秦艽种质资源研究

【作者】 曹晓燕；

【导师】 王喆之；

【作者基本信息】 陕西师范大学 ， 植物学， 2010， 博士

【摘要】 秦艽是我国传统常用中药材,能祛风湿、清湿热、止痹痛。中国药典(2005,一部)收录的秦艽原植物为龙胆科植物秦艽(又称大叶秦艽,以下统称大叶秦艽)(Gentiana macrophylla Pall.)、麻花秦艽(G straminea Maxim.)、粗茎秦艽(G crassicaulis Duthie ex Burk.)和达乌里秦艽(G.dahurica Fisch.)4种。除此之外,我国甘肃、河北等地种植的黄管秦艽(G.officinalis H.Smith)以及主产四川、青海等地的管花秦艽(G.siphonantha Maxim. Ex Kusnuz.)也常作为秦艽的代用品使用。本文在广泛收集秦艽种质资源的基础上,采用植物形态学、植物化学、药理药效学等方法对上述6种植物进行了系统的生物学性状、种子显微鉴别、AFLP分子标记、有效成分分析、HPLC指纹图谱及镇痛抗炎活性的比较,以期为秦艽的合理利用提供科学依据。论文的主要研究结果如下：1.在野外调查过程中发现,大叶秦艽、麻花秦艽、达乌里秦艽和粗茎秦艽的植物形态在种内存在丰富的变异,对4种秦艽的主要形态变异进行了观察、描述和统计；以主要形态差异为基础,收集、整理秦艽种质资源89份。2.对大叶秦艽、粗茎秦艽、麻花秦艽、达乌里秦艽、黄管秦艽和管花秦艽等6种秦艽的种子进行了比较,并对种子表面进行了扫描电镜观察,发现6种秦艽种子在颜色、长宽比、千粒重、种脐大小和沟纹长度等方面存在明显差异。3.利用AFLP技术对收集的89份种质资源进行了遗传多样性分析,7对AFLP引物组合从收集的89份秦艽种质中共扩增出1068条带,其中多态性条带1067条,每对引物平均扩增152.57条带,所有种质具有1条共有带,即E+ACT/M+CAA-385.5.4.7对引物从40份大叶秦艽种质、21份麻花秦艽种质、13份达乌里秦艽种质和12份粗茎秦艽种质中分别扩增产生159、220、219和154个特异位点,AFLP指纹图谱的鉴别效率分别为97.5%、95.2%、100%、100%。5.基于DICE相似系数的非加权类平均法(UPGMA)聚类分析结果显示：药典所收录的4种秦艽的聚类结果与形态学分种基本吻合。基于6种植物种间平均相似系数的聚类结果显示,黄管秦艽与达乌里秦艽聚在一起,显示其较近的亲缘关系,粗茎秦艽与其他秦艽可能存在较远的亲缘关系。6.在40份大叶秦艽种质中,采自陕西凤县的种质其龙胆苦苷含量较高。40份大叶秦艽主要生物学特性与龙胆苦苷含量的相关分析结果显示：根中龙胆苦苷含量和莲座叶叶形、顶端花絮密度具有极显著的相关性,莲座叶的叶形越窄其龙胆苦苷含量越高,顶端花序越密龙胆苦苷含量越高；根中龙胆苦苷含量和花萼颜色、茎的颜色具有显著相关性,颜色越深龙胆苦苷含量越高。7.利用HPLC测定了来自6个种的19批秦艽样品中龙胆苦苷、马钱酸、獐牙菜苦苷和獐牙菜苷等4种环烯醚萜苷类成分的含量,4种成分的含量在不同样品间变化很大,其中黄管秦艽和管花秦艽这两种常用替代品中龙胆苦苷的含量分别为9.63%和9.09%,均高于19批样品的平均值7.72%。19批样品的HPLC指纹图谱的相似系数在0.976～0.999之间,其中黄管秦艽和管花秦艽的相似度均高于平均值0.991。在药典所收录秦艽的4种原植物中,粗茎秦艽和大叶秦艽中4种环烯醚萜苷类成分的含量相对较高,达乌里秦艽的含量最低。云南粗茎秦艽不同采收期4种环烯醚萜营成分的测定结果显示,无论是2年生还是3年生栽培的粗茎秦艽,7月份龙胆苦苷的含量均为最高值。8.利用GC-MS对秦艽中脂肪酸成分进行了分析,棕榈酸、油酸和亚油酸3种脂肪酸的相对百分含量在6种秦艽中均占有绝对优势,达到84.29-96.46%。尽管每种脂肪酸的相对百分含量在6种秦艽中存在一定的差异,但MANOVA分析结果显示：6种秦艽在12种脂肪酸的相对百分含量上没有显著性差异。9.利用火焰原子吸收光谱法测定秦艽中大量和微量元素的含量,结果显示：6种植物在Ca, K、Na、Mg等4种大量元素的含量上存在较大差异,但Fe、Zn、Mn、Cu等4种微量元素的含量在6种植物中具有较高的一致性,即Fe>Zn>Mn>Cu<。主产陕西的大叶秦艽中Ca、Mg、K、Zn、Mn等5种元素的含量均高于药典所收录的其它3种秦艽的含量。10.镇痛抗炎活性研究结果显示,粗茎秦艽对由于腹腔注射冰醋酸引起急性腹膜炎而产生持久的疼痛刺激的镇痛效果最好,达乌里秦艽对热刺激引起的疼痛的缓解效果最好,大叶秦艽对二甲苯引起的炎症反应的抑制效果最佳,而麻花秦艽对棉球诱导的慢性炎症反应的抑制率最高。MANOVA分析结果显示,黄管秦艽和管花秦艽在镇痛、抗炎活性上与上述4种药典收录的秦艽没有显著性差异,因此,该实验从药理药效的角度支持黄管秦艽和管花秦艽在镇痛抗炎方面可作为秦艽的替代品。更多还原

