五味子鲜果多酚提取鉴定及其对受损HepG2细胞保护作用的研究
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摘要
五味子[Schisandra chinensis (Turcz.) Baill.]鲜果的果实属于浆果类果实,主产于中国东北,包括黑龙江、吉林、辽宁省的山区。目前五味子已作为功能性成分添加到各种食品中,如果红酒、啤酒、饮料、酸奶、果酱、蛋糕等。同时,五味子果实干燥后作为一种中药材,也被广泛应用于各种传统中药制剂中。近年来已有文献报道五味子具有多种生理活性,包括保肝、抗癌、抗肥胖、抗病毒、抗炎、以及抗糖尿病等作用。主要存在于五味子果籽中的木脂素被公认为是五味子主要的活性成分,具有抗氧化,保护肝脏、免疫调节,抗炎和抗癌等多种功效。但对于五味子鲜果多酚如花色苷的研究报道却很少。
本课题以五味子鲜果多酚为主要研究对象。第一部分采用随机质心映射优化法研究五味子鲜果果肉多酚及果籽原花青素的最佳提取条件,并测定其主要成分含量和抗氧化活性。分别通过三个提取目标的测定对鲜果多酚6因素和原花青素4因素的最佳组合条件进行考察,得到五味子果肉多酚的最佳提取条件为：67.3%乙醇,pH1.75,液料比8,提取温度55℃,提取时间4h,提取次数4次。其果肉多酚提取物得率最高为18.3mg/g,多酚最高含量为1.847mg/g, DPPH自由基清除率最高为88.9%。此外,含有花色苷0.179mg/g,糖9.573mg/g,蛋白质0.327mg/g。五味子果籽原花青素的最佳提取条件是65%乙醇,pH5.21,液料比9,提取时间8h。其果籽提取物得率最高为167.3mg/g,原花青素最高含量为7.12mg/g, DPPH自由基清除率最高为82.9%。
第二部分研究对比了盐酸和不同有机酸包括柠檬酸、草酸、乙酸、苹果酸、琥珀酸以及抗坏血酸在不用浓度下对五味子多酚提取物稳定性的影响；苹果酸、琥珀酸在高浓度下可显著升高提取物的吸光值(520nm)。异抗坏血酸明显降低提取物的吸光值(520nm)在多酚提取过程中,0.125M盐酸及0.125M草酸可明显提高WPE-S的多酚含量；添加0.125M的盐酸、草酸、苹果酸所得WPE-S、WPE-SC和WPE具有更高的抗氧化能力。
第三部分对五味子果肉多酚提取物进行分离纯化得到五味子花色苷提取物(Wuweizi anthocyanin extract,WAE),联用HPLC-PDA-MS和1H-13C NMR分析,最终确认五味子花色苷为矢车菊-3-O-葡萄糖苷(cyanidin-3-O-glucoside)和矢车菊-3-O-木糖鼠李糖苷(cyanidin-3-O-xylosylrutinoside),含量分别为10.5%和89.5%。
第四部分运用WST-1、流式细胞术以及RT-PCR法对比检测了五味子鲜果多酚提取物(WPE、WAE和WLE)对EtOH、CCl4和AAPH致损肝细胞的保护作用,WST-1细胞增殖检测证明五味子鲜果多酚提取物可以提高EtOH、CCl4和AAPH致损后肝细胞的细胞存活率。0.1mg/mL的WPE和WAE,0.01mg/mL的WLE与损伤组比较的细胞存活率明显上升。通过细胞周期和凋亡的流式细胞术检测,结果证明五味子鲜果多酚提取物可减少由EtOH、CCl4以及AAPH诱导的肝细胞凋亡率和坏死率。保护处理后,凋亡相关基因家族(TIMPs、Caspase-8、 Caspase-9、FADD等)呈现不同程度地抑制表达,而氧化应激基因家族(SOD、GPX1和CAT)以及SATA3信号则呈现高表达,均说明五味子鲜果多酚提取物对受损肝细胞具有明显的保护作用。
Wuweizi (Schisandra chinensis (Turcz.) Baill.) is a berry-like fruit, mainly grown in north-eastern China, including Heilongjiang, Jilin, Liaoning provinces. Wuweizi has been used as a functional ingredient in foods, such as wine, beer, beverages, yoghourt, jam, fruitcake and other products. Wuweizi is also used in Chinese traditional medicine. Recently, some new bioactivities of Wuweizi have been reported, such as hepatoprotection, anti-carcinogenicity, anti-obesity, anti-virus, anti-inflammation and anti-diabetes. Wuweizi has high content of lignan, which mainly exist in seeds which have been considered as the primary constituents for Wuweizi's antioxidant, hepatoprotective, immuo-modulating, anti-inflammatory and anticancer effects. Compared to the intensive studies on lignan, polyphenols in Wuweizi pulp, especially anthocyanins have not received much attention. There were a few of reports about anthocyanins extracted from Wuweizi.
