Moringa oleifera Hot Water Extract Protects Vero Cells from Hydrogen Peroxide-Induced Oxidative Stress by Regulating Mitochondria-Mediated Apoptotic Pathway and Nrf2/HO-1 Signaling

The present study discloses the identification of phenolic compounds in Moringa oleifera hot water extract (MOH) and the evaluation of its antioxidant activity on H2O2-induced oxidative stress in Vero cells. Upon analysis, MOH was found to contain phenolic compounds and indicated 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS+) radical scavenging with IC50 values of 102.52 and 122.55 µg/mL, respectively. The ferric reducing antioxidant power (FRAP) of MOH indicated a dose-dependent increase with a maximum absorbance at 125 μg/mL and the oxygen radical absorbance capacity (ORAC) of MOH was 1004.95 µmol TE/mg. Results showed that MOH dose-dependently reduced intracellular ROS generation in H2O2-stimulated Vero cells while increasing the cell viability. Fluorescence microscopy and flowcytometric analyses have supported the above findings. MOH markedly suppressed the H2O2-induced mitochondrial depolarization and apoptosis through suppression of the mitochondrial-mediated apoptosis pathway and activated the Nrf2/HO-1 signaling pathway by possibly involving H2O2 generation in cell media. Findings of western blot were supported by immunocytochemistry of Nrf2 nuclear translocation. Thus, MOH bioactivity would potentiate its applications in manufacturing functional food.


Introduction
Exposure to ionizing radiation, xenobiotics, and disease conditions generate oxidative stress in live cells, which has been linked to the pathogenesis of a variety of detrimental cellular responses [1]. As evidence accumulated over time has shown, oxidative and inflammatory processes in the human body are triggered by lifestyle-related factors such as exposure to contaminated air, smoke and alcohol consumption, exposure to ionization radiation, xenobiotics, and urbanization hypoxia [2]. Cell and tissue damage, and, hence, As oxidative stress has implications for human kidneys, Vero cells derived from African green monkey kidney fibroblasts were used in assessing oxidative stress using H 2 O 2 to generate ROS in cells [22]. The present study was carried out to evaluate the efficacy of the hot water extraction method to extract M. oleifera and evaluate the antioxidant activity of M. oleifera hot water extract (MOH) on H 2 O 2 -induced oxidative stress in Vero cells. The study was carried out by hypothesizing that MOH ameliorates the effects of H 2 O 2 -induced oxidative stress in Vero cells by suppressing the mitochondria-mediated apoptosis pathway and promoting the Nrf2/HO-1 signaling pathway.

Sample Collection and Extraction
Tender branches and leaves of M. oleifera were collected from Suncheon Bay Moringa Cooperative (Suncheon-si, Jeollanam-do, Korea), chopped into about one-centimeter pieces, air-dried under room temperature, and stored in air-tied polythene bags for further uses. A part of the air-dried material was pulverized into powder by using IKA MF10 laboratory pulverizer (Staufen, Germany). Based on the DPPH radical scavenging activity and flavonoid content, one of the previous studies revealed that the 100 • C water in a pressurized system was the optimum temperature for M. oleifera leaf extraction [23]. In this study, boiling water (900 mL) was used to extract 100 g of powder at 100 • C for 4 h. After centrifugation and filtration, the filtrate was frozen at −80 • C and freeze-dried to obtain a dry powder of hot water extract. The freeze-dried powder was kept in an air-tight container at −20 • C.

Compositional Analysis of MOH
The total polyphenolic content of MOH was determined using the method outlined by Singleton et al. (1999) [24]. A gradient concentration of gallic acid was used as the reference standard. The Lowry method with BSA as the reference standard was used to determine total protein content in MOH [25]. Carbohydrate content was measured according to the phenol-sulphuric method using d-glucose as the reference standard [26].

