Original papers
Protective Effects of Methanol Extract of Perilla Seed Meal against Oxidative Stress in HepG2 Cells
2018 Volume 24 Issue 4 Pages 583-590
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2018 Volume 24 Issue 4 Pages 583-590
This study aimed to evaluate the protective effect of methanol extract of perilla seed meal (PSE) against oxidative stress induced by tert-butylhydroperoxide (t-BHP) in HepG2 cells. The total phenolics and flavonoid contents of PSE were 41.36 ± 1.00 mg gallic acid equivalents/g of residue and 38.20 ± 0.17 mg catechin equivalents/g of residue, respectively. Quantitative analysis of the major constituent phenolic acids and tocopherols was achieved by using high-performance liquid chromatography. PSE (10–100 µg/mL) pretreatment significantly prevented t-BHP-induced cytotoxicity in HepG2 cells. Oxidative damage induced by t-BHP was accompanied by glutathione depletion, ROS generation, and lipid peroxidation, which were all significantly ameliorated by PSE. The cytoprotective effect of PSE was attributed to the modulation of cellular antioxidant defense enzymes such as glutathione peroxidase, superoxide dismutase, glutathione reductase, and catalase. Collectively, the bioactive compounds of PSE were able to prevent oxidative stress through the enhancement of the antioxidant indices.
The involvement of oxidative stress in the etiology of many pathologies, including cancer, inflammation and age-related diseases, is well established (Birben et al., 2012). Under normal conditions, the cellular levels of reactive oxygen species (ROS) are sufficiently low for them to be efficiently removed by defense systems (Alia et al., 2005). However, an imbalance in cellular pro-oxidant/antioxidant homeostasis can induce an excessive ROS production, which causes oxidative stress, and ultimately leads to cell death (Kehrer, 1993). Dietary antioxidants protect against oxidative stress through the modulation of protective enzymatic and non-enzymatic antioxidative biomolecules, which maintain intracellular redox homeostasis (Kim et al., 2013; Choi et al., 2010). Non-enzymatic antioxidants, which include glutathione (GSH), polyphenols, flavonoids, and tocopherols, interrupt free radical chain reactions (Nimse and Pal, 2015). The enzymatic antioxidant system is the main defense system against ROS and comprises glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT) (Martin et al., 2010). Accumulating evidence has indicated that many bioactive food components with antioxidant properties could counteract oxidative stress-related chronic diseases (Lobo et al., 2010).
Perilla (Perilla frutescens Linn, Britton), belonging to the family Labiatae, has been commonly used as an edible and medical plant in Asian countries including Korea, China, and Japan. Perilla seeds are used not only as a food flavoring, but also as a rich source of nutrients (Asif, 2012). Perilla seed oils contain a high content of polyunsaturated fatty acids (60 % α-linolenic acid, 15 % linoleic acid, and 15 % oleic acid), which provide various health benefits, such as a reduction in plasma lipid level (Chang et al., 2009; Shin and Kim, 1994). Perilla seed meal is also an important agricultural byproduct and generated in abundance during the process of perilla seed oil extraction (Zhu and Fu, 2012). Despite the abundance of bioactive compounds, such as phytic acid, and phenolic compounds in perilla seed meal, this material is mainly used as a protein source for animal feed (Tang et al., 2014). Although previous studies have predominantly focused on the separation and purification of proteins from perilla seed meal, neither the antioxidant activity nor the bioactive compounds of perilla seed have been reported (Zhu and Fu, 2012). Therefore, the present study evaluated the efficacy of a methanol extract of perilla seed meal (PSE) in the protection of human hepatoma HepG2 cells from tert-butylhydroperoxide (t-BHP)-induced oxidative damage. In addition, the antioxidant activities of perilla seed meal were compared with those of the equivalent mixture of bioactive compounds found in perilla seed meal to examine t-BHP-induced hepatotoxicity in HepG2 cells.
Materials and chemicals Rosmarinic acid, caffeic acid, tocopherol standards, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), thiobarbituric acid (TBA), 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), trichloroacetic acid, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), oxidized glutathione (GSSG), GR, β-nicotinamide adenine dinucleotide phosphate (NADPH), GPx, peroxidase, t-BHP, GSH, xanthine, xanthine oxidase, H2O2, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were obtained from Sigma- Aldrich. Dulbecco's phosphate-buffered saline (PBS), penicillin-streptomycin, ethylenediaminetetraacetic acid (EDTA), and Dulbecco's modified Eagle's medium (DMEM), were purchased from Gibco (Gaithersburg, MD, USA).
