Protective effects of mung bean (Vigna radiata L.) and pea (Pisum sativum L.) against high‐fat‐induced oxidative stress

Abstract Hyperlipidemia is closely related to oxidative stress, and it has been proved that the intake of legumes can protect the body from chronic diseases related to oxidative stress. In this study, we investigated the protective effects of mung beans and peas against high‐fat‐diet‐induced rats. It was found that, with 50% addition of mung beans or peas, the intake of mung beans and peas could significantly restore the levels of serum total cholesterol, low‐density lipoprotein cholesterol, and high‐density lipoprotein cholesterol. Liver staining also showed that high‐fat diet (HFD) led to liver lesions, whereas whole‐grain intake could significantly relieve these symptoms. Compared with the HFD group, the antioxidant defense system and antioxidant gene expression in administered legume groups improved markedly. Furthermore, the antioxidant activities of the two legume extracts were determined. Characterization showed that the ethanol extracts of mung beans and peas possessed high antioxidant activities, for their ability to scavenge ABTS and DPPH, reduce Fe3+ and their antilipid peroxidation capacity. Treatments with ethanol extracts at different doses could restore the levels of intracellular lipid, malondialdehyde, and antioxidant enzyme activities in oleic acid‐induced HepG2 cells. All these results suggested that mung beans and peas or their extracts may be utilized as good candidates of natural antioxidant agents.

body leads to excessive free radicals, which are harm to the body (Costa et al., 2015).
There are several mechanisms in human body for dietary antioxidants to protect against ROS. Increased intake of antioxidants could have the health effects such as reducing the incidence of cancer and cardiovascular diseases. Legume plays an important role in providing nutrients to people around the world (Summo, Centomani, Paradiso, Caponio, & Pasqualone, 2016). Consumption of legumes is correlated to a number of positive health benefits. These benefits are known to be associated with phytochemicals present in legumes (Magalhaes et al., 2017). Significant antioxidant activities and phenolic compounds in several consumed legumes have been reported, and a legume-based dietary can decrease the risk of oxidative stress-related chronic diseases (Liu, Liu, Chen, Chang, & Chen, 2013). Phenolic compounds including phenolic acids, flavones, isoflavones, and condensed tannins have been identified and characterized in food legumes (Cheng, 2009).
Mung bean (Vigna radiata L.) is one of the main leguminous plants rich in proteins, vitamins, and minerals (Mubarak, 2005). Mung bean is also abundant in bioactive substances (Mattila, Pihlava, & Hellstrom, 2005), such as flavonoids, alkaloids, and tannins. Flavonoids with antioxidant activity are one of the most studied compounds in recent years for their antioxidant, anticancer, antibacterial, anti-inflammatory, and hypolipidemic effects (Randhir & Shetty, 2007). Although many studies have been carried out to investigate the functional components and functions of mung beans at home and abroad in vitro, experimental data in vivo are still lacking.
Pea (Pisum sativum L.) has a nutritionally favorable composition with respect to macronutrients including low-fat-rich protein and high fiber. Moreover, peas have high contents of antioxidant components mainly include phenols, tocopherols, and carotenoids. Duenas, Estrella, and Hernandez (2004) found that peas contain a variety of phenolic compounds, especially in the seed coat. Some studies have shown that other antioxidants are present in peas, however, some of which are poorly characterized. The concentration of polyphenols in peas varied associated with the processing treatments.
Previous studies were mostly focused on the in vitro antioxidant effects of polyphenols in legumes, whereas few studies investigated the effects of intact legumes on antioxidant enzyme activity in vivo and legume extracts on oxidative stress in cells. Therefore, in this study, we assessed and compared the influence of intact mung beans and peas on the antioxidant defense system of rats induced with high-fat diet (HFD). The antioxidant components of mung bean and pea extracts were further validated in vitro or performed with HepG2 cell antioxidant experiments.

| Extract preparation
Mung beans and peas were milled into flour and thoroughly mixed, and then stored at 4°C for further analysis. Nutritional components of mung beans and peas were provided by China Agricultural University (Table 1). A portion of the processed samples were obtained for sequential extraction, and the rest was added into animal feeds.
The extraction method was carried out based on a previously described method with slight modifications (Faller, Fialho, & Liu, 2012).
Briefly, the flours were placed in a shaking water bath with n-hexane (1:4 ratio), which was used to extract lipids from the samples, at 58°C for 2 hr. After the lipids were removed, the grain flours were dried and extracted with deionized water (1:10 ratio) at 50°C for 2 hr and centrifuged at 4,500 r/min for 20 min. Supernatants were regarded as the water extracts, and the precipitates were freeze-dried. Then, the water-insoluble fractions were mixed with 80% ethanol (1:10 ratio) at 60°C for 2 hr and then centrifuged. The precipitates were discarded, and ethanol was evaporated by using a rotary evaporator.
The ethanol extracts were obtained after freeze-dried.

