【作者】 秦哲；

【导师】 杨志强； 余四九；

【作者基本信息】 甘肃农业大学 ， 临床兽医学， 2012， 博士

【摘要】 黄芪是临床常用中药，其主要成分黄芪多糖具有提高免疫力、抗病毒、抗菌、抗氧化、调节血糖、保肝护肾等多种药效作用。然而，由于生药黄芪中黄芪多糖提取得率较低而限制了其广泛应用。前期研究表明，生药黄芪经M-9菌株发酵后，发酵产物中粗多糖得率显著提高。但发酵黄芪的质量控制，药效成分的含量变化及其药理学作用有待研究。因此论文首次开展了发酵黄芪中毛蕊异黄酮葡萄糖苷和黄芪甲苷的含量测定，发酵后黄芪皂苷和多糖的最佳提取工艺条件的优化及发酵黄芪多糖对实验性大鼠肝纤维化的影响并运用数字基因表达谱分析其作用机理。为今后发酵黄芪的药理学深入研究、开发利用及益生菌发酵转化机理的阐述提供理论依据。1.高效液相色谱法测定发酵黄芪和生药黄芪中黄芪甲苷及毛蕊异黄酮葡萄糖苷含量参照2010版《中国药典》黄芪有效成分含量测定方法，应用高效液相色谱法分别对发酵黄芪、生药黄芪中黄芪甲苷和毛蕊异黄酮葡萄糖苷的含量进行测定，结果为0.049%、0.087%、0.030%和0.040%。含有等量黄芪的发酵黄芪和生药黄芪中黄芪甲苷的含量分别为0.063%和0.087%，毛蕊异黄酮葡萄糖苷的含量分别为0.038%和0.040%。结果提示，发酵前后毛蕊异黄酮葡萄糖苷含量差异不显著，而发酵后黄芪甲苷的含量降低了27.6%。这两种成分的含量变化可为发酵黄芪产品的质量控制提供参考。2.发酵黄芪和生药黄芪中黄芪多糖和皂苷的提取分离及含量测定采用综合提取黄芪皂苷和黄芪多糖的方法并筛选提取工艺。黄芪皂苷和黄芪多糖最佳提取工艺条件为：称取一定量黄芪粗粉，用20倍量80%乙醇于80℃回流提取黄芪皂苷，烘干药渣用于黄芪多糖提取。A.黄芪皂苷提取工艺：醇提液制成浸膏后取适量溶于50倍量纯水，上样于AB-8大孔树脂柱，经80%乙醇洗脱得皂苷富集部位中皂苷含量达81.23%。B.通过比较温浸法、传统水煎法、纤维素酶法及饱和生石灰水法，优选出温浸法提取黄芪多糖，提取工艺为：药渣用20量纯水，80℃温浸提取4.5h，离心，浓缩，Sevag法除蛋白，透析，醇沉，无水乙醇、丙酮、乙醚分别洗涤，烘干，得黄芪粗多糖。C.香草醛-硫酸法测得生药黄芪浸膏和发酵黄芪浸膏中皂苷含量分别为16.87%和10.81%，皂苷的提取率分别为0.0654%和0.0558%。苯酚-硫酸法测定多糖含量，发酵黄芪多糖（FAPS）含量及得率分别为66.59%，4.35%；生药黄芪多糖（APS）含量及得率分别为36.01%，2.73%。结果提示，黄芪经发酵后多糖含量及得率分别提高84.92%和59.34%，发酵后皂苷提取率降低17.20%。经薄层层析法测定FAPS中主要糖单组分为葡萄糖、甘露糖等，主要多糖组分为FAPS-2^-1的糖含量为95.41%，其数均分子量为1,564,267Da，重均分子量为1,753,456Da。3. FAPS对大鼠实验性肝纤维化的作用及机制研究采用皮下注射CCl4的方法制备大鼠肝纤维化模型。造模同时给予不同药物干预，研究FAPS对大鼠肝纤维化的作用。雄性SD大鼠60只，随机分为6组，发酵黄芪高剂量组（FAPSH）200mg·kg^-1·d^-1，发酵黄芪中剂量组（FAPSM）100mg·kg^-1·d^-1，发酵黄芪低剂量组（FAPSL）50mg·kg^-1·d^-1，秋水仙碱组（Col.）0.2mg·kg^-1·d^-1，CCl4模型组和对照组给予等体积生理盐水，持续8周。结果得出，FAPS各剂量组大鼠体重均高于模型组，FAPS可显著降低小鼠血清中的ALT、AST、ALP活性（P<0.01），可显著降低肝组织中Hyp含量（P<0.05）及血清中HA，LN，CⅣ，PⅢNP的水平，使降低的GSH-Px （P<0.01）、T-AOC （P<0.05）和GST值显著升高，使异常升高的GSH和MDA含量趋于正常。尤其是FAPSH组各项抗肝纤维化指标均优于FAPSM和FAPSL。通过H.E.、Masson染色及透射电镜观察大鼠肝组织病理变化发现，模型组大鼠肝细胞脂肪空泡化和纤维化病变明显，给予不同药物干预的各组肝组织病理变化有所减轻。结果提示，FAPS对CCl4所致大鼠实验性肝纤维化有一定的修复作用，作用程度与剂量成一定的相关性，FAPSH(200mg·kg^-1·d^-1)与Col.(0.2mg·kg^-1·d^-1)防治肝纤维化效果相当，其原理可能与其抗氧化作用有关。4.各组大鼠肝组织数字基因表达谱差异分析应用数字基因表达谱技术建立对照组、模型组、Col.、FAPSL、FAPSM和FAPSH的肝脏表达谱，初步分析差异基因的表达和功能。结果表明，模型组与对照组表达谱差异基因总共有2324个（表达量上调基因2162个，下调基因162个），差异基因通过多个细胞生物学过程中的218个信号通路参与肝纤维化的形成，特别是引起PPAR信号通路、TGF-β信号通路、MAPK信号通路、VEGF信号通路、Jak-STAT信号通路中多个基因表达量表达量下调。Col.组、FAPSL组、FAPSM组、FAPSH组肝脏表达谱与CC14模型组进行比较，结果表明FAPS可显著抑制TGF-β、PPAR信号通路多个关键基因的上调而抑制HSCs的活化，特别是SCD^-1基因表达量显著下调，表明FAPS可以显著降低肝脏SCD活性，推测FAPS对CCl4引起的肝纤维化的改善作用机制可能为通过抑制SCD活性，削弱单不饱和脂肪酸的生成来减少肝脏脂质蓄积从而抑制HSCs的活化。更多还原

【Abstract】 Astragalus is one of the commonly used traditional Chinese drugs. The maineffective components-astragalus polysaccharides （APS） of the crud drug has manypotency effects, such as improving immunity, antibiosis, anti-virus, hepatoprotection,protecting kidney and so on. While, the yield rate of astragalus polysaccharides fromastragalus is low, therefore its clinical use was restricted to some extent. Previousstudies have shown that the average polysaccharides yield rate of probiotics M-9fermented astragalus was improved significantly （P<0.01）. However, the qualitycontrol of the fermented astragalus, the contents of the active ingredients andpharmacologic effects of them were not clear yet, therefore, this study carried outquantitative determination of astragaloside Ⅳ and calycosin-7-glucoside of fermentedastragalus and crude drug astragalus by high performance liquid chromatography,extraction technology of astragalosides and polysaccharides, effects of the FAPS onexperimental hepatic fibrosis in rats and analysis of digital gene expression profiling（DGE） on the differences of gene expression in every group of rats liver tissue. Inorder to lay the theoretical foundation for the further pharmacological research,development and utilization of fermented astragalus and interpretation the mechanismof biotransformation.