Study of the anti-inflammatory and neuroprotective activity of plants used in Traditional Chinese Medicine (TCM)

Universidade do Minho Escola de Ciências Bruna Gabriela Araújo da Silva Study of the anti-inflammatory and neuroprotective activity of plants used in Traditional Chinese Medicine (TCM) junho de 2022 Study of the anti-inflammatory and neuroprotective activity of plants used in Traditional Chinese Medicine (TCM) Bruna Gabriela Araújo da Silva UMinho | 2022 Universidade do Minho Escola de Ciências Bruna Gabriela Araújo da Silva Study of the anti-inflammatory and neuroprotective activity of plants used in Traditional Chinese Medicine (TCM) Dissertação de Mestrado Mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas Trabalho efetuado sob a orientação da Professora Doutor Alberto Carlos Pires Dias junho de 2022 DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Creative Commons Attribution-ShareAlike 4.0 International CC BY-SA 4.0 https://creativecommons.org/licenses/by-sa/4.0/deed.en i AGRADECIMENTOS Ao fim de vários meses de trabalho, frustrações, mas principalmente de muitas aprendizagens, quero agradecer primeiro ao Prof. Alberto Dias, pela oportunidade e boa disposição. Obrigada pela sua confiança no meu trabalho e por estar sempre aberto a discutir e aceitar as minhas sugestões durante o desenvolvimento desta tese. Deixo, também o meu profundo agradecimento ao grupo do LBB, pela constante simpatia, carinho, pelos almoços e por todos os conselhos durante o desenvolvimento deste projeto. À Vera pela paciência ao ensinar-me o trabalho com as culturas celulares, ao Luís por estar sempre disponível para as minhas questões, à Bárbara por tentar sempre que eu me sinta em casa e por último à Luara pelas gargalhadas e por toda a colaboração durante os ensaios bioquímicos e na escrita desta dissertação. Em especial, um agradecimento à Rita pela sua disponibilidade em me ensinar os protocolos de RT-PCR, e pelos seus conselhos mesmo à distancia. Deixo também um agradecimento aos colegas do LCCA, por estarem sempre prontos para ouvir as minhas sugestões e dúvidas. Ainda, um agradecimento à Cecília Cristelo pela cedência do LPS, crucial para o desenvolvimento desta tese. Sem descurar dos meus, quero agradecer à Sara e a Cinda por sempre me apoiaram a seguir os meus objetivos. À minha Rita, o meu mais recente porto de abrigo, e que apesar de longe esteve sempre presente durante a realização desta tese. Aos migos, por ouvirem os meus desabafos, pelas brincadeiras e por serem sempre uns fofos, no fim vamos todos ao camada ! Por fim, aos meus mais que tudo: aos meus pais e ao meu irmão pelo constante esforço para verem os meus sonhos realizados; ao meu Ti, muito pela paciência ! Por me chamares à terra após os meus devaneios ansiosos, por tentares chamar sempre pelo meu lado racional. Por sempre acreditares em mim, por nunca me negares ajuda. Tu, mais que ninguém sabes o quão desafiante foram estes meses que teriam sido intoleráveis sem o teu apoio. Obrigada. ii STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. iii RESUMO Study of the anti-inflammatory and neuroprotective activity of plants used in Traditional Chinese Medicine (TCM) Apocynum venetum é uma planta conhecida pelo seu uso na medicina tradicional chinesa. Neste trabalho, um extrato metanólico de folhas de A. venetum foi submetido a uma digestão in vitro para realizar uma comparação entre as atividades anti-inflamatórias e antioxidantes do extrato antes e depois da digestão.Os extratos foram submetidos a ensaios in vitro incluindo DPPH, atividade sequestrante de radicais ABTS, FRAP e ICA, radical superóxido e NO e atividade de inibitória da COX-2. Os efeitos citoprotetores e citotóxicos de ambos extratos foram também avaliados em células BV-2 e HepG2. As propriedades anti-inflamatórias dos extratos foram avaliadas através da quantificação do NO e avaliação da expressão genética de genes pró-inflamatórios em células BV-2 induzidas por LPS. Os resultados revelaram que ambos os extratos tiveram atividade em todos os ensaios bioquímicos realizados, exceto o extrato digerido nos ensaios ICA e FRAP. Ambos extratos revelaram efeitos protetores contra os danos induzidos pelo t-BHP em células BV-2 e HepG2. A digestão não causou diferenças significativas nas propriedades protetoras do extrato contra os danos induzidos pelo t-BHP. Em relação às atividades anti-inflamatórias, o extrato não digerido foi capaz de diminuir a libertação de NO e inibir a expressão de genes pró-inflamatórios. Enquanto que o extrato digerido só revelou atividade anti-inflamatória através da diminuição da libertação do NO. Estes resultados mostram que A. venetum pode ser uma fonte de compostos ativos com propriedades anti-inflamatórias e antioxidante e que estas atividades são mantidas em algum nível mesmo após as modificações que ocorrem durante a digestão. Palavras-chave: Apocynum venetum, atividade antioxidante, digestão In vitro,Neuroproteção iv ABSTRACT Study of the anti-inflammatory and neuroprotective activity of plants used in Traditional Chinese Medicine (TCM) Apocynum venetum is a plant known for its wide use in traditional Chinese medicine. In this work, an A. venetum methanolic leaves extract was subject to an in vitro digestion to conduct a comparison between the anti-inflammatory and antioxidant activities of the extract after and before the performance of digestion. The extracts were subjected to in vitro assays including DPPH, ABTS radical scavenging activity, FRAP and ICA, superoxide anion radical (PMS-NADH system) and NO (Griess method) scavenging activity and inhibition of COX-2 activity. Cytoprotective and cytotoxicity effects of both extracts were also evaluated in BV-2 and HepG2 cells. The intracellular ROS levels were also measured, after the treatment with the extracts and t-BHP in the HepG2 cell line. The anti-inflammatory properties of the extracts were also evaluated through the measurement of NO levels and evaluation of gene expression in LPS-induced BV-2 cells. The results revealed that both extracts had activity in all biochemical assays performed except for the digested extract in ICA and FRAP assays. Results proved that the digestion processes impaired the antioxidant activity in the biochemical analysis. Both extracts did not induce significant cytotoxicity and revealed protective effects against the damages induced by t-BHP in both BV-2 and HepG2 cells, probably due to the decrease of ROS induced by t-BHP, as revealed in HepG2 cells. The digestion did not cause significant changes in the protective properties of the extract against t-BHP-induced damages. Regarding anti-inflammatory activities, the non-digested extract was able to decrease the release of NO and inhibit the gene expression of pro-inflammatory genes. While the digested extract only revealed anti-inflammatory activity through the decrease of NO release. Our results show that A. venetum may be a potential source of pharmacological active compounds related to inflammation and oxidative stress, and that A. venetum activities are kept at some level even after the modifications occurring in digestion. Keywords: Antioxidant activity, Apocynum venetum,In vitro digestion, Neuroprotection v CONTENTS Agradecimentos ii Resumo iv Abstract v List of Figures ix List of Tables xvii Acronyms xviii 1 general introduction 1 1.1 Apocynum venetum 2 1.2 Oxidative stress 3 1.2.1 Sources of Reactive Oxygen Species/ Reactive Nitrogen Species 3 1.2.2 Oxidative damage to biomolecules 4 1.2.3 Antioxidant systems 6 1.3 Phenolic compounds 8 1.4 Inflammation and neurodegeneration 11 1.5 Objectives and work-plan 16 2 systematic review of the pharmacological activities 18 2.1 Introduction 19 2.2 Methods 20 2.2.1 Search Strategy 20 2.2.2 Inclusion and exclusion criteria 20 2.3 Results 20 2.3.1 Antioxidant properties 21 2.3.2 Antidepressant properties 23 2.3.3 Anti-hypertensive properties 25 2.3.4 Hepatoprotective properties 27 2.3.5 Cardioprotective properties 29 2.3.6 Neuroprotective properties 32 2.3.7 Anxiolytic properties 33 2.4 Conclusion 34 3 preparation of the extracts and phytochemical characterization 35 vi list of figures xiii Figure 32 Cytotoxicity of AV and dAV on HepG2 cells. HepG2 cells were incubated with AV and dAV at 50 100, 500 µg dwr/ml for 4h of incubation (A) or for 24 h (B). Cellular viability was assessed with MTT assay. AV-non-digested and dAV-digested of Apocynum venetum methanolic leaves extract. Each bar represents the mean ± SD of the results obtained in three independent experiments. Asterisks mean significant differences from the control (cells in medium), as indicated: * p-value< 0.05; ** p-value< 0.01; *** p-value< 0.001; **** p-value< 0.0001. 79 Figure 33 Cytoptotective potential against tert-butyl hydroperoxide (t-BHP) - induced damages. HepG2 cells were co-incubated for 4 hours with both dAV (50 100, 500 µg dwr/ml) and t-BHP(A); or pre-incubated with the extracts (50 100, 500 µg dwr/ml) for 20 h and co-incubated with t-BHP for 4 hours (B). AV-non-digested Apocynum venetum methanolic leaves extract; Cellular viability was assessed by the MTT assay. Each bar represents the mean ± S.D. of the results obtained in three independent experiments. Asterisks mean significant differences from the cells only incubated with t-BHP 0.5 mM, as indicated: **** p-value< 0.0001. 80 Figure 34 Cytoptotective potential against tert-butyl hydroperoxide (t-BHP)-induced damages. HepG2 cells were co-incubated for 4 hours with dAV (50 100, 500 µg dwr/ml) and t-BHP (A); or pre-incubated with the extracts (50 100, 500 µg dwr/ml) for 20 h and co-incubated with t-BHP for 4 hours (B). dAV-digested Apocynum venetum methanolic leaves extract; Cellular viability was assessed by the MTT assay. Each bar represents the mean ± S.D. of the results obtained in three independent experiments. Asterisks mean significant differences from the cells only incubated with t-BHP 0.5 mM for 4 h), as indicated: * p-value< 0.05; ** p-value< 0.01; *** p-value< 0.001; **** p-value< 0.0001. 81 list of figures xiv Figure 35 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) microplate assay. HepG2 cells were labeled with DCFH-DA (25 µM) for 30 min. and then incubated with the AV and dAV at 50, 250, 500 µg dwr/ml during 4 hours and co-incubated with tert-butyl hydroperoxide (t-BHP) 0.5 mM (for 1h). Cells were then analyzed on a fluorescent plate reader with excitation and emission wavelengths of 485/535 nm. Values are presented as % fluorescent intensity. AV-non-digested and dAV-digested of Apocynum venetum methanolic leaves extract. Each bar represents the mean ± S.D. of the results obtained in three independent experiments. Asterisks mean significant differences from the cells only incubated with t-BHP 0.5 mM for 4 h), as indicated: * p-value <0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001. 83 Figure 36 Cytotoxicity of AV and dAV on BV-2 cells. BV-2 cells were incubated with AV and dAV at 50 100, 500 µg dwr/ml for 4h of incubation (A) or for 24 h (B). Cellular viability was assessed with MTT assay. AV-nondigested and dAV-digested of Apocynum venetum methanolic leaves extract. Each bar represents the mean ± SD of the results obtained in three independent experiments. Asterisks mean significant differences from the control (Cells in medium), as indicated: * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001. 85 Figure 37 Cytoptotective potential against Tert-butyl hydroperoxide (t-BHP)- induced damages. BV-2 cells were co-incubated with 4 hours with dAV (50 100, 500 µg dwr/ml) and t-BHP(A); or pre-incubated with the extracts (50 100, 500 µg dwr/ml) for 20 h and co-incubated with t-BHP for 4 hours (B); dAV-non-digested Apocynum venetum methanolic leaves extract; Cellular viability was assessed by the MTT assay. Each bar represents the mean ± S.D. of the results obtained in three independent experiments. Asterisks mean significant differences from the cells only incubated with t-BHP 0.5 mM for 4 h , as indicated: * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** pvalue < 0.0001. 86 list of figures xv Figure 38 Cytoptotective potential against tert-butyl hydroperoxide (t-BHP) 0.5 mM. BV-2 cells were co-incubated with 4 hours with both dAV (50 100, 500 µg dwr/ml) and t-BHP (A) or pre-incubated with the extracts (50 100, 500 µg dwr/ml) for 20 h and co-incubated with t-BHP for 4 h (B). dAV-digested Apocynum venetum methanolic leaves extract; Cellular viability was assessed by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Each bar represents the mean ± S.D. of the results obtained in three independent experiments. Asterisks mean significant differences from the cells only incubated with t-BHP 0.5 mM for 4 h, as indicated: ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001. 87 Figure 39 Nitric oxide (NO) measurement in lipopolysaccharides (LPS)-induced BV-2 cells supernatant. BV-2 cells in 12-well plates were pre-incubated (30 min.) with extracts ( 75 and 125 µg dwr/ml), and co-incubated with LPS (2 µg/ml) for 20h. Supernatants were collected for the measurement of nitric oxide (NO) by Griess reaction (Absorbance at 543 nm). dAV-digested and AV-non-digested Apocynum venetum methanolic leaves extract. Each bar represents the means ±SD of the results obtained in three independent ones experiments. Asterisks mean significant differences from the positive control ( cells incubated exclusively with LPS 2 µg/ml final concentration);*p-value < 0.01 *** p-value < 0.001; **** p-value < 0.0001. 89 list of figures xvi Figure 40 Real time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of genes in lipopolysaccharides (LPS)-induced BV-2 cells. BV-2 cells in 12-well plates were pre-incubated (30 min.) with extracts (125 µg dwr/ml), and co-incubated with LPS (2 µg/ml) for 20h. The cells were collected for RNA extraction and RT-PCR was performed. Gene expression of: (A) interleukin-1BETA (IL-1BETA); (B) interleukin 6 (IL-6); (C) cyclogenase-2 (COX-2); (D) inducible nitric oxide synthase (iNOS); were calculated using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression as an internal control. Cells incubated with medium and cells incubated with LPS were used as a negative and positive control, respectively. AV-non-digested and dAV-digested of Apocynum venetum methanolic leaves extract. Experiments were analyzed with the software Bio-Rad CFX Manager. Statistical differences are presented vs. positive control (cells with LPS): *p-value< 0.05; ** p-value< 0.01; ***p-value< 0.001; ****p-value< 0.0001. 91 LIST OF TABLES Table 1 Elution gradient for the analysis of non-digested Apocynum venetum leaves extract 40 Table 2 Phenolic quantification by external standard. 42 Table 3 Sequence of primers used in this study. 77 Table 4 Percentage of protection against tert-butyl hydroperoxide (t-BHP) effects after the pre-incubation of HepG2 cells with both AV-nondigested and dAV-digested of Apocynum venetum methanolic leaves extract (50 100, 500 µg dwr/ml) for 4h or 20 hours. The results are represent as mean ± S.D. of the results obtained in three independent experiments. 82 Table 5 Percentage of protection against tert-butyl hydroperoxide (t-BHP) effects after the pre-incubation of BV-2 cells with both AV-non-digested and dAV-digested of Apocynum venetum methanolic leaves extract (50 100, 500 µg dwr/ml) for 4h or 20 hours. The results are represent as mean ± S.D. of the results obtained in three independent experiments. 