Analysis of serum biochemical values
TG, TC, LDL-C, and HDL-C levels in the serum were analyzed using a Multi-Mode Microplate Reader (Synergy HTX systems; Biotex) according to the manufacturer's instructions.

Assessment of SOD, MDA, GSH-Px, CAT, and T-AOC
The activities of SOD, MDA, GSH-Px, CAT, and T-AOC were tested in line with the kit's instructions. Liver homogenates were prepared in an ice bath with tissue homogenizers to determine the liver antioxidant enzyme activities, and the supernatants were obtained via centrifugation at 12,000 r/min for 10 min.

| Histopathological investigations
The livers were collected, fixed, stained with hematoxylin and eosin, and observed under a light microscope (Shen et al., 2017).

| Gene expression in the liver
Firstly, the total RNA in rat liver was first extracted by TRIzol Kit following the manufacturer's instructions. Then, the total RNA was reverse-transcribed into cDNA. Real-time polymerase chain reaction was used to quantify the expression of mRNA, and the primers are listed in Table 2.

| Antioxidant activity in vitro
Because antioxidants have various mechanisms in the oxidation-reduction process, the use of a variety of assays to determine antioxidant activity may help us in better understanding these mechanisms.

Free radical scavenging activity on DPPH
The antioxidant potential of the obtained extracts was tested by using DPPH assay as described by Shen et al. (2017) with slight modifications.

Free radical scavenging ability on ABTS
The ABTS scavenging assay was performed following the method described by Zhao et al. (2006) with some modifications.

| Reducing power
The reducing power of the extracts was determined by using the assay described by Bamdad, Wu, and Chen (2011). Phosphatebuffered saline (PBS) was used as a blank reagent.

| Antilipid peroxidation capacity
The antilipid peroxidation capacity was assessed using the procedures described by Liu and Huang (2018) with some modifications.
PBS was used as a blank reagent.

| Determination of total phenolic content
Total phenolic content (TPC) in mung bean and pea was determined by a Folin-Ciocalteu assay with slight modifications (Guo, Li, Tang, & Liu, 2012) using gallic acid as the standard. The TPC was expressed as milligram gallic acid equivalents per gram dry legume (mg GAE/g) through the calibration curve of gallic acid.

| Cell culture and treatment
The cell culture was following by Faller et al. (2012) with slight modifications. OA was dissolved in 1% bovine serum albumin, and an OAinducing medium with 1 mM was obtained. The final concentration of ethanol extracts was 100, 200, and 400 μg/ml by adding extracts and OA-inducing medium into cell culture medium as a treatment group for 12 hr.

| MTT assay experiment
Cell viability was tested in line with the MTT Cell Proliferation and Cytotoxicity Assay Kit's instructions.

| Oil Red O staining and biochemical analysis in cells
After treatment with the extracts, lipid droplet accumulation cells were measured with Oil Red O staining (Xiao et al., 2016). The stained photographs were taken with a microscope (Olympus, Japan).

| Statistical analysis
At least three independent trials were conducted in all experiments, and the data were expressed as mean ± standard deviation.
The results were analyzed for variance using the GraphPad Prism software (version 6.01; GraphPad Inc.), and statistical significance of differences (p < .05) was evaluated using Dunnett's multiple comparisons test. Table 3 shows the growth changes in experimental rats after being fed with different diets for 4 weeks. In the present study, the final body weight and liver weight of rats fed with HFD were 12.26% and 70.71% higher than those rats fed with NFD (p < .05), and there was no significant difference between legume supplementation groups and HFD group. Compared with the HFD group, the liver index of rats in NFD, HFDM-L, and HFDM-L groups decreased significantly (p < .05). In addition, there were no significant changes in the food intake of each experimental group (p > .05), indicating that different diets had no significant effect on the food intake of rats.

