Protective Effect of Silybum marianum and Silibinin on Endothelial Cells Submitted to High Glucose Concentration

• Abstract
• Introduction
• Results and Discussion
• Material and Methods
• Reagents and materials
• Sample preparation
• HPLC analysis
• Cell culture
• Cell treatment with epicatechin, milk thistle extract, or silibinin
• Evaluation of cell viability and reactive oxygen species production
• Determination of reduced glutathione concentration, and glutathione peroxidase and glutathione reductase activity
• Determination of carbonyl groups
• Statistics
• Acknowledgements
• References

Abstract

Silybum marianum Gaertn. (Milk thistle) has been used since ancient times for the relief of liver diseases characterized by intense oxidative stress such as inflammatory liver disease and cirrhosis. As oxidative stress by hyperglycemia is involved in micro- and macrovascular complications of type 2 diabetes, our aim was to assess the protective effect of milk thistle seed extract against oxidative stress induced by a high glucose concentration on endothelial cells (EA.hy926 cells). High-performance liquid chromatographic analysis shows flavonolignans silychristin and silibinin A and B as major components. No cell toxicity was observed for concentrations up to 100 µg/mL of milk thistle extract for 24 h. Concentrations of 5–25 µg/mL of the extract were used to assess the protective effect on EA.hy926 cells treated with 30 mM glucose for 24 h. Oxidative damage by 30 mM glucose was shown as a significant decrease in reduced glutathione and a significant increase in protein carbonyls and antioxidant enzyme activities. S. marianum extract recovered reduced glutathione and balanced the elevated carbonyls and enzyme activity. Silibinin alone also recovered reduced glutathione and antioxidant enzymes. S. marianum protects endothelial cell against oxidative damage by modulating antioxidant enzyme activity, reduced glutathione, and protein carbonyl levels.

Introduction

Oxidation-reduction reactions are chemical processes needed within biological systems to obtain those products involved in cellular metabolic processes. These reactions imply the transference of electrons and may generate free radicals, such as ROS, including superoxide anion (O₂ ⁻), hydroxyl (·OH), and peroxyl (ROO·). The intrinsic antioxidant system comprises an extensive array of enzymatic and nonenzymatic antioxidants that counteract oxidizing agents and oxidative damaged molecules in order to prevent cellular injury. When ROS production is not controlled by intrinsic antioxidant systems, the generated oxidative stress may affect other biological processes and induce different diseases.

One of the first affected tissues is the vascular endothelium that has exposed itself to blood, this being the first step towards the development of vascular diseases, such as hypertension and atherosclerosis [1]. Oxidative stress may cause functional disorders of vascular endothelia and thus alter the function and structure of the vascular tissues [2]. In fact, damage to the vascular endothelium is the primary complication of type 2 diabetes mellitus leading to endothelial dysfunction and further complications such as diabetic nephropathy [3]. Therefore, prevention of oxidative stress is one of the main objectives nowadays of cardiovascular research, with natural products being a source of active substances with promising applications [4]. Several studies have demonstrated that antioxidants protect the endothelium against oxidative stress and then become an effective option to treat vascular diseases [5], [6].

Silybum marianum (L.) Gaertn. (Asteraceae) seeds and fruit have been used since Greco-Roman times as an herbal remedy for a variety of ailments, particularly liver and biliary diseases. Eclectic physicians in the United States in the late nineteenth and early twentieth centuries acknowledged the clinical benefits of the preparations of milk thistle for congestion of the liver, spleen, and kidneys [7]. The chemistry of milk thistle is well documented, with the main group of active constituents being a characteristic group of flavonol derivatives known as flavonolignans (silibinin, isosilibinin A, isosilibinin B, silychrystin, and silydianin), together with other polyphenolic compounds (apigenin, chrysoeriol, eriodictyol, taxifolin, quercetin, dihydrokaempferol, and kaempferol) [8], [9].

Extensive research has been conducted on the pharmacological activities of milk thistle, showing high scavenging capacity of ROS (antioxidant activity), regulation of cell membrane permeability, leukotriene inhibition, relief of drug-induced hepatic injury, and an antiaterogenic effect [10], [11]. Results demonstrate that silymarin (made up by flavonolignans silibinins A and B, isosilibinins A and B, silychristins A and B, and silydianin) and its components, mainly silibinin, are responsible for the pharmacological effects of milk thistle [12], [13]. However, most of this research has been focused on the health effects of S. marianum in liver injuries, whereas studies dealing with potential benefits of the plant extracts on damaged endothelial tissue have been scarce. Only recently an improvement of vascular function by silymarin in aged rats has been reported [14].