Preparation of methanol extract from perilla seed meal Perilla seed meal was obtained from a local market in Cheongju area, Korea. Perilla seed meal (10 g) was extracted with HPLC grade methanol (300 mL) by shaking 24 h at 25 °C. After shaking, the supernatant was filtered, and then, evaporated under vacuum at 35 °C. The perilla seed meal gave a yield of 10.13 %. The dried matters were redissolved in DMSO and the samples were stored at −10 °C.
Determination of total flavonoid and phenolic contents The total phenolic content of PSE was assayed by using Folin- Ciocalteu (FC) phenol reagent (Choi et al., 2007). The total flavonoid content was assayed by using the aluminum chloride colorimetric method (Sung and Lee, 2010).
Determination of phenolic acids in perilla seed meals Rosmarinic acid and caffeic acid were analyzed using an HPLC system equipped with a PU-2089 pump, an AS-2057 auto injector, and an MD-2010 UV-vis variable wavelength detector (JASCO Corp., Tokyo, Japan). The separation was carried on a C18 column (250 × 4.6 mm, 5 µm i.d., Shisheido, Tokyo, Japan) with mobile phases of 0.05 % trifluoroacetic acid in water (v/v) (eluent A) and methanol (eluent B). The flow rate was 1.0 mL/min and the sample injection volume was 20 µL. The gradient program was optimized to the following programs: 0 min, 50 % A; 35 min, 50 % A; 36 min, 0 % A; 40 min, 0 % A; 41 min, 50 % A; and 43 min, 50 % A. The elution was monitored at 340 nm. The chromatographic peaks of rosmarinic acid and caffeic acid were confirmed through the comparison of their UV spectra and retention times with those of the standards.
Tocopherols were analyzed by using an HPLC system equipped with a PU-2089 pump, an AS-2057 auto injector, and an FP-2020 fluorescence detector (JASCO Corp.). The separation was carried on a LiChrosphere® column (250 × 4 mm, 5 µm i.d., Merck, Berlin, Germany) with isocratic elution in a mobile phase of hexane/isopropanol (98.7:1.3, v/v). An excitation wavelength of 290 nm and an emission wavelength of 330 nm were used to detect peaks.
Cell culture Human hepatoma HepG2 cells were purchased from ATCC (Rockville, MD, USA) and grown in DMEM supplemented with 10 % heat-inactivated FBS, 50 µg/mL streptomycin, and 100 units/mL penicillin in a humidified atmosphere with 5% CO2 at 37 °C.
HepG2 cells were seeded onto 6-well plates at a density of 1.0 × 106 cells/well or 96-well plates at a density of 1.5 × 104 cells/well. After incubation for 24 h, the cells were treated with different concentrations of PSE (10, 25, 50 and 100 µg/mL) for 12 h and then the culture medium was removed. Oxidative stress was induced through the treatment of cells with 500 µM t-BHP for a further 2 h. The protective effect of PSE was evaluated by using the MTT assay. To determine the levels of reduced GSH, the cells were harvested and the cell lysates were collected. To determine malondialdehyde (MDA) levels and antioxidant enzyme activities, the cells were lysed for 20 s by using a sonicator, thereafter, the lysates were centrifuged at 9,000 g for 15 min at 5 °C and the supernatant was collected.
Cell viability and intracellular reactive oxygen species (ROS) Cell viability was evlauated by the MTT colorimetric assay. After treatment at the specified conditions, MTT reagent (0.5 mg/mL) was added and the cells were incubated for 4 h at 37 °C in an atmosphere of 5% CO2. The culture medium was then discarded and DMSO was used to dissolve the intracellular blue formazan product. The product was measured at 550 nm. Intracellular ROS levels were determined as previously described (Wang and Joseph, 1999).
Determination of lipid peroxidation and reduced GSH Lipid peroxidation was quantified through the measurement of the amount of intracellular MDA by using the TBA-reactive substance measurement assay (Buege and Aust, 1978). Briefly, the cell supernatant was added to an equal volume of thiobarbituric acid solution (0.375 % thiobarbituric acid, 15 % trichloroacetic acid, and 0.25 N HCl) and heated for 15 min in a boiling water bath. After centrifugation at 10,000 rpm for 5 min, the absorbance of the supernatants was measured at 535 nm using a spectrophotometer.