The polyphenol extracted from Wuweizi fresh fruits were studied in this project. Firstly, the extraction optimization and composition analysis of polyphenols in the fresh pulp of Wuweizi have been investigated in this study. The extraction processes of polyphenols from Wuweizi pulp and proanthocyanidins from Wuweizi seed were optimized using Random-Centroid Optimization (RCO) methodology. Six factors for polyphenols and four factors for proanthocyanidins, three extraction targets for polyphenols and proanthocyanidins were considered in the RCO program respectively. Three sets of optimum proposed factor values were obtained corresponding to three extraction targets respectively. The set of optimum proposed factor values for polyphenol extraction given was chosen in further experiments as following:liquid and solid ratio (v/w)8, ethanol67.3%(v/v), initial pH1.75, temperature55℃for4h and extraction repeated for4times. The Wuweizi polyphenol extract (WPE) was obtained with a yield of16.37mg/g and composition of polyphenols1.847mg/g, anthocyanins0.179mg/g, sugar9.573mg/g and protein0.327mg/g. The WPE demonstrated high scavenging activities against DPPH radicals of88.9%. The set of optimum proposed factor values for proanthocyanidins extraction given was chosen in further experiments as following:liquid and solid ratio (v/w)9, ethanol65%(v/v), initial pH5.21and the extraction time is4h. The Wuweizi seed extract (WSE) was obtained with a yield of167.3mg/g and composition of proanthocyanidins7.12mg/g and the scavenging activities against DPPH radicals of82.9%.
Secondly, the stability of Wuweizi polyphenols in Wuweizi has been studied. The study investigated the effect of organic acids including citric acid, oxalic acid, acetic acid, malic acid, succinic acid and erythorbic acid in comparison with HC1on the stability of Wuweizi anthocyanins crude extacts. Higher concentration (0.250M and0.125M) of malic acid, succinic acid increased the absorbance at520nm of anthocyanins significantly. Erythorbic acid has no obvious hypochromic effect on the WPE. In the extraction processing,0.125M of hydrochloric acid and oxalate could obviously increase the polyphenol content of extracts compared with the control without acid added; 0.125M of hydrochloric acid, oxalic acid, malic acid could increase the scavenging activities against DPPH radicals of WPE-S, WPE-SC and WPE, coarse solution, producing clearance rate is significantly higher than acid free blank group, have higher antioxidant ability. The extraction efficiency was evaluated with three parameters, including anthocyanin content, polyphenol content and DPPH radical scavenging activity. All three parameters of the anthocyanin extracts were increased significantly, with oxalic, malic and succinic acid, but not with citric and acetic acid.
Thirdly, Wuweizi anthocyanin crude extract was purified by XAD-7resin, and the profile and the structure of Wuweizi anthocyanin has been confirmed using HPLC-ESI-MS and 'H-13C NMR spectroscopy. Two types of anthocyanins were detected and identified as cyanidin-3-O-glucoside (11%of total anthocyanin content) and cyanidin-3-O-xylosylrutinoside (89%of total anthocyanin content).
Fourthly, the polyphenol extracts were prepared from Wuweizi fresh fruits, including the polyphenol extract (WPE), anthocyanin extract (WAE), and lignin extract (WLE), and their protective effects were investigated on injured HepG2cells by EtOH, CC14and AAPH, respectively. Cell proliferation was tested by the WST-1assay, which showed that the WPE, WAE and WLE treatments increased the cell numbers of HepG2cells injured by EtOH, CCl4in a dose-dependent manner. At the protective concentrations of0.1mg/mL of WPE and WAE, and0.01mg/mL of WLE, cell numbers were significantly increased when compared to the no-treatment control (p<0.05). The AnnexinV-propidium iodide assay showed Wuweizi extract treatments, could also decrease the apoptosis rate and necrosis rate. After injured HepG2cell treated by different Wuweizi extracts, the inhibition expression of apoptosis related gene families (TIMPs, Caspase-8, Caspase-9and FADD) were presented, but showed the high expression on oxidative stress gene families (SOD, GPX1and CAT) and SATA3gene signal. These results indicated that protective effects of Wuweizi polyphenol extracts were generalizable to HepG2cell.