Since hyperglycemia-induced oxidative damage to endothelial cells is a main cause of micro- and macrovascular complications of type 2 diabetes and the antioxidant capacity of milk thistle fruit extracts has been documented, the objective of this study was to test the chemoprotective effect of an extract on endothelial cells submitted to high glucose concentrations. Thus, in this study, a human endothelial cell line, EA.hy926, was used as a cell culture model of the endothelium and in vitro treatment with high glucose was used to reproduce an in vivo condition of hyperglycemia-induced oxidative stress in order to study the possible protective mechanisms exerted by a well-characterized extract of S. marianum on endothelial function. Moreover, the ability of silibinin as the main active flavonolignan of silymarin was also assayed.

Results and Discussion

The flavonolignans identified in S. marianum ethyl acetate extract by HPLC are listed in [Table 1].

A chromatographic profile of milk thistle extract shows the flavonolignans silychristin and sililbinins A and B as the major components ([Fig. 1]), with the total content of silymarin, calculated as silibinin, being 2.4 percent. These results are in agreement with those previously published [8], [9], ensuring the integrity and quality of the tested samples.

Beneficial effects of milk thistle in liver damage have been conferred to silymarin (a mixture of flavonolignans) and its components, mainly silibinin [12], [13]. However, there is no indication of the possible chemoprotective effect of these compounds in endothelial cells submitted to oxidative challenges such as high glucose exposure; therefore, we have conducted a study employing a S. marianum extract in cultured endothelium-derived cells. In this study, we tried to unravel the intimate cellular antioxidant mechanisms involved in the chemoprotection induced by S. marianum.

Since elevated doses of these plant compounds can also act as pro-oxidants in cell culture systems and evoke cellular injury [15], it is necessary to ensure that no direct cell damage is caused by concentrations within the physiological range of the tested antioxidant before investigating its protective effect. The concentration range used to study the effect of milk thistle on viability and redox status of EA.hy926 cells is not far from realistic in order to evaluate the effect at the biological level. Doses of 20 mg of S. marianum extract orally administered to rats have been proven effective in reducing carbon tetrachloride (CCl₄)-induced liver damage [16], and concentrations around 240 µM of some pure compounds have been reported in rat plasma after the consumption of the same dose (100 mg/kg) of grape seed polyphenol extract [17]. In the present study, EA.hy926 cells treated for 24 h with concentrations ranging from 5 to 50 µg/mL of milk thistle extract showed no increase in crystal violet staining, indicating that no cell damage is induced by any concentration of the extract ([Table 2]). Additionally, doses of 5, 10, 25, and 50 µg/mL of the extract did not increase ROS concentration, indicating no cellular stress or oxidative damage, which could influence the functional conditions of the cells to face stressful injury ([Table 2]). Therefore, endothelial cells treated with the S. marianum extract seem to be in a favorable condition to face an oxidative challenge.

Next, the protective effect of milk thistle on GSH concentration and GPx and GR activity was evaluated. As a positive control to evaluate and compare the putative protective effect of milk thistle extract against oxidative stress, epicatechin (EC) was used. This cocoa and tea flavan-3-ol have been proven to be a powerful protective compound against chemically-induced oxidative stress in hepatic [18], colonic [19], and pancreatic beta [20] cells. A dramatic depletion (about 50 %) of intracellular GSH levels was observed when 30 mM glucose was added for 24 h to EA.hy926 cells; however, co-treatment with 5 µM EC or 5–10 µg/mL of the extract completely prevented the depletion of GSH induced by high glucose ([Fig. 2 a]).

Our results agree with a previous study showing valuable biological activity of milk thistle extract in vivo. A high and significant increase in glutathione levels and the HDL/LDL risk factor was found in rats submitted to CCL4-induced liver fibrosis and necrosis which had been treated with an S. marianum extract [16], or rats with N-nitrosodimethylaminethe-induced degenerative hepatic changes treated with 100 mg/kg b. w. silibinin [21]. Additionally, other studies have reported comparable recoveries of GSH with antioxidant extracts from the wild berry Corema album [22], cocoa [18], green coffee [23], and cranberry [24] in different cell types submitted to oxidative stress. GSH is the main nonenzymatic antioxidant defense as a substrate in glutathione peroxidase-catalyzed detoxification of organic peroxides by reacting with free radicals and by repairing free radical-induced damage through electron transfer reactions. It should be emphasized that the loss of cellular GSH seems to have an important role in apoptotic signalling [25]. Therefore, maintaining a GSH concentration above a critical threshold while facing a stressful situation represents a crucial advantage for cell survival.