Total GSH was measured by the previously described method (Baker et al., 1990) with some modification. Briefly, the cell lysate was added to 5 % sulfosalicyclic acid for deproteinization and centrifuged at 10,000 g for 15 min at 5 °C. Twenty microliters of the supernatant was added to 180 µL of the reaction mixture containing 2.5 mM NADPH, 3 mM 5,5′-dithiobis (2-nitrobenzoic acid), and 400 units/mL GR. The absorbance at 412 nm was recorded every 10 s for 10 min by using a spectrophotometer. The concentration of GSH in the cell lysate was calculated using a standard curve. The results were expressed as a percentage of the results obtained with t-BHP untreated control cells.
Determination of antioxidant enzyme activities GR activity was measured by the decrease in absorbance that results from the oxidation of NADPH during the reduction of oxidized GSH. The GPx assay is based on the oxidation of GSH by GPx using t-BHP as a substrate, coupled to the disappearance of NADPH by GR (Günzler et al., 1974). CAT activity was determined by the concentration of H2O2 remaining after CAT action present in the sample (Fossati et al., 1980). SOD activity was determined by using WST-1 (Ukeda et al., 1999).
Statistical analysis The results are expressed as the mean ± standard deviation or standard error (SE) and were representative of at least three independent experiments. Statistical analysis of data was performed by Tukey's multiple comparisons test with GraphPad Prism 5.0 software (GraphPad Software, Inc. La Jolla, CA, USA). A p-value of <0.05 was considered significant.
Contents of bioactive compounds It had been reported that the antioxidant activity of plant materials was well correlated with the content of their bioactive compound such as polyphenols, flavonoids, and tocopherols (Choi et al., 2007). Thus, it is important to consider the effect of these bioactive compounds on the antioxidant activity of plant-based foods. Dietary polyphenols constitute one of the most numerous and widely-distributed groups of phytochemicals in the plant kingdoms (Tsao, 2010). They can be classified into different groups as a function of the number of phenolic rings that they contain and of the structural elements that bind these rings to one another. The main classes include phenolic acids and flavonoids, which are important antioxidants that act as chain-breakers in the free radical chain reaction and convert lipid radicals into more stable products (Pandey and Rizvi, 2009; Kornsteine et al., 2006). In the present study, the contents of the different types of bioactive compounds in PSE are presented in Table 1. The total phenolic and flavonoid contents of PSE were 41.36 ± 1.00 mg gallic acid equivalents (GAE)/g of residue and 38.20 ± 0.17 mg catechin equivalent (CE)/g of residue, respectively. Even though FC reagent has been used for the estimation of total phenolic content in a variety of plant extracts, the reducing agents such as tocopherol can interfere with the analysis and thus overestimate the content of phenolic compounds. In this respect, quantitative analyses of the major phenolic acids and tocopherols were performed using HPLC and the results are presented in Table 1 and Fig. 1A. Two major peaks were detected in the HPLC profile of PSE, which were attributable to rosmarinic acid and caffeic acid. Rosmarinic acid was the major phenolic acid (15.02 ± 0.51 mg/g of residue), followed by caffeic acid (70.60 ± 14.00 µg/g of residue), together, they accounted for more than 36 % of the total phenolics present in PSE. These results are in agreement with those previously reported by Lee et al. (2013), who suggested that rosmarinic acid was the major compound and constituted approximately 48 % of the total phenolic content in the 80 % methanol extract of perilla seeds. The phenolic acid content reported in our study was lower than that previously reported, the content of phenolic compounds in agricultural crops is dependent on many factors, such as climatic conditions, agricultural practices, environmental factors during growth, and place of growth (Lachowicz et al., 2017).
| Perilla seed meal extract | |
|---|---|
| Total phenolic contents (mg GAE/g residue) | 41.36 ± 1.00 |
| Total flavonoid content (mg CE/g residue) | 38.20 ± 0.17 |
| Phenolic compound | |
| Rosmarinic acid (mg/g residue) | 15.02 ± 0.51 |
| Caffeic acid (µg/g residue) | 70.60 ± 14.00 |
| Tocopherols | |
| α-tocopherol (µg/g residue) | 5.05 ± 0.08 |
| γ-tocopherol (µg/g residue) | 98.36 ± 0.09 |
| δ-tocopherol (µg/g residue) | 7.34 ± 0.10 |
Each value represents the mean ± standard deviation (n = 3).