87 xvii ACRONYMS TM Traditional Medicine HPLC-DAD high Performance Liquid Chromatography with a diode-array detector AV Apocynum venetum methanolic leaves extract dAV digested of Apocynum venetum methanolic leaves extract ROS reactive oxygen species RNS reactive nitrogen species SOD superoxide dismutase DNA deoxyribonucleic acid HAT hydrogen atom transfer GABA gamma-amino butyric acid PCR polymerase chain reaction NO nitric oxide RNA ribonucleic acid TAE tris-acetate-EDTA buffer cDNA complementary DNA ONOOperoxynitrite GPX glutathione peroxidase CAT catalase GSH reduced glutathione GSSG oxidized glutathione SET single electron transfer SET single electron transfer COX-2 cyclogenase-2 xviii xix ICOX-2 cyclogenase-2 inhibition DAMPS danger associated molecular patterns PAMPS pathogen associated molecular patterns PRRs pattern recognition receptors TLRs toll-like receptors Nrf2 nuclear factor erythroid 2-related factor 2 NF-KB nuclear factor kappa-light-chain-enhancer of activated B cells MAPK mitogen-activated protein kinase AP-1 activator protein-1 NOX nicotinamide adenine dinucleotide phosphate oxidase NADH nicotinamide adenine dinucleotide NADPH nicotinamide adenine dinucleotide phosphate iNOS inducible nitric oxide synthase nNOS nitric oxide synthase NOS nitric oxide synthase Keap1 kelch-like ECH-associated protein 1 ARE antioxidant response elements NDDs neurodegenerative diseases AD Alzheimer’s disease PD Parkinson’s disease HD Huntington’s disease CNS central nervous system BBB blood-brain barrier FST forced swimming test 5-HT serotonin NE norepinephrine DA dopamine xx MAO monoamine oxidase TST tail suspension test LPS Lipopolysaccharide D-GalN D-galactosamine TNF tumor necrosis factor CYP2E1 cytochrome P4502E1 IL-6 interleukin 6 IL-1 interleukin 1 IL-10 interleukin-10 IL-1BETA interleukin-1BETA TLR4 toll-like receptor 4 TLR2 toll-like receptor 2 TIR toll-interleukin-1 receptor JNK C-Jun NH2-terminal kinase BDNF brain-derived neurotrophic factor Bax Bcl-2 Associated X Protein Bcl-2 B-cell lymphoma 2 CREB cAMP-response element binding OGD oxygen and glucose deprivation MAPs microtubule-associated proteins CUMS chronic unpredictable mild stress MDA malondialdehyde AGEs advanced glycation endproducts mPTP mitochondrial permeability transition pore LDH lactate dehydrogenase MI/R myocardial ischemia/reperfusion Src proto-oncogene tyrosine-protein kinase xxi eNOS endothelium nitric oxide synthase PI3K phosphoinositide 3-kinase EPM elevated plus maze TBARS thiobarbituric acid reactive substance DPPH 2,2-diphenyl-1-picrylhydrazyl FRAP ferric-reducing antioxidant power assay LDL low-density lipoprotein HO-1 heme oxygenase-1 AGEs advanced glycation end-products PUFAs polyunsaturated fatty acids dwr dry weight residue IGF-1 insulin-like growth factor-1 NOi scavenging of nitric oxide DPPHr reduced 2,2-diphenyl-1-picrylhydrazyl ICA iron chelating activity ABTS 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid FRAP ferric reducing antioxidant potential DMSO dimethyl Sulfoxide PMS phenazine methosulphate NBT trizma hydrochloride solution SO superoxide anion SOi scavenging of superoxide anion NOi scavenging of nitric oxide PBS phosphate buffer MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] t-BHP Tert-butyl hydroperoxide SD Standard deviation xxii FBS fetal bovine serum TPTZ 2,4,6-Tris(2-pyridyl)-s-triazine HCl hydrochloric acid FeSO4 ferrous sulfate SNP sodium Nitroprusside APAP N-Acetyl-p-Aminophenol NED naphthylethylenediamine dihydrochloride Cyt-c cytochrome-c PI3K phosphoinositide 3-kinases AKt protein kinase B ERK1/2 extracellular signal-regulated kinase 1/2 AMPK AMP-activated protein kinase mTOR mammalian target of rapamycin DMEM Dulbecco’s Modified Eagle Medium FKN fractalkine SYK spleen tyrosine kinase p38 mitogen-activated protein kinase AS atherosclerosis TMPD N,N,N�,N�-Tetramethyl-p-phenylenediamine RT-PCR Quantitative real-time reverse transcription-polymerase chain reaction AA arachidonic acid mRNA Messenger ribonucleic acid DCFH non-fluorescent compound DCF 2´,7´-dichlorodihydrofluorescein GAPDH glyceraldehyde-3-phosphate dehydrogenase DCFH-DA 2´,7´-dichlorodihydrofluorescein diacetate EC50 statistical estimation of the concentration necessary to reach 50% of the observed effect 1.2. Oxidative stress 6 substance (TBARS) assay. Besides causing damage to proteins and therefore its function, lipid peroxidation has been associated with many inflammatory diseases and can also act as a cell death signal inducing programmed cell death [33]. 1.2.3 Antioxidant systems Given the toxic effects of ROS/RNS overload, living creatures have evolved many defense mechanisms to neutralize these species to achieve redox balance. Antioxidants are molecules that possess the ability to delay or prevent the oxidation of macromolecules [34–36]. The antioxidants system concerns antioxidants whose goal is to suppress the generation of free radicals and prevent oxidation propagation and repair enzymes such as lipases, and proteases, responsible for restoring the damage caused by ROS/RNS overload [34]. There are three major types of enzymatic antioxidants in the body SOD, glutathione peroxidase (GPX), and catalase (CAT) which act as the first line of defense. Superoxide dismutase scavenges superoxide radicals and converts them to water, thereby avoiding the formation of peroxinitrite from superoxide radical [36]. Both glutathione peroxidase and catalase can then remove 𝐻2𝑂2converting it to water. The first enzyme is the most abundant and is present in both mitochondria and cytosol. Glutathione peroxidase uses the reduced reduced glutathione (GSH) as a substrate to transfer electrons to peroxides, while oxidized glutathione (GSSG) is produced [20,37]. Apart from that GPX catalyzes the reduction of peroxide radicals to alcohols and oxygen. Thus, preventing the formation of lipid peroxyl radicals [38,39]. Additionally, transition metals such as iron and copper can be sequestrated by iron-binding protein, for instance, to avoid the generation of reactive free radicals [20]. Besides enzymatic, non-enzymatic antioxidants are part of the antioxidant system and can be obtained from the diet. Ascorbic acid (vitamin C), for instance, is present in many aliments and can rapidly be distributed to all tissues [20]. It effectively scavenges much relevant ROS/RNS causing the generation of ascorbyl radicals, which can then be regenerated to ascorbic acid. Additionally, ascorbic acid plays a distinguished role in restoring small molecules 1.2. Oxidative stress 7 such as alpha-tocopherol and urea so they can continue acting as antioxidants [40]. Vitamin C also possesses the ability to maintain the metal ions present in the active center of enzymes in a reduced state acting as a co-factor. Nonetheless, the reduction of these metal ions can result in a pro-oxidant effect, due to the intervention on Fenton reactions causing ROS generation [41]. Vitamin E (alpha-Tocopherol) has good solubility in lipid and can break the cycle of lipid peroxidation by reacting with peroxyl before it can attack lipid molecules [42]. Growing evidence has shown that phenolic compounds can also contribute to the regulation of ROS RNS levels and intervene in oxidative stress. The major defense lines and pro-oxidant reactions are represented in figure 1. Figure 1: Representations of the major pro-oxidant and antioxidant reactions in a biological system. Several sources can produce superoxide which can rapidly react with nitric oxide (NO) to produce peroxynitrite. Superoxide dismutase (SOD) can avoid this pathway through the catalyzation of superoxide to produce hydrogen peroxide. Which can then be neutralized by catalase or glutathione peroxidase. However, in presence of transition metal ions (iron and copper), hydroxyl free radicals may be produced from superoxide via the Fenton reaction. Reactive species are shown in red and antioxidant enzymes are shown in green boxes. SOS: superoxide dismutase; GSH: reduced glutathione; GS-SG: oxidized glutathione; NOS: nitric oxide synthase. Adapted from Biswas et al,2016 [43]. 1.3. Phenolic compounds 8 1.3 Phenolic compounds Phenolic compounds are secondary metabolites that are widely spread across plants, playing relevant roles in their existence [44,45]. These compounds perform different functions, most of them are related to plants’ defense against pathogens, including bacteria and viruses, and abiotic stresses like drought and salinity. Also, contributes to its characteristics such as flavor, and color, among others. Phenolic compounds are mostly found attached to other molecules such as sugars and proteins, the existence of its free forms is less common in plants [46,47] Bearing one or more aromatic rings with two or more hydroxyl groups as represented in figure 2, these compounds can be divided into two main classes: flavonoids and non-flavonoids [48,49]. The last ones comprise phenolic alcohols, hydroxybenzoic and hydroxycinnamic acids, and their derived esters and glycosides as well as other minor classes such as stilbenes [44,45]. Figure 2: Basic structures of phenolic acids. Adapted from Khoddami et al., 2013 [50]. Two aromatic rings enclosing a heterocyclic six-membered ring with oxygen are the base structure of flavonoids. The differences found on the pyran ring and the different patterns of methylation and hydroxylation on the rings allow the classifying of flavonoids into six groups: flavanones, flavonols, flavones, anthocyanins, and isoflavones [44,45] (see figure 3). The molecular structures of these phenolic compounds confer the capacity of inactivating free radicals. The ability to scavenge free radicals, donating hydrogen atoms, electrons, or chelate metal cations allows these compounds to protect the human body from some hazards such as oxidative stress [52]. This ability seems to allow compounds to intervene in several biological processes such as anti-inflammatory, anti-mutagenic, anti-carcinogenic, and antioxidant properties [44,53]. Besides the direct inactivating of free 1.3. Phenolic compounds 9 Figure 3: Flavonoid structure and main types of flavonoids. Adapted from Cosme et al., 2020 [51]. radicals, phenolic compounds can favor indirect antioxidant mechanisms, by modulating the activity of several antioxidant enzymes, mostly by the mediation of the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) [54]. Nrf2 is a positive regulator of a set of antioxidants and detoxication enzymes. At normal conditions kelch-like ECH-associated protein 1 (Keap1) promotes degradation of Nrf2 preventing their translocation to the nucleus where it binds to antioxidant response elements (ARE) in the promoter region. When oxidation occurs Keap1 loses the ability to degraded Nrf2. In this case, Nrf2 can easily target the nucleus leading to the activation of ARE, resulting in a cascade of reactions leading to ultimate anti-inflammatory and antioxidant effects (see figure 4) [54–57]. Furthermore, flavonoids may also inhibit key ROS/RNS-producing enzymes such as xanthine oxidase, NOS and cyclogenase-2 (COX-2) [58]. Recent studies have also revealed a connection between phenolic compounds and other inflammatory-pathways through the mediation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) and mitogen-activated protein kinase (MAPK) signaling [55,59,60]. Despite the valuable connection between phenolic compounds and anti-inflammatory and antioxidant activities found in several scientific works, there is still controversy on whether 1.3. Phenolic compounds 10 Figure 4: Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Nrf2 bounds to kelch-like ECH-associated protein 1 (Keap1) in cytoplasm during low stress states. Under oxidative stress situations Keap1 loses the ability to degradates Nrf2 which can then be translocated to the nucleus, where it binds to antioxidant response elements (ARE) leading to cytoprotective antioxidant gene transcription. As a result, enhanced antioxidant and anti-inflammatory activity. Created with BioRender.com. these compounds may have similar effects on the human body. Mainly due to the difficulty to know if the compounds responsible for those bioactivities can reach the targets at levels capable of inducing the desired effects. But also due to the sensibility of these compounds to the modifications that occur in the digestion processes [61,62]. Bioaccessibility is a term that refers to the first step to making an ingredient bioavailable, in other words, is the step where compounds/ ingredients are released from the food matrix to make them available for absorption. The compounds are the target of several modifications during digestion so that can be absorbed and enter the bloodstream which can be distributed to tissue to exercise its bioactivities. All these steps are part of the biovability [63–65]. During digestion are several changes that may alter the bioactivities of the initial food or compound when compared with the resulting metabolite. First, in the mouth, some bacteria present there together with saliva and chewing movements can cause the deglycosylation of some ingredients and phenolic compounds [64,65]. While others reach the stomach unmodified. Amylase, the most abundant enzyme in saliva, has no great impact on phenolic compounds. The stomach pH impairs the stability of phenolic compounds as well as other compounds. Some articles propose that some phenolic compounds can be absorbed 1.4. Inflammation and neurodegeneration 11 into the stomach through transporters in the stomach wall, but that is a small percentage. In the small intestine, some phenolic compounds may undergo hydrolysis becoming aglycones and due to their polarity, aglycones can easily pass through the portal vein to the liver where they undergo further reactions. Furthermore, the enzymes secreted by the pancreas and bile can contribute to the absorption of a phenolic compound through the formation of water-soluble micelles helping the digestion of apolar components. For instance, isoflavonoids and flavonoids can also be micellarized increasing their bioavailability [61,62,64–66]. In the colon, phenolic acids can be absorbed or modified by esterases and colon microbiota yielding absorbable metabolites [62]. The need to study the bioavailability of phenolic and other compounds, to understand the changes in their metabolite bioactivities, is thus highlighted through these modifications. There are several in vitro digestion protocols available to evaluate the final metabolites of the compounds and determine their bioavailability. Most protocols include the simulation of physiological conditions of the digestive process in the mouth, stomach, small intestine and, in some cases, intestinal fermentation. These procedures mainly consider factors such as digestive enzymes, pH, digestion time and saline concentrations [63]. 1.4 Inflammation and neurodegeneration The inflammatory response is a protective mechanism that aims to restore the normal physiology of the organism through the regeneration of damaged tissue/cell and/or removal of the pathogen that initiated the injurious stimuli [35,67,68]. The host cell can recognize the nature of the stimulus due to the presence of danger associated molecular patterns (DAMPS) and pathogen associated molecular patterns (PAMPS) that are recognized by pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) a family of cell-surface receptors. After the recognition of the stimulus, each TLRs activates distinct transcription factors that enters the nucleus to bound to the promoter’s region activating a series of genes. For instance, lipopolysaccharides (LPS) a major component of the cell walls of gram-negative bacteria (an endotoxin) activates specifically toll-like receptor 1.4. Inflammation and neurodegeneration 12 4 (TLR4) which recruits its downstream adaptors through interactions with the toll-interleukin-1 receptor (TIR) activating NF-KB and/or AP-1 which then expresses antimicrobial components to put an end in the initial stimulus (see figure 5) [67,68]. Transcription factor AP-1 is induced by the activation of MAPK pathways and can induce an inflammatory response The major signalling pathway that involved in TLRs recognition is NF-KB. Normally, this transcription factor is inhibited by 𝑘𝐵, which is degraded, after TLRs activation, allowing NF-KB to be translocated to the nucleus. There, NF-KB up-regulates the expression of genes encoding for pro-inflammatory cytokines such as interleukin 1 (IL-1) interleukin 6 (IL-6),tumor necrosis factor (TNF) (to increase vascular permeability among other functions),COX-2 (aiming the production of prostaglandins to promote further recruitment of inflammatory cells) and iNOS and nicotinamide adenine dinucleotide phosphate oxidase (NOX) (NO and ROS are used to phagocytosed microbes) [38,68–70]. Figure 5: Lipopolysaccharides (LPS) inflammatory response. LPS is recognized by toll-like receptor 4 (TLR4) which activates inflammatory pathways. 