Activity Guided Isolation Of Phenolic Compositions From Anneslea Fragrans Wall. And Their Cytoprotective Effect Against Hydrogen Peroxide Induced Oxidative Stress in HepG2 Cells Part 2

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2.7. Inhibitory Effect on Intracellular ROS Generation

Oxidative stress is a kind of imbalance between oxidation and antioxidation in the body [28]. Excessive accumulation of ROS can lead to oxidative stress that may cause damage to cells and tissues such as lipids, membranes, and DNA [29]. Antioxidants including polyphenols and flavonoids can help the body reduce these damages caused by ROS. Generally, H, O, is widely used to induce intracellular ROS disordered production and impair the antioxidant defense of cells [30]. In our present study, H2O2 was chosen to induce the abnormal accumulation of intracellular ROS and impair the antioxidant defense of cells. To evaluate the inhibitory effect on intracellular ROS generation in H, O2-induced HepG2 cells, the levels of intracellular ROS were tested by flow cytometry. Briefly, HepG2 cells were cultured in a 6-well plate(1 × 10° cells/well). After incubation for 24 h of 50 ug/ml of different extracts or 8 ug/mL of Vc (positive group), all the groups except for the control group were induced by HO, for 6 h. The intracellular ROSlevels of each compound were tested(Figure 3). The ROS generation ratio significantly increased to 215.64±9.80% in H2O,-the treated group compared with the control group (100%). Compared with the H, O,-treated group, compounds 2,5, and 6 remarkably suppressed intracellular ROS production (p<0.05), and their inhibitory effect was equal to the Vc group (Figure 3). Many phenolics have been proven to have a protective effect against intracellular ROS by H2O2 induction[31]. Additionally, compound 6 displayed the strongest suppressive effect on intracellular ROS production, which suggests that flavonoid compounds play a crucial role in inhibiting intracellular ROS production.

2.8. Cytoprotective Effect Against H2O2-Induced Cell Apoptosis

Apoptosis is a basic biological phenomenon of cells, which plays an important role in the regulatory mechanism of cells' proliferation, growth, and mutation, and the stability of the internal environment. Apoptosis, different from necrosis, is a special type of cell death. The disorder of the apoptotic process has bad effects on the body and causes many diseases [32]. H2O2, as an important signaling molecule, regulates the process of cell proliferation, growth, and apoptosis [5]. The present study measured the apoptosis of H, O,-induced HepG2cells and evaluated the cytoprotective effects of compounds 2, 5, and 6. After treating HepG2 cells with 1.0 mM H2O2, the apoptosis ratio was remarkably augmented (57.20±1.97%), compared with that of the control group (9.10±0.62%,p<0.05)(Figure 4). The ratios of apoptotic cells in the treated groups of compounds 2, 5, and 6 significantly decreased compared with the H2O2-treated group (model group, p<0.05)(Figure 4). Moreover, compound 6 had significant efficiency in protecting Hepa-2 cells from H2O2 toxicity, and the cell apoptosis ratio of 6(10.56±1.15%)was lower than that of the Vc group (positive control), which was equal to that of the control group(Figure 4). Meanwhile, compounds 2 and 5 showed a moderate cytoprotective effect with cell apoptosis ratios of 2(26.76±2.60%) and 5(27.64±0.83%). The differences in antioxidant capacity may be attributed to the number of phenolic hydroxyl moieties and the link positions.

3. Materials and Methods

3.1. Chemicals and Reagents

Methanol, acetonitrile, and formic acid for high-performance liquid chromatography (HPLC) were of HPLC grade and purchased from Merck(Darmstadt, Germany). Solvents for sample extraction including ethanol, dichloromethane, and n-butanol were of analytical grade. Deionized water was purified using a Milli-Q ultrapure water system (Millipore, Bedford, Massachusetts, MA, USA) and employed in all the experiments. Phenolic standard compounds of gallic acid, rutin, Trolox, and vitamin C were purchased from Chengdu Must Bio-Technology Co., Ltd.(Chengdu, China). Methylthiazol-2-yl-25-diphenyl tetrazolium bromide (MTT), Folin-Ciocalteu reagent, 2,2'-and-bis(3-ethylbenzene-thiazoline-6-sulfonic acid)(ABTS),2,2-diphenyl-1-picrylhydrazylradical (DPPH), 1,3,5-tri(2-pyridyl)-2,4,6-triazine (TPTZ),2',7'-dichlorofluorescein diacetate (DCFH-DA), and FeSO4-7HOwere purchased from Sigma-Aldrich (Shanghai, China). The NMR spectra were obtained using Bruker AV-400, and/or DRX-500 spectrometers. ESIMS spectra were recorded on An Agilent 1290 UPLC/6540 Q-TOF spectrometer.