High-Performance Liquid Chromatography (HPLC) Analysis of MOH
HPLC analyses were conducted with the aid of a Shimadzu system equipped with a gradient pump integrated into an SPD-M30A Photodiode Array Detector. The separation was achieved by a Luna PFP (2) 100A (150 × 3.0 mm, 3 m) column. The system was eluted with the gradient program of a binary solvent system consisting of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) mixture at a constant flow rate of 0.34 mL/min. The sample injection volume was 3 µL. A 5-min post-run at starting conditions was performed to equilibrate the column. The gradient program was started with 0% of B and then changed to obtain 25%, 45%, 65%, 85%, and 100% of eluent B at 0, 25, 50, 75, 100, and 125 min, respectively. For each predetermined phenolic compound that was evaluated, the mixed standard solution was prepared by diluting the mixed stock standard solutions in methanol to provide a concentration of 1.176 mg/mL. A solution of MOH at a concentration of 10 mg/mL in methanol was prepared, sent through a syringe filter, injected, and phenolic compounds were evaluated using UV absorbance at 270 nm. The phenolic compounds in the MOH were identified by comparing retention times and absorbance spectrum profiles with the standards of each detected compound. Quantification was carried out by comparing the chromatograms of the standard mixture with that of the sample (MOH). Triplicate independent analyses were conducted, and the results are presented as means ± standard deviations from three separate studies. All chromatographic processes were carried out at 35 • C, with data acquisition, peak integration, and calculations carried out using LCsolution version 1.24 SP2 software (Shimadzu, Kyoto, Japan).

Radical Absorbance Capacity of MOH
ABTS + and DPPH radical scavenging activity of MOH were determined using the methods described in one of the previous studies conducted by Um et al. (2017) [27]. In brief, the stock solution containing ABTS, and potassium persulfate (K 2 S 2 O 8 ) were mixed with each 50 µL of the MOH hydrolysates (250 µg/mL). Then the mixture was left to react for 10 min in the dark, and the absorbance was measured at 414 nm by using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, USA). To analyze the DPPH radical scavenging capacity, 100 µL of MOH hydrolysates (250 µg/mL) was added to 100 µL of DPPH solution (150 µM). The mixture was kept in dark at room temperature for 30 min, and then absorbance was measured at 517 nm by using a SpectraMax M2 microplate reader. Ferric chloride (10% in distilled water) was used to measure the Ferric reducing antioxidant power (FRAP) of MOH. Briefly, 0.1 M phosphate buffer (pH 6.6-7.0), 1% potassium ferricyanide, and sample or distilled water as a control mix in a 3:5:2 ratio, vortexed and incubated for 20 min at 50 • C. Thereafter, 10% Trichloroacetic acid was mixed and centrifuged at 3000 rpm for 10 min. The supernatant was taken, mixed with distilled water and 10% ferric chloride in a 5:5:1 ratio, and the absorbance was measured at 700 nm by using SpectraMax M2 microplate reader. Oxygen radical absorbance capacity (ORAC) assay was used to examine the antioxidant ability of MOH. In brief, 50 µL of 50 g/mL MOH in 75 mM phosphate buffer (pH 7.0) was mixed with 78 nM fluorescein (440 g/mL in 75 mM phosphate buffer) and incubated at 37 • C for 15 min. Then, 25 µL of AAPH was added to each well, and the emission at 538 nm under 485 nm excitation was recorded every 5 min for 2 h using SpectraMax M2 microplate reader. Trolox (1,5,10,20, and 40 µM in 75 mM phosphate buffer) was used as the standard. The antioxidant capacity of MOH was expressed as Trolox equivalents per µg/mL of MOH.

Cell Culture
Vero cells (The monkey kidney fibroblasts, KCLB, Seoul, Korea) were cultured and then sub-cultured every 3 days in a humidified atmosphere at 37 • C with 5% of CO 2 in DMEM supplemented with 1% P/S and 10% inactivated FBS. Cells were seeded in 24-well plates or 96-well plates accordingly for subsequent experiments.