𝐼𝑘𝐵, the nuclear factor kappa-lightchain-enhancer of activated B cells (NF-KB) inhibitor are destroyed allowing NF-KB to be translocated to the nucleus. Simultaneously, mitogen-activated protein kinase (MAPK) pathway can be activated, resulting in the phosphorylation and activation of activator protein1 (AP-1). NF-KB and AP-1 controls inflammatory responses through the induction of inflammatory cytokines.Created with BioRender.com. The production of NO and ROS during inflammatory response is a major clear-cut example of the mutual connection between redox processes and inflammation [35]. 1.4. Inflammation and neurodegeneration 13 Furthermore, inflammasome a multiprotein complex responsible for processing inactive precursor forms of pro-inflammatory cytokines is activated by ROS and ROS itself can activate NF-KB activation generating a vicious cycle [71–73]. Therefore, maintaining the control of inflammatory response is crucial to avoid an overload of both oxidative stress and inflammatory status [74,75]. The term “oxinflammation” has been proposed as a pre-pathological condition due to the above-described relationship. Characterized by a chronic inflammatory status which is coupled with an oxidative stress situation. This occurs when, after an initial stimulus the inflammatory response is not properly ended due to persistent stimulus or due to a failure in normal resolution mechanisms, resulting in a continuous vicious circle [72,76,77]. To date, several pathologies have been associated with this vicious cycle created by a mutual combination of chronic inflammatory state and abnormal redox balance. This includes not only genetically determined diseases, such as Rett and Down’s syndrome but also more common metabolic disorders, such as diabetes, cardiovascular disease, cancer, and neurodegenerative diseases (NDDs) [72,76]. Neuronal death underlies the symptoms of many NDDs, including, among others, Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD). The progressive loss of neurons, at specific regions of central nervous system (CNS), leads to a gradual loss of cognitive and/or motor skills, resulting in dementia, debilitation and premature death [78,79]. Depending on which group of neurons begins to undergo pathological changes, a clinical condition of the particular neurodegenerative disease phenotype will emerge. Thus, there is a wide variety of clinical courses and pathological manifestations in the different NDDs even so, an overlap of symptoms can also occur [80, 81]. Despite the variety of clinical courses, there are common features in the different NDDs such as programmed cell death, mitochondrial dysfunction, and accentuating inflammatory status [79,82]. The brain is considered one of the most vulnerable organs in the body to oxidative stress mainly due to its high oxygen consumption rate and the presence of large amounts of polyunsaturated fatty acids, which are highly susceptible to lipid peroxidation [83]. Also, iron 1.4. Inflammation and neurodegeneration 14 is easily found throughout the brain, since many enzymes require it for its proper functioning. This high iron content can promote the ROS generation contributing to oxidative stress [84]. Another critical aspect of the brain lies in the fact that it is mostly composed of terminally differentiated neurons and glia thus, its potential to replace damaged cells is too little [83]. Moreover, the neurons possess relatively low levels of glutathione, a relevant antioxidant [85]. All the features mentioned above make the brain highly susceptible to oxidative stress, which can then cause direct damage and initiate many processes related to cell apoptosis, mitochondrial dysfunction, and inflammation [35,86,87] . Furthermore, a crucial role of inflammation has been highlighted in some NDDs such as AD PD and HD, since has been proven that proteins such as amyloid-beta or alpha-synuclein can bind to toll-like receptor 2 (TLR2) present in both microglia and astrocytes. Thus, promoting the phagocytic activity to remove the protein aggregates. However, at the same time, inflammatory signaling pathways are being activated, which may create an ”Oxinflammation” status. Furthermore, those proteins can themselves activate inflammasomes promoting the activation of interleukins contributing to neuroinflammation. Despite the inhibition of this inflammatory status may not resolve the NDDs, the reduction of neuroinflammation may result in clinical benefits, therefore is important to unveil how an inflammatory response may be controlled in microglia [70,88–90]. Microglia, are CNS’s primary resident immune cells, have a myeloid origin and are able to self-renewal independent of hematopoietic stem cells [91–93]. Microglia can adapt to different phenotypes to respond to changes in their surroundings and two classic phenotypes of activated microglia are well characterized. The pro-inflammatory M1-like that releases destructive mediators and the anti-inflammatory M2-like phenotype, that produces beneficial mediators such as interleukin-10 (IL-10) and interleukin-4. Thus, a balance in these phenotypes is fundamental for immune homeostasis in the brain [94]. Three major roles can be associated with these cells (see figure 6): 1) housekeeping, functions that contribute to the maintenance of neurons and their well-being; 2) sentinel which includes the constant evaluation of changes in their environment; and lastly 3) defense function to protect against injurious agents [92]. 1.4. Inflammation and neurodegeneration 15 The housekeeping functions include the elimination of many products in the brain such as synaptic elements, dying or dead cells, or abnormally accumulated proteins. Additionally, microglia can release neurotrophic factors such as insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor (BDNF) which contributes to the protection of neurons [92, 95,96]. The microglia’s sentinel function is essential for its good performance in its housekeeping and defense functions [97]. Their defense function is accomplished due to the presence of PRRs that respond to a variety of molecular patterns. Activation of these receptors leads to the production and of antiand pro-inflammatory mediators including interleukins, free radicals, proteases (matrix metalloproteinases) among others [92,93,98]. The release of matrix metalloproteinases has been linked to the disruption of blood-brain barrier (BBB) by degrading junctional complex proteins which can contribute to the recruitment of circulating leukocytes [99,100]. This aims to help the neuro-immune system end with the initial insult, however, this can impair BBB permeability resulting in brain infarction [101]. Activated microglia can also induce neuronal apoptosis through the release of cathepsins and can cause neuronal excitotoxic through the direct release of glutamate [92, 102]. In healthy brains, microglia can perform all the mentioned functions aiming at the protection of the CNS and the end of the initial stimulus. However, dysregulation of any of these functions can initiate or propagate an ongoing neurodegeneration situation [92]. Figure 6: Three major roles associated with microglia: housekeeping, functions that contribute to the maintenance of neurons and their well-being; sentinel which includes the constant evaluation of changes in their environment; defense function to protect against injurious agents Sentinel. Created with biorender. 2.3. Results 22 Figure 8: Percentage of categorized pharmacological activities found in the literature of Apocynum venetum until January 3, 2022. (ABTS) among others [10,125–130], will not be discussed here because those analyses are not always traduced into bioactivities in the human body. An A. venetum leaves extract protected against damage 𝐻2𝑂2-induced damages in 293T cells through the increment of GSH, SOD and CAT expression [131]. Another study using an aqueous extract from A. venetum leaves revealed an inhibitory effect on TBARS formation indicating that A. venetum can rise low-density lipoprotein (LDL) resistance to oxidation, which can help to prevent atherosclerosis (AS) and cardiovascular diseases [115]. Two studies [8,132] using d-Galactose-induced aging in mice/d-Galactose-induced oxidative stress prove that treatment with A. venetum tea and polyphenol extract, respectively, protects against d-Galactose-induced damages. Pathological observations revealed that polyphenol extract [132] could inhibit oxidative damage to the skin, liver and spleen of mice induced by D‑galactose. Besides the reduction of GSH, SOD after the treatment with A. venetum an up-regulation of Nrf2 expression levels, was also reported in this study. The same article also reported for the first time, an effect on a stress protein expression, heme oxygenase-1 (HO-1) related to anti-inflammatory and antioxidant processes. The results achieved by A. venetum treatment was stronger than others caused by vitamin C a well-known antioxidant. Li et al. [8] prove that the A. venetum extract can alleviate 2.3. Results 23 hepatocyte edema and inflammatory infiltration. The tea administration not only up regulated GSH,SOD and CAT but also cause an increase of anti-inflammatory factors (IL-10) along with a down-regulation of pro-inflammatory factors (IL-6, TNF). In particular, the treatment could regulate the weight of the major organs and diminished the generation of NO and malondialdehyde (MDA) levels. Both works proved that A. venetum exhibited antioxidant effects and can have multiple beneficial effects. Nonetheless, further clinical studies are required to better comprehend its mechanisms. 2.3.2 Antidepressant properties Butterweck and colleagues [133], encouraged by the content of hiperoside and isoquercetin present in A. venetum leaves decided to assess the antidepressant capacity of A. venetum. The forced swimming test (FST), commonly used for antidepressant assessment essays, revealed that A. venetum leaves extract markedly shortened the immobility time of male rats and its effects were similar to those induced by the antidepressant imipramine. Then, the same authors tried to establish a relationship between imipramine and A. venetum through the analysis of regional levels of serotonin (5-HT), norepinephrine (NE) and dopamine (DA). However, both differences and similarities were found, nonetheless, these were the first evidence of the effects of A. venetum on monoamine levels [134]. Since then, many authors have been trying to find the mechanism by which A. venetum can influence monoamine levels. For instance, Zhou and colleagues [135], evaluated the effect of A. venetum leaves on the monoamine oxidase (MAO) an important enzyme in the monoamine neurotransmitters metabolism. Their findings showed that the aqueous phase of ethanolic extracts of A. venetum significantly inhibited the activity of MAO, however, the ethanolic extract was not able to cause a significant inhibition. The antidepressant properties of flavonoids isolated from A. venetum were evaluated by Yan and colleagues [136], revealing that quercetin, kaempferol-3-O-𝛽-D glucose and quercetin3-O𝛽-D-glucose possesses antidepressant activity. This may be due to the increase of NE, DA and 5-HT as well as reduction of 5-HT metabolism in mice brain and CNS. 2.3. Results 24 Another study [137] also reported an increase of NE and DA concentration, along with their metabolites, after the administration of A. venetum leaves extract. The antidepressant effects of the extract on FST and tail suspension test (TST) were prevented by the pretreatment with dopamine D1 and D2 receptor antagonists, indicating that the antidepressant effect of A. venetum is dependent on the interaction with dopaminergic systems. A study on PC12 cell lines [138], under the premise that the antidepressant effects of A. venetum are attributable to the presence of hyperoside in the extract evaluated the properties of this compound. The antidepressant effects of hypeoroside from A. venetum reported may be related to its cytoprotective action. This includes the increase of gene expression related to antidepressant activity after chronic treatment, such as BDNF and CREB and the decrease of intracellular 𝐶𝑎2+. Those cytoprotective actions were also verified after fluoxetine administration, a commonly used antidepressant. Decrease of intracellular 𝐶𝑎2+ was also reported by Zheng and colleagues [139] as a neuroprotective effect caused by A. venetum leaves extract against corticosterone-induced neurotoxicity in PC12 cells an in vitro model of depression. The up-regulating of BDNF and microtubule-associated proteins (MAPs) may also contribute to its neuroprotective effects. The administration of fluoxetine, which increases serotonin levels also up-regulates BDNF. Previous work also revealed a linkage between the increase of serotonin and BDNF [140]. However, according to Butterweck et al. [134] and Zheng [137]A. venetum leaves extract administration did not cause an increase of serotonin, so another mechanism must be involved in the up-regulation of BDNF provoked by the extract. Animal studies further illuminated the protective effects of A. venetum in a chronic unpredictable mild stress (CUMS) of depression. Changes caused by CUMS were significantly mitigated after extract administration in two different studies [141,142]. According to Wu and colleagues [141], after four weeks of administration, A. venetum leaves significantly reversed apoptosis induced by CUMS in both the hippocampus and cortical areas. The down-expression of cytochrome-C, caspase-3, and caspase-9 proteins along with the normalization of Bcl-2 Associated X Protein (Bax)/B-cell lymphoma 2 (Bcl-2) ratio seems to 2.3. Results 25 be responsible for the decrease of apoptosis. Furthermore, the administration led to a significant increase of BDNF and CREB expressions. Multiple reports [140–142] have noted the effects of A. venetum on BDNF expression. Despite the upstream signaling pathway-regulated expression remaining unclear [142], some evidence has linked the increment in oxidative stress to the decrease of BDNF expression in neurodegenerative disorders [143]. Moreover, the reduction of BDNF mRNA and protein levels has been prevented by the treatment with vitamin E, through its antioxidant activities [144]. Thus, probably the antioxidant effects of A. venetum may cause an indirect effect of BDNF expression. Nonetheless, the direct effect on the modulation of BDNF is also a topic worthy of study. The search for the main antidepressant compounds in A. venetum extract along with the scientific works on which ones can pass through BBB is also necessary. The MAPs seems to be related to several pathologies such as Alzheimer’s disease, so understanding the effects of A. venetum on increasing the expression of these microtubules may be relevant for the treatment of other diseases. Finally, studies are needed to understand the influence of A. venetum extract on the monoamine systems 5-HT, NE, and DA involved in clinical depression. 2.3.3 Anti-hypertensive properties Traditional Chinese medicine has labeled the dried leaves of A. venetum as “anti-hypertensive tea”. Kim et al. [115] administered A. venetum’ leaves extract at a dose of 70 mg/rat per day to a hypertensive animal model. The study showed that the treatment was able to decrease blood pressure and the observed effects were associated with an increase in glomerular filtration. Nonetheless, the decreasing effects were weaker than captopril, a common drug used in hypertension. Another study performed by Kwan et al.[145] proved that A. venetum leaves extract caused vascular relaxation effects in both rat aorta and superior mesenteric artery. The vasorelaxation effects in the rat aorta were completely reversed by using a NOS inhibitor and a𝐾+channel blocker proving the relevant roles of NO and 𝐾+channels in this effect. 