The enzymatic constituents of the antioxidant defense system play a crucial role against oxidative stress; thus, the significant increase in the activity of GPx and GR observed after 24 h of treatment with 30 mM glucose ([Fig. 2 b]) clearly indicates a positive response of the cell defense system to face the exposure to high glucose and overcome the oxidative insult. However, a rapid return of the antioxidant enzyme activities to basal values once the challenge has been surmounted will position the cell in a favorable condition to deal with a new glucose challenge [26]. The present study has shown that, similar to what observed in cells treated with the positive control EC, co-treatment of high glucose submitted endothelial cells with 5–25 µg/mL of S. marianum extract can efficiently return GPx and GR activities to basal values, preparing cells to further oxidative insults ([Fig. 2 b]). Accordingly, we have previously reported that a realistic treatment with an antioxidant-rich cocoa extract averted cell damage by preventing the permanently increased activity of GPx and GR induced by high glucose in liver cells [26]. These results, together with those of GSH, indicated that the prevention or delay of the appearance of conditions causing oxidative stress in the cell may also reflect the ability of a compound to modulate the cellular antioxidant defenses.

One of the most consistent biomarkers of oxidative damage to proteins is carbonyl groups [24]. The significant increase in carbonyl groups in EA.hy926 cells treated with high glucose concentrations (30 mM) indicates extensive damage to cellular proteins at cellular level. The co-treatment of cells with 5 µM EC or 5, 10, and 25 µg/mL milk thistle extract significantly reduced the percentage of carbonyl groups in response to high glucose, demonstrating a decreased protein oxidation under a stressful situation ([Fig. 3]). Similarly, administration of an S. marianum extract to rats submitted to liver damage induced by CCl₄ significantly reduced the levels of malondialdehyde, a biomarker of lipid peroxidation [16]. In addition, a comparable chemoprotective effect on oxidative markers in a condition of hyperglycemia has also been reported in livers of diabetic rats fed a diet rich in cocoa flavonoids [27].

Finally, we tested whether the claimed chemoprotective activity of milk thistle extract on the antioxidant defenses resulted in an increase of cell viability in cells submitted to the high-glucose insult. [Table 3] shows that treatment with either 5 µM EC or 1–50 µg/mL milk thistle extract completely preserved cell viability of high-glucose-damaged endothelial cells submitted to 30 mM glucose.

Thus, the protective mechanism of S. marianum extract on high-glucose-damaged endothelial cells can be illustrated in terms of regulation of the cellular redox status, i.e., scavenging of oxygen radicals by extract antioxidants favors recovering of GSH levels that reduces the need for elevated activity of antioxidant enzymes and, simultaneously, decreases oxidative damage to proteins and subsequent cell death. This group of results indicates that the integrity of EA.hy926 cells treated with milk thistle extract was notably protected against the oxidative insult.

Although S. marianum is a complex mixture of phytochemicals that has traditionally been used as a whole extract, namely silymarin, we wanted to further investigate whether a specific majoritarian component of the extract was mostly responsible and even sufficient for the protective effect. Thus, the major flavonolignan of the extract, silibinin, was tested for its chemoprotective effect on the same EA.hy926 cells submitted to the same high glucose challenge. In order to compare the results of both experiments, concentrations of silibinin corresponding approximately to those of the compound within the doses of S. marianum extract previously tested were used to assay its chemoprotective effect on antioxidant defenses and cell viability. The results obtained with the single compound were very similar to those with the whole extract, i.e., a recovery of GSH ([Fig. 4 a]) and of antioxidant enzymes, although in this case, only with the highest dose of silibinin ([Fig. 4 b]), and a full protection of cell viability challenged by the high glucose ([Table 4]). These results agree with those of other authors [21] and indicate that although an adding or synergic effect of the multiple phytochemicals within the extract should not be ruled out, the major flavonolignan of the mixture, silibinin, is responsible for most of the chemoprotective effect of the milk thistle extract on cultured endothelial cells.

In conclusion, S. marianum seeds are rich in flavonol derivatives, mainly the mixture known as silymarin. This work shows that realistic doses of S. marianum contribute to the antioxidant defense system of endothelial cells submitted to oxidative damage by high glucose via modulating the enzymatic activity and maintaining GSH and protein carbonyl levels at safety levels. Futhermore, the results support the main component of the extract, silibinin, as the major component responsible for the chemoprotective effect. Considering all of the data, it can be concluded that treatment of EA.hy926 with milk thistle or its major component silibinin practically normalizes cell biochemistry in spite of the oxidative challenge. Further experiments are needed to assess and define the mechanism of action of this biological activity in experimental animals previous to the potential test in humans in order to confirm S. marianum extract as a potential therapeutic in this field.

