0.653 红枣中生物活性物质在神经元的PC-12细胞氧化应激的保护作用

Food Sci. Biotechnol. 24(6): 2219-2227 (2015)DOI 10.1007/s10068-015-0296-4Protective Effects of Bioactive Phenolics from Jujube (Ziziphus jujuba) Seeds against H2O2–inducedOxidative Stress in Neuronal PC-12 CellsJuhee Choi1,2, Xiangxue An1,2, Bong Han Lee1,2, Jong Suk Lee3, Ho Jin Heo4, Taewan Kim5,Jang-Woo Ahn6, and Dae-Ok Kim1,2,*1Department of Food Science and Biotechnology, Kyung Hee University, Yongin, Gyeonggi 17104, Korea2Skin Biotechnology Center, Kyung Hee University, Suwon, Gyeonggi 16229, Korea3Gyeonggi Biocenter, Gyeonggi Institute of Science and Technology Promotion, Suwon, Gyeonggi 16229, Korea4Division of Applied Life Science, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Gyeongnam 52828, Korea 5Department of Food Science and Biotechnology, Andong National University, Andong, Gyeongbuk 36729, Korea6Depratment of Food Nutrition, Chungkang College of Cultural Industries, Icheon, Gyeonggi 17390, KoreaIntroductionZiziphus jujuba (jujube) is a tree of the Rhamnaceae family that isdomesticated worldwide and found primarily in the subtropicalregions of Asia and America (1). Jujube is consumed in both freshand dried forms, and is used in a variety of products including breads,cakes, candies, and compotes. In Korea, jujube is used as aningredient in porridge, rice cakes, and yakbap (a sweet Korean dishwhose name literally means medicinal rice). Jujube has also recentlycome into prominent use as an ingredient in processed foods such asdrinks and tea granules in Korea (2,3). Furthermore, jujube is alsoused as traditional medicine in Korea (4), where it is reported topossess numerous health-promoting benefits such as antioxidantactivity (5), anticancer effects (6), and hepatoprotective effects (7).Degenerative brain diseases such as Alzheimer’s disease (AD) andParkinson’s disease occur in elderly individuals. Senile plaques andneurofibrillary tangles are characteristic features of AD, which isassociated with profound memory loss, learning dysfunctions, andbehavioral disturbances (8,9). Although the etiology of AD remainsunclear, continued oxidative stress of polyunsaturated fatty acids,DNA, and proteins has been suggested to play an important role in itspathogenesis and progression (10). Indeed, hydrogen peroxide(H2O2), one of the major reactive oxygen species (ROS), is producedduring redox processes and results in lipid peroxidation, proteinoxidation, and DNA damage (11).Polyphenols as secondary metabolites are reported to exert a widerange of physiological effects, partly due to their varied structures, andcan protect plants from attacks by pathogens such as bacteria andviruses (12). Polyphenols comprise the major organoleptic characteristicsof plant-derived foods, including color and taste. Flavonoidsconstitute one of the most abundant groups among polyphenols andhave many interesting biological and pharmacological activities Received June 1, 2015Revised August 13, 2015Accepted August 19, 2015Published online December 31, 2015*Corresponding AuthorTel: +82-31-201-3796Fax: +82-31-204-8116E-mail: DOKIM05@khu.ac.krpISSN 1226-7708eISSN 2092-6456© KoSFoST and Springer 2015Abstract Ziziphus jujuba (jujube) seeds were extracted with 60% (v/v) aqueous ethanol assisted byhomogenization and sonication followed by fractionation into n-hexane, chloroform, ethyl acetate, n-butanol, and water. The ethyl acetate fraction exhibited the highest levels of total phenolics (102.05 mggallic acid equivalents/g fraction), total flavonoids (35.43mg catechin equivalents/g fraction), andantioxidant capacity (271.67 and 66.02mg vitamin C equivalents/g fraction in respective ABTS andDPPH assays) among the fractions tested. Ultra-high-performance liquid chromatography-electrosprayionization-tandem