2.3. Results 26 Nonetheless, the effects were not specifically confined to one type of 𝐾+ion channel. The authors further discarded the possibility of A. venetum acting on endothelial muscarinic receptors through the use of atropine, an inhibitor of those receptors which did not inhibit the observed effects. The same study also reported that the effects persisted even after a brief exposure and subsequent prolonged washing, suggesting a great affinity between the compounds found in the extract A. venetum and in the generating system of NO. Another explanation for these prolonged effects would be related to the inhibition of the superoxide anion or effects on its elimination, which prevents the formation of peroxynitrite and, thus, increases the bioavailability of NO allowing a prolonged vasorelaxation effect [145]. Lau et al. [146], explore the second hypothesis using angiotensin II and phenylephrine, which not only causes vasoconstriction via receptor coupled calcium signaling pathway but also via the production of ROS - due to the activation of NOX. The results proved that A. venetum could counteract these effects through the inhibition of superoxide anion radical formation and the release of NO. The researchers also proposed that the inhibition of angiotensin II contractility and superoxide production may be related to the ability of A. venetum to downregulated NOX subunit (p67 phox and gp 91 phox). Additionally, A. venetum proved to be a potent inhibitor of vasoconstriction through the stimulation of vascular receptors, such as alpha-adrenergic and angiotensin II in both endothelium-intact rat aortic rings. Another article studied the mechanisms by which A. venetum can induce NO release [147]. Firstly, the authors confirmed the NO-mediated vasorelaxation induced by A. venetum through the measurement of total NO metabolites in human endothelial cells and isolated rat aortas. The enhancement provoked by A. venetum treatment was blocked by the presence of protooncogene tyrosine-protein kinase (Src) kinase and phosphoinositide 3-kinases (PI3K) inhibitors. The increment of NO and the protein expression levels of phospho-protein kinase B (AKt) and pohospho-eNOS caused by the treatment were also abolished by the use of those inhibitors. Polyphenols are known to cause NO-mediated endothelium-dependent relaxation through the activation of PI3K/AKt pathway leading to eNOS activation and increase of endothelial NO generation [148]. Quercetin, a major compound found in A. venetum leaves protected against reduced NO production in diabetic mouses via activation of PI3K/AKt signalling pathways [149]. In this way, quercetin among other compounds can be responsible for the relationship 2.3. Results 27 between Src/PI3K signalling pathways and A.venetum properties. These findings settled the anti-hypertensive effects of A. venetum, unraveling the linkage between Src/PI3K signalling pathways and the effects of A. venetum [147]. Irie and colleagues [150] further evaluated the cardiotonic effects of different extracts of this plant in isolated guinea pig right atria. The extracts were able to induce a contractile response of the isolated atria and increased the pulse at a concentration of 1 mg/ml, a higher concentration than the concentration reported by kwan [145]. The vasorelaxation effects provoked by A. venetum extracts were not inhibited by propranolol, proving that the observed effects were not related to beta-receptors. The authors hypothesized a possible association between the observed effects and phosphodiesterase-3. However, more studies regarding the possible linkage between phosphodiesterase-3 and A. venetum properties are required. Long-term NO stimulation has been associated with the modulation of phosphodiesterase-3 expression in endothelial cells, therefore this could be also occurring after A. venetum treatment [151]. The anti-hypertensive effects described by the ingestion of A. venetum tea agrees with the studies herein analyzed. Probably, A. venetum can activate Src/PI3K/AKt signalling pathway, causing an activation of eNOS leading to the increase of NO generation. Moreover, their vasoprotective effects may also be related to the increase of NO bioavailability and probably due to its superoxide scavenging activities. 2.3.4 Hepatoprotective properties A water extract of A. venetum was firstly reported [152] as hepatoprotective against two injury models: 𝐶𝐶𝐿4and D-galactosamine (D-GalN) / LPS. The treatment with the extract was not able to reduce the production of TNF but was able to inhibit TNF-induced cell death. These results suggest that A. venetum hepatoprotective effects occur after the production of TNF. In the same work, the authors proved that all fifteen flavonoids isolated from A. venetum demonstrated inhibitory effects on TNF-induced cell death with different intensities. Since it 2.3. Results 28 is known that flavonoids have scavenging activity, it is presumed that by scavenging ROS, they can interfere with signal cascade initiated by TNF, causing the inhibition of cell death [152]. Zhang and colleagues [124] also revealed that total flavonoids from A. venetum (25, 50, and 100 µg/mL) exert a protective effect on carbon tetrachloride 𝐶𝐶𝐿4-induced hepatocyte damage both in vitro in vivo, mainly due to their antioxidant effects such as decreasing the MDA formation and increasing antioxidant enzymes activities in liver tissue. Further analysis proved that besides the ability to modulate the activity of antioxidant enzymes activities the treatment with A. venetum also decreases the expression levels of cytochrome-C and suppresses caspase-3 and caspase-8 cleavage. N-Acetyl-p-Aminophenol (APAP) is a toxic metabolite involved in liver injury, through oxidative stress responsible for mitochondrial dysfunction, caspase-3 activation, DNA fragmentation, and finally necrotic/apoptotic cell death. The pre-treatment with A. venetum leaves extracts significantly inhibited nuclear DNA damages, normalized the levels of GSH, hepatic MDA, and reestablished the activities of GPX and SOD in mice [153]. The same authors [154] further confirmed that hyperoside a major flavonoid found in A.venetum had hepatoprotective effects. Again, APAP was used to cause liver damage. Like the A. venetum extract, hyperoside was able to restore the activity of antioxidant enzymes and attenuated the effects caused by APAP. Hyperoside accelerated the detoxification of APAP through the enhancement of detoxification enzymes expression and activities, as well as the inhibition of cytochrome P4502E1 (CYP2E1) which is responsible for the formation of APAP reactive intermediate. These activities appear to be related to its ability to modulate the translocation of Nrf2 which is involved in inducing the expression of antioxidant enzymes and phase II enzymes. Isoquercetin is another major compound present in A. venetum, demonstrated similar hepatoprotective effects to the hyperoside when tested in rats with damages induced by APAP. The decrease iNOS, TNF, and IL-6 production proved the anti-inflammatory effects of isoquercetin against the inflammation caused by APAP. In line with these results, the authors studied the relationship of isoquercetin and MAPK pathways, revealing that the pre-treatment with the compound suppressed APAP-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), mitogen-activated protein kinase (p38), C-Jun NH2-terminal kinase (JNK), which activates MAPK pathway [111]. 2.3. Results 29 Further study by Jiang and colleagues [155] proved that A. venetum extract and its major compounds showed hepatoprotective effects against APAP-induced liver injuries, through the decreased Cytochrome P450 levels. This study correlates the positive effects of A. venetum with its major compounds and shows its positive effects. Further assessment should be done to understand the influence of A. venetum in the MAPK pathway to better understand its hepatoprotective properties. The influence of A. venetum anti-inflammatory properties in its hepatoprotective effects should also be a target of the study. In short, although the underlying mechanisms are not fully known, A. venetum appears to have good hepatoprotective effects and clinical studies should analyze this potential. 2.3.5 Cardioprotective properties An article regarding the activity of A. venetum tea against the formation of advanced glycation end products (AGEs) revealed protective effects [156]. AGEs formation is promoted by the presence of ROS, and besides being highly connected with diabetes, AGEs are involved in aging-related organ damage, cardiovascular diseases and neurodegenerative diseases such as AD [157]. Thereby, avoiding these end products can avoid some of these conditions [156]. The ability of A. venetum to inhibit AGEs may be related to its radical scavenging activity [158]. However, more research is needed to establish whether A. venetum attenuates the AGEs’s in vivo. A study using a polysaccharide-rich extract [159] from A. venetum showed significant anti-hypoglycemic effects and the ability to modulate gut microbiota in diabetes rats. The treatment led to a significant decrease in fasting blood glucose levels, total cholesterol, triacylglycerols, and low-density lipoprotein cholesterol. A study using a cardiotoxicity rat model further shed a light on the protective effects of A. venetum leaves extract on the injuries induced by pirarubicin [160]. The molecular mechanisms behind the injury provoked by pirarubicin are not fully understood, nonetheless, ROS and oxidative stress seem to play a major role. The experiment found out that the pre-treatment decreased cardiac markers for myocardial necrosis and MDA levels as well as increased SOD activity. Additionally, an increase in Bcl-2/Bax ratio and reduction of 2.3. Results 30 cleaved-Caspase-3 protein were also reported in the same study. The article also suggested for the first time the ability of the A. venetum extract to reduce the release of cytochrome-C and to reverse the opening of mitochondrial permeability transition pore (mPTP) caused by pirarubicin revealing anti-apoptotic properties [160]. The opening of this pore has been linked to mitochondrial depolarization which can be followed by progressive mitochondrial swelling speeding up apoptosis processes [161]. The mPTP has been proposed as a promising target for the treatment of PD due to its importance on mitochondrial dysfunction and excessive cell death, so further studies on the mechanism behind these effects may be relevant [162]. Another study [163], confirmed the ability of A.venetum to suppress the release of cytochrome-C and the closure of mPTP revealing cardioprotective effects. The antioxidant activities of A.venetum were partially responsible for the observed cardioprotective effects as well as the decrease of cleaved caspase-9 and cleaved caspase-3 expression implying that A. venetum may inhibit the activation of apoptotic pathways, through AKt/Bcl-2 signalling pathway [164]. Hyperoside a compound found in A.venetum also revealed anti-apoptotic effects against 𝐻2𝑂2 -induced apoptosis on endothelial cells through the decrease of intracellular 𝐶𝑎2+concentration and cleaved caspase-3. Additionally, effects on Bcl-2/Bax ratio were observed and suggests the regulation of p38 and a possible association with MAPK reveling anti-apoptotic properties and suggesting association with MAPK signalling pathway . In a myocardial ischemia/reperfusion (MI/R) model, treatment with A. venetum leaves extract also led to a reduction of cardiac markers. The authors associated the positive effects with the direct reduction of oxidative stress through the increment of SOD activity and decrease of MDA content [165]. However, these effects were blocked by the use of inhibitors of ERK1/2 and PI3K, implying a linkage between A. venetum and Akt/ERK1/2 signaling pathways. At the same time, the authors confirmed a previous hypothesis theorized by Lau et al. [146] that A. venetum treatment could significantly reduce gp91(phox) expression, a critical subunit of NOX inhibiting superoxide generation. Quercetin, a compound found in A. venetum, was able to down-regulate the expression of p47phox, p67phox, two NOX components, exhibiting anti-hypertensive and proving to be 2.3. Results 31 a preventive compound against atherosclerosis (AS) [119,166]. Apocynin, also found in A. venetum is a well-known nicotinamide adenine dinucleotide phosphate (NADPH) inhibitor [167] and signaling research found that it was able to restore endothelial dysfunction in streptozotocin-diabetic rats due to the down-regulation of p22(phox) and gp91(phox) expression, two subunits of NOX [168]. So, further research on the influence of these compounds on the observed effects after the treatment should be done. Another work explores the effects of A. venetum treatment with atherosclerosis. A reduction in total cholesterol and triglyceride concentration was observed after the treatment with A. venetum which suppressed the progression of the diseases [7]. Furthermore, the treatment inhibited the excessive collagen synthesis in the aorta and the over-proliferation of smooth muscle cells. The authors associated these effects with the inhibition of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway. Zhang et al. [116] further enlighten the effects of A. venetum on AS progression on rats. The treatment with A. venetum extract reduced blood lipid levels and suppressed the progression of the disease. The authors indicated that the reduction of MDA levels and serum inflammatory factors such as IL-6 as well as the increase of SOD levels are the mechanisms behind the observed effects. Additionally, the experiment found a significant reduction of the protein expression of fractalkine (FKN), spleen tyrosine kinase (SYK), and p38, a pivotal signaling pathway in the occurrence and development of AS. To sum up, all articles herein analyzed show that the antioxidant and anti-apoptotic activities induced by A. venetum seem to be relevant to its cardioprotective properties. This indicates that A. venetum could be a promising complementary treatment for cardiovascular diseases, especially in preventing diseases and promoting well-being. Nonetheless, more research is needed to enlighten the relationship between A. venetum and the different signaling pathways herein discussed. Furthermore, the study of the relationship between A. venetum and mPTP could be a promising field for the treatment of other diseases. 3.2. Material and methods 38 was subjected to a second methanolic extraction using the same conditions and protocol. Thereafter, both organic phases were joint and concentrated with the aid of a rotavap (Buchi RII) at 40∘C. Finally, the extracts were evaporated to dryness in a lyophilizer (Christ Alpha 2-4, B. Braun). The resulting powder was suspended in DMSO and stored at -4∘C until further analysis. 3.2.3 In vitro digestion of Apocynum venetum leaves extract The study of digestion effects on the bioactivities and phytochemical composition is an area that has seen a continuous increase in interest in recent years [186]. There are several protocols available to simulate digestion, which is cheaper and simpler than performing in vivo experiments. Here, an in vitro digestion including a mouth, gastric and intestinal phase was performed. The in vitro digestion of AV was conducted according to the methodology described by Magalhaes et al., [187]. First, 3 g of the extract was mixed with 9 ml of artificial saliva solution containing 2.38 mg/ml 𝑁2𝐻𝑃𝑂4, 0.19 mg/ml 𝐾𝐻2𝑃𝑂4, 8.0 mg/ml NaCl and alpha-amylase (1.5 mg/ml). The pH was adjusted to 6.75 at 37∘C with HCl 1 M ( pH calibrator, Hanna instruments). To simulate the chewing movements of the oral phase, the mixture was shaken at 80 rpm for 2 min ( incubator shaker, infors HT multitron Pro). Next, the pH was adjusted to 1.2 with HCl 5 M to initiate the gastric phase and 9 ml of artificial gastric fluid (3.2 mg/ml pepsin in 0.03 M NaCl was added. For the next 120 min. the mixture was incubated on a shaker at 150 rpm, at 37 ∘C. Finally the pH was adjusted back to 6.0 with 𝑁𝑎𝑂𝐻 5 M, and 1.5 ml NaCl (120 mM), 1.5 ml of KCl (5 mM) and 9 ml of artificial intestinal fluid (10 mg/ml of pancreatin and 11 mg/ml of bile extract in 9 ml of 0.1 M 𝑁𝑎𝐻𝐶𝑂3) were sequentially added and incubated at 37 ∘C at 150 rpm. After 1 hour of incubation, the extract obtained was centrifuged (Centrifuge falcons Sigma 2-16K) at 5000 rpm for 5 minutes, the pellet was discarded and the supernatant frozen at -80∘C (Deep freezer Sanyo and Panasonic) and subsequently lyophilized. Following this protocol, we obtained a second extract: digested of Apocynum venetum methanolic leaves extract (dAV). 