五味子质量控制方法及其提取物体外抗氧化活性研究
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摘要
五味子是我国传统著名中药，具有滋补强健、镇静安神、止咳平喘和保肝护肝等作用。长久以来，传统中医将五味子（Schisandra chinensis(Turcz.) Baill.）和华中五味子（Schisandra sphenanthera Rehd.et Wils.）的干燥成熟果实作为同种药材使用，统称为“五味子”。但是，大量现代研究表明，五味子和华中五味子果实化学成分和含量均有较大差别。因此，自《中国药典》（2000年版）开始将华中五味子果实单独收载为“南五味子”。《中国药典》（2010年版）中收载的五味子和南五味子的质量标准缺乏专属性且指标单一，不能真正反映其内在质量。本文采用成分分析、中药指纹图谱技术和DNA分子标记技术对五味子和南五味子进行了较为系统的研究，为二者的评价和质量控制提供更全面、科学、有效的方法。此外，本文还研究了五味子提取物（SCEs）对H2O2诱导氧化损伤的胰岛细胞RINm5F的保护作用，初步探索其对Ⅱ型糖尿病的治疗机制。本研究获得国家科技重大专项课题-重大新药创制“中药药效物质基础及物质资源库研究关键技术”资助，主要研究内容如下：
1.研究了五味子木脂素的最佳提取工艺，通过正交试验进行筛选，以五味子醇甲和总木脂素含量为指标，优选出五味子木脂素最佳提取工艺为：90%的乙醇为提取溶剂，料液比为1：15，超声提取20min，提取3次。
2.对五味子木脂素类成分进行研究，经多次硅胶柱层析，从五味子中共分离得到6个木脂素类单体化合物，经1H-NMR、13C-NMR鉴定其结构并用HPLC检测其纯度。6个单体化合物分别为五味子丙素（schisandrin C）、五味子乙素（schisandrin B）、五味子甲素（deoxyshisandrin）、五味子酯甲（schisantherinA）、五味子醇乙（schisandrol B）和五味子醇甲（schisandrin）。
3.建立了可同时分离检测五味子和南五味子中8种主要木脂素类成分的高效液相色谱（HPLC）方法，并用该方法对不同产地的五味子和南五味子进行分析。结果发现五味子中普遍存在五味子醇甲、五味子醇乙、五味子酯甲、五味子酚、五味子甲素、五味子乙素和五味子丙素，其中五味子醇甲活性较强且含量最高，建议采用五味子醇甲和7种木脂素总含量双指标控制五味子质量。五味子醇甲含量不低于4.0mg/g且7种木脂素总含量不低于14.0mg/g为优级品；五味子醇甲含量不低于2.9mg/g，且7种木脂素总含量不低于8.0mg/g为合格品。发现五味子酯甲、安五脂素和五味子甲素为南五味子的主要成分，建议采用上述3种木脂素总含量为指标控制南五味子质量，3种木脂素总含量不低于8.0mg/g为合格品。
4.首次采用胶束电动毛细管电泳法（MEKC）实现了五味子和南五味子中8种主要木脂素类成分的快速分离检测，本方法的特点和突出优势在于分析速度较现有方法大幅提高（本法9.0min，文献报道MEKC法18min以上，HPLC法30min以上）。用该方法对不同产地的五味子和南五味子进行分析，结果与HPLC法一致。
5.建立了五味子HPLC指纹图谱，确定了10个共有峰为特征峰，并指认了其中8种成分，用所建立的方法对20批样品进行指纹图谱分析。以指纹图谱中各特征峰相对峰面积为变量，进行系统聚类分析，将20批药材分为A、B两大类，A类为五味子，B类为南五味子；进一步将A类分为两小类，其中A1类为优级品，A2类为合格品；聚类结果与本文第3章中HPLC法测定结果一致。以9批五味子优级品药材为对照，建立共有模式，采用指纹图谱软件计算相似度，规定相似度大于0.850为五味子合格品药材，大于0.950为五味子优级品药材。对20批样品进行分析，所得结果与聚类分析和HPLC法结果一致。
6.建立了五味子高效毛细管电泳（HPCE）指纹图谱，确定了10个特征峰，并指认了其中8种成分，用所建立的方法对20批样品进行指纹图谱分析。根据聚类分析和相似度分析结果，可将20批样品分为五味子优级品、五味子合格品和南五味子，分类结果与本文第3章中HPLC法测定结果一致。
7.建立了五味子红外（IR）指纹图谱，并用该方法对20批样品进行指纹图谱分析。以9批五味子推荐药材的平均光谱作为红外参考光谱（R），采用“OPUS”软件进行相似度分析。9批五味子优级品药材指纹图谱与参考图谱（R）相似度为（97.33～99.64%），6批五味子合格品药材指纹图谱与参考图谱（R）相似度为（94.86～96.63%），5批南五味子药材指纹图谱与参考图谱（R）相似度较低（83.05～90.56%）。可见红外指纹图谱可有效区分五味子和南五味子药材，还可在一定程度上反映五味子药材的品质。
8.应用随机扩增DNA多态性分析（RAPD）技术对7批五味子和3批南五味子样品进行DNA指纹分析，从60条引物中筛选出5条重复性好、扩增条带多且清晰的引物。其中引物S28扩增结果多态性丰富、扩增条带特异性强，可区分五味子和南五味子。
