Antioxidant and anti‐inflammatory effects of Peanut (Arachishypogaea L.) skin extracts of various cultivars in oxidative‐damaged HepG2 cells and LPS‐induced raw 264.7 macrophages

Abstract This study was performed to investigate the distribution of phenolic compounds in the peanut skins of various cultivars, as well as their antioxidant and anti‐inflammatory effect (Arachishypogaea L. cv. K‐Ol, cv. Sinpalkwang, cv. Daan, cv. Heuksaeng) and extraction solvent. The major components of red peanut cultivars (K‐Ol, Sinpalkwang, and Daan) were identified as proanthocyanidin, catechin, gallic acid, coumaric acid, and hesperidine, whereas the major components of black peanut cultivar (Heuksaeng) were identified as anthocyanin, ferulic acid, and quercetin. The DPPH and ABTS radical scavenging activities, and FRAP values were the highest in Daan followed by Sinpalkwng, K‐Ol, and Heuksang. Furthermore, the skin extracts of red peanuts effectively improved cell viability, reactive oxygen species generation, MDA concentration, and antioxidant enzyme activity (GR, GPx, CAT, and superoxide dismutase) in oxidative stress‐induced HepG2 cells, and reduced the expression of pro‐inflammatory factors (NO, TNF‐α, IL‐6, and IL‐1β) in LPS‐stimulated RAW 264.7 macrophages. These results suggest that red peanut skin extracts could effectively mediate physiological activity and provide valuable information for the use of peanut byproducts as functional food materials.

2007). While acute inflammation is essential for combating infection and tissue repair, excessive and uncontrolled inflammation is often associated with chronic diseases, such as metabolic disorders, atherosclerosis, and certain types of cancer (Chakrabarti et al., 2014).
The role of food polyphenolics in the prevention of cardiovascular diseases and certain types of cancer is well recognized. Byproducts of seeds (Galali et al., 2020), such as the skins of brazil nut (John & Shahidi, 2010), almond (Wijeratne et al., 2006), and peanuts (Levy et al., 2017;Sarnoski et al., 2012) have been reported to be rich sources of polyphenolics and antioxidants.
Peanut (Arachishypogaea L.) is a critical oil crop that is widely cultivated in many countries, and it is an important food material that is consumed worldwide (Oldoni et al., 2016). Even though peanut skin is a small portion of the total seed, it was discarded 7.5 × 105 tons a year as a by-product. But peanut skin contains several abundant polyphenolic compounds, such as flavonoids, phenolic acids, procyanidins, and anthocyanins, in approximately 90-150 mg/g of the dried skin (Nepote et al., 2010). The Rural Development Administration (RDA) of Korea conducted research on the development of peanut varieties with various functional characteristics such as Sinpalkwang , Daan , K-Ol , and Heuksaeng . Sinpalkwang and Daan could be cultivated easily in high yields. Additionally, K-Ol was found to comprise greater than 80% of oleic acid among the typical fatty acid composition, and Heuksaeng had a characteristic black color and high containing anthocyanin content. These developed peanut cultivars have different characteristics and phenolic compound profiles in peanut skin, whereas there is a lack of investigation on antioxidant and anti-inflammatory effect in the HepG2 cell and RAW 264.7 macrophages. Therefore, the objective of this study is to (a) investigate the composition of phenolic compounds in the skins of four peanut cultivars, as well as their antioxidant and anti-inflammatory effects on tert-butyl hydroperoxide (TBHP)-induced oxidative stress in HepG2 cells and LPS-stimulated RAW 264.7 macrophages and (b) evaluate the correlation between the phenolic compounds and the antioxidant and anti-inflammatory characteristics to identify marker compounds in peanut skin.