Material and Methods

Reagents and materials

t-BOOH, (−)-epicatechin (> 95 % of purity), GR, GSH and oxidized glutathione, DCFH, OPT, NADPH, gentamicin, penicillin G, and streptomycin were purchased from Sigma-Aldrich Chemical. Bradford reagent was from BioRad Laboratories S. A. Methanol of HPLC grade, DMSO, light petroleum, and phosphoric acid of analytical grade were purchased from Panreac. Silymarin and silibinin (A and B diastereomers) of HPLC grade were purchased from Sigma-Aldrich. DMEM culture media and FBS were from Cultek. All other reagents were of analytical quality.

Sample preparation

The sample was supplied and authenticated by the Department of Aromatic and Medicinal Plants Research, National Institute of Agricultural and Food Technology (INIA, Madrid, Spain), and a voucher specimen was deposited at the Herbarium of the Faculty of Pharmacy, Universidad Complutense de Madrid, with voucher number MAF 164 283.

Seeds of S. marianum were finely separated from the plant fruits, dried in the shade, chopped, and extracted with ethyl acetate. Solvents were removed at 40 °C under reduced pressure to obtain the dried extract (2.5 % yield, w/w). For chromatographic analysis, the sample was prepared according the Eur. Ph. monograph [28], with slight modifications. In brief, 1.0 g of the powdered drug was treated with 100 mL of light petroleum and heated under reflux in a water bath for 1 h. The extract was then filtered and the defatted drug was allowed to dry at room temperature for 2 h. Then, 100 mL of methanol were added and heated under reflux in a water bath for 1 h. The extract was taken to a volume of about 30 mL in vacuo and filtered into a 50-mL flask, rinsing the extraction flask and the filter, and diluting to 50 mL with methanol.

HPLC analysis

A liquid chromatograph Agilent 1100 series was used. A Spherisorb C₁₈ (125 × 4 mm, 5 µm) column was used with a mobile phase consisting of A: phosphoric acid : methanol : water (0.5 : 35 : 65 V/V/V) and B: phosphoric acid : methanol : water (0.5 : 50 : 50 V/V/V) in gradient elution ([Table 5]). The flow rate was set at 0.8 mL/min. Silychristin, silydianin, silibinin A, silibinin B, isosilibinin A, and isosilibinin B were identified according to their retention time and their purity checked throughout their absorption spectra at 288 nm. The injection volume was 10 µL in triplicate [28].

Cell culture

EA.hy926, a human hybrid cell line, was a kind gift from Profs. Patricio Aller and Carmelo Bernabeu, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain. The cell line was cultured and passaged in Biowhittaker DMEM media supplemented with 10 % FBS. Cells were maintained in a humidified incubator containing 5 % CO₂ and 95 % air at 37 °C and grown in DMEM medium supplemented with 10 % FBS and 50 mg/L of each of the following antibiotics: gentamicin, penicillin, and streptomycin [29]. The culture medium was changed every other day in order to remove the non-adherent and dead cells, and the plates were usually split 1 : 3 when they reached confluence.

Cell treatment with epicatechin, milk thistle extract, or silibinin

EC was dissolved in 50 % methanol and diluted in DMEM media. An S. marianum extract stock solution of 10 mg/mL in distilled water was prepared and stored at − 20 °C. A stock solution of 1 mM silibinin in DMSO was also prepared and stored at − 20 °C. Stock solutions were diluted the day of the experiment with FBS-free DMEM medium to prepare the concentrations to test. To assay the direct effect of the extract, cells were incubated with the noted concentrations for 24 h. To induce a condition of damage by oxidative stress, EA.hy926 cells were treated with 30 mM glucose for 24 h and then tested for ROS production, antioxidant defenses, and carbonyl groups. To evaluate the protective effect of milk thistle against high glucose-induced toxicity, concentrations of the extract or silibinin (1, 5, or 10 µM) were added together with 30 mM glucose to the cell plates for 24 h.

Evaluation of cell viability and reactive oxygen species production

Cell viability was determined by using the crystal violet assay [30]. Concentrations of milk thistle extract ranging from 5 to 50 µg/mL were diluted in DMEM culture medium and added to the cell plates for 24 h to test the direct effect of the extract. Cells were seeded at a low density (10 000 cells per well) in 96-well plates, grown for 24 h, and incubated with crystal violet (0.2 % in ethanol) for 20 min. Plates were rinsed with water and 1 % sodium dodecylsulphate was added. The absorbance of each well was measured using a microplate reader at 570 nm. Intracellular ROS were quantified by the DCFH fluorometric assay using a microplate reader [20]. After being oxidized by intracellular oxidants, DCFH becomes dichorofluorescein and emits fluorescence. Cells were cultured in 24-well multiwells and treated with different concentrations of milk thistle (5, 10, 25, 50 µg/mL) for 24 h, then the DCFH probe was added for 30 min and the unabsorbed probe was removed, and fluorescence at 485 nm/530 nm was determined.

