilic, rod-shaped,

coliform, Gram-negative bacterium

Source: DSMZ

Listeria innocua ATCC 33090

DSM # 20649

Resistance: no resistances reported

Isolate (bovine brain), mesophilic,

rod-shaped, Gram-positive

bacterium

Source: DSMZ

Listeria monocytogenes ATCC 15313

DSM # 20600

Resistance: CST, NYT, PA

Intermediate resistance: PMB

Isolate (rabbit), mesophilic, rod-

shaped, Gram-positive human

pathogen

Source: DSMZ

Staphylococcus aureus ATCC 25923

DSM # 1104

Resistance: ATM, CST, NYT

Intermediate resistance: PA

Human clinical isolate, mesophilic,

Gram-positive human pathogen

Source: DSMZ

*abbreviations:

ATM: aztreonam; BAC: bacitracin; CLI: clindamycin; CST: colistin; DSMZ: German

Collection of Microorganisms and Cell Cultures GmbH; LIN: lincomycin; LZD: linezolid;

NYT: nystatin; OXA: oxacillin; PA: pipemidic acid; PEN-G: penicillin G; PMB: polymyxin

B; Q-D: quinupristin-dalfopristin (Synercid); TEC: teicoplanin; VAN: vancomycin

Supplementary Table S4:

Procedure of the COX reaction step of the COX inhibition library screening assay

Background

tubes (2)

COX 100% initial

activity tubes (2)

Sample (positive

control) tubes

COX solution

(heat inactivated)

10 μL - -

COX solution

10 μL

COX buffer

160 μL

Heme solution

10 μL

Plant extract

10 mg/mL DMSO

(or positive control)

- - 10 μL

DMSO

(sample vehicle)

10 μL 10 μL -

Incubate for 10 minutes at 37 °C

Arachidonic acid solution

10 μL

Incubate for

exactly

2.00 minutes at 37 °C

Saturated stannous

chloride solution

30 μL 30 μL 30 μL

Supplementary Figure S1:

Figure S1. Plate layout and assay setup during the resazurin bioassay; violet wells indicate inhibition of cell

viability; pink wells indicate bacterial growth; X: sterility control; Y: growth control; Z: positive control; 1-4:

extract/sample solutions

Supplementary Figure S2:

Figure S2: Schematic description of the resazurin bioassay for growth inhibition

References cited in supplementary files

1 BacDive. E. coli K12 BacDive ID: 4414, <https://bacdive.dsmz.de/strain/4414> (2020).

2 BacDive. Listeria innocua 58 BacDive ID: 6871, <https://bacdive.dsmz.de/strain/6871>

(2020).

3 BacDive. Listeria monocytogenes 53 XXIII BacDive ID: 6875,

<https://bacdive.dsmz.de/strain/6875> (2020).

4 BacDive. Staphylococcus aureus Seattle 1945 BacDive ID: 14448,

<https://bacdive.dsmz.de/strain/14448 > (2020).

126

Manuscript V:

"Pharmacological assessment of the antiprotozoal activity, cytotoxicity and

genotoxicity of medicinal plants used in the treatment of malaria

in the Greater Mpigi Region in Uganda"

Pages: 127-168

Personal contribution

In the following, my personal contribution to the presented study and manuscript is briefly

described: I designed most of the overall strategy of the study. I contributed to the collection

and processing of the plant material in Uganda. I performed the extraction procedures and

created the extract library. I contributed to the preparation of the extract solutions for all assays.

I contributed to the work involved in the heme biocrystallization inhibition assay, the

genotoxicity experiments, and the data analysis. I wrote the manuscript. A more detailed

author-contribution statement is given in the submitted article.

Information on publication

This study was submitted to Frontiers in Pharmacology on March 9, 2021, and is presented as

a preprint version. If accepted, it will be an open access article distributed under the Creative

Commons Attribution 4.0 International License (CC BY 4.0).

Schultz, F.; Osuji, O. F.; Nguyen, A.; Anywar, G.; Scheel, J. R.; Caljon, G.; Pieters, L.; Garbe,

L.-A.: Pharmacological assessment of antiprotozoal activity, cytotoxicity and genotoxicity of

medicinal plants used in treatment of malaria in the Greater Mpigi Region in Uganda. Frontiers

in Pharmacology, manuscript submitted on March 9, 2021 (in review)

127

Pharmacological assessment of the antiprotozoal activity, cytotoxicity

and genotoxicity of medicinal plants used in the treatment of malaria in

the Greater Mpigi Region in Uganda

Fabien Schultz1,2*, Ogechi Favour Osuji2, Anh Nguyen2, Godwin Anywar3, John R. Scheel4,5,

Guy Caljon6, Luc Pieters7, Leif-Alexander Garbe2,8

1Institute of Biotechnology, Faculty III - Process Sciences, Technical University of Berlin, Berlin,

Germany

2Department of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences,

Neubrandenburg, Germany

3Department of Plant Sciences, Microbiology and Biotechnology, Makerere University, Kampala,

Uganda

4Department of Global Health, University of Washington, Seattle, WA, USA

5Department of Radiology, University of Washington, Seattle, WA, USA

6Laboratory of Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical, Biomedical and

Veterinary Sciences, University of Antwerp, Belgium

7Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical

Sciences, University of Antwerp, Belgium

8ZELT - Neubrandenburg Center for Nutrition and Food Technology gGmbH, Neubrandenburg,

Germany

* Correspondence:

Fabien Schultz

Fabien.Schultz@mailbox.tu-berlin.de

Keywords: malaria, antiprotozoal, cytotoxicity, genotoxicity, medicinal plants, Uganda, Mpigi,

ethnopharmacology.

128

This is a provisional file, not the final typeset article

Abstract

We investigated the potential antimalarial and toxicological effects of 16 medicinal plants frequently

used by traditional healers to treat malaria, fever, and related disorders in the Greater Mpigi region in

Uganda. Species studied were Albizia coriaria, Cassine buchananii, Combretum molle, Erythrina

abyssinica, Ficus saussureana, Harungana madagascariensis, Leucas calostachys, Microgramma

lycopodioides, Morella kandtiana, Plectranthus hadiensis, Securidaca longipedunculata, Sesamum

calycinum subsp. angustifolium, Solanum aculeastrum, Toddalia asiatica, Warburgia ugandensis, and

Zanthoxylum chalybeum. In addition, the traditional healers indicated that P. hadiensis is used as a

ritual plant to boost fertility and prepare young women and teenagers for motherhood in some Ugandan

communities where a high incidence of rapidly growing large breast masses in young female patients

was observed (not necessarily breast cancer). We present results from various in vitro experiments

performed with 56 different plant extracts, namely, (1) evaluation of traditional use by identifying

promising plant extract candidates using a heme biocrystallization inhibition library screen; (2) follow-

up investigation of antiprotozoal effects of the most bioactive crude extracts against chloroquine-

resistant P. falciparum K1; (3) cytotoxicity counterscreen against human MRC-5SV2 lung fibroblasts;

(4) genotoxicity evaluation of the extract library without and with metabolic bioactivation with human

S9 liver fraction; and (5) assessment of mutagenicity of the ritual plant P. hadiensis. A total of seven

extracts from five plant species were selected for antiplasmodial follow-up investigations based on

their hemozoin formation inhibition activity in the heme biocrystallization assay. Among other

extracts, an ethyl acetate extract of L. calostachys leaves exhibited antiplasmodial activity against P.

falciparum K1 (IC50 value: 5.7 μg/mL), which was further characterized with a selectivity index of 2.6

(CC50 value: 14.7 μg/mL). The experiments for assessment of potential procarcinogenic properties of

plant extracts via evaluation of in vitro mutagenicity and genotoxicity indicated that few extracts cause

mutations. The species T. asiatica showed the most significant genotoxic effects on both bacterial test

strains (without metabolic bioactivation at a concentration of 500 μg/plate). However, none of the

mutagenic extracts from the experiments without metabolic bioactivation retained their genotoxic

activity after metabolic bioactivation of the plant extract library through pre-incubation with human S9

liver fraction. While this study did not show that P. hadiensis has genotoxic properties, it did provide

early-stage support for the therapeutic use of the medicinal plants from the Greater Mpigi region.

129

1 Introduction

In recent years, the prevalence of malaria infection and the incidence of related clinical disease has

significantly decreased in sub-Saharan Africa (Bhatt et al., 2015; Snow et al., 2017). Nevertheless,

malaria continues to be one of the most severe public health problems worldwide (CDC, 2021). In

2019, there were an estimated 229 million cases in 89 malaria-endemic countries associated with

409,000 deaths. Africa alone accounted for 94% of these cases (215 million) with 384,000 deaths

(WHO, 2020). Human malaria is caused by five species of protozoan parasites of the genus

Plasmodium that are transmitted through the bites of infected female Anopheles mosquitoes (Scholar,

2007; Kotepui et al., 2020). In Africa, 94% of malaria cases result from infection with the species

Plasmodium falciparum, which poses the greatest malaria threat globally (WHO, 2020; CDC, 2021).

Children under five years of age remain the most vulnerable group affected with 274,000 malaria

deaths worldwide in 2019. In addition, in moderate to high transmission countries in the WHO African

Region, malaria infection during pregnancy resulted in 822,000 children with low birthweight (Park et

al., 2020; WHO, 2020).

In 2019, about 5% of the world’s malaria cases were recorded in Uganda alone (WHO, 2020).

At our field study sites in the tropical Ugandan Greater Mpigi region and around Nakawuka village in

Wakiso district, traditional medicine and the use of plants are still the predominant form of primary

healthcare. In a recent study, we documented for the first time the traditional use of 16 medicinal plant

species that are frequently used in local traditional medical practices (Schultz et al., 2020b). Figure 1

lists these species and their traditional use in treatment of malaria and fever, reporting the cumulated

relative frequencies of citation in % (nௗ=ௗ39), which serve as the ethnopharmacological basis for this

study.

Figure 1. Stacked histogram for ethnopharmacological information from the Greater Mpigi region in Uganda, describing the medicinal

use of 16 plant candidates (with emphasis on treatment of malaria and fever). The figure shows the relative frequencies of citation (RFCs)

in %, a field assessment index, which was calculated from data obtained through an ethnobotanical survey of 39 traditional healers

(Schultz et al., 2020b). Individual RFCs indicate the importance of each plant species used for treatment relative to the total number of

informants interviewed in the study (n=39). RFCs vary from 0% (none of the survey participants uses this plant species to treat a specific

medical condition) to 100% (all survey participants use this plant species to treat a specific medical condition). Consequently, the higher

the cumulative RFC values (x-axis), the more common the traditional use of a plant species in treatment of medical conditions caused

by malaria infection.

130

This is a provisional file, not the final typeset article

When applying the novel Degrees of Publication (DoP) method for identification of species

that merit the costly lab studies in ethnopharmacological research, the majority of these 16 species

from the Greater Mpigi region were classified as being either highly understudied or understudied

(Schultz et al., 2021a). Therefore, the present study further investigated the selected species for

antimalarial activity and toxicity, contributing to an evaluation of traditional use and to drug discovery.

In this study, extracts of the 16 medicinal plants were initially investigated using a heme

biocrystallization assay as a prescreen prior to antiplasmodial experiments with parasite cultures. The

assay is based on monitoring the formation of the distinctive molecule hemozoin, which is a byproduct

of the digestion of hemoglobin by the plasmodium parasite (Hempelmann, 2007). During infection and

in the intraerythrocytic stage, the parasite metabolizes host hemoglobin within its digestive vacuole to

facilitate parasite growth and development (Kapishnikov et al., 2019). This process involves the

oxidation of hemoglobin to methemoglobin and subsequent hydrolysis by aspartic proteases to form

denatured globin and free heme (Fe3+) (ferriprotoporphyrin IX). Free heme is toxic to the parasite and

large quantities accumulate, reaching high concentrations that cause membrane disruption, lipid

peroxidation, and protein and DNA oxidation. To prevent this, the free heme is detoxified by

biocrystallization to form inert, insoluble hemozoin in the parasitic digestive vacuole (Schmitt et al.,

1993; Becker et al., 2004; Kumar and Bandyopadhyay, 2005; Kumar et al., 2007; Coronado et al.,

2014; Roy, 2017; Herraiz et al., 2019). The resulting hemozoin (biocrystallized heme) is a dark pigment

that represents the characteristic black crystalline spot observed in red blood cells during diagnosis of

patients infected with malaria (Egan, 2002; Combrinck et al., 2013; Coronado et al., 2014; Gupta et

al., 2017). Thus, sequestration of heme into hemozoin is essential for the survival of P. falciparum, and

the vital pathway of hemozoin pigment formation is one of the main targets of antimalarial drugs

(Hempelmann, 2007; Herraiz et al., 2019; Kapishnikov et al., 2019). The mechanism of action of many

antimalarial drugs on the market, such as quinacrine, amodiaquine, or chloroquine, is based on

disruption of the heme detoxification pathway within the parasite, making these drugs potent heme

biocrystallization inhibitors (Coronado et al., 2014; Herraiz et al., 2019). Interestingly, the mechanisms

underlying hemozoin formation are yet to be fully elucidated and are poorly understood (Chugh et al.,

2013; Herraiz et al., 2019).

There are a few reports of plants that, following long-term ingestion, are suspected to promote

formation of breast tumors through mutagenesis and/or growth stimulation by phytoestrogens (Stopper

et al., 2005; Bilal et al., 2014). The incidence rates of breast cancer are rapidly increasing in sub-

Saharan Africa, including Uganda (Azubuike et al., 2018). Anecdotal reports from health providers at

one of our other study sites, around Nakawuka village in Wakiso District, indicated a relatively high

prevalence of rapidly enlarging breast masses in young women in these small rural communities (not

necessarily cancer). Further discussions with local traditional healers and some affected women

suggested that the medicinal plant Plectranthus hadiensis might be responsible. P. hadiensis

(synonym: P. cyaneus), a member of the Lamiaceae family, is used regularly by local teenagers and

young women as a ritual and medicinal plant to boost fertility and to "prepare women for marriage",

beginning with their first menstruation. The leaves of the plant are dried in the shade and subsequently

pounded in a traditional mortar into powder. The women then mix the powder with Vaseline and apply

it around the labia and administer intravaginally. Because of this frequent and long-term exposure to

the plant and the potential connection to the undiagnosed rapidly growing large breast masses in

women from the Nakawuka village area, we sought to be the first to preliminarily investigate potential

genotoxic effects derived from P. hadiensis. Due inefficiencies in the healthcare structure and limited

social-economic abilities, access to non-traditional medical facilities for breast cancer detection and

treatment is scarce and often unaffordable to the rural Ugandan population (Foerster et al., 2019;

Nakaganda et al., 2021). The development of the mammary glands takes place in lifecycle windows

that were previously identified as "hot-spots" for breast cancer risk (Martinson et al., 2013). Since the

breasts are not fully developed when the first menstruation occurs and puberty involves a massive

131

proliferation of cells, this window of susceptibility makes teenagers and young women particularly

vulnerable for DNA damage caused by mutagens/carcinogens (Davis and Lin, 2011; Macias and

Hinck, 2012; Natarajan et al., 2020). The administration of P. hadiensis and its genotoxic

phytoestrogens could potentially serve as the first incident of cell growth perturbation that, in the long

term, allows for further incidents to give rise to tumor growth and breast cancer. Upon oral

administration of a (herbal) drug, the human body will eventually try to eliminate xenobiotics. This

often requires initial biotransformation for many drugs, which takes place in part in the gut wall but

primarily in the liver (van de Waterbeemd and Gifford, 2003; Stavropoulou et al., 2018). This process

is further illustrated in Figure 2. After oral intake, bioavailability of a drug or pharmacologically active

secondary plant metabolites is dependent on two bioprocesses: absorption and metabolism (Rein et al.,

2013; Chow, 2014). The oral bioavailability of a (herbal) drug is determined by the drug fraction of

the initial dose that a) was successfully absorbed by the gut wall; b) appeared in the hepatic portal vein;

and c) ultimately entered the blood circulation after first pass through the liver metabolism (van de

Waterbeemd and Gifford, 2003). It is important to note that the most essential liver enzymes for

bioactivation of procarcinogens and biotransformation of (herbal) drugs are the cytochrome P450

enzymes, which oxidize substances using iron and are linked to a variety of reactions, including

epoxidation, hydroxylation, S-oxidation, and O-dealkylation (Werck-Reichhart and Feyereisen, 2000;

van de Waterbeemd and Gifford, 2003; Stavropoulou et al., 2018).

Figure 2. Simplified illustration of the locations of metabolic elimination of xenobiotics inside the human body

In this study, we investigate a unique library, composed of 56 extracts derived from the 16

Ugandan plant species, for their pharmacological in vitro activity. The objectives of this study were a)

an evaluation of traditional use by identifying promising plant extract candidates using a heme

132

This is a provisional file, not the final typeset article

biocrystallization inhibition library screen; b) follow-up investigation of antiprotozoal effects of the

most bioactive crude extracts against chloroquine-resistant P. falciparum K1; c) cytotoxicity

counterscreen against human MRC-5SV2 lung fibroblasts; d) genotoxicity evaluation of the extract

library; and e) assessment of mutagenicity of the ritual plant P. hadiensis that might be connected to

an increased prevalence of breast cancer in young female patients in Eastern Uganda.

133

2 Results

2.1 Plant species and extractions

For each plant species, multiple extraction strategies were applied in order to look not only into the

chemistry yielded by traditional preparation but also to investigate chemistries only accessible by

alternative extraction methods and extractants. Consequently, plant extracts were regarded as chemical

libraries in this study. The extraction methods were (a) maceration in either methanol, ethanol, ethyl

acetate, or diethyl ether; (b) Soxhlet extraction using n-hexane and successively methanol; and

total of 56 different plant extracts from 16 medicinal plant species. Taxonomic information on the

species studied, local plant names in the Luganda language, sample identification numbers (extract

IDs), plant parts selected for investigation, extraction solvents used, and herbarium voucher specimen

numbers and locations are reported in Table 1.