mass spectrometry analysis of the ethyl acetate fraction revealed 12 phenoliccompounds: 3,4-dihydroxybenzoic acid, methyldopa, p-coumaric acid, dihydro-p-coumaric acid, rutin,isoquercetin, kaempferol 3-O-rutinoside, 3-O-cis-p-coumaroylalphitolic acid, 2-O-cis-p-coumaroylalphitolicacid, 3-O-trans-p-coumaroylalphitolic acid, 3-O-cis-p-coumaroylmaslinic acid, and 3-O-trans-p-coumaroylmaslinic acid. Phenolic compounds generally increased viability, reduced lactatedehydrogenase release, and attenuated intracellular oxidative stress in PC-12 cells. The results of thisstudy suggest that Z. jujuba seeds may serve as an effective source of antioxidative agents to reduceoxidative stress and enhance neuronal cell viability.Keywords: lactate dehydrogenase release, liquid-liquid extraction, tandem mass spectrometry, totalphenolics, vitamin C equivalent antioxidant capacity2220Choi et al.Food Sci. Biotechnol.including antiinflammatory, antitumor, antimicrobial, antiulcer, and antioxidative activity (12,13). Antioxidants are substance that are present at low concentrations compared to oxidant compounds and act through several distinct mechanisms including scavenging ROS or their precursors and inhibiting ROS formation (14). Cells and tissues in the body are constantly under attack by free radicals, which are produced by oxygen metabolism and exogenous/environmental factors such as UV radiation. Cells in the brain are relatively susceptible to lipid peroxidation because brain cells consume a large amounts of oxygen and have high concentration of polyunsaturated fatty acids (10), and naturally occurring flavonoids with antioxidant capacity may contribute to neuroprotective effects in the brain.Many studies have reported the antioxidant capacity and beneficial health effects of Z. jujuba , including towards AD (1,5-7,15). It was previously reported that Z. jujuba fruits had procyanidin B2, epicatechin,quercetin 3-O -rutinoside, quercetin 3-O -galactoside, kaempferol-glucosyl-rhamnoside, whereas Z. jujuba seeds contained saponarin,spinosin, vitexin, swertish, 6'''-hydroxybenzoylspinosin, and 6'''-feruloylspinosin (16). However, there is limited research on Z. jujuba seeds as by-product available in Korea. It is important to identify neuroprotective compounds from Z. jujuba seeds. Therefore, the aims of the present study were to evaluate the total phenolic and flavonoid contents, as well as the antioxidant capacity of different fractions of Z. jujuba seed extracts. In addition, we sought to evaluate the neuroprotective effects of the ethyl acetate fraction of Z. jujuba seeds against oxidative stress in neuronal PC-12 cells. Specifically, the major phenolic compounds present in the ethyl acetate fraction were identified using ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS), and their neuroprotective effects were also investigated.Materials and MethodsSample Ziziphus jujuba fruits were grown and harvested in Gyeongsan,Gyeongbuk, Republic of Korea in 2011. The seeds were carefully separated from the fruits, and then the seeds were cut into small slices and lyophilized. The lyophilized seeds were milled and sieved through a standard sieve (25 mesh). The resulting Z. jujuba seed powders were stored at −20o C until use.Reagents Ascorbic acid, ABTS, catechin, 2',7'-dichlorofluorescin diacetate (DCFH-DA), DPPH, dimethyl sulfoxide (DMSO), Folin-Ciocalteu’s phenol reagent, gallic acid, H 2O 2, MTT , acetylcholin-esterase (AChE), butyrylcholinesterase (BChE), acetylcholine iodide (ATCI), butyrylthiocholine chloride (BTCC), tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), phosphate buffered saline (PBS), p -coumaric acid, rutin (quercetin 3-O -rutinoside), isoquercetin (quercetin 3-O -glucoside),nicotiflorin (kaempferol 3-O -rutinoside), and