Antioxidant and hepatoprotective activities of five eggplant varieties

Food and Chemical Toxicology 48 (2010) 3017–3021

Contents lists available at ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Antioxidant and hepatoprotective activities of five eggplant varieties Pannarat Akanitapichat *, Kallayanee Phraibung, Kwunchai Nuchklang, Suparichart Prompitakkul Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand

a r t i c l e

i n f o

Article history: Received 14 April 2010 Accepted 28 July 2010

a b s t r a c t Eggplant is consumed throughout the world and varies in fruit color, shape, and size. In this study, five varieties of eggplant (purple colored moderate size, white-green colored moderate size, long green, green striped moderate size and pale-green colored small size, respectively, called SM1–SM5) were evaluated for total phenolic and flavonoid content, antioxidant activity and hepatoprotection against cytotoxicity of tert-butyl hydroperoxide (t-BuOOH) in human hepatoma cell lines, HepG2. Total phenolic content found in methanol extracts of SM1–SM5 ranged from 739.36 ± 1.59 to 1116.13 ± 7.30 gallic acid equivalents mg/100 g extract and total flavonoid content from 1991.29 ± 6.32 to 3954.20 ± 6.06 catechin equivalents mg/100 g extract. SM1 and SM2 which contained high total phenolic and flavonoid had better antioxidant activities than the other varieties. Pretreatment of HepG2 cells with 50 and 100 lg/mL of SM1–SM5 significantly increased the viability (p < 0.05) of t-BuOOH-exposed HepG2 cells by 14.49 ± 1.14% to 44.95 ± 2.72%. The antioxidant activities of the eggplant were correlated with the total amounts of phenolic and flavonoid (r = 0.53100.7961). Significant correlation was found between hepatoprotective activities and total phenolic/flavonoid content (r = 0.6371–0.8842) and antioxidant activities (r = 0.5846–0.9588), indicating the contribution of the phenolic antioxidant present in eggplant to its hepatoprotective effect on t-BuOOH-induced toxicity. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Eggplant Antioxidant Hepatoprotective

1. Introduction Production of reactive oxygen species (ROS) during normal cell metabolism is a normal and necessary process that provides important physiological functions. An imbalance between ROS production and antioxidant defenses results in oxidative stress which has been recognized as playing a prominent role in the causation of several age-related and chronic diseases such as cancer, diabetes, and neurodegenerative and cardiovascular diseases (Valko et al., 2006; Djordjevic, 2004; Willcox et al., 2004). Regarding liver diseases such as hepatocellular carcinoma, viral and alcoholic hepatitis and non-alcoholic steatosis in particular, reactive oxygen and nitrogen species play a crucial role in disease initiation and progression (Morisco et al., 2008; Nagata et al., 2007; Loguercio and Federico, 2003). Foods rich in antioxidants have been proposed as a tool to prevent and cure liver damage (Morisco et al., 2008). Eggplant, Solanum melongena L. (Solanaceae), a common vegetable grown in the subtropics and tropics, is consumed throughout the world and contains a variety of phytochemicals such as phenolics and flavonoids that provide important health benefits. Studies have shown that eggplant extract results in hypolipidemic activity in rats fed normal as well as high fat diets (Sudheesh et al., 1997), * Corresponding author. Tel.: +66 45 353630/353632; fax: +66 45 353626. E-mail addresses: (P. Akanitapichat).

[email protected],

[email protected]

0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.07.045

suppresses the formation of blood vessels required for tumor growth and metastasis (Matsubara et al., 2005), and inhibits inflammation (Han et al., 2003). The whole eggplant fruit possesses antioxidant activities and is ranked amongst the top 10 vegetables in terms of antioxidant capacity (Cao et al., 1996). Additionally, antioxidant activities and phenolic compounds are found in both the pulp and skin of eggplant (Huang et al., 2004). Extract from purple eggplant skin has been shown to possess a high capacity in the scavenging of superoxide radicals and inhibition of hydroxyl radical generation by chelating ferrous iron (Noda et al., 2000; Kaneyuki et al., 1999). Nasunin, an anthocyanin isolated from the skin of purple eggplant fruit, is one phenolic compound implicated in both inhibition of hydroxyl radical generation and superoxide scavenging activity. Eggplant is one of most common vegetables used in Thai cuisine. World-wide, the fruit of most commercial eggplant varieties is purple (Nothmann et al., 1976) and this is what was used in the studies mentioned above. In Thailand, eggplant varies in fruit color, shape, and size. Green and white fruit colors of different intensities are common for Thai eggplant. As no reports were found on studies of antioxidant and associated hepatoprotective activities of eggplant, especially in respect to different varieties, the present study aimed to: (1) determine the phenolic and flavonoid content that exist in five eggplant varieties from Thailand,

