Cytoprotective Effect of Acid-Hydrolyzed Sesame Leaves against Oxidative Stress in HepG2 Cells

: In this study, we investigated the cytoprotective effects of acid-treated sesame leaf extracts against oxidative damage in HepG2 cells. Treatment with 0.1 M HCl of sesame leaves signiﬁ-cantly increased their verbascoside content (4.398 g/100 g) compared to non-acid-treated leaves (3.950 g/100 g). Acid-treated sesame leaf extract (ASLE) showed no cytotoxicity in HepG2 cells. ASLE conferred a greater cytoprotective effect against oxidative insult than a methanol extract of sesame leaves (SLE), verbascoside, and a vehicle control group. ASLE treatment also signiﬁcantly inhibited reactive oxygen species generation in response to oxidative stress. Treatment with tert - butyl hydroperoxide increased malondialdehyde (MDA) levels, and depleted reduced glutathione (GSH). However, ASLE treatment signiﬁcantly ameliorates this MDA and GSH depletion. Moreover, ASLE increased the activities of antioxidant enzymes including catalase, glutathione reductase, and glutathione peroxidase. Phenolic compounds in ASLE and SLE were characterized using UPLC-Q-TOF/MS analysis. A total of 29 iridoid and phenol compounds were tentatively identiﬁed in ASLE, and 27 compounds were observed in SLE. These results suggest that acid treatment of sesame leaves enhances the protective effects of their extract against oxidative stress by modulating antioxidant enzymes in HepG2 cells.


Introduction
Excessive reactive oxygen species (ROS) exposure can drive the progression of metabolic disorders including diabetes, obesity, hypertension, and atherosclerosis [1].Metabolic diseases have become an important global health problem because of their increasing incidence.In response to oxidative stress, antioxidant enzymes, including glutathione reductase (GR), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), are induced and may play a pivotal role in endogenous defensive metabolism in the liver [2].Glutathione (GSH) is a well-known potential antioxidant molecule that can neutralize ROS.Oxidative stress depletes cellular GSH [3].In addition, excessive ROS generation leads to liver damage by inducing pro-inflammatory cytokine production [4].Based on numerous studies, the endogenous antioxidant defense system is generally considered to be protective against oxidative stress-induced damage [5,6].Reducing ROS levels could thus serve as an effective approach to managing and preventing liver diseases.
Sesame (Sesamum indicum L.) is an important oilseed crop that is widely cultivated in Asia and Africa [7].Sesame oil is used in cooking and in folk medicine.Sesame seeds contain polyphenolic compounds, lignans, minerals, and omega-3 fatty acids, which are responsible for their health benefits.In addition, sesame seeds possess hepato-protective properties against oxidative stress in rat hepatocytes [8].To date, limited information is available on sesame leaf contents and their functions.Previous studies have shown that sesame leaves contain abundant nutrients and phytochemicals [7,9].Verbascoside, also known as acteoside, is one of the main bioactive compounds in sesame leaves.This compound belongs to the family of phenylethanoid glycosides, which are commonly found throughout the plant kingdom and extracted from medicinal plants [10].Studies have demonstrated that verbascoside possesses a range of biological functions including antioxidant, anti-inflammatory, hepatoprotective, and neuroprotective effects [11][12][13].It is widely recognized that aglycones exhibit greater activity within the intestine than their glycoside counterparts, leading to increased bioavailability [14].Our previous studies have demonstrated that subjecting glycosidic flavonoids to acid treatment enhanced their bioactivity [15,16].Acid hydrolysis thus has the potential to enhance the bioactivity of phenolic compounds through deglycosylation and the release of bound phenolic compounds.Here, we investigated the cytoprotective effects of acid-treated sesame leaves, including their ability to modulate antioxidant enzyme activity against oxidative stress, using human HepG2 cells.

Sample Preparation
Sesame leaves were collected from a local farm (Yangsan, Kyungnam, Republic of Korea) in August 2022.The leaves were washed, lyophilized, and ground into a fine powder.To prepare a sesame leaf extract (SLE), 10 g of powder was added to 500 mL of methanol.This mixture was extracted using an ultrasonic bath for 1 h at 24 • C, and then filtered through filter paper (Advantec, No. 2, Toyo Roshi, Tokyo, Japan).Solvent was removed using a vacuum evaporator, and the residue was dissolved in DMSO and stored at −20 • C until use.
To prepare acid-treated sesame leaf extract (ASLE), we used a previously reported method with slight modifications [15].Sesame leaf powder (10 g) was added to 500 mL of an acidic solvent (100% methanol containing 0.1 M HCl).This mixture was extracted in an ultrasonic bath for 1 h at 24 • C. When the reaction was complete, the reactant was cooled and the pH was adjusted to 7 using sodium hydroxide (1.0 M).The neutralized solution was subjected to vacuum evaporation to remove organic solvents.Subsequently, the residue was dissolved in distilled water and the resulting aqueous solution was washed twice with ethyl acetate.The ethyl acetate layer was concentrated under reduced pressure.The resulting residue was dissolved in DMSO and stored at −20 • C until needed for use.