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3.2. Sample Preparation

Anneslea fragrans Wall. leaves were collected from the Lincang city of China in July 2020. The leaves were dried in a shaded room until constant weight and then were powdered with an electric grinder. The extraction and fractionation were performed as our previously reported method with slight modification [33]. The powdered sample (100 g)was mixed with 1000 mL of 80% aqueous ethanol solvent and ultrasonicated in an ultrasonic cleaning bath at 200 W three times(0.5 h per time). Then, the sonicated slurry was collected and centrifuged at 1500×g for 10 min by Eppendorf centrifuge (TGL-20B, Shanghai Anting Scientific Instrument Factory, Shanghai, China). The combined supernatant was concentrated at 50°Cby a rotary evaporator (Hei-VAP, Heidolph, Germany) and further dried by a vacuum drying lyophilizer(Alpha 1-2 LD plus, Christ, Germany). The crude ethanol extract(CE, 30 g) was re-suspended with water and sequentially partitioned with dichloromethane, ethyl acetate, and n-butanol solvents three times. After concentration and lyophilization, the dichloromethane fraction (DF), ethyl acetate fraction (EAF), an n-butanol fraction(BF), and residual water fraction (RWF)weighing 3.2 g,6.3g,7.2 g, and 8.2 g were obtained, respectively. According to the antioxidant activities of different fractions, the BF was chromatographed on glass columns(30 mm ×400 mm) wet-packed with 20 g(dry resin) of the selected hydrated resin D101. The bed volume (BV) of the resin was about 40 mL. After reaching the adsorptive saturation, the column was first washed by distilled water with 4×BV and then eluted by ethanol-water(0:100,20:80,50:50,80:20,100:0,v/y, each4× BV), to yield five subfractions (BF-A to E). Each part of the desorption solutions was concentrated to dryness under a vacuum. The CE, four fractions (DF, EAF, BF, and RWF), and five subfractions(BF-A to E) were stored in a refrigerator(-20 °C) for further experimentation.

3.3. Bio-Guided Isolation of Active Constituents

Under the guidance of antioxidant assays and HPLC analysis, the antioxidative fraction was further chromatographed for the isolation of pure compounds. In brief, the BF was subjected to a hydrated resin D101 column to yield five subfractions(BF-A to E). The BF-C(1.5g) was subjected to a silica gel column, eluting with DCM/MeOH(15:1), and then was separated using preparative TLC(DCM/MeOH, 10:1) to obtain compounds 6 (118 mg) and 7(10 mg). BF-D(1.1 g) was subjected to silica gel column(CHCl3/MeOH 10:1, 5:1)to give compounds 1(129 mg)and 4(15 mg).BF-E(1.2 g)was subjected to silica gel column (CHCl3/MeOH, 30:1, 10:1, 5:1)to yield compounds 1(216 mg),2(135mg), and a mixture.The latter was purified by silica gel column (CHCl3/MeOH,12:1)to afford compounds 3 (19 mg) and 5(15 mg) (Figure 5).

3.4.Structure Elucidation of Compounds 1-7

According to the antioxidant activities of different fractions, three subfractions, BF-C to E were submitted to column chromatography. In this way, the Bioactivity-guided fractionation of BF-C to E led to the isolation of seven individual phenolic compounds. Their structures were identified as confusoside(1)[34], vaccinifolin (2)[34],1-[4-(β-D-glucopyranosyloxy)-2-hydroxyphenyl]-3-(4-hydroxy-3-methoxyphenyl)-1-propanone (3) [35], (S)-naringenin-7-O-β-D-glucopyranoside (4)[22],2',3,4,4'-tetrahydroxydihydrochalcone(5)[26], (epi)-catechin (6)[26], and cornoside (7)[36](Figure 1B)by the analysis of 1D-NMR and ESI-MS data and comparison with the previously reported compounds in the literature. Among them, compounds 3,4, and 7 were isolated from this plant for the first time.

