Mentha Pulegium Linnaeus: From Chemistry to Cell Protection Against T-BHP-Induced Toxicity

Mentha pulegium Linnaeus: from chemistry to cell protection against t-BHP-induced toxicity Dissertation for Master degree in Analytical, Clinical and Forensic Toxicology Supervision: Professor Patrícia Valentão Co-supervision: Professor Paula B. Andrade João Tiago Alves Bernardo July 2014 THE REPRODUCTION OF THIS THESIS, IN ITS WHOLE, IS AUTHORIZED ONLY FOR RESEARCH PURPOSES, UPON WRITTEN DECLARATION FROM THE INTERESTED PART, WHICH COMPROMISES ITSELF TO DO SO. In loving memory of my grandmother Raquel Fernandes VII ACKNOWLEDGMENTS I would like to thank everyone that directly or indirectly have contributed for the success of this work and, somehow, supported me during this last two years: To Professor Patrícia Valentão, my supervisor, for all the support, encouragement and enthusiasm, and also by the availability and dedication that led to the realization of this work. I will never forget the right words said when they were needed. It has been a pleasure to work under your guidance and to learn so much with you. To Professor Paula Andrade, my co-supervisor, to whom I have a great admiration and respect, and made possible to integrate this research team. To all the collaborators of the laboratory of Pharmacognosy, with whom I learned so much and that were always there to hear my complaints, to share my laughs and to encourage me. To my parents, nothing would be the same without you. There are no words to describe how grateful I am for having such amazing examples. Thank you for making possible the realization of my dreams. To all my family, that has always been present. To my dear friends, Cristiana Viegas, Eunice Neves, José Barreira e Vitor Esteves, for all the support, for sharing a great part of my life, being present at the best and worst moments. Thinking of you makes me instantly smile and you know how I love to do it. To my house mattes, Luis and Tiago, for your fellowship and all the happy and joyful moments shared. VIII ABSTRACT Herbal teas are widely consumed for their valuable content on phytochemicals with antioxidant properties. This work represents the first attempt to establish a linkage between the chemical composition of Mentha pulegium L. infusion and its potential to prevent oxidative stress in cells. The phenolic profile was established by HPLC-DADESI/MSn, 12 out of 15 compounds being identified for the first time in this species. The infusion presented a total phenolic content of 122.92 mg/g (lyophilized extract), also demonstrated a remarkable antiradical activity against DPPH· (EC50= 39 µg/mL), O2·- (EC50= 23 µg/mL) and ·NO (EC50= 226 µg/mL) radicals. It also revealed to be very effective in protecting AGS and Caco-2 cell lines against tert-butylhydroperoxide induced toxicity as evaluated by the MTT assay, under a pre-treatment experimental model. Reduced glutathione detoxification mechanism was demonstrated to be involved in Caco2 cells resistance, while the same was not observed for AGS cells. The presence of phenolic compounds inside the cells, after infusion removal, was confirmed by DPBA phenol fluorescence dye staining. A direct antioxidant effect can contribute to the observed protective effect. Attending to all the results here presented, the consumption of M. pulegium infusion provides bioactive compounds with great importance to the maintenance of a proper antioxidant imbalance in gastrointestinal cells. KEYWORDS: Mentha pulegium L.; infusion; phenolic compounds; antioxidant effect; tertbutylhydroperoxide; IX XVI XVII INDEX OF TABLES Table 1. Examples of ROS activated pathways and their contribution to the cellular outcome. ........................................................................................................................ 24 Table 2. Mints classification according to the major monoterpenoid present in the essential oil and its metabolic pathway. ........................................................................... 37 Table 3. Regression equations, LOD and LOQ for phenolic compounds quantification. ........................................................................................................................................ 52 Table 4. Rt, UV and MS:  M-H  -, MS2  M-H  - and MS3  (M-H)→base peak  - data of phenolic compounds from M. pulegium infusion. ............................................................ 60 Table 5. Phenolic composition of M. pulegium infusion. ............................................. 62 XVIII XIX ABREVIATIONS AND SYMBOLS AChE Acetylcholinesterase ATP Adenosine triphosphate CAT Catalase DAD Diode-array detector DNA Deoxyribonucleic acid EC50 Half maximal effective concentration ESI Electrospray ionization GSH Glutathione reduced form GSx Total glutathione GSSG Oxidized glutathione HPLC High-performance liquid chromatography LDH Lactate dehydrogenase m/z Mass-to-charge ratio MS Mass spectrometry MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ROS Reactive oxygen species RNS Reactive nitrogen species SOD Superoxide dismutase XX Introduction 21 Chapter I - Introduction Introduction 22 1. Phytotherapy Medicinal plants are used as pharmacotherapy tools worldwide. Since remote times, different species were selected and applied to prevention and treatment of diverse pathologies. This ancient practice has been replaced by modern medicine brought with the 20th century. But it is a fact that for approximately 80% of world’s population, phytotherapy constitutes the first line of treatment, often being the only one existing. Moreover, in 1978 the World Health Organization recognized Traditional Medicine as a valid form of primary healthcare, being continuously working towards its regulation and correct use (1). Nature is a huge source of chemical compounds with pharmacologic and toxic activities, also having a key role in the development of novel synthetic drugs. In the past thirty years, major contributions have been made to diverse fields, such as neurodegenerative diseases, cancer treatment and anti-inflammatory agents (2). A revivalist trend as recently brought back phytotherapy to the spotlight. All over Europe this market has shown a big expansion, particularly in Germany, France and Italy (3). New approaches to Traditional Medicine, based on reverse-pharmacology, seem to allow the perfect linkage between popular knowledge, science and the positive health outcomes for consumers (4). Moreover the easy access, low prices, strong marketing and the wrong popular idea of absence of associated risks are the main boosters behind this consumption’s growth. These products are presented in pharmaceutical dosage forms like capsules, tablets and drops. More traditional forms can also be found at herbal stores (5). Medicinal plants are currently applied to cure and prevent respiratory, gastrointestinal and sleep disorders, to alleviate side effects of anticancer therapies, to fight depression, to enhance weight loss and dietary supplementation, in dermatology and cosmetics, among other uses (6). Such impact on public health justifies, per se, the need to understand and study, not only the chemical composition of these products, but also the risks and benefits resulting from their consumption. The increasing number of reports on interactions between herbal medicines and prescribed drugs reflects this reality. Additionally, adulteration of final products, misidentification of species, heavy metals contamination and the presence of pesticides are safety issues that must be taken into consideration (7). These events are complex to elucidate and have the disadvantage of both qualitative and quantitative variation of the chemical profile of the natural species, due to different growing conditions Introduction 23 and industrial and storage processes (8). For all the reasons presented above the pharmacological and toxic potentials of medicinal plants is an important and vast field for scientists to explore. 2. Cellular oxidative stress A homeostatic cell environment is necessary to maintain proper physiological functions. A vast number of free radicals, resulting from both endogenous processes and exogenous sources, is responsible for the significant alteration of cellular homeostasis, leading to oxidative stress. Nevertheless, their physiological roles cannot be underestimated (9). As examples, reactive oxygen species (ROS) include either oxygen radicals like superoxide (O2·-), hydroxyl (·OH), and peroxyl (RO2·) radicals, or non-radical oxidizing agents, such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), easily converted into radicals. Also, reactive nitrogen species (RNS) and reactive sulfur species (RSS), which are formed by thiols’ reaction with ROS, play an active role in disrupting the imbalance between pro-oxidants and antioxidants. The interest on these radical species has been increasing, since they are thought to be involved in different pathophysiological mechanisms behind neurodegenerative and cardiovascular diseases, diabetes, arthritis, osteoporosis and cancer (10). The cellular damage depends on the doses and time of ROS exposure, triggering different pathways and modulating the action of enzymes and transcription factors. Diverse effects can be observed, from cellular proliferation by mitogenic stimulation in low doses, to cellular cycle arresting or even cellular death by apoptosis or necrosis when exposed to high doses (Fig. 1). Introduction 24 Figure 1. Sources of radical species and possible cellular responses. The oxidative imbalance results from the simultaneous presence of endogenous or exogenous ROS and enzymatic antioxidant defenses like catalase (CAT), superoxide dismutase (SOD) glutathione peroxidase (GPx) or non-enzymatic like glutathione. Diverse defensive mechanisms can be triggered leading to a proliferative decrease up to cell death (adapted from (10)). While some of the pathways enhance cell survival, others are associated with cell death and many of them can act both ways depending on the circumstances (Table 1). However, it is important to understand that the ultimate response is a result of an integration of multiple stimuli (11). Table 1. Examples of ROS activated pathways and their contribution to the cellular outcome. From (11). Cellular outcomeb Signaling pathwaysa Enhanced survival Cell death p53 + +++ NFkB + +++ HSF1 +++ - PI3K/Akt +++ - ERK +++ ++ JNK ++ +++ p38 + + PLCɣ +++ - JAK/STAT +++ - c-Abl + +++ a p53: tumor suppressor protein 53; NFkB: nuclear factor kB; HSF1: heat shock transcription factor 1; PI3K: phosphoinositide 3-kinase; Akt: protein kinase B; ERK: extracellular signal-regulated kinases; JNK: cJun N-terminal kinases; p38: p38 mitogen-activated protein kinases; PLCɣ: phospholipase Cɣ; JAK: Janus protein kinase; STAT: signal transducers and activators of transcription; c-Abl: c-Abl tyrosine kinase. b (-): minimal or no evidence of influence; (+): some evidence for the outcome; (++): much evidence that the pathway promotes this outcome; (+++): predominant outcome for this pathway. Endogenous sources Mitochondria Peroxisomes Lipoxygenases NADPH oxidase Cytochrome P450 Antioxidant defenses Enzymatic systems CAT, SOD, GPx Non-enzymatic systems Glutathione Vitamins Exogenous sources UV light Ionizing radiation Chemotherapeutics Environmental toxins Inflammatory cytokines More Less ONOO - ·RO ·NO2·O2 ·NO ·OH O2 ·RO H2O2 ·O2Impaired physiological function Homeostasis Impaired physiological function Decreased proliferative response Defective host defenses Normal growth and metabolism Random cellular damage Specific signaling pathways Cell death Introduction 25 Continuous work to discover oxidative stress biomarkers has been developed in order to understand and establish correlations between the mechanisms behind this process, and the possible prevention of its effects (12). 2.1. Molecular damage 2.1.1. Lipid peroxidation Briefly, lipid peroxidation is a self-propagation chain reaction, representing one of the most harmful toxic phenomena to cellular viability. The polyunsaturated fatty acids (PUFA) located at the cell’s membrane are highly affected by this ROS-mediated process. Glycolipids, phospholipids and cholesterol are also well-known targets. The process is initiated when a hydrogen atom is abstracted from a methylene group, resulting on the formation of a carbon radical, which then suffers a rearrangement into a conjugated diene, capable of reacting with oxygen and originating a lipid peroxyl radical (ROO·). The chain reaction is propagated when these radicals further abstract hydrogen atoms from other lipids, to form lipid hydroperoxides (ROOH). Also peroxyl radicals can undergo a cyclisation reaction forming endoperoxides, precursors of malondialdehyde, an important marker of oxidative stress able to impair several physiological mechanisms and to react with DNA and proteins (13). Another toxic secondary lipid peroxidation product is 4hydroxy-2-nonenal (14). Lipid peroxidation-derived aldehydes can easily diffuse across membranes and covalent bind to proteins in cytoplasm and nucleus. The continuity of this process leads to the loss of membranes’ integrity and to possible severe cellular and tecidual injuries (14). 