Table 1. Information on collected plant species and different extracts investigated in the study

Scientific name Extraction

solvent Extract ID Family Local name in

Luganda Plant part Voucher specimen #

and location

Securidaca

longipedunculata

Fresen.

ethyl acetate1 eE001

Polygalaceae Mukondwe stem

AG196

(Makerere University

Herbarium, Uganda)

water1 wE001

n-hexane (sox.)1 hE001

methanol1 mE001

methanol

(sox. succ.)1

smE001

Microgramma

lycopodioides

(L.) Copel.

ethyl acetate1 eE002

Polypodiaceae Kukumba root

(rhizomes)

AG639

(Makerere University

Herbarium, Uganda)

aqueous1 wE002

methanol1 (sox.

succ.)

smE002

Ficus saussureana

DC.

ethyl acetate1 eE003

Moraceae Muwo stem

AG219

(Makerere University

Herbarium, Uganda)

aqueous1 wE003

n-hexane (sox.)1 hE003

methanol 1 mE003

methanol (sox.

succ.)1

smE003

134

This is a provisional file, not the final typeset article

Sesamum calycinum

subsp.

angustifolium

(Oliv.) Ihlenf. &

Seidenst.

ethyl acetate1 eE004

Pedaliaceae Lutungotungo leaves

AG205

(Makerere University

Herbarium, Uganda)

23173*

(Emory University

Herbarium, USA)

water1 wE004

hexane (sox.)1 hE004

methanol1 mE004

methanol (sox.

succ.)1

smE004

Leucas calostachys

Oliv.

ethyl acetate1 eE005

Lamiaceae Kakuba musulo leaves

AG195

(Makerere University

Herbarium, Uganda)

23175*

(Emory University

Herbarium, USA)

water1 wE005

hexane (sox.)1 hE005

methanol (sox.

succ.)1

smE005

Solanum

aculeastrum Dunal

ethyl acetate1 eE006

Solanaceae Kitengo root

AG193

(Makerere University

Herbarium, Uganda)

water1 wE006

hexane (sox.)1 hE006

methanol (sox.

succ.)1

smE006

Albizia coriaria

Oliv.

ethyl acetate2 eE007

Fabaceae Mugavu stem bark

AG203

(Makerere University

Herbarium, Uganda)

ethanol2 etE007

Erythrina

abyssinica DC.

ethyl acetate2 eE008

Fabaceae Jjirikiti stem bark

AG199

(Makerere University

Herbarium, Uganda)

ethanol2 etE008

Zanthoxylum

chalybeum Engl.

ethyl acetate2 eE009

Rutaceae Ntaleyaddungu stem bark

AG204

(Makerere University

Herbarium, Uganda)

ethanol2 etE009

ethanol3 etE017

diethyl ether3 dietE017

135

ethanol4 etE017a

diethyl ether4 dietE017a

Toddalia asiatica

(L.) Lam.

ethyl acetate2 eE010

Rutaceae Kawule

leaves

(80%)

bark

(20%)

AG190

(Makerere University

Herbarium, Uganda)

ethanol2 etE010

diethyl ether4 dietE010

ethanol4 etE010a

Harungana

madagascariensis

Lam. ex Poir.

ethyl acetate2 eE011

Hypericaceae Mukabiiransiko stem bark

AG230

(Makerere University

Herbarium, Uganda)

23180*

(Emory University

Herbarium, USA)

ethanol2 etE011

diethyl ether4 dietE011

ethanol4 etE011a

Morella kandtiana

(Engl.) Verdc. &

Polhill

ethanol2 etE012

Myricaeae Mukikimbo root

AG201

(Makerere University

Herbarium, Uganda)

23174*

(Emory University

Herbarium, USA)

ethanol4 etE012a

diethyl ether4 dietE012

Cassine buchananii

Loes.

ethyl acetate2 eE013

Celastraceae Mbaluka stem bark

AG198

(Makerere University

Herbarium, Uganda)

ethanol2 etE013

ethanol4 etE013a

Warburgia

ugandensis Sprague

ethanol4 etE014a

Canellaceae Abasi stem bark

AG220

(Makerere University

Herbarium, Uganda)

23181*

(Emory University

Herbarium, USA)

diethyl ether4 dietE014

Combretum molle

R.Br. ex G.Don

ethyl acetate2 eE015

Combretaceae Ndagi stem bark

AG191

(Makerere University

Herbarium, Uganda)

ethanol2 etE015

136

This is a provisional file, not the final typeset article

Plectranthus

hadiensis (Forssk.)

Schweinf. ex

Sprenger

diethyl ether4 dietE016

Lamiaceae Kibwankulata leaves

AG210

(Makerere University

Herbarium, Uganda)

hexane4 hE016

*Specimens have been digitized and are available for viewing at http://sernecportal.org/portal/; 1collected in Apr. 2016;

2collected in Oct. 2015; 3collected in Sep. 2013; 4collected in Sep. 2016; sox. = Soxhlet extraction; sox. succ. = successive

Soxhlet extraction.

2.2 Heme biocrystallization assay library screen

A library screen for in vitro inhibition of hemozoin formation was conducted to identify promising

extract candidates for the antiplasmodial follow-up studies. Here, a modified heme biocrystallization

assay served as an alternative technique to circumvent initial testing of antimalarial activity in parasite

cultures. The assay does not replace experiments with plasmodia but may serve as a prescreen followed

by antiplasmodial experiments.

ȕ-hematin, which is structurally and chemically identical to hemozoin, was included in the

heme biocrystallization assay. The extract library was first screened at a concentration of 1000 μg/mL.

Extracts displaying a hemozoin formation percent inhibition above 20 were tested at the next lower

concentration (100 μg/mL). Extracts showing no hemozoin formation inhibition activity or inhibition

values less than 20% were eliminated from the antimalarial investigations. Next, extracts exhibiting

inhibition values above 1% were regarded as active and subsequently tested at 10 μg/mL. This

experimental step was repeated at a final concentration of 1 μg/mL. Results are shown in Table 2.

Table 2. Results of the heme biocrystallization library screen; +: observed inhibition activity; -: negative; nt indicates that

a sample was not tested.

Scientific name Extract

Inhibition of in vitro hemozoin formation

1000 μg/mL 100 μg/mL 10 μg/mL 1 μg/mL

20–50

>50

1–20

20–50

>50

1–20

20–50

>50

1–20

20–50

>50

Securidaca

longipedunculata

eE001 - -

wE001 - -

hE001 nt

mE001 - -

smE001 - -

Microgramma

lycopodioides

eE002 nt

wE002 - -

smE002 - -

Ficus

saussureana

eE003 - -

wE003 - -

hE003 - -

mE003 - -

smE003 - -

eE004 + - + - - + - - - - -

137

Sesamum

calycinum subsp.

angustifolium

wE004 - -

hE004 + - - + - - + - - + -

mE004 - -

smE004 + - - - -

Leucas

calostachys

eE005 + - + - - - + - + - -

wE005 - -

hE005 - + + - - - - -

smE005 - + - - -

Solanum

aculeastrum

eE006 - -

wE006 - -

hE006 - -

smE006 - -

Albizia coriaria eE007 - -

etE007 - -

Erythrina

abyssinica

eE008 - + - - -

etE008 + - - - -

Zanthoxylum

chalybeum

eE009 + - + - - + - - - - -

etE009 + - + - - + - - - - -

etE017 - -

dietE017 nt

etE017a - -

dietE017a - -

Toddalia asiatica

eE010 nt

etE010 - -

dietE010 - -

etE010a - -

Harungana

madagascariensis

eE011 - -

etE011 + - - - -

dietE011 - -

etE011a - -

Morella

kandtiana

etE012 - -

etE012a - -

dietE012 nt

Cassine

buchananii

eE013 nt

etE013 - -

etE013a - -

Warburgia

ugandensis

etE014a - -

dietE014 + - - - + - + - - - -

Combretum molle eE015 - -

etE015 - -

Plectranthus

hadiensis

dietE016 + - - - -

hE016 - + + - - - + - + - -

chloroquine

hos

hate

positive

control - + - - + - - + - - +

DMSO solvent

control - -

138

This is a provisional file, not the final typeset article

The seven extracts from five plant species that inhibited heme biocrystallization at 10 μg/mL

and below were selected for further pharmacological investigation and subsequently included in the

in vitro antiplasmodial and cytotoxicity dose-response experiments. These seven extracts were the n-

hexane and the ethyl acetate extracts of S. calycinum subsp. angustifolium leaves (hE004, eE004), the

ethyl acetate extract of L. calostachys leaves (hE005), the ethanolic and ethyl acetate extracts of

Z. chalybeum stem bark (etE009, eE009), the n-hexane extract of P. hadiensis leaves (hE016), and the

diethyl ether extract of W. ugandensis stem bark (dietE014).

2.3 In vitro antiplasmodial activity

Seven extracts from five species identified by hemozoin formation inhibition library screening were

further tested for antiplasmodial activity against the chloroquine-resistant P. falciparum K1 strain. The

results are shown in Table 3. Generally, results obtained from the heme biocrystallization assay were

supported by the antiplasmodial experiments, since all seven extracts exhibited high to moderate

antiplasmodial activity, reaching half maximal inhibitory concentrations (IC50 values) below 25 μg/mL.

Table 3. Results of the in vitro dose-response studies, investigating the antiplasmodial activity and cytotoxic effects of

selected medicinal plant samples from the Greater Mpigi region against chloroquine-resistant P. falciparum K1 and human

MRC-5SV2 lung fibroblasts. The half maximal inhibitory concentration (IC50) and the half maximal cytotoxic concentration

(CC50) are stated in μg/mL. SEM: standard error of the mean; SI: selectivity index.

Extract ID Plant species Type of extract

P. falciparum K1 MRC-5SV2 cells

IC50 ± SEM CC50 ± SEM

dietE014 Warbur

ia u

andensis dieth

l ethe

0.5 ± 0.1 0.3 ± 0.1 0.6

eE005 Leucas calostach

s eth

l acetate 5.7 ± 1.2 14.7 ± 2.9 2.6

eE009 Zanthoxylum

chal

beum ethyl acetate 11.9 ± 3.7 24.1 ± 1.3 2.0

etE009 Zanthoxylum

chal

beum ethanol 12.4 ± 2.4 26.9 ± 1.0 2.2

hE004 Sesamum calycinum

subsp. an

usti

olium n-hexane 19.6 ± 5.0 26.6 ± 3.7 1.4

eE004 Sesamum calycinum

subsp. an

usti

olium ethyl acetate 21.9 ± 6.8 27.0 ± 3.8 1.2

hE016 Plectranthus hadiensis n-hexane 23.1 ± 3.9 7.3 ± 1.1 0.3

chloroquine positive control - (pure compound) 0.04 ± 0.0 >20 >500

tamoxife

positive control - (pure compound) nt 3.85 ± 0.14 -

DMSO solvent control - (pure compound) > 64 > 64 -

With an IC50 value of 0.5 μg/mL, the extract dietE014 (diethyl ether extract of W. ugandensis stem

bark) showed the highest antiplasmodial activity against the tested strain of malaria parasite. The ethyl

acetate extract of L. calostachys leaves (eE005) also exhibited strong antiplasmodial effects with a

calculated IC50 value of 5.7 μg/mL. The IC50 values of extracts eE009, etE009 hE004, eE004, and

hE016 ranged from 11.9 to 23.1 μg/mL, whereas the n-hexane extract of P. hadiensis leaves displayed

the lowest antimalarial activity among the plant extract candidates tested.

139

2.4 Cytotoxicity counterscreen and selectivity index

In an effort to assess the cytotoxicity of the five antimalarial plant species, the seven pharmacologically

active extracts previously introduced to the antiplasmodial assay were further studied in a human lung

fibroblast toxicity assay using the MRC-5SV2 cell line. The results of this counterscreen are shown in

Table 3 along with the calculated selectivity indices (SIs) for antiplasmodial activity. The SI enables

for characterization and optimization of efficacy and safety of drug candidates and is regularly used as

a vital parameter in drug discovery for assessing whether a safety-efficacy profile is appropriately

balanced for a given indication (Muller and Milton, 2012; Babii et al., 2018; Zhang et al., 2019; Touret

et al., 2020).

The majority of extracts displayed moderate cytotoxic effects and half maximal cytotoxic

concentrations (CC50 values) above 10 μg/mL, ranging from 27.0 μg/mL (eE004) to 14.7 μg/mL

(eE005). However, the diethyl ether extract of W. ugandensis stem bark (dietE014), which was the

most active extract in the antiplasmodial experiments, showed strong cytotoxic effects on the human

lung cells, displaying a CC50 value as low as 0.3 μg/mL. The recorded antiplasmodial activity of these

extracts was found not to be selective (SI <10), which may relate to the compositional complexity of

the extracts.

2.5 Investigation of the mutagenic effects of extracts

The plant extract library was further screened for genotoxicity and mutagenic effects via the Salmonella

reverse mutation assay without metabolic activation to detect direct mutagenic extracts at 500 μg/plate.

The strains used were S. enterica subsp. enterica Typhimurium strains TA98 and TA100. Strain TA98

is particularly susceptible to frameshift mutations, whereas strain TA100 is optimized for base-pair

substitution mutations (Isono and Yourno, 1974; Jurado et al., 1993; Mortelmans and Zeiger, 2000).

The results of the library screen for mutagenic effects of extracts (without metabolic activation) are

given in Table 4, showing the calculated mutagenicity indices (MIs) and the MI interpretations.

Absolute data such as mean values of His+ revertant colonies are provided in Supplementary Table S1.

Table 4. Results of the Salmonella reverse mutation assay showing the mutagenicity indices (MIs) and the MI

interpretations at 500 μg/plate; +: positive, mutagenic; +/-: weakly mutagenic; -: negative: non-mutagenic; nt: not tested;

GI: growth inhibition

Scientific name Extract

TA98 TA100

without

metabolic activation

with

metabolic activation

without

metabolic activation

with

metabolic activation

mutagenicity MI mutagenicity MI mutagenicity MI mutagenicity MI

Securidaca

longipedunculata

eE001 - 0.8 - 0.9 - 1.4 - 0.9

wE001 - 1.5 - 0.7 - 1.6 - 1.2

hE001 nt - 0.4 nt - 0.8

mE001 - 0.7 - 1.1 - 1.2 - 0.4

smE001 - 0.9 - 1.0 - 1.5 - 0.0

Microgramma

lycopodioides

eE002 - 1.2 - 0.9 - 1.4 - 1.0

wE002 - 1.2 - 1.1 - 1.1 - 0.7

smE002 - 1.4 - 0.8 +/- 1.7 1.0

eE003 - 0.9 - 0.9 - 1.0 - 0.7

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This is a provisional file, not the final typeset article

Ficus

saussureana

wE003 - 1.0 - 0.8 - 1.6 - 1.0

hE003 - 1.1 - 0.7 - 1.5 - 0.9

mE003 - 0.8 - 0.8 - 1.4 - 1.0

smE003 - 1.0 - 0.7 - 1.6 - 0.9

Sesamum

calycinum subsp.

angustifolium

eE004 - 0.8 - 0.8 - 1.3 - 1.0

wE004 - 1.0 - 1.0 +/- 1.7 - 1.1

hE004 + 6.5 - 0.6 - 1.5 - 1.0

mE004 - 1.0 - 1.1 - 1.4 - 1.0

smE004 - 0.7 - 1.0 - 1.5 - 1.1

Leucas

calostachys

eE005 - 0.9 - 0.6 - 1.4 - 0.7

wE005 - 1.0 - 0.9 +/- 1.7 - 1.1

hE005 - 1.2 - 0.8 - 1.5 - 1.0

smE005 - 1.0 - 0.8 - 1.6 - 1.0

Solanum

aculeastrum

eE006 - 0.8 - 0.8 +/- 1.9 - 1.0

wE006 nt - 1.1 nt - 1.0

hE006 + 10.7 - 0.6 - 1.6 - 0.9

smE006 - 0.8 - 0.7 +/- 1.7 - 1.0

Albizia coriaria

eE007 - 0.7 - 0.9 - 1.3 - 1.0

etE007 - 1.1 - 1.0 - 1.6 - 1.0

Erythrina

abyssinica

eE008 - 0.8 - 0.4 - 1.0 - 0.8

etE008 - 1.0 - 0.4 - 1.0 - 0.7

Zanthoxylum

chalybeum

eE009 - 1.1 - 1.0 +/- 1.8 - 1.0

etE009 - 0.8 - 1.0 - 1.5 - 1.0

etE017 nt nt nt nt

dietE017 - 0.7 nt +/- 1.8 nt

etE017a - 0.9 - 0.9 - 1.4 - 1.4

dietE017a - 0.7 - 0.7 - 1.2 - 0.9

Toddalia asiatica

eE010 + 3.8 - 0.9 + 3.9 - 1.0

etE010 +/- 1.8 - 0.4 + 3.1 - 0.7

dietE010 + 4.0 - 0.6 + 3.7 - 1.2

etE010a +/- 1.7 - 1.0 + 3.2 - 0.9

Harungana

madagascariensis

eE011 - 1.0 - 0.9 - 1.4 - 1.0

etE011 - 0.9 - 0.6 - 1.4 - 1.2

dietE011 - 0.7 - 0.4 - 1.1 - 0.6

etE011a - 1.3 - 0.8 - 1.4 - 0.7

Morella

kandtiana

etE012 - 0.8 - 1.1 - 1.5 - 1.0

etE012a - 1.0 - 1.1 - 1.4 - 0.9

dietE012 - 1.0 - 0.8 - 0.7 - 0.9

Cassine

buchananii

eE013 - 0.9 - 0.8 - 1.6 - 1.1

etE013 - 0.9 - 1.1 - 1.5 +/- 1.7

etE013a - 0.9 - 0.9 - 1.3 - 0.9

Warburgia

ugandensis

etE014a - 0.8 - 0.9 - 0.7 - 1.3

dietE014 nt nt nt nt

Combretum molle eE015 - 0.9 - 1.1 - 1.1 - 1.2

141

etE015 - 1.0 - 1.2 - 1.5 - 1.0

Plectranthus

hadiensis

dietE016 - 1.0 - 1.1 nt - 1.2

hE016 - 0.9 - 0.5 - 1.4 - 0.8

spontaneous

mutations

negative

control - 1.0 - 1.0 - 1.0 - 1.0

2-nitrofluoren positive

control + 18.9 nt nt nt

methyl

methanesulfonate

positive

control nt nt 6.6 nt

2-aminoflourene positive

control - 1.0 + 12.4 + 2.2 + 6.1

Among the 56 extracts from 16 plant species, four extracts from three species were identified

to cause significant direct mutagenic effects in the TA98 strain at a concentration of 500 μg/plate with

MIs ranging from 3.8 to 10.7. These extracts were the n-hexane extract of S. calycinum subsp.

angustifolium leaves (hE004, MI: 6.5; previously active in the antiplasmodial experiments against

P. falciparum K1), the n-hexane extract of Solanum aculeastrum roots (hE006, MI: 10.7), and the

diethyl ether and ethyl acetate extracts of Toddalia asiatica leaves and bark (eE010, MI: 3.6; dietE010;

MI: 4.0). Two more extracts of T. asiatica showed weak mutagenicity against the TA98 strain (without

metabolic activation). These were the ethanolic extracts etE10 (MI: 1.8) and etE010a (MI: 1.7),

resulting in all four T. asiatica extracts displaying either significant or weak genotoxicity against the

TA98 strain.

Interestingly, these four extracts of T. asiatica also exhibited significant mutagenic effects

during the experiments with the TA100 strain at 500 μg/plate, and mutation rates were tripled and

almost quadrupled. The MIs ranged from 3.1 to 3.9 (MIeE010: 3.9; MIetE010: 3.1; MIdietE010: 3.7; MIetE010a:

3.2). No other extracts were identified as causing significant mutagenicity in the TA100 strain.

However, seven extracts from five plant species displayed weak mutagenic effects. These were, in

descending order of the MI: 1) extract eE006 (ethyl acetate extract of S. calycinum subsp. angustifolium

leaves, MI: 1.9); 2) and 3) extracts eE009 and dietE017 (ethyl acetate and diethyl ether extracts of

Z. chalybeum stem bark, MIs: 1.8); 4)–7) extracts smE002 (methanolic extract of Microgramma

lycopodioides rhizomes, MI: 1.7), wE004 (aqueous extract of S. calycinum subsp. angustifolium leaves,

MI: 1.7), wE005 (aqueous extract of L. calostachys leaves, MI: 1.7), and smE006 (methanolic extract

of S. aculeastrum roots, MI: 1.7).

For further illustration, plate photos of revertant colonies for extract eE007 (Albizia coriaria,

non-mutagenic against test strain TA98) and hE006 (S. aculeastrum, mutagenic against test strain

TA98) are provided in Supplementary Figure S1.

2.6 Simulation of human liver metabolism and mutagenic effects

The experiments for investigation of genotoxicity of the 56 extracts were repeated after incorporating

a pre-incubation assay, aiming at in vitro simulation of human liver metabolism and assessment of

potential bioactivation or deactivation/detoxification of mutagenic compounds in the extracts. Prior to

the Salmonella reverse mutation assay, extracts were treated with a pooled hepatic S9 fraction, which

is the post-mitochondrial supernatant fraction of homogenized liver. It was prepared by

homogenization of human liver in isotonic KCl with subsequent separation by centrifugation at 9000 g

(Hamel et al., 2016). The human liver S9 fraction represents a rich source of drug metabolizing phase

I and phase II enzymes, including the cytochromes P450, UDP-glucuronosyltransferases,

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This is a provisional file, not the final typeset article

acetyltransferases, methyltransferases, glutathione S-transferases, sulfotransferases, epoxide

hydrolases, carboxylesterases, and flavin-monooxygenases. It is therefore widely used in the study of

drug interactions and xenobiotic metabolism (Jia and Liu, 2007; Cox et al., 2016; Vrbanac and Slauter,

2017).