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Confusoside (1). The molecular formula was assigned as C21H24O,, and the separation gave 129 mg of pale-yellow needles. 'IH NMR (500 MHz, DMSO-d6) and ESI-MS(m/z 421 [M+H])data were identical to the previously reported compound in the literature [34]. The identification was further supported by 13C NMR（125 MHz，DMSO-dg）∶δ204.4 （s，C=O),163.5(s,C-2'),163.3(s,C-4'),155.5(s,C-4),132.6(d,C-6'),130.9(s,C-1),129.2(d, C-2, 6),115.0(d,C-3/C-5),114.4(s, C-1'),108.3(d, C-5'),103.4(d,C-1"),99.6(d,C-3'),77.1(d, C-5"),76.4(d, C-3"),73.1(d,C-2"),69.5(d, C4'),60.5(t,C-6'),39.8(t,C-α),28.9(t, C-6).

Vacciniifolin (2) was obtained as yellow amorphous powder(153 mg) and assigned a molecular formula of C2H24O1o.'H NMR(500 MHz, DMSO-dg) and ESI-MS (m/z 437 [M+H])data were the same as the previous reported data [34]. The identification was further supported by 13CNMR（125 MHz，DMSO-de）∶δ204.4 （s， C=O），163.5（s， C-2'），163.3 (s, C-4),145.0(s, C-3),143.3(s, C-4),132.6(d, C-6),131.7(s, C-1),118.9(d, C-6),115.8(d, C-2),115.4(d, C-5),114.4(s, C-1'), 108.3(d, C-5'),103.4(d, C-1'),99.6(d,C-3'),77.1(d, C-5"),76.4(d,C-3'),73.1(d,C-2"),69.5(d,C-4'),60.5(t,C-6'),39.4(t,C-a),29.1(t, C-6).

1-[4-(β-D-Glucopyranosyloxy)-2-hydroxyphenyl]-3-(4-hydroxy-3-methoxyphenyl)-1-propanone (3). The compound was obtained as colorless needles(19mg), and the molecular formula was assigned as C22H2O10.'H NMR(400 MHz, DMSO-d6) and ESI-MS (m/z451 [M+ H]*) data agreed with the literature [35]. The identification was further supported by 13CNMR（100 MHz，DMSO-d）∶δ204.5（s，C=O），163.5（s，C-2'），163.3（s，C-4'），147.4（s，C-3),144.6(s,C-4),132.6(d,C-6),131.6(s,C-1),120.4(d,C-6),115.2 (d, C-5),114.5(s,C-1'), 112.6(d,C-2),108.3(d,C-5'), 103.3 (d,C-1"),99.5 (d, C-3'),77.0(d,C-5'),76.3(d, C-3"), 73.0(d,C-2"),69.5(d,C-4'),60.5(t,C-6'),55.5(q,C-OCH3),39.5(t, C-α),29.4(t,C-6).

(S)-Naringenin-7-O-β-D-glucopyranoside (4)had the molecular formula of C21H2.O10, which was obtained as 15 mg of white powder. IH NMR (500 MHz, DMSO-dg)and ESI-MS (m/z 435 [M+H]+)data were in agreement with the previous work[22]. The identification was further supported by13C NMR（125 MHz，DMSO-d）∶δ197.2 （s， C-4），165.3（s， C-7），165.2 (s, C-5), 162.9(s, C-9), 157.9(s, C-4'),128.6(s, C-1'), 128.4(d, C-2',6'),115.2 (d, C-3'/C-5'),103.2 (s,C-10),99.6(d,C-1'),96.5(d,C-6),95.4(d,C-8),78.7(d,C-2),77.0(d, C-5"),76.3 (d,C-3'),73.0(d,C-2"), 69.5 (d,C-4'),60.5(t,C-6'),42.0(t,C-3). 2'3,4,A'-Tetrahydroxydihydrochalcone (5) possessed the molecular formula as C15H4O5 and separated (15 mg) as white powder.'H NMR(400 MHz, DMSO-dg)and ESI-MS (m/z 275 [M+H]+) data were consistent with that reported in the literature [26]. The identification was further supported by 13C NMR（125 MHz， DMSO-d6）∶δ203.9 （s， C=O），164.7(s,C-2'),164.2(s,C-4'),144.9(s, C-3),143.3(s, C-4),133.0(d,C-6),131.8(s,C-1),118.9 (d,C-6),115.7(d,C-2),115.4(d,C-5),112.5 (s,C-1),108.2(d,C-5'),102.4(d,C-3'),39.5(t, C-a2),29.2(t, C-B). (Epi)-catechin(6) was isolated as white powder(118 mg)and established the molecular formula as CHO4.1H NMR (400 MHz, DMSO-dg) and ESI-MS (m/z 291 [M+ H]+)data agreed well with that reported in the literature [26]. The identification was further supported by13C NMR （125 MHz，DMSO-d6）∶δ156.4（s，C-7），156.1（s， C-5），155.3（s，C-9），144.8(s,C-3'),144.8(s,C-4'),130.5(s,C-1'),118.4(d,C-6'),115.0(d,C-5'),114.4(d,C-2'), 99.0(s,C-10),95.1(d,C-6),93.8(d, C-8),80.9(d, C-2),66.2 (d,C-3),27.8(t, C-4). Cornoside (7) was obtained as amorphous solid (10mg) and determined the molecular formula as C1H20O8.'H NMR (500 MHz,DMSO-dg)and ESI-MS (m/z317 [M+H]+)data corresponded with the published data [36]. The identification was further supported by 13CNMR（100 MHz，DMSO-dg）∶δ185.3（s， C-4），153.3（d，C-2），153.2 （d，C-6），126.4（d， C-3），126.4(d,C-5),102.8(d,C-1'),76.8(d,C-5'),76.6(d,C-3'),73.3(d,C-2'),70.0(d,C-4),67.3(s, C-1), 63.8(t, C-8), 61.0(t, C-6'), 39.7(t, C-7).