Cell Viability and ROS Production Analysis
Vero cells seeded in a 96-well plate were treated with a series of MOH concentrations and incubated for 1 h. Then, 10 µL of H 2 O 2 (1 mM) was added and incubated at 37 • C for 24 h. Cells were then subjected to MTT assay. The absorbance of formazan crystals dissolved in DMSO was measured by using SpectraMax M2 microplate reader at 570 nm. Effect of MOH on intracellular ROS levels in H 2 O 2 -induced Vero cells was measured by 2 ,7 -dichlorofluorescein diacetate (DCF-DA) assay. In brief, seeded cells were incubated for 24 h and then treated with a series of concentrations of MOH for one hour. Then, 10 µL of H 2 O 2 (1 mM) was added to each well and incubated for another 1 h. Finally, these cells' intracellular ROS levels were determined after adding 10 µL DCF-DA (500 µg/mL) by using a microplate reader, and images were captured by using Invitrogen™ EVOS™ M5000 fluorescence microscope (Thermo Fisher Scientific, Waltham, MA, USA).

Evaluation of Apoptotic Body Formation
The nuclear morphology of the H 2 O 2 -induced Vero cells was observed to identify the apoptotic body formation. For that, seeded cells were treated with a series of MOH concentrations, incubated for 1 h, and then treated with 1 mM H 2 O 2 . After 24 h, cells were stained with 10 µL of Hoechst 33,342 (0.5 mg/mL) and propidium iodide (2.5 µM). After 10 min of incubation, nuclear morphology was examined by using Invitrogen™ EVOS™ M5000 fluorescence microscope.

Cell Cycle Analysis
The cell cycle was investigated according to the procedure described in one of the previous studies [28]. In brief, the cells were rinsed with PBS and permeabilized in 70% ethanol for 30 min after harvesting. Then, the cell pellets were centrifuged again after being gently resuspended in PBS containing EDTA. For 30 min, the fresh pellet was gently resuspended in PBS solution containing PI, EDTA, and RNase A. The cells were then examined using a Beckman Colter CytoFLEX system flow cytometer (Brea, CA, USA).

Mitochondrial Depolarization Analysis by JC-1 Assay
Cultured cells were harvested after 4 h of sample treatment followed by stimulation and the mitochondria membrane potential was measured using MitoProbe JC-1 Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions followed by flow cytometric analysis.

Western Blot Analysis
Cells were seeded in 10 cm culture dishes for 24 h at 2 × 10 5 cells/mL concentration and stimulated with 1 mM H 2 O 2 after being treated with 15.6, 31.3, and 62.5 µg/mL of MOH for 2 h. Herein, 50 µM vitamin C was used as a positive control. Then, the cells were harvested for western blot analysis. Insoluble materials were removed from cell lysate by centrifugation followed by lysis. Cells were lysed by a nuclear and cytoplasmic extraction kit, NE-PER ® (Thermo Scientific, Rockford, IL, USA). The protein concentrations in cell lysate were estimated using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). After estimation was completed, 30 µg of protein of each lysate were subjected to electrophoresis on 10% polyacrylamide gels. Resolved protein bands were transferred onto nitrocellulose membranes (Merck Millipore, Dublin, Ireland), blocked with 5% skim milk in TBST, and then incubated with primary antibodies and HRP-conjugated secondary antibodies. Then, identified protein bands were visualized by an enhanced chemiluminescence (ECL) western blotting detection kit followed by imaging on the Core Bio Davinch-Chemi TM imaging system (Seoul, Korea).

Statistical Analysis
All the relevant data were presented as the mean ± standard error of the mean (SEM), while all statistical analyses were performed using the SPSS software (Version 24.0, Chicago, IL, USA). The values were evaluated and significant variations among data sets were obtained by using one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests, and p < 0.05 was considered as statistically significant.

Extraction Yield and Proximate Composition of MOH
According to the results indicated in Table 1, the polysaccharide content was relatively higher than the polyphenol composition and protein content of MOH on a dry basis (%).