2.1.2. Proteins oxidation Proteins are also a main target of oxidative stress and due to their ubiquitous location in cells the result of this process can be highly toxic. Besides ROS/RNS, they can also be damaged by sugars and aldehydes. Diverse intracellular pathways may be affected, since the modification of proteins can directly alter the expression of genes via modulation of transcription factors. Intrinsically related to proteins oxidation are the mechanisms of protein repair and elimination, with important roles on the pathophysiology Introduction 32 Glutathione S-transferases (GST) are known by their ability to detoxify xenobiotics and to inactivate endogenous reactive molecules. They constitute a second line of defense against oxidative stress since they catalyze the conjugation of GSH with oxidation end-products (28). Despite the de novo synthesis, GSH levels can be maintained through a reaction catalyzed by glutathione reductase (GR): The role of phenolic compounds, as representatives of exogenous antioxidants, will be discussed forward. 2.3. Antioxidant activity assessment Distinct methods and experimental models have been developed in order to assess the antioxidant activity of a given compound/extract (29). The more classic ones consisted on the measurement and presentation of the results as total antioxidant capacity or total antioxidant activity. The reductive and radical scavenging activities can be classified in three main groups: i) ability to reduce metal ions, including the ferric ion reducing antioxidant power (FRAP), ii) ability to reduce organic radicals, 2,2-diphenyl-1picrylhydrazyl (DPPH·) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS·+) radicals being the most commonly used, and iii) peroxyl radical reduction ability assays, like the oxygen radical absorbance capacity (ORAC) and total radical trapping antioxidant parameter (TRAP). Other parameters, such as metal ions chelating activity, inhibition of free radical generating enzymes and activation of internal antioxidant enzymes like CAT, SOD and GSH-dependent enzymes are also frequently applied. Nowadays, determinations based only on a chemical approach are used as screening tools for cell based systems, since they do not take into consideration bioavailability and metabolic variables (30). GSSG + NADPH + H+GR 2GSH + NADP+ Introduction 33 3. Phenolic Compounds Concerning to the most studied chemical families of natural bioactive compounds, polyphenols are certainly the best example. With ubiquitous distribution and comprising molecules from low molecular weight to more than 30000 Da, they are particularly known for their antioxidant (31), anti-inflammatory (32) and antimicrobial (33) properties. These secondary metabolites are biosynthesized via shikimate and acetate pathways. They can present simple structures like the phenolic acids, or complex polymeric ones characteristic of tannins. Additionally, in heterosides’ structure the aglycone can be linked to sugar residues either by their hydroxyl groups or by carbon atoms of the main skeleton. Although glucose is the most common sugar, others like rhamnose, galactose, xylose and glucuronic acid are frequently observed (34). 3.1. Flavonoids and hydroxycinnamic acids Flavonoids are phenolic compounds with a common structure known as 2-phenilbenzopirane (C6-C3-C6), consisting in two aromatic rings linked by three carbons, usually forming an oxygenated heterocycle (Fig. 7) (34). The A ring usually comes from a resorcinol or phloroglucinol via acetate pathway, while the B ring is biosynthesized by the shikimate pathway (34). These compounds can be sub-classified according to different substitutions in the main structure (Fig. 7). Introduction 34 Figure 7. Common structure of flavonoids (2-phenyl-benzopirane), and main sub-classes. Among the phelylpropanoid derivatives (C6-C3) the hydroxycinnamic acids are considered to be the most important compounds (Fig. 8) (35). Figure 8. Basic structure of hydroxycinnamic derivatives. 3.2. Phenolic compounds as antioxidants Behind the antioxidant properties of phenolics underlies a conjugation of a vast number of mechanisms. Their characteristic hydroxyl groups easily donate hydrogen atoms to radical species, stopping the chain reactions due to the formation of a nonB 1’ 4’ 3’ 2’ 5’ 6’ 7 81 4 2 3 6 5 C A 2-phenyl-benzopirane 4’ 3’ 8 7 6 54 3 2 1 Flavones Flavonols Flavanones Dihydroflavonols Flavan-3-ols Flavan-3,4-diols Chalcones 1 3 1’ 2’ 4’ Aurones Anthocyanidins Introduction 35 propagating radical. Another mechanism is by metal ions’ chelation, despite that sometimes it leads to pro-oxidant activity (36). Recent studies show that besides this more classic mechanisms, phenolic compounds can interact with biological systems by modulation of signal transduction pathways, or even interaction with cell receptors. Moreover, phenolic compounds seem to be able of endogenous cell antioxidants induction, like sulfotransferases, epoxide hydrolases and GSH and GSH-related enzymes, through the modulation of antioxidant responsive elements located in the promoters of diverse genes (28). Focusing on flavonoids, their antioxidant activity is intrinsically correlated to their chemical structure. Generally, aglycones are more potent antioxidants than their corresponding glycosides. The number and configuration of hydroxyl groups influences the antioxidant activity, B-ring configuration as 3’,4’-cathecol structure seeming to be the most determinant (Fig. 9) (37). Figure 9. Structure-activity relationship of flavonoids. A greater stability of the flavonoid radical is observed when there are present a carbonyl group at position 4 and a 2-3 double bound between A and B rings, due to the resonance effect of the aromatic nucleus (Fig. 9) (37). Furthermore, differences in the hydrophobicity and molecular planarity due to substitutions either by hydroxyl or methoxyl groups have been noticed. When a methoxyl group is present at the B-ring, the scavenging activity is diminished by the steric obstruction caused to the 3’,4’-cathecol structure, when compared to the ones with hydroxyl substitutions (37). Some of the same features can be applied to hydroxycinnamic derivatives. The highest antioxidant activity is related with the presence of more hydroxyl groups, especially on ortho positions, but contrary to flavonoids it is enhanced by methoxyl substitutions on the catechol group (38). The propenoic side chain in hydroxycinnamic 3’ 2’ 1’ 9 8 7 6 510 4 3 2 14’ 5’ 6’ Introduction 36 derivatives, instead of the carboxylic group of benzoic acid derivatives, provides additional stability by resonance on the phenoxyl radical, thus highest activity (38). 