| Sample preparation and extraction
Four types of peanut cultivars (Arachishypogaea L.) cv. Sinpalkwang, cv. K-Ol, cv. Daan, and cv. Heuksaeng were used in this study. Two peanut cultivars, cv. Sinpalkwang and K-Ol, were grown at the National Institute of Crop Science and Foundation of Agriculture methanol (80 ml), 80% acetone (80 ml), and distilled water (80 ml) at room temperature for 1 hr using an ultrasonic bath. The extracts were then filtered and concentrated using a rotary evaporator under vacuum and were then freeze-dried and stored at −20°C in an ultralow temperature freezer.

| Determination of phenolic compounds
The total polyphenol and flavonoid levels were measured according to the method reported by Dewonto et al (Dewanto et al., 2002).
The results are expressed as milligrams of gallic acid and catechin equivalents per gram of peanut skin (mg GAE/g peanut skin, mg CE/g peanut skin). Total proanthocyanidin levels were measured using the vanillin-sulfuric acid method (Takahama et al., 2010). The results are expressed as milligrams of catechin equivalents per gram of the peanut skin (mg CE/g peanut skin). Total anthocyanin content was determined based on a reported pH differential method; these results were expressed in milligrams of cyaniding-3-glucose equivalents per gram of peanut skin (mg C3GE/g peanut skin) (Tchabo et al., 2015).
The phenolic acid and anthocyanin composition of each extract was determined by high-performance liquid chromatography (HPLC), as described by Kim et al. (2016) and Choung (2008), respectively.

| Protective effect in oxidative-damaged HepG2 cell
Human hepatoma HepG2 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). The cell line was maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 50 μg/ml streptomycin at 37°C in an incubator with a 5% CO2 atmosphere. Human hepatoma HepG2 cell death was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay (Ishiyama et al., 1996). To determine cytoprotective effects against oxidative stress, HepG2 cells were seeded in a 96-well plate at a density of 1.5 × 104 cells/well. After 24 hr, the culture medium (200 ml/well) was replaced with an FBS-free medium (200 ml/well) containing various concentrations of extracts, after 12 hr, the culture medium containing the extract was discarded, and the cells were treated for 3 hr with the medium (200 ml/well) containing 200 mmol/L of TBHP to induce oxidative stress. We then evaluated the protective effect of extracts using the MTT assay. Intracellular ROS levels were quantified with a DCFH-DA fluorescent probe, as previously described (Wang & Joseph, 1999). The fluorescence intensity, corresponding to the intracellular ROS generation, was measured with a fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT, USA) for 2 hr at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. To determine lipid peroxidation and antioxidant enzyme activity, the cells were harvested and lysed for 10 s using a Vibra-Cell VCX 750 sonicator (Sonics&Materials, Inc., Newtown, CT, USA).
The lysates were centrifuged at 10,000 × g for 10 min at 4°C, and the supernatants were used for protein and lipid peroxidation, and antioxidant enzyme assay according to the Ham et al (Ham et al., 2015).

| Anti-inflammation activity in LPS-induced RAW 264.7 cell
RAW 264.7 macrophages were obtained from the Korean Cell Line Bank (Seoul, Korea). The cell line was maintained in DMEM, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 50 μg/ mL streptomycin at 37°C in an incubator with a 5% CO2 atmosphere.

| Statistical analysis
All data are expressed as means ± standard deviations. Significant differences among treatments were determined by one-way analysis of variance using Duncan's multiple range test, with SAS ver. 9.2 software (SAS Institute). The significance level was set to 0.05.

