Optimization of processing conditions on the quality of extracts and formulation of Arnica Montana.

UNIVERSIDAD DE VALLADOLID
ESCUELA DE INGENIERIAS INDUSTRIALES
Más e en Ingenie ía Química
OPTIMIZATION OF PROCESSING CONDITIONS
ON THE QUALITY OF EXTRACTS AND
FORMULATION OF ARNICA MONTANA
Au o :
Pequeño Alonso, Ál a o
Responsable de In e cambio en la U a
Ángel Ma ín
Uni e sidad de Ma ibo
Valladolid, julio 2022.
TFM REALIZADO EN PROGRAMA DE INTERCAMBIO
TÍTULO: Op imizacija pos opka eks akcije učinko in iz a nike A nica
Mon ana in o mulacija p oduk o
ALUMNO: Ál a o Pequeño Alonso
FECHA: 01/07/2022
CENTRO: FKKT
UNIVERSIDAD: Uni e sidad de Ma ibo
TUTOR: Assoc. P o . D . Maša Knez Ma e ci
Resumen
A nica Mon ana es una uen e p ome edo a de compues os bioac i os, p incipalmen e enoles, que
debido a su ac i idad an ioxidan e mues a bene icios pa a la salud en humanos. Pa a llega a una
e apa a macéu ica, las lo es de A nica Mon ana necesi an se p ocesadas pa a ob ene un p oduc o
iable. Pa a es e p opósi o, se ealiza on a ias ex acciones con sol en es: mace ación en ío,
ul asonidos, Soxhle y CO2 supe c í ico; los sol en es, e anol, me anol y una mezcla 95:5 ( : ) de
e anol y agua ue on usados. Se ealiza on análisis de espec ome ía ul a iole a/ isible pa a medi
la ac i idad an ioxidan e y el con enido enólico o al de los ex ac os. Finalmen e, un o ganogel ue
o mulado con al a es abilidad con el ex ac o, acei e de cannabis y ce a de sal ado de a oz, y ue
e aluado según su es abilidad. Una e isión bibliog á ica exhaus i a de la li e a u a sob e A nica
Mon ana, los mé odos de ex acción y los sol en es, compues os enólicos y o ganogeles se incluye
además en es e abajo. Es necesa io que el abajo u u o se cen e en la de e minación de sus
p opiedades eológicas y solubilidad en di e en es disol en es a a ias empe a u as.
Palab as cla e: A nica Mon ana, o ganogels, ex accion, enoles, sepa ación
Abs ac
A nica Mon ana is a p omising sou ce o bioac i e compounds, mainly phenols, which, due o hei
an ioxidan ac i i y, show heal h bene i s in humans. To each a s age o pha maceu ical use, A nica
Mon ana lowe s need o be p ocessed in o a iable p oduc . Fo his pu pose, a ious ex ac ions
echniques wi h di e en sol en s we e pe o med: cold mace a ion, ul asounds, Soxhle , and
supe c i ical CO2; he sol en s used we e e hanol, me hanol, and a 95:5 ( : ) mix u e o e hanol and
wa e . Analysis in ol ing ul a iole / isible spec ome y we e accomplished o measu e he
an ioxidan ac i i y and he o al phenolic con en o he ex ac s. Finally, an o ganogel was
o mula ed using he ex ac , cannabis oil, and ice b an wax and was es ed o i s s abili y. In he
ame o his wo k, o ganogel wi h a high s abili y has been o mula ed o he i s ime, since un il
now, scien i ic li e a u e does no p o ide any da a o such kind o o mula ion. Addi ional s udies
will be equi ed o de e mine i s heologic p ope ies and solubili y in di e en sol en s a a ious
empe a u e condi ions.
Key wo ds: A nica Mon ana, o ganogels, ex ac ion, phenols, isola ion
Ál a o Pequeño Alonso
Op imizacija pos opka eks akcije učinko in iz a nike
A nica Mon ana in o mulacija p oduk o
Magis sko delo
Ma ibo , 2022
OPTIMIZATION OF PROCESSING CONDITIONS ON THE
QUALITY OF EXTRACTS AND FORMULATION OF
ARNICA MONTANA
S uden : Ál a o Pequeño Alonso
S udy p og am: Mas e 's s udy p og am II. Deg ee in Chemical
Enginee ing
In ended p o essional i le: Mas e 's deg ee in Chemical Enginee ing
Supe iso : Assoc. P o . D . Maša Knez Ma e ci
Co-supe iso : P o . D . Željko Knez
Ma ibo , July 2022

OPTIMIZATION OF PROCESSING CONDITIONS ON THE QUALITY OF EXTRACTS AND FORMULATION OF
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1
Table o con en s
Table o con en s....................................................................................................................... 1
Acknowledgmen s .................................................................................................................... 2
Abs ac ..................................................................................................................................... 3
Table o ables........................................................................................................................... 4
Table o igu es ......................................................................................................................... 5
1 In oduc ion ....................................................................................................................... 7
1.1 P oblem de ini ion ..................................................................................................... 7
1.2 Objec i es .................................................................................................................. 9
2 A nica Mon ana ............................................................................................................... 10
2.1 Desc ip ion ............................................................................................................... 10
2.2 P ope ies and chemical composi ion ...................................................................... 12
2.3 Cu en and po en ial applica ions ........................................................................... 15
3 Ex ac ion p ocedu es ...................................................................................................... 17
3.1 Cold mace a ion ....................................................................................................... 18
3.2 Ul asound ex ac ion ............................................................................................... 18
3.3 Soxhle ex ac ion .................................................................................................... 19
3.4 Supe c i ical luid ex ac ions .................................................................................. 20
4 O ganogels ...................................................................................................................... 23
4.1 Desc ip ion o o ganogels ........................................................................................ 23
4.2 P ope ies and cha ac e iza ion o o ganogels ......................................................... 25
4.3 Applica ions o o ganogels ...................................................................................... 26
5 Expe imen al wo k .......................................................................................................... 27
5.1 Ex ac ion o compounds om A nica Mon ana plan ............................................ 27
5.2 UV/Visible spec opho ome y ................................................................................ 29
5.3 O ganogel o mula ion ............................................................................................. 30
6 Resul s ............................................................................................................................. 31
6.1 Ob en ion o A nica Mon ana ex ac ...................................................................... 31
6.2 UV/Visible spec opho ome y ................................................................................ 31
6.3 O ganogel o mula ion ............................................................................................. 33
7 Discussion o esul s ........................................................................................................ 34
8 Conclusions ..................................................................................................................... 36
9 Bibliog aphy .................................................................................................................... 38
10 Anex ............................................................................................................................. 43
10.1 Da a ables ................................................................................................................ 43
OPTIMIZATION OF PROCESSING CONDITIONS ON THE QUALITY OF EXTRACTS AND FORMULATION OF
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Acknowledgmen s
I would like o hank e e yone and e e y hing in ol ed in he
w i ing o his hesis.