Quantitation of Verbascoside and Caffeic Acid Content
Verbascoside and caffeic acid contents were assessed as reported earlier using highperformance liquid chromatography (Hitachi 5000 Chromaster, Hitachi Ltd., Tokyo, Japan) with a UV detector [17].Verbascoside and caffeic acid standards were obtained from Sigma-Aldrich.Phenomenex ultramex (5 µm, 250 mm × 4.6 mm, Luna., Torrance, CA, USA) was used with a mobile phase of acetonitrile (solvent A) and water (containing 0.2% phosphoric acid, solvent B) with a flow rate of 1.0 mL/min.Column oven temperature was 30 • C.
Data acquisition and processing were performed using Agilent MassHunter Qualitative Analysis software (Version 10.0, Agilent Technologies).Raw MS data files obtained from UPLC-Q-TOF/MS analysis were matched with the METLIN database B 08.00, and an in-house library of data was derived from prior studies for identification [7,19,20].

Cell Culture and Cytotoxicity
HepG2 cells were purchased from the Korean Collection for Type Cultures (Daejeon, Republic of Korea).SLE and ASLE cytotoxicities were assessed through MTT assay.HepG2 cells were seeded in 96-well plates at a density of 1 × 10 4 cells/well.The next day, cells were treated with extracts (25 and 50 µg/mL) for 24 h.Then, MTT assays were performed.
To evaluate extracts for cytoprotective effects against TBHP treatment, HepG2 cells were cultured in 96-well plates at a density of 1 × 10 4 cells/well in FBS-free medium for 18 h, and then incubated in FBS-free medium containing 0.5 mM TBHP and different concentrations of extracts (25 and 50 µg/mL).After 6 h, MTT assays were conducted.Absorbance was measured at 550 nm using a spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA).

Determination of Intracellular Reactive Oxygen Species Generation
Generation of intracellular ROS was measured using the fluorescent probe 2 ,7dichlorofluorescin diacetate (DCFH-DA) as previously described [21].In brief, HepG2 cells were seeded in 96-well black plates at a density of 5 × 10 4 cells/well, and pretreated with SLE and ASLE.After 1 h, the cells were washed twice with phosphate-buffered saline.Oxidative stress was induced through the addition of 0.5 mM TBHP to the culture medium for 1 h.The cells were washed twice with phosphate-buffered saline and were incubated with 25 µM DCFH-DA in serum-free medium for 1 h at 37 • C.Then, the cells were incubated in Hank's balanced salt solution.Fluorescence intensity was evaluated using a fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT, USA) for 90 min at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Quantitation of Glutathione and Lipid Peroxidation
HepG2 cells were seeded in 6-well plates at a density of 1 × 10 6 cells/well.The next day, cells were treated with different concentrations of each extract and TBHP for 6 h, and then harvested to measure malondialdehyde (MDA) and GSH levels.To determine cellular lipid peroxidation, MDA levels were measured using a thiobarbituric-acid-reactive substance assay [22].Intracellular GSH levels were determined as previously described, with slight modifications [23].The results are expressed as nmol of MDA/mg of protein using a molar extinction coefficient of 1.56 × 10 5 M/cm.

Measurement of Antioxidant Enzyme Activities
HepG2 cells were seeded in 6-well plates at a density of 1 × 10 6 cells/well to measure antioxidant enzyme activities.The next day, HepG2 cells were treated with each extract (50 µg /mL) and 0.5 mM TBHP for 6 h.Catalase (CAT) [24], glutathione reductase (GR) [25], and glutathione peroxidase (GPx) [26] activities were evaluated using previously reported methods.

Statistical Analyses
All results are representative of at least three independent experiments and are expressed as means ± standard deviation.Statistical analyses were conducted using one-way analysis of variance (ANOVA) with GraphPad Prism software (GraphPad Prism version 5.0 Software, San Diego, CA, USA), followed by Tukey's test.The threshold of significance was set at a p value of <0.05.