| Effects of legumes on serum lipid profile
As shown in Figure 1(a-d), dyslipidemia in HFD rats was observed.
Compared with the NFD group, the serum levels of TC, TG, and LDL-C in the HFD group increased markedly, and the HDL-C level decreased significantly. Compared with HFD rats, the TC levels in HFDM-L and HFDP-L rats increased by 33.57% and 23.37%, respectively. The HDL-C and TC levels of rats, fed with HFDM-H, were TA B L E 3 Effects of cooked mung bean and cooked pea supplementation for 4 weeks on body weights, liver weight, liver index, and food intake in high-fat diet rats (n = 8) 34.19% and 48.57% higher than those of HFD rats. Moreover, the LDL-C level was reduced by 32.28% (p < .05). Treatments with mung beans and peas revealed significant decline in serum lipid profile in a dose-dependent manner. With a higher dose, the levels of TC and LDL-C did go lower, and bigger of the HDL-C level. Interestingly, a higher dose of legumes did not have an effect on the levels of TG, though they were lower than the HFD ones. were significantly reduced, and their volumes were decreased. All these above indicated that mung bean and pea had certain effects in assisting the prevention of fatty liver.

| Effects of legumes on antioxidant status
Lipid peroxidation and antioxidant enzymes both in serum and in liver were determined (shown in Figure 3). It was found that high-fat diet As shown in Figure 4, the relative gene expression levels of CAT, SOD, heme oxygenase 1 (HO-1), transcription factor NF-E2-related factor 2 (Nrf2), and NAD(P)H quinone dehydrogenase 1 (NQO1) in HFDM-L and HFDP-L rats were markedly increased by a higher dose legume diet compared with the HFD group. In rats fed with diets containing 10% mung bean, the mRNA levels of HO-1 and SOD in the liver were 50.63% and 82.36% higher than those of rats fed with HFD (p < .05), respectively. The relative gene expression levels of Nrf2, NQO1, CAT, SOD, and HO-1 in rats fed with HFDM-H were 84.22%, 180.32%, 54.65%, 118.38%, and 79.35% higher than those in rats fed with HFD (p < .05), respectively. In HFDP-H rats, the expression levels of Nrf2, NQO1, CAT, SOD, and HO-1 were 80.11%, 165.92%, 53.57%, 97.54%, and 65.83% higher than those in the HFD group (p < .05).

| Antioxidant activity in vitro
The above results suggested that intact mung beans and peas can effectively improve oxidative stress injury induced by high-fat diet F I G U R E 1 Effects of mung beans and peas for 4 weeks on serum lipids in hyperlipidemia rats. Results are expressed as mean ± SD for eight independent experiments (animals) performed in duplicate. ***p < .001, **p < .01, and *p < .05 versus HFD in rats. However, the nutrients in these two kinds of legumes are complex, and it is difficult to determine which active ingredients play a key role in antioxidation. Therefore, in order to verify the main antioxidant components of mung beans and peas, these two kinds As shown in Table 1, the contents of ferulic acid and protocatechuic acid in mung bean and pea showed significant difference, while the other components did not. The content of ferulic acid in mung bean and pea was 17.6 and 2.33 mg/100 g, and the content of protocatechuic acid was 400 and 11.1 mg/100 g, respectively. F I G U R E 3 Effects of mung beans and peas for 4 weeks on serum (a-e) and liver tissue (f-j) antioxidant enzymes in hyperlipidemia rats. Results are expressed as mean ± SD for eight independent experiments (animals) performed in duplicate. ***p < .001, **p < .01, and *p < .05 versus HFD
Oil Red O staining of HepG2 cells is shown in Figure 7. As shown in Figure 8b, compared with the control group, OA could obviously F I G U R E 4 Effects of mung beans and peas on antioxidant gene expression in liver. #p < .05 versus NFD group, and *p < .05, **p < .01, and ***p < .001 versus HFD group