OPTIMIZATION OF PROCESSING CONDITIONS ON THE QUALITY OF EXTRACTS AND FORMULATION OF
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wo king in he Ul a iole /Visible ange (UV/Vis). This is a popula and widely used
echnique in analy ical chemis y; i is a powe ul analy ical echnique wi h e y high
sensi i i y and speci ici y, and being ela i ely inexpensi e and easily implemen ed, i is
widely used in di e se applied and undamen al applica ions. Wi h his p ocess, in o ma ion
abou he an ioxidan capabili ies and o al phenols con en , o he ex ac can be ob ained.
Gels a e de ined as semi-solid sys ems, o med by a h ee-dimensional s uc u e composed o
gela o molecules, wi h a liquid sol en phase immobilized (Zeng e al., 2021). O ganogels
use as an o ganic phase o o he 3D s uc u e; his allows hem o ha e highe boiling poin s,
which con e s hem highe s abili y, han s anda d gels. Also, o ganogels exhibi a se ies o
unique p ope ies and unc ionali ies, such as opical deli e ies o hyd ophobic d ugs (Zeng
e al., 2021) which makes hem in e es ing o pha maceu ical applica ions. Common o ganic
sol en s as well as mine al and ege able oils a e commonly epo ed o be used as o ganic
sol en s o o ganogel o mula ions (Esposi o e al., 2018); in his case, cannabis oil and ice
b an wax we e used as he o ganic phase and he gela o , espec i ely.
1.2 Objec i es
The gene al objec i e o his hesis is o s udy he pha maceu ical iabili y o A nica Mon ana.
Fo his, he a o emen ioned me hods o ex ac ion and sol en s will be used o he ob en ion
o A nica Mon ana ex ac . An analysis o he an ioxidan ac i i y and o al phenol con en o
he ex ac and phenolic p o ile, using UV/Visible spec ome y, will be pe o med. A e , an
o ganogel o med om he cannabis oil, ice b an wax, and he A nica Mon ana ex ac will
be p oduced and o mula ed.

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2 A nica Mon ana
2.1 Desc ip ion
A nica Mon ana, membe o he As e aceae amily, one o he la ges lowe ing plan amilies,
is an he baceous pe ennial plan g owing on moun ains o alpine meadows, pas u es and in
ligh o es s, i has b igh g een lea es wi h ounded ips a he base ha a e le el wi h he
g ound. Thei su ace is eined and agg ega ed in ose es. The op lea es a e opposing, and
smalle (Figu e 1). They bloom in o a s a o sun lowe o ma ion om June h ough Augus
(Han ahan, 2018). The lowe is na i e o he moun ains o Sibe ia and cen al Eu ope, bu i
has sp ead o e mos o Eu ope (Figu e 2) and can be ound in No h Ame ica and Russia.
Howe e , his species is conside ed endange ed in di e en Eu opean coun ies so i s
cul i a ion is inc easing bu i s ha es is no allowed in mos coun ies o p o ec he species
(K iplani e al., 2017).
Figu e 1.- Map o he popula ion o A nica Mon ana in Eu ope (Wikipedia, 2007)
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Figu e 2.- A nica Mon ana g owing in he moun ains o Swi ze land (S ude , 2006)
A nica Mon ana is popula as a adi ional homeopa hic ma e ial. The he b has been used in
olk emedies since he six een h cen u y. A wound healing e ec was al eady a ibu ed o
A nica in a manual o Tabe naemon anu in 1613, and i was a popula emedy o nume ous
ailmen s du ing he coming cen u ies. Since he middle o he 20 h cen u y, in coun ies like
Ge many, A nica is used o ea hema omas, sp ains, and con usions, in homoeopa hic
sys ems o medicine. A monog aph on A nica was es ablished by he Ge man Commission E,
a scien i ic ad iso y boa d o he Fede al Ins i u e o D ugs and Medical De ice specializing
on he bal medicines, indica ing, and limi ing, he applica ion o ex e nal use, due o possible
isks wi h he inges ion o A nica Mon ana based solu ions (EMA, 2013). Also, de ailed
w i en s udies elabo a ed by he Eu opean Pha macopoeia, a egional book which p o ides
common quali y s anda ds h oughou he pha maceu ical indus y, indica e A nica’s lowe s
as an app op ia e opical emedy wi h he igh elabo a ion. I is clea ly a widesp ead he b in
he homeopa hic ield, since he e a e o e one hund ed medicinal p epa a ions using A nica
ex ac s comme cially a ailable in Ge many. Romania is he main p oduce o wild-collec ed
A nica Mon ana lowe s, expo ing mainly o Ge many, I aly, F ance and Swi ze land
(Robe son e al., 2007). A esea ch om he Uni e si y o Koblenz-Landau in Ge many
es ima ed ha 50 me ic ons o d ied A nica lowe s a e used annually in Eu ope (Ucenic &
Mas o akis, 2007) I is es ima ed ha he demand in Ge many is 10 me ic ons o he
pha maceu ical sec o alone. In he Uni ed S a es, a nica is widely used in opical applica ion,
like in lamma ions (Fiume, 2001).