3.2. Material and methods 39 3.2.4 Extract acidic hydrolysis To confirm the compounds identified in the extract, an acidic hydrolysis of glycosides was conducted. Briefly, to 1 ml of the AV extract at a concentration of 20 mg dry weight residue (dwr)/ml in methanol 70%, 1 ml of HCl 2 M was added. The mixture was then incubated for 2 hours at 80 ∘C in a water bath (Gran). Next, 1 ml of 𝐻2𝑂was added, followed by the addition of 2 ml of diethyl ether. After that, two phases appeared in the flask, the top layer was discarded and this protocol was performed twice. Finally, the rest of the diethyl ether was left to be evaporated in the hood. To the obtained residue 2 ml of methanol 70% was added, achieving a final concentration of 10 mg/ml. 3.2.5 HPLC-DAD analysis Apocynum venetum leaves extract (AV) and the hydorlysed AV were analysed by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD). HPLC-DAD analysis was conducted according to Dias and colleagues [188]. Apocynum venetum methanolic leaves extract was dissolved in 200 µl of DMSO and 800 µl of methanol 70% were added achieving a final concentration of 10 mg dwr/ml. Both AV and hydrolyzed AV were filtrated and injected in the HPLC-DAD equipment (HITACHI, LabChrom Elite, Japan) controlled by the elite EZChrome software (Agilent technologies, California, United States). The detection occurred in the range between 250 nm and 600 nm. Chromatograms were recorded at 260, 280 and 350 nm for qualitative analysis of the compounds present in the extract. A column LichroCART (Lichrospher 100. Rp-18e. 5 µm, Merck, German) was used in this analysis. The mobile phase used solvent A (0.1% of formic acid in methanol) and solvent B (0.1% formic acid in ultrapure water). Elution was performed at a flow rate of 0.8 ml/min using a gradient developed for the analysis of the extracts described in table 1. The identification of phytochemical compounds present in Apocynum venetum extract was done through the 3.3. Results and discussion 40 comparison of their UV spectra and the retention time to standard reference compounds. Quercetin and chlorogenic acid were used as external standards to quantify the compounds present in the extracts. The quantification of compounds was performed according to Wang et al.,[189]. The values of the peak area obtained for standards and its known concentration were used to determine the response factor (peak area/concentration of standard). Next, the peak area of the compound found in the extract, under study, was divided by the response factor of the standard (area of peak/response factor). Finally, the obtained value was divided by the concentration of extract (10 mg/ml) to know the concentration of the compound in 1 mg of extract. Table 1: Elution gradient for the analysis of non-digested Apocynum venetum leaves extract 3.3 Results and discussion The analysis of the phytochemical composition of plants is fundamental for conducting quality control protocols and also to look for possible correlations between bioactive properties of plants and their composition [49,190]. HPLC-DAD analysis is a wide use technique to analyze and identified the compounds present in a sample and is based on a stationary phase and a mobile phase. HPLC-DAD uses a column often packed with reversed-phase C18 column material (stationary phase), a pump responsible for moving the mobile phase through the column, and a detector that shows the retention times of the molecules [49]. Each compound present in a sample has different molecular structures and functional groups that give them different degrees of affinity with the mobile and stationary phases. The higher the affinity to 3.3. Results and discussion 41 the stationary phase the slower would be the movement of that specific compound and the higher would be its retention time. The UV-detector further allows detecting the solute as they elute from the column [191, 192]. In this way, each compound has a distinct migration rate and can be identified through the chromatogram generated by the electrical signals recorded by the HPLC-DAD detector. The results obtained by HPLC-DAD revealed the presence of phenolic acids and flavonoids derivatives in the AV as shown in figure 9. Four major compounds were identified as derivatives of caffeoylquinic acid and quercetin due to their UV-Visible spectrum, retention time and wavelength of maximum absorption. Quantification of compounds is indicated in table 2. The Figure 9: HPLC-DAD Chromatogram (350 nm) of: non-digested Apocynum venetum leaves extract (10 mg/ml); Peaks: 1:caffeoylquinic acid derivatives; 2,3,4:quercetin derivatives. results show that quercetin derivatives are the major compounds found on the AV followed by derivatives of caffeoylquinic acid. The results are in concordance with those found in the literature which also reckoned flavonoids as the main compounds of AV. Nonetheless, other authors were able to identify other flavonoids such as kaempherol, and hyperoside in A.venetum leaves extract [9,135,143,193]. Some of these compounds have been linked to antioxidant activity. [52]. 3.3. Results and discussion 42 Table 2: Phenolic quantification by external standard. To ensure that the comparison with UV-Visible spectrum, retention time and wave-length of maximum absorption was a reliable method for the identification of the compounds acidic hydrolysis of the extract was performed to identify the free forms [50]. Flavonoids are often extracted from herbal samples with methanol, ethanol, acetone, water, or mixtures of these solvents [50]. The extraction may depend on the time, active compounds and the type of plant. Flavonoids are often found in plants bound to some sugar, creating its derivatives. To obtain the flavonoids aglycone is necessary to follow a protocol that subjects the extract to acidic hydrolisis at high-temperature conditions. Hydrolysis of flavonoid glycosides of A. venetum methanolic leaves extract was carried out in 2 M HCl at 80 ∘C for 2 h. These conditions completely hydrolyze 3-0-glucosides (such as hyperoside, isoquercitrin and rutin) within a few minutes while flavonol with glucose attached to the 7-hydroxyl is distinguished to more resistant to acid hydrolysis [194,195]. The HPLC-DAD analysis are showed in figure 10. Only quercetin was found in this analysis. Quercetin was not identified in the original AV HPLC-DAD analysis, confirming the identification of quercetin glycosides in AV. Caffeoylquinic acid derivatives were not identified in this analysis, due to their degradation in these acidic conditions as previous described by Biesaga et al., [196]. This analysis also proves that glycosides are cleaved in acidic conditions, which might occur in stomach digestion. According to the literature, the conditions occurring in digestion may impair the structure of phenolic compounds. Amylase found in saliva does not have great effects on phenolic compounds and they seem to be stable under gastric conditions due to the low pH that protects them from degradation [197– 200]. However, according to some sources some free forms of phenolic compounds can be 3.3. Results and discussion 43 absorbed such as phenolic acids and hydrolysis and deconjugation can also occur in the gastric conditions, depending on the phenolic compounds class [64,65,201]. The literature describes the condition of the small intestine as the most critical for phenolic compounds’ stability. The alkaline conditions as well as the presence of pancreatic esterase and bile acid cause degradation of these compounds [64,197–200]. Figure 10: HPLC-DAD Chromatogram (350 nm) of: hidrolised Apocynum venetum methanolic leaves extract (10 mg/ml); Peaks: 1:quercetin. Nevertheless, there are also studies reporting significant losses of phenolic compounds in oral and stomach phases and others reporting higher stability of those compounds in small intestine conditions. For instance according to [197] only 3% of the total loss of rutin occurred in the intestinal phase, while 44% to 95.7 % of the total loss of chlorogenic acid, took place in the small intestinal phase. The results obtained by the different articles regarding the effects of in vitro digestion are sometimes contradicted and challenging to analyze due to the different protocols performed. Furthermore, the food matrix seems to be a critical factor in the effects of digestion [63,197]. Thus, more efforts should be made to arrange a reliable uniform protocol of the modifications occurring in the digestion. Furthermore, a phytochemical analysis of the digested extract needs to be conducted, to analyze the effects of the in vitro protocol conducted on the composition of the extract. 3.4. Conclusions 44 3.4 Conclusions The study of bioavailability and metabolism of phenolic compounds has been increasing steadily during the past years. This information is crucial to a proper analysis of its potential health effects. Only by understanding which metabolites and at what concentrations can reach tissues and cells, is possible to truly evaluate the beneficial effects of a phenolic compounds-rich diet. The phytochemical analysis and the hydrolysis performed showed that AV is mainly composed of flavonoid derivatives and phenolic acids which are known to have antioxidant properties. Quercetin derivatives such as rutin, hyperoside, and isoquercitrin were the most abundant compounds herein analyzed, and are related to antioxidant and anti-inflammatory properties. CHAPTER 4 Biochemical in vitro analysis of the antioxidant and anti-inflammatory properties 45 46 4.1 Introduction The rising acceptability and accessibility of Traditional Medicine (TM) have encouraged many researchers to study the properties of several traditional medicinal plants [202,203]. At present, for instance, many traditional Chinese medicinal plants are under scientific evaluation to assess their bioactive efficacy and safety [203–205]. Many of those studies focus on the evaluation of the properties of phytochemicals classes such as alkaloids, phenols, tannins, steroids, cardiac glycosides, diterpenes, and saponins, found in those plants. The evaluation of biological and chemical properties of those compounds can be a valuable source to help the development of effective drugs [206]. A relevant bioactivity to be explored is the antioxidant capacity, which can be exercised in several ways: oxidizing agents of free radicals and/or non-radicals, chelating agents of transition metals, compounds that inhibit the generation of oxidants and/or that can stimulate the production of endogenous antioxidant compounds [207]. Several biochemical assays have been developed to evaluate these different antioxidant activities. These assays are normally related to the generation of different radicals acting through a variety of mechanisms [208– 210]. Usually, they are based on a spectrophotometric determination as is the case of DPPH or ABTS assay. Due to the variety of the protocols many standard compounds, such as quercetin and Trolox, are used to allow comparisons [211]. Besides the evaluation of antioxidant properties of extracts under study, anti-inflammatory properties were also target of study here. Since NO intervenes in inflammatory situations, the NO scavenging properties evaluated by Griess of the extracts can be considered an antiinflammatory property [35]. The ability of the extracts to inhibit the activity of COX-2 was also evaluated in this work. COX-2 is one of the most significant enzymes since its inhibition is the pharmacological mechanism of non-steroidal anti-inflammatory drugs. Thus, finding new safer, and perhaps more efficacious COX-2 inhibitors is an important area of research. The COX-2 is an inducible isoform of cyclooxygenase which are responsible for the production of prostaglandins [212]. The first step of COX-2 is the addition of molecular oxygen to arachidonic acid (AA) to form the unstable prostaglandin G2. Then, prostaglandin G2 is 4.2. Material and methods 47 converted to endoperoxide by the peroxidase function of COX-2, which is then non-enzymatic converted to prostaglandin E2. There are three ways to measure COX-2 activity: measurement of the final product of the reaction, prostaglandin E2, by ELISA; measurement of the oxygen uptake that occurs during the first step of the reaction using an oxygen sensor; and the third is to measure the second reaction spectrophotometrically. For this work, the last method was chosen [213]. Considering all this, this chapter aims to present the biochemical methodologies carried out to determine the antioxidant/anti-inflammatory activities of AV and the digested extract (dAV) by radical scavenging activity (DPPH and ABTS), FRAP method, ICA, superoxide anion radical (PMS-NADH system) and nitric oxide (Griess method) scavenging activities and inhibiton of COX-2) as well as to discuss the results obtained. 4.2 Material and methods 4.2.1 Chemicals and Reagents For the biochemical assays, DMSO �99.7% purity, quercetin, ( � 95% purity). 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferrozine (3-(2-Pyridyl)-5,6diphenyl-1,2,4-triazine-4�,4��-disulfonic acid sodium salt, 97% purity) all purchase on Sigma, (Darmstadt, Germany). Nicotinamide adenine dinucleotide (𝑁𝐴𝐷𝐻) was purchased at Roche Diagnostics, (Penzberg, Germany) and 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) ( �98% purity) to FLUKA, (Buchs, Switzerland). Phenazine methosulfate phenazine methosulphate (PMS) 99% purity and Trolox (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, � 97% purity) was purchased from Acros Organics, (Geel, Belgium). Persulfate solution, FeCl3.6H2O, and disodium salt of ethylenediaminetetraacetic acid (𝑁𝑎2𝐸𝐷𝑇𝐴) and ethanol were bought from Merck, ( Darmstadt, Germany). Trizma hydrochloride solution and hydrochloric acid (HCl) were purchased from Fisher, (Hampton, New Hampshire). 4.2. Material and methods 54 The control and the blank were performed following the same conditions, however the extract and ferrozine, were replaced by the equivalent amount of ultra-pure water. 𝑁𝑎2𝐸𝐷𝑇𝐴 was used as a positive control and as a chemical reference in comparison to the iron-chelating activity capacities of the extracts. After 15 minutes under dark conditions and at room temperature, the absorbance was measured at 515 nm, with a SpectraMaxPlus microplate reader. All measurements were done in triplicate and three independent assays were performed. The percentage of ICA was then estimated through the equation represented in figure 18. Due to the fact that 𝐸𝐶50 was not achieved using the higher concentration of Figure 18: Formula used for calculation of the ICA percentage. 2 mg dwr/ml of A. venetum, the 𝐸𝐶25was determined. The 𝐸𝐶25(concentration that caused chelation of 25% of the iron added) was estimated, after plotting the % ICA against tested concentrations. 4.2.7 Nitric oxide scavenging activity The ability of Apocynum venetum to scavenge nitric oxide production was measured according to the method of Colle et al., 2012 [221]. This assay is based on the fact that sodium Nitroprusside (SNP) decomposes in an aqueous solution at physiological pH producing nitric oxide radical. This radical can react with oxygen and generate stable products such as nitrate and nitrite, that can be quantified using Griess reagent. The presence of antioxidants inhibits 4.2. Material and methods 55 the production of nitric oxide radical generated from SNP which can then be quantified by Griess reaction (represented in figure:19) [214]. Figure 19: Scheme of the Griess diazotization reaction. Adapted from : Brizzolari et al., 2021 [222]. For this protocol a stock solutions of PBS (19 mM in deionized water, pH 7.4), phosphoric acid (5% in ultrapure water), SNP (20 mM in ultrapure water, protected from the light), sulfanilamide (1% in 5% phosphoric acid) and NED (0.1% in ultrapure water) were prepared. For this assay 50 µl SNP were added to 50 µl of the different concentrations of the AV (3 µg dwr/ml to 150 µg dwr/ml in PBS) to obtain a range of concentrations between 0.5 µg dwr /ml to 10 µg dwr/ml. Following this the samples were incubated for 60 minutes at room temperature. Then, 50 µl of Griess reagent (1% sulfanilamide in 5% phosphoric acid 0.1% NED in ultrapure water with 1:1 porportion) were added. After 15 minutes at dark conditions and room temperature, the absorbance was read at 546 nm using a SpectraMaxPlus microplate reader. The same procedure was used for the control and blank, however, the extract and Griess reagent were, respectively, replaced with the equivalent amount of PBS. A blank for the control was also performed, where the extract and Griess were replaced by PBS. Quercetin was used as a positive control. All measurements were done in triplicate and three independent assays were performed. The percentage of NOi was calculated according to the formula presented in figure 20. The 𝐸𝐶25value was determined as previously described. 