Composition of Antioxidant Phytochemicals in MOH
HPLC was used to examine phenolic compounds in MOH. The chromatograms obtained by the MOH were compared to seventeen distinct phenolic standards that had been predetermined ( Figure S1). HPLC analysis revealed the presence of fourteen out of seventeen phenolic standards in the MOH. D-mandelic acid, gentisic acid sodium salt hydrate, and syringic acid were not detected in the MOH, but 3-hydroxy benzoic acid was identified in the greatest concentration of 95.64 ± 0.36 µmol/100 g, and rutin hydrate was found in the lowest concentration, which was 1.11 ± 0.43 µmol/100 g, as shown in Table 2. Corresponding chromatograms are provided with Supplementary Materials ( Figure S1).

Antioxidant Activities of MOH
The decolorization of the ABTS + extent is determined by the percentage inhibition of the ABTS + radical cations [29,30]. DPPH absorbs at 515 nm in the radical form, but this absorption is reduced when it is reduced by an antioxidant or a radical species [29,30]. In the FRAP, the antioxidant activity of an extract is determined based on the ability to reduce ferric (III) iron to ferrous (II) iron. MOH indicated substantial ABTS + and DPPH radical scavenging activity with respective IC 50 values of 102.52 and 122.55 µg/mL ( Figure 1A,C). Scavenging activity increased with the MOH concentration, with a maximum scavenging activity of 60.05 ± 0.24% and 51.48 ± 0.41%, respectively, at 125 µg/mL. The MOH had a significantly low FRAP value compared to the positive control, vitamin C ( Figure 1B). The FRAP of the MOH indicated a significant and dose-dependent increase with a maximum absorbance of 0.2398 at 125 µg/mL ( Figure 1B). The ORAC of the MOH was 1004.95 µmol TE/mg of the sample, which is illustrated in Figure 1D.

Antioxidant Activities of MOH
The decolorization of the ABTS + extent is determined by the percentage inhibition of the ABTS + radical cations [29,30]. DPPH absorbs at 515 nm in the radical form, but this absorption is reduced when it is reduced by an antioxidant or a radical species [29,30]. In the FRAP, the antioxidant activity of an extract is determined based on the ability to reduce ferric (III) iron to ferrous (II) iron. MOH indicated substantial ABTS + and DPPH radical scavenging activity with respective IC50 values of 102.52 and 122.55 µg/mL ( Figure  1A,C). Scavenging activity increased with the MOH concentration, with a maximum scavenging activity of 60.05 ± 0.24% and 51.48 ± 0.41%, respectively, at 125 µg/mL. The MOH had a significantly low FRAP value compared to the positive control, vitamin C ( Figure  1B). The FRAP of the MOH indicated a significant and dose-dependent increase with a maximum absorbance of 0.2398 at 125 µg/mL ( Figure 1B). The ORAC of the MOH was 1004.95 µmol TE/mg of the sample, which is illustrated in Figure 1D.

Effects of MOH on Cell Viability and Intracellular ROS Production
According to measured intracellular ROS inhibition in Vero cells, the MOH indicated good antioxidant effects. As shown in Figure 2A, the MOH concentrations used were not cytotoxic towards the Vero cells up to 125 µg/mL. Therefore, the concentrations of 15.6 µg/mL, 31.3 µg/mL, and 62.5 µg/mL were used throughout the study. Contrary to this, the cell viability of H2O2-induced Vero cells was decreased compared to control cells, while MOH-treated cells showed an increase in dose-dependent cell viability (31.3-125 µg/mL) ( Figure 2C). Vitamin C was used as a positive control. H2O2 boosted the intracellular ROS production in Vero cells and showed a reduction in ROS production in a dose-

Effects of MOH on Cell Viability and Intracellular ROS Production
According to measured intracellular ROS inhibition in Vero cells, the MOH indicated good antioxidant effects. As shown in Figure 2A, the MOH concentrations used were not cytotoxic towards the Vero cells up to 125 µg/mL. Therefore, the concentrations of 15.6 µg/mL, 31.3 µg/mL, and 62.5 µg/mL were used throughout the study. Contrary to this, the cell viability of H 2 O 2 -induced Vero cells was decreased compared to control cells, while MOH-treated cells showed an increase in dose-dependent cell viability (31.3-125 µg/mL) ( Figure 2C). Vitamin C was used as a positive control. H 2 O 2 boosted the intracellular ROS production in Vero cells and showed a reduction in ROS production in a dose-dependent manner by MOH-pretreated cells ( Figure 2B). Figure 2D  dependent manner by MOH-pretreated cells ( Figure 2B). Figure 2D indicates the fluorescence microscopy analysis of the inhibitory effects of the MOH against H2O2-induced oxidative stress in Vero cells. According to the results, H2O2-stimulated cells unveiled higher green fluorescence for DCFH-DA compared to the control group. MOH-pretreated Vero cells indicated a dose-dependent reduction in the green fluorescence supporting the potential antioxidant activities of MOH against H2O2-induced intracellular ROS generation in Vero cells.

