Antioxidant and Hepatoprotective Effect of (â)-Epigallocatechin 3-O-(3

Article pubs.acs.org/JAFC

Antioxidant and Hepatoprotective Effect of (−)-Epigallocatechin 3‑O‑(3‑O‑Methyl) gallate (EGCG3″Me) from Chinese Oolong Tea Xin Zhang,* Zufang Wu, and Peifang Weng Department of Food Science and Engineering, School of Marine Science, Ningbo University, Ningbo 315211, People’s Republic of China ABSTRACT: (−)-Epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3″Me) has exhibited various biological activities in oolong tea. However, little information about its hepatoprotective activity is available. The objectives of the present study, therefore, were to determine the hepatoprotective activity of EGCG3″Me. First, high-purity EGCG3″Me was prepared from Chinese oolong tea by column chromatography. In antioxidant assay in vitro, EGCG3″Me exhibited potential antioxidant activity. For hepatoprotective activity in vitro, it was observed that EGCG3″Me effectively alleviated the changes induced by alcohol in a concentration-dependent manner. For hepatoprotective activity in vivo, the administration of EGCG3″Me at a dose of 100 mg/ kg BW per day significantly decreased the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) from 64.6 ± 3.17 and 97.6 ± 3.78 to 39.6 ± 2.72 and 59.6 ± 3.02 U/L, decreased the liver level of malondialdehyde (MDA) from 1.14 ± 0.08 to 0.77 ± 0.03 nmol/mg protein, and remarkably restored the liver activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) from 247 ± 20.1 U/mg and 6.12 ± 0.17 nmol/mg protein to 261 ± 9.98 U/mg and 8.10 ± 0.03 nmol/mg protein, respectively, in alcohol-induced liver injury mice. This suggested that the protective effect of EGCG3″Me against alcohol-induced liver injury is possibly via its antioxidant activity to protect biological systems against oxidative stress. KEYWORDS: oolong tea, EGCG3″Me, antioxidant activity, hepatoprotective effect

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INTRODUCTION

methylated derivatives of tea catechins for their antiallergic properties and potential applications. Alcohol-induced liver injury is one of the most common causes of liver diseases nowadays. Due to the increase of drinking and change of diet, the incidence of alcoholic liver disease (ALD) has become an important risk factor for morbidity and mortality in addition to viral hepatitis.6 Thus, more attention has been paid to the effective therapy for ALD and agents for protecting against alcohol-induced liver injury. It has been recognized that oxidative stress and generation of free radicals play critical roles in the development of ALD.7 Therefore, natural products with antioxidant activity, especially tea, have attracted great attention as potential functional ingredients to protect against hepatotoxin-induced liver injury.8,9 In China, tea has been used to detoxify alcoholic intoxication for more than a millennium, and considerable evidence has accumulated indicating that the extracts of tea can reduce alcohol toxicity.10 Various pathways involving multiple types of cells and enzymes are thought to be associated with the pathological process of alcohol-induced liver injury.11,12 Oxidative stress, caused by a decrease in antioxidant defense and an increase in prooxidant production, is known to play a major role in the pathogenesis of alcohol-induced liver injury.13 Because of its high antioxidant activity, tea has demonstrated antioxidant properties in alcohol intoxication and protective effects against ethanol-induced lipid peroxidation.10 Further-

Nowadays, tea is widely drunk across the world, and more and more people drink it due to its benefits to health. It has been reported that these beneficial effects have been attributed mainly to the presence of tea catechins.1 Tea catechins are involved in many biological activities and have antioxidative, antiviral, and antitumor properties.2,3 Moreover, many of the beneficial effects of tea catechins are attributed to the major tea catechin (−)-epigallocatechin gallate (EGCG), which has been reported to exert various biological effects.4 In addition, the unique O-methylated form of EGCG, (−)-epigallocatechin 3O-(3-O-methyl) gallate (EGCG3″Me, Figure 1), which is present only in limited oolong teas and green teas, has been reported to have potent antioxidant and inhibitory activities to allergies.5 Accordingly, great attention has been paid to the O-

Received: Revised: Accepted: Published:

Figure 1. Chemical structures of EGCG and EGCG3″Me. © 2014 American Chemical Society

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April 5, 2014 September 14, 2014 September 26, 2014 September 26, 2014 dx.doi.org/10.1021/jf5016335 | J. Agric. Food Chem. 2014, 62, 10046−10054

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concentrated, loaded onto the Toyopearl HW-40S column, respectively, and treated as described above. As results, the fractions containing EGCG and EGCG3″Me were concentrated and freezedried by a freeze-dry system respectively, affording EGCG and EGCG3″Me. Analysis and Characterization of EGCG3″Me. The contents of tea catechins were determined according to the reported method using an Agilent 1100 series HPLC (Agilent, Santa Clara, CA, USA).17 The separation was achieved on a TSKgel ODS-100Z column (4.6 × 150 mm, 5 μm; Tosoh). The mobile phase consisted of formic acid solution (pH 2.5, A) and methanol (B). Elution was performed with a linear gradient as follows: 0−15 min, A from 82 to 40%. The injection volume was 20 μL. The temperature of column oven was set at 40 °C, and the flow rate was set at 1.0 mL/min. The structures of tea catechin monomers isolated from oolong tea were characterized by ESI-MS/MS and 1H NMR. ESI-MS/MS analysis was performed by using a TSQ Quantum Ultra triple -quadrupole mass spectrometer (Thermo Scientific) in positive mode. The operation parameters of the ESI ion source were as follows: drying gas, N2; temperature, 270 °C; flow rate of drying gas, 4 L/min; pressure of nebulizer gas (N2), 9.0 psi; and capillary voltage, 4 kV. ESITOF-MS spectra and 1H NMR spectrum were recorded on an Applied Biosystems mass spectrometer. 1H NMR spectrum was recorded by a Bruker DRX-500 spectrometer operated at 300 K with D2O as solvent. Chemical shifts (δ) are given in parts per million, and J values are given in hertz. DPPH Assay. The DPPH free radical scavenging activity of each sample was determined according to the reported method.19 The percentage of DPPH• that was scavenged was calculated using