| Composition of phenolic compounds in peanut skin extracts
Plant phenolic compounds are currently among the most studied phytochemicals because of their biological functions, including antioxidant and anti-inflammatory characteristics (Koleckar et al., 2008).
The total polyphenol contents (TPC), total flavonoid contents (TFC), total proanthocyanidin contents (TPAC), and total anthocyanin TA B L E 1 Total polyphenol content (mg GAE/g), total flavonoid content (mg CE/g), total proanthocyanidin content (mg CE/g), and total anthocyanin contents (mg C3GE/g) of peanut skin extracts according to cultivars and extraction solvent contents (TAC) of the peanut skin according to cultivars and extraction solvents are shown in Table 1. Among the extraction solvents tested, 80% methanol and 80% acetone were found to be the most efficient solvent systems for extracting the polyphenolic compounds when compared with all other solvent systems used, and the levels of the TPCs ranged from 122.17 to 166.29 mg GAE/g peanut skin. The TFC results tended to be similar to TPC results. The recovery of the phenolic contents in different samples is influenced by the polarity of extraction solvents and the solubility of the particular compound in the solvent used for the extraction process (Sulaiman et al., 2011).
When considering the effect of the cultivar on the TPC and TFC, the highest TPC was observed for the Daan cultivar in the case of the peanut skin extracted with 80% ethanol, 80% acetone, and distilled water. In contrast, the highest TPC, which was significantly higher than that of others, was observed for Heuksaeng (166.29 mg GAE/g peanut skin) and Daan (166.20 mg GAE/g peanut skin) in the case of the peanut skin extracted with 80% acetone and methanol, respectively. The high polyphenol content of the Heuksaeng cultivar was related to the anthocyanin content of the black pigment peanut cultivar. Also, these results are in close agreement with those reported by Larrauri et al. (2016) They observed that the extraction yield of the roasted and blanched peanut skin with 70% ethanol and distilled water ranged from 9.8% to 18.0%, and total the polyphenol contents of these extracts ranged from 295 to 672 mg GAE/g.
Since the pigments of the peanut skin play an important role in physiological effects, there has been growing attention on the composition and medicinal use of peanut skin (Tatsuno et al., 2012).
Therefore, we analyzed the pigment characteristics, such as UV absorption, total proanthocyanidin content (TPAC), and total anthocyanin content (TAC) of the peanut skin extracts of the various cultivars ( Figure 1, Table 1). Peanut seeds have several skin colors, including red and black (Chukwumah et al., 2009). K-Ol, Sinpalkwng, and Daan are red-pigmented peanut cultivar, whereas Heuksaeng is a black-pigmented peanut cultivar. These differences in the colors of the peanuts affected the UV spectra, TPAC, and TAC of the peanut skin extracts. As shown in Table 1, the total proanthoyanidin content was the highest in Daan (20.07-48.91 mg CE/g peanut skin) and was generally higher in the red peanut cultivars than those in the black ones. Meanwhile, the total anthocyanin contents were highest in Heuksang (13.85-23.31 mg C3GE/g peanut skin).
Furthermore, the variations in the individual phenolics and anthocyanin profiles of the peanut skins according to their cultivar are shown in Table 2. Among the individual phenolics, the major compounds in the red peanut cultivars were catechin and ferulic acid, which accounted for approximately 85% of the total phenolics.

| Effect of peanut skin extracts on radical scavenging activity and reducing power
The effects of the cultivar and extraction solvents on the peanut skin extract's ABTS, DPPH radical scavenging activities, and reducing power (FRAP) are presented in Figure 2.  (Adhikari et al., 2019). In this study, we postulated that red peanut cultivars, containing a high amount of catechins and proanthocyanidins, which have a relatively large number of aromatic hydroxyl groups, showed higher antioxidant activity than the black peanut cultivar composed of anthocyanins.