4.2.8 Peroxidase assay for measurement of COX-2 activity COX-2 plays a crucial role in the development of inflammatory status. COX-2 is an inducible isoform capable of converting arachidonic acid (AA) to prostaglandin E2 which is the precursor 4.2. Material and methods 56 Figure 20: Formula used for calculation of the percentage of inhibition of nitric oxide NOi. of prostaglandins, thromboxanes, and prostacyclins [212,223,224]. These small molecules are involved in inflammatory conditions in certain cancers, in the brain, and in other physiologic stimuli. Thus, the inhibition of COX-2 can help to counteract an inflammatory status and may have positive effects on health conditions [225]. A peroxidase assay for measurement of COX-2 activity was used to evaluate the inhibitory potential of the extracts under study since it is a high-throughput and cheap method. In this protocol AA to generate hydroperoxide by COX-2, and TMPD an electron donor, which turns blue upon reduction, were used. TMPD is oxidated by the heme peroxidase producing a highly colored product that absorbs at 611 nm. Thus, if inhibitory compounds of COX-2 are present TMPD is less oxidized avoiding the production of the highly colored product [226]. The effects of A. venetum extracts on COX-2 was analyzed through the COX-2 peroxidase assay conduct according to the method described by Mogana Wiart [227]. Briefly 20 µl of extracts dissolved in DMSO were plated in triplicates at different concentrations ( 10, 25,50, 125 and 250 µg dwr /ml ) in a 96-well microplate followed by 20 µl of 10 U/mL of COX-2 enzyme solution. Then, 180 µl of endpoint assay mix containing 100 µM bovine hemin chloride, 10 mM of AA, 17 mM of TMPD, and 1 M of buffer Tris-HCl pH 8.1. After 5 minutes of incubation at room temperature in the dark, the absorbance was measured at 595 nm. Negative and positive controls in which the extract was replaced by 20 µl DMSO or COX-2 inhibitor ( 𝑚𝑒𝑡ℎ𝑦𝑙[5−𝑚𝑒𝑡ℎ𝑦𝑙𝑠𝑢𝑙𝑓 𝑜𝑛𝑦𝑙−1−(4−𝑐ℎ𝑙𝑜𝑟𝑜𝑏𝑒𝑛𝑧𝑦𝑙)−1𝐻−2−𝑖𝑛𝑑𝑜𝑙𝑦𝑙]𝑐𝑎𝑟𝑏𝑜𝑥𝑦𝑙𝑎𝑡𝑒), respectively were done. Blanks were also included where 180 µl endpoint assay mix and 20 µl of COX-2 4.3. Results and discussion 57 was replaced by buffer Tris-HCl. Results were expressed as cyclogenase-2 inhibition (ICOX-2) relatively to the negative control using the formula present in figure 21. The 𝐼𝐶50 (halfmaximal inhibitory concentration) value was then obtained, as previously described for both the extract and the COX-2 inhibitor. Figure 21: Formula used for calculation of Cyclogenase-2 inhibition (ICOX-2) percentage. 4.2.9 Stastical analysis In general, experiments were performed independently three times, with at least three replicas. Data were analyzed in GraphPad Prism v. 8.0 Software (San Diego, CA, USA). 𝐸𝐶50 and 𝐸𝐶25 values were also calculated in GraphPad via non-linear regression analysis. Data are shown as mean ± standard deviation (SD) of three independent experiments. Results were analyzed by ANOVA analyses. Differences were considered significant at a * p-value< 0.05; ** p-value< 0.01; *** p-value< 0.001; **** p-value< 0.0001. 4.3 Results and discussion 4.3.1 Antioxidant activities Various methods have been developed to measure the antioxidant activity of several samples such as compounds, plants, and food among others. These methods are widely used due 4.3. Results and discussion 58 to their rapidity, and convenience and are based mainly on three methods: hydrogen atom transfer (HAT), single electron transfer (SET), and the ability to chelate transition metals SET [207,228]. FRAP,ABTS are based on SET, DPPH is based on both, while ICA is based on the ability to chelate transition metals. Each method is performed in specific conditions such as pH, time, polarity, and temperature, therefore, the performance of only one assay will not reflect the total antioxidant capacity [214]. Furthermore, the fact that compounds found in plants are mostly bound to other molecules such as sugars and proteins can interfere with their antioxidant effects and may lead to lower results when compared to free forms of the compounds [46]. In this work, to study the antioxidant potential of AV and dAV several methodologies, including radical scavenging activities (DPPH and ABTS), ferric reducing antioxidant power,iron-chelating assay, superoxide anion radical (PMS-NADH system), nitric oxide (Griess method) scavenging activities and in vitro determination of COX-2 activity were used. DPPH radical can be reduced by an antioxidant through the donation of hydrogen to form a more stable molecule. This method has some limitations related to its nitrogen atom location at the center of the structure that can enable larger molecules to assess the radical proportions due to steric hindrance. Furthermore, samples with high content in carotenoids or other compounds with strong absorbance at the same wavelength as the DPPH may interfere with the results [207,229]. Despite these limitations and the fact that DPPH radical is not present in living organisms, DPPH based methods are widely used [207]. Quercetin is a well-known flavonoid and has been associated with several biological properties such as anti-inflammatory and antioxidant activities [185]. Studies show that quercetin can react stoichiometrically and rapidly with DPPH radicals, therefore was chosen as a standard compound and was used as a positive control in this assay [230]. The 𝐸𝐶50 value is a statistical estimation of the concentration necessary to reach 50% of the observed effect calculated by using a hyperbolic fit. The lower the value of 𝐸𝐶50, the higher the antioxidant potential of the sample [231]. Results are present in figure 22, both extracts under study revealed antioxidant activity towards DPPH radical. Quercetin, the standard used showed an 𝐸𝐶50 value of 3.1 ± 0.1 4.3. Results and discussion 59 µg/ml, showing a higher antioxidant activity than both extracts under study according to the ANOVA analysis performed (p-value < 0.0001). The AV inhibited 50 % of the radicals at a concentration of 118.7 ± 6.6 µg dwr/ml while the dAV had an 𝐸𝐶50 value of 314.5 ± 5.6 µg dwr/ml. The 𝐸𝐶50 value of the non-digested extract was signinficant lower than the value of the digested extract (pvalue < 0.0001) which shows an effect of the digestion process on Apocynum venetum antioxidant activity towards DPPH. Figure 22: Antioxdiant activity by DPPH. The results are presented as 𝐸𝐶50 values of AV, dAV and quercetin (standard reference ). Each bar represents the means ± SD of three independent experiments. Asterisks mean significantly differences from the quercetin ****p-value < 0.0001 determined by ANOVA analysis. DPPH scavenging potential of A. venetum have been already reported in literature, for instance Liang et al., 2010, [126] showed that A. venetum leaves methanolic extracts had a lower 𝐸𝐶50 in the DPPH assay than butylhydroxytoluene, the standard used in the study. Another study show that Apocynum venetum leaves were able to scavenge 22.4% to 55.9% of DPPH in the concentration range of 0.2–1.0 mg/mL [8]. The DPPH scavenging activity of Apocynum venetum have been attributed to the presence of quercetin glycosides. Isolated quercetin glycosides from Apocynum venetum also revealed activity towards DPPH radical,showing higher activity than trolox, the standard compound used in the study [10]. Results are also in line with results found by lee and colleagues [232], that reported a 4.3. Results and discussion 60 decrease of activity towards DPPH of onion quercetin and grape resveratrol after being subjected to an in vitro digestion. The ABTS assay is very similar to the DPPH method since both of them use a strongly colored stable radical which is not present in living organisms [207]. However, ABTS has the advantage of being freely soluble in both organic and aqueous solvents so it can be used to screen both hydrophilic and lipophilic compounds, unlike other antioxidant methods [229]. In this assay Trolox, a water-soluble analog of vitamin E was used as a standard compound. The results are shown in the figure 23. Figure 23: ABTS scavenging activity. The results are presented as 𝐸𝐶50 values of AV – non-digested and dAVdigested Apocynum venetum methanolic leaves extract and trolox (standard reference ). Each bar represents the means ± SD of three independent experiments. Asterisks mean significantly differences from the trolox ****p-value <0.0001 determined by ANOVA analysis. The 𝐸𝐶50 value for AV was 14.9 ± 0.5 µg dwr/ml and dAV was 73.5 ± 6.5 µg dwr/ml. The 𝐸𝐶50 of dAV was significant higher than the 𝐸𝐶50 of trolox (13.9 ± 0.1µg dwr/ml) (p-value= 0.0001) while the AV did not show significant differences in comparison with trolox according to the ANOVA analysis performed. Again, the digestion process impaired the antioxidant activity of the extract, leading to a significant increase of 𝐸𝐶50 value (p-value <0.0001) according to the ANOVA performed. 4.3. Results and discussion 61 Previous work has shown the capability of compounds found in Apocynum venetum to scavenge ABTS radical, however the analysis of Apocynum venetum leaves extract has not been addressed [12,127]. Here, we prove that Apocynum venetum has great antioxidant activity in ABTS, and despite the decrease of activity provoked by the digestion process, Apocynum venetum still showed antioxidant ability in the assay. Superoxide anions can be very harmful to cellular components due to their ability to generate hydroxyl radicals and their role in lipid peroxidation initiation [233]. Therefore, several compounds such as flavonoids have been screened for their scavenging activity of superoxide anions [234]. The scavenging activity of A. venetum, before and after performing in vitro digestion, was assessed through a non-enzymatic method. The results are present in figure 24, where AV exhibited a lower 𝐸𝐶50 value (13.33 ± 2 µg dwr/ml) than quercetin standard ( 𝐸𝐶50 value of 27.28 ± 3 µg/ml). The differences between these two values were significant according Figure 24: Superoxide scavenging activity. The results are presented as 𝐸𝐶50 values of AV – nondigested and dAVdigested Apocynum venetum methanolic leaves extract and quercetin (standard reference ). Each bar represents the means ± SD of three independent experiments. Asterisks mean significantly differences from the ***p-value <0.001 determined by ANOVA analysis. to ANOVA analysis (p-value = 0.0002) performed. The 𝐸𝐶50 of the dAV was 24.4 ± 1 µg 4.3. Results and discussion 62 dwr /ml which did not show significant differences from the quercetin 𝐸𝐶50 value. Regarding the effects of the digestion, a decrease of the ability to scavenge superoxide anion (SO) was also observed. The differences between the 𝐸𝐶50 of both extracts were significantly different with a p-value = 0.0009 (ANOVA analysis performed). The ability of Apocynum venetum to scavenge SO, was also revealed by Li et al. [8]. Regarding the effects of digestion, the decreased pattern was similar to the one observed in DPPH and ABTS. Transition metal ions such as 𝐹𝑒2+ are capable of perpetuating the formation of free radicals by the gain or loss of electrons. Therefore, chelating agents can prevent the formation of reactive oxygen species [235]. The ferric reducing antioxidant power of Apocynum venetum was determined using FRAP a convenient and reproducible method [236]. The AV was able to reduce the ferric-TPTZ complex in a concentration-dependent manner and showed chelating activity at concentrations of 25 µg dwr/ml, the results are shown in figure 25. The in vitro digestion also diminished the ability of Apocynum veentum to reduce the ferric-TPTZ complex and dAV was not able to chelate in the minor tested concentration, 25 µg dwr/ml, however, still reduced the complex in a concentration-dependent manner. Our results are in line with Record Lane [237] that also reported a decrease of antioxidant activity in FRAP analysis after the intestinal digestion of green and black tea. The iron-chelating activity of Apocynum venetum extracts was also measured and 𝑁𝑎2𝐸𝐷𝑇𝐴 was used as the positive control for this assay. Since 𝐸𝐶50 was not achieved using the higher concentration of 2 mg dwr/ml the 𝐸𝐶25 was determined. The 𝐸𝐶25 value is a statistical estimation of the concentration necessary to reach 25% of the observed effect calculated by using a hyperbolic fit. The screening of Apocynum venetum as chelating agent showed that the extract was able to chelate iron showing an 𝐸𝐶25 value of 396.1 ± 22.66 µg dwr/ml, the results are present in figure 26. The ANOVA analysis revealed that 𝑁𝑎2𝐸𝐷𝑇𝐴 𝐸𝐶25 value (2.5± 2 µg/ml) was significantly minor than AV (p-value < 0.0001), showing a higher ICA potential. The dAV did not show ICA in the performed assay, showing once again the effects caused by the in vitro digestion. 4.3. Results and discussion 63 Figure 25: Ferric reducing antioxidant power of non-digested,AV present in Aand digested extract of Apocyunm venetum dAV in B, respectively. Results are expressed as 𝐹𝑒2+ equivalents and data is represent mean ± SD for three independent experiments. As previously mentioned NO plays a relevant role in inflammatory processes and chronic expression of NO is associated with various carcinomas and inflammatory conditions [238]. In this assay, nitric oxide generated from sodium nitroprusside reacted with oxygen to form nitrite. The ability of Apocynum venetum extract to compete directly with oxygen in the reaction with nitric oxide translates its ability to scavenging NO generation. Quercetin was once again used as a positive control due to its role in antioxidant pathways [185]. The results are shown in figure 27. The 𝐸𝐶25 value was determined here instead of 𝐸𝐶50. The 𝐸𝐶25 value of AV was 13.3 ± 0.9 µg dwr/ml which was significantly minor than the quercetin (28.96 ± 1.7 µg/ml) according to the ANOVA analysis performed (p-value < 0.0001). The in vitro digestion performed caused a decrease of the ability to inhibit NO generation, revealing an 𝐸𝐶25 value of 27.70 ± 0.3 µg dwr/ml. There were no significant differences between dAV and quercetin. Nonetheless the 𝐸𝐶25 of dAV and AV were significant different (p-value < 0.0001). Despite this, the extract still revealed scavenging activity towards NO even after performing in vitro digestion. To sum up the performed in vitro digestion lead to a decrease of antioxidant activity in all tested assays performed here. So, we can conclude that the modifications occurring in simulated 5.2. Material and methods 70 cell line were also evaluated. For that purpose, the expression of genes was evaluated by Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and NO levels of cells supernatant were measured. Finally, before proceeding with the study of possible positive effects, plant extracts were screened for possible toxicities to avoid negative effects [244]. The liver plays a pivotal role in the metabolism and biotransformation of compounds, an activity that can cause hepatotoxicity which can lead to systemic toxicity [246]. Thus, the toxicities of extracts under study were evaluated using a HepG2 cell line (human hepatocyte carcinoma). This is a widely used model to measure cytotoxicity since this cell line retained all the enzymes responsible for the metabolism of xenobiotics [228,247]. So the cytotoxicity of dAV and AV were first evaluated in this cell line. Then the cytoprotective effects of both extracts were also evaluated in the HepG2 cell line through the measurement of cell metabolic activity (MTT assay) and ROS intracellular levels, through the use of 2´,7´-dichlorodihydrofluorescein diacetate (DCFH-DA) as a probe for redox state. 