Determination of reduced glutathione concentration, and glutathione peroxidase and glutathione reductase activity

The concentration of GSH was evaluated by a fluorometric assay previously described [20]. The method takes advantage of the reaction of GSH with OPT at pH 8.0, and fluorescence was measured at an emission wavelength of 460 nm and an excitation wavelength of 340 nm. The determination of GPx activity is based on the oxidation of GSH by GPx using t-BOOH as a substrate, coupled to the disappearance of NADPH by GR [20]. GR activity was determined by following the decrease in absorbance due to the oxidation of NADPH utilized in the reduction of oxidized glutathione [20]. Protein was measured by the Bradford reagent.

Determination of carbonyl groups

The protein oxidation of the cells was measured as carbonyl groups content in supernatants according to a published method [31]. Absorbance was measured at 360 nm and carbonyl content was expressed as nmol/mg protein using an extinction coefficient of 22 000 nmol/L/cm. Protein was measured by the Bradford reagent.

Statistics

Statistical analysis of data was as follows: prior to analysis the data were tested for homogeneity of variances by the test of Levene; for multiple comparisons, one-way ANOVA was followed by a Bonferroni test when variances were homogeneous or by the Tamhane test when variances were not homogeneous. The level of significance was p < 0.05. The SPSS version 21.0 program was used.

Acknowledgements

This work was supported by grant AGL2010-17579 from the Spanish Ministry of Science and Innovation (MICINN).

Conflict of Interest

The authors declare no financial or commercial conflicts of interest.