3018

P. Akanitapichat et al. / Food and Chemical Toxicology 48 (2010) 3017–3021

(2) measure the in vitro antioxidant activities, (3) determine hepatoprotective activities against the cytotoxicity of tert-butyl hydroperoxide (t-BuOOH) in human liver cells, (4) and determine correlations between antioxidant activities, hepatoprotective effects and total phenolic and flavonoid content, and between antioxidant activities and hepatoprotective effects. 2. Materials and methods 2.1. Chemicals and reagents 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,20 -azinobis(3-ethylbenzothiazoline-6sulphonic acid diammonium salt (ABTS), catechin, dimethyl sulfoxide (DMSO), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and t-BuOOH were obtained from Sigma/Aldrich (St. Louis, MO, USA). Folin–Ciocalteu reagent, glacial acetic acid, sodium hydroxide, sodium carbonate and sodium nitrite were purchased from Carlo Erba Reagenti (Milano, Italy). Gallic acid was supplied by Fluka Chemie AG (Buchs, Switzerland). Aluminium chloride was obtained from BDH (Poole, UK). The Human hepatoma cell line, HepG2 was purchased from Cell Lines Service (Eppelheim, Germany). Minimum Essential Medium Eagle (MEM), fetal bovine serum (FBS), phosphate buffer saline and trypsin were obtained from GIBCO (Grand Island, NY, USA). All other chemicals and reagents were of analytical grade. 2.2. Plant materials Five varieties of S. melongena (eggplant), arbitrarily labeled as SM1–SM5 (Fig. 1), were purchased fresh from a local market in Warinchamrab, Ubon Ratchathani, Thailand.

was recovered. The extraction was repeated with 400 mL of methanol. The two filtrates were combined and dried in a vacuum using a rotary evaporator (yield 30– 36%). All dry extracts were sealed in a glass bottle and stored at 4 °C until used.

2.4. Determination of total phenolic and flavonoid content 2.4.1. Total phenolic content Total phenolic content was determined using the Folin–Ciocalteu colorimetric method reported previously (Dewanto et al., 2002) with some modification. Appropriately diluted test extracts (200 lL) were mixed with 125 lL of Folin–Ciocalteu reagent followed by the addition of 250 lL of 7% aqueous sodium carbonate. Water was then added to adjust the final volume to 2 mL. After standing in the dark at room temperature for 40 min, the absorbance of the mixture was read at 760 nm against reagent blank using a Spectronic Genesys 10 UV scanning spectrophotometer (Milton Roy, New York, NY). Quantification was done with respect to the standard of gallic acid. The total phenolic content was expressed as gallic acid equivalents (GAE) in mg per 100 g extract.

2.4.2. Total flavonoid content Total flavonoid content was measured using the aluminium chloride colorimetric method as described by Dewanto et al. (2002) with minor modification. Briefly, 500 lL of the solanum extract or (+)-catechin standard solution was mixed with 1.25 mL of distilled water followed by the addition of 75 lL of a 5% sodium nitrite solution. After 6 min, 150 lL of a 10% aluminium chloride solution was added and allowed to stand for another 5 min before adding 500 lL of 1 M sodium hydroxide. The reaction volume was brought to 3 mL with the addition of distilled water. The absorbance was measured immediately versus the blank at 510 nm. The total flavonoid content was expressed as catechin equivalents (CE) in mg per 100 g extract from a calibration curve of catechin standard solution.