4. The genus Mentha Mentha genus is one of the most important and studied within Lamiaceae (Labiatae) family, in the Order Lamiales. Its species are commonly known as mints due to their characteristic menthol flavor (39). Mints have an important commercial value, since they are used in industry, medicine and agriculture. In the Northwest of the Iberian Peninsula they have a particular application in gastronomy as aromatic herbs, being also consumed as herbal teas (40, 41). The systematics of this genus is not consensual. Regarding the number of species included, it varies depending on the type of study, being rather morphological, cytological, anatomical, phytochemical or genetic (42). The complex classification is based on the ease of hybridization favored by gynodioecy, that despite the majority of infertile specimens created, has a strong capacity of dissemination by vegetative propagation in Nature and crop species (43). The last proposed classification refers to the presence of 18 species and 11 hybrids, distributed by four sections: Mentha, Eriodontes, Tubulosae and Pulegium (44). Concerning mints as sources of bioactive compounds, they provide a distinct number of molecules like alkaloids, flavonoids, phenolic acids and terpenoids (45). The most part of the scientific studies approaching the pharmacological and toxicological potential of these species focus on their essential oils. Different activities, such as insecticidal (46), antimicrobial (47), anti-inflammatory (48), analgesic (49), and antioxidant (50), are well established. Also, essential oils are highly toxic and the ingestion of large amounts results on acute toxicity with allergic reactions and hepatic injuries (51). One traditional way for mints classification is based on the analysis of the chemical composition of the essential oils, identifying which is the major monoterpenoid present and respective metabolic pathway. There by, the species can be categorized in three groups: one with major expression of the menthol pathway, other with major expression of the carbon pathway, and the last with major expressing of the linalool pathway (Table 3). Introduction 37 Table 3. Mints classification according to the major monoterpenoid present in the essential oil and its metabolic pathway (adapted from (42)). Menthol pathway Carvon pathway Linalool pathway Mentha aquatica L. Mentha spicata L. Mentha x piperita (L.) Huds var. lavanduliora ined. Mentha arvenses L. Mentha x villosa Huds. Mentha longifólia L. Mentha suaveolens Rhr. Mentha x aquatica var. citrata (Ehrh.) Frensen. Mentha pulegium L. Mentha x piperita L. Mentha x verticillata L. Mentha pulegium L. has particular interest in the Mediterranean and Portuguese cultures (Fig. 10). It is an herbaceous species with height ranging between 20 and 40 cm, strongly aromatic and little elliptic oblongated leaves (8 to 30 mm length); it has characteristic spherical verticillated inflorescences, ciliate calyx-teeth and a lilac corolla measuring around 5 cm; it can be easily found in Europe and Western Asia, with large distribution in continental Portugal and Azores; its habitat consists in high humid areas, mostly near water courses (52). Figure 10. Mentha pulegium Linnaeus. In phytotherapy, the flowered aerial parts and the essential oil are used to treat and prevent diverse gastrointestinal disorders like the lack of appetite, intestinal cramps, low intestinal motility, flatulence and dyspepsia, and to alleviate cold and flu-related symptoms. External application is due to their antiseptic and healing properties. In Portugal, popularly known as “poejo”, it is used in gastronomy as an aromatic herb in the regions of Trás-os-Montes and Alentejo, consumed as an alcoholic beverage (“licôr de Introduction 38 poejo”) and herbal tea, being also used as parasites and insects’ repellent at livestock farms (53). M. pulegium essential oil, or pennyroyal oil, has emmenagogue and abortifacient properties. It is used by women to induce menstruation and abortion (54). The ingestion of high doses can lead to acute intoxication and death, owing to the presence of pulegone and its bioactive metabolite menthofuran, both strong hepatotoxic agents (55). 4.1. Mentha pulegium L. chemical profile 4.1.1. Terpenoids The vast class of terpenoids includes more than 40.000 different known structures. Synthesized by plants, they have a huge impact in their life cycle as primary and secondary metabolites. Among other biological functions, they play important roles as membrane’s constituents, hormones, photosynthetic pigments, in communication and as natural mechanisms of defense. Widely found in medicinal plants’ essential oils, they are of great importance to the pharmaceutical and food processing industries (56). Terpenoids are biosynthesized by mevalonate (cytosolic) and non-mevalonate (plastidic) pathways, both converging on the isoprene formation, the basic functional unit. The addition of isopentenyl pyrophosphate units to its isomer dimethylallyl pyrophosphate results in new precursors of other complex terpenoids, via prenyltranferases. Based on the number of isoprene units (5 carbons) present on the structure, terpenoids can be subclassified into several groups (Fig. 11). Introduction 39 Figure 11. Terpenoids biosynthesis. From the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), higher terpenoids precursors are formed. Geranyl pyrophosphate (GPP) is the monoterpenoids’ precursor, farnesyl pyrophosphate (FPP) is the sesquiterpenoids’ precursor and geranylgeranyl pyrophosphate (GGPP) is the diterpenoids’ precursor. Squalene, being the precursor of triterpenoids, results from de condensation of two FPP units. Two GGPP units condense to form tetraterpenoids (adapted from (56)). As previously described, mints can also be classified by the major monoterpenoids present in their essential oils. In the essential oil of M. pulegium pulegone, menthone, piperitenone and piperitone (Fig. 12) are the main monoterpenoids, biosynthesized by the menthol pathway. According to the oxygenated monoterpenoid present, this species can also be classified in 3 distinct chemotypes: pulegone type, piperitone/piperitenone type and isomenthone/neo-isomenthol type (44). Figure 12. Major monoterpenoids present in the essential oil of M. pulegium. Other classes, such as hydrocarbonated monoterpenes (limonene, α-pinene and camphene), sesquiterpenes (β-borbene), oxygenated sesquiterpenes (palustrol and αcadinol), oxygenated diterpenes (epimanoyl oxide), among others, can also be found in FPP (C15) GPP (C10) DMAPP (C5)IPP (C5) Isoprene (C5) Monoterpenoids (C10)Sesquiterpenoids (C15) Triterpenoids (C30) Diterpenoids (C20) Tetraterpenoids (C40) FPP GGPP GGPP (C20) OPP Pulegone Menthone Piperitone Piperitenone Introduction 40 the aerial parts of the plant (57). Recently, new terpenoids have been identified in the leaves, but no significant antioxidant or antimicrobial activity was described (58). As essential oils are obtained from natural sources, their chemical composition can vary according to the physical-chemistry characteristics of soil, climate and maturation stage of the species (59). 