3.5.Determination of Total Phenolic(TPC) and Total Flavonoid Contents(TFC)

The TPC and TFC of four fractions(DF, EAE, BF, and RWF) and five subfractions (BF-A to E)were measured according to our previously reported method [37]. For TFC,1.0 mL of each sample (with the concentration at 1.0 mg/mL) dissolved in methanol was mixed with 0.5 mL of Folin-Ciocalteu reagent in a centrifuge tube and incubated for 1 min. Then, 20%Na2CO3 solution (m/o)(1.5 mL) and deionized water(7.0 mL) were added to the tube and kept at 70°C in a water bath for 10 min. After being cooled to room temperature, 200 uL of the solution was transferred to a 96-well microplate and the absorbance was determined at 765 nm by a SpectraMax M5 microplate reader(Molecular Devices, Sunnyvale, CA, USA).

For TFC,1.2 mL of sample solutions(with the concentration at 1.0mg/mL) were mixed with 0.3 mL of NaNO, (5%m/o) and 3.8 mL of 70%aqueous ethanol and incubated for 8 min.Subsequently,0.3 mL 10% aqueous Al(NO3),4 mL4% aqueous NaOH, and 0.4 mL 70% aqueous ethanol was added to the mixture and allowed to react at room temperature for 30 min. Then， 200 μL of the solution was transferred to a 96-well microplate， for which the absorbance was measured at 510 nm by using a microplate reader. The TFC and TPC were expressed as milligrams of gallic acid equivalents (mg GAE/gextract) and rutin equivalents per gram of extract (mg RE/g extract).

3.6.Determination of Antioxidant Actiovity

The antioxidant activity of four fractions (DF, EAF, BF, and RWF) and five subfractions (BF-A to E)were evaluated in a combination of DPPH and ABTS radical scavenging assays and FRAP assay based on the method described in our previous study [33]. For DPPH assay, 50μL of the sample solution （50，100，200ug/mL）was mixed with 0.2 mL DPPH solution (0.1 mmol/L) in a 96-well plate and allowed to incubate for 30 min. The absorbance was measured at 517 nm with SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA， USA）. For ABTS， 25 μL of the sample solution（50， 100， 200 μg/mL）were added to 0.2mLABTSsolution(7 mmol/L).The mixture was kept in the dark for6min, and then, the absorbance was recorded at 734 nm. For FRAP， 20 μL sample solution（50，100，200ug/mL）was mixed with0.18 mL of FRAP reagent (7 mmol/L). After incubating for 10 min in the dark at 37°C, the absorbance was determined at 593 nm. All the tests were performed in triplicate. The results of DPPH, ABTS, and FRAP values were expressed as umol Trolox equivalents per gram of extract （μmol TE/g extract）.