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2.2 P ope ies and chemical composi ion
I has been in es iga ed by many esea che s ha lowe s o he plan a e mainly ich in ac i e
cons i uen s (Han ahan, 2018; K iplani e al., 2017; Pawlaczyk e al., 2014). A nica Mon ana
con ains up o 1% o a ola ile oil ha is pa ly composed (app oxima ely 50%) o a y acids,
especially palmi ic, linoleic, my is ic, and linolenic acids (Bandaiphe & Kennedy, 2004). The
lowe s o A nica species con ain especially sesqui e pene lac ones. Sesqui e pene a e a class
o e penes ha consis o h ee isop ene uni s, and occu na u ally in insec s and plan s as
semiochemicals, ha is, as chemicals ha a ec he beha io o he indi idual and hose
a ound i when eleased. Sesqui e pene lac ones ha e a lac ose ing and occu mainly in in
plan s o he amily As e aceae.
Beside sesqui e pene lac ones (wi h a na u al a iabili y o 0.3 o 1%), o he componen s
include essen ial oil compounds (0.2 o 0.35%), la onoids (0.4 o 0.6%), hyd oxycouma ins
and phenyl ac ylic acids (K iplani e al., 2017). The lowe s also con ain 13 helenanolides,
which a e sesqui e pene lac ones; hese ha e been iden i ied as helenalin, 11,13-
dihyd ohelenalin , and 11 es e de i a i es. In i o s udies ha e shown ha he mos ac i e
componen s o A nica, as well as o o he membe s o he As e aceae amily, a e helenalin
and o he sesqui e pene lac ones such as 11a,13-dihyd ohelenalin and chamissonolid. (Fiume,
2001). The mos ele an cons i uen s ha ha e been isola ed a e he helenalins (Figu e 3).
Helenalin is a sesqui e pene esponsible o he oxici y o he A nica Mon ana, causing skin
i i a ion and especially se e e gas oen e i is and in e nal bleeding o he diges i e ac , i
inges ed in la ge amoun s. Because o his, A nica ex ac s a e no ecommended o in e nal
adminis a ion, al hough helenalins possess some an i-in lamma o y and an i-neoplas ic
e ec s (Sch öde e al., 1990).
Figu e 3.- Skele al o mula o helenalin (Concellos, 2008)
Phenolic compounds a e a g oup o small molecules cha ac e ized by hei s uc u es ha ing
a leas one phenol uni . Phenol i sel is a benzene ing ha is subs i u ed wi h a hyd oxyl g oup
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(Figu e 4). Thus, i s sys ema ic name is hyd oxybenzene. Based on hei chemical s uc u es,
phenolic compounds can be di ided in o di e en subg oups, such as phenolic acids,
la onoids, annins, couma ins, lignans, quinones, s ilbens, and cu cuminoids. The ca ego ies
o phenolic compounds and hei ep esen a i e compounds a e shown in Figu e 5.
Figu e 4.- Phenol uni (Mama i, 2021)
Figu e 5.- Ca ego ies o phenolic compounds (Coope & Nicola, 2014)
Fla onoids (Figu e 6) a e some o he mos common phenolics, widely dis ibu ed in plan
issues, and o en esponsible alongside he ca o enoids and chlo ophylls o hei blue, pu ple,
yellow, o ange and ed colo s. The la onoid amily includes la ones, la onols, iso-
la onols, an hocyanins, an hocyanidins, p oan hocyanidins and ca echins. All la onoids a e
de i ed om he a oma ic amino acids, phenyalanine and y osine, and ha e h ee- inged
s uc u es. Phenolic acids a e one o he o he main phenolic classes wi hin plan s and occu
in he o m o es e s, glycosides, o amides, bu a ely in ee o m. Va ia ion in phenolic acids
is in he numbe and loca ion o hyd oxyl g oups on he a oma ic ing. Phenolic acids ha e
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wo pa en s uc u es: hyd oxycinnamic and hyd oxybenzoic acid (Figu e 6).
Hyd oxycinnamic acid de i a i es include e ulic, ca eic, p-couma ic and sinapic acids, while
hyd oxybenzoic acid de i a i es consis o gallic, anillic, sy ingic and p o oca echuic acids
(Khoddami e al., 2013).
Figu e 6.- S uc u e o phenolic acids and la onoids (Khoddami, 2013).
They a e e y impo an an ioxidan s due o hei hyd oxyl g oups ha con e ee adical
sca enging abili y o plan p oduc s. This means hey can neu alize ha m ul ee adicals ha
would o he wise damage body cells and inc ease he isk o condi ions like cance , diabe es,
and hea disease (Ćujić e al., 2016). Polyphenols a e seconda y me aboli es o plan s and a e
gene ally in ol ed in de ense agains ul a iole adia ion o agg ession by pa hogens. A s udy
done in 2004 showed ha e hanolic ex ac s o A nica Mon ana con ain high concen a ions
o phenolic acids and la onoids con en , con e ing hem a good an ioxidan ac i i y, which
jus i ies i s use in he ea men in skin diso de s (C aciunescu e al., 2012). Acco ding o
epidemiological s udies, he in ake o phenolic compounds is in e sely co ela ed
wi h he isk o co ona y hea disease (do Ca mo e al., 2021). A numbe o polyphenolic
compounds, especially ca echins, ha e been ound o be po en an ioxidan s and o be e ec i e
in p e en ing cance (Šu o ská e al., 2014) while annins ha e been epo ed o exe o he
physiological e ec s; hey can educe blood p essu e, accele a e blood clo ing, lowe se um-
lipid le els, modula e immuno esponses and cause li e nec osis (Khoddami e al., 2013).