9.利用H2O2诱导氧化损伤的胰岛细胞RINm5F为模型，通过MTT法、检测细胞内和培养液中SOD活力及MDA含量来研究五味子提取物（SCEs）的抗氧化活性及其机制。
1）SCEs对胰岛细胞有增殖作用，并呈剂量依赖性。
2）SCEs对H2O2诱导氧化损伤的胰岛细胞有保护作用。与模型组相比，SCEs高、中剂量组细胞存活率明显增加，分别提高了63.4%和45.6%。这说明SCEs具有浓度依赖性的抗氧化保护作用，且以SCEs高剂量组效果最为明显。
3）与模型组相比，SCEs高、中剂量组培养液和细胞内MDA含量明显减少（P﹤0.05），显示细胞氧化损伤程度减轻。
4）与模型组相比，SCEs高、中剂量组培养液和细胞内SOD活力明显增强（P﹤0.05），说明细胞的抗氧化能力增强。
Schisandra chinensis (Turcz.) Baill.(Schisandraceae) is a well-known medicinal plantin Traditional Chinese Medicine. The fruits have been used for centuries as an antitussive,tonic and sedative agent, and in traditional medicine to improve the liver function of patientswith viral hepatitis. In traditional Chinese medicine, the dried ripe fruits of both Schisandrasphenanthera and Schisandra chinensis have long been used as Wuweizi, even though theirchemical constituents and contents of the bioactive components are quite different. Since2000, they have been accepted as two different crude drugs, Schisandrae chinensis Fructusand Schisandrae sphenantherae Fructus, respectively, by the Chinese Pharmacopoeia. In theChinese Pharmacopoeia (2010), the quality standards of S. chinensis Fructus and S.sphenantherae Fructus were not specific and could not reflect their inherent quality. In thispaper, chemical analytical methods, fingerprint techniques and DNA molecular markerstechniques were used to study S. chinensis Fructus and S. sphenantherae Fructussystemically, in order to develop a comprehensive, scientific and effective method for theevaluation and quality control for them. Moreover, we studied the protective effect of SCEson the oxidative damage of RINm5F induced by H2O2, and explored the therapeuticmechanism of SCEs for type2diabetes.
1. According to the orthogonal experimental design, the optimum extracting conditionsof lignans from the S. chinensis Fructus was showed as following: extracting solvent was90%ethanol, solid-liquid ratio was1:15, supersonic extracting time was20min andextracting thrice.
2. Six lignans: schisandrin C, schisandrin B, deoxyshisandrin, schisantherin A,schisandrol B and schisandrin were isolated by silica gel chromatography from S. chinensisFructus, and their structures were elucidated on1H-NMR、13C-NMR and HPLC.
