Polysaccharide isolated from wax apple suppresses ethyl carbamate-induced oxidative damage in human hepatocytes 莲雾多糖对氨基甲酸乙酯诱导人肝细胞氧化损伤的抑制作用研究 1 2 equal first-author https://orcid.org/0000-0002-2373-2437 1 2 equal 2 4 1 2 3 corresp Wei CHEN, zjuchenwei@zju.edu.cn Wei CHEN, zjuchenwei@zju.edu.cn zjuchenwei@zju.edu.cn 浙江大学医学院附属邵逸夫医院中医科， 中国杭州市， 310016 浙江大学医学院附属邵逸夫医院中医科， 中国杭州市， 310016 Department of Traditional Chinese Medicine, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China Department of Traditional Chinese Medicine, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China 浙江大学食品科学与营养学系， 中国杭州市，310058 浙江大学食品科学与营养学系， 中国杭州市，310058 Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China 浙江大学宁波创新中心， 中国宁波市，315100 浙江大学宁波创新中心， 中国宁波市，315100 Ningbo Innovation Center, Zhejiang University, Ningbo 315100, China Ningbo Innovation Center, Zhejiang University, Ningbo 315100, China 泰国瓦莱拉大学联合健康科学学院生物医学科学， 泰国Nakhon Si Thammarat，80161 泰国瓦莱拉大学联合健康科学学院生物医学科学， 泰国Nakhon Si Thammarat，80161 Biomedical Sciences, School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat 80161, Thailand Biomedical Sciences, School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat 80161, Thailand Wax apple ( Syzygium samarangense ) has received growing research interest for its high nutritional and medicinal value due to its constituents such as polysaccharide, organic acids, flavonoids, minerals, and other substances. In this study, wax apple polysaccharide (WAP) was isolated from this plant and its protective effect against ethyl carbamate (EC)‍-induced oxidative damage was evaluated in human hepatocytes (L02 cells). Firstly, a series of analyses such as high-performance liquid chromatography (HPLC), high-performance gel permeation chromatography (HPGPC), Fourier transform infrared spectroscopy (FT-IR), gas chromatography/mass spectrometry (GC/MS), and 1 H and 13 C nuclear magnetic resonance (NMR) were conducted to identify the structure of WAP. Thereafter, in vitro cell experiments were performed to verify the protective effects of WAP against EC-induced cytotoxicity, genotoxicity, and oxidative damage in L02 cells. Our results revealed that WAP is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, and fucose in a molar ratio of 2.20:‍3.94:‍4.45:‍8.56:‍8.86:‍30.82:‍39.78:‍1.48. Using a combination of methylation and NMR spectroscopic analysis, the primary structure of WAP was identified as Ara f -(1→, Glc p -(1→, →2)‍-Ara f -(1→, →3)‍-Gal p -(1→, →3)‍-Ara f -‍(1→, and →6)‍-Gal p -‍(1→. Cell experiments indicated that WAP exhibited significant protective effects on EC-treated L02 cells via suppressing cytotoxicity and genotoxicity, reducing reactive oxygen species (ROS) and O 2 •- formation, as well as improving mitochondrial membrane potential (MMP) and glutathione (GSH). In a nutshell, WAP has the potential as an important therapeutic agent or supplement for hepatic oxidative damage. Meanwhile, further studies are needed to prove the above effects in vivo at the biological and clinical levels. 莲雾（ Syzygium samarangense ）因其含有多糖、有机酸、类黄酮、矿物质等功能活性成分而具有很高的营养和药用价值，受到越来越多研究者的关注。本研究从莲雾中分离出水溶性多糖（WAP），并在人肝细胞（L02细胞）中评估了其对氨基甲酸乙酯（EC）诱导的氧化损伤的保护作用。首先采用高效液相色谱法（HPLC）、高效凝胶渗透色谱（HPGPC）、傅里叶变换红外光谱（FT-IR）、气相色谱/质谱（GC/MS）、 1 H和 13 C 核磁共振（NMR）等一系列分析方法来鉴定WAP的结构。随后进行体外细胞实验验证WAP对EC诱导的L02细胞中的细胞毒性、遗传毒性和氧化损伤的保护作用。结果表明，莲雾的WAP由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖和岩藻糖组成，其摩尔比为2.20:3.94:4.45:8.56:8.86:30.82:39.78:1.48。结合甲基化和NMR光谱分析，WAP的一级结构为Ara f -(1→、Glc p -(1→、→2)-Ara f -(1→、→3)-Gal p -(1→、→3)-Ara f -(1→和→6)-Gal p -(1→。体外细胞实验表明，WAP通过抑制细胞毒性和遗传毒性，减少活性氧（ROS）和超氧根离子（O 2 •- ）的形成，对EC诱导的L02细胞表现出显著的保护作用。综上所述，WAP具有作为肝脏氧化损伤的重要治疗剂或补充剂的潜力，但仍需通过体内生物学和临床研究进一步证实上述作用。 莲雾多糖 多糖表征 氨基甲酸乙酯 肝脏氧化应激 Wax apple polysaccharide Polysaccharide characterization Ethyl carbamate Hepatic oxidative stress 1 Introduction 1 Liver is the primary organ affected by reactive oxidants from food, the environment, and chemicals. In severe cases, these agents first cause hepatocyte apoptosis via initiating oxidative-dependent cell signaling pathways, and then induce hepatic pathologies such as non-alcoholic fatty liver disease, cirrhosis, and even hepatocellular carcinoma ( Cichoż-Lach and Michalak, 2014 Cichoż-Lach and Michalak, 2014 ; Meng et al., 2020 Meng et al., 2020 ; Karim et al., 2022 Karim et al., 2022 ). Reactive oxygen species (ROS) including superoxide anion (O 2 •- ), hydroxyl (·OH), hydrogen peroxide (H 2 