Va ia ion in he chemis y o phenolics in a sample is ela ed o he concen a ion o simple
and complex polyphenolic compounds and he di e en p opo ions o phenolic acids,
la onoids, an hocyanins and p oan hocyanins (among o he s). Thus, i is di icul o choose
a single me hod o p epa a ion and ex ac ion o phenolics o many plan p oduc s. The mos
common echniques o ex ac phenolics employ sol en s, ei he o ganic o ino ganic. Se e al
pa ame e s may in luence he yield o phenolics, including ex ac ion ime, empe a u e,

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sol en - o-sample a io, he numbe o epea ex ac ions o he sample, as well as sol en ype.
Sol en s, such as me hanol, e hanol, p opanol, ace one, e hyl ace a e, and hei combina ions
ha e also been used o he ex ac ion o phenolics, o en wi h di e en p opo ions o wa e .
Fo example, phenolic compounds can be e icien ly ex ac ed om legumes using an
e hanol/wa e (70:30 : ) sys em (Casazza e al., 2010). Ex ac ion o phenolics om
ae ial pa s o Po en illa a osanguinea showed ha 50% aqueous e hanol was mo e e icien
han pu e o 50% aqueous o ms o me hanol, and ace one (Kalia e al., 2008).
Supe c i ical luid ex ac ion has p o en o be a p omising echnique o ob aining high-quali y
phenolic compounds wi h minimal loss o i s p ope ies (Ži ek e al., 2022). Ca bon dioxide is
he mos widely used sol en due o i s cha ac e is ics, such as mode a e c i ical condi ions
and high a ailabili y. I is also non oxic, in lammable and chemically s able. Howe e , CO2
as he ex ac ing sol en is no e icien o he ex ac ion o phenolic compounds because o
i s low pola i y in compa ison o mos phenols, which can be sol ed using a pola co-sol en ,
as elabo a ed in he nex sec ion.
Despi e a e y la ge numbe o published in es iga ions, quan i ica ion o a ious phenolic
s uc u al g oups emain di icul . While lowe molecula weigh compounds a e qui e easily
analyzed by UV– isible spec opho ome y and mass spec ome y, analysis o polyme ic
compounds emains a challenge, as hei di e si y esul s in poo esolu ion and de ec ion.
Tannins a e majo examples o such s uc u es o which me hod de elopmen is needed
(Khoddami e al., 2013). Spec opho ome y is one o he ela i ely simple echniques o
quan i ica ion o plan phenolics. The Folin-Ciocal eu me hod is a classic me hod used in
spec opho ome y o measu e o al phenolics in plan ma e ials (H nčič e al., 2019).
2.3 Cu en and po en ial applica ions
Focusing on he medical aspec s o he plan , i is widely used wo ldwide as a homeopa hic
emedy. The lowe s o he plan show g ea e medicinal alue and a e used as an iphlogis ic,
ino opic, an ibio ic, an i-in lamma o y, immunomodula o y, an ipla ele , u e o onic,
an i heuma ic and analgesic in eb ile condi ions (K iplani e al., 2017). Clinical s udies
conduc ed in humans ha e wo ked on he opical and in e nal applica ions o A nica Mon ana
ex ac s, and hei homeopa hic p oduc s. They ha e shown g ea po en ial in he ea men o
a ious in lamma o y and mic obial condi ions. A nica ex ac s ha e been s udied p ima ily
o hei opical use while s udies on in e nal uses a e limi ed o homeopa hic p oduc s, due o
A nica’s oxici y (Koo e al., 2000; K iplani e al., 2017).
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In he Eu opean Union, se e al alcoholic p epa a ions om A nica Mon ana, a e unde
egula ion as adi ional he bal medicinal p oduc s, his means, equi ing egis a ion and an
au ho iza ion be o e accessing he ma ke . This solu ions a e o a ious s eng hs; he ones
p epa ed om he whole plan a e 1:5, e hanol 60% ( / ); and 1:10, e hanol 60% o 70% ( / )
and he liquid ex ac o esh lowe s a e 1:20, e hanol 50% (m/m) (Gayle & B inckmann,
2015). These p epa a ions a e ma ke ed o cu aneous use only and a e indica ed o he elie
o b uises, sp ains, and localized muscula pain. In Ge many alone, he e a e 843 egis e ed
medicinal p oduc s lis ing A nica Mon ana as an ac i e ing edien , o which 360 p oduc s a e
homeopa hic medicines (Han ahan, 2018).
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3 Ex ac ion p ocedu es
The ini ial s age in sepa a ing he desi ed na u al p oduc s om he aw ing edien s is
ex ac ion. The mos common app oach in plan ex ac ion is sol en -based me hods. The
ollowing phases a e in ol ed in he ex ac ion o na u al p oduc s: (1) he solid ma ix o he
aw plan ma e ial is pene a ed by he sol en ; (2) he sol en s dissol e he solu e; (3) in he
solid ma ix, he solu e di uses ollowing he mass ans e mechanisms; and (4) he solu es
a e collec ed as he inal p oduc ex ac ed. A lo o ac o s a e he e o be conside ed when
wo king wi h he ex ac ion o plan ma e in an e icien manne .