3. A High Performance Liqnid Chromatography (HPLC) method has been developedfor the simultaneous determination of eight lignans, schizandrin, schisandrol B, schisantherinA, schisanhenol, anwulignan, deoxyschizandrin, schizandrin B and schizandrin C, in S.chinensis Fructus and S. sphenantherae Fructus, and20batches of samples from different areas were analyzed by the developed HPLC method. The results showed that seven lignancomponents, that is schizandrin, schisandrol B, schisantherin A, schisanhenol,deoxyschizandrin, schizandrin B and schizandrin C, exists widely in S. chinensis Fructus,and schizandrin is the highest amount component with strong activity. So it was suggestedthat the content of schizandrin and the total content of the seven lignans could be as doubleindexes for quantitative examination of S. chinensis Fructus, and the contents of schizandrinand seven lignans should be4.0and14.0m g/g, respectively, for superior drugs, and theyshould be2.9and8.0mg/g, respectively, for certified drugs. Schisantherin A, anwulignanand deoxyschizandrin were present as the main components in S. sphenantherae Fructus, itwas suggested that the total content of the three lignans could be as the indexe forquantitative examination of Fructus Schisandrae Sphenantherae, and the total content shouldbe8.0mg/g for certified drugs.
4. A simple, rapid and sensitive Micellar Electrokinetic Chromatography (MEKC)method has been developed for the simultaneous quantitative analysis of eight lignans,schizandrin, schisandrol B, schisantherin A, schisanhenol, anwulignan, deoxyschizandrin,schizandrin B and schizandrin C, in S. chinensis Fructus and S. sphenantherae Fructus. Toour best knowledge, this analysis may be the first example involving the simultaneousdetection of the eight lignans in S. chinensis Fructus and S. sphenantherae Fructus by theMEKC method. Compared to published methods, the analysis time of this method wasgreatly decreased with good separation efficiency.20batches of samples from different areaswere analyzed by the developed MEKC method and HPLC method, and the results weresimilar.
5. A High Performance Liqnid Chromatography (HPLC) fingerprint of S. chinensisFructus has been developed and20batches of samples from different areas were analyzed bythe developed method. According to the comparative study of fingerprints, we found10communal peaks and identify8peaks.20batches of samples were classified as3clusters bycluster analysis, A1and A2were superior and certified drugs, respectively. B was S.sphenantherae Fructus. The results were similar to HPLC method.9batches of superiordrugs were confirmed to establish the mutual model. The quality of samples were assessedby "Similarity Evaluation System for Chromatographic Fingerprint of TCM2004". It wassuggested that the Similarity should be0.950and0.850for superior drugs and certified drugs.20batches of samples from different areas were analyzed by the developed method,and the results were similar to HPLC method.
6. A High Performance Capillary Electrophoresis (HPCE) fingerprint of S. chinensisFructus has been developed, and20batches of samples were analyzed by the developedmethod. They were classified to be superior drugs, certified drugs and S. sphenantheraeFructus, respectively, based on the results of cluster analysis and similarity analysis, and theresults were similar to HPLC method.
7. A Infrared Spectrum (IR) fingerprint of S. chinensis Fructus has been developed, and20batches of samples were analyzed by the developed method.9batches of superior drugswere confirmed to establish the reference spectrum. The quality of samples were assessed by"OPUS" based on the results of similarity analysis. The similarity between referencespectrum and the fingerprint of superior drugs, certified drugs, S. sphenantherae Fructuswere97.33～99.64%,94.86～96.63%and83.05～90.56%, respectively. The results showedthat the IR fingerprint can provides a rapid identification and quality control method of S.chinensis Fructus.
8.7batches of S. chinensis and3batches of S. sphenanthera were analyzed by RandomAmplified Polymorphic DNA (RAPD) techniques.5RAPD primers were selected from60primers, and its strip was neat, distinct and stability. Among these selected primers only1primer could identify S. chinensis and S. sphenanthera.
9. In this study, we observe the effects of SCEs on the rate of cell vitality, the activity ofSOD and content of MDA in RINm5F cells and cells culture fluid injured by hydrogenperoxide (H2O2), in order to discuss the possible mechanisms of the antioxidant activity ofSCEs.
1) SCEs enhanced RINm5F cells growth in a dose-dependent manner in vitro;
2) SCEs could protect the RINm5F cells injured by H2O2, compared with model group,high-dose group and middle-group enhanced the cell viability significantly, the cell viabilityincreased by63.4%and45.6%, respectively. The result showed that the antioxidant activityin vitro of SCEs is in dose dependent manner, and the high-dose group is the most notable.