2.5. Evaluation of antioxidant activity 2.3. Preparation of plant extracts Fresh eggplant of each variety was cleaned, air dried for 2–3 h, and ground to a powder. The powdered sample (40 g) was then extracted with 400 mL of methanol for two and half days at room temperature with occasional shaking and the filtrate

All the stock eggplant extracts were prepared in DMSO at a concentration of 5 mg/mL, and were diluted in DMSO to various concentrations (150–1500 lg/mL) At least six different concentrations of the test extract were used to evaluate antioxidant activity.

Fig. 1. Different varieties of Solanum melongena (eggplant) used in this study.

3019

P. Akanitapichat et al. / Food and Chemical Toxicology 48 (2010) 3017–3021 2.5.1. DPPH radical scavenging activity Radical scavenging activity of each eggplant extract was estimated using a stable DPPH radical (DPPH) assay (Brand-Williams et al., 1995). Eight hundred microliters of DPPH solution (100 lM) was mixed with 200 lL of various concentrations (150–1500 lg/mL) of the test extract. The mixture was left to stand in the dark at room temperature for 20 min. The absorbance was measured spectrophotometrically at 515 nm. Results were expressed as % radical scavenging activity.

highest total flavonoid content. SM3, the variety with the lowest total phenolic content, also had the lowest content of total flavonoid. Among all eggplant varieties studied, significant differences in total phenolic content were found in comparisons between SM1, SM2, SM3 and SM4 (p < 0.05), and significant differences from each other were found in total flavonoid content (p < 0.05).

% Radical scavenging activity ¼ ð1  absorbancesample =absorbancecontrol Þ  100 The concentration of extract required to scavenge free radical by 50% was estimated from the graph plotting percent radical scavenging activity against final extract concentration, and defined as 50% efficient concentration (EC50). 2.5.2. ABTS radical cation scavenging activity Radical scavenging activity of each eggplant variety was measured using an improved ABTS assay (Re et al., 1999). The ABTS radical cation (ABTS+) stock solution was prepared by the reaction of 7 mM ABTS and 2.45 mM potassium persulfate and incubated for 16 h in the dark at room temperature. The ABTS+ solution was then diluted to obtain an absorbance of 0.712 ± 0.018 at 734 nm. One thousand microliters of the ABTS+ solution was reacted to 150 lL of of the test extract at varying concentrations (150–1000 lg/mL) for 6 min and the absorbance was immediately measured at 734 nm. Results were expressed as % radical scavenging activity. 2.6. Hepatoprotective effect HepG2 cells were grown in a humidified incubator containing 5% CO2 and 95% air at 37 °C. They were cultured in MEM medium supplemented with 5% (v/v) FBS, 0.1 mM non-essential amino acid, and 2 mM glutamine. Only cells in exponential growth were used for the experiments. Cells (5  104 /well) were seeded in 96-well plates and incubated at 37 °C for 24 h. The cultured medium was replaced with serum-free medium and supplemented with test extracts. Twenty hours later, cells were placed in a medium containing 300 lM t-BuOOH and incubated for 4 h. Cell viability was determined using the MTT assay (Mosmann, 1983) and results were expressed as % cytoprotection.

% Cytoprotection ¼ % viability of cells pretreated with eggplant extracts  % viability of cells treated with t  BuOOH alone

2.7. Statistical analysis All statistical analyses were performed using SPSS version 15.0 software for Windows. Results were subjected to ANOVA, and differences between means were located using Bonferroni multiple-comparison test. Correlations among data obtained were calculated using Pearson’s correlation coefficient (r). p Values 60.05 were considered statistically significant.