（1） Quantitative determination of astragaloside Ⅳ and calycosin-7-glucoside offermented astragalus and crude drug astragalus by high performance liquidchromatographyAstragaloside Ⅳ and calycosin-7-glucoside contents of fermented astragalus andcrude drug astragalus were determined by high performance liquid chromatography（HPLC） according to the pharmacopoeia of the people’s republic of China （2010）. Theresults were0.049%、0.087%、0.030%and0.040%respectively. Results showed thatcontent of astragaloside Ⅳ of fermented astragalus （with equal quantity of crude drugastragalus） and crud drug astragalus were0.063%and0.087%, the content ofcalycosin-7-glucoside of fermented astragalus（with equal quantity of crude drugastragalus） and crude drug astragalus was0.038%and0.040%. Changes ofCalycosin-7-glucoside content before and after fermentation was not significant （P<0.05） after the quality conversion, while the content of astragaloside Ⅳ infermented astragalus was reduced by27.6%comparing with crude drug astragalus.Therefore, content of calycosin-7-glucoside and astragaloside Ⅳ could be used forquality control of fermented astragalus.（2） Extraction, isolation and quantitative determination of the astragalosides andpolysaccharides of fermented astragalus and crude drug astragalusMethod of comprehensive extraction of astragalus polysaccharides andastragalosides was carried out to screen the extraction process. The results showedthat the optimum extracting conditions of astragalosides and polysaccharides were asfollows: Weighed a certain amount of astragalus powder and soaked in10times of themedicine amount of80%ethanol at room temperature for12h in a closed container,then it was reflux extracted at80℃twice,1h per time. The mixture of twice medicalliquids was used in extracting astragalosides and the herbal residues were dried andused in extracting polysaccharides.（A） Process for extraction of astragalosides:Solution of ethanol extracting was filtrated and concentrated by reducing pressuredistillation into liquid extract. Dissolved a certain amount of liquid extract in50timesof the medicine amount （mL/g） of purified water then were absorbed by AB-8macropore resin, there was eluted with4times in column volume of80%ethanol. Theeluates were concentrated by reducing pressure distillation and derived astragalosidesenrichment site.（B） Through comparision the extraction method of immersionmethod, traditional decoction method, assisted extraction with cellulase and saturatedlimewater method. The immersion method was optimized for polysaccharides’extraction: Herbal residues were extracted by20times water （purified water to herbalresidues amount, mL/g） at80℃for4.5h, got water extracts, then was centrifuged,condensed, deproteinized by Sevag method, dialysed, added ethyl alcohol to4timesof the volume of the resulting dialyzed filtrate, the mixture was stirred and depositedfor12h and then centrifuged, the precipitate was washed with absolute ethyl alcohol,acetone, and diethyl ether,2times separately. Polysaccharides was oven dried at60℃.