an in vitro toxicology assay kit of lactate dehydrogenase (LDH) were purchased from SigmaChemical Co. (St. Louis, MO, USA). Methyldopa and 3,4-dihydroxy-benzoic acid (protocatechuic acid) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Dihydro-p -coumaric acid was purchased from Alfa Aesar Co., Inc. (Ward Hill, MA, USA). AAPH was obtained from Wako Pure Chemical Industries, Ltd. (Osaka,Japan). Dulbecco’s phosphate buffered saline (DPBS), Roswell Park Memorial Institute (RPMI)-1640 medium, fetal bovine serum (FBS),Hank’s balanced salt solution (HBSS), and penicillin/streptomycin were purchased from Welgene Inc. (Daegu, Korea). All other reagents were of analytical or HPLC grade.Extraction and fractionation Phenolics from the seed powder were extracted using 60% (v/v) aqueous ethanol assisted by homogenization and sonication. First, 20g seed powder and 100mL of 60% (v/v)aqueous ethanol were mixed and homogenized using a Polytron homogenizer (PT 10/35; Kinematica, Kriens-Luzern, Switzerland) at 15,000rpm for 2min. The homogenized mixture was further sonicated for 20min with continual N 2 purging. Whatman #2 filterpaper (Whatman International Limited, Kent, England) was used to filter the sample into a chilled Büchner funnel. The remaining residue in the funnel was re-extracted using the same procedure described above. The two filtrates were then combined and evaporated to dryness under reduced pressure using a rotary evaporator (Eyela,Tokyo, Japan) in a water bath at 37o C. The solid obtained after evaporation was dissolved in 1L of deionized water (DW), which was then subjected to liquid–liquid extraction with the same volume of four organic solvents in the sequence of n -hexane, chloroform, ethyl acetate, and n -butanol to separate the phenolics according to solvent polarities. The fractions were then evaporated to dryness and kept at 20o C until analysis. Three independent extractions were performed, and each of the resulting extracts was subjected to fractionation.Determination of total phenolics The total phenolic abundance of the five Z. jujuba seed extract fractions were determined color-imetrically using Folin-Ciocalteu’s phenol reagent (17). Briefly, each fraction (0.2 mL) was mixed with 2.6 mL of DW , to which 0.2 mL Folin-Ciocalteu’s phenol reagent was added. After 6 min, 2.0mL of 7%(w/v) Na 2CO 3 solution was added to the reaction mixture, and absorbance was measured at 750 nm after a reaction time of 90 min.The total phenolic abundance of each fraction was expressed as mg gallic acid equivalents (GAE)/g fraction. Each fraction was analyzed in triplicate.Determination of total flavonoids The total flavonoid levels of the five Z. jujuba seed extract fractions were measured using a colorimetric assay (18). First, 500µL of each fraction was mixed with 3.2 mL DW to which 150µL of 5% (w/v) NaNO 2 was added. Five minutes later,150µL of 10% (w/v) AlCl 3 was added, followed by 1 mL of 1 M NaOH at a total reaction time of 6 min. The absorbance of the mixture was then measured immediately at 510 nm. The total flavonoid levels ofNeuroprotective Effects of Jujube Seeds 2221December 2015 | Vol. 24 | No. 6the fractions were expressed as mg catechin equivalents (CE)/g fraction. Each fraction was analyzed in triplicate.Determination of ABTS radical scavenging activity The antioxidant capacity of five fractions of Z. jujuba seed extracts was measured using an ABTS radical assay (19). First, a solution of ABTS radicals was adjusted to an absorbance of 0.650±0.020 at 734 nm. Reactions between ABTS radicals and appropriately diluted fractions were then carried out at 37o C for 10 min. The antioxidant capacity of each fraction was expressed as mg vitamin C equivalents (VCE)/g fraction.Determination of DPPH radical scavenging