O 2 ), peroxyl (RO 2 • ), alkoxyl (RO·‍), and others have important roles in balancing cell homeostasis ( Gowd et al., 2018a Gowd et al., 2018a , 2019 2019 ; Shishir et al., 2019 Shishir et al., 2019 ). However, excessive production of ROS has been associated with the imbalance of the cellular redox system and was further linked to oxidative stress ( Karim et al., 2018 Karim et al., 2018 , 2022 2022 ). Ethyl carbamate (EC) is known as a by-product in several fermented foods, alcoholic beverages, and others like bread or soy sauce ( Gowd et al., 2018b Gowd et al., 2018b ). Some studies have suggested the potential carcinogenic effect of EC in experimental animals ( Liu et al., 2017 Liu et al., 2017 ; Wang et al., 2021 Wang et al., 2021 ), while there is limited evidence of such effect in humans. In 2010, the International Agency for Research on Cancer (IARC) recognized EC as a group 2A carcinogenic substance ( IARC, 2010 IARC, 2010 ). Previous studies by our lab and others indicated that EC treatment induces ROS overproduction, mediates the imbalance of the cellular redox system, and thereby generates toxicity in cells ( Chun et al., 2013 Chun et al., 2013 ; Chen et al., 2016b Chen et al., 2016b , 2016c 2016c , 2017 2017 ). Accumulating evidence indicates that natural antioxidants such as polyphenols, sterols, peptides, and polysaccharides, have positive effects against toxicant-induced oxidative damage ( Sun et al., 2004 Sun et al., 2004 ; Chen et al., 2013 Chen et al., 2013 ; da Silva Marineli et al., 2014; Jin et al., 2016 Jin et al., 2016 ; Wang et al., 2016 Wang et al., 2016 ; Zhang et al., 2017 Zhang et al., 2017 ; Lu et al., 2021 Lu et al., 2021 ). Owing to their antioxidant properties, several natural polysaccharides from plants, marine flora, bacteria, fungi, fauna, etc. were shown to exert potential protective effects against oxidative damage ( Zhang et al., 2003 Zhang et al., 2003 ; Ng et al., 2006 Ng et al., 2006 ; Kodali and Sen, 2008 Kodali and Sen, 2008 ; Guo et al., 2010 Guo et al., 2010 ; Chen W et al., 2011 Chen W et al., 2011 ). The above studies also revealed that the reduction of capacity, radical scavenging activity, and messenger RNA (mRNA) expression of some antioxidant enzymes (glutathione peroxidase ( GPx ) and superoxide dismutase ( SOD )) was associated with the antioxidant activity of polysaccharides ( Zhang et al., 2003 Zhang et al., 2003 ; Ng et al., 2006 Ng et al., 2006 ; Kodali and Sen, 2008 Kodali and Sen, 2008 ; Guo et al., 2010 Guo et al., 2010 ). Wax apple ( Syzygium samarangense ), originating from the Greater Sunda Islands, Malay Peninsula, Andaman and Nicobar Islands, is now cultivated in Guangdong and Guangxi Provinces, China. It has been shown to contain an abundance of phytochemicals, such as flavonoids, ellagitannins, proanthocyanidins, anthocyanidins, triterpenoids, and chalcones ( Srivastava et al., 1995 Srivastava et al., 1995 ; Resurreccion-Magno et al., 2005 Resurreccion-Magno et al., 2005 ; Simirgiotis et al., 2008 Simirgiotis et al., 2008 ; Huang et al., 2016 Huang et al., 2016 ), which have potential health benefits such as antioxidant, liver protection, hypoglycemic, antibacterial, and anti-tumor ( Wang et al., 2019 Wang et al., 2019 ; Li et al., 2020 Li et al., 2020 ; Yang, 2022 Yang, 2022 ). Wax apple and jambo ( Syzygium jambos (L.) Alston) belong to the same genus. Recently, an arabinogalactan was found in jambo ( Tamiello et al., 2018 Tamiello et al., 2018 ); however, there have been no reports on polysaccharides in wax apple. In this work, we isolated a new polysaccharide from wax apple and evaluated its protective effect against EC-induced oxidative damage in normal human hepatocytes (L02 cells). Firstly, we characterized the chemical properties of this polysaccharide, such as molecular weight (MW), monosaccharide composition, linkages, functional groups, and internal chemical bonds. Thereafter, we evaluated the in vitro antioxidant activity (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical scavenging activity and ferric reducing antioxidant power (FRAP)). Subsequently, we investigated the protective effect of wax apple polysaccharide (WAP) against EC-induced oxidative damage in L02 cells. 