Effects of Acid-Hydrolyzed Sesame Leaves on Verbascoside Content
In this study, acid hydrolysis was employed to increase verbascoside and caffeic acid content in sesame leaves.Verbascoside and caffeic acid contents increased with acid treatment (Table 1).The highest verbascoside content (4.398 g/100 g) was observed in 0.1 M HCl-treated leaves.The highest caffeic acid yield (0.534 g/100 g) was also obtained from 0.1 M HCltreated leaves.However, treatment of 0.5 M HCl into sesame leaves decreased both verbascoside and caffeic acid compared to 0.1 M HCl treatment.The sesame leaf verbascoside content was previously determined to be in the 0.13-4.86g/100 g range [5].The observed broad variability in phytochemical content may be due to climatic, seasonal, and geographic conditions; harvest time; and storage conditions [27].Phytochemicals that exist in plant leaves mainly crosslink with cellulose, lignin, and hemicellulose by glycosidic bonds [28].Verbascoside is a phenylethanoid glycoside composed of hydroxytyrosol, caffeic acid, and glucose.On the other hand, acid hydrolysis breaks glycosidic bonds.For that reason, we anticipated the increased content of caffeic acid and verbascoside after acid hydrolysis.Our result showed that acid hydrolysis increased the contents of both verbascoside and caffeic acid.Moreover, acid hydrolysis combined with ultrasonic extraction was an efficient method for increasing the bound phenolic contents from leaves of Rubus idaeus L. [29].On the other hand, verbascoside, the primary bioactive constituent of dietary supplements in Japan, is derived from Sesamum indicum leaves [6].It also possesses antioxidant, antitumor, antibacterial, and neuroprotective activities [30][31][32][33].In a previous study, acid-treated rutin was converted to quercetin, which exerted an enhanced cytoprotective activity against oxidative stress and anti-adipogenic and anti-inflammatory activities [15].These results thus showed that acid treatment effectively increased verbascoside and caffeic acid content, and 0.1 M HCl treatment provided the most efficient conditions for extracting verbascoside and caffeic acid.

Effects of Acid-Hydrolyzed Sesame Leaves on Cell Viability and Cytoprotection in HepG2 Cells
The cytotoxicity of sesame leaves extracts was assessed using the MTT assay.None of the extracts showed cytotoxicity at concentrations up to 50 µg/mL in HepG2 cells after 24 h (Figure 1A).  (1Rt: retention time in minutes. (2)m/z calculated: m/z calculated using software. (3)m/z observed: m/z observed in experiment. (4)SLE: sesame leaf extract, ASLE: acid-treated sesame leaf extract.

Effects of Acid-Hydrolyzed Sesame Leaves on Cell Viability and Cytoprotection in HepG2 Cells
The cytotoxicity of sesame leaves extracts was assessed using the MTT assay.None of the extracts showed cytotoxicity at concentrations up to 50 µg/mL in HepG2 cells after 24 h (Figure 1A).To explore the extracts' protective efficacy on TBHP-treated HepG2 cells, cells were exposed to TBHP and each extract for 6 h.TBHP treatment decreased cell viability by approximately 50% compared to control cells.Treatment with verbascoside, SLE, and ASLE significantly increased HepG2 cell viability after oxidative stress challenge (Figure Figure 1.Cytotoxic effect of sesame leaves (A) and cytoprotective effect of various concentrations of sesame leaf methanolic and acid-treated extracts (25 and 50 µg/mL) against 0.5 mM tert-butylhydroperoxide-induced cytotoxicity (B).a~e Means with different letters are significantly different according to Duncan's multiple range test with significance threshold at p < 0.05.NS, not significant; CON, control; TBHP, tert-butyl hydroperoxide; VS, verbascoside (50 µM); SLE, sesame leaf extract; ASLE, acid-treated sesame leaf extract.
To explore the extracts' protective efficacy on TBHP-treated HepG2 cells, cells were exposed to TBHP and each extract for 6 h.TBHP treatment decreased cell viability by approximately 50% compared to control cells.Treatment with verbascoside, SLE, and ASLE significantly increased HepG2 cell viability after oxidative stress challenge (Figure 1B).An amount of 50 µg/mL of ASLE resulted in the highest cell viability.Oxidative stress increases hepatocyte cell death.Dietary interventions using natural antioxidants have thus been utilized to reduce hepatocyte oxidative stress and protect against cellular damage.Several flavonoids, including luteolin, quercetin, and kaempferol, exert well-known protective effects against oxidative insults [39].An earlier study showed that verbascoside effectively mitigated ROS-induced cytotoxicity in HT-29 cells through ROS scavenging [12].Hydroxytyrosol can neutralize free radicals and inhibit LDL oxidation in vitro [40][41][42].ASLE contained various phytochemicals including ferulic acid, luteolin, and morin, as well as verbascoside (Table 2).It seems that these phytochemicals were attributed to the increased cell viability compared to treatment with verbascoside alone.These results show that all treatments, including verbascoside, SLE, and ASLE, provided cytoprotective effects against oxidative stress, and the extract of acid-hydrolyzed sesame leaves provided the most effective cytoprotection.Tricin SLE, ASLE (1) Rt: retention time in minutes. (2)m/z calculated: m/z calculated using software. (3)m/z observed: m/z observed in experiment. (4)SLE: sesame leaf extract, ASLE: acid-treated sesame leaf extract.

