【作者】 田峦鸢；

【导师】 陈家春；

【作者基本信息】 华中科技大学 ， 药理学， 2009， 博士

【摘要】 紫红獐牙菜Swertia punicea Hemsl．为龙胆科（Gentianaceae）獐牙菜属（Swertia L．）植物，在我国，民间应用紫红獐牙菜入药的历史悠久，该植物已被民间用于治疗急性黄疸型肝炎、胆囊炎、肺炎、流感、骨髓炎等疾病。据报道，本属印度獐牙菜和日本獐牙菜具有明显的抗糖尿病作用。本课题对紫红獐牙菜的活性物质和资源质量的评价进行了研究。首先，为阐明紫红獐牙菜抗乙肝病毒活性物质基础，本研究对紫红獐牙菜进行了系统的药理和化学成分研究。本研究采用乙肝病毒基因组转染的人肝癌细胞（Hep G₂）2，2，15细胞为细胞模型，对紫红獐牙菜各个不同极性部位进行了活性筛选。紫红獐牙菜乙醇提取物和其乙酸乙酯部位对HBsAg和HBeAg的表达表现出了明显的抑制作用，证实了紫红獐牙菜具有抗HBV作用，并确定乙酸乙酯部位为活性部位。本研究通过系统的化学成分研究，从紫红獐牙菜中分离并鉴定了20个单体化合物。其中Comp．18（1-hydrate，3-deoxy-α-D-tagatofuranose）为新化合物，Comp．16（Campestroside）、17（Uridine）首次从该植物中分离得到，Comp．12（7-[（β-D-xylopyranosyl）oxy]-1，8-dihydroxy-3-methoxyxanthone）、13（Norswertianolin）、14（Tetrahydrosertianolin）、15（8-hydroxy-10-hydrosweroside）为首次从该属植物分离得到。本研究对紫红獐牙菜活性部位中分离得到的单体化合物和其他含量较大的化合物采用相同模型进行了体外抗乙肝病毒活性筛选。实验结果表明：Comp．2、5、10对HBsAg有抑制作用，选择指数（SI）分别为1．2、14．2、6．8；Comp．7、8、9、11对e抗原有抑制作用，选择指数（SI）分别为4．5、6．2、6．9、2．4。另一方面，为了阐明紫红獐牙菜抗糖尿病活性的物质基础，本实验利用高脂高糖饲料+STZ诱导的2型糖尿病小鼠为模型，对紫红獐牙菜各提取部位和部分化学成分进行了活性的筛选。并对得到的活性成分抗糖尿病活性的可能机制进行了探讨。本研究采用正常BABL／c小鼠和小剂量STZ加以高脂饲料诱导的高血糖BABL／c小鼠，对紫红獐牙菜中的部位进行了抗高血糖药理活性筛选。从紫红獐牙菜中分离出乙醚、乙酸乙酯、正丁醇及水溶性四个不同极性部位，测定给予上述四个部位后小鼠的空腹血糖、糖耐量和胰岛素抵抗指数，筛选活性部位。结果表明乙酸乙酯部位为主要的降血糖活性部位，其抗糖尿病的可能机制为改善胰岛素抵抗。本研究对从乙酸乙酯部位分离得到的两个含量较高的（?）山酮化合物Methylswertianin和Bellidifolin采用STZ+高脂饲料诱导的高血糖小鼠进行了进一步的活性筛选。结果表明Methylswertianin和Bellidifolin高低剂量组均能明显降低STZ引起的高血糖小鼠的血糖。Methylswertianin和Bellidifolin各剂量组小鼠在口服葡萄糖1小时内血糖水平迅速升高，1小时以后血糖水平开始迅速下降，表明Methylswertianin和Bellidifolin明显提高了糖尿病小鼠的糖耐受能力。Methylswertianin和Bellidifolin的各剂量组中血浆的TC和LDL浓度与模型组比较都明显的降低（P＜0．01），TG浓度在Methylswertianin高低剂量组和Bellidifolin高剂量组也表现出明显的降低（P＜0．01）。同时，Methylswertianin和Bellidifolin的高剂量组的HDL浓度也有所降低（P＜0．05）。但是Methylswertianin和Bellidifolin各剂量组的相对HDL水平与模型对照组比较都有很明显的提高，甚至高过了Rosiglitazone对照组。Methylswertianin和Bellidifolin均有显著缓解胰岛素抵抗作用，通过对胰岛素指数的计算，发现各实验组对高血糖小鼠的胰岛素抵抗均有非常显著作用（P＜0．01）。本实验在对Methylswertianin和Bellidifolin抗糖尿病活性机制研究中发现，Methylswertianin和Bellidifolin能明显曾强胰岛素受体-a（insulin-receptorαsubunit，InsR-α）、胰岛素底物1（insulin-receptor substrate-1，IRS-1）和磷脂酸肌醇3激酶（phosphatidylinositol 3-kinase，PI3K）三个胰岛素信号传导分子在肾小管上皮细胞胞浆中的表达，从而增强胰岛素信号传导，改善胰岛素抵抗。此外，Methylswertianin和Bellidifolin还能增加肝糖原的含量，增加肝脏中葡萄糖激酶的活性并降低葡萄糖-6-磷酸酶的活性，因而降低胰岛素抵抗导致的高血糖。证实了改善胰岛素抵抗为其抗糖尿病机制。最后，为了更好的开发和利用紫红獐牙菜的药用价值，我们对紫红獐牙菜的资源与质量评价进行了研究。本研究用高效液相色谱法对采收于湖北西部10个地点的紫红獐牙菜进行了HPLC指纹图谱研究。结果表明紫红獐牙菜指纹图谱具有比较好的稳定性和重复性，10批紫红獐牙菜样品指纹图谱的特征性和专属性很强，13个共有峰的稳定性很好。通过相似度分析方法解析，表明紫红獐牙菜的指纹图谱相似程度良好，符合指纹图谱相似度要求，可以用于紫红獐牙菜的鉴定和质量评价．该方法的建立为紫红獐牙菜质量的全面控制提供了参考。本研究用高效液相色谱法对Swertiamarin、Mangiferin、Swertianolin和Bellidifolin四种活性成分进行了分析。结果表明在选定的色谱条件下四种成分分离良好，在检测浓度范围内线性良好。最低检测限和定量限分别大于0．86μg／ml和1．73μg／ml。日内和日间精密度RSD％分别小于2％和4．6％，加样回收率为97．94％-99．84％。建立了獐牙菜属植物中四种不同化学结构类型抗肝炎活性成分的含量测定方法。本方法简单、准确，能成功的的运用于同时测定獐牙菜属植物不同部位中Swertiamarin、Mangiferin、Swertianolin和Bellidifolin四种活性成分的含量，以及獐牙菜植物的鉴定。本研究制定了紫红獐牙菜的质量标准。该标准中采用的方法准确可靠、专属性强、重复性好，能有效的评价紫红獐牙菜的质量。更多还原