2 Materials and methods 1 2.1 Materials and reagents 2 Fresh wax apples (cultivar: Heijingang (in Chinese)) were collected in July 2017 from Guangxi Province, China. Based on weight uniformity, shape, and visual color, suitable fruits (average weight (120±30) g) were selected for experimental analysis. Fruits with immature size or shape or color defect were excluded. The selected fruits were thoroughly washed with distilled water, left to dry in a clean dark place, and then stored at -80 ℃. The chemicals 2,4,6-tris(2-pyridyl)‍- S -triazine (TPTZ), ABTS, 3-‍(4,‍5-dimethyl-2-thiazolyl)‍-2,‑5-diphenyl-2H-tetrazolium bromide (MTT), 2',‍7'-‍‍‍ dichlorofluorescein diacetate (DCFH-DA), dihydroethidium (DHE), Rhodamine 123 (Rh123), Mito-Tracker Green (MTG), naphthalene-2,3-dicarboxal-dehyde (NDA), and Hoechst 33258 were purchased from Sigma Chemical (St. Louis, MO, USA). Glucose, fucose, arabinose, rhamnose, mannose, galacturonic acid, galactose, glucuronic acid, erythritol, and glycerin were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). All reagents used in this study were of analytical grade unless stated otherwise. 2.2 Extraction of polysaccharide from wax apple 2 The peeled wax apples (1 kg) were homogenized and the dissolved contents were extracted using 4 L distilled water at 90 ℃ for 2 h. The extraction process was repeated three times. The supernatant was collected and mixed with ethanol (final concentration of 95%, volume fraction) to obtain the crude polysaccharide. And then it was dissolved in distilled water and mixed with 1/4 volume of Sevag solution to remove the proteins. The mixture was shaken for 20 min and then centrifuged at 8000 g for 10 min. The supernatant was collected and the centrifugation process was repeated six times. Next, the polysaccharide solution was placed in a dialysis bag (MW cut-off: 3.50 kDa), and then dialyzed for 48 h in tap water and then for 24 h in ultra-pure water. The obtained WAP solution was lyophilized and stored at -80 ℃ until further use. 2.3 Determination of total sugar 2 The total sugar content of WAP was measured using the phenol-sulfuric acid method, with arabinose acting as the standard ( DuBois et al., 1956 DuBois et al., 1956 ). 2.4 Analysis of monosaccharide composition and molecular weight 2 Briefly, trifluoroacetic acid (2 mol/L) was employed to hydrolyze the WAP (5 mg) at 110 ℃ for 8 h, followed by complexation with 1-phenyl-3-methyl-5-pyrazolone (PMP; 0.5 mol/L, dissolved in methanol). Afterwards, high-performance liquid chromatography (HPLC) equipped with a Promosil C18 column (4.6 mm×250 mm, 5 μm) was used to analyze the obtained product. Phosphate buffer (KH 2 PO 4 -NaOH, 0.05 mol/L, pH 6.9) and acetonitrile were employed as mobile phases A and B, respectively. A linear gradient program was set as follows: 17% (mobile phase B) from 0 to 15 min, 17% to 25% (mobile phase B) from 15 to 18 min, 25% (mobile phase B) from 18 to 23 min, 25% to 17% (mobile phase B) from 23 to 25 min, and 17% (mobile phase B) from 25 to 30 min. The sample injection volume was 10 μL and the flow rate was fixed at 1.0 mL/min. The chromatograms were detected at the wavelength of 245 nm. Commercially available standards were used for the qualitative and quantitative determination of monosaccharides in the samples. In order to identify the MW of WAP, this study employed high-performance gel permeation chromatography (HPGPC) equipped with a Waters-1525 HPLC system matched with Waters ultra-hydrogel 500 column (7.8 mm×300 mm, column temperature 30 ℃). The sample was detected by the differential refraction index detector (RID) at 40 ℃. A sample volume of 50 μL was injected into the HPGPC system and operated at 30 ℃ with the 0.5 mL/min flow rate using 0.2 mol/L NaCl solution. Dextran standard with different molecular weights (1, 5, 12, 50, 80, 150, 270, 410, and 670 kDa) was used for the calibration curve. 2.5 FT-IR analysis 2 The Fourier transform infrared spectroscopy (FT-IR) spectra of WAP were analyzed within the range of 4000–400 cm -1 with 2 cm -1 resolution (Nicolet iS 10, Thermo Fisher Scientific, Massachusetts, USA) ( Ren et al., 2017 Ren et al., 2017 ). 