4.1.2. Phenolic compounds As for the phenolic profile of M. pulegium, a lot of work has to be done. A few number of compounds has been identified and the literature on this subject is scarce and lacks consistency. Most of the studies available are directed to the total quantification of phenolic compounds and not to their structural characterization, leaves being the most studied material. The presence of flavonoids and phenolic acids has already been reported for this species’ extracts. Phenolic acids, such as caffeic, vanillic, ferulic, rosmarinic and lithospermic, and the flavonoids luteolin, apigenin, naringenin, (+)-catechin and diosmetin 7-O-rutinoside are some examples of the compounds already identified (Fig. 13) (60-65). Also jaceosidin, pectolinarigenin and pedalitin were found in M. pulegium leaves’ alcoholic extracts (66). Introduction 41 Figure 13. Chemical structures of some phenolic compounds identified in M. pulegium extracts. 4.2. Pharmacologic and toxic activities 4.2.1. Antioxidant activity As above mentioned, there are available a variety of in vitro assays to assess the antioxidant activity of extracts, sometimes making hard to correlate the results obtained. Here will be presented some examples of works concerning diverse extracts of M. pulegium. The potential to scavenge DPPH· radical was evaluated for the methanolic extracts of flowered aerial parts of mint species (67). When compared to the other mints, M. pulegium showed a lower ability; nevertheless, 76% of scavenging activity was found at 0.4 mg/mL, a very promising result. Neither the chemical profile of each extract nor the phenolic quantification were determined, rendering impossible an attempt to link the activities with the compounds present. Ferulic acid Caffeic acid Rosmarinic acid Lithospermic acid Apigenin Luteolin 7-O-Glucoside Naringenin (+)-Catechin Experimental section 48 Chapter III - Experimental section Experimental section 49 1. Materials and methods 1.1. Standards and reagents Sodium pyruvate, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), βnicotinamide adenine dinucleotide reduced form (NADH), tert-butylhydroperoxide (t-BHP), sodium nitroprusside dehydrate (SNP), phenazine methosulfate (PMS), nitroblue tetrazolium chloride (NBT), N-(1-naphthyl)ethylenediamine dihydrochloride, sulfanilamide, amphotericin B (250 µg/mL), transferrin (4 mg/mL), non-essential amino acids, methanol, formic acid, sodium hydroxide, acetonitrile, formaldehyde, perchloric acid, dimethyl sulfoxide (DMSO), 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI), 2-aminoethyl diphenylborinate (DPBA), GSH, GSSG, glutathione reductase (EC 1.6.4.2), caffeic, chlorogenic and rosmarinic acids were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ferulic acid, luteolin 7-O-glucoside and apigenin 7-O-glucoside was from Extrasynthese (Genay, France) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) from Alfa Aesar (Karlsruhe, Germany). Dulbecco’s Modified Eagle Medium (DMEM), Dulbecco’s phosphate buffered saline (DPBS), Hank's Balanced Salt Solution (HBSS), fetal bovine serum (FBS), Pen-Strep solution (Penicillin 5000 units/mL and Streptomycin 5000 mg/mL) and trypsin-EDTA were purchased from Gibco (Invitrogen, Paisley, UK). Water was deionized using a Milli-Q water purification system (Millipore, Bedford, MA, USA). Human epithelial colorectal adenocarcinoma (Caco-2) and Human epithelial gastric adenocarcinoma (AGS) cell lines were acquired from American Type Culture Collection (ATCC). 1.2. Samples and extract preparation Specimens were kindly collected and provided by Direção Regional de Agricultura entre Douro e Minho (DRAEM). The samples were dried at 30 ºC for 24 hours followed by powdering, and stored protected from light and humidity. In order to mimic the herbal tea usually prepared for human consumption, 200 mL of boiling water were added to 4.00 g of the powdered material and left to stand for 15 min. After filtration the obtained extract was frozen at -20 ºC and then lyophilized in a Labconco 4.5 Freezone apparatus (Kansas City, MO, USA). Experimental section 50 1.3. HPLC-DAD-ESI/MSn qualitative analysis of phenolic compounds HPLC-DAD-ESI/MSn chromatographic analysis was carried out on a Kinetex column (5 µm, C18, 100 Å, 150 x 4.6 mm; Phenomenex, Macclesfield, UK). The mobile phase consisted of two solvents: water-formic acid (1%) (A) and acetonitrile-formic acid (1%) (B), starting with 10% B and using a gradient to obtain 30% B at 20 min. The flow rate was 1 mL/min and the injection volume 20 µL. Spectral data from all peaks were accumulated in the range of 240-400 nm and chromatograms were recorded at 330 nm. The analyses were carried out in an Agilent HPLC 1100 series equipped with a diode array detector and mass detector in series (Agilent Technologies, Waldbronn, Germany). The HPLC consisted of a binary pump (model G1312A), an auto sampler (model G1313A), a degasser (model G1322A) and a photodiode array detector (model G1315B). The HPLC system was controlled by ChemStation software (Agilent, v. 08.03). The mass detector was an ion trap spectrometer (model G2445A) equipped with an electrospray ionization interface and was controlled by LCMSD software (Agilent, v. 4.1). The ionization conditions were adjusted at 350 °C and 4 kV for capillary temperature and voltage, respectively. The nebulizer pressure and flow rate of nitrogen were 65.0 psi and 11 L/min, respectively. The full scan mass covered the range from m/z 100 up to m/z 1000. Collision-induced fragmentation experiments were performed in the ion trap using helium as the collision gas, with voltage ramping cycles from 0.3 up to 2 V. Mass spectrometry data were acquired in the negative ionization mode. MSn was carried out in the automatic mode on the more abundant fragment ion in MS(n-1). 1.4. HPLC-DAD quantitative analysis of phenolic compounds HPLC-DAD conditions were the same as those described above, except for the Waters Spherisorb ODS2 (25.0 x 0.46 cm; 5 μm particle size) column. 