| Effect of peanut skin extract on endogenous antioxidant defense system and biomarker of oxidative stress in TBHP-induced HepG2 cell
The hepatoprotective effects of the peanut skin extracts on oxidative stress-induced HepG2 cell were investigated to determine protective effects, ROS inhibition, malondialdehyde (MDA) concentration, and antioxidant enzyme activity (glutathione reductase; GR, glutathione peroxidase; GPx, SOD, catalase; CAT). Before measuring the cytoprotective effects of the peanut skin extract on TBHPinduced HepG2 cells, the cytotoxicity was estimated by MTT assay Figure 3a). Next, we evaluated the cytoprotective effects of the peanut skin extracts of the four different cultivars against TBHPinduced oxidative damage (Figure 3b). These results demonstrate that the treatment with 500 μM TBHP significantly decreased cell viability by 53.68% compared to that in control cells. However, TA B L E 2 Phenolic acid contents (μg/g peanut skin) and anthocyanin contents (mg/g peanut skin) of peanut skins, according to their cultivar Note: Different small letters in the same item indicates a significant difference (p < .05) among different cultivars.
Abbreviation: ND, Not detected. pretreatment of the peanut skin extract at 2.5-10 μg/ml significantly increased cell viability in a dose-dependent manner, regardless of the identity of the cultivar. Especially, the highest protective effects from the TBHP-induced stress in the HepG2 cells were observed in the peanut skin extracts of Daan and Sinpalkwng; these extracts increased cell viability to 101.19% and 95.97%, respectively, at a concentration of 10 μg/ml.
After evaluating the protective effects of the peanut skin extracts against oxidative stress, the levels of the ROS were analyzed in the TBHP-induced HepG2 cells (Figure 3c,d). Accumulation of the cellular ROS is known to be a significant cause of intracellular damage and causes age-related and metabolic diseases (Halliwell et al., 1992). Therefore, determination of the ROS released from the cell provides a good index of oxidative damage of the cell (Alía et al., 2006). As shown in Figure 3c,d, the TBHP treatment rapidly stimulated oxidative cellular stress and damage, elevating the release of ROS in HepG2 cell by 210.01% compared to the control cell without TBHP. However, the pretreatment of peanut skin extracts at 2.5-10 μg/ml significantly decreased the intracellular ROS level in the TBHP-induced HepG2 cell in a dose-dependent manner.
These extracts of Daan and Sinpalkwng reduced the ROS level from 210.01% for the TBHP-induced cellular oxidative stress to 130.18% and 129.19%, respectively, at a concentration of 10 μg/ml.
Next, the intracellular MDA concentration in the TBHPinduced HepG2 cell protein was analyzed as a lipid peroxidation index (Suttnar et al., 2001). Additionally, the intracellular antioxidant enzymes play critical roles in the defense mechanism against oxidative damage (Alía et al., 2006). Oxidative stress (TBHP, 1 mM) treatment in HepG2 cells stimulated a significant increase of MDA concentration and the enzyme activities of CAT, SOD, GR, and GPx (Table 3). However, these increases were reversed by pretreatment with the peanut skin extracts at 10 μg/ml.  Note: Lipid peroxidation of 1 malondialdehyde (MDA, nmol mg protein) and antioxidant enzyme activity of 2 glutathione reductase (GR, μmol min mg −1 protein), 3 glutathione peroxidase (GPx, μmol min mg −1 protein), 4 catalase (CAT, μmol min mg −1 protein), and 5 SOD, unit mg -1 protein) were evaluated in HepG2 cells treated for 6 hr with the samples, followed by treatment for 4 hr with 500 μM TBHP. Values are means ± SD (n = 3).
Significant at ***p < .001 oxidative stress in the HepG2 cells. Hepatoprotective effects of proanthocyanidin and catechin from various plants against oxidative stress have been investigated in many studies. These protective effects against oxidative stress are in consistent with the other in vitro studies carried out with various cell types (Praphasawat et al., 2011).
Pretreatment of the HepG2 cells with the oligomeric procyanidin fraction could reduce the decrease in cell viability induced by TBHP by reducing ROS generation and malondialdehyde formation (Kim et al., 2013). Furthermore, it has been reported that the MDA levels were reduced by various plant polyphenols such as catechin, epicatechin, proanthocyanidin, following oxidative stress. It has also been reported that flavonoids have the ability to interact with bio-membranes and protect them from free radicals38, and that procyanidins trigger the upregulation of a battery of antioxidant and detoxification enzymes, which boost cellular defenses against oxidative stress (Saija et al., 1995). From the results of this study, we postulate that the red peanut cultivars, which contain high amounts of catechins and proanthocyanidins, possess the ability to impart strong protection against oxidative damage in HepG2 cells, by modulating ROS production, MDA generation, and antioxidant enzyme activities.