【Abstract】 Swertia punicea Hemsl.,a perpnnial dried herb of Gentianaaeae,is used as a folktraditional medicine in China for a long time.It is frequently used for the treatment ofhepatitis,cholecystitis,pneumonia,pandemic influenza,inflammation of bone,et al.Moreover,plants of Swertia,S.chirayita and S.japonica,were reported to show significantanti-diabetic activity.In this study,material basis for anti-hepatitis B virus (HBV) andanti-diabetic activities of Swertia punicea Hemsl.was investigated.Moreover,the sourceand quality of Swertia punicea Hemsl.were evaluated for its development of medicalvalues.Firstly,to clarify the material basis for anti-HBV activity of Swertia punicea Hemsl.,activities of fractions and components from Swertia punicea Hemsl.were screened in HepG2 2.2.15 cell,a kind of HBV-producing human hepatoblastoma cell line,and theconsitituents of Swertia punicea Hemsl were studied.In this study,ethanol extract and ethyl acetate fraction of Swertia punicea Hemsl.significantly inhibited the expression of HBsAg.,which confirms the anti-HBV activity ofSwertia punicea Hemsl.and the ethyl acetate fraction was the active fraction.Study on consitituents of Swertia punicea Hemsl afforded 20 compounds,identified as,Comp.18 (1-hydrate,3- deoxy-α-D-tagatofuranose) was a new compound,CompoundsComp.16 (Campestroside),17 (Uridine)were isolated from this genus for the first time andcompounds Comp.12 (7-[(β-D-xylopyranosyl)oxy]-1,8-dihydroxy-3-methoxyxanthone),13(Norswertianolin),14 (Tetrahydrosertianolin),15 (8-hydroxy-10-hydrosweroside) wereobtained from this plant for the first time.Compounds from ethyl acetate fraction were screened for anti-HBV activity in thisstudy,as well as some compounds from other fractions.Compound 2,5 and 10 inhibitedthe expression of HBsAg and their selected index (SI)was 1.16,14.2 and 6.8,respectively.Compound 7,8,9 and 11 inhibited the expression of HBsAg and their SI was 4.5,6.2,6.87and 2.36,respectively. Moreover,to study the material foundation of anti-diabetic activity of Swertia puniceaHemsl.,we studied anti-diabetic activities of fractions and constituents of Swertia puniceaHemsl.in STZ-induced type 2 diabetic mice.As a following study,the potential mechanismof active constituent (s) was investigated.The hyperglycemia BABL/c mice which were induced by low dose of streptozotocin(STZ) and high calorie food are used to screen the hypolglycaemic activity ingredients fromS.punicea Hemsl.The effects of four fractions (ether,ethyl acetate,n-butanol and thewater-soluble),obtained from S.punicea Hemsl.,on FBG,glucose tolerance and insulinresistance were studied.The result indicated S.punicea Hemsl.was beneficial to type 2diabetes and its potential mechanism could be improvement of insulin resistance.In this study,STZ-induced type 2 diabetic male BABL/c mice treated with or withoutmethylswertianin and bellidifolin at different doses (orally,200 and 100 mg/kg/d) for 4weeks were analyzed.The results proved that methylswertianin and bellidifolinsignificantly reduced fasting blood glucose (FBG).The administration of methylswertianinand bellidifolin also improved the oral glucose tolerance and lowered fasting serum insulin(FINS).Moreover,the administration of methylswertianin and bellidifolin lowered theserum total cholesterol (TC),low density lipoprotein cholesterol (LDL) and triglyceride(TG) levels