Effect of MOH on H2O2-Induced Apoptosis
In the Hoechst (33342) and PI nuclear double staining, cells with equally stained nuclei are considered viable cells, where chromatin condensation and fragmentation indicate apoptosis, and red to orange nuclei indicate necrotic death of cells [31]. As Figure 3A indicates, H2O2-induced Vero cells indicated nuclear condensation and fragmentation. Dose-dependent treatment of the MOH reduced the formation of the apoptotic bodies by reducing the condensation of chromatin and nuclear fragmentation. H2O2-induced Vero cells without pretreatment of the MOH showed necrotic cells. These results were compatible with the results of the cell cycle analysis conducted with flow cytometry. Cell cycle analysis was performed after PI staining to test if the cells were undergoing apoptosis. The hypodiploid cell population in the Sub-G1 phase was analyzed by flow cytometry.

Effect of MOH on H 2 O 2 -Induced Apoptosis
In the Hoechst (33342) and PI nuclear double staining, cells with equally stained nuclei are considered viable cells, where chromatin condensation and fragmentation indicate apoptosis, and red to orange nuclei indicate necrotic death of cells [31]. As Figure 3A indicates, H 2 O 2 -induced Vero cells indicated nuclear condensation and fragmentation. Dose-dependent treatment of the MOH reduced the formation of the apoptotic bodies by reducing the condensation of chromatin and nuclear fragmentation. H 2 O 2 -induced Vero cells without pretreatment of the MOH showed necrotic cells. These results were compatible with the results of the cell cycle analysis conducted with flow cytometry. Cell cycle analysis was performed after PI staining to test if the cells were undergoing apoptosis. The hypodiploid cell population in the Sub-G 1 phase was analyzed by flow cytometry. According to the findings ( Figure 3B), an increase in the Sub-G 1 apoptotic cell population (32.72%) further indicates H 2 O 2 -induced apoptosis compared to the nonstimulated control (1.78%). The MOH treatment dose-dependently reduced the Sub-G 1 apoptotic cell population.

Effect of MOH on Mitochondrial Depolarization and Expression Levels of the Proteins Related to Mitochondrial Apoptosis Pathway
The JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolyl carbocyanine iodide) has been used for many years as a specific membrane-permeable dye for measuring mitochondrial membrane potential. Emission of the red color indicates healthy mitochondria. The decreased potential of the mitochondrial membrane causes a significant shift in the blue laser excitation from orange to green fluorescence emissions at 488 nm. Based on the data shown in Figure 4A Results from nuclear double staining with Hoechst and PI and JC-1 assay, predicted that apoptosis in H 2 O 2 -induced Vero cells happened through a mitochondria-mediated apoptotic pathway. To examine the mitochondria-mediated apoptosis, levels of Bcl-xL, Bcl-2, Bax, caspase 3, p53, cleaved PARP, cleaved caspase 9, and cytochrome c were investigated by using western blotting. Figure 4B shows that Bcl-xL and Bcl-2 were suppressed and Bax, caspase 3, p53, cleaved PARP, cleaved caspase 9, and cytochrome c were increased by H 2 O 2 in Vero cells. Hence, MOH controls the mitochondria-mediated apoptosis by increasing the levels of Bcl-xL and Bcl-2 and decreasing the levels of BAX, Caspase 3, p53, cleaved PARP, cleaved caspase 9, and cytochrome c ( Figure 4B). The suppression of apoptosis was mediated via the mitochondrial apoptosis pathway.