Chromatographic profiles of the methanol extract of perilla seed meal. (A) Peaks of the caffeic acid and rosmarinic acid standards were detected by the HPLC system (B) Peaks of the α-tocopherol, α-tocopherol and δ-tocopherol standards were detected by the HPLC system.
The concentration and chromatographic profiles of the tocopherols in PSE are shown in Table 1 and Fig. 1B, respectively. The most abundant isomer was γ-tocopherol (98.36 ± 0.09 µg/g of residue), which accounted for approximately 89 % of the total tocopherol content, followed by δ-tocopherol (7.34 ± 0.10 µg/g of residue) and α-tocopherol (5.05 ± 0.08 µg/g of residue), which was consistent with previous reports that the predominant tocopherol in perilla seed was γ-tocopherol (Shin and Kim, 1994). α-Tocopherol is believed to be the main contributor to vitamin E activity, whereas γ-tocopherol is considered the most efficient for the prevention of food autooxidation, with a higher antioxidant capacity than α-tocopherol in some model system (Wagner et al., 2004). Therefore, perilla seed peel may be considered an excellent source of natural antioxidants, such as phenolics, flavonoids, and tocopherols.
Protective effect of PSE against t-BHP-induced cytotoxicity in HepG2 cells Human hepatoma HepG2 cells have been widely used in many studies as a human hepatic model to evaluate the biological activity of dietary compounds (Martin et al., 2010; Alia et al., 2006). In this study, the cytotoxicity of HepG2 cells in the presence of PSE was investigated. The treatment of PSE at 10, 25, 50 and 100 µg/mL for 24 h did not affect the cytotoxicity of HepG2 cells (Fig. 2A). t-BHP is an organic peroxide that is used to induce oxidative stress in a variety of cells (Martin et al., 2001). The treatment of HepG2 cells with 500 µM t-BHP for 2 h caused a significant decrease (aproximately 50 %) in cell viability. However, pre-treatment with PSE for 12 h significantly decreased the t-BHP-induced reduction of cell viability (Fig. 2B). These results indicated that PSE treatment largely protected against t-BHP-induced cytotoxicity in HepG2 cells.
Effect of methanol extract of perilla seed meal (PSE) on cell viability of HepG2 cells exposed to t-BHP-induced oxidative stress. (A) Cells were treated with various concentration of PSE for 24 h. (B) Cells were pretreated with PSE for 12 h before treatment with 500 µM t-BHP. #P < 0.05, significant difference compared with the control cells, *P < 0.05, significant difference compared with the t-BHP-treated group. NS: not significant.
Protective effect of PSE against t-BHP-induced cellular oxidative stress in HepG2 cells In hepatocytes, exposure to t-BHP results in excessive ROS generation and depletion of the antioxidant system. Excessive ROS generation causes oxidative stress and initiates the disruption of biomolecules such as lipids, proteins, and DNA, which leads to the loss of cellular functions (Davies, 1989); consequently, the direct evaluation of ROS levels provides a good indication of the extent of oxidative damage to living cells (Wang and Joseph, 1999). In the present study, HepG2 cells treated with 500 µM t-BHP showed a significant increase in ROS generation compared with control cells. However, pretreatment with PSE significantly ameliorated the increased ROS generation and protected cells in a dose-dependent manner (Fig. 3A).
Effect of methanol extract of perilla seed meal (PSE) on ROS production, GSH depletion, and lipid peroxidation against t-BHP-induced oxidative stress in HepG2 cells. The cells were pretreated with PSE for 12 h before treatment with 500 µM t-BHP. (A) Cellular ROS production was determined by the fluorescence analysis of DCFH-DA. (B) Cellular GSH levels were determined by the DTNB-GSSG reductase recycling assay. (C) Cellular lipid peroxidation was determined by the MDA levels. #P < 0.05, significant difference compared with the control cells, *P < 0.05, significant difference compared with the t-BHP-treated group.