5.2 Material and methods 5.2.1 Chemicals and Reagents For the cell culture assays, high glucose Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA solution, antibiotic–antimycotic solution, t-BHP, LPS (lipopolysaccharides from Escherichia coli, O111:B4), DCFH-DA, sodium bicarbonate, and MTT ( � 97.8% purity), were purchased from Sigma, (Darmstadt, Germany). GRS Total RNA Kit – Blood Cultured Cells, Xpert cDNA Synthesis Mastermix and Xpert Fast SYBR Green Master Mix were purchased from GrisP, (Porto, Portugal). Primers for mouse interleukin-1BETA (IL-1BETA), IL-6, iNOS, COX-2, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and NF-KB were purchased from StabVida, (Oeiras, Portugal). 5.2. Material and methods 71 5.2.2 Cell culture Two cell lines were used in this work: HepG2 (human hepatoma) and BV-2 (mouse microglia). BV-2 cell line was kindly donated by Dr. Annika Höhn (German Institute of Human Nutrition, Germany) and HepG2 was obtained from the American Type Culture Collection (ATCC). Both cells were kept in DMEM (13.4 g/l, 3.7 g/l sodium bicarbonate, pH 7.1-7.3) supplemented with 10% (v/v) heat-inactivated FBS, and 1% (v/v) antibiotic–antimycotic solution. After filtering in a laminar flow chamber into an autoclaved flask using a 0.1 Whatman nitrocellulose membrane filters Sigma, (Darmstadt, Germany) the medium was kept at 4 ∘C. Cultures were maintained in 25 cm2flasks in a humidified incubator containing 95% air and 5% CO2 at 37∘C (HERACELL 150i, Thermo scientific). Cell morphology was monitored regularly by observation under a microscope (Inverted microscope Olympus CK2). BV-2 cells were sub-cultured three times a week with trypsin 0.05% and HepG2 cells two times a week with trypsin 0.25%. To perform assays a cell suspension with a cell density of 2𝑥105cell/ml for both cell lines was used to plate on a 96 (200 µl / well). For the 12 and 48 plates both cells were plated at 2.5𝑥105500 µl and 1 ml/ well, respectively. After plating, cells were left for adhesion for 24 hours. 5.2.3 MTT assay Cell metabolic activity (viability) was determined using the MTT assay as described before [187]. This assay is based on the ability of viable cells to convert the water-soluble yellow dye MTT into an insoluble purple formazan through the action of mitochondrial reductase present in the cell. Following this, formazan can be solubilized and can be quantified by optical density at 570 nm. The amount of formazan formed is proportional to the number 5.2. Material and methods 72 of functional mitochondria and therefore proportional to the number of metabolic active cells which is an indicator for the number of viable cells [248,249]. The protocol was performed after cell incubation with the extracts and compounds under study. The medium was aspirated and 100 µl of a solution with a 1:10 proportion of MTT ( 5 mg/ml in PBS) and DMEM (without FBS) was added to each well. Immediately after, cells were kept protected from light and incubated for 1.5 hours at 37 ∘C in a 5% CO2 incubator. After incubation time, the supernatant was aspirated and 100 µl of DMSO: ethanol solution (1:1) was added to re-suspend formazan crystals. Finally, absorbance was read at 570 nm in the SpectraMaxPlus microplate reader. The results were analyzed and expressed as % of cell viability according to the formula presented in figure 29. . Figure 29: Formula used to calculate % of cell viability. 5.2.4 Cytoxicity assay To assess the potential cytotoxicity of Apocynum venetum BV-2 and HepG2 cells were seeded in 96-well culture plates at density of 2𝑥105cells /ml (200 µl/well) in complete DMEM (with FBS). After the incubation period (24 hours), the medium was discarded, and cells were incubated with different concentrations of the extracts (concentrations tested were 500, 100, 50 µg dwr/ml) for 4h or 24h. In each experiment cells in DMEM (without the extracts) or with 10% DMSO in DMEM were used as normal growth and death controls, respectively. Cells with 0.05% DMSO were used as solvent control (to make sure that DMSO present in the extracts was not harmful to cells). 5.2. Material and methods 73 Cell viability was determined using the MTT assay previously described (see section 5.2.3). The cell viability was expressed as a percentage of the control (cells just with DMEM). 5.2.5 Cytoprotective assay against an oxidative insult To evaluate the antioxidant cytoprotective effects of the extracts t-BHP was used as an oxidative insult [247]. This is a compound commonly used to induce oxidative stress in cells to evaluate the cytoprotective effects of the extracts under study the following protocol was carried out [247,250,251]. Cells were seeded in the same conditions as for the citoxicity assay and were first preincubated with extracts for 20h at 500, 100, 50 µg dwr/ml concentrations, and then coincubated with t-BHP (0.5 mM) for additional 4 hours. Additionally, cells were co-incubated with the extract and t-BHP for 4 hours. Cellular viability was assessed by the MTT assay (see section 5.2.3). In each experiment cells in DMEM (not incubated with the extracts or oxidant insult; normal growth), 0.05% DMSO in DMEM (solvent control) and in DMEM with t-BHP (0.5 mM), were used as controls. The cell viability was expressed as percentage of the control (cells just with DMEM). Results were plotted as the means ± SD of three independent experiments performed in triplicates for each experimental condition. All parameters were graphically represented using the software GraphPad 8 (Prism, USA). 5.2.6 Evaluation of intracellular ROS through DCFH-DA in HepG2 cells The t-BHP is easily diffused through the cell membrane and generates intracellular alkoxyl and peroxyl radicals causing several damages to the cell and consequently cell death as previously discussed [252]. Intracellular levels of ROS were measured through the DCFH-DA, a commonly used method. DCFH-DA is a non-fluorescent compound capable of entering the cell to the cytoplasm where it is hydrolyzed by intracellular esterases to the non-fluorescent compound 5.2. Material and methods 74 (DCFH). Then, DCFH can be oxidized by free radicals and converted into a fluorescent compound 2´,7´-dichlorodihydrofluorescein (DCF). Thus, the measurement of the fluorescent directly measures the redox state of a cell [253] (see figure 30). Figure 30: Mechanism of action of DCFH-DA probe inside the cell. DCFH-DA (2´, 7´ − 𝑑𝑖𝑐ℎ𝑙𝑜𝑟𝑜𝑑𝑖ℎ𝑦𝑑𝑟𝑜𝑓 𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑖𝑛𝑑𝑖𝑎𝑐𝑒𝑡𝑎𝑡𝑒), a cell permeable non-fluorescent dye enters the cell where is hydrolyzed into DCFH ( 2´, 7´ − 𝑑𝑖𝑐ℎ𝑙𝑜𝑟𝑜𝑑𝑖ℎ𝑦𝑑𝑟𝑜𝑓 𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑖𝑛) through the action of esterases. DCFH is then oxidized by peroxidases and intracellular reduced oxygen species (ROS) into its fluorescent form DCF(2´, 7´ − 𝑑𝑖𝑐ℎ𝑙𝑜𝑟𝑜𝑓 𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑖𝑛) which can then be quantified. HepG2 cells were seeded in 48-well plates at a density of 2.5𝑥105cell/ml (500 µl/well). After the adhesion time (24 hours) cells were incubated with 500 µl of DCFH-DA (25 µM in PBS) and incubated for 30 min. at 37 ºC and 5% CO2, in the dark. DCFH-DA solution was then removed, and cells were washed with warm PBS. Next, cells were pre-incubated at previous conditions for 4 hours with the extracts at 50, 250, and 500 µg dwr/ml of both AV and dAV. Then, t-BHP was added at 0.5 mM and left for incubation for 1 hour. After this, cells were washed with warm PBS and lysate with a DMSO/PBS solution (9:1 solution), at room temperature, for 10 min. in a shaker. In each experiment cells in DMEM (not incubated with the extracts or oxidant insult; normal growth), 0.05% DMSO in DMEM (solvent control) and in DMEM with t-BHP (0.5 mM), were used as controls. Changes in intracellular levels of ROS were monitored using a fluorescent microplate reader (Fluoroskan Ascent FL Microplate Fluorometer and Luminometer, ThermoScientific) at excitation and emission wavelengths of 485 nm and 538 nm, respectively. The results were expressed as a percentage and normalized 5.2. Material and methods 75 relative to the control (cells just with DMEM) according to the equation present in figure 31. Figure 31: Equation used to determine the percentage of fluorescence intensity. 5.2.7 Quantification of NO produced by LPS-induced BV-2 cells In this procedure, cells were plated in a 12-well plate, 2.5𝑥105cells/ml (1 ml per well) and after the adhesion time (24 hours), cells were pre-incubated with the extracts under study for 30 min.. After that, cells were supplemented with LPS (2 µg/ml final concentration) and incubated for 20 hours. Then, the supernatants of the culture medium were collected and 50 µl were transferred to a 96-well plate. Next, 50 µl of 1% (w/v) sulfanilamide in phosphoric acid buffer 2 % (w/v) was added to each well, and cells were incubated at room temperature in the dark for 10 minutes. Next, 50 µL of 0.1% (w/v) NED in phosphoric acid buffer 2 % (w/v) was added and incubated under the same conditions. The absorbance was then, read at 543 nm in the SpectraMaxPlus microplate reader. Cells in the medium, without any addition of LPS were used as negative control. As a positive control, cells were only incubated with LPS, without a pre-incubation with extracts. The cells incubated with the extracts and LPS were recovered for ribonucleic acid (RNA) extraction to evaluate gene expression. 5.2. Material and methods 76 5.2.8 Quantification of gene expression of pro-inflammatory genes (RTPCR) To perform RNA extraction, BV-2 cells were recovered. Plated BV-2 cells after performing an pre-incubation period with AV and dAV at a concentration of 125 µg/ml for 30 min. and coincubation with LPS for 20 hours were washed with warm sterile PBS (37∘C) and trypsinized (0.05 %). Cells were then recovered in DMEM medium with 10 % FBS to marked eppendorfs (for each condition) and kept on ice. Then, eppendorfs were centrifuged (centrifuge 5418R Eppendorf Fischer Scientific) at 1000 rpm for 5 minutes, and the yielded pellet was washed with PBS ice-cold. Once again, eppendorfs were centrifuged in the same conditions and the resulting pellet was kept at -80∘C for further assays. Total RNA was isolated from recovered BV-2 cells using the GRS Total RNA Kit – Blood Cultured Cells (grisPin), following standard protocol. RNA purity and concentration were evaluated using a Nanodrop spectrophotometer (ND-100, Thermo Fischer) through the analysis of 260/280 and 260/230 ratios. The integrity of RNA was confirmed through the performance of electrophoresis in a 1% agarose gel (agarose in tris-acetate-EDTA buffer (TAE) 0.5x buffer). The addiction of cybersafe dye (grisPin) in the samples allowed the visualization of RNA bands and a specific marker Bioron Ladder 1 kb was used to quantify RNA bands. The electrophoresis was performed at 100 V voltage and the gel was visualized in ChemiDoc (ChemiDoc Imaging System VWR. Geno-Smart). Isolated RNA was reverse-transcribed into complementary DNA (cDNA) (0.5 µg of each sample) using the Xpert cDNA synthesis master mix following the standard protocol. The samples were run in a thermocycler (C1000, Bio-Rad) under the following conditions: 5 minutes at 65 ∘C, 2 minutes on ice, 10 minutes at 25 ∘C after the addiction of RNA transcriptase, 15 minutes at 50 ∘C and lastly 5 minutes at ∘C, to inactive reverse transcriptase enzyme. To each cDNA sample 55 µl of RNase free water was added reaching a final concentration of 6.7 ng/ µl and samples were kept on ice to the posterior step. Quantitative real-time polymerase chain reaction (PCR) was carried out with Xpert Fast SYBR Green Master Mix (GrisP) and performed in 96-well plates using a CFX96 Real-Time 5.2. Material and methods 77 Detection System (Bio-Rad). First, a primer solution containing both forward and reverse primers at a concentration of 10 µM was made for each primer under study. Then a reaction mix was done by mixing 5 µl of blue master mix (Xpert Fast SYBR Green Master Mix), 0.6 µl of each primer solution at 10 µM and 3.4 µl of nuclease-free water to each well. Finally, 1 µl of cDNA was added to each well. The conditions used in the cycler were 15 min at 95∘C and 45 cycles of 15 s at 95∘C, 30 s at 55∘C and 30 s at 72∘C. Experiments were done in triplicates and analyzed with the software Bio-Rad CFX Manager (Bio-Rad), using GAPDH as an internal control. For each well, melting curves were analyzed and the results were optimized IL-1BETA, IL-6, iNOS and COX-2 were the genes under evaluation and the specific primers were designed with Quantprime Software (see table:3). Table 3: Sequence of primers used in this study. 5.2.9 Statistical analysis All experiments were performed at least in triplicate, with data presented as the means ± SD of three independent experiments and analyzed using GraphPad 8 (Prism, USA). Comparisons between control and treatment groups were made using one-way ANOVA, the p-value was considered significant when t * p-value< 0.05; ** p-value< 0.01; *** p-value< 0.001; **** p-value< 0.0001. All analysis were performed using the software GraphPad 8 (Prism, USA). 5.3. Results and discussion 78 5.3 Results and discussion 5.3.1 Cytotoxicity and Cytoprotective assay against an oxidative insult of extracts in HepG2 cells The liver is the main detoxifying organ in the human body being responsible for drug metabolism. Hence hepatic cells are important to understanding possible side effects of drugs [254]. The HepG2 cell line is a human liver cancer cell line well adapted to different environments and a well-known model to study the action of many chemicals and medications [255]. Therefore, this cell line was used, to evaluate the possible toxicity of the extracts, under study, through the analysis of the cells metabolically active. Cytotoxicity of AV and dAV were evaluated through the MTT assay, at three different concentration (50, 100 and 500 µg dwr/ml) and during 4 and 24 hours of incubation, mimicking acute and chronic cell exposure (figure 32A and figure 32B, respectively). Results showed that both AV and dAV did not induce toxicity for any tested concentrations after 4 h of incubation as shown in figure 32A. At 24 hours of incubation, there were also no significant differences in cellular viability when comparing the cell viability percentage of the different extract concentrations with the control (cells with medium) (figure 32B). According to the results present in figure 32, the in vitro digestion did not induce changes on % cell viability. Previous work had already elucidated on cytotoxic effects of Apocynum venetum in the HepG2 cell line. Our results are in line with Zhang and colleagues [124] that revealed that an ethanolic extract of Apocynum venetum leaves showed no cytotoxicity effects on HepG2 cells even up-value 400 µg dwr/ml during 24 hours of incubation. The cytotoxicity effects were evaluated both through MTT assay and lactate dehydrogenase (LDH) release assay. The same study also revealed non-symptoms of toxicity or mortality during the experiments on mice with the same extract after 14 consecutive days of treatment. On the other hand, an article published by Chong Li [131] revealed an inhibitory effect in the growth of HepG2 cells within a concentration range between 50 and 200 µg dwr/mL, after 5.3. Results and discussion 79 Figure 32: Cytotoxicity of AV and dAV on HepG2 cells. HepG2 cells were incubated with AV and dAV at 50 100, 500 µg dwr/ml for 4h of incubation (A) or for 24 h (B). Cellular viability was assessed with MTT assay. AV-non-digested and dAV-digested of Apocynum venetum methanolic leaves extract. Each bar represents the mean ± SD of the results obtained in three independent experiments. Asterisks mean significant differences from the control (cells in medium), as indicated: * p-value< 0.05; ** p-value< 0.01; *** p-value< 0.001; **** p-value< 0.0001. 