土茯苓化学对照品及质量综合评价研究

【作者】 李磊；

【导师】 乔延江；

【作者基本信息】 北京中医药大学 ， 中药学， 2007， 博士

【摘要】 土茯苓为百合科植物光叶菝葜(Smilax glabra Roxb.)的干燥根茎。有除湿,解毒,通利关节之功效。临床用于湿热淋浊,带下,痈肿,瘰疬,疥癣,梅毒及汞中毒所致的肢体拘挛,筋骨疼痛。本论文对其进行了化学成分、化学对照品、质量标准及药理作用四个方面的研究。土茯苓药材化学成分研究。从土茯苓中分离得到20个化合物,经IR、UV、MS、1H-NMR、13C-NMR、HMBC、HSQC、DEPT等手段及化学方法鉴定出17个化合物。它们分别为槲皮素、落新妇苷、白藜芦醇、β-谷甾醇、豆甾醇、(-)表儿茶素、柚皮素、莽草酸、琥珀酸、阿魏酸、胡萝卜苷、花旗松素、棕榈酸、5-O-咖啡酰莽草酸、5-羟甲基糠醛、木蜡酸、尼克酰胺,其中木蜡酸为首次从该植物中分离得到。采用气相色谱-质谱(GC-MS)联用技术对土茯苓挥发性成分进行分析,鉴定出21个化合物,主要为芳香族化合物。土茯苓化学对照品研究。通过优化提取分离条件,建立了落新妇苷化学对照品的制备工艺,该工艺能大量、快速地获得落新妇苷,并且建立了该对照品的质量标准。共累积得到2 g(纯度≥98 %)以上的落新妇苷对照品。土茯苓药材质量标准研究。以总黄酮含量为指标,采用正交试验设计,对乙醇浓度、溶剂用量、提取时间3因素进行优化,确定了土茯苓总黄酮提取工艺。建立了土茯苓中总黄酮的定量分析方法,证明了选择落新妇苷作为土茯苓总黄酮含量测定对照品的合理性和科学性。采用高效液相色谱法同时测定10批不同来源的土茯苓药材中落新妇苷和白藜芦醇的含量。对10批不同来源的土茯苓药材进行了HPLC指纹图谱分析,相似度结果较好(相似度大于0.900),确定了10个共有峰,并用HPLC-MS和HPLC-1H NMR法对部分特征峰进行指认。采用指纹图谱方法鉴别了土茯苓正品及其常见的3种混淆品。对土茯苓4个提取部位以及化合物落新妇苷进行了抗菌、抗氧化、抗乙肝病毒、抗SIVmac251病毒和抗肿瘤的实验研究。结果表明,土茯苓乙酸乙酯部位对金黄色葡萄球菌、大肠杆菌、绿脓杆菌、志贺氏痢疾杆菌均有抑制作用;土茯苓水煎液和落新妇苷对这5种菌均没有抑制作用。土茯苓各提取部位以及落新妇苷均有较好的抗自由基活性;对乙肝病毒以及HepG2细胞都没有抑制作用。此外,土茯苓乙醇提取物对Hela细胞表现出一定的抑制作用;土茯苓乙醇提取物以及正丁醇部位对SIVmac251病毒有一定的抑制作用。上述研究结果为土茯苓的质量评价提供了科学依据;为评价土茯苓整体功效与上述各药理指标之间的关联关系,应用贝叶斯网络对其清热解毒作用进行预测,进一步证实了土茯苓具有清热解毒的功效。本论文围绕着国家“863”课题—土茯苓化学对照品的研究,对土茯苓进行化学成分研究,确定落新妇苷为土茯苓的指标性成分,建立落新妇苷化学对照品的制备工艺及其质量标准,在此基础上建立土茯苓药材的质量标准,并对其进行了药理活性初步研究,为土茯苓药材的进一步开发利用提供科学依据。更多还原