more, the most abundant tea catechin, EGCG, has also been shown to exhibit hepatoprotective effects against liver injury by ethanol and carbon tetrachloride (CCl 4 )-induced liver injury.14,15 To the best of our knowledge, most studies on EGCG3″Me have focused on its antioxidant and inhibitory activities to allergies, and little information about the hepatoprotective effect of EGCG3″Me against alcohol-induced toxicity is available. Therefore, the main objective of the present study was to evaluate the hepatoprotective effects of EGCG3″Me. First, high-purity EGCG3″Me was prepared through extraction from Chinese oolong tea and simple purification procedures by using polyamide and Toyopearl HW-40S column chromatography. Then, the hepatoprotective effects of EGCG3″Me were evaluated by the antioxidant and hepatoprotective activity in alcohol-induced hepatotoxicity in the HepG2 cell line and mice.

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MATERIALS AND METHODS

Chemicals. Toyopearl HW-40S resin was purchased from Tosoh Corp. (Tokyo, Japan), and polyamide resin was obtained from Qingdao Ocean Chemical Co., Ltd. (Qingdao, China). 2,2-Diphenyl1-picrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salts (ABTS) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) was obtained from Fluka (Buchs, Swiltzerland). Standards of EGCG and EGCG3″Me were prepared according to our reported methods.16,17 Human hepatic cancer HepG2 cells were obtained from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), penicillin, and streptomycin were purchased from Sigma Chemical Co. Fetal bovine serum (FBS), trypsin, and RPMI-1640 media were purchased from Gibco/Invitrogen (Grand Island, NY, USA). Male Kunming mice were obtained from the Experimental Animal Centre of Academy of the Military Medical Sciences (Beijing, China). Analytical kits for aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA) were obtained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). All other chemicals and reagents were of analytical grade. Tea Samples. Oolong tea was purchased from a local market in Guangzhou, China. The fresh tea leaves picked were given a solar withering process for 2 h, followed by indoor withering for 4 h, and then 5 cycles of hand rolling for 20 min. The final steps consisted of 2 h of fermentation, followed by 5 min of killing, 5 min of kneading, and 5 h of roasting.18 The oolong tea manufacturing process was replicated five times. After the manufacture, the water content of oolong tea is 4.05 ± 0.24%. The oolong tea sample was ground into powder using a milling machine, and the material that passed through a 40 mesh sieve was kept in sealed polyethylene bags at −20 °C until use. Preparation of Tea Catechins. EGCG and EGCG3″Me were prepared from oolong tea according to the reported method with some modifications.16,17 Briefly, 2 kg of tea powder was extracted with 32 L of distilled water at 96 °C for 40 min. Upon extraction, the extract was centrifuged at 4500g for 15 min, and the resulting insoluble residue was treated again as described above. The supernatants, which were extraction solutions of 2 times, were combined and concentrated by using rotary evaporators under vacuum. The resulting residue was dissolved, filtered, and applied to a column wet-packed polyamide resin. After reaching adsorptive saturation, the column was washed by distilled water and then eluted by 80% ethanol. The effluent of the ethanol solution was collected and concentrated, affording the crude extract for further purification. The crude extract was dissolved and loaded onto a Toyopearl HW40S column pre-equilibrated with 80% ethanol. The column was eluted with 80% ethanol, and the elution was monitored by measuring the absorbance at 280 nm and autocollected. The eluted fractions were analyzed by high-performance liquid chromatography with a diode array detector (HPLC-DAD), and the desired fractions were collected,

scavenging activity (%) = (Abscont − Abssamp)/Abscont × 100 where Abscont is the absorbance of the control and Abssamp is the absorbance of the sample. The DPPH free radical scavenging activity was expressed by IC50 value, which is the concentration of extract required to decrease the absorbance at 517 nm by 50%. Measurements were performed at least in triplicate. ABTS Assay. The ABTS free radical scavenging activity of each sample was determined according to the method described by Stratil et al.20 The radical cation ABTS•+ was generated by persulfate oxidation of ABTS, and the ABTS•+ scavenging rate was calculated to express the antioxidant ability of the sample. IC50 values calculated denote the concentration of sample required to decrease the absorbance at 734 nm by 50%. FRAP Assay. The ability to reduce ferric ions was measured using a modified version of the method described by Benzie and Strain.21 The antioxidant capacity based on the ability to reduce ferric ions of sample was calculated from the linear calibration curve and expressed as millimoles of FeSO4 equivalents per gram of sample (DW). Cell Culture. The human hepatoma cell line HepG2 was cultured in the RPMI-1640 medium supplemented with FBS (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL). This was incubated at 37 °C in a humidified 5% CO2 incubator; cells were diluted to a density of 1 × 105 cells/mL and fed every 2 days with fresh medium. Under these conditions the generation time of HepG2 cells was approximately 36 h. At subconfluence the Petri dishes were carefully washed with serum-free culture medium and treated for the following investigations. In Vitro Cytotoxicity Assay. The CTC50 (50% cytotoxic concentration) was determined by estimating mitochondrial synthesis using the tetrazolium assay.22 Briefly, HepG2 cells were pipetted into a 96-well flat-bottom plate (100 μL/well) at a density of 1 × 105 cells/ mL. After 12 h of incubation at 37 °C in a humidified 5% CO2 incubator, nonadherent cells were removed by washing with serumfree culture medium. Then, fresh medium (100 μL/well, control group) or test sample (100 μL/well, EGCG3″Me at a final concentration of 25, 50, 100, or 200 μg/mL) was added to each well, and the cells were incubated for 24, 48, and 72 h, respectively. After incubation, MTT solution (10 μL/well, 5 mg/mL) was added to each well, and the plate was incubated for an additional 4 h at 37 °C. 10047