本课题以五味子鲜果多酚为主要研究对象。第一部分采用随机质心映射优化法研究五味子鲜果果肉多酚及果籽原花青素的最佳提取条件,并测定其主要成分含量和抗氧化活性。分别通过三个提取目标的测定对鲜果多酚6因素和原花青素4因素的最佳组合条件进行考察,得到五味子果肉多酚的最佳提取条件为：67.3%乙醇,pH1.75,液料比8,提取温度55℃,提取时间4h,提取次数4次。其果肉多酚提取物得率最高为18.3mg/g,多酚最高含量为1.847mg/g, DPPH自由基清除率最高为88.9%。此外,含有花色苷0.179mg/g,糖9.573mg/g,蛋白质0.327mg/g。五味子果籽原花青素的最佳提取条件是65%乙醇,pH5.21,液料比9,提取时间8h。其果籽提取物得率最高为167.3mg/g,原花青素最高含量为7.12mg/g, DPPH自由基清除率最高为82.9%。
第二部分研究对比了盐酸和不同有机酸包括柠檬酸、草酸、乙酸、苹果酸、琥珀酸以及抗坏血酸在不用浓度下对五味子多酚提取物稳定性的影响；苹果酸、琥珀酸在高浓度下可显著升高提取物的吸光值(520nm)。异抗坏血酸明显降低提取物的吸光值(520nm)在多酚提取过程中,0.125M盐酸及0.125M草酸可明显提高WPE-S的多酚含量；添加0.125M的盐酸、草酸、苹果酸所得WPE-S、WPE-SC和WPE具有更高的抗氧化能力。
第三部分对五味子果肉多酚提取物进行分离纯化得到五味子花色苷提取物(Wuweizi anthocyanin extract,WAE),联用HPLC-PDA-MS和1H-13C NMR分析,最终确认五味子花色苷为矢车菊-3-O-葡萄糖苷(cyanidin-3-O-glucoside)和矢车菊-3-O-木糖鼠李糖苷(cyanidin-3-O-xylosylrutinoside),含量分别为10.5%和89.5%。
第四部分运用WST-1、流式细胞术以及RT-PCR法对比检测了五味子鲜果多酚提取物(WPE、WAE和WLE)对EtOH、CCl4和AAPH致损肝细胞的保护作用,WST-1细胞增殖检测证明五味子鲜果多酚提取物可以提高EtOH、CCl4和AAPH致损后肝细胞的细胞存活率。0.1mg/mL的WPE和WAE,0.01mg/mL的WLE与损伤组比较的细胞存活率明显上升。通过细胞周期和凋亡的流式细胞术检测,结果证明五味子鲜果多酚提取物可减少由EtOH、CCl4以及AAPH诱导的肝细胞凋亡率和坏死率。保护处理后,凋亡相关基因家族(TIMPs、Caspase-8、 Caspase-9、FADD等)呈现不同程度地抑制表达,而氧化应激基因家族(SOD、GPX1和CAT)以及SATA3信号则呈现高表达,均说明五味子鲜果多酚提取物对受损肝细胞具有明显的保护作用。
Wuweizi (Schisandra chinensis (Turcz.) Baill.) is a berry-like fruit, mainly grown in north-eastern China, including Heilongjiang, Jilin, Liaoning provinces. Wuweizi has been used as a functional ingredient in foods, such as wine, beer, beverages, yoghourt, jam, fruitcake and other products. Wuweizi is also used in Chinese traditional medicine. Recently, some new bioactivities of Wuweizi have been reported, such as hepatoprotection, anti-carcinogenicity, anti-obesity, anti-virus, anti-inflammation and anti-diabetes. Wuweizi has high content of lignan, which mainly exist in seeds which have been considered as the primary constituents for Wuweizi's antioxidant, hepatoprotective, immuo-modulating, anti-inflammatory and anticancer effects. Compared to the intensive studies on lignan, polyphenols in Wuweizi pulp, especially anthocyanins have not received much attention. There were a few of reports about anthocyanins extracted from Wuweizi.