2.6 ‍ Characterization of WAP using methylation and NMR 2 The reduction of WAP was achieved following the method of Song et al. (2018) Song et al. (2018) . Firstly, 20 mg of sample was methylated following the protocol of Ciucanu and Kerek (1984) Ciucanu and Kerek (1984) . Then, the dried methylated sample was hydrolyzed with 2 mol/L trifluoroacetic acid at 110 ℃ for 8 h. Methanol was used to remove the excess trifluoroacetic acid from the sample, which was then reduced by sodium borohydride at room temperature for 2 h. Subsequently, acetylation of the sample was performed with acetic anhydride to yield alditol acetates via interacting between pyridine and acetic anhydride at 100 ℃ for 2 h. Then, gas chromatography-mass spectrometry (GC-MS; GC7890B-MS7000C, Agilent Technologies Co., USA) was used to analyze the partially methylated alditol acetates (PMAAs). The obtained data were used to determine the methylated sugar linkages according to the standard data in the Complex Carbohydrate Research Center (CCRC) Spectral Database ( https://glygen. https://glygen. ‍ccrc.‍uga.‍edu/ccrc/specdb/ms/pmaa/pframe.‍html). The acetylated derivatives were loaded into an HP-5 capillary column, and the temperature program was set as follows: the initial column temperature was set at 80 ℃ and kept constant for 1 min, increased to 220 ℃ at the rate of 10 ℃/min kept constant for 1 min, and then increased from 220 to 300 ℃ at the rate of 20 ℃/min. The temperature of the interface was 250 ℃. Helium was used as the carrier gas. WAP was dissolved in deuterium oxide (D 2 O) and examined at 30 ℃. The chemical shift of the sample was expressed in ppm (1×10 -6 ). A Bruker DRX-600 nuclear magnetic resonance (NMR) spectrometer (Bruker, Rheinstetten, Germany) was implemented to record the 1 H and 13 C NMR spectra of sample. WAP was dissolved in D 2 O and examined at 30 ℃. 2.7 Antioxidant properties of WAP 2 2.7.1　‍ABTS radical scavenging activity of WAP 3 The radical scavenging effect of WAP was evaluated using ABTS radical cation (ABTS + ) assay with a slight modification ( Bao et al., 2016 Bao et al., 2016 , 2021 2021 ). Briefly, WAP solution (50 μL) was incubated with ABTS + working solution (150 μL), and then kept at room temperature for 20 min in the dark. The absorbance was measured at 734 nm. The ABTS radical scavenging rate (RSR) of WAP was calculated using Eq. (1) Eq. 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The ABTS radical scavenging activity was expressed as the concentration of WAP that yields 50% of radical scavenging (IC 50 ). 2.7.2　FRAP of WAP 3 FRAP assay was conducted according to Bao et al. (2016) Bao et al. (2016) with a slight modification. In brief, 100 μL of WAP solution was added to 100 μL of FRAP solution, and then incubated at 37 ℃ for 5 min. The absorbance was measured at 593 nm. FeSO 4 was selected as standard. The data were expressed as μmol Fe 2+ /mg. 2.8 Culture of normal human hepatocytes (L02 cells) 2 Normal human hepatocytes (L02 cells) were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured with 1640 medium containing 10% (volume fraction) of the calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. They were kept in an incubator at 37 ℃ with 5% CO 2 ( Chen et al., 2014 Chen et al., 2014 ). 2.9 Cell viability assay and nuclear detection 2 MTT assay was performed according to a method described by Zhang et al. (2017) Zhang et al. (2017) to evaluate cell viability. At first, cells were seeded at a density of 6×10 3 cells/well into a 96-well plate. After 24 h of incubation, different concentrations (2.5, 5, 10, 25, 50, 100, 200, and 250 μg/mL) of WAP were added to cells and incubated for another 24 h. Thereafter, cells were washed and added with MTT solution (0.5 mg/mL) for 4 h. After the removal of medium and washing the cells, dimethyl sulfoxide (DMSO, 200 μL) was added to cells and the absorbance was measured at 490 nm using Tecan Infinite M200 microplate reader (Molecular Devices, California, USA). The protective effect of WAP on EC-induced cytotoxicity was estimated as follows. After seeding cells into a 96-well culture plate at a density of 6×10 3 cells/well, the plate containing the cells was incubated for 24 h and then treated with different concentrations of WAP. Following 24 h of treatment, EC was added for another 24 h, and absorbance was recorded at 490 nm using Tecan Infinite M200 microplate reader (Molecular Devices). For nuclear detection, Hoechst 33528 (fluorescence probe) was used according to a protocol described by Zhang et al. (2017) Zhang et al. (2017) . Firstly, cells were seeded into a 24-well plate at a density of 4×10 4 cells/well. After 24 h of cell culture, different concentrations (2.5, 5, 10, 25, 50, 100, 200, and 250 μg/mL) of WAPs were added, followed by incubation for 24 h. Thereafter, cells were treated with EC (60 mmol/L) for another 24 h. Then, all cells were washed and incubated with Hoechst 33258 (10 μmol/L) for 30 min at 37 ℃. After washing properly, cell nuclei were detected under a Nikon Eclipse Ti-S inverted fluorescence microscope (Nikon, Natori, Japan). 