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Finally, 100 μL of 10% sodium dodecyl sulfate (SDS) in 0.01 N HCl was added to each well, and the plate was kept overnight for the dissolution of formazan crystals. The absorbance of each well at 570 nm was measured. The inhibition rate was calculated according to the following formula:

respectively fed 0.5 mL of EGCG3″Me solution in three different doses (25, 50, and 100 mg/kg BW) once daily. For positive control and EGCG3″Me treatment groups, they were fed 0.5 mL of 50% alcohol solution before given bifendate and EGCG3″Me, respectively. All administrations were conducted by gastric gavage for 14 consecutive days. After 10 h of fasting following the last administration, the mice were killed by decapitation. Blood samples were collected immediately and centrifuged at 3000 rpm for 10 min at 4 °C to afford the sera. The sera were stored at −70 °C until the determination of AST and ALT activities. The liver was excised and homogenized in 0.1 g/mL wet weight of ice-cold physiological saline. The homogenate was centrifuged at 3000 rpm for 10 min, and the supernatant was collected for determination of SOD and GSH-Px activities and the level of MDA. Biochemical Assays of Alcohol-Induced Toxicity in Mice. The activities of AST and ALT in serum and the levels of MDA, SOD, and GSH-Px activities in liver were determined by using commercial reagent kits from Nanjing Jiancheng Bioengineering Institute according to the instruction manuals. Histopathological Evaluation. Livers from mice in different groups were fixed in 10% neutral formalin solution, dehydrated in graded alcohol, and embedded in paraffin. Thin sections of 5 μm thickness of liver tissue were cut and mounted on glass slides and then were counter-stained with hematoxylin−eosin (H&E).25 Then, thin sections of liver were made into permanent slides and were examined for possible histopathological changes under a high-resolution microscope with photographic facility. Statistical Analysis. Data were analyzed by SPSS and expressed as mean ± standard deviation (SD) for at least three replicates. Significance was determined at P < 0.05 by ANOVA followed by Duncan’s multiple-comparison tests.

inhibition rate (%) = 100 − (mean OD of individual test group /mean OD of control group) × 100 A concentration−response curve was generated using inhibition rate (%) and EGCG3″Me sample concentration (μg/mL). The CTC50 value is calculated from the concentration−response curve. Alcohol-Induced Toxicity in HepG2 Cell Line. To each well of the 96-well flat-bottom plate was added 100 μL of diluted HepG2 cell suspension (1.0 × 105 cells/mL). After 12 h, when a partial monolayer was formed, the supernatant was removed and the monolayer was washed once with serum-free culture medium. Bifendate, a synthetic intermediate of schisandrin C with protective effect against druginduced liver injury in rodents, is often used for the treatment of hepatitis as well as regarded as a positive control for exploring other hepatoprotective drugs.23 Therefore, bifendate was adopted for the evaluation of the hepatoprotective effect of EGCG3″Me as positive control. The cells were treated with 100 μL of toxicant (medium containing 1% (v/v) alcohol), 100 μL of positive control (medium containing 1% (v/v) alcohol and bifendate (10, 20, and 40 μg/mL)), 100 μL of sample (medium containing 1% (v/v) alcohol and EGCG3″Me (10, 20, and 40 μg/mL)), and 100 μL of control (medium, medium and EGCG3″Me (40 μg/mL)), medium, and bifendate (40 μg/mL) for various time intervals of 3, 6, 12, and 24 h, and the following assays were carried out to observe time-dependent changes. Cell Viability. Cell viability after alcohol treatment was evaluated by using a MTT-based colorimetric method as mentioned under in Vitro Cytotoxicity Assay. Briefly, after the cells were treated with 1% (v/v) alcohol for 24 h, MTT solution was added to each well, and the plate was incubated for an additional 4 h at 37 °C. Finally, 100 μL of 10% SDS in 0.01 M HCl was added to each well, and the plate was kept overnight for the dissolution of formazan crystals. The absorbance of each well at 570 nm was measured, and the viability rate was calculated according to the formula below:

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RESULTS Preparation of EGCG3″Me. In the present study, EGCG3″Me was prepared from Chinese oolong tea by extraction and purification of column chromatography.17 As shown in Figure 2, peaks 1−13 in the HPLC chromatogram of oolong tea infusion were identified to be gallic acid, (−)-gallocatechin, theobromine, (−)-epigallocatechin, (−)-catechin, theophylline, EGCG, caffeine, (−)-epicatechin, GCG

viability (%) = (Abssamp /Abscont ) × 100% Biochemical Assays of Alcohol-Induced Toxicity in HepG2 Cell Line. The 96-well flat-bottom plates were centrifuged at 500g for 5 min at 4 °C, and the culture medium and cells were collected separately. The medium (0.2 mL) was used for measuring AST and ALT activities. AST and ALT activities in sera were measured using commercial reagent kits from Nanjing Jiancheng Bioengineering Institute. The level of MDA and activity of GSH-Px in HepG2 cells were also determined by using commercial reagent kits from the same institute according to the instruction manuals. Animals and Experimental Design. The hepatoprotective effect of EGCG3″Me against alcohol-induced acute liver damage in mice was evaluated according to the reported method with proper modifications.24 Furthermore, all procedures involving animals were conducted in strict accordance with Chinese legislation on the use and care of laboratory animals during the entire experimental period. Male Kunming mice (8 weeks old) with body weights (BW) of about 20 g were used in the present study. They were maintained in a room with a controlled temperature of 22 ± 0.5 °C and a normal day/night cycle and allowed free access to basal pellet diet and tap water. After adapting to the environment for 7 days, these mice were randomly divided into six groups of eight each: (I) normal control group; (II) alcohol model control group; (III) positive control group; (IV−VI) EGCG3″Me treatment groups. Mice in the normal control group and alcohol model control group were given 1.0 mL of physiological saline and 50% alcohol solution, respectively, once daily. Bifendate and EGCG3″Me were both dissolved in physiological saline. Mice in the positive control group were given 0.5 mL of bifendate (10 mg/kg BW) per day. Mice in EGCG3″Me treatment groups were

Figure 2. HPLC chromatograms of tea catechins, gallic acid, caffeine, theobromine, and theophylline from oolong tea (1, gallic acid; 2, (−)-gallocatechin; 3, theobromine; 4, (−)-epigallocatechin; 5, (−)-catechin; 6, theophylline; 7, EGCG; 8, caffeine; 9, (−)-epicatechin; 10, GCG; 11, EGCG3″Me; 12, (−)-epicatechin gallate; 13, (−)-catechin gallate). 10048



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((−)-gallocatechin gallate), EGCG3″Me, (−)-epicatechin gallate, and (−)-catechin gallate according to the retention times and online DAD spectra of authentic standards. In our previous study, EGCG3″Me as standard was obtained from Japanese green tea. Here, we found that Chinese oolong tea from Guangdong province contained relatively higher contents of EGCG, GCG, and EGCG3″Me. The contents of EGCG, GCG, and EGCG3″Me in Chinese oolong tea and Japanese green tea were 49.6 ± 0.82, 3.26 ± 0.23, and 11.9 ± 0.48 mg/g and 41.7 ± 0.91, 2.05 ± 0.12, and 8.07 ± 0.29 mg/g, respectively. Therefore, the infusion of oolong tea was loaded onto polyamide and Toyopearl HW-40S columns, respectively, affording high purity (>99%) of EGCG3″Me. The structure of the resulting EGCG3″Me was characterized by ESI-TOF-MS and 1H NMR. Positive-mode MS for it gave molecular ion signals at m/z 495.2 for [M + Na]+ and at m/z 473.2 for [M + H]+. The results indicated that its molecular weight is 472.2, corresponding to that of EGCG3″Me. 1 H NMR data for EGCG3″Me: 7.01 (1 H, d, H-2″), 6.98 (1H, d, H-6″), 6.49 (2H, s, H-2′, H-6′), 5.87 (1H, d, H-8), 5.83 (1H, d, H-6), 5.56 (1H, s, H-2), 5.35 (1H, bs, 3-OH), 5.24 (1H, d, H-3), 3.82 (3H, s, OCH3), 3.10 (1H, dd, H-4 ax), 2.85 (1H, dd, H-4 equiv). The 1H NMR spectrum was similar to that of EGCG except for the presence of a methoxyl proton signal at δ 3.82 (3H, s) and two aromatic proton signals at δ 6.98 (1H, d) and 7.01 (1H, d).26 On irradiation of the methoxyl proton signal, a nuclear Overhauser effect was observed at the aromatic proton signal at δ 6.98. From these data it was assumed to be EGCG3″Me and was assigned by comparison of the reported NMR data.27 Determination of Antioxidant Activity of EGCG3″Me. DPPH free radical scavenging activities of EGCG3″Me and EGCG are shown in Figue 3A. EGCG3″Me showed high scavenging activity on superoxide radical, and it increased with the increase of sample concentration. At concentrations ranging from 0.2 to 0.8 mg/mL, the scavenging activity increased rapidly with the increase of concentration; after that, it increased slowly. The EGCG3″Me showed DPPH radical scavenging activity with an IC50 of 0.37 mg/mL. The free radical scavenging activity of EGCG3″Me was less than that of EGCG (IC 50 = 0.31 mg/mL), which exhibited high antioxidative property. The antioxidant abilities of EGCG3″Me and EGCG determined by using the ABTS method are shown in Figure 3B. In our results, EGCG exhibited considerably higher activity than EGCG3″Me. In addition, the ABTS•+ scavenging rates of EGCG3″Me and EGCG increased with the increase of concentration in our study. The trends for ferric ion reducing activities of EGCG3″Me and EGCG are shown in Figure 3C. For EGCG3″Me and EGCG, the absorbance increased clearly, due to the formation of the Fe2+−TPTZ complex with increasing concentration. Similar to the results of the DPPH and ABTS assays, the reducing activity for EGCG was higher than that of EGCG3″Me. Determination of Hepatoprotective Activity of EGCG3″Me. To ascertain the extent of toxicity caused by 1% of alcohol in HepG2 cells, we quantified the cell viability (Figure 4A) and AST (Figure 4B) and ALT (Figure 4C) levels at various time intervals of 3, 6, 12, and 24 h. MDA formed (Figure 4D) and GSH-Px content (Figure 4E) were also determined. It can be seen that no significant changes in cell