• References

• 1 Favero G, Paganelli C, Buffoli B, Rodella LF, Rezzani R. Endothelium and its alterations in cardiovascular diseases: life style intervention. Biomed Res Int 2014; 2014: 801896
  PubMedSearch in Google Scholar
• 2 Polovina MM, Potpara TS. Endothelial dysfunction in metabolic and vascular disorders. Postgrad Med 2014; 126: 38-53
  CrossrefPubMedSearch in Google Scholar
• 3 Paneni F, Beckman JA, Creager MA, Cosentino F. Diabetes and vascular disease: pathophysiology, clinical consequences and medical therapy: part I. Eur Heart J 2013; 34: 2436-2443
  Search in Google Scholar
• 4 Ríos JL, Francini F, Schinella GR. Natural products for the treatment of type 2 diabetes mellitus. Planta Med 2015; 81: 975-994
  Thieme ConnectPubMedSearch in Google Scholar
• 5 González J, Valls N, Brito R, Rodrigo R. Essential hypertension and oxidative stress: New insights. World J Cardiol 2014; 6: 353-366
  CrossrefPubMedSearch in Google Scholar
• 6 Song P, Zou MH. Redox regulation of endothelial cell fate. Cell Mol Life Sci 2014; 71: 3219-3239
  CrossrefPubMedSearch in Google Scholar
• 7 Abenavoli L, Capasso R, Milic N, Capasso F. Milk thistle in liver diseases: past, present, future. Phytother Res 2010; 24: 1423-1432
  CrossrefPubMedSearch in Google Scholar
• 8 Kim Y, Kim E, Lee E, Lee ED, Kim JH, Jang SW, Kim YG, Kwon JW, Kim WB, Lee MG. Comparative bioavailability of silibinin in healthy male volunteers. Int J Clin Pharmacol Ther 2003; 41: 593-596
  CrossrefPubMedSearch in Google Scholar
• 9 Sweetman S. Martindale: the complete Drug Reference. 36th ed. London: Pharmaceutical Press; 2009: 1452
  Search in Google Scholar
• 10 Saller R, Melzer J, Reichling J, Brignoli R, Meier R. An updated systematic review of the pharmacology of silymarin. Forsch Komplementmed 2007; 14: 70-80
  CrossrefPubMedSearch in Google Scholar
• 11 Kwon DY, Suk JY, Kim SJ, Kim YS, Choi DW, Kim YC. Alterations in sulfur amino acid metabolism in mice treated with silymarin: a novel mechanism of its action involved in enhancement of the antioxidant defense in liver. Planta Med 2013; 79: 997-1002
  Search in Google Scholar
• 12 Lee D, Liu Y. Molecular structure and stereochemistry of silybin A, silybin B, isosilybin A, and isosilybin B, isolated from Silybum marianum (Milk Thistle). J Nat Prod 2003; 66: 1171-1174
  CrossrefPubMedSearch in Google Scholar
• 13 Quercia V, Pierini N, Incarnato G, Terracciano M, Papetti P. Identification and assay by HPLC of the flavonoid components of Silybum marianum in medicinal specialities. Fitoterapia 1980; 51: 83-88
  PubMedSearch in Google Scholar
• 14 Demirci B, Dost T, Gokalp F, Birincioglu M. Silymarin improves vascular function of aged ovariectomized rats. Phytother Res 2014; 28: 868-872
  CrossrefPubMedSearch in Google Scholar
• 15 Halliwell B. Cell culture, oxidative stress, and antioxidants: avoiding pitfalls. Biomed J 2014; 37: 99-105
  PubMedSearch in Google Scholar
• 16 Shaker E, Mahmoud H, Mnaa S. Silymarin, the antioxidant component of Silybum marianum extracts prevents liver damage. Food Chem Toxicol 2010; 48: 803-806
  CrossrefPubMedSearch in Google Scholar
• 17 Ferruzzi MG, Lobo JK, Janle EM, Cooper B, Simon JE, Wu QL, Welch C, Ho L, Weaver C, Pasinetti GM. Bioavailability of gallic acid and catechins from grape seed polyphenol extract is improved by repeated dosing in rats: implications for treatment in Alzheimerʼs disease. J Alzheimers Dis 2009; 18: 113-124
  CrossrefPubMedSearch in Google Scholar
• 18 Rodríguez-Ramiro I, Ramos S, Bravo L, Goya L, Martin MA. Procyanidin B2 and a cocoa polyphenolic extract inhibit acrylamide-induced apoptosis in human Caco-2 cells by preventing oxidative stress and activation of JNK pathway. J Nutr Biochem 2011; 22: 1186-1194
  CrossrefPubMedSearch in Google Scholar
• 19 Rodríguez-Ramiro I, Martín MA, Ramos S, Bravo L, Goya L. Comparative effects of dietary flavanols on antioxidant defences and their response to oxidant-induced stress in Caco2 cells. Eur J Nutr 2011; 50: 313-322
  CrossrefPubMedSearch in Google Scholar
• 20 Martín MA, Fernández-Millán E, Ramos S, Bravo L, Goya L. Cocoa flavonoid epicatechin protects pancreatic beta cell viability and function against oxidative stress. Mol Nutr Food Res 2014; 58: 447-456
  CrossrefPubMedSearch in Google Scholar
• 21 Ezhilarasan D, Karthikeyan S. Silibinin alleviates N-nitrosodimethylamine-induced glutathione dysregulation and hepatotoxicity in rats. Chin J Nat Med 2016; 14: 40-47
  PubMedSearch in Google Scholar
• 22 León-González A, Mateos R, Ramos S, Martín MA, Sarriá B, Martín-Cordero C, López-Lázaro M, Bravo L, Goya L. Chemo-protective activity and characterization of phenolic extracts from Corema album . Food Res Int 2012; 49: 728-738
  CrossrefPubMedSearch in Google Scholar
• 23 Baeza G, Amigo-Benavent M, Sarriá B, Goya L, Mateos R, Bravo L. Green coffee hydroxycinnamic acids but not caffeine protect human HepG2 cells against oxidative stress. Food Res Int 2014; 62: 1038-1046
  CrossrefPubMedSearch in Google Scholar
• 24 Martín MA, Ramos S, Mateos R, Marais J, Bravo L, Khoo C, Goya L. Chemical characterization and chemo-protective activity of cranberry phenolic extracts in a model cell culture. Response of the antioxidant defences and regulation of signaling pathways. Food Res Int 2015; 71: 68-82
  CrossrefPubMedSearch in Google Scholar
• 25 Ramos S, Rodríguez-Ramiro I, Martín MA, Goya L, Bravo L. Dietary flavanols exert different effects on antioxidant defences and apoptosis in Caco-2 and SW480 colon cancer cells. Toxicol In Vitro 2011; 25: 1771-1781
  CrossrefPubMedSearch in Google Scholar
• 26 Cordero-Herrera I, Martín MA, Goya L, Ramos S. Cocoa flavonoids protect hepatic cells against high glucose-induced oxidative stress: relevance of MAPKs. Mol Nutr Food Res 2015; 59: 597-609
  CrossrefPubMedSearch in Google Scholar
• 27 Cordero-Herrera I, Martín MA, Goya L, Ramos S. Cocoa intake ameliorates hepatic oxidative stress in young Zucker diabetic fatty rats. Food Res Int 2015; 69: 194-201
  CrossrefPubMedSearch in Google Scholar
• 28 EDQM. European Pharmacopoeia. Silybum marianum. 7th edn. Strasbourg, France: EDQM Eds.; 2008: 1860
  Search in Google Scholar
• 29 Bouis D, Hospers GA, Meijer C, Molema G, Mulder NH. Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research. Angiogenesis 2001; 4: 91-102
  CrossrefPubMedSearch in Google Scholar
• 30 Granado-Serrano AB, Martín MA, Izquierdo-Pulido M, Goya L, Bravo L, Ramos S. Molecular mechanisms of (−)-epicatechin and chlorogenic acid on the regulation of the apoptotic and survival/proliferation pathways in a human hepatoma cell line. J Agric Food Chem 2007; 55: 2020-2027
  CrossrefPubMedSearch in Google Scholar
• 31 Granado-Serrano AB, Martín MA, Bravo L, Goya L, Ramos S. A diet rich in cocoa attenuates N-nitrosodiethylamine-induced liver injury in rats. Food Chem Toxicol 2009; 47: 2499-2506
  CrossrefPubMedSearch in Google Scholar