2.10 Determination of intracellular ROS and O 2 • - 2 An oxidation-sensitive fluorescence probe, DCFH-DA, was implemented to determine the intracellular ROS production ( Chen et al., 2016a Chen et al., 2016a ). Briefly, L02 cells were treated exactly as described in Section 2.9 (nuclear detection part). Subsequently, cells were washed with phosphate-buffered saline (PBS) and then incubated with DCFH-DA (10 μmol/L) for 30 min at 37 ℃. Thereafter, cells were washed with PBS immediately before examination through fluorescence microscope (Nikon). O 2 •- production in L02 cells was examined using a DHE probe. The data were obtained from five microscopic images evaluated using ImageProPlus 6.0 software (Media Cybernetics, Inc., Maryland, USA) and were expressed as mean fluorescence intensity. 2.11 Evaluation of mitochondrial mass and MMP 2 Mitochondrial mass and mitochondrial membrane potential (MMP) were assessed according to an earlier protocol ( Chen et al., 2016a Chen et al., 2016a ). In short, L02 cells were treated exactly as in Section 2.9 (nuclear detection part). Subsequently, cells were washed with PBS and then incubated with MTG (1 μmol/L) for 30 min at 37 ℃. Thereafter, cells were washed with PBS immediately before examination through a fluorescence microscope (Nikon). For MMP analysis, L02 cells were treated with 10 μmol/L Rh123 probe. The data were obtained from five microscopic images evaluated by ImageProPlus 6.0 software (Media Cybernetics, Inc.) and were expressed as mean fluorescence intensity. 2.12 Analysis of cellular glutathione 2 Cellular glutathione (GSH) was detected using NDA probe according to a slightly modified method by Li et al. (2018) Li et al. (2018) . In brief, L02 cells were treated exactly as in Section 2.9 (nuclear detection part). Subsequently, cells were washed with PBS and then incubated with NDA (50 μmol/L) for 30 min at 37 ℃. Thereafter, cells were washed with PBS immediately before examination through a fluorescence microscope (Nikon). The data were obtained from five microscopic images evaluated by ImageProPlus 6.0 software (Media Cybernetics, Inc.) and were expressed as mean NDA fluorescence intensity. 2.13 Statistical analysis 2 Data are expressed as mean±standard deviation (SD), whereas multiple comparisons were performed by two-tailed unpaired Student’s t -tests with GraphPad Prism version 7.0 (GraphPad Software, California, USA). P < 0.05 was considered as indicative of statistical significance. All analyses were carried out in triplicates. 3 Results and discussion 1 3.1 Chemical properties of WAP 2 Our total carbohydrates assay (phenol-sulfuric acid method) revealed that WAP contained (81.23±2.25)% carbohydrates. After acid hydrolysis and subsequent PMP derivatization, the monosaccharide composition of WAP was analyzed by HPLC. WAP was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, and fucose in a molar ratio of 2.20:‍3.94:‍4.45:‍8.56:‍8.86:30.82:39.78:‍1.48. Among all monosaccharides in WAP, arabinose and galactose were the major constituents. The MW of WAP were 5.34 and 40.34 kDa. Based on the results of carbohydrate content and monosaccharide composition, WAP was acknowledged as an ideal polysaccharide. 3.2 FT-IR spectral and linkage analysis of WAP 2 In order to characterize the functional groups and chemical bonds of polysaccharides, we conducted the FT-IR of WAP within the wavelength of 4000–400 cm -1 ( Meng et al., 2017 Meng et al., 2017 ). As shown in Fig. 1, a major broad stretching peak was found at 3388.12 cm -1 , which indicated the hydroxyl group with stretching vibration. The characteristic peaks of methyl and methylene C‍–‍H stretching vibration were found at 2921.02 and 2851.33 cm -1 , respectively. Moreover, the characteristic peaks of C=O of acetyl and carboxylic acid ester bonds were found at 1738.12 and 1639.56 cm -1 , respectively. The presence of pyranose was confirmed by the peaks of 1141.39 and 1074.27 cm -1 , while the presence of β‍-type and α‍-type glycosidic linkages in WAP was confirmed by the bands of 893.77 and 832.14 cm -1 , respectively ( Liu et al., 2016 Liu et al., 2016 ; Luo et al., 2016 Luo et al., 2016 ). The above results indicated that WAP represents the typical absorption groups of an ideal polysaccharide. 