Figure 3. Antioxidant activities of EGCG and EGCG3″Me made from Chinese oolong tea determined by DPPH free radical-scavenging assay (A), ABTS assay (B), and FRAP assay (C).

viability, AST and ALT levels, lipid peroxidation, and glutathione depletion were observed until an exposure period of 3 h when compared with normal control, thus overruling any direct solvent-mediated damage by alcohol in HepG2 cells. Thereafter, a time-dependent significant (P < 0.05) increased leakage of AST and ALT and loss of cell viability were observed. Similarly, a significant (P < 0.05) increased lipid peroxidation with concurrent decrease in GSH-Px content was noted compared with normal control when cells were incubated for 6, 12, and 24 h. The cytotoxicity of EGCG3″Me was studied by in vitro cytotoxicity assay, and it showed the CTC50 value of EGCG3″Me was 94.2, 156, and 190 μg/mL for 24, 48, and 72 h, respectively, which showed EGCG3″Me had no cytotoxicity for the HepG2 cell line in this study. Table 1 depicts the protective effect of EGCG3″ and bifendate on alcohol-induced toxicity in the HepG2 cell line. A significant decrease in cell viability and significant increases in AST and ALT levels were observed in HepG2 cells exposed to alcohol as compared with the normal control group. Both EGCG3″Me and bifendate treatments along with alcohol significantly increased the viability of cells. The middle concentration of bifendate (20 μg/mL) was more effective 10049



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Figure 4. Time-dependent changes observed in (A) cell viability, (B) AST and (C) ALT leakage, and (D) MDA and (E) GSH-Px levels after exposure to 1% of alcohol in HepG2 cells. Group 1, normal control; group 2, alcohol control (1%, v/v). Results are expressed as mean ± SEM (n = 5). Level of significance P < 0.05. aCompared with normal control.

Table 1. Protective Effect of EGCG3″Me and Bifendate on Alcohol-Induced Toxicity in HepG2 Cell Linea group

exptl group

(I) control 1 normal control 2 EGCG3″Me control (40 μg/mL) 3 bifendate control (40 μg/mL) (II) toxin treatment 4 alcohol control (1%, v/v) (III) EGCG3″Me treatment 5 alcohol (1%, v/v) + EGCG3″Me (10 μg/mL) 6 alcohol (1%, v/v) + EGCG3″Me (20 μg/mL) 7 alcohol (1%, v/v) + EGCG3″Me (40 μg/mL) (IV) bifendate treatment 8 alcohol (1%, v/v) + bifendate (10 μg/mL) 9 alcohol (1%, v/v) + bifendate (20 μg/mL) 10 alcohol (1%, v/v) + bifendate (40 μg/mL)

cell viability (%)

AST (U/L)

ALT (U/L)

MDA (nmol/mg protein)

GSH-Px (nmol/mg protein)

99.9 ± 0.24 99.5 ± 0.19 99.8 ± 0.30

17.9 ± 0.34 17.8 ± 0.22 18.0 ± 0.18

12.0 ± 0.21 12.0 ± 0.31 12.2 ± 0.14

2.08 ± 0.11 2.14 ± 0.13 2.16 ± 0.20

32.5 ± 0.15 32.0 ± 0.19 32.1 ± 0.24

48.5 ± 0.69a

53.9 ± 0.85a

36.2 ± 1.47a

7.91 ± 0.38a

10.3 ± 0.43a

61.4 ± 1.19b 66.8 ± 1.04c 75.3 ± 1.75d

42.8 ± 1.44b 39.1 ± 0.31c 33.0 ± 0.60d

29.2 ± 1.13b 27.4 ± 0.85c 20.7 ± 1.38f

6.80 ± 0.36b 6.22 ± 0.24c 5.31 ± 0.16f

26.7 ± 0.78f 22.7 ± 1.74e 16.7 ± 1.03c

67.8 ± 1.43c 78.3 ± 2.30e 87.3 ± 1.61f

38.9 ± 0.75c 32.2 ± 0.81e 24.7 ± 1.05f

26.6 ± 1.21d 21.4 ± 0.63e 16.5 ± 1.09g

6.02 ± 0.35d 5.52 ± 0.61e 4.87 ± 0.22g

22.6 ± 1.26e 17.7 ± 0.69d 13.8 ± 0.43b

a Results are expressed as mean ± SEM (n = 5). No significant difference was noted between groups 1, 2, and 3. Data in the same column (group 4− 10) with different letters are significantly different at P < 0.05 among different treatments.