The selec ion o he sol en is c ucial o sol en ex ac ion. Alcohols (e hanol and me hanol)
a e uni e sal sol en s o phy ochemical in es iga ion, since hey a e pola sol en s, and mos
o he compounds o in e es s in plan ex ac ions, like polyphenols, a e pola oo (EMA,
2013). Acco ding o law o simila i y, he close he pola i y alues o he sol en s and solu es,
mo e e ec i e he ex ac ion will be.
No mally, inc easing ime and empe a u e p omo e analy e solubili y; howe e , plan
phenolics a e gene ally deg aded o unde go undesi able eac ions such as enzyma ic
oxida ion by ex ended ex ac ion imes and high empe a u es. This will esul in a lowe yield
and he p esence o impu i ies in he inal ex ac (Huang e al., 2013).
Ex ac ion e iciency is usually di ec ly co ela ed o how ine he pa icles a e. A smalle
solu e pa icle size inc eases he su ace a ea he sol en can ac on, imp o ing sol en
di usi i y in he solu e. I he pa icle size is oo small, howe e , he e will be ouble in he
ollowing il e ing. Usually, he me hods o educe he pa icle size o he ma e ial a e
mechanical (g inding, milling, cu ing, e c.).
Polyphenol eco e y om plan s may be accomplished using a a ie y o ex ac ion p ocesses,
which can be b oadly classi ied as con en ional and con empo a y. Classic ex ac ion
p ocedu es include cold mace a ion, and Soxhle ex ac ion. Wi h inc easing ene gy
consump ion and he d i e o imp o e e iciency, indus ies and esea ch ins i u ions a e
challenged o ind ways which can simpli y ope a ion p ocedu e, mee low-cos equi emen s,
and achie e good quali y. In ecen yea s, no el app oaches o ex ac ing bioac i e subs ances
ha e been de eloped, such as ul asound-assis ed ex ac ion, and supe c i ical luid ex ac ion.
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3.1 Cold mace a ion
This is a e y simple ex ac ion me hod, whe e he plan ma e is pu in o con ac wi h he
sol en and i s s i ed, wi h he pu pose o ans e ing he solid compounds in o he liquid
solu ion. The ex ac ion is done a oom empe a u e and p essu e, so he ex ac ions imes a e
longe han he o he me hods, bu i is use ul o he ex ac ion o he molabile componen s,
like plan ma e .
A s udy in 2015 achie ed a high yield o o al phenols chokebe y ui using 50% e hanol, a
solid–sol en a io o 1:20, which migh mean ha mace a ion was a simple and e ec i e
me hod o he ex ac ion o phenolic compounds om chokebe y ui (Ćujić e al., 2016).
In 2018, Soxhle ex ac ion was compa ed wi h cold mace a ion o ind ou which one ga e a
be e an imic obial ac i i y o lico ice ex ac agains S ep ococcus mu ans; he indings
sugges ed ha using 100% e hanol as he sol en , lico ice ex ac kep i s an imic obial ac i i y
be e when i was ob ained using cold mace a ion (Sankeshwa i e al., 2018). The ola ile
ac ions ob ained om Lonice a mac an hoides using di e en ex ac ion me hods we e
compa ed in a s udy done in 2015. Al hough he p e e ed me hod was hyd odis illa ion, cold
mace a ion showed ad an ages o e he a ie y o cons i uen s ob ained in he inal ex ac
(Wu e al., 2015).
3.2 Ul asound ex ac ion
An ex ac ion me hod whe e high-powe , low- equency ul asound wa es a e used in a
solu ion o he plan ma e and he sol en . These wa es c ea e ca i a ion bubbles in he
solu ion which implode on he su ace o he solid, b eaking i down and c ea ing a b eakage
in he plan cell walls, eleasing he bioac i e compounds om he plan ma e o he sol en
in he p esence o mass ans e g adien . This ca i a ion accele a es he dissolu ion and
di usion, as well as imp o ing he mass and hea ans e , which imp o es he ex ac ion
e iciency. Ul asound-assis ed ex ac ion equen ly uses less sol en , akes less ime, and
yields a g ea e ex ac ion yield, and i is a sa e op ion o he mos-sensi i e ma e ials han
o he hea -based me hods. I is a p omising p ocessing echnology o ex ac plan bioac i e
compounds such as phenolics, la onoids, hymols, saponins and p o eins (Yuso e al.,
2022).
Many esea che s p e e o use ul asonic ene gy o he ex ac ion o phenolic compounds
om plan ma e ial. Me hanol was showed o be he mos app op ia e sol en in ex ac ing
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In addi ion o he na u e o ne wo ks o sol en s, he c osslinking mechanism o o ganogels is
also an impo an classi ica ion basis o o ganogels ca ego izing o ganogels in o physical and
chemical o ganogels. O ganogels a e o med by c osslinking junc ion poin s and 3D ne wo ks.
Physical o ganogels a e o med by s ong non-co alen in e ac ions (hyd ogen bonding, an
de Waals o ces, elec os a ic...) in he c osslinking poin s. Chemical o ganogels a e
cons uc ed wi h co alen c osslinks du ing he gela ion p ocess, using p ocesses like
copolyme iza ion eac ions. Wi h physical o ganogels, he in e molecula non-co alen
in e ac ions a e hei p ima y mechanisms o gela ion, which gi e ise o weak polyme
ne wo ks. This o ganogels a e gene ally less s able wi h low mechanical s eng h. In he o he
hand, chemical o ganogels a e mo e s able and ha e be e mechanical p ope ies due o he
s ong co alen ly c osslinked ne wo k (Zeng e al., 2021).