activity The antioxidant capacity of five fractions of Z. jujuba seed extracts was determined using a DPPH radical assay (19). First, the absorbance of fresh DPPH radicals in 80% (v/v) aqueous methanol was set at 0.650±0.020 at 517 nm. Reactions between DPPH radicals and appropriately diluted fractions was carried out at 23o C for 30 min. The antioxidant capacity of each fraction was expressed as mg VCE/g fraction.UHPLC-ESI-MS/MS analysis Phenolics were qualitatively analyzed using a high-resolution LTQ-Orbitrap XL mass spectrometer connected to an Accela system (Thermo Fisher Scientific, Waltham, MA, USA).An ACQUITY BEH C 18 column (150×2.1mm, 1.7µm; Waters Corp.,Milford, MA, USA) was used for polarity-based separation of phenolics.The flow rate and injection volumes were 0.4mL/min and 2µL,respectively. The solvent gradient conditions of binary mobile phases consisting of solvent A (DW with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid) were as follows: 99% A/1% B at 0min, 82% A/18% B at 6 min, 30% A/70% B at 18 min, 10% A/90% B at 20 min, 0% A/100% B at 20.1 min, 0% A/100% B at 22 min, and 99%A/1% B at 25min. Phenolics were detected at 200-600nm. The optimized MS conditions in negative ESI mode were as follows: spray voltage, 4.0kV; capillary voltage, 35V; and temperature, 300o C.Xcalibur software was used for data acquisition. Identification of phenolic compounds was based on comparison of retention times,UV spectra, high-resolution masses, and MS/MS fragment ions with those of available standard compounds.Cell Culture PC-12 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 medium containing 10% FBS, 100 units/mL penicillin, and 100µg/mL streptomycin in a humidified incubator with a 5% CO 2atmosphere at 37o C.Determination of cell viability Cellular viability was determined using an MTT reduction assay. PC-12 cells were seeded at a density of 1×104 cells per well in 96-well plates and then treated with 300µM of H 2O 2 for 1 h. The cells were then incubated with 0.5 mg/mL MTT for 3h at 37o C, and the resulting formazan products were dissolved by the addition of DMSO. The amount of MTT formazan products was determined by measuring the absorbance using a microplatereader (Infinite M200; Tecan Austria GmbH, Grödig, Austria) at 570nm test wavelength with reference at 630 nm. Cytotoxicity was expressed as the percentage (%) of viable cells relative to stress-treated cells cultured without antioxidant compounds.Measurement of LDH PC-12 cells were seeded at a density of 1×104 cells per well in 96-well plates and treated with 300µM H 2O 2for 1 h. An in vitro toxicology assay kit was used for the LDH assay.Specifically, damage to plasma membranes was evaluated by measuring the amount of the intracellular enzyme LDH released into the medium. The absorbances of the test and reference wavelengths were measured at 490 and 690 nm, respectively.Measurement of intracellular oxidative stress Levels of intracellular oxidative stress were evaluated using the fluorescent probe DCFH-DA. PC-12 cells were seeded at 2×104 cells per well in 96-well plates with 100µL of RPMI-1640 medium containing FBS and incubated for 24 h. Cells were then treated with various concentrations of the ethyl acetate fraction and its phenolics for 24 h. Next, cells were incubated with 50µM DCFH-DA in HBSS for 30 min and treated with 300µM H 2O 2 in HBSS for 1 h. The fluorescence was then measured using a microplate reader (Infinite M200; T ecan Austria GmbH) with excitation and emission wavelengths of 485 and 530 nm, respectively.Anticholinesterase activity Anticholinesterase activity was determined using AChE and BChE assays performed in a 96-well plate. ATCI and BTCC substrates were used for AChE and BChE inhibitory assays,respectively, and DTNB