【作者】 严寒静；

【导师】 余世孝；

【作者基本信息】 中山大学 ， 生态学， 2007， 博士

【摘要】 中药何首乌( Radix Polygoni Multifiori )为蓼科植物何首乌( Follopia multiflora( Thunb.) Harald.)的干燥块根,具有补肝肾、益精血、乌须发、强筋骨之功效,临床应用十分广泛。但是,不同产地的何首乌在活性成分含量和抗衰老作用存在显著差异,造成何首乌药材质量极不稳定,直接影响到临床疗效;何首乌种内变异规律不明,对何首乌种内居群的种质资源的鉴定和评价未曾作过深入研究;何首乌药材的质量标准过于单一,未能体现中药的复杂性;对于影响药材质量的因素未曾进行详尽的研究。本文采用植物分类学、植物解剖学、植物生态学、中药成分分析和DNA分子标记技术,比较研究何首乌的变异规律;寻找优良的种质资源;建立更加完善的何首乌定性与定量质控方法,为何首乌药材的生产、收购、鉴定分析提供科学依据,确保临床用药的安全、有效。1.根据叶的性状指标,采用聚类分析的方法,将10个何首乌种质资源种群分为三个大的类群,即缙云山、井冈山、德庆、恩施、施秉、峨眉山、靖西七个种源的何首乌,地理位置均分布于北纬23°～30°之间;济源、登封种源为一类群,其地理位置位于北纬34°～35°,属于纬度相对较高的地区;田阳种源自成一类群。呈现出纬度地带性。2.通过扫描电镜和光学显微镜对叶表皮和茎表皮微形态进行观察,叶表皮细胞外壁角质膜纹饰为密集的条纹,但角质条纹在气孔周围的排列方向、条纹的粗细、弯曲程度有明显变化。田阳种源何首乌叶下表面叶脉处表皮细胞外表面具有角质乳突;茎外表面的角质条纹集结扭曲呈乳突状突起,表现出与其他种源何首乌的差异。在光学显微镜下观察了不同产地何首乌叶表皮结构特征,应用多元回归方法对不同产地何首乌的叶表皮特征与气候因子的关系进行了探讨。发现上表皮有少量气孔器分布;在下表皮,气孔器类型为非典型不等细胞型和不规则型,有少量腺鳞分布,气孔器密度241.7(64– 573)个/mm2,气孔指数为17.1(7.5-26.5)%,气孔器长31.1(20 - 44)μm,气孔器宽23.1(16-38)μm。何首乌下表皮的气孔密度、气孔指数、气孔器长、宽、气孔极区角质加厚存在着明显的差异;气孔器长、气孔器宽以及气孔密度均与纬度关系密切,随纬度的升高,气孔器长、气孔器宽呈减小的趋势,气孔密度呈增加的趋势,R2值分别为0.619、0.729、0.772,表明纬度对气孔器的长和宽有着一个协同影响的作用。3.对何首乌药材横切面的观察发现10个种源的何首乌药材可分为2大类群:田阳种源何首乌自成一类群,与对照药材存在明显差别,主要表现在断面的纤维性;其他种源何首乌为一类群,与2005版药典记载特征相符。4.采用ICP法测定了不同地区何首乌植物及其生境土壤中的15种元素(Al、B、Ba、Ca、Cu、Fe、K、Mg、Mn、Ni、P、Sr、Ti、Zn),分析比较了各种元素在不同地区何首乌体内和生境土壤中的分布规律及不同地区何首乌对无机元素的吸收富集能力。结果表明,不同地区何首乌和土壤中无机元素含量存在着显著差异,德庆、井冈山、靖西三地何首乌锰元素含量较高;不同地区何首乌对元素的富集系数无显著性差异,而何首乌对不同无机元素的富集系数则有显著差异,主要表现在对Ca、K、Mg、Sr的吸收富集能力较强。5.采用TLC方法对不同产地何首乌进行薄层色谱指纹图谱的研究。以甲醇提取活性成分蒽醌类和2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷,以石油醚-乙酸乙酯-甲酸(15:5:1)、三氯甲烷-甲醇-水(6.5:2.25:0.42,冰醋酸调pH=4)两种展开系统进行薄层色谱分离。扫描后以峰高进行聚类分析,建立了何首乌的TLC指纹图谱。6.用HPLC法测定了5种有效成分,并通过相关系数分析、逐步回归、主成分分析法探讨17个环境因子对5种有效成分的影响。研究结果表明仅结合大黄素和结合大黄素甲醚与环境因子有显著相关性,而2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、游离大黄素、游离大黄素甲醚与环境因子无显著相关性。最冷月均温、积温、有效K、Zn与结合大黄素呈极显著相关(除有效K的P<0.05,其它指标P<0.01)。最冷月均温、积温、有机质与结合大黄素甲醚呈负显著相关(最冷月均温P<0.01,其它P<0.05)。逐步回归显示积温是影响结合大黄素和结合大黄素甲醚的主要环境因素。化学成分的差异主要源于何首乌适应特定环境形成的特定基因型。对何首乌化学成分进行主成分分析,将10个来源的何首乌划分为四个类群,德庆和峨眉山所产何首乌品质最佳。7.采用ITS测序法研究何首乌种内DNA遗传物质的变异规律,测定了9个产地,10个居群的何首乌新鲜叶片核糖体DNA ITS区序列。得到各样品rDNA的ITS及5.8S rDNA完全序列,18S和26S rDNA部分序列,共约648bp ,其中ITS1长度为195bp,5.8S长度为164bp,ITS2长度为189bp。序列间共有22个变异位点。更多还原