3. Results and discussion 3.1. Total phenolic and flavonoid content Polyphenols, the large group of phytochemicals, are known to act as antioxidants (Bors et al., 2001; Rice-Evans et al., 1997). Flavonoid is a typical phenolic that possesses antioxidant activities. Table 1 shows the total phenolic and flavonoid content of the five eggplant varieties (SM1–SM5) from Thailand. The total amounts of phenolics and flavonoids found in SM1–SM5 were in the range of 739.36 ± 1.59–1116.13 ± 7.30 mg GAE/100 g extract and 1991.29 ± 6.32–3954.20 ± 6.06 mg CE/100 g extract, respectively. SM2 had the highest total phenolic content whereas SM1 had the

3.2. Antioxidant activities Antioxidant activities in the present study were assessed by employing DPPH and ABTS assays. The DPPH and ABTS are decolorization assays which measure the relative antioxidant abilities of natural extracts to scavenge free radicals (DPPH and ABTS+ , respectively) generated in the assay system (Apak et al., 2007). Both assays are the most commonly used antioxidants methods due to excellent reproducibility under certain assay conditions. However, they show significant differences in their response to antioxidants. In the present study, scavenging activities of the DPPH and ABTS+ for all eggplant varieties studied were observed in concentration-dependent patterns (data not shown). EC50 values which are eggplant concentrations required to scavenge 50% free radical are presented in Table 1. A low EC50 translates to a higher antioxidant activity. SM2 was the most effective DPPH scavenger with the lowest EC50 value (61.44 ± 4.14 lg/mL), followed by SM1, SM4, SM3 and SM5. A statistically significant difference (p < 0.05) in antioxidant activity determined by the DPPH assay was found between SM2, SM3, and SM4. No significant difference was found between SM2 and SM1, and between SM3 and SM5 (p > 0.05). Similar to the scavenging activity of DPPH, SM5 possessed the lowest activity in scavenging ABTS+. SM1 was the strongest ABTS+ scavenger, followed by SM2, SM4, and SM3. Among all eggplant varieties studied, antioxidant activities measured by the ABTS assay were significantly different from each other (p < 0.05). 3.3. Potential hepatoprotective effect against cytoxicity of t-BuOOH in human liver cells In this study, the hepatoprotective effect was evaluated in HepG2 cells. The human hepatoma cell line HepG2 is considered a good model to study in vitro toxicity to the liver since it retains many of the specialized functions which are characteristics of normal human hepatocytes (Knasmüller et al., 1998). To select appropriate extract concentrations of SM1–SM5 to be used for cytoprotective study, non-cytotoxic concentrations were first determined. In HepG2 treatment with SM1–SM4 (10, 100 and 300 lg/mL), and SM5 ((10 and 100 lg/mL), the cell viability was P85% (data not shown), indicating that SM1–SM5 showed no cytotoxicity at 6100 lg/mL. Therefore, extract concentrations of 50 and 100 lg/mL were used to further evaluate whether SM1–SM5 were able to protect HepG2 cells against t-BuOOH-induced toxicity. tertButylhydroperoxide (t-BuOOH) is an organic hydroperoxidant that can be metabolized to free radical intermediates, which can subse-

Table 1 Total phenolic and flavonoid content, and antioxidant activities of the five different varieties of eggplant. Variety

Total phenolic content (mg GAE/100 g extract)

Total flavonoid content (mg CE/100 g extract)

SM1 SM2 SM3 SM4 SM5

1002.67 ± 8.33a 1116.13 ± 7.30b 739.36 ± 1.59c 930.70 ± 5.45d 943.64 ± 4.49d

3954.20 ± 6.06a 3146.79 ± 3.67b 1991.29 ± 6.32c 2696.17 ± 9.14d 3324.19 ± 7.29e

Antioxidant activities DPPH (EC50, lg/mL)

ABTS (EC50, lg/mL)

66.74 ± 4.60a 61.44 ± 4.14a 152.58 ± 1.68b 84.95 ± 2.33c 159.33 ± 2.87b

53.18 ± 0.71a 75.01 ± 2.43b 94.68 ± 1.98c 82.84 ± 2.16d 105.63 ± 1.90e

Values are mean (n = 3) ± SD. Values with the different superscript letter are statistically different (p < 0.05). GAE – gallic acid equivalents. CE – catechin equivalents.