3.3 Methylation results and NMR data of WAP 2 At first, the fully methylated product of WAP was hydrolyzed via acid and then converted to PMAA, which was analyzed by GC-MS. Table 1 Table 1 showed the related linkage patterns and corresponding molar ratios in WAP. From the peak of GC chromatogram, the molar ratios of all sugar residues were calculated. According to the results, WAP mainly consisted of Ara f -(1→, Glc p -‍(1→, →2)‍-Ara f -‍(1→, →3)‍-Gal p -(1→, →3)-Ara f -‍(1→, and →6)‍-Gal p -‍(1→ in a molar percentage of 9.3%, 6.2%, 19.4%, 16.6%, 26.0%, and 22.5%, respectively. The data were consistent with the monosaccharide composition results of HPLC. Ara: arabinose; Gal: galactose; Glc: glucose; Me: methyl. Methylated sugar Linkage type Molar ratio (%) Mass fragment ( m / z ) 2,3,5-Me 3 -Ara f Ara f -(1→ 9.3 43, 57, 71, 85, 101, 117, 131, and 161 2,3,4,6-Me 4 -Glc p Glc p -(1→ 6.2 43, 57, 71, 85, 101, 117, 129, 145, 161, and 205 3,5-Me 2 -Ara f →2)-Ara f -(1→ 19.4 43, 57, 71, 85, 101, 117, 129, 145, 161, and 190 2,4,6-Me 3 -Gal p →3)-Gal p -(1→ 16.6 43, 57, 71, 85, 101, 117, 129, 143, 149, 161, 173, and 233 2,5-Me 2 -Ara f →3)-Ara f -(1→ 26.0 43, 57, 71, 85, 99, 117, 129, 149, 161, 173, and 233 2,3,4-Me 3 -Gal p →6)-Gal p -(1→ 22.5 43, 49, 57, 71, 87, 99, 101, 117, 129, 161, 189, and 233 The 1 H and 13 C NMR spectra of WAP were shown in Fig. S1. The standardization of chemical shifts of H 2 O ( δ 4.79 ppm) was performed according to a previous report of 1 H NMR spectrum ( Gottlieb et al., 1997 Gottlieb et al., 1997 ). As shown in Fig. S1a, in the anomeric region of the 1 H NMR spectrum, δ 4.97, 5.00, 5.02, 5.04, 5.05, 5.06, 5.07, 5.10, 5.11, 5.12, 5.14, 5.15, 5.16, 5.17, 5.18, 5.20, 5.21, 5.28, and 5.32 ppm were recognized for α‍-anomeric protons, and the others ( δ 4.31, 4.32, 4.36, 4.39, 4.41, 4.43, 4.46, 4.48, 4.50, 4.51, 4.54, 4.56, and 4.57 ppm) were identified as β‍-anomeric protons ( Tamiello et al., 2018 Tamiello et al., 2018 ). The anomeric 1 H signals mainly occurred within the range of δ 4.31–4.57 ppm, which indicated that the configurations of WAP were mostly β form. Fig. S1b presented the 13 C NMR spectrum of WAP. The existence of the C1 signal ( δ 102.87, 100.46, 96.22, 96.07, 95.90, 95.87, 95.36, 92.02, and 91.73 ppm) indicated the presence of pyranose forms in WAP ( Gorin and Mazurek, 1975 Gorin and Mazurek, 1975 ; Agrawal, 1992 Agrawal, 1992 ). Furthermore, the 13 C signal range of δ 64–70 ppm indicated the presence of →6) glycosidic linkages in WAP. The signals at δ 102.87/4.48 (C1/H1) were related to β‍‍-D‍-Gal p units ( Yan et al., 2013 Yan et al., 2013 ). Overall, the data indicated that WAP represents all characteristics of an ideal polysaccharide. 3.4 In vitro antioxidant effects of WAP 2 Previous studies have revealed the good antioxidant effect of wax apple extracts ( Simirgiotis et al., 2008 Simirgiotis et al., 2008 ; Sulaiman and Ooi, 2014 Sulaiman and Ooi, 2014 ; Esua et al., 2017 Esua et al., 2017 ). Moreover, polysaccharides from plants and fungi showed remarkable antioxidant activity ( Zeng et al., 2014 Zeng et al., 2014 ; Xie et al., 2016 Xie et al., 2016 ; Yan et al., 2016 Yan et al., 2016 ). Therefore, the antioxidant activity of WAP was verified by two in vitro antioxidant assays, while vitamin C was used as positive control. Regarding the ABTS scavenging activity, the IC 50 values of WAP and vitamin C were (0.29±0.01) and (0.01±0.00) mg/mL, respectively. In terms of FRAP, the antioxidant activity levels of WAP and vitamin C were (0.51±0.01) and (14.15±0.18) μmol Fe 2+ /mg, respectively. These results indicated that WAP from wax apple has potent antioxidant effects. 