was used as the color developing reagent. For the AChE inhibition assay, 20µL of each fraction (10 mg/mL) and 20µL AChE (0.2 U/mL) were added to 150µL PBS. After incubation for 5 min at 37o C, 30µL DTNB (10 mM) and 20µL ATCI substrate (15mM) were added. The BChE inhibition assays was performed using a similar protocol, except that instead of AChE and ATCI, 0.2 U/mL BChE and 10mM BTCC were used. After 20min at 37o C,absorbance was measured at 415 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Tacrine was used as a positive control. AChE and BChE inhibitory activities were calculated using the following formula: Inhibition (%)=(1−sample absorbance/control absorbance)×100. All samples were tested in triplicate.Statistical analysis All experiments were performed in triplicate.Statistical analyses were performed using SAS software (version 8.2,SAS Institute, Inc., Cary, NC, USA). One-way analysis of variance was performed to evaluate differences between average values. Significant differences were verified by Duncan’s multiple range test at a 95%confidence level.Results and DiscussionTotal phenolics, total flavonoids, and antioxidant capacity The2222Choi et al.Food Sci. Biotechnol.total phenolic and flavonoid contents, as well as antioxidant capacity of the five fractions are listed in Table 1. The contents of total phenolics among the fractions decreased in the following order:ethyl acetate>n -butanol>water>chloroform>n -hexane. The ethyl acetate fraction had a significantly higher phenolic content (p <0.05)than the other fractions. Consistently, phenolic acids in the insoluble-bound form in Z. jujuba seeds have been reported to contribute to the total phenolic content (15).The total flavonoid contents of the five fractions decreased in the following order: ethyl acetate>n -butanol>water>chloroform>n -hexane (Table 1). The ethyl acetate fraction showed the highest total flavonoid content at 35.43 mg CE/g fraction, which was significantly higher than the other fractions (p <0.05). Furthermore, the ethyl acetate fraction had an approximately 5.6 fold higher total flavonoid content than the n -hexane fraction.In the ABTS assay for the five fractions, the ethyl acetate fraction had the highest antioxidant capacity at 271.67mg/g fraction, whereas the n -hexane fraction had the lowest at 21.00mg/g fraction. The antioxidant capacity measured using the ABTS radical assay decreased in the following order: ethyl acetate>n -butanol>water>chloroform >n -hexane. Likewise, the antioxidant capacity was significantly (p <0.05)different among fractions, with the ethyl acetate fraction having an approximately 12.9 fold higher antioxidant capacity than the n -hexane fraction. Similar to the ABTS assay results, the ethyl acetate fraction had the highest antioxidant capacity at 66.02 mg/g fraction according to the DPPH assay. The antioxidant capacity of five fractions decreased in the following order: ethyl acetate>n -butanol>water>chloroform>n -hexane. The antioxidant capacity measured in both the ABTS and DPPH assays was significantly different among fractions (p <0.05).Consistently, seed extracts of dried jujube (Z. jujuba ) obtained with 70% ethanol have been shown to have antioxidant capacity as measured by both ABTS and DPPH radical assays as well as have superoxide dismutase-like activity (20). The brain cells are relatively vulnerable to lipid peroxidation due to plenty of highly oxidizable polyunsaturated fatty acids and large amounts of oxygen consumption (10,21). Furthermore, cells of the brain have relatively low amounts of enzymatic and non-enzymatic scavengers of ROS associated with the apoptosis of neuronal cells (21). Therefore, antioxidative flavonoids may contribute to neuroprotective effects in the brainagainst oxidative