3) The contents of MDA in RINm5F cells and cells culture fluid of high-dose group andmiddle-group were significant lower than that of model group (P﹤0.05), the result showedthat SCEs can obviously alleviate the oxidative injury of RINm5F cells.
4) The activity of SOD in RINm5F cells and cells culture fluid of high-dose group andmiddle-group were significant higher than that of model group (P﹤0.05), the result showedthat SCEs can obviously enhance the antioxidant ability of RINm5F cells.
1.研究了五味子木脂素的最佳提取工艺，通过正交试验进行筛选，以五味子醇甲和总木脂素含量为指标，优选出五味子木脂素最佳提取工艺为：90%的乙醇为提取溶剂，料液比为1：15，超声提取20min，提取3次。
2.对五味子木脂素类成分进行研究，经多次硅胶柱层析，从五味子中共分离得到6个木脂素类单体化合物，经1H-NMR、13C-NMR鉴定其结构并用HPLC检测其纯度。6个单体化合物分别为五味子丙素（schisandrin C）、五味子乙素（schisandrin B）、五味子甲素（deoxyshisandrin）、五味子酯甲（schisantherinA）、五味子醇乙（schisandrol B）和五味子醇甲（schisandrin）。
3.建立了可同时分离检测五味子和南五味子中8种主要木脂素类成分的高效液相色谱（HPLC）方法，并用该方法对不同产地的五味子和南五味子进行分析。结果发现五味子中普遍存在五味子醇甲、五味子醇乙、五味子酯甲、五味子酚、五味子甲素、五味子乙素和五味子丙素，其中五味子醇甲活性较强且含量最高，建议采用五味子醇甲和7种木脂素总含量双指标控制五味子质量。五味子醇甲含量不低于4.0mg/g且7种木脂素总含量不低于14.0mg/g为优级品；五味子醇甲含量不低于2.9mg/g，且7种木脂素总含量不低于8.0mg/g为合格品。发现五味子酯甲、安五脂素和五味子甲素为南五味子的主要成分，建议采用上述3种木脂素总含量为指标控制南五味子质量，3种木脂素总含量不低于8.0mg/g为合格品。
4.首次采用胶束电动毛细管电泳法（MEKC）实现了五味子和南五味子中8种主要木脂素类成分的快速分离检测，本方法的特点和突出优势在于分析速度较现有方法大幅提高（本法9.0min，文献报道MEKC法18min以上，HPLC法30min以上）。用该方法对不同产地的五味子和南五味子进行分析，结果与HPLC法一致。
5.建立了五味子HPLC指纹图谱，确定了10个共有峰为特征峰，并指认了其中8种成分，用所建立的方法对20批样品进行指纹图谱分析。以指纹图谱中各特征峰相对峰面积为变量，进行系统聚类分析，将20批药材分为A、B两大类，A类为五味子，B类为南五味子；进一步将A类分为两小类，其中A1类为优级品，A2类为合格品；聚类结果与本文第3章中HPLC法测定结果一致。以9批五味子优级品药材为对照，建立共有模式，采用指纹图谱软件计算相似度，规定相似度大于0.850为五味子合格品药材，大于0.950为五味子优级品药材。对20批样品进行分析，所得结果与聚类分析和HPLC法结果一致。
6.建立了五味子高效毛细管电泳（HPCE）指纹图谱，确定了10个特征峰，并指认了其中8种成分，用所建立的方法对20批样品进行指纹图谱分析。根据聚类分析和相似度分析结果，可将20批样品分为五味子优级品、五味子合格品和南五味子，分类结果与本文第3章中HPLC法测定结果一致。
7.建立了五味子红外（IR）指纹图谱，并用该方法对20批样品进行指纹图谱分析。以9批五味子推荐药材的平均光谱作为红外参考光谱（R），采用“OPUS”软件进行相似度分析。9批五味子优级品药材指纹图谱与参考图谱（R）相似度为（97.33～99.64%），6批五味子合格品药材指纹图谱与参考图谱（R）相似度为（94.86～96.63%），5批南五味子药材指纹图谱与参考图谱（R）相似度较低（83.05～90.56%）。可见红外指纹图谱可有效区分五味子和南五味子药材，还可在一定程度上反映五味子药材的品质。
8.应用随机扩增DNA多态性分析（RAPD）技术对7批五味子和3批南五味子样品进行DNA指纹分析，从60条引物中筛选出5条重复性好、扩增条带多且清晰的引物。其中引物S28扩增结果多态性丰富、扩增条带特异性强，可区分五味子和南五味子。
9.利用H2O2诱导氧化损伤的胰岛细胞RINm5F为模型，通过MTT法、检测细胞内和培养液中SOD活力及MDA含量来研究五味子提取物（SCEs）的抗氧化活性及其机制。
1）SCEs对胰岛细胞有增殖作用，并呈剂量依赖性。
2）SCEs对H2O2诱导氧化损伤的胰岛细胞有保护作用。与模型组相比，SCEs高、中剂量组细胞存活率明显增加，分别提高了63.4%和45.6%。这说明SCEs具有浓度依赖性的抗氧化保护作用，且以SCEs高剂量组效果最为明显。
3）与模型组相比，SCEs高、中剂量组培养液和细胞内MDA含量明显减少（P﹤0.05），显示细胞氧化损伤程度减轻。
4）与模型组相比，SCEs高、中剂量组培养液和细胞内SOD活力明显增强（P﹤0.05），说明细胞的抗氧化能力增强。