20 µL of the lyophilized extract dissolved in water (25 mg/mL) were injected into a HPLC-DAD unit (Gilson), spectral data were acquired with a Gilson-DAD and processed on Unipoint system software (Gilson Medical Electronics, Villiers le Bel, France). Phenolic compounds quantification was achieved by the absorbance recorded at 320 nm and 350 nm for phenolic acids and flavonoids, respectively, in relation to external calibration curves (Table 4). Since commercial standards were not available for all the compounds, caffeic acid glucosides, trimmers and tetramer were quantified as caffeic acid, ferulic acid glucosides as ferulic acid, luteolin and apigenin derivatives as luteolin 7-O-glucoside and apigenin 7- Experimental section 51 O-glucoside, respectively. Caffeoylquinic acids were quantified as 5-O-caffeoylquinic acid. Rosmarinic acid and its derivatives were quantified as rosmarinic acid. Experimental section 52 Table 4. Regression equations, LOD and LOQ for phenolic compounds quantification. Compoundsa Regression equation R2 Range of concentrations (mg/mL) LODb (mg/mL) LOQc (mg/mL) 5-Caffquin Ac 7.2 x 108 x - 3.1 x 106 0.994 1.2 x 10-1 – 1.5 x 10-2 4.3 x 10-4 1.3 x 10-3 Caffeic Ac 1.5 x 109 x + 7.5 x 106 0.992 1.6 x 10-1 – 8.0 x 10-3 6.4 x 10-4 1.9 x 10-3 Ferulic Ac 1.4 x 109 x + 6.2 x 106 0.994 1.3 x 10-1 – 6.0 x 10-3 4.7 x 10-4 1.4 x 10-3 Rosmarinic Ac 8.2 x 108 x + 6.1 x 106 0.994 2.1 x 10-1 – 1.1 x 10-2 1.3 x 10-3 1.4 x 10-3 Lut-7-Gluc 4.6 x 108 x + 2.0 x 106 0.999 1.20 x 10-1 – 6.0 x 10-3 1.1 x 10-4 3.4 x 10-4 Apig-7-Gluc 8.5 x 108 x - 1.5 x 106 0.997 1.4 x 10-1 – 1.7 x 10-2 4.3 x 10-4 1.3 x 10-3 a Caffquin: caffeoylquinic; Ac: acid; Lut: luteolin; Apig: apigenin; Gluc: glucoside. b Limit of detection. c Limit of quantification. Experimental section 53 1.5. Antiradical activity 1.5.1. DPPH• scavenging assay DPPH· is a free radical able to accept an electron or hydrogen atom becoming a non-radical and hardly oxidizable species. The scavenging activity was determined spectrophotometrically (83). The assay was performed in 96-well plates and a set of extracts dilutions was prepared. The reaction mixture consisted on 25 µL of extract (redissolved in water) and 200 µL of DPPH· methanolic solution (150 mM). The plates were incubated for 30 min with light absence, and the absorbance at 515 nm was determined with a Multiskan Ascent plate reader (Thermo Electron Corporation, Vantaa, Finland). Three experiments were performed in triplicate. 1.5.2. Superoxide radical scavenging assay Antiradical activity procedure was in accordance with previously reported (83). The superoxide radical was generated by the NADH/PMS system, followed by reduction with NBT to form a formazan blue dye. The reaction mixture consisted of 50 µL of sample, 50 µL NADH (166 µM), 150 µL NBT (43 µM) and 50 µL of PMS (2.7 µM). The plate reader was set in kinetic function, and the absorbance was determined for 2 min after PMS addition at 562 nm. All components were dissolved in phosphate buffer (19 mM, pH 7.4). Three experiments were performed in triplicate. 1.5.3. Nitric oxide scavenging assay ·NO radical was generated by a SNP solution at pH 7.4 with posterior reaction with oxygen to produce nitrite, that was determined by Griess reagent (83). To each well were added 100 µL of sample and SNP (20 mM), followed by 60 min of incubation at room temperature under light exposure. Finally 100 µL of Griess reagent Experimental section 54 (1% sulfanilamide and 0.1% naphthyethylenediamine in 2% H3PO4) were added and the absorbance of the produced chromophore after the diazotization reaction at 540 nm was determined with a plate reader. Three experiments were performed in triplicate. 1.6. Cellular assays 1.6.1. Cell culture and treatments Cells were maintained in DMEM with 10% FBS and 2% Pen-Strep in an incubator at 37 ºC, with 5% CO2 and controlled humidity, in 75 cm2 flasks. Additionally Caco-2 cell medium was supplemented with amphotericin B (1%), transferrin (0.15% µL) and nonessential amino acids (1%). Once confluence was achieved, cells were washed twice with 10 mL HBSS and 3 mL of trypsin-EDTA were added, followed by 8 min of incubation. 200 µL of the prepared cellular suspension (150000 cells/mL) was seeded in 96 well-plates and incubated for 3 days before carrying out the viability assays. Preliminary assays were performed to determine the appropriate t-BHP concentration and exposure time to assess the antioxidant activity of the infusion (data not shown). Cells were seeded under the same conditions as described above. After 24 h, the medium was completely removed and t-BHP was added to the final concentration of 0.5 mM, with an exposure time of 6 h. MTT and lactate dehydrogenase (LDH) assays were then carried out to evaluate the effect of the infusion against the induced toxicity. 1.6.2. MTT assay The yellow tetrazolium salt MTT is converted to an insoluble purple formazan product, by mitochondrial dehydrogenases of metabolically active cells. Cells were incubated with different concentrations of the infusion for 24 h. After medium removal, 200 µL of MTT (0.5 mg/mL in DMEM) were added to each well, followed by 30 min of incubation. The extent of reduction to formazan was than quantified spectrophotometrically by measuring the absorbance at 510 nm, and compared to controls (84). Experimental section 55 1.6.3. LDH assay LDH is a cytosolic enzyme released into the culture medium after loss of integrity of the cellular membrane. LDH activity can be spectrophotometrically determined at 340 nm, by following NADH oxidation during the conversion of pyruvate to lactate (84). The medium was collected after 24 h of cellular exposure to the infusion. The reaction mixture consisted of 50 µL of sample, 200 µL NADH (0.15 mg/mL) and 25 µL pyruvate (0.15 mg/mL), all prepared in phosphate buffer (0.1 M, pH 7.4). 1.6.4. Determination of total and oxidized glutathione Total glutathione levels were determined by the DTNB-GSSG reductase recycling assay, after protein precipitation with perchloric acid (0.5%), and GSSG was determined after sample pre-treatment with 2-vinylpyridine for 1 h at 4 ºC, with agitation (85). The absorbance was read at 405 nm in kinetic function for 3 minutes. The experiments were conducted with and without exposure to t-BHP after incubation with the infusion. Proteins were quantified spectrophotometrically at 595 nm with Bradford reagent, using bovine serum albumin as standard. 1.6.5. Intracellular polyphenols’ staining and fluorescence microscopy The procedure was based on a previous work, with some modifications (86). Caco2 and AGS cells were seeded at 75000 cells/mL, in 24-multiwell plates, and treated with infusion concentrations of 0.31 and 0.16 mg/mL respectively, for 24 h. Also a negative control and positive controls with apigenin (25 µM) and rosmarinic acid (25 µM) were evaluated under the same conditions. Cell’s fixation procedure consisted on removing the culture medium and adding 600 µL of the pre-warmed 3.7% formaldehyde solution, with 10 min of incubation at room temperature. The solution was then removed and each well was rinsed with 600 µL HBSS. Nuclear staining was achieved with 1 µg/mL DAPI (with excitation at 364 nm and emission at 454 nm) for 30 min, followed by 0.1% (w/v) DPBA (excitation at 490 nm, emission at 530 nm) staining for 1 min. Fluorescence was detected by microscopy (Nikon Experimental section 56 TS100 microscope, Tokyo, Japan) and digital images were generated with a Nikon DS-Fi1 camera and NIS-Elements D 3.2 software (Nikon Instruments INC, New York). 