3020

P. Akanitapichat et al. / Food and Chemical Toxicology 48 (2010) 3017–3021

Fig. 2. Hepatoprotective effects of five eggplant varieties against t-BOOH-induced toxicity. Percent cytoprotection is calculated as percent viability of cells pretreated with eggplant extracts – percent viability of cells treated with t-BuOOH alone (mean ± SD, n = 3). Bars with no letters in common are significantly different (p < 0.05).

quently initiate lipid peroxidation, affect cell integrity, and form covalent bonds with cellular molecules, resulting in cell injury (Rush et al., 1985). t-BuOOH-induced toxicity in HepG2 cells has increasingly been used as a model to study the cytoprotection of natural antioxidants (Lima et al., 2007; Alía et al., 2006; Kinjo et al., 2003; Thabrew et al., 1997). In the present study, the possible protection of eggplant extracts against t-BuOOH-induced loss of viability was evaluated by preincubating cells with and without eggplant extracts for 20 h, followed by treatment with the toxicant for 4 h. When t-BuOOH only was used to treat HepG2 cells, the cell viability was dose-dependently decreased (data not shown). Treatment with 300 lM t-BuOOH alone resulted in 52.33 ± 0.50% viability. As shown in Fig. 2, pretreatment with SM1–SM5 (50 and 100 lg/mL) significantly increased viability (p < 0.05) of t-BuOOH-exposed HepG2 cells by 14.49 ± 1.14–44.95 ± 2.72%. Cytoprotective activity at 50 lg/mL of each eggplant variety was weaker than that observed at 100 lg/mL. At 50 lg/mL, SM2 had the strongest cytoprotection, followed by SM1, SM4, SM3 and SM5. Similarly, at 100 lg/mL, SM2 exhibited the most potent hepatoprotective eggplant, followed by SM1, SM5, SM4 and SM3. In addition, the hepatoprotective effect of SM2 was insignificantly different from that of gallic acid at equal concentration (p > 0.05). 3.4. Relationship among phytochemical content, antioxidant activities, and hepatoprotective effects Previous studies have demonstrated the strong relationship between total phenolic content and antioxidant activities found in different varieties of crops such as guava, onion and raspberry (Thaipong et al., 2006; Yang et al., 2004; Liu et al., 2002). A recent study showed total phenolic content in 33 varieties of S. melongena (eggplant) and two varieties of S.aethiopicum were positively correlated with antioxidant activities using superoxide scavenging assay (Hanson et al., 2006). In this study, a significant correlation was observed between total phenolic content found in SM1–SM5 and the DPPH (1/EC50) antioxidant activities (r = 0.7960, p < 0.001). On the contrary, no significant correlation was found between total phenolic content and the 1/EC50 ABTS values (r = 0.4572, p > 0.05). Moreover, the total flavonoid content was significantly correlated with the 1/EC50 values of both DPPH and ABTS (r = 0.5310–0.6737, p < 0.05). These results indicate that total phenolic/flavonoid content may be at least partially responsible for the antioxidant activities of eggplant tested. Additionally, the high diversity in the

number of phenolic compounds as well as the proportions of phenolic compounds contained in individual eggplant varieties (Stommel and Whitaker, 2003) probably explains the differences in the antioxidant activities of SM1–SM5 observed in the present study. The relationship between phytochemical content and hepatoprotective activities of SM1–SM5 was studied. A strong correlation was observed between total phenolic content and the hepatoprotective activities at both 50 and 100 lg/mL (r = 0.7568–0.8842, p < 0.01). Additionally, total flavonoid content was correlated with the hepatoprotective activities of eggplant extract at only 100 lg/ mL (r = 0.6371, p < 0.05). This finding suggests that in pretreated HepG2 cells with eggplant extracts at higher concentrations, total phenolic compounds play a major role in the hepatoprotection against toxicity of t-BuOOH. However, at lower concentrations of eggplant pretreatment, other phenolic compounds, but not flavonoid, have influence on hepatoprotective activities. To relate the in vitro antioxidant activities and hepatoprotective effects of SM1–SM5, the relationship between these two activities was also investigated. Strong correlations were observed between 1/EC50 DPPH values and the hepatoprotective activities at 50 and 100 lg/mL (r = 0.8505–0.9588, p < 0.001). Similarly, the 1/EC50 ABTS values were significantly correlated with the hepatoprotective activities at both 50 lg/mL (0.7316, p < 0.01) and 100 lg/mL (0.5846, p < 0.05). The antioxidant mechanisms in biological systems include direct quenching free radicals to terminate the radical chain reaction, chelating transition metals, acting as reducing agents, or stimulating the antioxidative enzyme activities (Lima et al., 2006). The significant correlation between hepatoprotective activities and phytochemical content, and antioxidant activities of eggplant demonstrated in the present study indicate that its hepatoprotective activities are probably due to phenolic/flavonoid compounds present in eggplant in conjunction with its antioxidant activities to scavenge free radicals generated by t-BuOOH in HepG2 cells. 4. Conclusion The present study demonstrated the in vitro antioxidant activities of five eggplant varieties (SM1–SM5) and hepatoprotective effects against cytotoxicity of t-BuOOH in HepG2 cells. The total phenolic and flavonoid content was also evaluated. SM1, SM2 and SM4 with high total phenolics and flavonoids had better anti-