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Table 3 shows the effects of EGCG3″Me pretreatment on alcohol-induced alteration in serum enzyme activities of AST and ALT and hepatic SOD, GSH-Px, and MDA levels in mice. Apparently, significant increases in AST and ALT activities were observed in sera of the alcohol model group when compared with the normal control group, indicating that the alcohol-induced hepatotoxicity in mice was well-established. The elevated AST and ALT levels were reduced significantly in a concentration-dependent manner in mice pretreated with EGCG3″Me. Compared with those in the model control group, the levels of AST and ALT decreased 38.9 and 38.7%, respectively, at a dose of 100 mg/kg BW per day of EGCG3″Me. There was also a significant decrease in SOD and GSH-Px activities and an increase in MDA level in the alcohol model group when compared with the normal control group. As shown in Table 3, the administration of EGCG3″Me significantly enhanced SOD and GSH-Px activities but decreased MDA levels when compared with the model control group. At a high dose of EGCG3″Me, SOD and GSH-Px activities in the liver were restored by 5.67 and 32.4% in alcohol-induced liver injury mice. However, there was no significant difference of SOD activity for mice pretreated by EGCG3″Me at a medium or high dose. The level of MDA was significantly reduced in a concentration-dependent manner in the mice pretreated with EGCG3″Me. At a dose of 100 mg/kg BW per day of EGCG3″Me, the level of MDA in mice was 0.77 ± 0.03 nmol/mg protein, which was 32.5% lower than that of the alcohol model group. The effects of EGCG3″Me and bifendate on liver histopathology of alcohol-treated mice are presented in Figure 5. In the normal control group (Figure 5I), the liver sections showed normal hepatic cells with well-preserved cytoplasm, prominent nucleus, and clear central vein (CV), whereas in the model control group, massive inflammation, infiltration, and many ballooned hepatocytes (blue arrow) were observed around the CV (Figure 5II). For the positive control group, bifendate (10 mg/kg) exhibited a significant protective effect evidenced by showing a clear CV and similar liver tissue compared with the normal group (Figure 5III). In addition, similar protection from alcohol-induced inflammation and infiltration in liver tissues was observed by the pretreatment with EGCG3″Me in a dose-dependent manner (Figure 5IV− VI). In Figure 5IV, a moderate inflammation area with color changes around the CV and fewer ballooned hepatocytes were observed; in Figure 5V, less inflammation and infiltration around CV and trace ballooned hepatocytes were presented, whereas Figure 5IV shows a clear CV and similar tissue structure when compared with the normal control group.

when compared with all concentrations of EGCG3″Me. Increased release of intracellular enzymes AST and ALT was observed at >3 h exposure to alcohol. This indicated membrane damage and instability owing to oxidative injury created by alcohol. Likewise, alcohol caused an increase in the level of MDA with a concurrent decrease in GSH-Px content in HepG2 cells. The alcohol-induced changes in the HepG2 cells were ameliorated by cotreatment of the toxin along with both EGCG3″Me and bifendate. It was observed that when treated with different concentrations (10, 20, and 40 μg/mL) of EGCG3″Me, the cells showed a significant restoration of the altered biochemical parameters toward normal compared with the toxin-treated group in a concentration-dependent manner. Effects of EGCG3″Me pretreatment on alcohol-induced alteration in body weight and relative organ weight in mice are shown in Table 2. It can be seen that body weights of mice Table 2. Effects of EGCG3″Me Pretreatment on AlcoholInduced Alteration in Body Weight and Relative Organ Weight in Micea relative weight (g/g body weight, %) group

body weight (g)

I II III IV V VI

35.3 34.4 34.6 35.2 36.3 35.1

± ± ± ± ± ±

liver

2.04 1.28 1.39 2.23 1.87 2.08

4.38 5.62 4.39 5.47 4.45 4.58

± ± ± ± ± ±

0.20a 0.33e 0.34a 0.27d 0.16b 0.19c

spleen 0.24 0.37 0.27 0.34 0.26 0.27

± ± ± ± ± ±

0.02a 0.04d 0.03b 0.03c 0.02b 0.02b

thymus 0.21 0.28 0.20 0.27 0.21 0.22

± ± ± ± ± ±

0.02a 0.01c 0.01a 0.02c 0.02b 0.02b

a

Groups I, II, and III represent the normal control, model control, and positive control, respectively. Groups IV, V, and VI represent the groups pretreated with EGCG3″Me at doses of 25, 50, and 100 mg/kg BW per day, respectively. Each value is presented as the mean ± SD (n = 8). Different lower case letters indicate significant difference from the model group at the level of P < 0.05.

were not affected by the treatment of alcohol, bifendate, or EGCG3″Me, for no differences were observed for body weights among different groups. However, significant increases of the relative weights of liver, spleen, and thymus were observed for the mice in the alcohol control group, indicating that alcohol induced hypertrophy of these organ tissues in mice. On the contrary, the pretreatment of bifendate showed a remarkable decrease in elevated relative organ weights when compared with the model control group. Moreover, the administration of EGCG3″Me significantly reduced the relative weight of liver and spleen and, notably, a similar effect was observed for the relative weight of thymus in mice pretreated with EGCG3″Me at a medium or high dose.