| D ISCUSS I ON
Numerous studies showed that elevated serum levels of TC, LDL-C, and TG could increase the incidence of atherosclerosis and cardiovascular disease. Conversely, elevated HDL-C levels could reduce these risks (Assmann & Gotto, 2004). In this study, elevated serum TG and TC levels were observed in the HFD rats, which are consistent with previous reports (Xiao et al., 2010).
However, the increases in LDL-C and TC levels and decreases in HDL-C could be significantly inhibited in the HFDM-H rats, suggesting that mung beans and peas could be good candidates for blood lipid reducing. Yeap et al. (2015) found that fermented mung beans significantly reduced the serum levels of TC, TG, and LDL in mice compared with nonfermented mung beans. This might be for the reason that γ-aminobutyric acid present in fermented mung bean may contribute to reducing the lipid in hypercholesterolemic mice. Abulnaja and Rabey (2015) found that the liver and heart tissues of rats fed with 2% cholesterol would lead to necrosis of hepatocytes and cytoplasmic blebbing. These findings observed in our study suggested that hypercholesterolemia was associated with pathological changes in vital organs.
Additions of mung beans and peas could protect liver and other tissues, and higher dose of legumes could almost return them to the normal states. Furthermore, high-fat diet may induce the production of reactive oxygen species (ROS) (Wang, Shimada, Kato, Kusada, & Nagaoka, 2015). The biological effects of ROS were controlled by antioxidant enzyme defense mechanism in vivo.
Supplementation with grain legumes could significantly enhance the activities of the main antioxidant enzymes in the serum and F I G U R E 6 The total phenolic content of ethanol extracts. There were significant differences in the different expressions of superscripts (a, b), p < .05 F I G U R E 7 The Oil Red O staining of mung bean and pea ethanol extracts liver of HFD rats, and then improve the lipid oxidation induced by HFD. These findings were consistent with the researches in animals (Feillet et al., 2009) and humans (Patel et al., 2007), whose oxida- activities (Duenas et al., 2004). In this study, the DPPH scavenging capacity of mung bean ethanol extract was higher than the pea extract, which was agreed with the results of Cheng (2009).
The higher DPPH scavenging capacity might be related to a higher content of natural antioxidants in mung beans, such as flavonoids (Nithiyanantham, Selvakumar, & Siddhuraju, 2012). ABTS radical scavenging activity of extracts is important and is exclusively measured by the ability of an antioxidant compound to be involved in a hydrogen atom transfer, which neutralizes generated ABTS + .
The previous study showed that the ABTS scavenging ability of the pea methanol extract was 6,155.37 μmol/g (Xu & Chang, 2008).
The total reducing power of legume extracts is also determined. Zhao, Du, Wang, and Cai (2014) found that the reducing power of undiluted ethanol extracted from mung bean was 0.96, and after diluting for five times higher, the reducing power of the raw mung bean extract was 0.23. Differences in the reducing power would be observed among the legume extracts, and the same results were also observed in this study. In addition, the absorbance of the reducing power determination increased with increasing extract concentrations. Many studies showed that phenolic compounds have strong antioxidant capacity (Randhir & Shetty, 2007). When reacting with oxide substances, these compounds can be used as hydrogen or electron donors. The total phenolic content of mung bean was reported to be 8.14 mg GAE/g (Yang, Cheng, Wang, Wang, & Ren, 2011), and 1.91 mg GAE/g for pea. Previous studies showed that the total flavonoid content of mung bean was 1.34 mg CE/g (Sreerama, Takahashi, & Yamaki, 2012) and that of pea was 1.10 mg CE/g. Furthermore, saponins in legumes also have antioxidant activities for their capacities of free radical capture (Kim et al., 2012).
It should be pointed out that antioxidant substances, such as phe-

| CON CLUS ION
Results showed that after intact mung bean and pea supplementation, the antioxidant defense system and gene expression of rats were improved in varying degrees compared with HDF group. The water and ethanol extracts of mung beans and peas have strong DPPH scavenging activity, a moderate ABTS scavenging activity, and high antilipid peroxidation ability and reducing power. Meanwhile, the ethanol extracts of mung beans and peas in HepG2 cells have also strong antioxidant activities. These results suggested that mung beans and peas have potent antioxidant activity, but the specific components of the extracts were not clear. Therefore, further research and discussion were needed. And this study is also worthy of further research and development in food and pharmaceutical industries.  19SG45) .

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L S TATEM ENTS
The authors declare that there is no conflict of interest regarding the publication of this article. The research described herein was performed on male SD rats. This study was performed in strict accordance with protocols approved by Institutional Animal Care and Use Committee of University of Shanghai for Science and Technology, Shanghai, China.