P. Akanitapichat et al. / Food and Chemical Toxicology 48 (2010) 3017–3021

oxidant and hepatoprotective activities than SM3. Significant correlations were found between antioxidant activities and total amounts of phenolics/flavonoids, between hepatoprotective activities and total amounts of phenolics/flavonoids, and between hepatoprotective effects and antioxidant activities.

Conflict of Interest The authors declare that there are no conflicts of interest.

References Alía, M., Ramos, S., Mateos, R., Granado-Serrano, A.B., Bravo, L., Goya, L., 2006. Quercetin protects human hepatoma HepG2 against oxidative stress induced by tert-butyl hydroperoxide. Toxicol. Appl. Pharmacol. 212, 110–118. Apak, R., Güçlü, K., Demirata, B., Özyürek, M., Çelik, S., Bektasßog˘lu, B., Berker, K., Özyurt, D., 2007. Comparative evaluation of various total antioxidant capacity assays applied to phenolic compounds with the CUPRAC assay. Molecules 12, 1496–1547. Bors, W., Michel, C., Stettmaier, K., 2001. Structure–activity relationships governing antioxidant capacities of plant polyphenols. Methods Enzymol. 335, 166–180. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT – Food Sci. Technol. 28, 25–30. Cao, G., Sofic, E., Prior, R.L., 1996. Antioxidant capacity of tea and common vegetables. J. Agric. Food Chem. 44, 3426–3431. Dewanto, V., Wu, X., Adom, K.K., Liu, R.H., 2002. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J. Agric. Food Chem. 50, 3010–3014. Djordjevic, V.B., 2004. Free radicals in cell biology. Int. Rev. Cytol. 237, 57–89. Han, S.W., Tae, J., Kim, J.A., Kim, D.K., Seo, G.S., Yun, K.J., Choi, S.C., Kim, T.H., Nah, Y.H., Lee, Y.M., 2003. The aqueous extract of Solanum melongena inhibits PAR2 agonist-induced inflammation. Clin. Chim. Acta 328, 39–44. Hanson, P.M., Yang, R.-Y., Tsou, S.C.S., Ledesma, D., Engle, L., Lee, T.-C., 2006. Diversity in eggplant (Solanum melongena) for superoxide scavenging activity, total phenolics, and ascorbic acid. J. Food Compos. Anal. 19, 594–600. Huang, H.Y., Chang, C.K., Tso, T.K., Huang, J.J., Chang, W.W., Tsai, Y.C., 2004. Antioxidant activities of various fruits and vegetables produced in Taiwan. Int. J. Food Sci. Nutr. 55, 423–429. Kaneyuki, T., Noda, Y., Traber, M.G., Mori, A., Packer, L., 1999. Superoxide anion and hydroxyl radical scavenging activities of vegetable extracts measured using electron spin resonance. Biochem. Mol. Biol. Int. 47, 979–989. Kinjo, J., Hirakawa, T., Tsuchihashi, R., Nagao, T., Okawa, M., Nohara, T., Okabe, H., 2003. Hepatoprotective constituents in plants. 14. Effects of soyasapogenol B, sophoradiol, and their glucuronides on the cytotoxicity of tert-butyl hydroperoxide to HepG2 cells. Biol. Pharm. Bull. 26, 1357–1360. Knasmüller, S., Parzefall, W., Sanyal, R., Ecker, S., Schwab, C., Uhl, M., MerschSundermann, V., Williamson, G., Hietsch, G., Langer, T., Darroudi, F., Natarajan, A.T., 1998. Use of metabolically competent human hepatoma cells for the detection of mutagens and antimutagens. Mutat. Res. 402, 185–202.