1.7. Statistical analysis Statistical analysis was performed using Graphpad Prism 6 Software (San Diego, CA, USA). Quantification of phenolic compounds was achieved from three determinations and results are shown as the mean (± SD). The EC50 values were calculated from three independent assays performed in triplicate. Cellular based assays consisted of six independent experiments and results are presented as mean values (± SEM). Data from different groups was compared using one-way ANOVA Tukey’s multiple comparisons test. A level of statistical significance at p<0.05 was used. Results and discussion 57 Chapter IV - Results and discussion Results and discussion 64 4. Cellular viability and antioxidant protection against t-BHP induced toxicity Originally, Caco-2 cells derive from human colon carcinoma and AGS cells from human gastric carcinoma. Both cell lines were selected for this study as models of cellular response to xenobiotics since, when consumed, M. pulegium infusion directly contacts with gastric and intestinal epithelia. A preliminary experiment was conducted in order to assess the range of concentrations for which the exposure to the infusion was not able to affect cellular viability (Fig. 16). Figure 16. Effect of M. pulegium infusion on Caco-2 and AGS cell lines viability after 24 h of exposure, assessed by MTT reduction and LDH leakage assays. Values show mean ± SEM of six independent assays performed in triplicate (**p<0.01, ****p<0.0001 and #p<0.05 compared to the respective controls). When comparing the two cell lines, AGS cells demonstrated to be more sensitive, since viability started to significantly decrease when exposed to 0.31 mg/mL of infusion, while for Caco-2 this happened only at 1.25 mg/mL (Fig .16). Moreover the results obtained by the MTT reduction assay were more expressive than the ones by LDH leakage for both cell lines, thus suggesting that mitochondrial damage happens prior to membrane’s damage. The following step was to evaluate the potential of M. pulegium infusion to protect the cells against the toxicity caused by t-BHP. This hydroperoxide is frequently applied to induce oxidative stress, the toxicity to hepatocytes being a result from either the metabolism through cytochrome p450 or reduction of GSH. As result, several biomarkers 0.04 0.08 0.16 0.31 0.63 1.25 0 50 100 150 L D H M TT [M . pu leg iu m ] m g/m L % o f co n trol ** Caco-2 0.02 0.04 0.08 0.16 0.31 0.63 0 50 100 150 L D H M TT [M . pu leg iu m ] m g/m L % o f co n tro l # **** ** AGS 0.04 0.08 0.16 0.31 0.63 1.25 0 50 100 150 L D H M TT [M . pu leg iu m ] m g/m L % o f co n trol ** Results and discussion 65 can be monitored, like the increased levels of alanine transaminase, aspartate aminotransferase, LDH and malondialdehyde, and also GSH depletion (92). t-BHP is reduced by GPx leading to the formation of t-butanol, with GSH depletion and increased GSSG: Cytochrome p450 or Fe2+ and Fe3+ ions can also catalyze its transformation into alcoxyl and peroxyl radicals that can trigger lipid peroxidation process (92): Additionally, alterations in the intracellular calcium homeostasis, metal chelation and DNA strand breaks are also some phenomena underlying its toxicity (92) In this work, cells were treated with M. pulegium infusion for 24 h prior to t-BHP exposure (0.5 mM, 6 h). Cellular viability was again determined by MTT reduction and LDH leakage assays (Fig. 17). t-BuOOH t-BuOH GSH NADP+ NADPH GSSG GRGPx alcoxyl radical peroxyl radical Results and discussion 66 Figure 17. Caco-2 and AGS cellular viability assessed by MTT reduction and LDH leakage assays, after exposure to M. pulegium infusion, with and without t-BHP-induced toxicity. Cells were pre-treated with the infusion for 24 h. Insulted cells were further exposed to t-BHP (0.5 mM) for 6 h. , Values show mean ± SEM of six independent experiments performed in triplicate (***p<0.001, ****p<0.0001 and ##p<0.01, ####p<0.0001 compared to the respective controls). control 0.04 0.08 0.16 0.31 0.63 1.25 0 50 100 150 [M . pu leg iu m ] (m g/m L ) L D H leak age (% ) w ithou t t-B H P w ith t-B H P control 0.04 0.08 0.16 0.31 0.63 1.25 0 50 100 150 **** [M . p u leg iu m ] (m g/m L ) M T T red uction (% ) **** w ithout t-B H P w ith t-B H P *** # # Caco-2 control 0.02 0.04 0.08 0.16 0.31 0.63 0 50 100 150 **** [M . p u leg iu m ] (m g/m L ) M T T red uction (% ) **** w ithout t-B H P w ith t-B H P *** # # # # # # control 0.02 0.04 0.08 0.16 0.31 0.63 0 50 100 150 [M . pu leg iu m ] (m g/m L ) L D H leak age (% ) w ithou t t-B H P w ith t-B H P AGS con trol 0.02 0.04 0.08 0.16 0.31 0.63 0 50 100 150 [M . pu leg iu m ] (m g /m L ) L D H leak ag e (% ) w ithout t-B H P w ith t-B H P Results and discussion 67 The infusion clearly exerted a dose-dependent protective effect in the MTT reduction assay. In fact, total protection, when compared to controls, was almost achieved with the infusion at the concentrations of 1.25 and 0.63 mg/mL for Caco-2 and AGS cells, respectively (Fig. 17), despite that at this concentrations the infusion induced cytotoxicity by itself (Fig. 16). Hence, it can be assumed that from the pre-treatment with the infusion and post exposure to the toxicant did not result a synergic toxic effect. Contrarily to what was expected, an increase of LDH leakage was not observed in neither cell lines (Fig. 17). In a previous work using LDH leakage as a biomarker for necrosis, and DNA fragmentation as a biomarker for apoptosis, it was reported that a concentration of t-BHP of 0.4-0.5 mM provides a transition point below which apoptosis is favored and beyond which necrosis is favored (93). Taking this into consideration, it can be inferred that for the experimented t-BHP exposure conditions (0.5 mM, 6 hours) cell death was triggered by apoptosis, thus not being observed the increase of extracellular LDH, which is related with a cellular membrane damage. The phenolic composition of M. pulegium infusion here reported (Table 5) can be implicated in the antioxidant protective effect observed. Other flavonoids, like rutin and quercetin, also tested in a similar pre-treatment experimental model, were very effective at preventing Caco-2 DNA damage induced by t-BHP (94). The authors attributed such results to the metal iron chelating and free radical scavenging properties of those compounds. With HepG2 cells, but using a co-exposition model, it was demonstrated that other phenolics like luteolin, quercetin, luteolin 7-O-glucoside, caffeic and rosmarinic acids also protected the cells against t-BHP, by preventing lipid peroxidation and GSH depletion (95). 