Results, including calculated MIs, are given in Table 4. Mean values of His+ revertant colonies

are reported in Supplementary Table S1. None of the extracts previously identified in the screening

without metabolic activation as mutagenic retained their genotoxic activity after pre-incubation of the

plant extract library with human S9 liver fraction at a concentration of 500 μg/plate. This observation

was irrespective of the strain tested and whether extracts displayed significant or weak mutagenic

properties in the experiments without metabolic activation. In addition, none of the other extracts in

the library displayed significant mutagenicity in both strains after metabolic activation. Only one

extract, the ethanolic extract of Cassine buchananii stem bark (etE013), exhibited a weak genotoxic

effect on test strain TA100 after contact and treatment with the human hepatic S9 fraction, reporting

an MI of 1.7.

143

3 Discussion

In this study, seven extracts from five plant species used in the Ugandan Greater Mpigi region in the

treatment of malaria were identified from a library of 56 extracts and selected for antiplasmodial

follow-up investigations due to their hemozoin formation inhibition activity in the in vitro heme

biocrystallization assay. The extracts that were further studied were extracts hE004 and eE004 (a n-

hexane and an ethyl acetate extract of S. calycinum subsp. angustifolium leaves), eE005 (an ethyl

acetate extract of L. calostachys leaves), etE009 and eE009 (an ethanolic and an ethyl acetate extract

of Z. chalybeum stem bark), hE016 (an n-hexane extract of P. hadiensis leaves), and dietE014 (a diethyl

ether extract of W. ugandensis stem bark). The modified heme biocrystallization assay proved to be an

effective method for pre-screening of natural product libraries since all seven extracts subsequently

displayed significant antimalarial activity in the antiplasmodial experiments against

chloroquine-resistant P. falciparum K1. The results of this study therefore further add to the scientific

basis for their effectiveness in traditional use in the Greater Mpigi region in Uganda as previously

described (Schultz et al., 2020b). Furthermore, antimalarial activity was studied and verified for the

first time for the majority of the species investigated, which was previously determined by the DoP

method (Schultz et al., 2021a). It needs to be pointed out that only those extracts were selected for

antiplasmodial follow-up experiments that acted as hemozoin formation inhibitors in the prescreen at

concentrations of 10 μg/mL or lower. It is therefore possible that extracts of other species in the library

might also possess antimalarial properties on basis of other mechanisms of action which were not

covered in this study.

The strongest antiplasmodial activity was displayed by the diethyl ether extract of

W. ugandensis stem bark (dietE014), showing an IC50 value as low as 0.5 μg/mL. The plant is an

evergreen tree, also known as the pepper-bark tree and East African greenheart, that grows in drier

highland forest and lower rainforest areas throughout East Africa (Katende et al., 1995; Dharani and

Yenesew, 2010; Dharani, 2019). In the Sango bay area in Southern Uganda, it is considered a

threatened species by the locals due to poor harvesting techniques and unsustainable harvesting

intensities of the stem bark (Ssegawa and Kasenene, 2007). However, in addition to the strong

antimalarial properties displayed by extract dietE014, the results of the cytotoxicity experiments

against MRC-5SV2 lung fibroblasts indicate even more potent cytotoxicity (CC50: 0.3 μg/mL). This led

to a relatively low calculated SI of 0.6, making this plant species and its extract potentially less suitable

for selection for further studies on the isolation and discovery of novel antimalarial drug leads. In

another study, the leaves of W. ugandensis were reported to exhibit comparable cytotoxic activity

against brine shrimp larvae (LC50: 24.5 μg/mL) as cyclophosphamide (LC50: 16.3 μg/mL), a standard

anticancer drug that was used as a positive control (Mbwambo et al., 2009). W. ugandensis was also

cited as being used by Ugandan healers in traditional therapy of several types of cancer (breast cancer,

cervical cancer, intestinal cancer, prostate cancer, skin cancer, throat cancer, and leukemia) in the

Greater Mpigi region (Schultz et al., 2020b). Therefore, it will be interesting to further investigate the

plant’s cytotoxic properties in selectively destroying related cancer cells. Interestingly, extract

dietE014 could not be evaluated for genotoxicity because it displayed growth inhibitory activity against

both Salmonella strains used in this study. These anti-Salmonella properties have previously been

described for apolar extracts of W. ugandensis stem bark from Kenya with MIC values ranging from

31 μg/mL to 488 μg/mL, depending on the Salmonella strain (Peter et al., 2015). In another study,

antiplasmodial properties were reported for chloroform, ethyl acetate, aqueous, and methanolic extracts

of W. ugandensis stem bark against P. knowlesi (Were et al., 2010). The most promising extract in this

study was the apolar chloroform extract with an IC50 value of 3.1 μg/mL. In the same study, this extract

was further investigated for chemosuppression of P. berghei growth in BALB/c mice at

200 mg/kg/day, reaching in vivo chemosuppression of 69% (curative) and 49% respectively

(prophylactic). However, all mice treated with the chloroform extract had died by the end of the assays

144

This is a provisional file, not the final typeset article

(12 days) when used for prophylaxis, whereas no deaths were observed in uninfected mice and the

positive controls. Another study investigating W. ugandensis stem bark, harvested in Ethiopia, tested

extracts (petroleum ether, dichloromethane, acetone, methanolic) against chloroquine-sensitive P.

falciparum 3D7 (Wube et al., 2010). The petroleum ether and dichloromethane extracts exhibited

strong antiplasmodial activity (IC50: 6.9 and 8.1 μg/mL) whereas the acetone and methanolic extracts

were inactive. Both pharmacologically active extracts showed cytotoxic effects on KB cells (ED50: 2.7

and 5.6 μg/mL), thus achieving a less promising SI. Six coloratane and six drimane sesquiterpenes

were further isolated from the dichloromethane extract, of which two compounds exhibited some

plasmodicidal activity against the chloroquine-resistant P. falciparum K1 strain (IC50: 7.3 μM and 7.9

μM). None of the isolated compounds tested were assessed for cytotoxicity in the study. In the

literature, there are also a few studies investigating the antileishmanial activity of W. ugandensis, which

were initiated due to its widespread traditional use to treat this neglected disease in Kenya (Ngure et

al., 2009a; Ngure et al., 2009b; Githinji et al., 2010).

The extract exhibiting the second strongest antiplasmodial activity against

chloroquine-resistant P. falciparum K1 in the plant extract library was the ethyl acetate extract eE005,

obtained from L. calostachys leaves, achieving an IC50 value of 5.7 μg/mL. Sample eE005 was also the

most promising extract in the study due to its relatively low cytotoxicity against MRC-5SV2 cells (CC50:

14.7 μg/mL). Consequently, a SI of 2.6 was calculated which was the highest selectivity index in this

study. Interestingly, the same plant extract eE005 was recently identified by the authors as a strong

selective cyclooxygenase-2 (COX-2) inhibitor (IC50: 0.66 ΐg/mL) with a promising selectivity ratio

(COX-2/COX-1) of 0.1 (Schultz et al., 2021c). Potentially generating fewer side effects due to

decreased COX-1 and increased COX-2 inhibition, sample eE005 seemed to be much more potent in

the study than comparable commercial COX-2 inhibitor drugs, such as Aspirin and ibuprofen. At the

same time, eE005 only displayed low inhibitory activity against S. aureus (MIC: 500 ȝg/mL) and no

antibacterial growth inhibition effects on multidrug-resistant Listeria innocua, Escherichia coli (MICs:

>ௗ500 ȝg/mL), Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter baumannii,

Pseudomonas aeruginosa, and Enterobacter cloacae (MICs: >ௗ256 ȝg/mL) at the highest

concentrations tested (Schultz et al., 2020a; Schultz et al., 2021c). Moreover, the n-hexane extract of

the leaves (hE005) showed significant quorum quenching activity against the accessory gene regulator

(agr) system in S. aureus (Schultz et al., 2020a). L. calostachys was recently identified using the DoP

method as a highly understudied medicinal species that merits further ethnopharmacological and

pharmacognostic research in the lab (Schultz et al., 2021a). In fact, only three other studies have been

published that examine potential bioactive properties of the plant. All of these focus on the potential

antiplasmodial properties of L. calostachys crude extracts. The first bioactivity study reported no

significant antiplasmodial activity of L. calostachys (Muregi et al., 2004) while the second study

reported antiplasmodial activity of a methanolic extract (IC50: 3.45 ȝg/mL) and an aqueous extract

(IC50: 0.79 ȝg/mL) against P. knowlesi (Nyambati et al., 2013). The last of these studies reported low

antiplasmodial activity of a chloroform and a methanolic extract against chloroquine-sensitive

P. falciparum D6 (IC50: 40.2 and 88.4 ȝg/mL) (Jeruto et al., 2015). The species L. calostachys was

recently reviewed more in detail in the discussion sections of two of our recent publications (Schultz

et al., 2021a; Schultz et al., 2021c). As far as to the author’s knowledge, there have been no studies

published on the identification or isolation of pharmacologically active secondary plant metabolites

from L. calostachys to date, highlighting the vital need to further study this interesting medicinal plant.

According to the ethnobotanical survey among Ugandan traditional healers (Schultz et al.,

2020b), the traditional methods of preparation most often cited were boiling ground plant parts in water

(aqueous decoctions) or suspending in water (cold infusions), followed by oral administration.

Aqueous extracts were therefore included into this study. However, other types of solvents were

included in a ‘pre-fractionation’ process during initial extraction procedures in order to investigate not

only the chemistry yielded by traditional preparation but also chemistries only accessible by alternative

145

extraction methods. The aim of this strategy was to depict the full range of polarity during the

extractions (from aqueous extracts via methanolic, ethanolic, ethyl acetate, diethyl ether to n-hexane

extracts with decreasing polarity), using new, unextracted material for each type of extraction. Thus,

plant material and individual extracts derived from it were regarded as chemical libraries that merit

pharmacological investigation of the totality of ingredients provided. Similar to the results of

pharmacological studies of extracts derived from the library that were published previously (Schultz et

al., 2020a; Schultz et al., 2021c), the apolar extracts exhibited the strongest antimalarial effects in the

in vitro assays. This phenomenon might be explained by the fact that the extracts produced were fine-

filtered prior to evaporation of the solvent and drying of extracts, ultimately leading to the removal of

tiny solids that would still be present in traditional preparations. Aqueous decoctions and infusions

with ground plant material are usually not filtered by the traditional healers, meaning that patients

ingest these tiny solids. It is likely that apolar antimalarial secondary plant metabolites remain in the

traditional herbal remedy as a result; hence, these active ingredients only occur in the apolar extracts

screened in this study. Another possible explanation could be that lipophilic compounds are also

extracted to a certain degree during boiling (potentially assisted by bipolar surface-active secondary

metabolites present in the plant material).

In a previous screening of the same plant extract library for antiinflammatory activity, nine

extracts were identified as potent COX-2 inhibitors (Schultz et al., 2021c). Interestingly, five of the

seven extracts that were reported as having strong antiplasmodial activity in the present study were

among the nine COX-2 inhibiting extracts (dietE014, eE005, etE009, hE004, eE004). In addition, other

active antimalarial extracts were eE009 and hE016, derived from Z. chalybeum and P. hadiensis,

whereas etE009 and dietE016 acted as COX-2 inhibitors. Thus, all five medicinal plant species reported

to possess antiplasmodial activity also showed significant inhibition of COX-2. This suggests that the

potential molecular mechanism of action may be similar. Heme plays a major role in both assays. On

the one hand, heme is a vital co-factor for COX isoenzymes and has been introduced externally to the

COX reaction in the antiinflammatory assays (Chandrasekharan and Simmons, 2004; Schultz et al.,

2021c). On the other hand, toxic free heme is released during plasmodial degradation of hemoglobin

and subsequently detoxified by heme biocrystallization (Schmitt et al., 1993; Roy, 2017). Due to the

heme biocrystallization pre-screen conducted in this study, it is likely that inhibition of hemozoin

formation is the mechanism of action for the reported antiplasmodial activity of plant extracts. In past

studies on the antimalarial drug chloroquine, scientists hypothesized that the hemozoin formation is

inhibited due to the drug’s ability to establish complexes with free heme (Cohen et al., 1964; Chou et

al., 1980; Egan, 2001; Egan, 2004). This has been confirmed in a recent study by (Kapishnikov et al.,

2019), in which the mode of action of quinoline antimalarial drugs in red blood cells infected by

P. falciparum was revealed in vivo using correlative X-ray microscopy. The authors report that an

excess of drug molecules in the digestive vacuole of the parasite leads to formation of a complex with

the free heme, making it unavailable for biocrystallization. The drug molecule also covers and blocks

a substantial number of the available docking sites on the surface of the hemozoin crystals that are

formed in the digestive vacuole of the parasite. These processes cause membrane puncture and spillage

of heme into the interior of the parasite due to the complex being driven toward the digestive vacuole

membrane (Kapishnikov et al., 2019). In terms of the COX-2 inhibition activity of the medicinal plant

extracts from the Greater Mpigi region, potential chelation of the co-factor free heme and complex

formation with active secondary metabolites may be responsible for the reported inhibition of

cyclooxygenase-2 and resultant antiinflammatory properties.

Genotoxicity is a major cause of the initiation and development of many types of cancer. It is

defined as the ability of different substances to produce damage to genetic material, i.e., to cause DNA

mutations but also to damage those cellular components that are responsible for the functionality and

behavior of chromosomes within the cell (Bhattachar, 2011; Nagarathna et al., 2013; SáoczyĔska et al.,

2014). Therefore, genotoxicity assays are in vitro and in vivo tests that have been developed to detect

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genotoxic, mutagenic, and potentially carcinogenic substances that induce genetic damage, point

mutations in genes, large deletions or rearrangements of DNA, cellular transformation, and

chromosomal breakage (Tice et al., 2000; Tejs, 2008; Samiei et al., 2015). Unlike marketed drug

compounds in the pharmaceutical industry, plants used in traditional medicine systems have often

never been investigated for potential genotoxic effects (Verschaeve, 2015). To increase awareness of

potential health hazards and to determine the safety of traditionally used herbal remedies, the plant

extracts were studied using the widely accepted Salmonella reverse mutation assay (Ames test). Plants

with mutagenic properties should be considered potentially unsafe, especially for long-term use

(Verschaeve, 2015). In the European Union, the Ames test is used as part of well described strategies

as an initial experimental method for assessment of the short-term genotoxicity and mutagenicity of

chemical agents. With identification of potential mutagens (‘positive’ results) using the Ames test,

further studies with different in vitro and in vivo assays are conducted for a detailed understanding and

confirmation of carcinogenic effects (Samiei et al., 2015; Verschaeve, 2015). The Ames test employs

several histidine dependent bacterial strains of Salmonella to identify agents that are capable of causing

genetic damage that leads to gene mutations. These strains possess preexisting mutations in the

histidine operon, acting as hot spots for mutagens, causing DNA damage via different mechanisms of

action (Ames et al., 1973; Mortelmans and Zeiger, 2000). When grown on minimal media agar plates

in the presence of mutagens, the genes’ function for cells synthesizing histidine may be restored

(reverse mutation) and the mutated cells form colonies that are counted (Mortelmans and Zeiger, 2000;

Tejs, 2008).

The experiments for assessment of potential procarcinogenic properties of plant extracts via

evaluation of in vitro mutagenicity and genotoxicity identified four extracts from three species that

caused significant direct mutagenic effects against test strain TA98 and four extracts from one species

with significant direct mutagenic effects against test strain TA100. The species T. asiatica showed the

most significant genotoxic effects on both test strains (without metabolic bioactivation at a

concentration of 500 μg/plate). TA98 is susceptible to frameshift mutations and the diethyl ether and

ethyl acetate extracts of T. asiatica leaves and bark (eE010, MI: 3.6; dietE010, MI: 4.0) resulted in

high genotoxicity. Two additional extracts of this species also displayed weak mutagenicity (etE10,

MI: 1.8; etE010a, MI: 1.7). In addition, all four extracts of T. asiatica exhibited strong mutagenic

effects on the TA100 test strain at 500 μg/plate, nearly quadrupling the (base-pair substitution)

mutation rates. The woody liana or shrub T. asiatica is a commonly used medicinal plant throughout

Africa and Southeast Asia. It has previously been identified as a ‘highly studied’ medicinal plant

species using the DoP method (Dharani and Yenesew, 2010; Schultz et al., 2021a). As far as the authors

are aware, no study has yet assessed the potential genotoxic effects of T. asiatica, making this study

the first report of mutagenic effects for the species. The fact that it is widely used in different traditional

medicine systems, and that, after S. longipedunculata, it is the second most studied species in the plant

extract library justifies further future investigations assessing its toxicity and potential carcinogenic

properties using more advanced toxicological methods. Plant extracts are complex mixtures, and it is

difficult to speculate which of the secondary plant metabolites are responsible for the mutagenic effects

of some of the extracts. The results of this study warrant the phytochemical characterization of

potentially genotoxic plant extracts and the isolation of mutagenic compounds.

Without metabolic bioactivation, the n-hexane extract of S. calycinum subsp. angustifolium

leaves (hE004) produced the strongest genotoxic effects measured in this study, reaching a calculated

MI of 6.5 when being in contact with the TA98 test strain. The absence of mutagenic activity against

test strain TA100 and after metabolic bioactivation against both strains indicates that frameshift

mutations were caused and that genotoxic secondary plant metabolites were successfully

detoxified/deactivated by human liver enzymes. Interestingly, extract hE004 was also the fifth most

active extract in the evaluation of the library for antiplasmodial activity against P. falciparum K1 (IC50

value: 19.6 ȝg/mL; TI: 1.4). The antimalarial activity of S. calycinum subsp. angustifolium leaves was

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reported for the first time in this study, which represents another scientific evidence supporting its

therapeutic use in the Ugandan Greater Mpigi region. The authors previously stated that extract hE004

acts as a potent antiinflammatory COX-2 inhibitor (IC50 value: 3.65 ȝg/mL) and as an agr system

quorum sensing inhibitor (IC50 values: 2, 2, 16, and 32 ȝg/mL (agr I–IV)). In these recent publications,

S. calycinum subsp. angustifolium was extensively reviewed in the discussions (Schultz et al., 2020a;

Schultz et al., 2021a; Schultz et al., 2021c).

Apart from extract hE004, none of the other extracts that were previously identified as

possessing antimalarial effects showed significant mutagenic activity in the genotoxicity assays (with

and without metabolic bioactivation). Interestingly, none of the mutagenic extracts from the screen

without metabolic bioactivation retained their genotoxic activity after metabolic bioactivation of the

plant extract library with human S9 liver fraction. What should be emphasized is that none of the 56

extracts in the library displayed significant genotoxic activity against either strain after metabolic

bioactivation, indicating that effective deactivation of potential procarcinogens occurred during in vitro

simulation of human liver metabolism. Nevertheless, more thorough screening for potential harmful

genotoxic effects via other methods is needed in order to recommend the plants used in the Greater

Mpigi region as being safe long-term use.

The ritual plant P. hadiensis was also investigated for genotoxicity due to the elevated

prevalence of rapidly growing large breast masses in young women in the Nakawuka village area,

Wakiso district, Uganda. As far as the authors are aware, this study is the first investigation of potential

genotoxic properties of this plant. The results show that extracts of P. hadiensis had no

mutagenic/genotoxic activity against either test strain regardless of whether extracts underwent

metabolic bioactivation with human S9 liver fraction prior to assaying. On the contrary, previous

studies have shown cytotoxic and anticancer effects of P. hadiensis (Minker et al., 2007; Mothana et

al., 2010; Menon and Gopalakrishnan, 2015). For example, terpenoids isolated from the shoots induced

apoptosis in human colon cancer cells via the mitochondria-dependent pathway (Menon and

Gopalakrishnan, 2015). In the Greater Mpigi region, P. hadiensis has mainly been reported as being

used to treat skin cancer (relative frequency of citation: 36%) as well as, with low informant consensus,

abdominal cancer, leukemia, brain cancer, breast cancer, intestinal cancer, lung cancer, prostate cancer,

throat cancer, and uterine cancer (Schultz et al., 2020b). The increased incidence of rapidly growing

breast mass diagnoses (not necessarily cancer, but possibly) is a) most likely associated with genetic

predisposition; b) could be the result of phytoestrogen properties of this plant; or c) could be caused

by some other unknown environmental stimulus. In the future, medical studies should emphasize the

fact that inherited genetic variations are a common phenomenon in relatively isolated ethnicities and

minority groups (Chlebowski et al., 2005; Brennan, 2017). However, phytoestrogens derived from P.

hadiensis may contribute to more rapid growth of tumors, particularly benign masses. If P. hadiensis

would be the cause alone, it would be very potent and most likely act genotoxic in the Salmonella

reverse mutation screen. Yet, cancer development and cause are highly complex, and P. hadiensis

could still act as an external stimulus in relatively isolated communities with high prevalence of

inactivated tumor suppressor genes in a so-far unknown mechanism. Further research is needed to

reliably rule out P. hadiensis as the causative agent for the rapidly enlarging masses observed in young

female patients. In addition, it is important to recognize that potentially benign masses can grow rapidly

and may mimic cancer from the health provider and patient’s perspective (Heilmann et al., 2012). This

can result in erroneous treatment (e.g., mastectomy) in resource-limited settings unless tissue diagnosis

is performed first.