Effect of MOH on Activation of Nrf2/HO-1/NQO1 Signaling Pathway
Activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling pathway has been shown to reduce ROS generation in cells in previous research [32]. The presence of green fluorescence in the nucleus can be used to identify Nrf2 nuclear translocation in an immunostaining experiment. The strong green fluorescent signal implies that Nrf2 nuclear translocation is increasing. Figure 5A shows that pretreatment with the MOH enhanced Nrf2 signaling in H 2 O 2 -stimulated Vero cells by boosting Nrf2 nuclear translocation in a dose-dependent manner. According to the results of western blot analysis, H 2 O 2 -stimulation raised the levels of nuclear Nrf2 as well as cytosolic HO-1 and NQO1 in Vero cells, followed by a substantial and dose-dependent increase in the levels of nuclear Nrf2 ( Figure 5B).

Discussion
The existence of selected phenolic components in Moringa oleifera hot water extract (MOH) and its antioxidant activity against H2O2-induced oxidative stress in Vero cells

Discussion
The existence of selected phenolic components in Moringa oleifera hot water extract (MOH) and its antioxidant activity against H 2 O 2 -induced oxidative stress in Vero cells were investigated in this study. The hot water extraction method was selected to extract M. oleifera because of its efficiency with a reduced cost. In addition, the hot water extraction method is useful in industry and can be used to extract ingredients for food and cosmetics [33]. Based on the outcomes of the study, the extraction yield of MOH was 35.67 ± 0.44%, while the previous study reported a yield of 28.26% per 90 min soaking at 121 ± 0.5 • C water [33]. According to the current observations, MOH had a relatively high polysaccharide content compared with protein while having a comparatively low polyphenol content. The present study reports polyphenol content of 5.24 ± 0.07% (w/w) in MOH, while Sreelatha, S. and Padma, P.R. (2009) reported 4.58% in Soxhlet extraction for 18-20 h of M. oleifera leaves [34]. Polyphenols are recognized as antioxidants due to their hydroxyl groups' capacity to scavenge free radicals, which have a significant in vitro effect [34,35]. Glucosinolates, and potentially alkaloids, in addition to polyphenols, are thought to be responsible for the bioactive effects of M. oleifera as reported before [36]. Pawlowska, E. et al. particularly mentioned the presence of two hydroxyl groups in their B ring structure in some dietary polyphenols, such as quercetin and cyanidin-3-glucoside affect directly to scavenge ROS in cells [37]. Young Chool Boo reported that the phenolic compounds derived from a variety of plants can lower the levels of ROS in cells and/or enhance cellular antioxidant capacity and anti-inflammatory effects [38]. Aside from direct ROS scavenging, dietary phenols can reduce oxidative stress through a variety of mechanisms such as inducing Nrf2 activation [37]. Kanner J.'s review contains evidence that polyphenols can inhibit cellular protein tyrosine phosphatases and activate cell signaling through transcription of the Nrf2 axis to adapt and protect cells against oxidative stress by producing H 2 O 2 at low levels (1-10µM) [39]. In this study, the presence of gallic acid, 2,3,4-trihydroxy benzoic acid, 3,4-dehydroxy benzaldehyde, 4-hydroxy benzoic acid, catechin hydrate, vanillic acid, 3-hydroxy benzoic acid, chlorogenic acid, p-coumaric acid, 3,4 dimethoxy benzoic acid, sinapic acid, rutin hydrate, trans-cinnamic acid, and quercetin in the MOH were confirmed based on the results obtained from HPLC analysis (Supplementary Material Figure S1). According to the results, the total polyphenolic content on a weight basis was found to be 136.28 mg/100 g. Quercetin-like phenolics were present in the MOH, which can be implicated to have positive benefits on human health and to alleviate oxidative stress [40]. Based on the findings, a quantitative difference was observed between total polyphenolic content as determined by proximate composition analysis and HPLC analysis. This suggests that MOH may contain a range of additional polyphenolic compounds, which was not revealed by HPLC analysis in this study. Due to the presence of phenolic compounds in MOH, H 2 O 2 production in