During oxidative stress, fatty acids in cell membranes damaged by ROS form lipid hydroperoxides and, in turn, readily decompose to secondary products (Li et al., 2013). Among these products, elevated levels of MDA have been found in various diseases and are thought to be related to free radical reactivity; consequently, the compound has been widely used as an index for oxidative stress (Suttnar et al., 2001). In this study, HepG2 cells treated with t-BHP for 2 h showed a significant increase in MDA level, indicating lipid oxidation. Again, pre-treatment with PSE for 12 h markedly prevented the lipid oxidation caused by t-BHP (Fig. 3B).
Cells naturally contain an extensive array of non-enzymatic and enzymatic antioxidants that counteract potentially hazardous oxidizing agents (Cuello et al., 2007). GSH, the main intracellular non-enzymatic antioxidant defense system, plays a key role in the protection against oxidative damage as a substrate in the GPx-catalyzed detoxification of injurious peroxides to maintain the redox balance of cells (Martin et al., 2010). It is usually assumed that GSH depletion reflects oxidative stress in cells, whereas a balanced GSH concentration could be expected to protect cells against oxidative damage (Alia et al., 2006). In this study, 500 µM t-BHP induced a marked decrease of cytoplasmic GSH, but this decrease was prevented by a 12 h pretreatment with PSE (Fig. 3C). These results clearly showed that PSE reduced intracellular GSH depletion, lipid peroxidation and ROS generation, which supports the maintenance of the normal redox status of cells and ameliorates oxidative damage.
Regulation of antioxidant enzyme activities by PSE Cellular antioxidant enzymes, including GR, GPx, CAT, and SOD, play key roles in the defense system against oxidative damage; the changes in the activities of antioxidant enzymes in response to oxidative stress can be considered as index of the antioxidant response (Martin et al., 2010). SOD catalyzes the conversion of superoxide radicals into hydrogen peroxide and oxygen and CAT is responsible for the reduction in hydrogen peroxide produced from the metabolism of fatty acids in peroxisomes (Wu et al., 2011). In the GSH system, the detoxification of ROS and hydroperoxides is indicative of the oxidation of GSH to GSSG by GPx. GSSG is then reduced to GSH by GR, which oxidizes NADPH to NADP+ and causes the subsequent depletion of the GSH pool (Kretzschmar, 1996). To determine whether PSE prevented t-BHP-induced damage through the modification of antioxidant enzyme activity, HepG2 cells were pretreated with 10–100 µg/mL PSE for 12 h before the addition of t-BHP to induce oxidative stress. HepG2 cells treated with t-BHP for 2 h showed a significant increase in the enzyme activity of GR, GPx, SOD, and CAT (Table 2). However, pre-treatment of cells with PSE for 12 h almost completely prevented the increases induced by t-BHP These results imply that the cytoprotective effect of PSE against t-BHP-induced oxidative stress in HepG2 cells may be attributable to the modulation of cellular antioxidant defense enzymes.
| Treatment | GR | GPx | CAT | SOD |
|---|---|---|---|---|
| Control | 1.57 ± 0.36 | 78.46 ± 2.59 | 52.63 ± 3.78 | 7.50 ± 0.10 |
| t-BHP | 4.07 ± 0.97### | 176.35 ± 9.66### | 73.78 ± 6.97### | 17.38 ± 2.27### |
| t-BHP + PSE 10 µg/mL | 2.52 ± 0.15* | 138.83 ± 11.17*** | 71.81 ± 8.37*** | 16.02 ± 1.41 |
| t-BHP + PSE 25 µg/mL | 2.43 ± 0.50* | 131.57 ± 2.66*** | 61.67 ± 4.67*** | 11.46 ± 1.77** |
| t-BHP + PSE 50 µg/mL | 1.87 ± 0.10** | 137.00 ± 5.98*** | 57.53 ± 5.24*** | 9.72 ± 0.88*** |
| t-BHP + PSE 100 µg/mL | 1.59 ± 0.14*** | 110.99 ± 9.14*** | 54.79 ± 5.63*** | 8.85 ± 0.93*** |
Antioxidant enzyme activities of glutathione reductase (GR; nmol/min/mg of protein), glutathione peroxidase (GPx; nmol/min/mg of protein), catalase (CAT; µmol/min/mg of protein), and superoxide dismutase (SOD; units/min/mg of protein) were evaluated in HepG2 cells treated for 2 h with 500 µM tBHP. Data are the mean ± SE values (n = 3). Different letters in the same column indicate a significant difference by Duncan's test (P < 0.05). #P < 0.05, significant difference compared with the control cells, *P < 0.05, significant difference compared with the t-BHP-treated group.