Genotoxicity studies in general and the Salmonella reverse mutation assay in particular have

some limitations that need to be discussed. First, some plant extracts may potentiate the genotoxicity

of a known mutagen, making them become co-mutagenic. This also means that a plant extract may

also have an antimutagenic action when administered with a known mutagen. Moreover, many

carcinogens may possess genotoxic properties in one tissue but have antigenotoxic/anticarcinogenic

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properties in a different tissue, making investigations and experimental design complicated and

complex (Verschaeve, 2015). The same applies to the dose of the drug ("the dose makes the poison").

Due to the high sensitivity of the Salmonella reverse mutation assay, the risk of false negatives is

relatively low. However, the relatively low specificity means that false (misleading) positives may

occur more often, which is problematic. Thus, further studies utilizing other in vitro methods, such as

the mouse lymphoma L5178Y cell Tk (thymidine kinase) gene mutation assay (MLA), the

micronucleus assay or the mammalian cell metaphase chromosome aberration assay, and in vivo

follow-up, e.g., via the transgenic mouse test or the UDS test, should be conducted as recommended

in the ICH guideline S2 (R1) issued by the European Medicines Agency (EMA, 2013; Verschaeve,

2015).

The present study can be regarded as a contribution to drug discovery since most of the past

discoveries of drug leads taking the ethnopharmacological approach were based on in vitro screening

studies from initial studies, which were then followed up on via bioassay-guided fractionation

strategies, investigations of the mechanisms of action, and in vivo antimalarial and toxicity

experiments. The study identified several medicinal plant species and extracts derived from them that

displayed significant antiplasmodial in vitro activity. The obtained pharmacological data did provide

early-stage support for the therapeutic use of the medicinal plants in rural Uganda as all these plants

are frequently used by traditional healers to treat malaria and related fever (Schultz et al., 2020b). The

promising results obtained in this study suggest that such studies will be vital for future research work

on the medicinal plants from the Greater Mpigi region. Yet, there may be synergistic effects between

multiple active secondary plant metabolites which could potentially jeopardize bioassay-guided

isolation efforts. After this initial screening study, further research work is also required to

phytochemically characterize the bioactive extracts of the most promising antimalarial medicinal plant

species.

All results of this study were reported back to the traditional healers who originally participated in

the ethnobotanical survey, and a video article describing a successful two-day workshop method for

transfer of lab results was published recently (Schultz et al., 2021b). The authors believe that it is the

responsibility of the ethnopharmacologist to facilitate bidirectional knowledge transfer and feedback

once a study is completed.

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4 Materials and Methods

4.1 Ethnobotanical information

Ethnobotanical information, e.g., use reports, on the 16 medicinal plant species for medical treatment

was obtained by means of a survey among 39 traditional healers from 29 different villages in the

Greater Mpigi region in Uganda. The results of this study were previously published (Schultz et al.,

2020b) and serve as a basis for the pharmacological experiments. Additional information on the ritual

use of P. hadiensis in Nakawuka village (Wakiso district) has been obtained through anecdotal reports

by local healthcare providers and female patients in 2016.

4.2 Collection of plant material

Plant sample and voucher specimen material were collected under the guidance of the traditional

healers in 2013, 2015, and 2016. Best practice collection procedures were followed at all times

(Alexiades and Sheldon, 1996; Martin, 2004; Heinrich et al., 2018). Assignments of plant family

correspond to The Angiosperm Phylogeny Group IV guidance (The Angiosperm Phylogeny, 2016).

Plant identification and assignment of scientific names were performed following the current standards

in the field of ethnopharmacology (Weckerle et al., 2018). Scientific names were cross-checked and

taxonomically validated with https://www.theplantlist.org. Voucher specimens were prepared for all

species investigated in this study and were deposited in indexed herbaria at the Makerere University

Herbarium (MUH) in Kampala, Uganda. Select specimens were additionally deposited at the Emory

University Herbarium (GEO) in Atlanta, GA, USA. These herbarium voucher specimens were made

digitally available on the SERNEC portal (http://sernecportal.org/portal/index.php). Voucher specimen

numbers are provided in Table 1.

4.3 Preparation of extracts and extract IDs

The collected plant material was roughly chopped, then shade dried, and taken to the laboratory.

Samples were ground prior to extraction. The extraction procedure has previously been described by

(Schultz et al., 2020a) (flow sheet in the supplementary material Figure S2 of the cited article). Briefly,

the extraction methods applied were either maceration, Soxhlet extraction, or aqueous decoction.

Extractions were performed using different solvents (water, methanol, ethanol, ethyl acetate, diethyl

ether, n-hexane), enabling selective extraction of biomolecules of different polarities from the samples

("pre-fractionation" approach). New dried material was always used, except for the methanolic Soxhlet

extractions which were performed successively from extracted dry material after Soxhlet extraction

with n-hexane. This way, pharmacological investigation of the totality of ingredients provided by the

individual medicinal plant was assured. All extracts were dried using a rotary vacuum evaporator or

under a nitrogen stream and then stored at í20°C in the dark. Labeling of the crude extracts was

performed according to their extraction solvent and the collection number (EXXX) that was assigned

to each individual plant species during the field studies, ranging from E001 to E017. In terms of the

maceration procedure, the extractants used were a) methanol (sample ID: mEXXX); b) ethanol

(etEXXX); c) ethyl acetate (eEXXX); and d) diethyl ether (dietEXXX). Crude extracts produced via

Soxhlet extraction were prepared using e) n-hexane (hEXXX); and f) methanol (smEXXX). In the

Ugandan Greater Mpigi region, traditional healers often prepare and administer herbal drugs as

aqueous decoctions (Schultz et al., 2020b). To simulate this original method of preparation, the plant

material was additionally boiled in g) water at 95°C for 30 min while being stirred (wEXXX).

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4.4 Sample preparation

Dry extracts were dissolved in DMSO (Carl Roth) at a sample concentration of 10 mg/mL, using a

vortex mixer or a sonicator. For some samples, moderate or low solubility was observed at RT. These

samples were further treated with sonication as well as a slow gradual temperature increase until fully

dissolved (up to 55°C max.). Extract solutions were stored at í20°C in the dark prior to use in the

assays.

4.5 Chemical heme biocrystallization assay

The heme biocrystallization assay was adapted from previously published studies (Baelmans et al.,

2000; Heshmati Afshar et al., 2011; Sarker and Nahar, 2012), and modified. Figure 3 illustrates the

modified heme biocrystallization assay. Briefly, a 3 mM hematin porcine (Sigma Aldrich) solution

(freshly dissolved in 0.1 M NaOH), a 10 mM oleic acid (Carl Roth) solution, and buffer A (3.91 g/L

NaCl, 0.3 g/L KCl, 0.297 g/L MgSO4 · 7 H2O, 0.901 g/L glucose, 7.1 g/L Na2HPO4, pH 5) were

prepared. 100 μL of hematin solution was pipetted into each reaction tube (Eppendorf vial) and mixed

with 10 μL 1M HCl, 10 μL 10 mM oleic acid solution, and 100 μL test sample solution. A total of 780

μL 500 mM sodium acetate buffer of pH 5 was added to each reaction tube, resulting in a total reaction

volume of 1000 μL. As a positive control, chloroquine diphosphate (Sigma Aldrich) was used. The

negative control contained buffer and DMSO without test extracts. All tubes were incubated for 16 h

at 37°C in a slowly rotating 360° vertical rotator setup inside an incubator. The incubation was

terminated by centrifugation at 14,000 rpm at 21°C for 10 min and the supernatant was carefully

removed. Next, the hemozoin pellets were repeatedly washed with 1 mL of 2.5% (w/v) SDS in buffer

A (pH 7.4), while applying sonication (15 min, 21°C). This procedure was repeated until the

supernatant was clear (usually 3–6 washes). A final wash was performed with 0.1 M sodium

bicarbonate buffer (pH 9.4, Merck) and the supernatant was carefully removed. The pellets were then

resuspended in 1 mL of 0.1 M NaOH. The tubes were vortexed, filtered through syringe filters, and for

determination of hemozoin content, the absorbance was measured photospectrometrically at 400 nm

(1 cm quartz cuvette). The % of inhibition (I%) was calculated using the following formula:

% inhibition = ((absorbancevehicle control í absorbancesample) / absorbancevehicle control) × 100

Data analysis and calculation of IC50 values was performed using Microsoft Excel®. All experiments

were conducted in triplicate and presented as the mean values with standard deviations.

Figure 3: Flow scheme describing the heme biocrystallization assay procedure

151

4.6 In vitro antiplasmodial bioassay

For the assessment of antiplasmodial activity, the chloroquine-resistant P. falciparum K1 strain was

selected. A lactate dehydrogenase (LDH) assay was used to evaluate parasite multiplication as

previously described (Makler et al., 1993; Cos et al., 2006; Mesia et al., 2010). Briefly, the plasmodia

were cultured in RPMI-1640 medium supplemented with 10% O+ human blood serum and 4% washed

O+ human erythrocytes and maintained at 37°C under an atmosphere of 3% O2, 4% CO2, and 93% N2.

96-well microtiter plates were incubated for 72 h under the same incubation parameters after addition

of extract solutions (twofold serial dilution) to the malaria parasite inoculums (1% parasitemia and 2%

hematocrit). The highest test concentration was 64 ȝg/mL. After incubation, the plates were frozen and

stored at í20°C. After thawing, in separate plates, 20 μL of hemolyzed parasite suspension from each

thawed well was added to 100 μL Malstat® reagent mixed with 10 μL of a 1/1 (v/v) solution of nitro

blue tetrazolium (0.1 mg/mL) and phenazine ethosulfate (2 mg/mL) solutions. The plates were allowed

to react in the dark for 2 h and any color change was measured photospectrometrically at 655 nm.

Chloroquine diphosphate was used as a positive control. DMSO was used as solvent control. Results

were expressed as % reduction in parasitemia compared to the infected controls. IC50 values were

calculated from the dose–response curves. Each sample was tested in triplicate and experiments were

repeated at least once on a separate day. Data analysis and calculation of IC50 values was performed

using Microsoft Excel®.

4.7 Cytotoxicity against human lung fibroblasts

A human diploid embryonic lung cell line (MRC-5SV2) was used for assessment of cytotoxicity.

Tamoxifen was included as a positive control for cytotoxicity, and DMSO served as negative control.

The assay procedure was previously described (McMillian et al., 2002; Mesia et al., 2010). Briefly,

MRC-5SV2 lung fibroblasts were cultured in a minimum essential medium containing 5% FCSi, 20

mmol/L L-glutamine, and 16.5 mmol/L sodium hydrogen carbonate. A total of 190 μL of cell

suspension (3 x 104 MRC-5SV2 cells) was added to each well of a 96-well microtiter plate, containing

10 μL pre-diluted sample extracts. Experiments were performed in twofold sample dilutions, starting

at 64 ȝg/mL. The plate was incubated for three days at 37°C under an atmosphere of 5% CO2. After

incubation, resazurin was added into the wells and the plate was incubated for another 4 h at 37°C. The

overall cell viability or proliferation was assessed fluorimetrically at 550 nm (excitation) and 590 nm

(emission). The data was processed as % reduction in cell viability in the treated cultures compared to

the untreated control cultures, and CC50 values were calculated from the dose–response curves. The SI

for antimalarial activity was calculated by dividing the CC50 for extract cytotoxicity by the IC50 for its

respective antiplasmodial activity. Data analysis and calculation of IC50 values was performed using

Microsoft Excel®.

4.8 Genotoxicity screening

To assess any potential genotoxic and mutagenic effects of plant extracts, the Salmonella reverse

mutation test (Ames mutagenicity test), which has been described previously (Maron and Ames, 1983;

Mortelmans and Zeiger, 2000; Tejs, 2008), was performed with modifications. The bacterial strains

used in the assay were the two histidine-dependent S. enterica subsp. enterica Typhimurium strains

TA98 and TA100. Strain IDs, characteristics, and sources are reported in Supplementary Table S3.

Both strains were maintained on tryptic soy agar (TSA) after streaking from freezer stock and overnight

incubation at 37°C. Overnight liquid cultures were prepared in tryptic soy broth (TSB) at 37°C and

with constant shaking at 120 rpm for 16 h. The strains were checked for characteristic spontaneous

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revertants and reversion of its mutants caused by the positive controls (reference mutagenic substances)

on each day of use. Genotypes were confirmed using the following methods: a) R-factor: Ampicillin

resistance or sensitivity was checked by spreading the S. enterica subsp. enterica wild type (DSM

11320, control), strain TA98, and strain TA100 on ampicillin-supplemented TBA plates (shown in

Supplementary Figure S2); b) uvrB mutation/UV sensitivity: Presence of the uvrB mutation in the test

strains was confirmed by spreading liquid cultures in parallel stripes on TBA plates. The plates were

then treated with UV light for 10 s, 30 s, 60 s, 2 min, 5 min, and 10 min, with half of each plate covered

with aluminum foil; and c) rfa mutation/crystal violet sensitivity: A total of 0.1 mL of an overnight

culture was added to a sterile tube containing 2 mL of warm TBA, previously kept at 45°C. After

vortexing for 3 s, the mixture was plated, a sterile filter paper disc (diameter: 6 mm) was placed on the

agar, and 10 μL of crystal violet solution (1000 μg/mL H2O) was transferred to the disc. All plates

were incubated at 37°C for 24 h and subsequently checked.

The plant extract dose introduced to this assay was 500 μg/mL. Prior to use, the extract solutions

were sterile filtered through 0.22 μm syringe filters (Carl Roth). A 100 μL aliquot of the test strain

suspension (overnight culture), 50 μL of extract solution, 50 μL of DMSO, and 500 μL of 0.2 M

phosphate buffer (pH 7.4) were added to 2.0 mL top agar (TBA containing 10% traces of biotin and

histidine) and vortexed. More detailed information is given in Supplementary Table S4. The mixture

was then poured over the surface of a minimal glucose agar petri dish and incubated at 37°C for 48 h.

After incubation, the number of mutants (revertant colonies) was counted and the reversion frequency

was determined as means with standard deviations. The known mutagenic substance 2-nitrofluorene

(2-NF; Sigma Aldrich; 1000 μg/mL DMSO) served as a positive control for strain TA98 and methyl

methanesulfonate (MMS; Sigma Aldrich; 200 μL MMS mixed with 1.8 mL DMSO) was used for strain

TA100. The final concentrations were 10 μg 2-NF/plate and 2μL MMS/plate respectively. Each

experiment was conducted in triplicate and on different days.

Absence of toxicity was assessed by observing the background bacterial growth. The

mutagenicity index (MI) describes the magnitude of the mutagenic induction and was calculated by

dividing the mean value of His+ revertant colonies counted on the sample treatment plates (spontaneous

mutations and induced mutations) by the mean value of His+ revertant colonies on the negative control

treatment plates (spontaneous mutations only). Data analysis and calculation of MI values was

performed using Microsoft Excel®. The MI was evaluated using by a modified "twofold rule" that has

previously been described (Zeiger et al., 1992; Mortelmans and Zeiger, 2000; Mathur et al., 2006). The

following criteria were used for interpretation of the MI:

Positive result (+): An extract sample was considered a mutagen if it has a

reproducible twofold increase of the reverse mutation rate

(MI ௗ2) (significantly mutagenic).

Weakly mutagenic result (+/-): A sample extract was considered a weak mutagen if a

reproducible, close to twofold increase of the reverse mutation

rate was determined (1.7ௗௗMIௗ<ௗ2.0).

Negative result (-): A sample extract was considered a non-mutagen if no increase or

an increase below 170% of the reverse mutation rate was

observed in at least two biological replicates (MI <ௗ1.7).

4.9 Human S9 liver fraction treatment of extracts

Human S9 liver fraction was incorporated into the Salmonella reverse mutagenicity assay to also assess

the human metabolic activation (or deactivation) of potentially mutagenic plant extracts. Pooled

hepatic S9 fraction represents the post-mitochondrial supernatant fraction from homogenized human

liver, and S9 fractions from the same batch only were used for the experiments (Lot# SLBR5681V,

153

S2442, Sigma Aldrich, always stored at í70°C). A sterile 0.1 M ß-nicotinamide adenine dinucleotide

phosphate disodium salt (NADP, Sigma Aldrich) solution was freshly prepared and kept on ice. A 1 M

glucose-6-phosphate solution and a MgCl2 (123 mg/mL) – KCl (81.4 mg/mL) salt solution were

aseptically prepared. Subsequently, a 4% S9 mixture was freshly prepared by first mixing 69.125 mL

of sterile H2O, 87.5 mL of sterile 0.2 M phosphate buffer (pH 7.4), 875 μL of glucose-6-phosphate

solution, 3.5 mL of MgCl2 – KCl salt solution, and 7 mL of NADP solution, and then adding 7.0 mL

of pooled human S9 liver fraction. The S9 mixture was kept on ice and quickly introduced to the

following steps.

Sample extracts and controls were treated with the S9 mixture via a pre-incubation assay. A total

of 50 μL of extract solution and 50 μL DMSO were added to 500 μL S9 mixture, vortexed, and

incubated at 37°C with constant shaking at 100 rpm for exactly 30 min. The mutagenic substance

2-aminofluorene (2-AF, 1 mg/mL DMSO) was used as a positive control for both Salmonella strains.

After incubation and metabolic activation of samples, the samples were introduced to the Salmonella

reverse mutagenicity assay by adding 2.0 mL TBA top agar and 100 μL of aliquot of the test strain

suspension (overnight culture) as described in section 4.8. Each plate contained 20 μL human liver S9

fraction. Further information is given in Supplementary Table S4. Figure 2 was created using

biorender.com software.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial

relationships that could be construed as a potential conflict of interest.

Author Contributions

F.S. wrote the manuscript. F.S. and L.P. designed the overall strategy of the study. F.S. and G.A.

collected and processed the plant material. G.A. prepared herbarium voucher specimens and

taxonomically identified the plant species. F.S. performed the extraction procedures and created the

extract library. F.S. and O.F.O. prepared the extract solutions for all assays and performed the heme

biocrystallization inhibition assay. G.C. and L.P. performed the antiplasmodial and the cytotoxicity

experiments. F.S., A.N. and O.F.O. conducted the genotoxicity experiments. F.S., O.F.O., A.N. and

L.P. analyzed the data. J.S. contributed toward the background on P. hadiensis in Wakiso district.

L.A.G. provided oversight of lab work and directed the study. All authors have read and agreed to the

published version of the manuscript.

Funding

This work was supported by two grants from the BMBF—German Federal Ministry of Education and

Research (13FH026IX5, 13FH066PX5; PI: L.A.G. and Co-I: F.S.). We acknowledge support for the

Article Processing Charge from the German Research Foundation (DFG, 414051096) and the Open

Access Publication Fund of Neubrandenburg University of Applied Sciences (HSNB). The content is

solely the responsibility of the authors and does not necessarily reflect the official views of the DFG,

HSNB, or BMBF.