Schisandra chinensis (Turcz.) Baill.(Schisandraceae) is a well-known medicinal plantin Traditional Chinese Medicine. The fruits have been used for centuries as an antitussive,tonic and sedative agent, and in traditional medicine to improve the liver function of patientswith viral hepatitis. In traditional Chinese medicine, the dried ripe fruits of both Schisandrasphenanthera and Schisandra chinensis have long been used as Wuweizi, even though theirchemical constituents and contents of the bioactive components are quite different. Since2000, they have been accepted as two different crude drugs, Schisandrae chinensis Fructusand Schisandrae sphenantherae Fructus, respectively, by the Chinese Pharmacopoeia. In theChinese Pharmacopoeia (2010), the quality standards of S. chinensis Fructus and S.sphenantherae Fructus were not specific and could not reflect their inherent quality. In thispaper, chemical analytical methods, fingerprint techniques and DNA molecular markerstechniques were used to study S. chinensis Fructus and S. sphenantherae Fructussystemically, in order to develop a comprehensive, scientific and effective method for theevaluation and quality control for them. Moreover, we studied the protective effect of SCEson the oxidative damage of RINm5F induced by H2O2, and explored the therapeuticmechanism of SCEs for type2diabetes.
1. According to the orthogonal experimental design, the optimum extracting conditionsof lignans from the S. chinensis Fructus was showed as following: extracting solvent was90%ethanol, solid-liquid ratio was1:15, supersonic extracting time was20min andextracting thrice.
2. Six lignans: schisandrin C, schisandrin B, deoxyshisandrin, schisantherin A,schisandrol B and schisandrin were isolated by silica gel chromatography from S. chinensisFructus, and their structures were elucidated on1H-NMR、13C-NMR and HPLC.
3. A High Performance Liqnid Chromatography (HPLC) method has been developedfor the simultaneous determination of eight lignans, schizandrin, schisandrol B, schisantherinA, schisanhenol, anwulignan, deoxyschizandrin, schizandrin B and schizandrin C, in S.chinensis Fructus and S. sphenantherae Fructus, and20batches of samples from different areas were analyzed by the developed HPLC method. The results showed that seven lignancomponents, that is schizandrin, schisandrol B, schisantherin A, schisanhenol,deoxyschizandrin, schizandrin B and schizandrin C, exists widely in S. chinensis Fructus,and schizandrin is the highest amount component with strong activity. So it was suggestedthat the content of schizandrin and the total content of the seven lignans could be as doubleindexes for quantitative examination of S. chinensis Fructus, and the contents of schizandrinand seven lignans should be4.0and14.0m g/g, respectively, for superior drugs, and theyshould be2.9and8.0mg/g, respectively, for certified drugs. Schisantherin A, anwulignanand deoxyschizandrin were present as the main components in S. sphenantherae Fructus, itwas suggested that the total content of the three lignans could be as the indexe forquantitative examination of Fructus Schisandrae Sphenantherae, and the total content shouldbe8.0mg/g for certified drugs.