The polyphenol extracted from Wuweizi fresh fruits were studied in this project. Firstly, the extraction optimization and composition analysis of polyphenols in the fresh pulp of Wuweizi have been investigated in this study. The extraction processes of polyphenols from Wuweizi pulp and proanthocyanidins from Wuweizi seed were optimized using Random-Centroid Optimization (RCO) methodology. Six factors for polyphenols and four factors for proanthocyanidins, three extraction targets for polyphenols and proanthocyanidins were considered in the RCO program respectively. Three sets of optimum proposed factor values were obtained corresponding to three extraction targets respectively. The set of optimum proposed factor values for polyphenol extraction given was chosen in further experiments as following:liquid and solid ratio (v/w)8, ethanol67.3%(v/v), initial pH1.75, temperature55℃for4h and extraction repeated for4times. The Wuweizi polyphenol extract (WPE) was obtained with a yield of16.37mg/g and composition of polyphenols1.847mg/g, anthocyanins0.179mg/g, sugar9.573mg/g and protein0.327mg/g. The WPE demonstrated high scavenging activities against DPPH radicals of88.9%. The set of optimum proposed factor values for proanthocyanidins extraction given was chosen in further experiments as following:liquid and solid ratio (v/w)9, ethanol65%(v/v), initial pH5.21and the extraction time is4h. The Wuweizi seed extract (WSE) was obtained with a yield of167.3mg/g and composition of proanthocyanidins7.12mg/g and the scavenging activities against DPPH radicals of82.9%.
Secondly, the stability of Wuweizi polyphenols in Wuweizi has been studied. The study investigated the effect of organic acids including citric acid, oxalic acid, acetic acid, malic acid, succinic acid and erythorbic acid in comparison with HC1on the stability of Wuweizi anthocyanins crude extacts. Higher concentration (0.250M and0.125M) of malic acid, succinic acid increased the absorbance at520nm of anthocyanins significantly. Erythorbic acid has no obvious hypochromic effect on the WPE. In the extraction processing,0.125M of hydrochloric acid and oxalate could obviously increase the polyphenol content of extracts compared with the control without acid added; 0.125M of hydrochloric acid, oxalic acid, malic acid could increase the scavenging activities against DPPH radicals of WPE-S, WPE-SC and WPE, coarse solution, producing clearance rate is significantly higher than acid free blank group, have higher antioxidant ability. The extraction efficiency was evaluated with three parameters, including anthocyanin content, polyphenol content and DPPH radical scavenging activity. All three parameters of the anthocyanin extracts were increased significantly, with oxalic, malic and succinic acid, but not with citric and acetic acid.
Thirdly, Wuweizi anthocyanin crude extract was purified by XAD-7resin, and the profile and the structure of Wuweizi anthocyanin has been confirmed using HPLC-ESI-MS and 'H-13C NMR spectroscopy. Two types of anthocyanins were detected and identified as cyanidin-3-O-glucoside (11%of total anthocyanin content) and cyanidin-3-O-xylosylrutinoside (89%of total anthocyanin content).
Fourthly, the polyphenol extracts were prepared from Wuweizi fresh fruits, including the polyphenol extract (WPE), anthocyanin extract (WAE), and lignin extract (WLE), and their protective effects were investigated on injured HepG2cells by EtOH, CC14and AAPH, respectively. Cell proliferation was tested by the WST-1assay, which showed that the WPE, WAE and WLE treatments increased the cell numbers of HepG2cells injured by EtOH, CCl4in a dose-dependent manner. At the protective concentrations of0.1mg/mL of WPE and WAE, and0.01mg/mL of WLE, cell numbers were significantly increased when compared to the no-treatment control (p<0.05). The AnnexinV-propidium iodide assay showed Wuweizi extract treatments, could also decrease the apoptosis rate and necrosis rate. After injured HepG2cell treated by different Wuweizi extracts, the inhibition expression of apoptosis related gene families (TIMPs, Caspase-8, Caspase-9and FADD) were presented, but showed the high expression on oxidative stress gene families (SOD, GPX1and CAT) and SATA3gene signal. These results indicated that protective effects of Wuweizi polyphenol extracts were generalizable to HepG2cell.
引文
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