Intracellular Antioxidative Activities of Acid-Hydrolyzed Sesame Leaves
To evaluate the effect of acid hydrolysis in sesame leaves on redox effectors including ROS, MDA, and GSH, and antioxidant enzymes, including GPx, CAT, and GR, sampletreated HepG2 cells were collected after 24 h.As shown in Figure 2, TBHP increased intracellular ROS generation.All extract-treated samples showed markedly decreased ROS generation compared to that in the TBHP-alone group.ASLE treatment decreased ROS generation to almost basal levels.We then measured GSH and MDA levels to gauge the HepG2 cell response to oxidative stress.TBHP significantly decreased GSH levels compared to control cells (Figure 3A).However, all extract-treated samples showed increased GSH levels compared to those treated with TBHP alone.ASLE treatment most strongly preserved GSH levels in HepG2 cells facing oxidative stress.The MDA level was also significantly elevated in TBHP-treated cells relative to control cells (Figure 3B), and verbascoside, SLE, and ASLE significantly abated the TBHP-mediated elevation of MDA levels.Remarkably, treatment with SLE and ASLE reduced MDA levels to near or below control values.To evaluate the effect of sesame leaf acid hydrolysates on antioxidant enzyme activity, GPx, CAT, and GR levels in HepG2 cells were evaluated.TBHP treatment decreased antioxidant enzyme activities, and verbascoside, SLE, and ASLE markedly restored GR and GPx activities in TBHP-treated cells (Figure 4A,B).CAT activity was increased only by ASLE (verbascoside and SLE did not increase this activity) (Figure 4C).
as well as verbascoside (Table 2).It seems that these phytochemicals were attributed to the increased cell viability compared to treatment with verbascoside alone.These results show that all treatments, including verbascoside, SLE, and ASLE, provided cytoprotective effects against oxidative stress, and the extract of acid-hydrolyzed sesame leaves provided the most effective cytoprotection.