Correspondence

• References

• 1 Favero G, Paganelli C, Buffoli B, Rodella LF, Rezzani R. Endothelium and its alterations in cardiovascular diseases: life style intervention. Biomed Res Int 2014; 2014: 801896
  PubMedSearch in Google Scholar
• 2 Polovina MM, Potpara TS. Endothelial dysfunction in metabolic and vascular disorders. Postgrad Med 2014; 126: 38-53
  CrossrefPubMedSearch in Google Scholar
• 3 Paneni F, Beckman JA, Creager MA, Cosentino F. Diabetes and vascular disease: pathophysiology, clinical consequences and medical therapy: part I. Eur Heart J 2013; 34: 2436-2443
  Search in Google Scholar
• 4 Ríos JL, Francini F, Schinella GR. Natural products for the treatment of type 2 diabetes mellitus. Planta Med 2015; 81: 975-994
  Thieme ConnectPubMedSearch in Google Scholar
• 5 González J, Valls N, Brito R, Rodrigo R. Essential hypertension and oxidative stress: New insights. World J Cardiol 2014; 6: 353-366
  CrossrefPubMedSearch in Google Scholar
• 6 Song P, Zou MH. Redox regulation of endothelial cell fate. Cell Mol Life Sci 2014; 71: 3219-3239
  CrossrefPubMedSearch in Google Scholar
• 7 Abenavoli L, Capasso R, Milic N, Capasso F. Milk thistle in liver diseases: past, present, future. Phytother Res 2010; 24: 1423-1432
  CrossrefPubMedSearch in Google Scholar
• 8 Kim Y, Kim E, Lee E, Lee ED, Kim JH, Jang SW, Kim YG, Kwon JW, Kim WB, Lee MG. Comparative bioavailability of silibinin in healthy male volunteers. Int J Clin Pharmacol Ther 2003; 41: 593-596
  CrossrefPubMedSearch in Google Scholar
• 9 Sweetman S. Martindale: the complete Drug Reference. 36th ed. London: Pharmaceutical Press; 2009: 1452
  Search in Google Scholar
• 10 Saller R, Melzer J, Reichling J, Brignoli R, Meier R. An updated systematic review of the pharmacology of silymarin. Forsch Komplementmed 2007; 14: 70-80
  CrossrefPubMedSearch in Google Scholar
• 11 Kwon DY, Suk JY, Kim SJ, Kim YS, Choi DW, Kim YC. Alterations in sulfur amino acid metabolism in mice treated with silymarin: a novel mechanism of its action involved in enhancement of the antioxidant defense in liver. Planta Med 2013; 79: 997-1002
  Search in Google Scholar
• 12 Lee D, Liu Y. Molecular structure and stereochemistry of silybin A, silybin B, isosilybin A, and isosilybin B, isolated from Silybum marianum (Milk Thistle). J Nat Prod 2003; 66: 1171-1174
  CrossrefPubMedSearch in Google Scholar
• 13 Quercia V, Pierini N, Incarnato G, Terracciano M, Papetti P. Identification and assay by HPLC of the flavonoid components of Silybum marianum in medicinal specialities. Fitoterapia 1980; 51: 83-88
  PubMedSearch in Google Scholar
• 14 Demirci B, Dost T, Gokalp F, Birincioglu M. Silymarin improves vascular function of aged ovariectomized rats. Phytother Res 2014; 28: 868-872
  CrossrefPubMedSearch in Google Scholar
• 15 Halliwell B. Cell culture, oxidative stress, and antioxidants: avoiding pitfalls. Biomed J 2014; 37: 99-105
  PubMedSearch in Google Scholar
• 16 Shaker E, Mahmoud H, Mnaa S. Silymarin, the antioxidant component of Silybum marianum extracts prevents liver damage. Food Chem Toxicol 2010; 48: 803-806
  CrossrefPubMedSearch in Google Scholar
• 17 Ferruzzi MG, Lobo JK, Janle EM, Cooper B, Simon JE, Wu QL, Welch C, Ho L, Weaver C, Pasinetti GM. Bioavailability of gallic acid and catechins from grape seed polyphenol extract is improved by repeated dosing in rats: implications for treatment in Alzheimerʼs disease. J Alzheimers Dis 2009; 18: 113-124