【Abstract】 Smilax glabra Roxb. (Family Liliaceae) is the Chinese medicinal herb called tufuling. It has antipyretic, detoxifying, and diuretic actions. It may be beneficial in treatments for brucellosis, syphilis, furunculosis, eczema, dermatitis, nephritis, cystitis, mercury and silver poisoning. In this thesis, a relatively systematic study on chemical constituents, chemical reference, quality control method and biological activity of Smilax glabra Roxb. was performed.Firstly, a study on chemical constituents of Smilax glabra Roxb. was carried out. 20 compounds were isolated and purified from the EtOH extracts of Smilax glabra Roxb., and 17 of them were identified on the basis of spectral data and physicochemical properties, the structures had been elucidated as follows: quercetin, resveratrol, daucosterol,β-sitosterol, astilbin, naringenin, succinic acid, shkimic acid, taxifoliol, ferulic acid, stigmasterol, (-)-epicatechin, palmitic acid, 5-O-caffeoylshikimic acid, 5-hydroxymethylfurfural, lignoceric acid, nicotinamide. Among the compounds, lignoceric acid was isolated from the plant for the first time. The composition of essential oil of Smilax glabra Roxb. was analyzed by gas chromatography-mass spectrometry(GC-MS). 21 compounds were identified, most of them were aromatics.Secondly, it is for the first time that the preparation method and quality standard of the chemical reference of Smilax glabra Roxb.were investigated. The preparation method developed is simple, and the yield and purification of the chemical reference obtained are higher. The chemical reference of 2 g has been accumulated in the course of study.Thirdly, a study on quality control method of Smilax glabra Roxb. was performed. Extraction methods were evaluated for the extraction efficiency of general flavonoids. In this experiment, three factors were studied by orthogonal design, including ethanol concentration, consumption of solvent, and time of extraction. Then, a method for determining general flavonoids in Smilax glabra Roxb. was developed, it is for the first time that we discovered that selection of astilbin as an index is more rational and scientific in the determination of general flavonoids in Smilax glabra Roxb.. Two bioactive constituents namely astilbin and resveratrol in 10 samples were determined with high performance liquid chromatography. The two constituents were completely separated in 20 min. The chromatographic peak areas presented a good linearity with the concentration for this two constituents. To study and develop the fingerprint of Smilax glabra Roxb. by high performance liquid chromatography (HPLC). The results showed that the fingerprints of the samples from 10 different sources were of high similarity with correlation coefficients above 0.9. Peaks existing in all 10 fingerprints were assigned as“common peaks”for Smilax glabra Roxb.. There are 10“common peaks”in the fingerprint. The components were identified by HPLC-MS and HPLC-1H NMR. By comparing the fingerprints, Smilax glabra Roxb. was well distinguished from the commercial adulterants including Heterosmilax japonica Kunth., Heterosmilax yunnanensis Gagnep. and Smilax China L.Finally, the pharmacological activities of Smilax glabra Roxb. on antibiosis, antioxidation, anti-HBV, anti-SIVmac251 and antitumor were studied. The result showed that the EtOAc fraction had some antibacterial activity on Escherichia coli, Staphylococcus aureus, Pseudominas aeruginosa and Shigella dysenteriae; there was no significant effect in the water extraction and compound astilbin. In antioxidation study, compound astilbin and all the fractions extracted from Smilax glabra Roxb. showed different free radical scavenging activity. All the samples mentioned above had no effect on the hepatitis B virus and HepG2 cells. The study demonstrated that the EtOH extract inhibited proliferation of Hela cells. In anti-SIVmac251 study, the result showed that the EtOH extract and the n-BuOH fraction had inhibitory activities against SIVmac251. The research offered a scientific basis for quality control of Smilax glabra Roxb.. To evaluate the association between its Traditional Chinese Medicine effect and pharmacological activities, the effect of“Clearing heat and Resolving toxin”of Smilax glabra Roxb. was predicted by Bayesian belief network. Finally, the effect of“Clearing heat and Resolving toxin”of Smilax glabra Roxb. was further confirmed by this method.In this thesis, the chemical constituents, chemical reference, quality control method and biological activity of Smilax glabra Roxb. were researched, which provide the scientific basis for the further development of Smilax glabra Roxb..更多还原