【Abstract】 Radix Gentianae Macrophyllae, a traditional Chinese medicine, has been frequently used to dispel rheumatism and ease pain. There are four species of Gentiana (G. macrophylla, G. straminea, G. dahurica and G. crassicaulis) recorded as the original plants in the Chinese Pharmacopoeia. Besides, G. officinalis and G. siphonantha,two other Gentiana species which are mainly planted in Gansu and Hebei, Sichuan and Qinghai respectively, are often used as the substitutes. In this study, the main morphological variations were observed and recorded on the basis of extensive collection of germplasm resources. The DNA fingerprints of the collective germplasms were established by AFLP.Six species of Gentiana including G. macrophylla,G. straminea, G. dahurica and G. crassicaulis, G. officinalis and G. siphonantha were compared in morphological features, chemical composition, analgesic and inflammatory activities, which would provide the scientific information for the further rational utilization of Gentiana resources. The main results showed as follows:1.Abundant morphological variations were observed and recorded in G. macrophylla, G. straminea, G. crassicaulis and G. dahurica.On this basis,89 germplasms were collected according to the main morphological characteristics.2. The seeds of the six Gentiana species were compared and the seed surface was observed by scanning electron microscopy. Obvious differences were observed among the six species containing the exterior color, aspect ratio,1000-grain weight, hilum size and the striation length.3.Totally,1068 bands containing 1067 polymorphic bands were generated by AFLP from the 89 Gentiana germplasms using the seven primer combinations. The average number of bands amplified by each primer pair was 152.27 and all the 89 germplasms shared one common band, viz. E+ACT/M+CAA-385.5.4. Seven primer combinations generated 159,220,219 and 154 specific bands in the germplasms of G. macrophylla,G. straminea, G. dahurica and G. crassicaulis, respectively. AFLP fingerprint identification efficiency was 97.5%,95.2%,100% and 100%, respectively.5. The cluster analysis was carried out using the non-weighted group average method (UPGMA) based on DICE similarity coefficient. The result showed that the clustering of the four Gentiana species listed in the Chinese Pharmacopoeia was basically consistent with the morphological classification. The cluster analysis based on the average similarity coefficient among the six different species revealed that the genetic relationship was the closest between G. officinalis and G. dahurica, and G. crassicaulis was distant from other 5 species.6. In the 40 germplasms of G. macrophylla, the contents of gentiopicroside in the germplasms from Fengxian were higher than those from other areas. The correlation analysis between the main biological characteristics and the content of gentiopicroside showed that the content of gentiopicroside in roots had a very significant correlation with the shape of leaves and the density of flowers. The germplasms with narrower leaves or denser inflorescences had the higher content of gentiopicroside. Moreover, the content of gentiopicroside had significant correlation with the color of stems and calyx. The deeper was the color, the higher content of gentiopicroside.7. Four iridoid and secoiridoid glycosides including loganic acid, swertiamarin, gentiopicroside and sweroside were simultaneously detected in the six Gentiana species by HPLC. Contents of the four marker compounds varied among different samples even those from the same species. The contents of gentiopicroside in G. siphonantha and G. officinalis were 9.09% and 9.63% respectively, which were higher than the average content (7.72%) of the 19 batches of samples. The HPLC fingerprint similarity indexes was between 0.996 and 0.994, in which the similarity of both G. siphonantha and G. officinalis were higher than 0.991, the average similarity index. Among the four Gentiana species listed in the Chinese Pharmacopoeia, the contents of the four iridoid and secoiridoid glycosides were relatively higher in G. macrophylla and G. crassicaulis, while the contents in G. dahurica were the lowest. The contents of the four iridoid and secoiridoid glycosides in G. crassicaulis at different harvest time were determined. The content of gentiopicroside in July was the highest for both three years old and two years old roots.8. The total ion chromatograms of fatty acids in the roots of six Gentiana species showed high similarity. The relative percentage content of the three main fatty acids (palmitic acid, oleic acid and linoleic acid) accounted for 84.29-96.46% in the six species. Though the relative percentage content of each fatty acid differed in the six species, no significant difference was found by MANOVA analysis.9. Macro and trace elements were analyzed by FAAS. Though the contents of four macro elements including Ca, K, Na and Mg differed in the roots of the six Gentiana species, the contents of the four trace elements containing Fe, Zn, Mn and Cu displayed high consistency, viz. Fe>Zn>Mn>Cu. The contents of Ca, Mg, K, Zn and Mn in G. macrophylla in Shaanxi were all higher than those in other three Gentiana species listed in the Chinese Pharmacopoeia.10. The results of analgesic and inflammatory activities showed that G crassicaulis had the best analgesic effect for the lasting pain stimulus caused by intraperitoneal injection of acetic acid; G. dahurica had the best effect for pain relief caused by thermal stimulation; G. macrophylla had the best anti-inflammatory effect for the inflammation induced by xylene; G. straminea showed the highest inhibition rate on the cotton ball-induced chronic inflammation. As indicated by MANOVA analysis, no significant difference in the analgesic and inflammatory activities was found among the six Gentiana species. The results supported from the perspective of pharmacological effect that G. siphonantha and G. officinalis could be used as the alternate original plants for Radix Gentianae Macrophyllae.更多还原