Vero cell-cultured media after treatment of MOH for 1 h was analyzed. According to the results obtained from the analysis, it revealed that MOH produces a maximum quantity of 0.235 ± 0.033 µM of H 2 O 2 in cell culture media at 62.5 µg/mL of MOH concentration (Supplementary Material Figure S3). In this study, TLC analysis (Supplementary Material Figure S2) verified the existence of antioxidative chemicals in MOH seen from the change of color in KMnO 4 -stained TLC. The discoloration of KMnO 4 is caused by a reduction in the oxidative state of Mn from +7 to +2 [41]. Furthermore, the occurrence of nucleophiles, aldehydes, and ketones was confirmed by p-anisaldehyde and Vanillin stains, which commonly undergo Aldol condensation and acetalization [42]. UV light with a short wavelength and long wavelength (254 nm and 365 nm, respectively) to enable visualization of TLCs, can be used to identify extended conjugated (aromatic) systems [42]. Bright purple or blue fluorescent represents highlyconjugated compounds, which may be comprised of a single or multiple aromatic rings. Accordingly, the presence of single or multiple aromatic rings in the MOH was confirmed by UV-enabled visualization of TLCs. Unsaturated and aromatic compounds were visualized in a brown color by iodine stains. Saponins and phenols were visualized by the 10% sulphuric acid in ethanol and the ferric chloride stains, respectively [42].
The ABTS + diammonium salt radical cation decolorization test is one of the commonly used spectrophotometric methods to evaluate the antioxidant activity of numerous substances, including plant extracts [43]. 2 ,7 -dichlorodihydrofluorescein (DCFH) is highly sensitive to ROS and can be oxidized to a highly fluorescent 2 ,7 -dichlorofluorescein (DCF) by ROS [44]. DCF can be seen with green fluorescence in fluorescence microscopic observations. During reactions between DPPH radicals and antioxidative extracts or antioxidative compounds, the absorption reduction with a characteristic wavelength is widely used to evaluate antioxidative activity that can give hydrogen [45]. The ABTS assay revealed that ABTS+ radical scavenging activity increases dose-dependently with the MOH. Antioxidant capabilities in fruit, vegetables, and other plant materials as well as their products, have been determined using the DPPH and ORAC assays. Because it uses a biologically relevant radical source, the ORAC assay is believed to be more relevant [46]. The finding of the ABTS assay was strengthened by DPPH scavenging activity and an ORAC value of the MOH. cell membrane permeability and disclosed that polyphenolic compounds (particularly myricetin in the relevant study) can diffuse through the cell membrane into the cells, where they prevent the production of different ROS compounds in the polar intracellular environment [47]. In that sense, the antioxidant effect of the MOH on cells can be justified since it contains numerous phenolic compounds.
Chromatin condensation, membrane surface hemorrhage, phosphatidylserine excretion, DNA fragmentation, and eventual apoptotic body formation are specific morphological and biochemical features associated with apoptosis [48]. Results obtained from the nuclear double staining analysis with Hoechst and PI in this study indicated the effects of MOH on the reduction of the events of apoptosis, including the formation of the apoptotic bodies and DNA damage. The above observations can be presented as evidence of the protective potential of MOH on Vero cells against oxidative stress induced by H 2 O 2 . Moreover, the above finding was confirmed by the reduction in Sub-G 1 apoptotic cells' accumulation along with the results of the JC-1 assay. Collectively, the above evidence suggested the effectiveness of MOH in ameliorating apoptosis in H 2 O 2 -induced Vero cells and the connection between the inhibition of mitochondrial dysfunction and H 2 O 2 -induced apoptosis progression. Based on the above outcomes, further experiments were designed to obtain a better understanding of the role of MOH in apoptosis pathway regulation.