4.2 P ope ies and cha ac e iza ion o o ganogels
O ganogels p esen iscoelas ic p ope ies gene ally ollowing he Maxwell model o
isoelas ici y. A lowe shea a es, o ganogels beha e like solid-like o mula ions (Kobo i e
al., 2007). They a e he mo e e sible, ha is, hey ha e he capaci y o eco e he s able
he modynamic solid-like s uc u e a e being hea ed. When an o ganogel is hea ed up abo e
hei mel ing empe a u e, he e is an inc ease in he he mal ene gy inside he low ene gy
he mos able ma ix, dis up ing he molecula connec ions in he ne wo k. When he sys ems
a e cooled down, he physical in e ac ions be ween o ganogela o molecules e e se back o
hei s able o m (Zeng e al., 2021). O ganogels wi h pha maceu ical applica ions a e
biodeg adable in na u e, especially he mos ecen o mula ions based on biomolecules, like
ege able oils, cocoa bu e , o mango bu e (Sagi i e al., 2013). The bio-deg ada ion a e can
be egula ed by adap ing he concen a ion o biocompa ible componen s and he used o
ad anced o mula ion echnologies.
Recen ly syn hesized o ganogels a e in ensi ely cha ac e ized ho ough a ious echniques
and me hodologies o con i m hei s abili y and e iciency. The mos common and simple is
a quali a i e e alua ion me hod based on a simple in e sion (upside down) o he con aine
wi h he o mula ion; he gel s a e is de e mined when he o mula ed sys em does no low
anymo e unde he e ec o i s own weigh . Va ious pa ame e s, such as he g a i a ional low,
ime, empe a u e and concen a ion o he gelling agen can hen a ec he isualiza ion and
he de e mina ion o he gel o ma ion. This me hod has been used in mul iple s udies ela ed

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o he cha ac e iza ion o o ganogels using ege able oils as sol en s (Li e al., 2022; Lupi e
al., 2016; Pa el e al., 2015)
4.3 Applica ions o o ganogels
The applica ions o o ganogels a e s ongly dependen on hei p ope ies. The ela i ely poo
s abili y o physical o ganogels makes hem app opia e o d ug deli e y. Fo example, some
biocompa ible molecules/ing edien s could elease om he gelled ma ix in a con olled
manne by simple disin eg a ion o he gel ne wo k. The chemical o ganogels, wi h a mo e
s able in e nal s uc u e, makes hem mo e app op ia e o long-las ing and demanding
applica ions such as indus ial an i- ouling and an i-icing applica ions (Zeng e al., 2021).
Focusing on he pha maceu ical applica ions o he physical o ganogels, hey ha e an
ad an age in d ug deli e y due o hei so ness, biocompa ibili y, and bioac i i y. O ganogels
allow en apping a a ie y o bioac i e agen s and allow o a ious deli e y modes,
sugges ing g ea po en ial o such use o d ug deli e y pla o ms. Leci hin o ganogels, o
example, ha e been ex ensi ely in es iga ed as ehicles o cu aneous adminis a ion due o
he low skin i i a ion and he biocompa ible na u e o leci hin. Also, i was ound ha leci hin
o ganogles ha e high and compa ibili y wi h a wide ange o o ganic liquid including a ious
edible oils and nonpola sol en s as well as pha maceu ical molecules (B inksma e al., 2000).
O ganogels p epa ed wi h soy bean, ege able oil p o ed o be an e ec i e cu aneous d ug
deli e y sys ems, hanks o he hyd ophobic an i-in lamma o y na u e o he s uc u e, he
ac i e compound encapsula ed in his o ganogel was slowly and sa ely eleased in he absence
o ligh (Pa k e al., 2016).
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5 Expe imen al wo k
The gene al pu pose o he expe imen al wo k was o ob ain A nica Mon ana ex ac o
measu e i s an ioxidan ac i i y and o al phenol con en . Also, an o ganogel o med by
cannabis oil and ice b an wax was o mula ed, wi h he ex ac ob ained by supe c i ical CO2
ex ac ion. A nica Mon ana ex ac s we e ob ained wi h di e en me hods and sol en s: cold
mace a ion, ul asound ex ac ion, Soxhle , and supe c i ical CO2 ex ac ion, using e hanol,
me hanol, and a 95:5 ( / ) mix u e o e hanol and wa e . A e i s an ioxidan ac i i y and o al
phenol con en we e measu ed using UV– isible spec opho ome y. A e , he ex ac was
immobilized in an o ganogel o med by ice b an wax and cannabis oil.
5.1 Ex ac ion o compounds om A nica Mon ana plan
D ied A nica Mon ana lowe s we e c ushed in o dus using an elec ical blende . The
esul ing ma e ial was hen dilu ed in he sol en in a lask and s i ed o a o he mixing. The
sol en s used we e e hanol, me hanol, and a 95:5 solu ion o e hanol and wa e ; in e e y case,
he amoun used was 150 mL o sol en .
The cold mace a ion ex ac ion was pe o med a oom empe a u e and wi h magne ic s i ing,
o 90 minu es. The ul asound ex ac ion ( igu e 10) was pe o med a oom empe a u e and
p essu e o 60 minu es. The Soxhle ex ac ion ( igu e 9) was pe o med hea ing he solu ion
up o 80ºC deg ees o 180 minu es. The supe c i ical CO2 ex ac ion was pe o med in an
au ocla e a 250 ba , which was subme ged in a wa e ba h a 40º C, o 180 min. A e each
ype o ex ac ion, he ex ac was collec ed in a ound lask, and an e apo a ion was pe o med
using a o a apo o sepa a e he sol en om he ex ac . A e comple ion, he ex ac was
emo ed om he ound lask using a me al spoon and ans e ed o a smalle con aine
( igu e 11), which we e s o ed in a idge a 2 ºC.