and increased the relative high density lipoprotein cholesterol (HDL)concentrations (HDL/TC).methylswertianin and bellidifolin appeared to improve insulinresistance by enhancing insulin signaling.The expression levels of insulin-receptorαsubunit (InsR-α),insulin-receptor substrate-1 (IRS-1),phosphatidylinositol 3-kinase (PI3K)were increased after the administration of methylswertianin and bellidifolin.Meanwhile,methylswertianin and bellidifolin increased hepatic glycogen content,decreasedglucokinase (GK) activities and increased glucose-6-phosphatase (G6Pase) activities.Inconclusion,these result indicated that methylswertianin and bellidifolin could be useful fortreating type 2 diabetes and the mechanism probably was the improvement of insulinresistance (IR).Lastly,To develop the medical values of Swertia punicea Hemsl.,the resource andquality of Swertia punicea Hemsl.were evaluated.The study aimed to establish HPLC fingerprint of Swertia punicea Hemsl.from Hubei Province The chronmtograms of 10 batches of Swertia punicea Hemsl.samples fromdifferent producing area were determined under such conditions as gradient elution withmethanol -0.05%aquaintance-acetonitrile,current speed at 0.8 ml/min and determinedwavelength at 256 nm.The characteristic fingerprint consisted of 13 common peaks ofSwertia punicea Hemsl.from Hubei province was established through comparing withcontrast medicinal materials.Analysis on the fingerprint by similarity analysis indicatedthat the fingerprint could be used to identify of Swertia punicea Hemsl.and evaluate itsquality.The method was stable and reliable with good reproducibility and could be used toestablish fingerprint of Swertia punicea Hemsl.and supply the reference for overall controlof its quality.In this study,a new HPLC method by using ultraviolet absorbance detection withdetected wavelength at 254 nm was presented for the simultaneous determination of fouractive components,namely Swertiamarin,Mangiferin,Swertianolin and Bellidifolin in 7species of Swertia plants (Gentianaceae).The samples were separated and detected on thecolumn of Alltima C18(250 mm×4.6 mm,5μm)under the condition of gradient elution.The limit of detection (S/N=3) is less than 0.86μg/ml and the limit of quantification(S/N=10) is less than 1.73μg/ml..All calibration curves show good linear regressionwithin test ranges.The RSD for intra- and inter-day of four investigated compounds werelower than 2% and 4.6%,respectively.The mean recovery was between 97.94% and99.84%.The method was establishded to simultaneous determination of the fouranti-hepatitis constituents relating to different kinds of chemical structures in Swertia plants.So,the method was successfully applied to quantify the four active components in differentparts of the herb and identify the specific species of genus Swertia.Moreover,the quality standard of Swertia punicea Hemsl.was established.The method,used in standard,were simple,accurate,specific and reproducible and can be used as areliable method for quality evaluation of Swertia punicea Hemsl.更多还原