The mitochondria-mediated caspase activation apoptosis pathway is one of the major signaling pathways that mediate apoptosis in the mammalian cells, with the characteristic permeabilization of the mitochondrial external membrane and consequent release of cytochrome c to the cytoplasm [49]. Many studies have reported that the regulation of this pathway depends on various factors including the type of apoptotic stimulant as well as the type of cell undergoing apoptosis [50]. Suppression of the above pathway reduces apoptosis, thereby minimizing the relevant responses such as limiting non-communicable illnesses. Bcl-2 family proteins including antiapoptotic Bcl-2 and Bcl-xL, and pro-apoptotic proteins including Bax, Bak, Bok, Bad, Bid, Bik, Bim, Bmf, Puma, and Noxa are identified as major responsible proteins that control mitochondrial outer membrane permeabilization [50]. The activation of caspase 9 causes the progression of apoptosis. Caspase 9 becomes cleaved caspase 9 when it meets cytochrome c. Mitochondrial pathway-mediated apoptosis is further aggravated by pro-apoptotic stimuli inducing p53, activated caspase-9 and, consequently, activating downstream effector caspases such as caspase −3, −6, and −7. The catalytic activity of PARP is inhibited by effector caspases by cleaving PARP. The present study clarified the effect of H 2 O 2 on the initiation of mitochondria-mediated apoptosis proteins and the dose-dependent attenuation effects of MOH.
The Nrf2-Keap1 system is a physiological thiol-based sensor-effect system in eukaryotes that responds to oxidative stress by maintaining redox homeostasis [5]. Nrf2 has been identified as the primary regulator of the transcription of several antioxidant and cytoprotective genes, whereas regulating oxidative stress and securing physiological homeostasis requires the regulation of antioxidant gene expression. Nrf2 is typically found in the cytosol due to its interaction with a cytosolic actin-binding protein called Keap1 (Kelch-like ECH-associated protein 1), also known as INrf2 (inhibitor of Nrf2) [51]. When cells encounter stress, such as exposure to mild oxidants, Nrf2 dissociates from Keap1, becomes stabilized, and translocases into the nuclei, and promotes the antioxidant response element-driven antioxidant gene transcription of numerous antioxidant genes, including NQO1, HO-1, glutathione S-transferase A2, and glutamate-cysteine ligase [52]. The results of the immunofluorescence analysis of Nrf2 nuclear translocation in H 2 O 2 -induced Vero cells reveal that Nrf2 activation and nuclear translocation were increased by H 2 O 2 stimulation and dose-dependently upregulated by the MOH in Vero cells. Apart from the scavenging of ROS in the cytosol by phenolic compounds, several studies have revealed that the nanomolar range of H 2 O 2 generated in the cell growth media could be penetrating cells, activating Nrf2 signaling, and supporting to adapt to changes in the environment and oxidative stress [39,53]. Moreover, H 2 O 2 is acknowledged as the most important ROS in the redox regulation of biological processes [5]. The activation of Nrf2 signaling in Vero cells can be explained by the fact that MOH generated low levels of H 2 O 2 in the cell growth medium after MOH incubation. Furthermore, western blot analysis, which was conducted to investigate the effect of MOH on H 2 O 2 -induced Vero cells, shows a significant increment of expression levels of nuclear Nrf2, cytosolic HO-1, and NQO1 with the MOH strengthened the antioxidant activity of the MOH on H 2 O 2 -induced Vero cells.

Conclusions
Based on the present study, hot water extract of M. oleifera (MOH) has various phenolic compounds that possess potent antioxidant activity and have an ability to generate a low level of H 2 O 2 in Vero cell-cultured media, which collectively ameliorate H 2 O 2 -induced oxidative stress and apoptosis in Vero cells by suppressing the mitochondria-mediated apoptosis pathway and regulating the Nrf2/HO-1 signaling pathway. Further analysis of MOH bioactivity and safety would potentiate its applications in the manufacturing of functional foods with beneficial bioactivities centered on their antioxidant activity.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/foods11030420/s1, Figure S1: HPLC chromatography of MOH, Figure S2: Thin-layer chromatography (TLC) analysis of MOH, Figure S3: H 2 O 2 production levels in Vero cellcultured media after treatment of MOH for one hour.