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Figu e 9.- Se -up o he Soxhle ex ac ion
Figu e 10.- Se -up o he ul asound ex ac ion
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Figu e 11.- Final ex ac o A nica Mon ana
5.2 UV/Visible spec opho ome y
The UV/Visible spec ome y was pe o med using he equipmen in igu e 12. Fo he
an ioxidan ac i i y, a solu ion o 0.01 g ams o ex ac and DPPH (2,2-diphenyl-pic yl-
hyd azil) dilu ed in dis illed wa e we e used; o he o al phenols con en , a solu ion o 0.05
g ams o ex ac wi h Folin-Ciocal eu eagen dilu ed in dis illed wa e was used. A e le ing
he solu ions es in a da k chambe o 2 minu es, hey we e hea ed o igge he eac ion.
The spec opho ome y analysis begins wi h a blank sample o pu e me hanol, and hen he
o al phenols and an ioxidan ac i i y o each sample we e measu ed, in ha o de .
Figu e 12.- U /Visible spec ome e used in he analysis
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5.3 O ganogel o mula ion
The o ganogel was o mula ed using he ex ac ob ained wi h he supe c i ical CO2 using
e hanol, since i had he highes amoun o an ioxidan ac i i y. Cannabis oil was used as
o ganic phase, and ice b an wax was used o he gela o : i s , he oil and ex ac we e mixed,
and hen, he wax was in oduced in he mix u e. The compoenen s we e mixed a 40ºC du ing
5 min. The o ganogels ob ained we e es ed quali a i ely by hei ex u e, colo , and s abili y
o e ime (Figu e 13).
Figu e 133.- O ganogels o mula ed om ice b an wax, cannabis oil, and A nica Mon ana ex ac

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6 Resul s
6.1 Ex ac ion o A nica Mon ana
The labo a o y ex ac ion esul s a e summa ized in Table 1 and Figu e 14. The collec ed da a
shows he aw A nica Mon ana used o he ex ac ion, he amoun o ex ac ob ained a e
he ex ac ions and e apo a ions, and he yield ob ained. The bes sol en o e all was he
me hanol, wi h he highes yields o ex ac in each ype o ex ac ion. The mos e ec i e
ex ac ion me hod, independen ly o he sol en , when looking a quan i a i e yield o ex ac
ob ained was Soxhle ex ac ion amongs all he ex ac ion echniques; ollowed closely by he
supe c i ical ex ac ion me hod. Cold mace a ion and ul asounds a e he leas e ec i e
me hods o hem all, wi h he lowes yields.
Figu e 14.- Ex ac ion yield in % o he A nica Mon ana samples
6.2 UV/Visible spec opho ome y
The spec ome e measu es he ligh abso bed by he sample, calcula ing he ansmi ance and
abso bance o he A nica Mon ana ex ac . The appa a us wo ks in he ange o 800 o 200 nm
o de ec ul a iole ligh (100 o 400 nm) and isible ligh (380 o 700 nm) spec um.
The an ioxidan ac i i y and o al phenol con en a e p esen ed in igu es 15 and 16,
espec i ely. A calib a ion cu e was p epa ed using galic acid dilu ed in dis illed wa e . The
o al phenolic con en was exp essed as mg GA pe g o ex ac . The ex ac ob ained wi h he
27.42% 29.50%
18.50%
5.04%
13.00%
8.96%
4.04%
11.68%
7.04%
19.50% 21.00%
16.90%
0%
5%
10%
15%
20%
25%
30%
35%
E OH MeOH E OH+H2O
Ex ac ion yield (%)
Sol en
Soxhle Cold mace a ion Ul asounds Supe c i ical CO2
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supe c i ical CO2 using e hanol as cosol en had he highes an ioxidan ac i i y, howe e he
highes con en o phenolic compounds was p esen in he samples using a mix u e o e hanol
and wa e as a sol en , being he Soxhle ex ac ion he one ha yielded mo e o hese phenolic
compounds.
Figu e 15.- An ioxidan ac i i y (DPPH)
Figu e 16.- To al phenol con en (mg GA / g ex ac )
18.55
21.69
25.86
16.77
14.61
22.08
19.25 19.32
21.60
10.97
20.24 21.02
0.00
5.00
10.00
15.00
20.00
25.00
30.00
E OH MeOH E OH+H2O
To al phenolic con en (mg GA / g ex ac )
Sol en
Soxhle Cold Mace a ion Ul asounds Supe c i ical CO2
6.25
14.2
8.11
31.23
18.15
7.73
26.49
22.81
6.98
37.45
21.77 20.82
0
5
10
15
20
25
30
35
40
E OH MeOH E OH+H2O
DPPH (%)
Sol en s
Soxhle Cold Mace a ion Ul asounds Supe c i ical CO2
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Resul s in Figu e 14 indica e ha he bes sol en in he e ms o he highes yield was
me hanol, wi h he highes yields o ex ac in each ype o ex ac ion, wi h he highes yield,
29.5%, in Soxhle ex ac ion. E hanol ga e a ela i ely high yield in Soxhle ex ac ion oo,
27.42%. This con i ms ha pola sol en s a e a good choice o he sol en in he ex ac ion
o A nica Mon ana due o hei high pola i y, making hem he indica ed sol en s o
polyphenolic componen s me hanol being he mos e ec i e sol en due o i s high pola i y
and selec i i y, as o he s udies con i m (Ži ek e al., 2022). Appa en ly, he high empe a u e
(80 ºC) o he Soxhle ex ac ion, con ibu ed o he highes yield o ex ac ; low empe a u e
me hods, like ul asound ex ac ion and cold mace a ion, yielded he lowes amoun o ex ac
when using any sol en .