3.5 Effects of WAP on EC-induced cytotoxicity and genotoxicity in L02 cells 2 In our study, L02 cells were used to evaluate the effect of WAP on EC-induced toxicity. Firstly, different concentrations (2.5, 5, 10, 25, 50, 100, 200, and 250 μg/mL) of WAP were analyzed for cytotoxicity on L02 cells. According to Fig. 2a, no cytotoxicity was detected for the different concentrations of WAP on L02 cells; hence, it was confirmed that WAP was suitable for further investigation. EC was selected at a concentration of 60 mmol/L due to its cytotoxicity of 55.45%, which was suitable for the subsequent investigation of the protective effect. When L02 cells were pretreated with different concentrations (2.5, 5, 10, 25, 50, 100, 200, and 250 μg/mL) of WAP for 24 h, the cell viability recovered dose-dependently compared to EC-treated group (Fig. 2b). Specifically, when L02 cells were treated with lower concentrations of WAP (2.5, 5, 10, and 25 μg/mL), no effective protection was observed against EC-induced cytotoxicity. Higher concentrations of WAP (50, 100, 200, and 250 μg/mL) significantly improved the cell viability at 60.77%, 63.75%, 65.10%, and 62.10%, respectively, compared to the EC (60 mmol/L) group. These data revealed that WAP could resist EC-induced cytotoxicity at a suitable concentration (50, 100, 200, and 250 μg/mL). Previous studies found that EC exposure can cause genotoxicity in cells ( Jiao et al., 2014 Jiao et al., 2014 ; Chen et al., 2016b Chen et al., 2016b ), which is an important perspective of cytotoxicity, as shown by the condensation of chromatin and nuclear fragmentation. Therefore, we evaluated the protective effect of WAP against EC-induced genotoxicity. Hoechst 33258 is a specific fluorescent dye that can bind with genomic DNA at high sensitivity; therefore, it was used in the present study. According to Fig. 2c, the fluorescence intensity of EC-treated cell nuclei was brighter than that of control group. In the WAP-treated group, the decrease in fluorescence intensity (by contrast) was observed compared to the EC-treated group. These results suggested that WAP can prevent the EC-induced DNA damage in L02 cells. 3.6 Protective effect of WAP against EC-induced ROS and O 2 • - generation 2 Previous studies associated EC cytotoxicity with ROS ( Chen et al., 2016b Chen et al., 2016b ; Cui et al., 2016 Cui et al., 2016 ). Accumulating evidence indicates that cellular redox disorders are caused by ROS overproduction, and treatment with antioxidant-rich components could suppress the ROS by regulating the cellular redox system ( Uttara et al., 2009 Uttara et al., 2009 ; Rodríguez-Ramiro et al., 2011 Rodríguez-Ramiro et al., 2011 ). ROS production in L02 cells can be analyzed by the 2',‍7'- dichlorofluorescein (DCF) fluorescence assay. According to Figs. 3 3 a and 3 3 b, EC treatment applied to L02 cells significantly increased the ROS production (124.02% mean fluorescence intensity) compared to the control (100.00%). On the contrary, pretreatment with WAP at 5, 10, 25, 50, 100, 200, and 250 μg/mL resulted in the significantly decreased mean fluorescence intensity of ROS, namely, 112.01%, 113.72%, 105.69%, 101.67%, 106.75%, 98.77%, and 90.65%, respectively (Figs. 3 3 a and 3 3 b). As a whole, WAP exhibited good performance in scavenging intracellular ROS. Cellular O 2 •- , a one-electron reduced product from the reduction of mitochondrial electron transport in the respiratory chain, was evaluated in previous studies ( Pervaiz and Clement, 2007 Pervaiz and Clement, 2007 ; Chen W et al., 2011 Chen W et al., 2011 ). As compared with the control (100.00%), EC-treated L02 cells showed significantly higher fluorescence intensity of O 2 •- (122.96%), which was suppressed dose-dependently in response to WAP treatment (Figs. 3 3 c and 3 3 d). WAP at 200 and 250 μg/mL significantly decreased the mean fluorescence intensity, with values of 98.60% and 95.64%, respectively. It can be concluded that WAP could provide protection against EC-induced intracellular ROS and O 2 •- overproduction and accumulation in L02 cells. 3.7 Prevention of EC-induced mitochondrial dysfunctions in L02 cells by WAP pretreatment 2 Mitochondria, as crucial organelles for the regulation of cellular energy production and metabolism, are associated with ROS production and regulating the cell redox potential ( Zorov et al., 2014 Zorov et al., 2014 ). The number, structure, and compartments of mitochondria are indicative of cell state. Therefore, the protective effects of WAP on mitochondrial mass and MMP in the EC-treated L02 cells were evaluated. EC-treated L02 cells lowered mitochondrial mass (79.35%) compared to the control (100.00%), while pretreatment with different concentrations of WAP did not improve the mitochondrial mass (Figs. 4a and 4b). As is known, mitochondrial membrane dysfunctions such as MMP decline and mitochondrial membrane lipid peroxidation are associated with ROS generation ( Lin and Beal, 2006 Lin and Beal, 2006 ). Thus, we further investigated the protective effect of WAP on MMP. According to our work, the exposure of L02 cells to EC (60 mmol/L) for 24 h significantly suppressed the MMP level, whereas the mean fluorescence intensity of EC-treated cells was only 71.71% compared to the control (100.00%) (Figs. 4c and 4d). In contrast, the pretreatment of L02 cells with different concentrations of WAP (10, 25, 50, 100, 200, and 250 μg/mL) dose-dependently increased the mean fluorescence intensity of Rh123, namely, with 79.57%, 83.35%, 99.73%, 101.29%, 99.96%, and 94.82%, respectively. These results suggested that EC treatment induced the collapse of MMP, which was effectively ameliorated by WAP (Fig. 4d). 