5. Total and oxidized GSH determination Attempting to disclosure a possible mechanism behind the antioxidant activity observed, total glutathione (GSx) and GSSG levels were quantified under the same model of pre-treatment with the infusion, followed by t-BHP-induced toxicity (Fig. 18). Results and discussion 68 Figure 18. Total glutathione (GSx) and oxidized glutathione (GSSG) determination. Caco-2 and AGS cells were pre-treated with M. pulegium infusion for 24 h. GSx and GSSG levels were quantified with and without t-BHP exposure. Values show mean ± SEM of three independent assays performed in triplicate (**p<0.01***p<0.001 and ****p<0.0001 compared to respective controls and ####p<0.0001 comparison with and without t-BHP). co ntrol 0.02 0.04 0.08 0.16 0.31 0.63 0 50 100 150 [M . pu leg iu m ] (m g /m L ) L D H leak age (% ) w ithout t-B H P w ith t-B H P Caco-2 C on tro l 0.04 0.08 0.16 0.31 0.63 0.0 0.5 1.0 1.5 2.0 [M . pulegium ] (m g /m L ) G SS G (n m o l/m g protein ) without t-BHP with t-BHP **** **** **** **** **** ### # AGS C on tro l 0.02 0.04 0.08 0.16 0.31 0 1 2 3 4 5 [M . pulegium ] (m g /m L ) G SS G (n m o l/m g protein ) without t-BHP with t-BHP ### # ** Con tro l 0.04 0.08 0.16 0.31 0.63 0 20 40 60 **** [M . pulegium ] (m g /m L ) G Sx (n m o l/m g p rotein ) *** w ithout t-B H P w ith t-B H P ** *** Con tro l 0.02 0.04 0.08 0.16 0.31 0 20 40 60 80 [M . pulegium ] (m g /m L ) G Sx (n m o l/m g p rotein ) w ithout t-B H P w ith t-B H P Results and discussion 69 The results revealed a different response of the two cell lines. In Caco-2 cells, no increase of GSx levels was observed when compared to respective controls without infusion treatment. However, when post-exposed to t-BHP, GSx levels increased very significantly in a concentration-dependent response for infusion concentrations above 0.04 mg/mL (Fig. 18). This increase may be the result of a synergic effect over the ɣglutamylcysteine synthetase enzyme, the rate limiting enzyme responsible for glutathione synthesis, which is known to be more active in response to cellular treatments with phenolic compounds, leading to de novo synthesis (28). As expected, after t-BHP exposure higher GSSG levels were observed when compared to the respective control, and it was demonstrated that the infusion was able to prevent GSH oxidation (Fig. 18). Taking together these results with the ones obtained for the MTT reduction assay (Fig. 17), it can be noticed that the infusion prevented GSH oxidation even at concentrations at which it was not able to avoid cell death (0.04 to 0.16 mg/mL). As so, this mechanism is certainly important, but not the only one behind the protective effect observed. In contrast, under the same experimental conditions, the infusion was not able to exert the same effect in AGS cells (Fig. 18). GSx levels were not significantly altered by neither the infusion nor the toxicant exposures. However, for the concentration of 0.31 mg/mL, a significant decrease of the GSSG levels was observed (Fig. 18), in accordance with the verified viability increase (Fig. 17). The results indicate that for AGS cells, t-BHP reduction by GSH was not the most relevant toxicity mechanism. 6. DPBA staining of intracellular polyphenols DPBA is capable of binding to flavonoids and phenolic acids in reactive sites after the loss of an ethanolamine chain, with adduct formation and fluorescence emission (96). One of its applications is related to flavonoids staining in plant cells, to understand the mechanisms of their transport and to follow their localization (97). To sustain if the phenolic compounds present in M. pulegium infusion were able to enter into the cells, a DPBA staining technique was applied. Nuclear staining with DAPI was also used to check on nuclear morphology. Based on the phenolic profile here reported, phenolic acids were the major represented class, rosmarinic acid being the one at the highest concentration (Table 5). So, in addition to the positive apigenin control (25 µM), as described in a previous work (98), a rosmarinic acid control (25 µM) was also tested. Results and discussion 70 The results showed that after 24 h of exposure to the infusion, the phenolic compounds could yet be targeted inside Caco-2 and AGS cells (Fig. 19). Figure 19. Fluorescence microscopy of intracellular DPBA staining of phenolic compounds and DAPI nuclear staining. Caco-2 and ÁGS cells were treated with M. pulegium infusion at the concentrations of 0.31 and 0.16 mg/mL, respectively, for 24 h. Three independent experiments were performed, and a representative field was selected. Based on the positive controls, the detected fluorescence was derived by DPBA linkage to flavonoids and phenolic acids (Fig. 19). In agreement with the results reported in a previous work with leukemia cells (98), when incubated with apigenin, AGS and Infusion DAPI DPBA Control Apigenin Rosmarinic acid AGS DAPI DPBA Caco-2 Results and discussion 71 Caco-2 cells seem to undergo an apoptotic process where chromatin condensation and vacuolization are clearly observed (Fig. 19). As for rosmarinic acid controls, in comparison to the respective negative controls some slight morphological alterations can be observed, but in a smaller scale when compared to apigenin (Fig. 19). It has been reported that despite its antioxidant properties, rosmarinic acid can also act as antiproliferative and as an apoptotic inducer agent (99-101). The viability assays did not demonstrate cellular toxic effects for the experimented infusion concentrations (Fig. 16). However, nuclear morphology alterations may be earlier visible than other toxicological end-points. This works also sustained that the phenolic compounds were capable to pass through the cellular membrane, even without prior hydrolyses. More important, since the protective effect was observed under a pre-treatment model, the phenolic compounds were still inside the cells when exposed to t-BHP. Besides GSH defenses, phenolic compounds might also act directly over the toxicant. As far as we know, no previous work focusing intracellular phenolic compounds staining and related antioxidant potential, of neither isolated compounds nor herbal extracts, in Caco-2 and AGS cells was performed. Conclusions 72 Chapter V - Conclusions Conclusions 73 With this work it was established for the first time the phenolic profile of M. pulegium infusion. Also, 12 new compounds were identified for the first time in this species. Rosmarinic acid was the major compound. The chemical profile found supports the very effective scavenging activity against DPPH·, ·NO and O2·- radicals. The infusion demonstrated to protect Caco-2 and AGS cells against t-BHP-induced toxicity, in a concentration-dependent manner, probably due to GSH induction and prevention of GSSG formation. It was also demonstrated that, even being glycosylated, the phenolic compounds were able to pass through the cellular membrane and act as direct antioxidants. 80 66. Zaidi F, Voirin B, Jay M, Viricel MR. 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