| Effect of peanut skin extracts on nitric oxide (NO) production and pro-inflammatory cytokine secretion in LPS-stimulated RAW 264.7 macrophages
The anti-inflammatory effect of the peanut skin extracts was evaluated by measuring the levels of nitric oxide (NO), and the secretion of the pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β.
Before evaluating the anti-inflammatory activities of the peanut skin extracts on the LPS-stimulated RAW 264.7 cells, the cytotoxicity was estimated by MTT assay (Figure 3a). The effects of peanut skin extracts on the inhibition of NO production are presented in Figure 3b.
NO production following LPS-treatment for 24 hr (39.74 μM) significantly increased (p < .001) compared with that noted in the unstimulated RAW 264.7 cells (7.93 μM). However, the peanut skin extract (40 μg/ml) reduced the NO level by 70, 84, 81, and 30%, compared to the LPS-stimulated controls of K-Ol, Sinpalkwng, Daan, and Heuksang cultivar, respectively. Furthermore, TNF-α, IL-6, and IL-1β are important pro-inflammatory cytokines that are linked to the pathogenesis of many infectious and inflammatory diseases, including cancer (Chakrabarti et al., 2014). As shown in Figure

| Correlation analysis
The correlation between the functional compounds and key factors on the antioxidant and anti-inflammatory characteristics of peanut skin extracts was analyzed for various cultivars with different colors, and is expressed by the value of the correlation coefficient (R) presented in Table 4. In general, major functional compounds of the red peanut cultivars, such as flavonoids, proanthocyanidins, catechin, gallic acid, coumaric acid, and hesperidin, showed a sig-

| CON CLUS ION
This study was investigated the composition and distribution of phenolic compounds of peanut cultivars (Sinpalkwang, K-Ol, Daan, Heuksaeng.), as well as the antioxidant and anti-inflammatory effects of the skins of the four peanut cultivars. The total polyphenol contents were not significantly different between the Daan of red peanut cultivars and Heukasang of the black cultivar. In contrast, there is difference in the content of individual phenolics that made F I G U R E 5 Effect of peanut skin extracts of different cultivars on (a) TNF-α, (b)IL-6, and (c) IL-1β concentration in LPS-stimulated RAW 264.7 macrophages. Different capital letters and small letter in the same items indicate a significant difference (p < .05) among different cultivars and extraction solvent, respectively up the total polyphenol content. The total flavonoid content, total proanthocyanidin content, catechin, gallic acid, coumaric acid, and hesperidin were generally higher in red peanut cultivars (Sinpalkwng, K-Ol, Daan) than in black peanut cultivars (Heukasang). Meanwhile, the anthocyanidin, ferulic acid, and quercetin contents were generally higher in black peanut cultivars than in red peanut cultivars. The ABTS and DPPH radical scavenging activities, and FRAP values were the highest in Daan, followed by Sinpalkwng, K-Ol, and Heuksang, and were generally higher in red peanut cultivars than in black ones.
In addition, our results show that the skin extracts of red peanut modulated cell viability, ROS generation, MDA concentration, and antioxidant enzyme activity (GR, GPx, CAT, and SOD) in oxidative stress-induced HepG2 cells, and inhibited pro-inflammatory factors (NO, TNF-α, IL-6, and IL-1β) in LPS-stimulated RAW 264.7 cells.
Therefore, this study provides valuable information on the application of functional materials for improving the utilization of phenolic compounds extracted from peanut skins, depending on the different cultivars, as effective physiological agents.

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

E TH I C A L A PPROVA L
Neither animal nor human testing was involved in this study.