Table 3. Effects of EGCG3″Me Pretreatment on Alcohol-Induced Alteration in Serum Enzyme Activities of AST and ALT and Hepatic SOD, GSH-Px, and MDA Levels in Micea group I II III IV V VI

AST (U/L) 28.6 64.6 33.8 51.6 47.6 39.6

± ± ± ± ± ±

2.72a 3.17f 2.44b 1.98e 2.72d 2.72c

ALT (U/L) 19.9 97.6 53.5 82.5 67.1 59.6

± ± ± ± ± ±

1.43a 3.78f 3.18b 2.71e 3.44d 3.02c

SOD (U/mg) 275 247 268 255 259 261

± ± ± ± ± ±

11.4a 20.1e 10.4b 21.8d 10.4c 9.98c

GSH-Px (nmol/mg protein) 10.3 6.12 8.89 6.76 6.99 8.10

± ± ± ± ± ±

0.26a 0.17f 0.22b 0.09e 0.05d 0.03c

MDA (nmol/mg protein) 0.58 1.14 0.78 1.02 0.88 0.77

± ± ± ± ± ±

0.03a 0.08e 0.03b 0.03d 0.04c 0.03b

a

Groups I, II, and III represent the normal control, model control, and positive control, respectively. Groups IV, V, and VI represent the group pretreated with EGCG3″Me at doses of 25, 50, and 100 mg/kg BW per day, respectively. Each value is presented as the mean ± SD (n = 8). Different lower case letters are significantly different from the model group at the level of P < 0.05. 10051



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antioxidant activity, which may also have excellent hepatoprotective ability as EGCG. Recently, the in vitro cytotoxicity and hepatoprotective activity of plant extracts have gained importance for primary level screening. HepG2 cells possess many morphological and biochemical features of normal hepatocytes. Because it retains many of the phenotypic and genotypic characteristics of liver cells, this cell line has been used in various studies related to medicinal plants for their liver-protecting property.31,32 Therefore, the present study was designed to ascertain the hepatoprotective activity of EGCG3″Me against alcohol in the HepG2 cell line. Alcohol-mediated liver injury in vitro is reported in the literature to be caused by two components, a direct solvent effect or the generation of free radicals and subsequent lipid peroxidation depending on its concentration, the duration of exposure, and the sensitivity of the in vitro system to which it is added.33,34 During alcohol exposure, superoxide radical is believed to play a central role in alcohol-induced liver injury. It can be produced readily by numerous processes and reacts through catalytic pathways in cells to form potent oxidants, including hydroxyl radical, hydrogen peroxide, and peroxynitrite.35 In this study, our results in conjunction with other reports proved that hepatotoxin caused a time-dependent production of reactive oxygen species (ROS) and subsequent lipid peroxidation in HepG2 cells, which was found to be maximum after an incubation period of 24 h.36 Therefore, HepG2 cells were incubated with alcohol for 24 h to study the protective effects of EGCG3″Me against alcohol-induced toxicity. Increased release of intracellular enzymes AST and ALT was observed at 24 h of exposure to alcohol. This indicated membrane damage and instability owing to oxidative injury created by the hepatotoxin. Likewise, alcohol treatment caused significant increase in the level of MDA, with a concurrent decrease in SOD and GSH-Px contents in HepG2 cells. When treated with different concentrations of EGCG3″Me, HepG2 cells showed a significant restoration of the altered biochemical parameters toward the normal compared with model control group. The possible underlying mechanism for the hepatoprotective effect of EGCG3″Me in a dose-dependent manner is its ability to inhibit lipid peroxidation and maintenance of GSH-Px and SOD by virtue of its antioxidative powers. Due to the nontoxic and hepatoprotective nature of EGCG3″Me, it was thought pertinent to assess its hepatoprotective potential in vivo using a mouse model. Alcohol administration in animal models has been used as a test for potential hepatoprotective agents frequently. In the present study, a remarkably increased relative organ weight was observed in the model control group compared with the normal control group (Table 2). Furthermore, we found a dramatic elevation of AST and ALT activities in the model control group when compared with other groups (Table 3). The results indicated that alcohol induced hepatotoxicity in animal organs and disorders of enzyme activities. In recent times, much evidence has demonstrated that increases of serum AST and ALT activities caused by hepatotoxin are related to hepatic structural damage because these entities are normally localized to the cytoplasm and are released into the circulation after cellular damage occurred.37,38 However, both relative organ weights and AST and ALT activities decreased dramatically in mice pretreated with bifendate when compared with those in the model control group (Table 2), and similar results have

Figure 5. Photomicrographs of liver sections stained with hematoxylin−eosin: (I) liver from a mouse in normal control group pretreated with physiological saline; (II) liver from a mouse in model control group pretreated with physiological saline followed by alcohol treatment; (III) liver from a mouse in positive control group pretreated with bifendate (10 mg/kg) followed by alcohol treatment; (IV) liver from a mouse in the group pretreated with EGCG3″Me (25 mg/kg) followed by alcohol treatment; (V) liver from a mouse in the group pretreated with EGCG3″Me (50 mg/kg) followed by alcohol treatment; (VI) liver from a mouse in the group pretreated with EGCG3″Me (100 mg/kg) followed by alcohol treatment. CV, central vein.

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DISCUSSION Oolong tea is reported to have a wide range of health benefits, and its polyphenols have been considered as promising active ingredients responsible for the effects. However, previous studies have not connected the hepatoprotective effects to the phenolic constituents of oolong tea. In this study, we for the first time explored the hepatoprotective efficacy of EGCG3″Me from Chinese oolong tea. EGCG3″Me is generally prepared from limited oolong tea or green tea by extraction with water and purification with HP-20 column chromatography and preparative HPLC.26 In the present study, EGCG3″Me of high purity was successfully prepared from Chinese oolong tea through water extraction and simple purification procedures of polyamide and Toyopearl HW-40S column chromatography. Obviously, it is a simple and efficient procedure for the preparation of EGCG3″Me compared with reported methods. In our study, we demonstrated that the in vitro antioxidant activity of EGCG is greater than that of EGCG3″Me. EGCG is the main component of tea polyphenols, which has been shown to have significant biological activity. However, for the low bioavailability, EGCG is rapidly metabolized to a variety of derivative products after absorption, including glycosylation, sulfation, and methylated derivatives.28 These derivatives are likely to play an important role in vivo and show higher biological activity.29,30 Currently, the function, mechanism, and biotransformation of these derivatives in vivo are still to be investigated and clarified. In this test, EGCG3″Me showed high 10052