4. A simple, rapid and sensitive Micellar Electrokinetic Chromatography (MEKC)method has been developed for the simultaneous quantitative analysis of eight lignans,schizandrin, schisandrol B, schisantherin A, schisanhenol, anwulignan, deoxyschizandrin,schizandrin B and schizandrin C, in S. chinensis Fructus and S. sphenantherae Fructus. Toour best knowledge, this analysis may be the first example involving the simultaneousdetection of the eight lignans in S. chinensis Fructus and S. sphenantherae Fructus by theMEKC method. Compared to published methods, the analysis time of this method wasgreatly decreased with good separation efficiency.20batches of samples from different areaswere analyzed by the developed MEKC method and HPLC method, and the results weresimilar.
5. A High Performance Liqnid Chromatography (HPLC) fingerprint of S. chinensisFructus has been developed and20batches of samples from different areas were analyzed bythe developed method. According to the comparative study of fingerprints, we found10communal peaks and identify8peaks.20batches of samples were classified as3clusters bycluster analysis, A1and A2were superior and certified drugs, respectively. B was S.sphenantherae Fructus. The results were similar to HPLC method.9batches of superiordrugs were confirmed to establish the mutual model. The quality of samples were assessedby "Similarity Evaluation System for Chromatographic Fingerprint of TCM2004". It wassuggested that the Similarity should be0.950and0.850for superior drugs and certified drugs.20batches of samples from different areas were analyzed by the developed method,and the results were similar to HPLC method.
6. A High Performance Capillary Electrophoresis (HPCE) fingerprint of S. chinensisFructus has been developed, and20batches of samples were analyzed by the developedmethod. They were classified to be superior drugs, certified drugs and S. sphenantheraeFructus, respectively, based on the results of cluster analysis and similarity analysis, and theresults were similar to HPLC method.
7. A Infrared Spectrum (IR) fingerprint of S. chinensis Fructus has been developed, and20batches of samples were analyzed by the developed method.9batches of superior drugswere confirmed to establish the reference spectrum. The quality of samples were assessed by"OPUS" based on the results of similarity analysis. The similarity between referencespectrum and the fingerprint of superior drugs, certified drugs, S. sphenantherae Fructuswere97.33～99.64%,94.86～96.63%and83.05～90.56%, respectively. The results showedthat the IR fingerprint can provides a rapid identification and quality control method of S.chinensis Fructus.
8.7batches of S. chinensis and3batches of S. sphenanthera were analyzed by RandomAmplified Polymorphic DNA (RAPD) techniques.5RAPD primers were selected from60primers, and its strip was neat, distinct and stability. Among these selected primers only1primer could identify S. chinensis and S. sphenanthera.
9. In this study, we observe the effects of SCEs on the rate of cell vitality, the activity ofSOD and content of MDA in RINm5F cells and cells culture fluid injured by hydrogenperoxide (H2O2), in order to discuss the possible mechanisms of the antioxidant activity ofSCEs.
1) SCEs enhanced RINm5F cells growth in a dose-dependent manner in vitro;
2) SCEs could protect the RINm5F cells injured by H2O2, compared with model group,high-dose group and middle-group enhanced the cell viability significantly, the cell viabilityincreased by63.4%and45.6%, respectively. The result showed that the antioxidant activityin vitro of SCEs is in dose dependent manner, and the high-dose group is the most notable.
3) The contents of MDA in RINm5F cells and cells culture fluid of high-dose group andmiddle-group were significant lower than that of model group (P﹤0.05), the result showedthat SCEs can obviously alleviate the oxidative injury of RINm5F cells.
4) The activity of SOD in RINm5F cells and cells culture fluid of high-dose group andmiddle-group were significant higher than that of model group (P﹤0.05), the result showedthat SCEs can obviously enhance the antioxidant ability of RINm5F cells.
引文
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