Intracellular Antioxidative Activities of Acid-Hydrolyzed Sesame Leaves
To evaluate the effect of acid hydrolysis in sesame leaves on redox effectors including ROS, MDA, and GSH, and antioxidant enzymes, including GPx, CAT, and GR, sampletreated HepG2 cells were collected after 24 h.As shown in Figure 2, TBHP increased intracellular ROS generation.All extract-treated samples showed markedly decreased ROS generation compared to that in the TBHP-alone group.ASLE treatment decreased ROS generation to almost basal levels.We then measured GSH and MDA levels to gauge the HepG2 cell response to oxidative stress.TBHP significantly decreased GSH levels compared to control cells (Figure 3A).However, all extract-treated samples showed increased GSH levels compared to those treated with TBHP alone.ASLE treatment most strongly preserved GSH levels in HepG2 cells facing oxidative stress.The MDA level was also significantly elevated in TBHP-treated cells relative to control cells (Figure 3B), and verbascoside, SLE, and ASLE significantly abated the TBHP-mediated elevation of MDA levels.Remarkably, treatment with SLE and ASLE reduced MDA levels to near or below control values.To evaluate the effect of sesame leaf acid hydrolysates on antioxidant enzyme activity, GPx, CAT, and GR levels in HepG2 cells were evaluated.TBHP treatment decreased antioxidant enzyme activities, and verbascoside, SLE, and ASLE markedly restored GR and GPx activities in TBHP-treated cells (Figure 4A,B).CAT activity was increased only by ASLE (verbascoside and SLE did not increase this activity) (Figure 4C).Excessive intracellular ROS levels indicate an oxidative insult in living cells, and predict damage to lipids, proteins, and DNA [39].Cellular MDA is a product of lipid peroxidation in cell membranes resulting from reactions between unsaturated fatty acids and radical species [43].Oxidative stress-induced liver damage leads to reduced levels of glutathione (GSH), which plays a central role in defending cells against oxidative stress and detoxifying their environment [44].Numerous dietary phytochemicals have been studied and have shown significant effects on antioxidant enzymes under oxidative stress.
dict damage to lipids, proteins, and DNA [39].Cellular MDA is a product of lipid peroxidation in cell membranes resulting from reactions between unsaturated fatty acids and radical species [43].Oxidative stress-induced liver damage leads to reduced levels of glutathione (GSH), which plays a central role in defending cells against oxidative stress and detoxifying their environment [44].Numerous dietary phytochemicals have been studied and have shown significant effects on antioxidant enzymes under oxidative stress.For example, flavonoids and phenolic-acid-rich jujube extracts increase cell viability against oxidative stress by decreasing ROS production and MDA and GSH depletion via nuclear factor erythroid 2-related factor 2 activation [45].Liao et al. previously reported that diosmetin exerts a strong antioxidant activity by inhibiting ROS generation and restoring intracellular antioxidant enzyme activity in response to oxidative stress [46].In addition, verbascoside significantly suppressed ROS and MDA levels and increased GPx and SOD activities to counter oxidative stress [47].The alterations in GR, CAT, and GPx activities in response to TBHP + sample treatment, compared with TBHP treatment alone, clearly indicate the capacity of the cell defense system to react to oxidative stress.Collectively, these results suggest that phytochemicals in sesame leaves exert cytoprotective effects.In addition, acid hydrolysis of sesame leaves enhances antioxidant capacity by modulating antioxidant enzymes and/or scavenging free radicals.dict damage to lipids, proteins, and DNA [39].Cellular MDA is a product of lipid peroxidation in cell membranes resulting from reactions between unsaturated fatty acids and radical species [43].Oxidative stress-induced liver damage leads to reduced levels of glutathione (GSH), which plays a central role in defending cells against oxidative stress and detoxifying their environment [44].Numerous dietary phytochemicals have been studied and have shown significant effects on antioxidant enzymes under oxidative stress.For example, flavonoids and phenolic-acid-rich jujube extracts increase cell viability against oxidative stress by decreasing ROS production and MDA and GSH depletion via nuclear factor erythroid 2-related factor 2 activation [45].Liao et al. previously reported that diosmetin exerts a strong antioxidant activity by inhibiting ROS generation and restoring intracellular antioxidant enzyme activity in response to oxidative stress [46].In addition, verbascoside significantly suppressed ROS and MDA levels and increased GPx and SOD activities to counter oxidative stress [47].The alterations in GR, CAT, and GPx activities in response to TBHP + sample treatment, compared with TBHP treatment alone, clearly indicate the capacity of the cell defense system to react to oxidative stress.Collectively, these results suggest that phytochemicals in sesame leaves exert cytoprotective effects.In addition, acid hydrolysis of sesame leaves enhances antioxidant capacity by modulating antioxidant enzymes and/or scavenging free radicals.For example, flavonoids and phenolic-acid-rich jujube extracts increase cell viability against oxidative stress by decreasing ROS production and MDA and GSH depletion via nuclear factor erythroid 2-related factor 2 activation [45].Liao et al. previously reported that diosmetin exerts a strong antioxidant activity by inhibiting ROS generation and restoring intracellular antioxidant enzyme activity in response to oxidative stress [46].In addition, verbascoside significantly suppressed ROS and MDA levels and increased GPx and SOD activities to counter oxidative stress [47].The alterations in GR, CAT, and GPx activities in response to TBHP + sample treatment, compared with TBHP treatment alone, clearly indicate the capacity of the cell defense system to react to oxidative stress.Collectively, these results suggest that phytochemicals in sesame leaves exert cytoprotective effects.In addition, acid hydrolysis of sesame leaves enhances antioxidant capacity by modulating antioxidant enzymes and/or scavenging free radicals.

Conclusions
In conclusion, acid hydrolysis increased the verbascoside and caffeic acid content extractable from sesame leaves.Among the tested treatments, ASLE exposure most strongly preserved cell viability against oxidative stress.ASLE also effectively reduced ROS generation, GSH depletion, and the elevation in malondialdehyde (MDA) levels in HepG2 cells.ASLE significantly increased antioxidant enzyme activity in response to oxidative damage in HepG2 cells.Twenty-nine and twenty-seven bioactive compounds were tentatively

Table 1 .
Sesame leaf verbascoside and caffeic acid content quantitated by acid hydrolysis.
a-c Means within a column with different superscript letters are significantly different according to Duncan's multiple range test with significance threshold at p < 0.05.

Table 2 .
Identification of compounds in acid-treated sesame leaves using UPLC-QTOF/MS.