3.8 Effect of WAP on EC-treated GSH depletion in L02 cells 2 GSH is known as an antioxidant molecule with a potential role of maintaining the cellular redox system ( Shila et al., 2005 Shila et al., 2005 ; Bhat et al., 2015 Bhat et al., 2015 ). Excessive ROS production is associated with the depletion of intracellular GSH. This is because a decreased level of intracellular GSH cannot eliminate an excessive ROS level. Thus, intracellular GSH level was measured using an NDA fluorescence probe in our research in the presence or absence of WAP. As confirmed by Fig. 5, cellular GSH depletion occurred after EC treatment, while the mean fluorescence intensity of EC-treated group was only 50.76% compared to the control (100.00%) (Fig. 5a). On the other hand, WAP pretreatment significantly increased the intracellular GSH content compared to the EC-induced group. Different concentrations of WAP (25, 50, 100, 200, and 250 μg/mL) yielded dose-dependent fluorescence intensity, such as 69.72%, 73.38%, 79.28%, 79.14%, and 80.37%, respectively (Fig. 5b). The above data indicated that WAP had a positive effect against EC-induced GSH depletion, which helped to maintain the redox balance in L02 cells. 4 Conclusions 1 Oxidative stress plays an important causative role in hepatic oxidative diseases such as nonalcoholic fatty liver disease, alcoholic liver disease, and hepatitis type C, which can be ameliorated by the intake of polysaccharides or polysaccharide-rich foods. WAP has recently attracted increasing attention regarding its potential therapeutic effect on oxidative dysfunction. This study isolated a polysaccharide from wax apple and evaluated its protective effect against hepatic oxidative disease. WAP was shown to contain several monosaccharides such as mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, and fucose. The molar ratios of these monosaccharides were determined to be 2.20:3.94:4.45:8.56:8.86:‍30.82:‍39.78:‍1.48. Cell-level studies indicated that WAP pretreatment could suppress EC-induced oxidative damage in human hepatocytes via preventing cytotoxicity and DNA damage, scavenging intracellular ROS and O2 •- , attenuating mitochondrial membrane potential collapse, and reducing GSH depletion. Collectively, our findings suggest that WAP could be an important component or supplement for the treatment of oxidative stress-mediated clinical complications such as EC-induced hepatic oxidative damage. At the same time, further studies are necessary to prove the effect in vivo at the biological and clinical levels. References Agrawal PK , 1992 . NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides . 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This work was supported by the Zhejiang Provincial Key R&D Program of China (No. 2021C02018) and the Open Project of Wencheng Joint Research Center of Big Health Industry of Zhejiang University (No. Zdwc2205), China. 10.1631/jzus.B2200629 1673-1581（2023）07-0574-13 鲍涛,Naymul KARIM,柯慧慧等.莲雾多糖对氨基甲酸乙酯诱导人肝细胞氧化损伤的抑制作用研究[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(07):574-586. 鲍涛,Naymul KARIM,柯慧慧等.莲雾多糖对氨基甲酸乙酯诱导人肝细胞氧化损伤的抑制作用研究[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(07):574-586. This work was supported by the Zhejiang Provincial Key R&D Program of China (No. 2021C02018) and the Open Project of Wencheng Joint Research Center of Big Health Industry of Zhejiang University (No. Zdwc2205), China. 2022-12-06 2023-02-27 2023-07-15 2023-07-14 2023-06-07 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 39916358 Tao BAO, Naymul KARIM, Huihui KE, Jitbanjong TANGPONG, and Wei CHEN declare that there are no conflicts of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines Supplementary information 1 Fig. S1 浙江大学学报（英文版）（B辑：生物医学和生物技术） 7 24