6.3 O ganogel o mula ion
The mos s able o ganogel esul ed o be he one wi h a a io 90:9:1 (cannabis oil: ice b an
wax, ex ac ), meaning 4.6 g ams o oil, 0.46 g ams o wax, and 0.051 g ams o ex ac . Rice
wax was hea ed up o he mel ing empe a u e and he mix u e o compounds has been
homogenized a 40ºC o 5 min.
O ganogel has been es ed o i s s abili y a e exposu e o he ligh a ai empe a u e o 10
days. No sepa a ion o phases o colo change has been obse ed.
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7 Discussion o esul s
In he expe imen al pa o he s udy, di e en ex ac ion me hods and sol en s we e used o
ob ain A nica Mon ana ex ac s, and hei an ioxidan ac i i y and phenol con en was
measu ed using UV/Vis spec opho ome y. The combina ion o a highly pola and selec i e
sol en like me hanol and a high empe a u e high ex ac ion ime me hod like Soxhle
ex ac ion showed he bes esul s in ex ac ion yield (29.5%), bu he high empe a u es o
Soxhle deg aded he phenolic compounds, esul ing in low an ioxidan ac i i y (14.2).
Supe c i ical ex ac ion using CO2 and e hanol was a mo e e ec i e way o p ese e
an ioxidan ac i i y on he ex ac s (37.45), due o he mode a e empe a u es and sho e
ex ac ion imes. The o al phenol con en was highe wi h he Soxhle ex ac ion, using
e hanol and wa e (25.86), bu was closely ollowed by he o he echniques, indica ing ha
he choice o sol en is mo e impo an han he echnique i a high amoun o phenolic
compounds is he main objec i e; s ill, his sol en combina ion was he one wi h he lowes
an ioxidan ac i i y (8.11), making i no so desi able o he pha maceu ical use o he ex ac .
Focusing on he an ioxidan ac i i y, i can be seen in Figu e 15 ha supe c i ical CO2
ex ac ion using a co-sol en p esen ed he highes DPPH ac i i y independen on co-sol en s
used; in he case o me hanol, i is sligh ly lowe o ul asound ex ac ion (22.81). Soxhle
ex ac ion ga e low an ioxidan ac i i y, p obably due o he high empe a u es and imes (80
ºC o 180 minu es) used du ing he ex ac ion, which esul ed in he mal deg ada ion o he
phenolic compounds con e ing he an ioxidan ac i i y o he ex ac s. All o his is o be used
as a ea i ma ion ha supe c i ical luid ex ac ion is a gen le , mo e e icien ex ac ion,
equi ing only mode a e empe a u es, and a oiding he use o ha m ul o ganic sol en s, while
p ese ing he p ope ies o he ex ac phenolic compounds.
Figu e 16 indica es a high quan i y o phenolic compounds in he samples ob ained using a
mix u e o e hanol and wa e as ex ac ion sol en . Soxhle had he highes con en o 25.86
mg GA / g ex ac ou o all he ex ac ion me hods using ha sol en , bu i was closely
ollowed by he es o he ex ac ion me hods a a ound 22 mg GA / g ex ac o all o hem.
This migh indica e ha he mix u e o e hanol and wa e is a good sol en o e all o he
ex ac ion o phenolic compounds. This is p obably due o he glycosidic bond ha should be
in e up ed o ex ac he phenolics.
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Sanjaya, R. E., Tedjo, Y. Y., Ku niawan, A., Ju, Y. H., Ayuci a, A., & Ismadji, S. (2014).
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10 Anex
10.1 Da a ables
Table 1.- Ex ac ion yield (%)
Ma e ial
Ex ac
Yield
g
g
%
Soxhle
E OH
12.07
3.31
27.42%
E OH+W
12
3.54
29.50%
MeOH
12
2.22
18.50%
Cold mace a ion
E OH
25
1.26
5.04%
E OH+W
25
3.25
13.00%
MeOH
25
2.24
8.96%
Ul asounds
E OH
25
1.01
4.04%
E OH+W
25
2.92
11.68%
MeOH
25
1.76
7.04%
Supe c i ical CO2
E OH
10
1.95
19.50%
E OH+W
10
2.1
21.00%
MeOH
10
1.69
16.90%
Table 2.- An ioxidan ac i i y o he ex ac s (DPPH, %)
DPPH (%)
S anda d
De ia ion
Soxhle
E OH
6.25
0.06
E OH+W
14.2
0.34
MeOH
8.11
0.06
Cold
Mace a ion
E OH
31.23
0.14
E OH+W
18.15
0.15
MeOH
7.73
0.28
Ul asounds
E OH
26.49
0.19
E OH+W
22.81
0.06
MeOH
6.98
0.26
Supe c i ical
CO2
E OH
37.45
0.18
E OH+W
21.77
0.22
MeOH
20.82
0.13
OPTIMIZATION OF PROCESSING CONDITIONS ON THE QUALITY OF EXTRACTS AND FORMULATION OF
ARNICA MONTANA
44
Table 3.- To al phenol con en (mg GA / g ex ac )
To al phenol
con en
mg GA / g ex ac
Soxhle
E OH
18.55
E OH+W
21.69
MeOH
25.86
Cold
Mace a ion
E OH
16.77
E OH+W
14.61
MeOH
22.08
Ul asounds
E OH
19.25
E OH+W
19.32
MeOH
21.60
Supe c i ical
CO2
E OH
10.97
E OH+W
20.24
MeOH
21.02
(ime članice UM)
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Izmenja a MAG
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Maša Knez Ma e ci
Željko Knez
Ál a o Pequeno Alonso
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Ma ibo , 13.07.2022