（C） Vanillin-sulfuric acid method for determing astragalosides content, results showedthat content of crude drug astragalus extract and fermented astragalus extract were16.87%and10.81%, extraction ratio of astragalosides from crude drug astragalus and fermented astragalus were0.0654%,0.0558%. Astragalosides content of AB-8derived astragalosides enrichment site was81.23%. Phenol-sulfuric acid method fordeterming polysaccharides content, results showed that content of fermentedastragalus polysaccharides （FAPS） and yield rate were66.59%and4.35%; crude drugastragalus polysaccharides （APS） and yield rate were36.01%and2.73%. Thecomponent sugars of FAPS-2and FAPS-3were confirmed by thin layerchromatography （TLC）. Results showed that FAPS were composed of mannose（D-Man） and glucose （D-Glu）, et al. Content of the main FAPS fraction FAPS-2^-1was95.41%, its weight average molecular weight （Mw） was1,753,456Da and thenumber average molecular weight （Mn） was1,564,267Da. Results showed thatpolysaccharides content in fermented astragalus was increased by84.92%, the gainratio was increased by59.34%, while the extract ratio of astragalosides was reducedby17.20%.（3） Effects and mechanism of the FAPS on experimental hepatic fibrosis in ratsSixty male SD rats were divided into six groups randomly, liver fibrosis modelwas made by subcutaneous injection of CCl4in5groups of the rats for8weeks.Simultaneously, administered FAPSH200mg·kg^-1·d^-1, FAPSM100mg·kg^-1·d^-1, FAPSL50mg·kg^-1·d^-1, colchicine （Col.）0.2mg·kg^-1·d^-1to the groups of treatment, and themodel group and control group were administered with the equal volume ofphysiological saline solution. Rat’s body weight in groups of FAPS were higher thanthe model group, after treating with FAPSH, ALT, AST, and ALP activity in serumwere significantly reduced compared with model group （P<0.01）, content of Hyp inliver tissue （P<0.05） and PⅢNP, CⅣ, LN, HA in serum were also reducedsignificantly. FAPSHcould effectively increase the low level of GSH-Px （P<0.01）,GST in serum and T-AOC （P<0.05） in liver tissue. High levels of GSH and MDA inliver tissue were tended to normal. H.E. staining, Masson trichrome staining andelectric microscope were used to observed the histopathological changes of livertissues, pathological examination showed that hepatocyte fatty vacuolation, apoptosis,necrosis and liver fibrosis in CCl4model group were serious, pathological changeswere improved in the treated groups. Functions of anti-liver fibrosis of FAPS were proportionated to its dosage. FAPSHanti-liver fibrosis effect was equivalent to Col.,and the mechanism may be related to antioxidation.（4） Analysis of digital gene expression profiling （DGE） on the differences of geneexpression in every group of rats liver tissueDifferences of gene expression and function in every group of rats liver tissuewere analyzed by DGE. Results showed that, there were162down-regulated genesand2162up-regulated genes between CCl4model group and control group.Differentially expressed genes took part in the formation of liver fibrosis through218signaling pathways of multiple cell biological processes. Multiple genes, particularlyin the PPAR signaling pathway, TGF-β signaling pathway, MAPK signaling pathways,VEGF signaling pathway and the Jak-STAT signaling pathway were down-regulated,which built the basis for screening new anti-liver fibrosis drugs.Comparing with the DGE of CCl4model group, results showed that FAPS couldinhibited the increasing of several key genes in the TGF-βand PPAR signalingpathways significantly, so that inhibited HSCs activity. Especially, expression ofSCD-1decreased significantly which showed that FAPS could inhibit the activity ofSCD. It is indicated that the mechanism of anti-liver fibrosis of FAPS was related withdecreasing the activity of SCD, the generation of monounsaturated fatty acids wasweaken, led to the reduction of hepatic lipid accumulation, so that inhibited theactivation of HSCs.更多还原