【Abstract】 The dry roots of Follopia multiflora( Thunb.) Harald. are used as Radix Polygoni Multifiori,which is a common ingredient in Chinese prescriptions. It is used for dizziness with tinnitus, premature greying of the hair, soreness and weakness of the knee and lower back, and for limb numbness. The crude extracts of F. multiflora containing phospholipids, anthraquinones, and bianthraquinonyl glucosides exhibit prominent actions in many clinical therapies, particularly on cardiovascular diseases. Radix Polygoni Multifiori that distributing in different places had significant differences in content of bioactive components and in the effect of lowering anti-decrepitude. So the quality of Radix Polygoni Multifiori was not stable. So far, there was no report about the genetic variation and the evaluation of idioplasmatic resources of Radix Polygoni Multifiori. The quality standards of Radix Polygoni Multifiori were too simple. In the present research, the botanically taxonomic and anatomic approaches, chemical analytical methods and the molecular marking techniques were used to find out the variations of F. multiflora in the tissue structures, chemical components and genetic characters, to search for the good resources and to establish the specific methods for identification of Radix Polygoni Multifiori. Data of this research will be useful for the quality control of Radix Polygoni Multifiori, and assure that the safety and efficiency of crude drugs in clinical practice.1.Base on the cluster analysis method, 10 places (Jinyunshan, Jinggangshan, Deqing, Enshi, Shibing, Emeishan, Jingxi, Jiyuan, Dengfeng and Tianyang) of F. multiflora were divided into three groups by leaf characters. Those growing in north China were the firsr group, such as Jiyuan’s and Dengfeng’s. Those in south China were the second group, and Tianyang’s was the third group. The distribution of three groups showed latitude zonation.2.The morphological features of leaf abaxial epidermis and micromorphological features of stem were observed with scanning electron microscope and light microscope. Cuticular membrane near stomata apparatuses was serried striate, while the way of arraying, thickness of stripe, grade of curve had no significant difference. Especially, F. multiflora from Tianyang had papilla on the spot of nervation in leaf abaxial epidermis and stem. The results indicated that characters of foliar surface under SEM are very useful evidence in identifying F. multiflora from different places.The epidermal characters of mature leaves of F. multiflora from different places were observed with light-microscope. Multi-regression analysis was used to determine the relationship between the leaves morphological characters of F. multiflora distributing in different locations and climate factors of different locations. The characters of leaf surface including the stomata density, stomata index, stomata width and stomata length were determined and compared. As a result, stomata were rarely observed at the upper epidermis. The stomata apparatuses on the lower epidermis were non-typical anisocytic type and anomocytic type. Stomata length was 31.1(20 - 44)μm, stomata width 23.1(16-38)μm, stomata index 17.1(7.5-26.5)%, stomata density 241.7(64– 573)个/mm2, respectively. The variations of anatomical characters for the lower of leaves of F. multiflora from different districts were definitely. The results of multi-regression analysis showed that the stomata length, width and stomata density were significant correlated to the latitude. With the latitude increasing, the length and width of stomata consistent decreased, while stomata density consistent increased.3.The medicinal materials from 10 places can be classified into two categories according to microstructure features of root. The root of F. multiflora from Tianyang had special characters, more fiber in the transverse section of root. The characters of transverse section of others were the same with the F. multiflora recorded in Chinese Pharmacopoeia.4.The contents of 15 inorganic elements (Al, B, Ba, Ca, Cu, Fe, K, Mg, Mn, Ni, P, Sr, Ti, Zn ) in soil and F. multiflora collected from 10 places were detected with ICP. The contents of inorganic element and their accumulation in F. multiflora between different places were compared. The results showed the contents of these elements in soil or plant had significant difference between 10 places. The content of Mn in plants from Deqing, Jinggangshan, and Jingxi is higher than that from other places. As to the accumulation coefficient of F. multiflora to these inorganic elements, there is no significant difference between 10 places. The accumulation coefficient of. F. multiflora to various inorganic elements had significant difference. Ca, K, Mg and Sr especially accumulated in F. multiflora.5.The effective components extracted by MeOH were separated by two kinds of mobile phase on TLC. Then the TLC fingerprints were scanned by Camag TLC scanner, and the results were analyzed by cluster analysis. The TLC chromatograms were constituted 14 characteristic common peaks include anthraquinone and stilbene glucoside.6.The bioactive components of F. multiflora determined its quality. Through measuring 17 environmental factors and 5 bioactive components (measured by HPLC), the influence of environmental factors on the bioactive components of F. multiflora (collected from ten places) was studied. Correlation, stepwise regression and principal component analysis were used to analyze the influence. Results showed that average temperature of Jan.,accumulated temperature, available potassium and Zn significant correlated with emodin. Average temperature of Jan.,accumulated temperature and organic matter significant negatively correlated with physcion. Stepwise regression showed that accumulated temperature was the main factor influencing the contents of emodin and physcion. But the environmental ecological factors did not significant correlated with 2,3,5,4′-Terahydroxystilbene-2-O-β-D-glucoside , emodin-8-O-β-D-glucoside and physcion-8-O-β-D-glucoside. Results indicated that the difference of bioactive components is mainly influenced by local specialized genotype. The HPLC data had been analyzed by principal components analysis, and results showed that F. multiflora from ten different places could be classified into four categories. The quality of F. multiflora from Deqing and Emeishan were the best.7. PCR products were sequenced directly and the sequences were analyzed with PAUP4.0. In order to find the patterns of the rDNA ITS sequence variation of F. multiflora, and to search of the method for distinguishing the Chinese crude drug Radix Polygoni Multifiori by molecular biological technique. The total length of ITS sequence is 648bp in the different samples. The lengths of three fragments, ITS1, 5.8S, ITS2, are 195bp, 164bp, 189bp respectively. There are twenty-one variable sites. The rDNA internal transcribed spacer is a good marker to distinguish the F. multiflora..更多还原