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Acknowledgments

Greatest thanks to the Ugandan traditional healers in the Greater Mpigi region and neighboring regions

who provided the ethnobotanical information that forms the foundation of this study and who provided

guidance during the collection of plant materials. Thanks to research assistant Tidjani Cisse for

assisting during the extractions of plant material. Thanks to student assistant Kristine Kossol for

assistance in graphic design. The authors acknowledge An Matheeussen, Natascha Van Pelt and Pim-

Bart Feijens for their excellent technical assistance with the in vitro biological evaluation. Thanks to

Dr. Akram Salam for fruitful discussions and advice after drafting the manuscript.

Data Availability Statement

The data presented in this study are available in this article and in the Supplementary Material.

Ethnobotanical data are available in Schultz, F.; Anywar, G.; Wack, B.; Quave, C. L.; Garbe, L.-A.,

Ethnobotanical study of selected medicinal plants traditionally used in the rural Greater Mpigi region

of Uganda. J. Ethnopharmacol. 2020, 256, 112742.

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Simple Test System Combining Liver Homogenates for Activation and Bacteria for

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Pharmacological assessment of antiprotozoal activity,

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Fabien Schultz1,2*, Ogechi Favour Osuji2, Anh Nguyen2, Godwin Anywar3, John R.

Scheel4,5, Guy Caljon6, Luc Pieters7, Leif-Alexander Garbe2,8

1Institute of Biotechnology, Faculty III - Process Sciences, Technical University of Berlin, Berlin, Germany

2Department of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences, Neubrandenburg,

Germany

3Department of Plant Sciences, Microbiology and Biotechnology, Makerere University, Kampala, Uganda

4Department of Global Health, University of Washington, Seattle, WA, USA

5Department of Radiology, University of Washington, Seattle, WA, USA

6Laboratory of Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences,

University of Antwerp, Belgium

7Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of

Antwerp, Belgium

8ZELT - Neubrandenburg Center for Nutrition and Food Technology gGmbH, Neubrandenburg, Germany

* Correspondence:

Fabien Schultz (F[email protected]erlin.de)

Supplementary information

163

Table of contents

Supplementary Table S1:

Results of the Salmonella reverse mutation assay showing the mean

values of His+ revertant colonies page 3

Supplementary Figure S1:

Plate photos of revertant colonies for extract eE007 (Albizia coriaria,

non-mutagenic against test strain TA98) and hE006 (Solanum

aculeastrum, mutagenic against test strain TA98) page 4

Supplementary Table S2:

Overview of pipetting instructions during the Ames test procedure

with metabolic activation (pre-treatment assay) page 5

Supplementary Table S3:

Information on bacterial strains used in the study page 5

Supplementary Figure S2:

Results of growth comparison of his- mutants with the wild type

regarding +/- ampicillin genes page 6

Supplementary Table S4:

Overview of pipetting instructions during the Ames test procedure

without metabolic activation page 6

References cited in supplementary files page 7

164

Supplementary Table S1:

Table S1. Results of the Salmonella reverse mutation assay showing the mean values of His+ revertant colonies

at 500 μg/plate; nt: not tested; GI: growth inhibition

Scientific name Extract

Mean values of His+ revertant colonies

TA98 TA100

Without

metabolic activation

With

metabolic activation

Without

metabolic activation

With

metabolic activation

Securidaca

longipedunculata

eE001 41 447 445 926

wE001 82 328 497 1164

hE001 nt 186 nt 824

mE001 36 551 369 369

smE001 46 479 487 43

Microgramma

lycopodioides

eE002 61 452 445 993

wE002 64 527 336 721

smE002 76 411 537 947

Ficus saussureana

eE003 47 427 316 677

wE003 53 416 501 1011

hE003 60 336 487 909

mE003 45 383 428 995

smE003 51 345 517 893

Sesamum

calycinum subsp.

angustifolium

eE004 41 397 423 984

wE004 51 481 521 1039

hE004 343 321 465 941

mE004 52 560 449 1019

smE004 36 481 488 1125

Leucas

calostachys

eE005 48 276 429 735

wE005 52 460 537 1083

hE005 61 388 477 967

smE005 53 417 519 988

Solanum

aculeastrum

eE006 45 377 597 968

wE006 nt 537 Nt 939

hE006 567 288 497 852

smE006 43 337 540 944

Albizia coriaria

eE007 39 463 416 979

etE007 59 492 516 996

Erythrina

abyssinica

eE008 44 204 328 819

etE008 54 186 304 721

Zanthoxylum

chalybeum

eE009 56 487 560 1009

etE009 44 499 473 943

etE017 nt nt nt nt

dietE017 37 nt 581 nt

etE017a 46 468 445 1372

dietE017a 39 333 385 853

Toddalia asiatica eE010 204 435 1228 985

165

etE010 94 219 963 732

dietE010 214 319 1180 1203

etE010a 91 509 995 889

Harungana

madagascariensis

eE011 52 435 431 985

etE011 47 320 451 1131

dietE011 38 186 336 565

etE011a 68 385 439 644

Morella kandtiana

etE012 45 523 471 973

etE012a 54 564 439 848

dietE012 52 391 220 901

Cassine

buchananii

eE013 47 391 496 1053

etE013 50 531 488 1632

etE013a 48 445 396 919

Warburgia

ugandensis

etE014a 40 437 217 1254

dietE014 0 (GI) 0 (GI) 0 (GI) 0 (GI)

Combretum molle eE015 46 543 352 1212

etE015 53 573 477 955

Plectranthus

hadiensis

dietE016 51 543 nt 1212

hE016 50 268 447 821

spontaneous

reverse mutations

negative

control 53 496 315 983

2-NF positive

control 1000

MMS positive

control 2064

2-AF positive

control 54 6130 704 6001

Supplementary Figure S1:

Figure S1. Plate photos of revertant colonies for extract eE007 (Albizia coriaria, non-mutagenic against test strain

TA98) and hE006 (Solanum aculeastrum, mutagenic against test strain TA98)

166

Supplementary Table S2:

Table S2. Overview of pipetting instructions during the Ames test procedure with metabolic activation

Buffer

control

Sample DMSO

(vehicle

control)

Positive control

TA 98

Positive control

TA 100

Pre-incubation assay

DMSO

50 μL 100 μL 90 90

S9 mixture 600 μL 500 μL 500 μL 500 μL 500 μL

Plant extract solution

(10 mg/ml)

50 μL

2-AF solution

10 μL 10 μL

Incorporation into the Ames test

Overnight culture

(TA98 or TA100)

100 μL 100 μL 100 μL 100 μL 100 μL

Top agar 2.0 mL 2.0 mL 2.0 mL 2.0 mL 2.0 mL

Supplementary Table S3:

Table S3. Information on bacterial strains used in the study

Species Strain IDs Characteristics* Ref.

Salmonella enterica

subsp. enterica

Typhimurium

TA 98

CIP 103798

Optimized for frameshift

mutations, DNA target: –C–G–C–

G–C–G–C–G–; his D3052 rfa

ǻ(gal chl bio uvrB) / pKM101

Source: Centre de Ressources

Biolo

iques de l'Institut Pasteu

1-4

Salmonella enterica

subsp. enterica

Typhimurium

TA 100

CIP 103799

Optimized for base-pair

substitution mutations, DNA target:

–G–G–G–; hisG46 rfa ǻ(gal chl bio

uvrB) / pKM101

Source: Centre de Ressources

Biolo

iques de l'Institut Pasteu

3,5,6

Salmonella enterica

subsp. enterica

DSM-No.

11320

Wild type 7,8

167

Supplementary Figure S2:

Figure S2. Results of growth comparison of his- mutants with the wild type regarding +/- ampicillin genes

Supplementary Table S4:

Table S4. Overview of pipetting instructions during the Ames test procedure without metabolic activation

Buffer

control

Sample DMSO

(vehicle

control)

Positive control

TA 98

Positive control

TA 100

Overnight culture

(TA98 or TA100)

100 μL 100 μL 100 μL 100 μL 100 μL

DMSO

50 μL 100 μL 90 90

Buffer 600 μL 500 μL 500 μL 500 μL 500 μL

Plant extract solution

(10 mg/ml)

50 μL

MMS solution

10 μL

2-NF solution

10 μL

Top agar 2.0 mL 2.0 mL 2.0 mL 2.0 mL 2.0 mL

168

References cited in supplementary files

1 FBRCMi. CIP 103799 - Salmonella enterica enterica Typhimurium,

<https://brclims.pasteur.fr/brcWeb/souche/detail/1/15636> (1993).

2 BacDive. Salmonella enterica CIP 103798 BacDive ID: 139109,

<https://bacdive.dsmz.de/search?search=CIP+103798> (2020).

3 Mortelmans, K. & Zeiger, E. The Ames Salmonella/microsome mutagenicity assay. Mutation

Research/Fundamental and Molecular Mechanisms of Mutagenesis 455, 29-60,

doi:https://doi.org/10.1016/S0027-5107(00)00064-6 (2000).

4 Isono, K. & Yourno, J. Chemical carcinogens as frameshift mutagens: Salmonella DNA

sequence sensitive to mutagenesis by polycyclic carcinogens. Proc. Natl. Acad. Sci. U. S. A.

71, 1612-1617, doi:10.1073/pnas.71.5.1612 (1974).

5 FBRCMi. CIP 103799 Salmonella enterica enterica Typhimurium,

<https://brclims.pasteur.fr/brcWeb/souche/detail/1/15636> (1993).

6 BacDive. Salmonella enterica CIP 103799 BacDive ID: 139108,

<https://bacdive.dsmz.de/search?search=CIP+103799> (2020).

7 DSMZ. Salmonella enterica subsp. enterica DSM 11320,

<https://www.dsmz.de/collection/catalogue/details/culture/DSM-11320> (1996).

8 BacDive. Salmonella enterica Zoosaloral H BacDive ID: 5113

<https://bacdive.dsmz.de/strain/5113> (2020).

169

Manuscript VI – Video article with accompanying short written article:

"Transferring ethnopharmacological results back to traditional healers

in rural indigenous communities –

The Ugandan Greater Mpigi region example"

Pages: 170-180

Personal contribution

In the following, my personal contribution to the presented study and manuscript is briefly

described: I designed the overall strategy of the study, conducted the workshop, and wrote the

manuscript for the video article. I contributed to the organization of the workshop and the

interpretation of the video footage. I wrote the short article. A more detailed author-

contribution statement is given in the submitted article.

Information on publication

This study was submitted to the Video Journal of Education and Pedagogy on February 18,

2021, and is presented as a preprint version. If accepted, it will be an open access article

distributed under the Creative Commons Attribution 4.0 International License (CC BY 4.0)

and will also be hosted on my YouTube channel for science outreach: Ethnopharmacology –

Fab_Ethnopharm.

Schultz, F.; Dworak-Schultz, I.; Olengo, A.; Anywar, G.; Garbe, L.-A.: Transferring

ethnopharmacological results back to traditional healers in rural indigenous communities – The

Ugandan Greater Mpigi region example. Video Journal of Education and Pedagogy,

manuscript submitted on February 18, 2021 (in review)

170

Access link to video article

Transferring ethnopharmacological results back to traditional healers in rural

indigenous communities – The Ugandan Greater Mpigi region example

http://dx.doi.org/10.14279/depositonce-12048

171

Transferring ethnopharmacological

results back to traditional healers in rural

indigenous communities – The Ugandan

Greater Mpigi region example

Fabien Schultz,a,b* Inken Dworak-Schultz,c Alex Olengo,d Godwin Anywar,d Leif-Alexander Garbeb,e

a Institute of Biotechnology, Faculty III - Process Sciences, Technical University of Berlin, Gustav-Meyer-Allee 25, Berlin,

13355, Germany

b Department of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences, Brodaer Str. 2,

Neubrandenburg, 17033, Germany

c ARUDEVO, Lwengo, Uganda

d Department of Plant Sciences, Microbiology and Biotechnology, Makerere University, P.O Box 7062, Kampala, Uganda

e ZELT - Neubrandenburg Center for Nutrition and Food Technology gGmbH, Seestraße 7A, Neubrandenburg, 17033,

Germany

* Corresponding author: [email protected]

Abstract

In the field of ethnopharmacology, scientists often conduct field studies, surveying indigenous

communities to identify and collect understudied natural remedies such as medicinal plants that are

yet to be investigated pharmacologically in a laboratory setting. The Nagoya Protocol and the

Convention on Biological Diversity provided international agreements on financial benefit sharing and

recognized each nation’s sovereignty over the biodiversity resources within its borders. However,

what has yet only been poorly defined in these agreements are the non-financial benefits for local

intellectual property right owners and the bidirectional transfer of knowledge back to the traditional

healers who originally provided the respective ethnomedicinal information. Unfortunately,

ethnopharmacologists still rarely return to the local communities after the sample collection,

laboratory analysis, and publication of results in scientific journals. In this video article, we present a

method for transferring results back to traditional healers in rural indigenous communities, taking our

previous studies among 39 traditional healers in the Greater Mpigi region in Uganda as an example.

Our approach is based on a two-day workshop, and the results are presented as original footage in

the video article. Our work demonstrated a successful method for ensuring bidirectional benefit and

communication while fostering future scientific and community-work collaborations. We believe it is

the responsibility and moral duty of ethnopharmacologists to contribute to knowledge transfer and

feedback once a study is completed.

Keywords

ethnopharmacology; traditional medicine; traditional healers; workshop; Mpigi; transfer of results;

Uganda; medicinal plants

172

1 Introduction

Across the globe and throughout history, plants have been used by humans as a source of medicine

and natural remedies.1,2 The World Health Organization defines traditional medicine as "the

knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different

cultures, used in the maintenance of health and in the prevention, diagnosis, improvement or

treatment of physical and mental illness."3 Traditional medicine continues to be of great importance

to all human beings.4 In the developing world, over 80% of the population still relies on medicinal

plants as their primary source of healthcare.1,2,5,6 Even in the modern Western pharmaceutical

industry, traditional medicine still plays a key role in drug discovery.7-11 For instance, nearly 50% of all

drugs that are currently FDA-approved have been derived directly or indirectly from natural

sources.12,13

Here, the science of ethnopharmacology seeks to investigate the medicinal use of plants, animals,

macrofungi, microorganisms, and minerals through pharmacological, socio-cultural, and

anthropological methods. Ethnopharmacology is a highly interdisciplinary field of research,14,15

encompassing a) field studies (such as ethnobotanical studies in local communities, interviews,

surveys, and the first time documentation of medicinal use, ritual use, or religious aspects), b) the

pharmacological assessment of recorded and collected medicinal species in a laboratory setting (so-

called “bioactivity studies”), and c) drug discovery of pharmacologically active natural products via

pharmacognostic approaches. These activities may be expanded to include community work, as we

believe ethnopharmacologists should also act as advocates for the respective indigenous communities

with whom they collaborate.

Throughout history, the intellectual property rights of indigenous peoples have not been recognized,

and questions concerning the ownership of biodiversity following the development and

commercialization of pharmaceuticals have arisen. The Nagoya Protocol and the Convention on

Biological Diversity provided international agreements on financial benefit sharing and recognized

each nation’s sovereignty over the biodiversity resources within its borders.15-18 But what about non-

monetary benefits? What about the transfer of knowledge in both directions? Unfortunately, even

today, ethnopharmacologists rarely return to the local communities after a study has been completed

and published.19,20 Thus, the successful collection of plant samples and ethnopharmacological

information from traditional healers and other community members often marks the end point of this

one-sided collaboration, despite the fact that this data will still be analyzed, interpreted, and published

(see Figure 1). This problem was previously addressed in a book by Herman et al.21

Laboratory studies may follow, leading to unique significant discoveries that would certainly be of

interest to the local study participants and could even empower them locally while fostering an equal

partnership.22-24 If the scientists ever return, then in many cases it is only because of an entirely new

study, aimed at extracting new information for their research. We believe that ethnopharmacologists

therefore have the great responsibility of keeping this collaboration and the communication with their

local informants bidirectional. Information and knowledge should be shared, creating a benefit for

both the scientists and the local study participants.

173

Figure 1: Objectives of ethnopharmacological research, showing the fieldwork and laboratory steps. But what comes

next?

1.1 Previous ethnopharmacological studies

In this video article, we would like to introduce a method for transferring the results of laboratory

analyses and ethnobotanical surveys back to traditional healers. Our approach is based on a two-day

workshop, using our previous studies from the Greater Mpigi region in Uganda as an example. A total

of 16 medicinal plant species were investigated as part of an ethnobotanical survey among 39

traditional healers from the Greater Mpigi region. This past study has recently been published in the

Journal of Ethnopharmacology.20 Traditional healers from 29 different villages, including one from a

small island in Lake Victoria, were interviewed. The study involved the first-time documentation of

preparation and administration methods and the identification of a total of 75 medical disorders that

are treated with these medicinal plants. In this study, information was obtained using questionnaires

that were specifically designed to collect in-depth data on each species. Figure 2 shows three

photographs from this field study.

Figure 2: Illustrations of Ugandan plant diversity and traditional African community life

174

In another follow-up study that was published, we applied the Degrees of Publication (DoP) method

as a novel tool for literature assessment in ethnopharmacological research.6 There are numerous field

assessment tools in use today. However, none of these tools are able to help researchers determine

which species merit the costly lab studies that would be required for their further investigation, e.g.,

pharmacological assays and the isolation of bioactive natural product compounds. The introduction

of the DoP method has filled this gap. In the context of the aforementioned ethnobotanical survey,

the DoP method made it possible to classify six of the 16 medicinal plant species as “highly

understudied” and three as “understudied.”6

At the Makerere University herbarium, taxonomic identification was accomplished based on the

herbarium voucher specimens that were prepared during fieldwork. Following the fieldwork activities,

samples of all 16 plant species were taken to the laboratory, where extracts were produced.7 Various

pharmacological investigations followed on the basis of the use reports provided by the traditional

healers. These included antimalarial, antibacterial, and antiinflammatory bioassays, among others

(see Figure 3). These unique investigations led to a large number of interesting results.

Figure 3: Illustration of our approach to ethnopharmacological lab studies aimed at the evaluation of traditional use and

drug discovery; HPLC: high performance liquid chromatography, GC/LC-MS: gas chromatography/liquid chromatography-

mass spectrometry, NMR: nuclear magnetic resonance spectroscopy.

During our fieldwork, we explicitly asked the 39 traditional healers about their motivation for

collaborating with us.20 The specific questions that we asked were: “What are your future expectations

from our scientific findings, and what do you expect from us researchers?” Participants were invited

to give multiple responses. Their answers were fascinating. Despite the fact that these traditional

healers live in relatively poor circumstances, only 5% stated that they would like to benefit financially

from the scientific information gained. On the contrary, there was a high demand for feedback on the

175

results of the survey and the laboratory studies, which we regard as the transfer of knowledge. The

majority of the traditional healers said they would like to improve and continue their collaboration

with us researchers. Nine percent were interested in a collaboration for improving their treatment of

patients, and 11% wanted to strengthen their collaboration with us. The second most common

expectation was to receive feedback on the findings of the pharmacological studies that followed the

fieldwork. In addition, more than a quarter of the traditional healers mentioned that they would be

interested in finding out whether scientific evidence could be found for the claimed medicinal

properties of the investigated plants, as such evidence would boost their confidence in the respective

treatments.20 Their responses indicated that there is a vital need for feedback from

ethnopharmacologists after a study is completed, as well as a strong interest in continued

collaboration and participation in the research.

2 Methods

The results of the laboratory work and ethnobotanical survey were shared with the traditional healers

through a two-day workshop in November 2019. All of the traditional healers who had initially

participated in the field study were invited to the workshop.20 In order to contact them again, we used

the network of the NGO PROMETRA Uganda (www.prometraug.com). Together with PROMETRA, we

organized transport and food. The workshop took place at the local PROMETRA headquarters in

Buyijja, Buwama sub county, in the Greater Mpigi region. Written informed consent for the filming

and publishing of the video footage was requested and obtained from all participants shown in this

video article.