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that EGCG3″Me effectively alleviated the changes induced by alcohol in a concentration-dependent manner. On the basis of the in vivo alcohol-induced liver injury model, we demonstrated that the administration of EGCG3″Me significantly decreased AST and ALT levels in the serum and the liver level of MDA and remarkably restored the liver activities of SOD and GSHPx in alcohol-induced liver injury mice. Accordingly, this suggested that EGCG3″Me had a significant hepatoprotective effect on alcohol-induced liver injury possibly via its antioxidant activity to protect biological systems against oxidative stress. These results provided evidence that EGCG3″Me might be an important active substance responsible for the hepatoprotective effect of Chinese oolong tea; therefore, EGCG3″Me could be developed as a potential functional food ingredient for liver injury and the maintenance of human health.

been obtained by the pretreatment of EGCG3″Me. The results suggested that EGCG3″Me might have a protective effect against alcohol-induced liver injury in mice. The accumulation of alcohol-induced radicals leads to a free radical mediated lipid peroxidation and cell necrosis, which causes liver injury, and the level of MDA provides an indirect index of lipid peroxidation. In the present study, the increase in MDA levels suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent formation of excessive free radicals. In our study, the pretreatment of EGCG3″Me inhibited alcohol-induced hepatotoxicity effectively by reducing the formation of MDA in the liver. At a dose of 100 mg/kg BW per day of EGCG3″Me, the MDA level reached the same level as the positive control group. This suggested that EGCG3″Me might have a hepatoprotective effect by decreasing oxidative stress in the liver of alcoholinduced mice. These results are also in good agreement with histopathological results. As shown in Figure 5, marked and widespread inflammation and infiltration areas were depicted in the model control group, whereas those of the bifendate treatment group and EGCG3″Me treatment group with a high dose reversed alcohol-induced liver injury, indicating that EGCG3″Me was effective against alcohol-induced hepatotoxic effect. In the model control group, SOD and GSH-Px activities were very low, whereas the mice of the positive control group showed higher activities when compared with the model control group. Notably, the activities of SOD and GSH-Px were also elevated markedly by the pretreatment of EGCG3″Me, especially at a dose of 100 mg/kg BW per day. It has been reported that the decrease in the activity of SOD, the increase in the lipid peroxidation, and the breakdown of the GSHdependent antioxidant defense system are obviously seen along with the liver damage induced by hepatotoxin.39 Oxidative stress has been considered as a major molecular mechanism involved in alcohol toxicity; in our study, EGCG3″Me showed excellent antioxidant and hepatoprotective activities in a dosedependent manner, and the present results were in good agreement with other results.37−39 According to the results stated above, it is believed that the hepatoprotective effect of polyphenol is related to its antioxidant activity. Cui et al. reported the protective effects of polyphenol-enriched extract from green tea, including epigallocatechin, rutin, and epicatechin, against CCl4-induced liver injury in mice, and emphasized the importance of a high content of polyphenols for guarding intact endogenous cellular antioxidant defense systems.40 The most abundant tea catechin, EGCG, has also been shown to exhibit hepatoprotective effects against liver injury by ethanol and carbon tetrachloride.14,15 Although EGCG has also been shown to exhibit hepatoprotective effects against liver injury, some studies demonstrated that high doses of EGCG can lead to mild liver injury.41 During digestion and transfer across the small intestine, and in the liver, EGCG is rapidly metabolized by phase II enzymes to various O-sulfated, O-glucuronidated, and O-methylated forms, which may play an important role in vivo.28 In the present study, the antioxidant and hepatoprotective effects of EGCG3″Me from Chinese oolong tea were confirmed. In conclusion, high-purity EGCG3″Me was successfully prepared from Chinese oolong tea by extraction with hot water and simple purification procedures of polyamide and Toyopearl HW-40S column chromatography. In an in vitro antioxidant study, EGCG3″Me exhibited potential antioxidant activity. For hepatoprotective activity in vitro, it was observed

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AUTHOR INFORMATION

Corresponding Author

*(X.Z.) Phone: +86-574-87609573. Fax: +86-574-87608347. Email: [email protected]. Funding

This work is sponsored by K. C. Wong Magna Fund in Ningbo University. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED EGCG, (−)-epigallocatechin gallate; EGCG3″Me, (−)-epigallocatechin 3-O-(3-O-methyl) gallate; GCG, (−)-gallocatechin gallate; ALD, alcoholic liver disease; CCl4, carbon tetrachloride; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2′-azinobis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salts; TPTZ, 2,4,6-tri(2-pyridyl)-s-triazine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; FBS, fetal bovine serum; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde; HPLC-DAD, high-performance liquid chromatography with diode array detector; ESI-MS/MS, electrospray ionization tandem mass spectrometry; ESI-TOF-MS, electrospray ionization time-of-flight mass spectrometry; NMR, nuclear magnetic resonance; CTC50, 50% cytotoxic concentration; SDS, sodium dodecyl sulfate; OD, optical density or absorbance; BW, body weight; H&E, hematoxylin−eosin; SD, standard deviation; SEM, standard error of the mean; CV, central vein

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Antioxidant and Hepatoprotective Effect of (â)-Epigallocatechin 3-O-(3

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