48 h of incubation. However, there are no more scientific reports regarding this incubation period on HepG2 cells. After the study of cytotoxicity of the extracts, the cytoprotective effects against t-BHP were analyzed. The t-BHP is a commonly used compound for evaluating the cytoprotection of natural antioxidants. This oxidant insult is able to induce ROS, accelerate lipid peroxidation and cause DNA damages leading to cell death [256]. Cytoprotective results of AV in HepG2 for both 4 hours and 24 hours are represented in figure 33A and 33B, respectively. First, the results confirmed that t-BHP causes cell death, inducing more than 50 % of cell death in the concentration used (0.5 mM). Furthermore, results show that incubation with AV had a positive effect on cell viability when compared with the control ( cells incubated with t-BHP 0.5 mM), revealing protective effects against the damage induced by the insult. For the 4 hours of incubation, the increase in cell viability caused by the extract occurred in a dosedependent manner revealing significant differences from all tested concentrations. The minor concentration showed a p-value of 0.0418, the concentration of 100 µg dwr/ml revealed a 5.3. Results and discussion 86 Figure 37: Cytoptotective potential against t-BHP-induced damages. BV-2 cells were co-incubated with 4 hours with dAV (50 100, 500 µg dwr/ml) and t-BHP(A); or pre-incubated with the extracts (50 100, 500 µg dwr/ml) for 20 h and co-incubated with t-BHP for 4 hours (B); dAV-non-digested Apocynum venetum methanolic leaves extract; Cellular viability was assessed by the MTT assay. Each bar represents the mean ± S.D. of the results obtained in three independent experiments. Asterisks mean significant differences from the cells only incubated with t-BHP 0.5 mM for 4 h , as indicated: * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001. To compare the differences in cytoprotective effects of digested and non-digested extracts the percentage of protection was calculated through the difference between the cell viability % of cells incubated with t-BHP and the obtained % for each study concentration the results are present in table 5 . As it happened in HepG2 cells no significant differences were found between dAV and AV protective activities against t-BHP, proving that the protective activities against t-BHP-induced damages remains after the digestion process. This is a good indicator for the use of Apocynum venetum as a medicinal tea. 5.3.4 Quantification of NO produced by LPS-induced BV-2 cells As previously mentioned (see section 5.1), LPS has the ability to simulate inflammatory conditions on BV-2 cells and can trigger an inflammatory response. After stimulation with LPS microglia initiates an inflammatory response which triggers the release of pro-inflammatory mediators, such as ROS, NO, IL-1BETA, IL-6 among others (See section 5.3. Results and discussion 87 Figure 38: Cytoptotective potential against tert-butyl hydroperoxide (t-BHP) 0.5 mM. BV-2 cells were co-incubated with 4 hours with both dAV (50 100, 500 µg dwr/ml) and t-BHP (A) or pre-incubated with the extracts (50 100, 500 µg dwr/ml) for 20 h and co-incubated with t-BHP for 4 h (B). dAV-digested Apocynum venetum methanolic leaves extract; Cellular viability was assessed by the MTT assay. Each bar represents the mean ± S.D. of the results obtained in three independent experiments. Asterisks mean significant differences from the cells only incubated with t-BHP 0.5 mM for 4 h, as indicated: ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001. Table 5: Percentage of protection against tert-butyl hydroperoxide (t-BHP) effects after the preincubation of BV-2 cells with both AV-non-digested and dAV-digested of Apocynum venetum methanolic leaves extract (50 100, 500 µg dwr/ml) for 4h or 20 hours. The results are represent as mean ± S.D. of the results obtained in three independent experiments. 1.4) [245,260]. The amount of NO produced by LPS-induced BV-2 cells can be determined by using a method based on Griess reaction [262], which measures the amount of nitrite present in the culture supernatant a metabolite of NO [249,263]. The anti-inflammatory ability of the extracts under study was evaluated by its ability to inhibit the NO production induced by LPS. For that, the NO levels in the supernatant were 5.3. Results and discussion 88 measured by Griess reaction after the incubation of BV-2 cells with extracts and LPS (20h). The treatment with LPS caused a significant increase in NO levels when comparing to the cells in DMEM (see fig:39). Remarkably, the tested concentration of both AV and dAV (75 and 125 µg dwr/ml ) significantly inhibited NO production induced by LPS in comparison with BV-2 cells only incubated with LPS (2 µg/ml final concentration) (p-value <0.0001). In general, the digestion process caused a significant reduction of anti-inflammatory activity of the extract revealing a p-value of 0.0175 and the p-value of 0.0133 for 75 µg dwr/ml and 125µg dwr/ml, respectively according to the ANOVA analysis. The decrease of the ability of the extract to scavenge NO after the performed digestion was reported in section 4.3. So, the decrease of anti-inflammatory properties of dAV revealed in these results may be related to the decrease in the ability to scavenge NO. Nonetheless, the inhibition of NO can also be related to the ability of the extract to intervene in inflammatory pathways, which will be discussed in the following section. Our results are in line with the ones described by Magalhaes et al., [187], that also reported a decrease of anti-inflammatory activities of Lycium ruthenicum and Lycium barbarum after performing in vitro digestion. 5.3.5 Quantification of gene expression of pro-inflammatory genes (RTPCR) RT-PCR is an efficient method capable of carrying out a sensitive and reproducible quantification of Messenger ribonucleic acid (mRNA) levels. First, the RNA is transverse-transcribed into cDNA by RNA transcriptase, which is amplified in the next phase. A fluorescent probe is embedded in the new formed PCR product, and through the measurement of fluorescence emitted by those probes the levels of mRNA transcription is achieved. The SYBR green molecule, which possesses a high affinity for double-strand DNA was used to estimate the levels of gene expression. For this method is crucial to use a reference gene to enable the normalization of gene expression, due to the differences in the 5.3. Results and discussion 89 Figure 39: Nitric oxide (NO) measurement in lipopolysaccharides (LPS)-induced BV-2 cells supernatant. BV-2 cells in 12-well plates were pre-incubated (30 min.) with extracts ( 75 and 125 µg dwr/ml), and co-incubated with LPS (2 µg/ml) for 20h. Supernatants were collected for the measurement of NO by Griess reaction (Absorbance at 543 nm). dAVdigested and AV-non-digested Apocynum venetum methanolic leaves extract. Each bar represents the means ±SD of the results obtained in three independent ones experiments. Asterisks mean significant differences from the positive control ( cells incubated exclusively with LPS 2 µg/ml final concentration);*p-value < 0.01 *** p-value < 0.001; **** p-value < 0.0001. amount and quality of each sample and the ones occurring in the previous phases (BV-2 recovery,RNA preparation and cDNA synthesis) [264–266]. The expression levels of genes encoding for IL-6, IL-1BETA, COX-2 and iNOS were evaluated after the pre-incubation of BV-2 cells with AV and dAV (125 µg dwr/ml 30 min.) and co-incubation with LPS (2 µg /ml final concentration for 20 hours) through RT-PCR and are present in figure 40. As expected the treatment with LPS significantly increased the expression of IL-1BETA (figure 40A), IL-6(figure 40B), COX-2 (figure 40C) and iNOS (figure 40D) genes, in comparison with control cells (cells not incubated with LPS). The preincubation with the AV resulted in a significant decrease of all genes expression in comparison to the negative control (cells in DMEM). This significant reduction was to a less extent for the iNOS gene (figure 40D) revealing a p-value of 0.0034. For the IL-6 the AV showed a significant decrease with a p-value < 0.0001, for the IL-1BETA a p-value= 0.0002 was found 5.3. Results and discussion 90 and for the COX-2 a p-value= 0.0034. Notably, for these two genes, the reduction of the gene expression levels led to similar values to the control, proving the great anti-inflammatory activities of AV. Previous work have already unveil the ability of Apocynum venetum or some compounds found in this plant to decrease the mRNA levels of genes related to inflammatory pathways in mice [8], in rats [116] and in LPS-stimulated RAW264.7 cells [267]. However, work regarding BV-2 cell line was not yet published. However, the pre-incubation with dAV, did not show a significant decrease for any of the genes under study (figure 40). Revealing that the anti-inflammatory activity of Apocynum venetum may be more susceptible to the modifications occurring in the digestion process than its antioxidant activities. The decrease of the inflammatory activities after digestion, through the decrease of ability to counteract the expression of genes, involved in inflammatory pathways, have been also described in the literature [187,268]. These results are in line with the results obtained for the quantification of NO produced by LPS-induced BV-2 cells. Where the dAV also showed less activity towards NO inhibition. Nonetheless, the decrease of mRNA levels of iNOS by AV also shows that the extract is capable of modulating the activity of enzymes. Proving that both scavenging activity and the modulation of iNOS expression may contribute to the decrease of NO reported here. Despite the decrease in NO scavenging activity and the ability to modulate iNOS after the digestion, the dAV was still capable to counteract the NO levels induced by LPS. To sum up, the results indicate that AV can modulate the expression of the genes encoding pro-inflammatory mediators in LPS-stimulated BV-2 cells. Although, these effects were diminished after the digestion process. This first confirms the need for more studies on the effects of digestion on bioactivities of plants and second to search for ways to avoid its negative effects. 5.4. Conclusion 91 Figure 40: Real time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of genes in lipopolysaccharides (LPS)-induced BV-2 cells. BV-2 cells in 12-well plates were preincubated (30 min.) with extracts (125 µg dwr/ml), and co-incubated with LPS (2 µg/ml) for 20h. The cells were collected for RNA extraction and RT-PCR was performed. Gene expression of: (A) interleukin-1BETA (IL-1BETA); (B) interleukin 6 (IL-6); (C) cyclogenase-2 (COX-2); (D) inducible nitric oxide synthase (iNOS); were calculated using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression as an internal control. Cells incubated with medium and cells incubated with LPS were used as a negative and positive control, respectively. AV-non-digested and dAV-digested of Apocynum venetum methanolic leaves extract. Experiments were analyzed with the software Bio-Rad CFX Manager. Statistical differences are presented vs. positive control (cells with LPS): *pvalue< 0.05; ** p-value< 0.01; ***p-value< 0.001; ****p-value< 0.0001. 5.4 Conclusion Following the premise that the blockage of over-activation of microglia may be a key step to intervene on NDDs and consequently cause a delay in the onset of disease symptoms, the Apocynum venetum was studied in this work. Both antioxidant and inflammatory capacities of 5.4. Conclusion 92 AV and dAV were evaluated to also understand the effects of digestion on Apocynum venetum properties. Apocynum venetum cytotoxic and cytoprotective effects were screened in both HepG2 and BV-2. The results proved that both extracts did not induce toxicity and even protected cells from damages induced by t-BHP. The protection was more prominent in HepG2 cells and the analysis of cellular ROS levels in HepG2 corroborated the activity of the extracts to eliminate ROS-induced by t-BHP. Furthermore,studies regarding antioxidant enzymes should be done to understand if the extracts can modulate the antioxidant activity. This is a hypothesis to justify the increment of the cytoprotective effects over time observed here, which is supported by results in the literature describing the ability of A. venetum to modulate antioxidant enzymes’ activity [8,124,132]. When comparing the results of the AV and dAV, it is possible to conclude that the performed in vitro digestion did not impair the protective activities against t-BHP-induced damages observed in both cell lines. These results validate the use of Apocynum venetum as an antioxidant beverage and further illuminates the antioxidant properties of Apocynum venetum on HepG2 cells and for the first time on Bv-2 cells. Regarding the anti-inflammatory effects, Apocynum venetum methanolic leaves extracts proved to be able to reduce NO levels induced by LPS and to modulate the levels of gene expression related to inflammatory status such as IL-1BETA, IL-6, COX-2 and iNOS through RT-PCR analysis. The AV was able to significantly reduce the gene expression of all evaluated genes. However, the digested extract in the tested concentration was not able to significantly reduce the mRNA levels of genes related to inflammation. Despite this, the digested extract still proved to be able to reduce NO levels, proving that some anti-inflammatory effects remained after the performed digestion. Perhaps because a dose-response was found in the quantification of NO, if a higher concentration of the extract was chosen, a more effective activity on the modulation of gene expression could be found for the dAV. Nonetheless, the results presented in this work demonstrated the potential of both AV and dAV to revert at some levels to neuroinflammatory/oxidative stress situations. Further research aiming to enlighten how the 5.4. Conclusion 93 extracts are able to modulate gene expression should be done, to understand the involved inflammatory pathways. CHAPTER 6 Final remarks 94 95 Currently, a renewal of interest in traditional medicines has been occurring. This led to a growth of interest by the scientific community in phytopharmaceuticals resulting in several efforts toward the understanding of natural products’ composition and properties. In this work we evaluate the ability of A. venetum towards the reduction of neuroinflammation/oxidative stress. Following the hypothesis that attenuating these inflammatory/oxidative processes, mainly in microglia may bring positive outcomes for NDDs. We further study the effects of digestion on those activities through the performance of a in vitro digestion. The cytotoxicity and cytoprotective effects of Apocynum venetum were evaluated on HepG2 since it is a commonly used cell line to evaluate toxicities. The first step of this work regarded the biochemical analysis of Apocynum venetum antioxidant/ inflammatory activities. In these analyzes both extracts, the non-digested and digested extract revealed antioxidant activities in almost all performed assays except for the dAV in ICA. AV antioxidant activity was significantly higher than the control in SO,NO scavenging activities and COX-2 inhibitory activity. Here, all reported activities were decreased after the in vitro digestion. Nevertheless, antioxidant activities remained as well as the ability to inhibit COX-2 activity. This is a relevant result due to the interest in finding new COX-2 inhibitors. Nonetheless, this is only a preliminary result, other analyzes such as polarographic, or prostaglandin detection should be done. In the fourth chapter, both AV and dAV were capable of protecting against t-BHP oxidative stress-induced toxicity in both HepG2 and BV-2. These cytoprotective effects can be related to the decrease of ROS levels. Since a reduction of intracellular ROS, up to basal levels was found in HepG2 after pre-incubation with the extracts and measurement of redox state by DCFH-DA. 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