One of the possible reasons why so few scientific findings are transferred back to indigenous peoples

and traditional healers is that scientific articles are often incomprehensible and inaccessible to them.25

Therefore, one major challenge was to explain the scientific results in a way that was appropriate to

their level of understanding. Western researchers face the same challenge in the context of science

outreach activities among the general public in their home countries. However, when such outreach

activities are organized in other countries, there should also be an emphasis on cultural

appropriateness. Our strategy for conveying complicated scientific results was to explain the assays

and results figuratively and in a simplified manner, using everyday examples.

Another challenge was the local language Luganda, which is why multiple translators were present at

the workshop.

The workshop was divided into three parts:

• Day 1 (morning): Transfer of results in a classroom setting using a laptop and projector (results

explanation)

• Day 1 (afternoon): Group discussion and questions from healers in the garden

• Day 2 (all day): Group visits to some of the healers’ homes

The healers visited on Day 2 were traditional healers who had participated in another study within the

Greater Mpigi region, and the video footage was intended as an example of how the bidirectional

communication and continued collaboration could be established.

176

3 Results

The results from each of the three parts of the workshop were presented separately in the video article

as video impressions.

Day 1 (morning): Transfer of results in a classroom setting using a laptop and projector

For example, after we asked the workshop participants for permission to film them, one of the

traditional healers asked us how the video would benefit them. Responses were given in the original

video footage shown in the video article. During the first part, we presented the outcome of the initial

questionnaires, as well as various results from our diverse lab studies (antimalarial, anti-inflammatory,

antivirulence, antibiotic, cytotoxicity, and genotoxicity assays). Figure 4 shows a photo of the organizer

of the workshop, Fabien Schultz, and some of the 39 traditional healers during the results-explanation

part of the workshop. The limitations of in vitro studies were explained during the presentation of lab

results, along with the explanation that follow up studies were needed in the future in order to fully

verify the pharmacological effects in vivo. The presented lab studies provided initial scientific evidence

for the potential therapeutic efficacy of the medicinal plants used by the healers in the Greater Mpigi

region.

Figure 4: Photo illustrating the transfer of results in a classroom setting (day 1, workshop part 1)

Day 1 (afternoon): Group discussion and questions from healers in the garden

The traditional healers wanted to know more about the testing methods and what they could learn

from them when it comes to preparing their medicines, which led to a very interesting discussion. The

questions from the healers focused on the limitations of science, the scope of laboratory experiments,

regional differences in the concentrations of active ingredients in plants, and even the role of

spiritualism in traditional medicine. Figure 5 shows a picture from the group discussion under a tree

in the garden.

177

Figure 5: Photo showing the group discussion (day 1, workshop part 2)

Day 2 (all day): Group visits to some of the healers’ homes

This video footage gave impressions on how the traditional healers find and harvest some of the forest

plants investigated in the laboratory studies, how they process these plants, how they sell them on

the market, and how they treat patients. The group visits to their homes led to bidirectional

communication, strengthening trust and interest in future collaborations. New research topics were

identified via brainstorming methods, and traditional healers expressed their interest.

Figure 6: Traditional healer Salongo Kato Konde explains how to harvest Toddalia asiatica in the local forest (photo

taken during visits to the homes of individual healers )

178

4 Conclusion

Conducting a workshop for traditional healers and indigenous communities is an efficient way to

transfer the results of ethnobotanical and ethnopharmacological studies back to local study

participants. This video article, showing a workshop along with visits to some of the healers’ homes,

demonstrated a successful method of how bidirectional benefits and communication is possible as a

starting point, fostering future scientific and community-work collaborations. Of course, our approach

and workshop concept may not be suitable for all local and indigenous peoples, cultural backgrounds,

or situations that may emerge during various aspects of ethnopharmacological research throughout

the world. It is meant as an example, and as always, scientists’ individual approaches need to be

adapted to the given circumstances. What remains a fact is that very few scientific findings are

transferred back to the traditional healers and indigenous peoples who originally laid the foundation

for the advanced ethnopharmacological research endeavors. For example, it is possible that

subsequent laboratory studies could even reveal that some medicinal plants pose a threat to local

communities, because they are toxic and harmful to patients as a direct outcome of treatment. We

believe that ethnopharmacologists should contribute to improving local herbal drug use, help reduce

health hazards derived from medicinal plants, care and advocate for local communities, and create

and maintain good relationships for future collaborations.

Consequently, we feel that ethnopharmacologists should commit to transferring the results of their

studies back to their informants, for example, through medicinal plant workshops.

Conflicts of Interest

This study was performed according to the international, national, and institutional rules considering

the Convention on Biodiversity and the Nagoya Protocol. Written informed consent was requested

and obtained from all participants of the workshop and all participants shown in this video. The

authors declare that the research was conducted in the absence of any commercial or financial

relationships that could be construed as a potential conflict of interest. The content is solely the

responsibility of the authors and does not necessarily reflect the official view of the funding agencies.

The funding agencies had no role in the study design, data collection and analysis, decision to publish,

or preparation of the manuscript/video article.

Acknowledgements

Our greatest thanks to the traditional healers who participated in the workshop, and to PROMETRA

Uganda for providing a location and a network of contacts within the previously surveyed

communities. Thanks to student research assistants Ssemukasa Steven and Muyanja Joseph for

assisting during the workshop. Special thanks to the Neubrandenburg University of Applied Sciences

for supporting F.S.’s fieldwork activities in Uganda in terms of working hours. We acknowledge

support for the Article Processing Charge from the German Research Foundation (DFG, 414051096)

and the Open Access Publication Fund of Neubrandenburg University of Applied Sciences (HSNB).

Author Contributions

Fabien Schultz (F.S.) designed the overall strategy of the study, conducted the workshop, and wrote

the manuscript for this video article. Inken Dworak-Schultz (I.D.S.) edited the video, produced the

voice-over, and acted as head of video production. F.S. and Godwin Anywar (G.A.) organized the

workshop. I.D.S., G.A., and Alex Olengo (A.O.) filmed during the workshop, at the lab, and at the

traditional healers’ homes. A.O. translated the interviews from Luganda to English. F.S. and I.D.S.

interpreted the video footage. Leif-Alexander Garbe directed the study. All of the authors watched,

revised, and approved the final video article.

179

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Discussion and conclusions

The scientific findings of the six individual studies were already discussed thoroughly in the

discussion sections of each manuscript. In these sections, reported pharmacological effects for

individual plant species were interpreted with regard to their context, including detailed

statements on similar findings by other lab groups and literature reviews. This assessment will

not be repeated in this chapter. In the following, the totality of the findings will be summarized

and discussed, the most promising plant species for drug discovery will be highlighted, the

limitations of my PhD studies will be stated, and an overview of future studies that may build

on the results of this PhD thesis to further explore the traditional use, pharmacology, and

phytochemistry of the plant species will be provided.

Initially, the 16 plant species were selected for the research because preliminary

interviews with a small number of traditional healers near the study site indicated that all of

these species seemed to be frequently applied for treatment of bacterial infections, wounds,

malaria, inflammation, and cancer. These claims of a high level of traditional use of the species

were successfully confirmed via an ethnobotanical survey among a larger number of local

traditional healers while also covering a larger study area. Therefore, this first phase of the

PhD studies made the documentation of traditional use in the Greater Mpigi region possible,

resulting in use reports for 75 medical disorders treated with the 16 medicinal plants, many for

the first time ("Publication I"). The results obtained are important for future generations

because the accurate documentation of plant use, including parts used, and methods of

preparation and administration, eventuates in the conservation of traditional knowledge, which

can facilitate future drug discovery research endeavors, among other benefits. The loss of

culturally and scientifically valuable medical knowledge is a global problem affecting all of

us. Such observations have recently been expressed by both policymakers and researchers

(Bussmann et al., 2018). The demand for more effective novel drugs derived from natural

product sources is higher and more vital than ever, making field studies for documentation of

herbal remedies and subsequent pharmacological lab investigations both urgently required and

justified. Thus, the high level of traditional use of these species warranted extensive lab studies

following the ethnopharmacological approach. Plant material was repeatedly collected during

multiple field research phases throughout my PhD research and processed for lab experiments.

The development of the DoP method allowed for extensive bibliographic and

comparative assessment of the 16 medicinal plant species, including the determination of the

degree to which each species had been studied before and the evaluation of the quality of the

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journals in which results had been published ("Publication I"). The novel method made it

possible to screen, sort, and classify published literature according to a scheme that was called

the "research chain of ethnopharmacology," ranging from ethnobotanical studies

(DoPtraditional use) and pharmacological assays (DoPbioactivity) to pharmacognostic research

(DoPstructure elucidation). The literature analysis resulted in the review of a total of 634 peer-

reviewed scientific articles published between 1960 and 2019. In fact, the majority of the 16

plants that were taken to the lab were identified as either "highly understudied" (Cassine

buchananii, Ficus saussureana, Leucas calostachys, Microgramma lycopodioides, Morella

kandtiana, and Sesamum calycinum subsp. angustifolium) or "understudied" (Albizia coriaria,

Plectranthus hadiensis, and Solanum aculeastrum). As a conclusion, there are many field

assessment tools in the field of ethnopharmacology. However, strategies for the selection of

medicinal plants remain a challenge as existing tools miss that critical point of how to really

determine which species to include in costly lab studies, such as pharmacological assays and

isolation of bioactive secondary metabolites. The new DoP method finally addresses this

problem not only by assessing what is already known about a species, but also by estimating

the quality of the evidence. The tool will be of great value when it comes to avoiding the

reproduction of results and costs, while fostering a more time-efficient approach to

ethnopharmacological research and drug discovery. Gaps in the literature can now be filled

more strategically, making research more efficient and targeted.

The three "bioactivity studies" ("Publication III," "Publication IV," and

"Manuscript V") sought to investigate the pharmacological properties of the 16 medicinal

plants and up to 86 extracts that were produced from them. During the ethnobotanical

interviews, the traditional healers provided ethnobotanical information on the medicinal use of

the species, and frequently cited was their application in the treatment of bacterial infections,

wounds, malaria, inflammation, pain, and fever. The pharmacological in vitro models for the

assessment of the medicinally used species and the initiation of early drug discovery stages

were therefore geared towards these use reports provided by the traditional healers, as

described individually in "Publication III," "Publication IV," and "Manuscript V." These lab

investigations led to a high quantity of interesting results. Antibacterial, antivirulence,

antiinflammatory, and antimalarial activity was reported in vitro for the first time for the

majority of the species investigated, which had been previously assessed by the DoP method

("Publication II"). The study results indicated that the pharmacological potential of plant

material highly depends on the species, which plant part is used, the time and location of

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harvest, and the solvents used for extraction. In general, the results often supported the

therapeutic use of the majority of the species in the Greater Mpigi region. For example, 13 out

of 16 species traditionally used in the treatment of infections and wounds showed significant

antibiotic or antivirulence effects in our pharmacological in vitro models against a panel of

clinical isolates of multidrug-resistant ESKAPE pathogens. S. calycinum subsp. angustifolium,

L. calostachys, S. aculeastrum, M. kandtiana, Warburgia ugandensis, and Zanthoxylum

chalybeum simultaneously displayed both quorum-sensing inhibition and growth inhibition

effects on the strains investigated, while exhibiting no cytotoxic effects against human

keratinocytes (HaCaT cells) at the highest test concentration of 512 μg/mL. Potential

antibacterial properties, e.g., growth inhibition effects of extracts, were studied twice against

different strains of human pathogens, and the results were published in two manuscripts

("Publication III" and "Publication IV"). Here, the most active extracts were obtained from

Harungana madagascariensis stem bark (MIC values: Staphylococcus aureusATCC 25923:

13 μg/mL; S. aureusATCC 49230: 32 ȝg/mL; Enterococcus faecium: 32 ȝg/mL; Listeria innocua:

40 μg/mL; Listeria monocytogenes: 150 μg/mL) and from Z. chalybeum stem bark (MIC

values: S. aureusATCC 25923: 13 μg/mL; S. aureusATCC 49230: 16 ȝg/mL; E. faecium: 32 ȝg/mL).

In vitro antiinflammatory effects were reported for most of the plant species, and a total

of 15 out of the 16 medicinal plants exhibited significant inhibitory activity in the COX-2

model ("Publication IV"). The results provide an early-stage scientific evidence for the

therapeutic use of these natural remedies in the treatment of inflammatory disorders, such as

fever, redness or pain. The only plant that did not display COX-2 inhibition activity at the

highest test concentration of 50 ȝg/mL was Combretum molle. There were nine extracts

identified as the most active COX-2 inhibitors in the library (IC50 < 20 μg/mL). These extracts

were produced from the following species: L. calostachys, S. aculeastrum, S. calycinum subsp.

angustifolium, P. hadiensis, M. kandtiana, Z. chalybeum, and W. ugandensis. The calculation

of EC50 values for DPPH radical scavenging activity (antioxidant activity) and the

determination of the TPCs displayed no correlation regarding COX-2 inhibition activity in

active extracts, which led to the assumption that the mechanism of action is likely not based on

reactive oxygen species scavenging, e.g., due to presence of elevated levels of polyphenols, as

sometimes suspected for other antiinflammatory (herbal) drugs (Schinella et al., 2002; Priya et

al., 2008; Allegra, 2019; Eom et al., 2020).

However, the results of the antimalarial experiments suggest that the potential

molecular mechanism of action of COX-2 inhibiting plant extracts may be similar to their

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antiplasmodial and hemozoin formation inhibition activity. Each of the 16 plants were cited to

be used for the treatment of malaria by at least four of the 39 traditional healers (Schultz et al.,

2020b). Based on their displayed hemozoin formation inhibition activity in the in vitro heme

biocrystallization assay, seven extracts from five plant species (S. calycinum subsp.

angustifolium leaves, L. calostachys leaves, Z. chalybeum stem bark, P. hadiensis leaves, and

W. ugandensis stem bark) were introduced to the antiplasmodial follow-up investigations

against the chloroquine-resistant Plasmodium falciparum K1 strain ("Publication IV"). In

general, the modified heme biocrystallization assay proved to be an effective method for pre-

screening sample libraries for antimalarial effects in absence of Plasmodia, since all seven

extracts also exhibited significant antiplasmodial activity, justifying their selection. The

recorded antiplasmodial activity did not seem to be selective (selectivity indices (SIs) <10),

which may be caused by the compositional complexities of the extracts. Interestingly, these

five plant species were also the species that had displayed the highest COX-2 inhibition activity

in the previous study ("Publication III"). In both assays (COX-2 inhibition assay and heme

biocrystallization assay), free heme plays a vital role. Free heme was introduced externally to

the COX reaction during the antiinflammatory assays as an essential co-factor for COX

isozymes (Chandrasekharan and Simmons, 2004; Schultz et al., 2021c). It is also released

during the degradation of hemoglobin by the malaria parasite and then detoxified by heme

biocrystallization (Schmitt et al., 1993; Roy, 2017). In the past, scientists had hypothesized that

the inhibition of hemozoin formation occurs due to the ability of the drug chloroquine to form

complexes with free heme (Cohen et al., 1964; Chou et al., 1980; Egan, 2001; Egan, 2004). A

recent in vivo study investigated the potential mode of action of quinoline antimalarial drugs

via correlative X-ray microscopy (Kapishnikov et al., 2019). The major finding was that drug

molecules in the digestive vacuole of the plasmodia indeed establish a complex with the free

heme, making it unavailable for biocrystallization, while also covering and blocking available

docking sites on the surface of the hemozoin crystals produced by healthy parasites. This

intervention by the drug molecules caused membrane puncture and spillage of heme, eventually

leading to the death of the plasmodia (Kapishnikov et al., 2019). Thus, the potential chelation

of the co-factor free heme and complex formation with affine secondary plant metabolites,

making it unavailable for the enzyme, may be one possible explanation for the reported

COX-2 inhibition and resultant antiinflammatory properties.

In the antimalarial investigations, the extract displaying the strongest antiplasmodial

activity was a diethyl ether extract of W. ugandensis stem bark, with an IC50 value of 0.5 μg/mL.

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However, due to strong cytotoxic effects against human MRC-5SV2 lung fibroblast, the

calculated SI was relatively low, making this plant potentially less suitable for prioritizing in

future studies for isolation and discovery of antimalarial drug leads. In the ethnobotanical

survey, the traditional healers stated that they use W. ugandensis for the treatment of various

types of cancer (Schultz et al., 2020b). Therefore, it will be interesting to conduct further

research on the plant's cytotoxic effects, especially when looking into the selective elimination

of related cancer cells.

The genotoxicity assessment for potential mutagenic/procarcinogenic properties of

plant extracts in the library showed significant direct mutagenic effects of four extracts from

three species against test strain TA98 (susceptible to frameshift mutations) and four extracts

from T. asiatica leaves against test strain TA100 (susceptible to base-pair substitution

mutations) at 500 μg/plate. Here, all four T. asiatica extracts in the library nearly quadrupled

the base-pair substitution mutation rates. Two of these extracts from T. asiatica were also

among the extracts that displayed high mutagenic effects against TA98 (MIs: 3.6 and 4.0),

while the other two exhibited low, but detectable mutagenicity (MIs: 1.7 and 1.8). To my

knowledge, this had been the first assessment of potential genotoxic properties for the 16

medicinal plant species, as well as the first report of mutagenic effects for Toddalia asiatica.

However, after in vitro simulation of human liver activity (elimination of xenobiotics) via pre-

incubation treatment of extracts with human S9 liver fraction, none of the extracts continued

to display mutagenic effects on the test strains. This indicates that effective deactivation of

potential procarcinogens occurred during the treatment with the human liver enzymes, and as

a result, previously mutagenic extracts did not retain their genotoxic activity.

Consequently, the ritual plant P. hadiensis, which was suspected to be linked to the

elevated prevalence of potential breast cancer (rapidly growing masses; not necessarily breast

cancer) in young female patients in the Ugandan Wakiso district, did not display

mutagenic/genotoxic properties against either test strain regardless of whether metabolic

bioactivation was initiated. On the contrary, extracts and isolated terpenoids from P. hadiensis

were previously reported to possess anticancer (cytotoxic) effects (Minker et al., 2007;

Mothana et al., 2010; Menon and Gopalakrishnan, 2015). At our field study site in the Greater

Mpigi region, the plant is also used in the treatment of multiple types of cancer, especially skin

cancer (RFC: 36%) (Schultz et al., 2020b). The elevated incidence of local rapidly growing

breast masses in young women needs to be further investigated. However, without local

patients undergoing biopsies and without further investigations regarding the origins of disease,

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it can only be speculated that it might be associated with genetic predisposition or that it could

be induced by some unknown environmental stimulus. Inherited genetic variations commonly

occur in relatively isolated ethnicities (Chlebowski et al., 2005; Brennan, 2017). However, the

cause and development of cancer is highly complex, meaning that P. hadiensis could still play

a role in pathogenesis. This needs to be considered especially in puberty and shortly thereafter

due to the previously described window of susceptibility for breast cancer, as a first incident

of cell growth perturbation may be caused (Davis and Lin, 2011; Macias and Hinck, 2012;

Martinson et al., 2013; Natarajan et al., 2020). Phytoestrogens present in the plant could

contribute to tumor growth (Stopper et al., 2005; Bilal et al., 2014). In communities with a high

prevalence of inactivated tumor suppressor genes, P. hadiensis could potentially act as such an

external stimulus in a yet unknown mechanism, while still being inactive in the Salmonella

reverse mutation assay. Further studies are therefore needed in order to fully evaluate the safety

of P. hadiensis in ritual use.

Three medicinal plant species need to be highlighted as one of the major results of this

PhD thesis. They were identified due to their displayed potent pharmacological activities paired

with a low degree of being studied so far, thus promising a high potential for future discovery

of novel drug leads. These highlighted plant species are L. calostachys (leaves), S. calycinum

subsp. angustifolium (leaves), and S. aculeastrum (root bark). In summary, the DoP analysis

classified L. calostachys and S. calycinum subsp. angustifolium as "highly understudied," and

S. aculeastrum as "understudied" ("Publication II"), indicating a vital need for the study of

these species' pharmacological properties.