3021

Lima, C.F., Fernandes-Ferreira, M., Pereira-Wilson, C., 2006. Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels. Life Sci. 79, 2056–2068. Lima, C.F., Valentao, P.C.R., Andrade, P.B., Seabra, R.M., Fernandes-Ferreira, M., Pereira-Wilson, C., 2007. Water and methanolic extracts of Salvia officinalis protect HepG2 cells from t-BHP induced oxidative damage. Chem. Biol. Interact. 167, 107–115. Liu, M., Li, X.Q., Weber, C., Lee, C.Y., Brown, J., Liu, R.H., 2002. Antioxidant and antiproliferative activities of raspberries. J. Agric. Food Chem. 50, 2926–2930. Loguercio, C., Federico, A., 2003. Oxidative stress in viral and alcoholic hepatitis. Free Radical Biol. Med. 34, 1–10. Matsubara, K., Kaneyuki, T., Miyake, T., Mori, M., 2005. Antiangiogenic activity of nasunin, an antioxidant anthocyanin, in eggplant peels. J. Agric. Food Chem. 53, 6272–6275. Morisco, F., Vitaglione, P., Amoruso, D., Russo, B., Fogliano, V., Caporaso, N., 2008. Foods and liver health. Mol. Aspects Med. 29, 144–150. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Nagata, K., Suzuki, H., Sakaguchi, S., 2007. Common pathogenic mechanism in development progression of liver injury caused by non-alcoholic or alcoholic steatohepatitis. J. Toxicol. Sci. 32, 453–468. Noda, Y., Kneyuki, T., Igarashi, K., Mori, A., Packer, L., 2000. Antioxidant activity of nasunin, an anthocyanin in eggplant peels. Toxicology 148, 119–123. Nothmann, J., Rylski, I., Spigelman, M., 1976. Color and variations in color intensity of fruit of eggplant cultivars. Sci. Hortic. 4, 191–197. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med. 26, 1231–1237. Rice-Evans, C., Miller, N., Paganga, G., 1997. Antioxidant properties of phenolic compounds. Trends Plant Sci. 2, 152–159. Rush, G.F., Gorski, J.R., Ripple, M.G., Sowinski, J., Bugelski, P., Hewitt, W.R., 1985. Organic hydroperoxide-induced lipid peroxidation and cell death in isolated hepatocytes. Toxicol. Appl. Pharmacol. 78, 473–483. Stommel, J.R., Whitaker, B.D., 2003. Phenolic acid content and composition of eggplant fruit in a germplasm core subset. J. Am. Soc. Hortic. Sci. 128, 704–710. Sudheesh, S., Presannakumar, G., Vijayakumar, S., Vijayalakshmi, N.R., 1997. Hypolipidemic effect of flavonoids from Solanum melongena. Plant Foods Hum. Nutr. 51, 321–330. Thabrew, M.I., Hughes, R.D., McFarlane, I.G., 1997. Screening of hepatoprotective plant components using a HepG2 cell cytotoxicity assay. J. Pharm. Pharmacol. 49, 1132–1135. Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L., Hawkins Byrne, D., 2006. Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. J. Food Compos. Anal. 19, 669– 675. Valko, M., Rhodes, C.J., Moncol, J., Izakovic, M., Mazur, M., 2006. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 160, 1–40. Willcox, J.K., Ash, S.L., Catignani, G.L., 2004. Antioxidants and prevention of chronic disease. Crit. Rev. Food. Sci. Nutr. 44, 275–295. Yang, J., Meyers, K.J., van der Heide, J., Liu, R.H., 2004. Varietal differences in phenolic content and antioxidant and antiproliferative activities of onions. J. Agric. Food Chem. 52, 6787–6793.