3.7.HPLC Analysis

The BF and compounds 1-7 were analyzed on an Agilent 1260 HPLC system coupled with a diode array detector. Before analysis, the freshly prepared sample solution was filtered through a 0.45 um nylon membrane. The separation was performed using a Reprosil-Pur Basic C18 column （5 μm， 4.6×250 mm， Germany）maintained at 35°C. The injection volume was 5.0 μL， the flow rate was 1.0 mL/min， and the detection wavelength was set at 280 nm. The mobile phases were acidified water with 0.1% formic acid (phase A)and acetonitrile (phase B), the linear gradient elution was performed as follows:0-3 min, 20% B;3-10 min, 40%B;10-15 min, 60% B;15-20 min,100%B.

3.8. Cell Culture and Cell Viability

Human liver cancer HepG2 cells were purchased from Kunming Cell Bank (Kunming, China). HepG2 cells were grown in DME supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum in an atmosphere of 5%CO,/95% air at 37°C.When the cells were incubated to an appropriate density (approximately 80%), they were treated with the positive control (Vc) and the isolated compounds for further experiments.

Cell viability was determined by MTT assay for evaluating the cytotoxicity of each sample [29]. The cells at a density of 1×104 cells per well were seeded in a 96-well plate and allowed to incubate for 24 h. Each compound (prepared as four doses from 50 to 200 μg/mL） was added to each well for 20 h. Then， the cells were treated using MTT solution with a final concentration of 4 h. The medium with MTT was removed, and 200 μL of the DMSO was added to dissolve the formazan. The absorbance was recorded at 570 nm by a microplate reader. The results demonstrated that each compound was nontoxic to HepG2 cells at the tested concentrations.

3.9.Inhibition of ROS Generation in H2O2-Induced HepG2 Cells

H,O,-induced HepG2 cells were employed to determine the inhibitory effect on ROS production [3]. HepG2 cells (1.0×10° cells per well)were seeded in a 6-well plate and co-cultured with isolated compounds with 50 μg/mL and Vc（8ug/mL）. After incubation for 20h， the medium was removed， and 2 μL of H， O，（0.5 mM）was added to each well for another6 h. At the end of the experiment， the cells were labeled with 2 μL DCFH-DA（10 mM）in the dark at 37°C for 20 min. The absorbance was recorded by flow cytometry (Guava easy yet 6-2L, Millipore, Billerica, Massachusetts, MA, USA).

3.10.Determination of Cell Apoptosis

The protective effect of each compound on H2O2-induced apoptosis of HepG2 cells was determined using a human annexin VFITC/PI apoptosis kit [38]. HepG2 cells were pre-treated with or without isolated compounds for 48 h. After incubation，100 μL of the binding buffer was added to the cells， and the cells reacted in the dark with 10 μL annexin V-FITC for 5 min at room temperature and with 10 μL propidium iodide （PI） in an ice bath for 5 min, successively. Cell apoptosis was immediately analyzed using flow cytometry.

3.11.Statistical Analysis

All the experiments were performed in triplicate. All the values are expressed as mean ± standard deviation (SD). The differences within and between the groups were analyzed using a one-way analysis of variance(ANOVA)followed by Tukey's test. The difference was considered statistically significant at p<0.05. All analyses were performed using Origin 2019b software (OriginLab, Northampton, MA, USA).

4. Conclusions

In this study, different fractions from A.fragrans leaves were fractionated, and their TPC and TFC were analyzed. Under antioxidant activity guided isolation, compounds 1-7, including four flavonoid glycosides(1-4) and two flavonoids (5 and 6), were isolated and identified from A. fragrans leaves, which suggested that this species is rich in flavonoid compounds. Compounds 2, 5, and 6 showed significant antioxidant activity in DPPH, ABTS radical scavenging, and FRAP assays. Furthermore, they visibly prevented oxidative stress damage through a decrease in ROS content and cell apoptosis in H2O2-induced HepG2 cells. According to these results, polyphenol compounds, especially flavonoids, have considerable antioxidant capacity because of their phenolic hydroxyl groups. Furthermore, compound 2, possessing the glycoside moiety and three phenolic hydroxyl groups, was the main antioxidant component with the highest content from A. fragrans leaves. Compound 6 displayed the best antioxidant activity, which may be a major contribution to the activity of A.fragrans. Furthermore, the extracts of A.fragrans could be served as a feasible natural source of antioxidants in promising health beverages. The study on the compounds from A. fragrans leaves suggests that these could be served as an antioxidant healthy tea for treating oxidative stress-induced cell damage and could serve as nutritional supplements applied in the food and health industry.

This article is extracted from Molecules 2021, 26, 3690. https://doi.org/10.3390/molecules26123690 https://www.mdpi.com/journal/molecules