In the antiinflammatory assays, an ethyl acetate extract of L. calostachys leaves

displayed the highest COX-2 inhibition activity in the library (IC50: 0.66 μg/mL), while also

reaching the most promising selectivity ratio of active extracts with 0.1 (COX-2/COX-1). This

was the first report of a strong in vitro antiinflammatory activity of L. calostachys. It should be

emphasized that other than NSAIDs, e.g., Aspirin or Ibuprofen, this is not a pure substance,

but a crude extract, containing hundreds or thousands of compounds. Compared to Aspirin

(IC50: 210 μg/mL) and Ibuprofen (IC50: 46 μg/mL), the L. calostachys extract acted as a much

more potent COX-2 inhibitor, while also displaying a much higher selectivity for COX-2

(COX-2/COX-1 ratios of Aspirin and Ibuprofen: 42 and 46) (Mitchell et al., 1993; Mitchell

and Evans, 1998; Mitchell and Warner, 1999; Varrassi et al., 2020). This indicates fewer side

effects of the extract compared to Aspirin and Ibuprofen due to less COX-1 and increased

COX-2 inhibition, but it needs to be further studied in the future. The same ethyl acetate extract

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from L. calostachys leaves also displayed the second strongest antiplasmodial properties

against chloroquine resistant P. falciparum K1 in the plant extract library, reaching an IC50

value of 5.7 μg/mL, and the highest selectivity index in the library (SI: 2.6) when taking

cytotoxicity against human MRC-5SV2 lung fibroblast into account ("Manuscript V"). In

addition, an n-hexane extract of L. calostachys leaves also exhibited significant quorum

quenching activity against the agr system in MRSA ("Publication II"), which merits further

pharmacognostic and ethnopharmacological research. As far as I know, no studies have been

published reporting the identification or isolation of pharmacologically active natural product

molecules from L. calostachys to date.

In this PhD thesis, an n-hexane extract from S. calycinum subsp. angustifolium leaves

and an ethyl acetate extract from S. aculeastrum root bark displayed strong QSI activity in

absence of growth inhibition when screening against the four S. aureus accessory gene

regulator (agr) alleles ("Publication III"). To my best knowledge, this was the first time

antivirulence effects had been investigated and reported for any of the 16 medicinal plant

species. Directly targeting quorum sensing and į-toxin production in S. aureus allowed for the

successful in vitro early-stage evaluation of the traditional application of S. calycinum subsp.

angustifolium and S. aculeastrum as anti-infective herbal drugs. The QSI activity reported for

these extracts were reporter gene subtype-dependent, reaching IC50 values of 4, 1, 16, and

64 ȝg/mL (agr I-IV), and 4, 2, 16, and 32 ȝg/mL respectively. Targeting the agr system allows

for the disruption of a wide variety of virulence factors, instead of trying to inhibit each

virulence factor individually. Furthermore, the selective inhibition of quorum sensing

pathways, compared to simply trying to kill the bacterial pathogen, may provide an efficient

alternative to conventional antibiotics in the future (Alksne and Projan, 2000; Kane et al.,

2018). As virulence factors are often not relevant to the overall survival of the pathogen, future

QSI therapies could theoretically facilitate a less selective pressure towards resistance when

fighting pathogens that tend to acquire resistance mechanisms during strong selective pressure

of conventional antibiotic therapy (Clatworthy et al., 2007; Maeda et al., 2012; Borges and

Simões, 2019). Both extracts significantly attenuated the į-toxin biosynthesis in two

high-toxin-producing S. aureus model strains, which confirmed the antivirulence activity of

the extracts. At the same time, both extracts displayed no cytotoxicity against human HaCaT

keratinocytes at the highest test concentration of 512 ȝg/mL. Apart from the antivirulence

effects of S. calycinum subsp. angustifolium leaves and S. aculeastrum root bark, extracts of

these two plants also showed strong antiinflammatory and, in the case of S. calycinum subsp.

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angustifolium, antimalarial activity in the test models ("Publication IV" and "Manuscript V").

On the one hand, the same ethyl acetate and an n-hexane extract of S. aculeastrum were the

second and third most potent COX-2 inhibitors in the plant extract library, reaching IC50 values

of 1.74 and 3.19 ȝg/mL (COX-2/COX-1 selectivity ratios: 0.2 and 0.8). The same n-hexane

and an ethyl acetate extract obtained from S. calycinum subsp. angustifolium, on the other hand,

exhibited significant COX-2 inhibition activity (IC50 values: 3.65 and 6.05 ȝg/mL). These

results further support the traditional use of the two plants in the treatment of inflammation,

pain, fever, swelling, and redness in the Greater Mpigi region. In addition, the same extracts

from S. calycinum subsp. angustifolium showed antiplasmodial properties against chloroquine-

resistant P. falciparum K1 (IC50 values: 19.6 and 21.9 ȝg/mL).

Finally, the video article ("Manuscript VI") introduces and visualizes an example of a

successful method for the transfer of ethnopharmacological results of field and lab studies back

to local communities. The 39 Ugandan traditional healers initially requested feedback from the

resulting studies during the ethnobotanical survey ("Publication I"), which was offered through

a two-day workshop in the Greater Mpigi region after the lab studies had been completed.

Unfortunately, in the field of ethnopharmacology, scientists still rarely report back to their local

informants once a study is completed (Maregesi et al., 2007; Schultz et al., 2020b). However,

there is a vital need for feedback and a strong interest in further collaboration after the

completion of studies, as the responses in the questionnaires of the survey strongly suggested.

The workshop has proved to be an efficient method for transferring results back to indigenous

and local study participants after ethnobotanical and ethnopharmacological studies. The video

article included visits to the homes of some of the healers, as well as extensive discussions in

the classroom and in nature, demonstrating how bidirectional benefit and communication is

possible in order to foster future scientific and community work collaborations.

Limitations and future directions

One limitation of the newly introduced DoP method is that it focusses on a logical approach to

the field of ethnopharmacology that can be applied in most but not all cases. The method

defines progress in ethnopharmacological research in a certain order, beginning with field

studies, continuing with the pharmacological assessment of the herbal drugs via lab

experiments, and finally progressing to the stage of natural product isolation and structure

elucidation studies. Another limitation is the problem of non-reproducibility of

pharmacological data. This issue is very complex and has often its origins in poor study design

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and lack of scientific rigor (Mullane et al., 2015; Percie du Sert et al., 2020). For example,

scientists might be keen to validate a traditional use up to the point of testing "bioactive"

extracts at ridiculous concentrations until an activity can be reported. The DoP method

estimates the quality of a scientific publication by assessment of the journal, where a study has

been published in, based on whether its publishing house is a member of the Committee on

Publication Ethics (COPE) or not. However, this approach does not guarantee adequate study

design or reproducibility of pharmacological data, but merely makes it less likely.

A major limitation of the pharmacological experiments presented in this PhD thesis is

the circumstance that pharmacologically active secondary plant metabolites within the crude

extracts were not identified / structure elucidated which would have gone beyond the scope of

extensive initial screening studies (and PhD studies) with such a high number of extracts. In

the future, bioassay-guided fractionation or other drug discovery strategies are needed to

phytochemically characterize the most active antibiotic, quorum quenching, antiinflammatory,

and antimalarial extracts. This could result in the isolation and discovery of promising natural

product molecules aimed at the development of novel drugs. Future studies will also require

research into the mechanisms of action of the herbal drugs and the compounds responsible for

the pharmacological effects. However, the pharmacological effects of bioactive extracts

reported in this PhD thesis might only occur when multiple active ingredients are in synergistic

relationships within the plant extracts, and natural product compound isolation procedures may

result in a loss of pharmacological activity. Therefore, synergy studies may also be required as

part of future investigations that may result from this PhD research. Another aspect of future

studies on the medicinal plant species will be the further in vitro and in vivo investigation of

toxicity, which will be vital for safety assessment.

When considering the great amount of promising plant extracts presented in this

dissertation, it is obvious that the subsequent isolation and the identification of novel drugable

compounds within this high biological diversity of secondary metabolites (and species, in

general) will be strongly limited by time, human resource, funding, and technical equipment

investments. Unfortunately, the time-consuming and labor-intensive strategy of bioassay-

guided fractionation and stepwise purification of natural products frequently results in the

"rediscovery" of already known molecules. This is one of major drawbacks in drug discovery

from natural products (Michel et al., 2013; Ito and Masubuchi, 2014; Hubert et al., 2017). One

potential approach to alleviate this drawback is the concept of dereplication. It is based on the

quick identification and disqualification of known natural product molecules in complex

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bioactive extracts via hyphenated techniques in order to make focusing on the discovery of

novel unknown compounds possible (Carnevale Neto et al., 2016; Hubert et al., 2017). Future

pharmacognostic efforts and follow-up studies on extracts identified in this dissertation could

therefore encompass techniques of plant metabolomics, e.g., LC/GC-MS-based methods,

strategically following the concept of dereplication.

In general, another limitation of the research approach is proceeding to a new stage of

research, which will be in vivo studies for the diverse pharmacological effects reported in vitro.

Conducting extensive in vivo studies with the entire extract library during the PhD research

would not have been a good use of lab and animal resources and could have been considered

unethical. As this thesis presented initial screening studies, it was not considered necessary at

this point to undertake in vivo studies investigating the extensive extract library as a totality

(prior to identification of the most promising plants and extracts and publication of in vitro

evidence). However, this will be a vital step for future research on the identified plants and

pharmacological effects. One area to examine further via in vivo studies could be the strongest

quorum sensing inhibitors in the plant extract library. These were leaf extracts of the species

S. aculeastrum and S. calycinum subsp. angustifolium, which are also used in wound treatment

in the Greater Mpigi region ("Publication I"). In two studies, QSI-active fractions from the

European Chestnut (Castanea sativa) and the Brazilian Peppertree (Schinus terebinthifolia)

significantly reduced dermonecrosis after infection of C5BL/6 and BALB/c mice with a

virulent MRSA strain (Quave et al., 2015; Muhs et al., 2017). It will be highly interesting to

further investigate the QSI effects of the two extracts from S. aculeastrum and S. calycinum

subsp. angustifolium in an in vivo skin lesion/abscess model. In addition, it will be essential to

further study the ability of these two extracts in potentiating the efficacy of conventional (by

now nearly ineffective) antibiotics, as well as in limiting the severity of disease by potential

deactivation of other virulence pathways, e.g., secretion systems or biofilm formation.

The assessment of antioxidant activity (DPPH assay) is another limitation of this

dissertation because all plants possess antioxidant properties, e.g., due to their polyphenols. As

stated in the "Rules of 5" of the Journal of Ethnopharmacology (JEP, 2021), this results in

assays having little meaning, and clear evidence needs to be provided via in vivo activity.

However, in correspondence with the determination of the TPC for each extract in the library,

this chemical assay was used to estimate and exclude the potential mechanism of action of

COX-2 inhibiting extracts, which seems like an appropriate approach at this stage of research.

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To my knowledge, the potential genotoxic effects of T. asiatica had not been

investigated and reported previously. However, the DoP analysis resulted in the identification

of T. asiatica as the second most studied species (after S. longipedunculata) and as a highly

applied species throughout the African continent. It is therefore of high importance that its

toxicity and potential carcinogenic properties be assessed using dose-response models and

other more advanced toxicological methods. In general, none of the tested plant extracts from

the library displayed significant genotoxic properties after metabolic bioactivation with human

S9 liver fraction. Nevertheless, in order to recommend the Ugandan plants for long-term use,

additional studies using other in vitro methods, such as the mammalian cell metaphase

chromosome aberration assay, the micronucleus assay, or the mouse lymphoma L5178Y cell

Tk (thymidine kinase) gene mutation assay (MLA), are required as recommended in the ICH

guideline S2 (R1) issued by the European Medicines Agency (EMA, 2013). In the case of

genotoxic "hits," studies using in vivo methods, e.g., the transgenic mouse test or the UDS test,

should be performed (EMA, 2013; Verschaeve, 2015). The Salmonella reverse mutation assay

also has some limitations that should be discussed. For example, the assay does not distinguish

between carcinogenic effects in varying tissues, as detected procarcinogens may become

inactive or even anticarcinogenic in a different tissue. The risk of false negatives in the

Salmonella reverse mutation assay is low due to its high sensitivity. However, there is relatively

low specificity, which leads to potentially misleading results (false positives). In general, this

makes genotoxicity studies highly complex and the experimental design complicated

(Verschaeve, 2015). Moreover, to reliably rule out P. hadiensis as a causative agent for the

rapidly enlarging breast masses in young women, further studies targeting a more mechanistic

pharmacological assessment are required, as this study provided an early-stage screening only.

In the future, the collaboration with Ugandan traditional healers will be further

strengthened. The applied approach for the transfer of results back to the healers might not be

suitable for all indigenous communities, local study participants, cultural backgrounds, and

situations during field research worldwide. The presented scientific approach to

ethnopharmacological education, outreach, and feedback discussions via a workshop

represents a potential best-practice example. However, ethnopharmacologists always need to

adapt to the given circumstances (often based on their experience and "gut feeling").

The extract library that was created during my PhD research (86 extracts from 16 plant

species) was further expanded in the context of my other projects. New samples were collected

to explore and to generate scientific evidence for self-medication in wild chimpanzees and

192

mountain gorillas at four new Ugandan study sites, as well as to investigate "forgotten" German

medicinal plants that were commonly used in medieval times. At the time of submission of this

PhD thesis, the library comprises 404 unique extracts from 87 species, including several insect

and mushroom species that have never been studied in a laboratory. In the future, collected

sample material and the individual extracts derived from it will continue to be regarded as

chemical libraries that merit pharmacological screenings. The investigation of the totality of

ingredients provided within these extracts, including strategies to isolate natural product

compounds or to identify synergistic effects, is currently being planned.

193

Awards, achievements, plenary talks, conference and poster presentations,

honorary positions, and press features linked to the PhD studies

Awards and achievements

2020 Society of Economic Botany Student/Post-doc Award, to be awarded at the SEB2022

conference in Mona, Jamaica

2019 Field research awarded a The Explorers Club Flag Expedition ("Self-medication in wild

chimpanzees and mountain gorillas – Discovery of novel natural remedies")

2019 German Society of Plant Sciences Young Researcher Travel Grant (Deutsche Botanische

Gesellschaft, Rostock, Germany, €350)

2019 Appointment as The Explorers Club Term Member (after nomination, 07/2019 – ongoing)

2019 ISE2019 Best Poster Award (2019 Congress of the International Society for

Ethnopharmacology, Dresden, Germany, €200)

2019 ISE Young Investigators Fellowship of the International Society for Ethnopharmacology

2018/2019 Fulbright Scholarship (visiting researcher at Emory University, School of Medicine, Atlanta,

Georgia, USA)

2018 GA Society of Medicinal Plant and Natural Products Research Travel Grant (Shanghai, China,

€1200)

2018 Edmund H. Fulling Award (Best Oral Presentation at the Joint Conference Society of

Economic Botany & Society of Ethnobiology, Madison, WI, USA, US$500)

2018 National Science Foundation Award in honor of outstanding scholarship and commitment,

NSF, USA (US$500)

2018 Best Poster Award in “Health & Nutrition” at 109th AOCS Annual Meeting (Minneapolis,

MN, USA, US$120)

2018 AOCS Outstanding Young Researcher Award (€750)

2017 American Society of Pharmacognosy General Student Award (US$600)

2017 DAAD PhD travel grant (Portland, OR, USA; €1450)

2016 Social Responsibility Award 2016 at the statewide idea contest of the federal state of

Mecklenburg-Western Pomerania, Germany (€500)

2016 AOCS European Section Travel Grant for Outstanding Young Researchers (€750)

2016 German Academic Exchange Service PhD Student Travel Grant Rwanda / Uganda (€1200)

2015 Exceptional PhD Student Award of the Ulrich Florin Foundation (€750)

Honorary positions held

Elected 2018-2020 and 2021-2022 Young Researcher Representative and Board Member of the International

Society of Ethnopharmacology

Visiting scholar at the Quave Lab - Medical Ethnobotany & Drug Discovery, School of Medicine, Emory

University, Atlanta, GA, USA

(Fulbright alumnus, 09/2018 – 02/2019)

Guest scientist in ethnobotany/ethnopharmacology at Makerere University, Uganda

(07/2014 – ongoing)

194

Scientific advisor of the ARUDUVO charity / NGO in Uganda (www.arudevo.com)

(10/2013 – ongoing)

Skype-A-Scientist Lecturer

(2020 – ongoing)

Co-chair of the AOCS (American Oil Chemist's Society) Student Common Interest Group (CIG)

(10/2016 – 09/2018)

Honorary lecturer in phytochemistry and traditional medicine at Kampala University, Uganda

(03/2015 – 04/2018)

Honorary lecturer at the BEKADEV Training Agency in Ntale, Massaka District, Uganda

(08/2013 – 10/2013)

Session chairing, webinar organization and moderation, participation in award juries,

and membership in international conference organizing committees

Schultz, F.: 7th Convention of Society for Ethnopharmacology (SFE) and International Symposium on

"Combating Covid-19 - Ethnopharmacology & Traditional Food and Medicine"; Co-organization and co-

chair/moderator of the Young Researcher Session on Diversity in Ethnopharmacological Research, virtual,

December 18, 2020

2020 Congress of the International Society for Ethnopharmacology, Thessaloniki, Greece, Member of the

International Organizing Committee – Young Researcher Events, November 23-24, 2020

Schultz, F.: Young Ethnopharmacologist Award 2020. Jury member, 7th International Congress of the Society

for Ethnopharmacology (SFEC 2020), New Delhi, India. February 17, 2020

2019 Congress of the International Society for Ethnopharmacology, Member of the International Organizing

Committee Dresden, Germany. June 11-14, 2019

Schultz, F.: African Ethnobiology. Session chair of Technical Session at Joint Conference Society of Economic

Botany & Society of Ethnobiology, Madison, Wisconsin, USA. June 5, 2018

Schultz, F.; Picklo, M.: General Health and Nutrition Session. Session organizer and chair of Technical Session

at 109th AOCS Annual Meeting, Minneapolis, Minnesota, USA. May 9, 2018

109th AOCS Annual Meeting, Minneapolis, Minnesota, USA. Member of the International Organizing

Committee – Student Common Interest Group, May 5-9, 2018

AOCS – American Oil Chemist's Society Webinars, Riding common sense, courage, and confidence — a

journey through the multicultural and diverse world, Kaustuv Bhattacharya - Principal Application Specialist for

Oils & Fats at DuPont Nutrition & Health, co-organizer and moderator, January 31, 2018

Research featured by the press

Der Heilkraft afrikanischer Pflanzen auf der Spur. (English: Investigating the healing power of African plants)

Nordkurier. February 2021 https://www.nordkurier.de/neubrandenburg/der-heilkraft-afrikanischer-pflanzen-auf-

der-spur-1042393002.html

Beitrag im Rahmen zu „Antibiotika – das ist wichtig zu wissen!“ (English: Contribution to “Antibiotics – what

you need to know) TV report, ARD buffet. ARD, November 6, 2020:

https://www.ardmediathek.de/daserste/video/ard-buffet/sendung-vom-06-11-2020/das-

erste/Y3JpZDovL3N3ci5kZS9hZXgvbzEzMzgwNDc/

Neue Strategien gegen multiresistente Krankheitserreger. (English: New strategies against multidrug-resistant

pathogens) TV report, UM6 – Das Ländermagazin, RBB - Rundfunk Berlin-Brandenburg, September 25, 2020

Heilpflanzen: Alternative zu Antibiotika? (English: Medicinal plants: An alternative to antibiotics?) TV report,

Nordmagazin. NDR - Norddeutscher Rundfunk, September 24, 2020:

https://www.ardmediathek.de/ndr/video/nordmagazin/heilpflanzen-alternative-zu-antibiotika/ndr-mecklenburg-

195

vorpommern/Y3JpZDovL25kci5kZS85Y2RiZjBhYS0zZjQ0LTRmZGEtYTNlYy02Mzg3OWRkMTZmNTA/

Neue Wirkstoffe im Kampf gegen antibiotikaresistente Keime. (English: Novel drug leads to combat antibiotic-

resistant germs) NDR radio report/interview. Norddeutscher Rundfunk, September 2020

Zwischen multiresistenten Keimen, Heilpflanzen und Pickleball. (English: In between multidrug-resistant germs,

medicinal plants and pickleball) Fulbright News, Our Fulbrighters, February 19, 2019

Heilmittel aus dem Dschungel (English: Natural remedies from the jungle). NDR radio interview. Norddeutscher

Rundfunk MV, November 2017

Vergessene Medizin (English: Forgotten medicine). Newspaper article. Nordkurier, November 2017

Science beyond borders. Magazine article. INFORM magazine, American Oil Chemist's Society, October issue,

2017

Invited plenary talks

Schultz, F.: Ethnopharmakologie in Deutschland - Was die Selbstmedikation von Schimpansen und

Berggorillas, traditionelle HeilerInnen in Uganda und die Hochschule Neubrandenburg verbindet. (English:

Ethnopharmacology in Germany – What connects chimpanzees and mountain gorillas, traditional healers in

Uganda and the Neubrandenburg University of Applied Sciences?) Virtual presentation, GDL-Online-Kongress

2020, Gesellschaft Deutscher Lebensmitteltechnologen, October 26, 2020

Schultz, F.: Zur Arbeit eines Ethnopharmakologen – Bericht zur Erforschung von Menschenaffen, Heilpflanzen

und des Wissens von HeilerInnen in Uganda. (English: The work of an ethnopharmacologist – Report on

research on the Great Apes, medicinal plants, and the knowledge of traditional healers in Uganda). With guests,

including an expert panel discussion, streamed online on YouTube. Wangelin Botanical Gardens, Wangelin,

Germany, September 29, 2020

Schultz, F.: Local knowledge and ethnopharmacological research in Uganda – Studying medicinal plant use in

humans and other great apes. Annual Symposium of the Swiss Ethnobiology Network. Lucerne, Switzerland,

March 15, 2020 (cancelled on the day of the symposium due to the COVID-19 pandemic restrictions)

Schultz, F.: Ethnopharmacological research across borders – Uganda, Germany, Belgium, USA, and back to

Uganda. 7th International Congress of the Society for Ethnopharmacology (SFEC 2020), New Delhi, India.