Protective effect of PSE phytochemicals against t-BHP-induced cellular oxidative stress in HepG2 cells To examine whether the observed protective effects of PSE were attributable to its constituent phytochemicals, a mixture of equivalent proportions of each phytochemicals was formulated (rosmarinic acid 2.47 µg/mL, caffeic acid 0.012 µg/mL, γ-tocopherol 0.012 µg/mL, α-tocopherol 0.0008 µg/mL, and δ-tocopherol 0.0008 µg/mL) to correspond 100 µg/mL of PSE. The equivalent phytochemical mixture conferred notable protective activity against t-BHP-induced cytotoxicity in HepG2 cells (Fig. 4A). However, the magnitude of the protective effect was less than that of PSE. Among the phytochemicals tested, rosmarinic acid exerted the highest protective effect in t-BHP treated HepG2 cells (Fig. 4B). Rosmarinic acid is known to possess marked antioxidant properties as a reactive species scavenge, which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triple oxygen or decomposing peroxides (Renzulli et al. 2004). In addition, Rosmarinic acid has been reported to exert hepatoprotective effects against t-BHP induced cell damage in HepG2 cells through the prevention of intracellular GSH depletion (Lima et al., 2006). These results demonstrated that rosmarinic acid in PSE plays a crucial role in the reduction of oxidative stress in HepG2 cells. A pharmacokinetic study previously reported that the absolute bioavailability of rosmarinic acid in rats was estimated as 1.69 %, 1.28 % and 0.91 % after oral administration rosmarinic acid at the doses of 12.5, 25, and 50 mg/kg (Wang et al., 2017). In addition, a in vivo study reported that the activities of SOD, CAT, and GPx and MDA levels in the liver and kidney of mice administrated 200 mg/kg of rosmarinic acid were enhanced against aging mice (Chen et al., 2014). These studies suggest that the ingestion of around rosmarinic acid 200 mg/kg may reach approximately 1.82 µg/mL of rosmarinic acid in plasma and have antioxidant effect in physiological condition. Therefore, rosmarinic acid of PSE may also be beneficial in reducing oxidative stress-related chronic diseases. However, further studies are required to assign effect of rosmarinic acid and its metabolites in vivo. The protective effects from α-, γ-, and δ-tocopherol were not observed against t-BHP-induced cytotoxicity in HepG2 cells. These findings indicated that other components, in addition to the major phytochemicals identified in PSE, may function either independently or synergistically as cytoprotective compounds against t-BHP-induced oxidative stress.
Effect of phytochemicals in methanol extracts of perilla seed meal (PSE) on cell viability against t-BHP-induced oxidative stress in HepG2 cells. (A) Cells were treated with 100 µg/mL PSE or the equivalent phytochemical mixture for 12 h before treatment with 500 µM t-BHP. (B) The cells were pretreated with 100 µg/mL PSE or the equivalent phytochemical mixture corresponding to PSE (rosmarinic acid, 2.47 µg/mL; caffeic acid, 0.012 µg/mL; γ-tocopherol, 0.012 µg/mL; α-tocopherol, 0.0008µg/mL; or δ-tocopherol, 0.0008 µg/mL) for 12 h before treatment with 500 µM t-BHP. Cell viability was determined by the MTT assay. #P < 0.05, significant difference compared with the control cells, *P < 0.05, significant difference compared with the t-BHP-treated group. $P < 0.05, significant difference compared with the PSE group.
In conclusion, the present study demonstrated that PSE strongly protected cells against t-BHP-induced oxidative stress through the modulation of intracellular GSH depletion, lipid peroxidation, ROS generation, and the activities of antioxidant enzymes. PSE is a rich source of active antioxidant compounds, including rosmarinic acid, which may contribute to the protective effect of PSE against t-BHP-induced hepatotoxicity in HepG2 cells. The results of this study suggest that PSE may not only be a potential useful dietary phytochemical, but also provide a source of antioxidants that can be used for the development of functional foods.
Acknowledgements This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through Agri-Bio Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA)(117037-2).