stress (14,21).The cumulative results above showed that the ethyl acetate fraction had the highest levels of total phenolics, total flavonoids, and antioxidant capacity. Therefore, we used the ethyl acetate fraction in subsequent experiments to examine whether it and its major phenolics would be able to contribute to neuroprotective effects in PC-12 cells.Identification of the phenolics in the ethyl acetate fraction using UHPLC-ESI-MS/MS The specific phenolics identified in the ethyl acetate fraction are listed in Table 2. Twelve phenolics were found in the ethyl acetate fraction analyzed using UHPLC-ESI-MS/MS (Fig. 1).High resolution MS data and MS/MS spectral characteristics of the phenolics in the ethyl acetate fraction were compared to commercially obtained standards and published data.Peaks 1-4 had m/z 153.0195, 210.0773, 163.0411, and 165.0559of [M-H]−, which were tentatively identified as respective 3,4-dihydroxybenzoic acid, methyldopa, p -coumaric acid, and dihydro-p -coumaric acid based on the high-resolution mass, UV spectra, and MS/MS ion products ions. Peak 5 had an m/z 609.1453 of [M-H]−,which produced an MS/MS fragment ion with an m/z value of 301(quercetin), indicative of a loss of rutinose. Peak 5 was confirmed by MS/MS spectral library search with the ion spectra of an authentic standard, and was tentatively identified as rutin (quercetin 3-O -rutinoside). Peak 6 had an m/z of 463.0878 of [M-H]−, which produced an MS/MS fragment ion with an m /z value of 301(quercetin) indicating the loss of a hexose group. These results and related observations (22) confirmed the assignment of peak 6 as isoquercetin (quercetin 3-O -glucoside). Peak 7 had an m/z 593.1504of [M-H]−, which produced MS/MS fragment ion with m/z value of 285 indicating the loss of rutinose. Peak 7 was tentatively identified as kaempferol 3-O -rutinoside based on the UV spectra, retention time, and MS/MS fragment patterns of an authentic standard. Peaks 9-13 had m /z values of 617.3836-617.3841, which were tentatively identified as p -coumaroyl derivatives of maslinic acid and alphitolic acid on the basis of literature data (23).Thus, the phenolics identified in the ethyl acetate fraction included 3,4-dihydroxybenzoic acid, methyldopa, p -coumaric acid, dihydro-p -coumaric acid, rutin, isoquercetin, kaempferol 3-O -rutinoside, 3-O -cis -p -coumaroylalphitolic acid, 2-O -cis -p -coumaroylalphitolic acid, 3-Table 1. Total phenolic and flavonoid contents, and antioxidant capacities of Ziziphus jujuba seed extract fractionsFractions Total phenolics (mg gallic acid equiv./gfraction)Total flavonoids (mg catechin equiv./gfraction)Antioxidant capacity(mg vitamin C equiv./g fraction)ABTS DPPH n -Hexane 7.93±0.47d 6.35±0.91c 21.00±1.30e 17.44±2.40e Chloroform 30.80±2.00c 7.26±0.20c 35.20±1.33d 20.30±0.55d Ethyl acetate 102.05±2.42a1)35.43±1.21a 271.67±28.95a 66.02±1.20a n -Butanol 41.44±2.13b 10.25±0.87b 128.22±52.76b 37.92±1.46b Water40.33±2.50b8.99±0.53b72.45±3.90c34.91±1.26c1)Data are presented as the mean±standard deviation (n =3); Means with different superscripts in the same column show significant difference by Duncan’s multiple range test (p <0.05).Neuroprotective Effects of Jujube Seeds 2223December 2015 | Vol. 24 | No. 6O -trans -p -coumaroylalphitolic acid, 3-O -cis -p -coumaroylmaslinic acid, and 3-O -trans -p -coumaroylmaslinic acid (Fig. 1 and Table 2). In addition, we identified several other compounds including an unknown molecule (peak 8) in the ethyl acetate fraction that have not been previously described and which remain to be identified. Therefore,the ethyl acetate fraction and seven identified phenolics (3,4-dihydroxybenzoic acid, methyldopa, p -coumaric acid, dihydro-p -coumaric acid, rutin, isoquercetin, and kaempferol 3-O -rutinoside)were further evaluated whether they would mediate protection against oxidative stress induced by H 2O 2 in vitro in neuron-like PC-12cells.Effect of the ethyl acetate fraction and its phenolics on the viability of PC-12 cells against oxidative stress as determined using MTT assay The MTT assay was used to determine both cell viability and cytotoxicity based on mitochondrial dehydrogenase activities in the living PC-12 cells. The cytotoxicity of the ethyl acetate fraction and its identified phenolics on PC-12 cells was first evaluated to determine appropriate concentrations for subsequent analysis of their ability to prevent oxidative stress. In this initial testing, we considered that treatment with either the ethyl acetate fraction or the identified phenolic compounds resulting in a cell viability of 80% would be indicative of a lack of cytotoxicity. The ethyl acetate fraction and seven pure compounds (3,4-dihydroxybenzoic acid, methyldopa, p -coumaric acid, dihydro-p -coumaric acid, rutin, isoquercetin, and kaempferol 3-O -rutinoside) had no cytotoxicity up to 200mg/L and 100µM, respectively (data not shown).We next evaluated the protective effects of the ethyl acetate fraction, 3,4-dihydroxybenzoic acid, methyldopa, p -coumaric acid,dihydro-p -coumaric acid, rutin, isoquercetin, and kaempferol 3-O -rutinoside against H 2O 2-induced oxidative stress cell (Fig. 2). Treatment with H 2O 2 at 300µM for 1h decreased the viability of PC-12 cells to 38% compared with cells without H 2O 2 treatment (data not shown).Pretreatment with the ethyl acetate fraction prior to exposuretoFig. 1. UHPLC chromatogram of the phenolics present in the ethyl acetate fraction of the Ziziphus jujuba seed extract detected at 280 nm. Peak 1,3,4-dihydroxybenzoic acid; Peak 2, methyldopa; Peak 3, p -coumaric acid; Peak 4, dihydro-p -coumaric acid; Peak 5, rutin; Peak 6, isoquercetin; Peak 7, kaempferol 3-O -rutinoside; Peak 8, unidentified substance; Peak 9, 3-O -cis -p -coumaroylalphitolic acid; Peak 10, 2-O -cis -p -coumaroylalphitolic acid; Peak 11, 3-O -trans -p -coumaroylalphitolic acid; Peak 12, 3-O -cis -p -coumaroylmaslinic acid; and Peak 13, 3-O -trans -p -coumaroylmaslinic acid.Table 2. Identification of phenolic compounds in the ethyl acetate fraction of Ziziphus jujuba seed extract determined using UHPLC-ESI-MS/MS Peak No.Retention time(min)Selected ion Molecular ion(m/z )Formula MS/MS ions (m/z )Identification1 2.46 [M-H]-153.0195 C 7H 6O 4 3,4-Dihydroxybenzoic acid a1)2 5.15 [M-H]-210.0773 C 10H 13NO 4124Methyldopa a3 6.04 [M-H]-163.0411 C 9H 8O 3p -Coumaric acid a4 6.72 [M-H]-165.0559 C 9H 10O 3Dihydro-p -coumaric acid a5 7.07 [M-H]-609.1453 C 27H 30O 16301 Rutin a6 7.29 [M-H]-463.0878 C 21H 20O 12301 Isoquercetin a7 7.89 [M-H]-593.1504 C 27H 30O 15285 Kaempferol 3-O -rutinoside a 8 10.44 [M-H]-517.2663 C 25H 41O 11291, 249 Unidentified substance9 19.78 [M-H]-617.3839 C 39H 54O 6497, 453 3-O -cis -p -Coumaroylalphitolic acid b2)10 19.99 [M-H]-617.3839 C 39H 54O 6573, 497, 4532-O -cis -p -Coumaroylalphitolic acid b 11 20.27 [M-H]-617.3838 C 39H 54O 6573 3-O -trans -p -Coumaroylalphitolic acid b 12 20.48 [M-H]-617.3841 C 39H 54O 6573, 497, 453 3-O -cis -p -Coumaroylmaslinic acid b 1320.96[M-H]-617.3836C 39H 54O 6497, 4533-O -trans -p -Coumaroylmaslinic acid b1)a Identification was obtained by comparison with high resolution MS and MS/MS data. 2)bIdentification was inferred from published literature.2224Choi et al.Food Sci. Biotechnol.H 2O 2 significantly (p <0.05) increased cell viability in a dose-dependent manner (Fig. 2A). Methyldopa increased PC-12 cell viability by 60.7-174.3% compared to control cells treated with H 2O 2. Similarly, p -coumaric acid at 50µM and dihydro-p -coumaric acid at 25µM increased viability by