February 15, 2020

Schultz, F.: Key Plants in Traditional Medical Systems of the World: Africa. Invited Botanical Medicine and

Health Guest Lecture, Undergraduate Program Medicine, School of Medicine, Emory University, Atlanta, GA,

USA. December 4, 2018

Schultz, F.: Assessment of ethnopharmacological activities of 16 medicinal plants traditionally used in western

Uganda and eastern Democratic Republic of Congo. Afrikatagung Deutscher Geowissenschaftler 2018 (2018

Africa Colloquium of German Geoscientists), Neubrandenburg, Germany. June 22, 2018

Schultz, F.: Bioactive plant compounds: From barley to African medicinal species. Invited symposium, Leibniz-

Institute of Plant Biochemistry, Halle, Germany. November 18, 2016

Schultz, F.: Natural oxygenated fatty acids as biological pesticides and food preservatives for an improved food

safety. 1st East African Conference on Food Science and Technology, Kigali, Rwanda. March 9, 2016

Oral presentations

Schultz, F.: The science of ethnopharmacology – Quo vardis? Event organized by the International Career

Center, Neubrandenburg University of Applied Sciences, October 21, 2020

Schultz, F.: Traditionelles Wissen in Uganda - Erforschung der natürlichen Heilmittel von Menschen und

anderen Großaffen. 1-hour virtual talk, Rotary Club Moritzburg Brücke/Dresden, April 20, 2020

Schultz, F.: Do mountain gorillas and chimpanzees use drugs? Skype a Scientist virtual talk,

Hamburg/Washington D.C., USA. March 27, 2020

196

Schultz, F.; Anywar, G.; Osuji, O. F.; Wack, B.; Garbe, L.-A.: African medicinal plants traditionally used in

western and central Uganda: Investigation of antiinflammatory activity in the COX / PGH2 and 15-LOX /

15(S)-HpETE pathways. 2019 Congress of the International Society for Ethnopharmacology, Dresden,

Germany. June 13, 2019

Schultz, F.; Garbe, L.-A.: African Ethnopharmacology & Research at HSNB. DigiMeP symposium (Digital

resource management for ethnobotanically relevant medicinal plant species of Morocco and Burkina Faso),

Neubrandenburg University of Applied Sciences, Neubrandenburg, Germany. March 19, 2019

Schultz, F.; Quave, C.L.: Anti-virulence activity of medicinal plants traditionally used in West-Central Uganda

against multidrug-resistant ESKAPE pathogens. Whitehead Biomedical Research Building, Emory University,

Atlanta, GA, USA. January 25, 2019

Schultz, F., Garbe, L.-A., Quave, C.-L.: Antibacterial screening and quorum quenching activity of African

medicinal plant extracts. Kubanek Group, School of Biological Sciences, Georgia Institute of Technology,

Atlanta, GA, USA. November 12, 2018

Schultz, F.: Ethnopharmacology and research in Uganda. Quave Group, Center for Human Health, Emory

University, Atlanta, GA, USA. October 26, 2018

Schultz, F., Dworak-Schultz, I.: Meet-A-Scientist Fulbright Talk and Discussions. 256 Students visited in 7

classes at Ashford Elementary School, Atlanta, GA, USA. October 3, 2018

Schultz, F.; Anywar, G.; Osuji, O.F.; Nguyen, A.; Pieters, L.; Garbe, L.-A.: Assessment of antimalarial

properties and potential genotoxicity of African medicinal plants traditionally used in western and central

Uganda. 66th Annual Meeting of the Society for Medicinal Plant and Natural Product Research (GA) jointly

with the 11th Shanghai International Conference on Traditional Chinese Medicine and Natural Medicines (S-

TCM), Shanghai, China. August 28, 2018

Schultz, F.: Forschung an der HSNB - Auf der Suche nach neuen Heilmitteln im afrikanischen Dschungel

(English title: Research at HSNB – The quest for new drugs from the African jungle). Invited high school

lectures, Tag der Technik 2018, State Mecklenburg-Vorpommern, Neubrandenburg, Germany, June 29, 2018

Schultz, F., Garbe, L.-A.: Innovative research ideas and methodologies for finding natural remedies from plants,

Welcoming reception, research delegation of the West Pomeranian University of Technology, Szczecin, Poland.

Neubrandenburg, Germany. June 19, 2018

Schultz, F.; Anywar, G.; Osuji, O. F.; Garbe, L.-A.: East and Central African medicinal plants as inflammatory

inhibitors in the 15-LOX / 15-Hydroxyeicosatetraenoic acid and COX / PGH2 pathways. Joint Conference

Society of Economic Botany & Society of Ethnobiology, Madison, Wisconsin, USA. June 5, 2018

Schultz, F.; Anywar, G.; Osuji, O. F.; Garbe, L.-A.: East and Central African medicinal plants as anti-

inflammatory inhibitors in the 15-LOX / 15-Hydroxyeicosatetraenoic acid and COX / PGH2 pathways. 109th

AOCS Annual Meeting, Minneapolis, Minnesota, USA. May 9, 2018

Schultz, F.; Anywar, G.; Garbe, L.-A.: Investigation of bioactive compounds from medicinal plants collected in

the tropical rainforests of East Africa. Applied Microbiology Research Group, Prof. Meyer, Technical

University of Berlin. November 13, 2017

Schultz, F.: Forgotten medicine: Is the natural cure for malaria, bacterial infectious diseases and even cancer

hidden in the African jungle? (Original title: Vergessene Medizin: Befinden sich natürliche Heilmittel für

Malaria, bakterielle Infektionskrankheiten und sogar Krebs unentdeckt im afrikanischen Dschungel?).

Seniorenhochschule Neubrandenburg - wissenschaftliches Veranstaltungs- und Bildungsangebot für ältere

Erwachsene. Neubrandenburg University of Applied Sciences, Neubrandenburg, Germany. November 9, 2017

Schultz, F.; Anywar, G.; Garbe, L.-A.: Investigation of bioactive properties of medicinal plants collected in the

tropical rainforests of East Africa. 2017 Annual Meeting of the American Society of Pharmacognosy, Portland,

Oregon, USA. July 30, 2017

Schultz, F.; Anywar, G.; Garbe, L.-A.: Bioactive lipids from novel medicinal plants of the tropical rainforests of

East Africa. 108th AOCS Annual Meeting, Orlando, Florida, USA. May 3, 2017

197

Schultz, F.; Garbe, L.-A.: Ethnobotanical, phytochemical and biological study of selected tropical medicinal

plants from Uganda. Visit of the NatuRA Research Group and Prof. Luc Pieters, discussions and mentoring,

University of Antwerp, Antwerpen, Belgium. December 9, 2016

Schultz, F.; Garbe, L.-A.: Auf dem Weg zum Doktorhut – Neue Medikamente aus Heilpflanzen des

ostafrikanischen Regenwaldes. 25 Jahre HSNB – Vorlesungen an besonderen Orten, Regional Museum,

Neubrandenburg, Germany. October 13, 2016

Schultz, F.; Dworak, I.; Garbe, L.-A.: ARUDEVO – Forschen und Helfen in Ostafrika. Rotary Club

Uckermünde-Pasewalk-Stettiner Haff. September 1, 2016

Schultz, F.: AfricanBioactives – From Research to Starting my own Company. Idea contest statewide finals

2016, Federal state of Mecklenburg-Western Pomerania, Germany. July 13, 2016

Dworak, I.; Schultz, F.: Presenting ARUDEVO. Idea contest statewide finals 2016, Federal state of

Mecklenburg-Western Pomerania, Germany. July 13, 2016

Schultz, F.: AfricanBioactives – Future Research Opportunities. Local idea Contest 2016, Federal state of

Mecklenburg-Western Pomerania, Germany. July 5, 2016

Schultz, F.; Dworak, I.: African Rural Development Volunteers. Local idea contest 2016, Federal state of

Mecklenburg-Western Pomerania, Germany. July 5, 2016

Schultz, F.; Garbe, L.-A.: Trihydroxy fatty acids as biological fungicides in crop and food industry. 107th

AOCS Annual Meeting, Salt Lake City, Utah, USA. May 5, 2016

Schultz, F.; Garbe, L.-A.: Plant Oxylipins as novel bioactive ingredients in African food science and agriculture.

Kampala University, Kampala, Uganda. March 12, 2016

Schultz, F.; Garbe, L.-A.: Scientific activities in lipid biochemistry research in Germany and Africa. Young

researchers meeting and course in lipid biochemistry, Denmark Technical University, Copenhagen, Denmark.

November 23-25, 2015

Schultz, F.; Garbe, L.-A.: Current research topics on food technology in Germany and Africa. 20 years food

technology gala, Neubrandenburg University of Applied Sciences, Germany. October 16, 2015

Poster presentations

Schultz, F.; Anywar, G.; Osuji, O.F.; Wack, B.; L.; Garbe, L.-A.: Medicinal plants traditionally used in the

Greater Mpigi Region, Uganda: Investigation of antiinflammatory activity in the COX / PGH2 and 15-LOX /

15(S)-HpETE pathways. Botanikertagung 2019 International Plant Science Conference, Rostock, Germany.

September 18, 2019

Schultz, F.; Dworak-Schultz, I.; McLennan, M.; Anywar, G.; Hobaiter, C.; Kalema Zikusoka, G.; Benian, G.;

Pieters, L.; Garbe, L.-A.: Discovery of yet unknown medicinal natural remedies from the Ugandan rainforests:

Exploring self-medication in wild chimpanzees & mountain gorillas. Botanikertagung 2019 International Plant

Science Conference, Rostock, Germany. September 18, 2019

Schultz, F.; Anywar, G.; Osuji, O. F.; Nguyen, A.; Pieters, L.; Garbe, L.-A.: Investigation of antimalarial,

genotoxic and antibacterial in vitro bioactivity of African medicinal plants traditionally used in western and

central Uganda. Botanikertagung 2019 International Plant Science Conference, Rostock, Germany.

September 18, 2019

Schultz, F.; Dworak-Schultz, I.; McLennan, M.; Anywar, G.; Hobaiter, C.; Kalema Zikusoka, G.; Benian, G.;

Pieters, L.; Garbe, L.-A.: Exploring self-medication in wild chimpanzees & mountain gorillas: Discovery of yet

unknown medicinal natural remedies. 2019 Congress of the International Society for Ethnopharmacology,

Dresden, Germany. June 12, 2019

Schultz, F.; Anywar, G.; Osuji, O. F.; Nguyen, A.; Pieters, L.; Garbe, L.-A.: Investigation of antimalarial,

genotoxic and antibacterial properties of African medicinal plants traditionally used in western and central

198

Uganda. 2019 Congress of the International Society for Ethnopharmacology, Dresden, Germany. June 12, 2019

Schultz, F.; Anywar, G.; Osuji, O.F.; Wack, B.; L.; Garbe, L.-A.: Antiinflammatory activity of selected

Ugandan plants traditionally used as painkillers. DigiMeP symposium (Digital resource management for

ethnobotanically relevant medicinal plant species of Morocco and Burkina Faso), Neubrandenburg University of

Applied Sciences, Neubrandenburg, Germany. March 19, 2019

Schultz, F.; Anywar, G.; Osuji, O. F.; Nguyen, L.; Pieters, L.; Garbe, L.-A.: Strategies and pharmacological

assays for evaluation of tradition use in Ugandan medicinal plants. DigiMeP symposium (Digital resource

management for ethnobotanically relevant medicinal plant species of Morocco and Burkina Faso),

Neubrandenburg University of Applied Sciences, Neubrandenburg, Germany. March 19, 2019

Schultz, F.; Dworak, I.: ARUDEVO – A NGO in Lwengo District, Central Uganda. DigiMeP symposium

(Digital resource management for ethnobotanically relevant medicinal plant species of Morocco and Burkina

Faso), Neubrandenburg University of Applied Sciences, Neubrandenburg, Germany. March 19, 2019

Schultz, F.; Anywar, G.; Osuji, O.F.; Wack, B.; L.; Garbe, L.-A.: African medicinal plants traditionally used in

western and central Uganda: Investigation of antiinflammatory activity in the 15-LOX / 15(S)-HpETE and COX

/ PGH2 pathways. 1st Botanical Research Symposium & Celebration, Emory herbarium, Emory University,

Atlanta, Georgia, USA. September 21, 2018

Schultz, F.; Anywar, G.; Osuji, O. F.; Nguyen, L.; Pieters, L.; Garbe, L.-A.: Investigation of antimalarial,

genotoxic and antibacterial properties of African medicinal plants traditionally used in western and central

Uganda. 1st Botanical Research Symposium & Celebration, Emory herbarium, Emory University, Atlanta,

Georgia, USA. September 21, 2018

Schultz, F.; Anywar, G.; Osuji, O.F.; Wack, B.; L.; Garbe, L.-A.: Investigation of antiinflammatory properties

of traditionally used East and Central African medicinal plants in the 15-LOX / 15-Hydroperoxyeicosatetraenoic

acid and COX / PGH2 pathways. 66th Annual Meeting of the Society for Medicinal Plant and Natural Product

Research (GA) jointly with the 11th Shanghai International Conference on Traditional Chinese Medicine and

Natural Medicines (S-TCM), Shanghai, China. August 27, 2018

Schultz, F.; Anywar, G.; Osuji, O. F.; Nguyen, L.; Pieters, L.; Garbe, L.-A.: Investigation of antimalarial,

genotoxic and antibacterial properties of African medicinal plants traditionally used in western and central

Uganda. 2018 Biotechnology Day, TIB, Technical University of Berlin, Germany. July 13, 2018

Schultz, F.; Anywar, G.; Garbe, L.-A.: Assessment of ethnopharmacological activity of medicinal plants

traditionally used in Central and East Africa, Summer celebrations of the North-East-German Science

Community, Germany. July 3, 2018

Schultz, F.; Anywar, G.; Osuji, O. F.; Nguyen, L.; Pieters, L.; Garbe, L.-A.: Investigation of antimalarial,

genotoxic and antibacterial properties of African medicinal plants traditionally used in western and central

Uganda. Joint Conference Society of Economic Botany & Society of Ethnobiology, Madison, Wisconsin, USA.

June 6, 2018

Schultz, F.; Anywar, G.; Osuji, O. F.; Nguyen, L.; Pieters, L.; Garbe, L.-A.: Investigation of bioactive lipids

from African medicinal plants collected in the tropical rainforests of Uganda. 109th AOCS Annual Meeting,

Minneapolis, Minnesota, USA. May 9, 2018

Schultz, F.; Osuji, O. F.; Nguyen, A..; Anywar, G.; Pieters, L.; Garbe, L.-A.: Investigation of bioactive

properties of medicinal plants collected in the tropical rainforests of East Africa: An overview. 2017

Biotechnology Day, TIB, Technical University of Berlin, Germany. July 14, 2017

Schultz, F.; Anywar, G.; Garbe, L.-A.: Novel bioactive molecules from medicinal plants of the tropical rain

forests of East Africa. Afrikatagung Deutscher Geowissenschaftler (Africa Colloquium of the German

Geoscientists), Berlin, Germany. June 23-24, 2017

Schultz, F.; Dworak, I.: ARUDEVO - Helping and Performing Research in East Africa. Afrikatagung Deutscher

Geowissenschaftler (Africa Colloquium of German Geoscientists), Berlin, Germany. June 23-24, 2017

Schultz, F.; Anywar, G.; Garbe, L.-A.: Novel bioactive lipids from medicinal plants of the tropical rain forests

of East Africa, Technological, Industrial and Political Conference 2017 – Nutrition, Neubrandenburg, Germany.

January 12, 2017

199

Schultz, F.; Dworak, I.: ARUDEVO - Helping and Performing Research in East Africa. Technological,

Industrial and Political Conference 2017 – Nutrition, Neubrandenburg, Germany. January 12, 2017

Schultz, F.; Mengdehl, M.; Garbe, L.-A.: Antifungal bioactivity of trihydroxy acids - chemo-enzymatic

synthesis, metabolomics and mechanism of antifungal action. GDL-Congress Food technology, Lemgo,

Germany. October 20-22, 2016

Schultz, F.; Anywar, G.; Garbe, L.-A.: Novel bioactive molecules from medicinal plants of the tropical rain

forests of East Africa. 25 Jahre HSNB – Vorlesungen an besonderen Orten, Neubrandenburg, Germany.

October 13, 2016

Schultz, F.; Dworak, I.: ARUDEVO – African Rural Development Volunteers. 25 Jahre HSNB – Vorlesungen

an besonderen Orten, Regional Museum, Neubrandenburg, Germany. October 13, 2016

Schultz, F.; Müller, M.F.; Caesar, J.; Sadykova, Z.; Mengdehl, M.; Garbe, L.A.: Antifungal bioactivity of

trihydroxy acids - chemo-enzymatic synthesis, metabolomics and mechanism of antifungal action. 2016

Biotechnology Day, TIB, Technical University of Berlin, Germany. July 14, 2016

Schultz, F.; Mengdehl, M.; Garbe, L.-A.: Antifungal bioactivity of trihydroxy acids - chemo-enzymatic

synthesis, metabolomics and mechanism of antifungal action. 11th Molecular Biology of Fungi Conference,

Berlin, Germany. October 7-9, 2015

Schultz, F.; Mengdehl, M.; Garbe, L.-A.: Chemo-enzymatic synthesis and metabolomics of trihydroxy fatty

acids, 2015 Biotechnology Day, TIB, Technical University of Berlin, Germany. July 16, 2015

Courses attended

PhD course “Lipid biochemistry, technologies and analysis”, 4 ECTS points, Denmark: Technical University of

Denmark (Nov 2015)

Practical lab course “Angewandte Biotechnologie aus Sicht der Mikrobiologie (0335 L 059)“, Master

Biologische Chemie, Prof. Vera Meyer, Germany: Technical University of Berlin (Oct 2015)

200

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