  CrossrefPubMedSearch in Google Scholar
• 18 Rodríguez-Ramiro I, Ramos S, Bravo L, Goya L, Martin MA. Procyanidin B2 and a cocoa polyphenolic extract inhibit acrylamide-induced apoptosis in human Caco-2 cells by preventing oxidative stress and activation of JNK pathway. J Nutr Biochem 2011; 22: 1186-1194
  CrossrefPubMedSearch in Google Scholar
• 19 Rodríguez-Ramiro I, Martín MA, Ramos S, Bravo L, Goya L. Comparative effects of dietary flavanols on antioxidant defences and their response to oxidant-induced stress in Caco2 cells. Eur J Nutr 2011; 50: 313-322
  CrossrefPubMedSearch in Google Scholar
• 20 Martín MA, Fernández-Millán E, Ramos S, Bravo L, Goya L. Cocoa flavonoid epicatechin protects pancreatic beta cell viability and function against oxidative stress. Mol Nutr Food Res 2014; 58: 447-456
  CrossrefPubMedSearch in Google Scholar
• 21 Ezhilarasan D, Karthikeyan S. Silibinin alleviates N-nitrosodimethylamine-induced glutathione dysregulation and hepatotoxicity in rats. Chin J Nat Med 2016; 14: 40-47
  PubMedSearch in Google Scholar
• 22 León-González A, Mateos R, Ramos S, Martín MA, Sarriá B, Martín-Cordero C, López-Lázaro M, Bravo L, Goya L. Chemo-protective activity and characterization of phenolic extracts from Corema album . Food Res Int 2012; 49: 728-738
  CrossrefPubMedSearch in Google Scholar
• 23 Baeza G, Amigo-Benavent M, Sarriá B, Goya L, Mateos R, Bravo L. Green coffee hydroxycinnamic acids but not caffeine protect human HepG2 cells against oxidative stress. Food Res Int 2014; 62: 1038-1046
  CrossrefPubMedSearch in Google Scholar
• 24 Martín MA, Ramos S, Mateos R, Marais J, Bravo L, Khoo C, Goya L. Chemical characterization and chemo-protective activity of cranberry phenolic extracts in a model cell culture. Response of the antioxidant defences and regulation of signaling pathways. Food Res Int 2015; 71: 68-82
  CrossrefPubMedSearch in Google Scholar
• 25 Ramos S, Rodríguez-Ramiro I, Martín MA, Goya L, Bravo L. Dietary flavanols exert different effects on antioxidant defences and apoptosis in Caco-2 and SW480 colon cancer cells. Toxicol In Vitro 2011; 25: 1771-1781
  CrossrefPubMedSearch in Google Scholar
• 26 Cordero-Herrera I, Martín MA, Goya L, Ramos S. Cocoa flavonoids protect hepatic cells against high glucose-induced oxidative stress: relevance of MAPKs. Mol Nutr Food Res 2015; 59: 597-609
  CrossrefPubMedSearch in Google Scholar
• 27 Cordero-Herrera I, Martín MA, Goya L, Ramos S. Cocoa intake ameliorates hepatic oxidative stress in young Zucker diabetic fatty rats. Food Res Int 2015; 69: 194-201
  CrossrefPubMedSearch in Google Scholar
• 28 EDQM. European Pharmacopoeia. Silybum marianum. 7th edn. Strasbourg, France: EDQM Eds.; 2008: 1860
  Search in Google Scholar
• 29 Bouis D, Hospers GA, Meijer C, Molema G, Mulder NH. Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research. Angiogenesis 2001; 4: 91-102
  CrossrefPubMedSearch in Google Scholar
• 30 Granado-Serrano AB, Martín MA, Izquierdo-Pulido M, Goya L, Bravo L, Ramos S. Molecular mechanisms of (−)-epicatechin and chlorogenic acid on the regulation of the apoptotic and survival/proliferation pathways in a human hepatoma cell line. J Agric Food Chem 2007; 55: 2020-2027
  CrossrefPubMedSearch in Google Scholar
• 31 Granado-Serrano AB, Martín MA, Bravo L, Goya L, Ramos S. A diet rich in cocoa attenuates N-nitrosodiethylamine-induced liver injury in rats. Food Chem Toxicol 2009; 47: 2499-2506
  CrossrefPubMedSearch in Google Scholar