approximately 38.5 and 37.7%, respectively (Fig. 2B). Isoquercetin and 3,4-dihydroxybenzoic acid increased PC-12 cell viability by 33.5-67.4 and 28.5-43.0% compared to control cells treated with H 2O 2, respectively (Fig. 2B). Kaempferol 3-O -rutinoside at 25µM increased viability by approximately 11.7% (Fig. 2B). On the other hand, cells treated with rutin exhibited no significant difference compared with cells treated with H 2O 2 (Fig. 2B). Similar to these results, rutin was shown to have no significant (p <0.01) protection on H 2O 2-induced cytotoxicity in RAW 264.7 macrophages (24).Effect of the ethyl acetate fraction and its phenolics on the viability of PC-12 cells against oxidative stress as determined using LDH release assay Polyunsaturated fatty acids in the cellular membrane are vulnerable to oxidative stress. Lipid peroxidation in the plasma membrane ruptures cellular integrity, leading to the release of cytoplasmic LDH into the medium. In this way, quantitative analysis of LDH release in a cell culture medium can be used to determine relative cell viability. Pretreatment of PC-12 cells with the ethyl acetate fraction decreased LDH release into culture medium in a dose-dependent manner (Fig. 3A). Specifically, 200mg/L of ethyl acetate fraction decreased LDH release from PC-12 cells by up to 37.7% compared with the stress control cells (100%) (Fig. 3A).Consistently, pretreatment of PC-12 cells with 3,4-dihydroxybenzoicacid, methyldopa, p -coumaric acid, dihydro-p -coumaric acid, rutin,isoquercetin, and kaempferol 3-O -rutinoside at 100µM resulted in LDH release of 15.5, 58.6, 80.9, 92.0, 70.1, 96.4, and 62.3% compared with the stress control (100%), respectively (Fig. 3B). Isoquercetin wasshown to have no protection of LDH release under oxidative stress. Methyldopa and dihydro-p -coumaric acid at 25µM decreased LDH release at approximately 46.3 and 41.7% compared with the stress control (100%), respectively (Fig. 3B). Treatment with 3,4-dihydroxybenzoic acid at 50µM produced the lowest level (6.1%) of LDH release than the other six compounds (Fig. 3B). 3,4-Dihydroxy-benzoic acid prevented LDH release in H 2O 2-induced oxidative stress in PC-12 cells (25). Importantly, administration of a water extract from Z. jujuba seeds to the rats reduces LDH activity in serum (26).Taken together, these results suggest that the phenolic compounds present in Z. jujuba seed might protect mitochondrial function and plasma membrane integrity partly due to the attenuation of oxidative stress in PC-12 cells, resulting in neuronal cell protection.Effect of the ethyl acetate fraction and its phenolics on intracellular oxidative stress in PC-12 cells Intracellular oxidative stress levels in neuronal PC-12 cells were measured using the DCFH-DA assay.Pretreatments of PC-12 cells with the ethyl acetate fraction decreased intracellular oxidative stress induced using 300µM H 2O 2 in a dose-dependent manner (Fig. 4A). Specifically, at 200mg/L, the ethyl acetate fraction attenuated intracellular oxidative stress to approximatelyFig. 3. Protective effects of the ethyl acetate fraction (A) and its pure phenolics (B) on the viability of PC-12 cells against oxidative stress as determined using LDH release assay. Data (bar) are presented as the mean±standard deviation (n =3). Means with different small letters on bars show significant difference by Duncan’s multiple range test (p <0.05).Fig. 2. Protective effects of the ethyl acetate fraction (A) and its pure phenolics (B) on the viability of PC-12 cells against oxidative stress as determined using MTT reduction assay. Data (bar) are presented as the mean±standard deviation (n =3). Means with different small letters on bars show significant difference by Duncan’s multiple range test (p <0.05).。