【作者】 范适；

【导师】 饶力群； 左家哺；

【作者基本信息】 湖南农业大学 ， 植物学， 2007， 博士

【摘要】 叶下珠（Phyllanthus urinaria L.）为大戟科叶下珠属植物,是我国传统的中草药。在我国长江流域和南方诸省都有分布,是一种资源十分丰富的植物。现代药理实验证明叶下珠有抗乙肝病毒和保肝护肝的药理作用,且无毒副作用。但其发挥药效的物质基础还不清楚,同时,中药受产地、采收期、药用部位、加工方法等因素的影响,导致药材质量的明显差异,从而影响临床疗效。本研究采用现代分离纯化技术逐级分离叶下珠的有效成分,同步采用体外细胞学活性筛选（采用新生大鼠原代肝细胞四氯化碳化学性损伤药理模型,MTT法）及动物试验,对叶下珠保肝护肝的活性成分进行追踪,并对得到的单体化合物进行四谱（IR、UV、MS、NMR）鉴定。同时,应用HPLC法对叶下珠药材及叶下珠胶囊色谱指纹图谱进行了系统的研究,建立叶下珠药材及叶下珠胶囊的标准指纹图谱,为叶下珠植物药的开发应用提供理论依据及为我国中药现代化打下基础。主要研究结果如下:1.采用体外细胞学活性筛选法,首次对叶下珠保肝护肝有效成分进行了追踪分离研究,得到了叶下珠保肝护肝有效部位,从有效部位分离得到5个单体化合物,通过紫外光谱、红外光谱、核磁共振、质谱等波谱分析,鉴定叶下珠保肝护肝有效成分为柯里拉京（corilagin）。2.叶下珠保肝护肝有效部位（PE）抗小鼠四氯化碳化学性肝损伤的试验结果表明:PE能够明显降低CCl₄致小鼠肝损伤所致ALT、AST增高,减轻肝细胞变性、炎症细胞浸润等病变,降低肝中MDA生成量,提高SOD活力,提高ALB的合成量,提高A/G值,因此,PE具有明显的保肝护肝活性。3.本研究首次对叶下珠不同产地、不同采收期、不同部位的药材进行了系统的HPLC指纹图谱研究,确定18个共有峰构成叶下珠药材的特征指纹图谱,确认了其中四个特征峰:2号峰为没食子酸,3号峰为原儿茶酸,6号峰为咖啡酸,8号峰为柯里拉京。所建立的特征指纹图谱具有好的专属性、稳定性、重现性,且能反映不同产地、不同采收期、不同部位药材的差异。使用计算机辅助相似度评价软件进行数据处理,对不同产地的叶下珠药材进行了相似度分析,结果表明:除3号药材（浙江杭州）和8号药材（云南昆明）外,其它各产地药材的相似度都在0.9以上。叶下珠不同采收期的7批药材相似度良好,都在0.94以上。比较各个特征峰的表观丰度,发现特征峰的表观丰度（即成分的含量）大多数随生长期而增高,至10月5日达到最高,过后则明显降低,建议叶下珠药材的采收期为每年10月上旬。对叶下珠不同部位的药材相似度进行了评价,叶、果部分与共有模式的相似度在0.94以上,而根、茎部分与共有模式的相似度在0.9以下,应该是由于各成分在含量上的差异所致。对各特征峰的表观丰度进行比较,发现各特征峰在叶中的含量最高,其次为果、茎,最低为根。由于根所占比例不到20%,且各成分含量又很低,因此,建议只采收地上部分。4.建立了叶下珠药材保肝护肝有效成分柯里拉京含量的HPLC测定方法。在0.458～4.580 mg/g质量浓度范围内,柯里拉京的色谱峰面积与样品浓度呈良好的线性关系,回归方程为:Y=302 172X+22 305,相关系数r=0.995 9,加样回收率为97.98%。不同产地药材含量差异较大,平均为2.288 mg/g,产地为广东中山的含量最高,达4.224 mg/g,产地为浙江杭州的含量最低,仅为0.982 mg/g。采收期为10月5日时柯里拉京的含量最高,为4.090 mg/g。不同部位中以叶的含量最高,达4.437 mg/g,其次为果、茎,根的含量最低,仅为叶的1/5。5.建立了叶下珠胶囊HPLC指纹图谱。确定17个共有峰构成叶下珠胶囊的特征指纹图谱,共有峰面积在90%以上,确认特征指纹图谱中的3个特征峰:3号峰为没食子酸,4号峰为原儿茶酸,8号峰为柯里拉京,10批叶下珠胶囊具有良好的相似度（在0.9以上）,说明叶下珠胶囊一致性好,质量稳定。对比并分析了叶下珠胶囊与对照药材的HPLC指纹图谱的相关性,结果表明,叶下珠胶囊与对照药材化学成分基本一致,但某些特征峰的表观丰度相差较大,应该是叶下珠胶囊加工过程所致。6.建立了叶下珠胶囊保肝护肝有效成分柯里拉京含量的HPLC测定方法。在8.400～42.000 mg/g质量浓度范围内,柯里拉京的色谱峰面积与样品浓度呈良好的线性关系,回归方程为:Y=199 850X-608 070,相关系数r=0.999 8,加样回收率为98.51%。各批叶下珠胶囊柯里拉京的含量较稳定,在17.19 mg/g～21.84 mg/g之间,平均含量为19.82 mg/g。更多还原

【Abstract】 Phyllanthus urinaria L. is a traditional Chinese herb medicine. It can be found in south of Yangtze River. The pharmacological experiments prove anti-HBV, hepatoprotective effect and non-toxicity of Phyllanthus urinaria L. But its effective constituents are not clear. The obvious difference in quality and clinical effect is caused by the factors, such as habits, harvest time, parts and processing method. Modern separating techniques, screening bioactivity in vitro（the mode of CCl₄-induced hepatic injury in newborn rats, MTT assay） and animal experiments were employed in this study. To screen the hepatoprotective constituents, the extracts from Phyllanthus urinaria L. were isolated and purified. The active compounds were identified by using IR, UV, MS, NMR. Meanwhile, HPLC fingerprint of Phyllanthus urinaria L. and Yexiazhu Capsules provided the theory basis for the application of Phyllanthus urinaria L.and lay a good foundation for the modernization of traditional Chinese medicine.The rusults were as follows:1. By screening hepatoprotective activity in vitro, isolation and purification of hepatoprotective constituents were studied for the first time. 5 compounds were isolated from the hepatoprotective fraction. the hepatoprotective active component was identified to corilagin by UV, IR, NMR, MS.2. The pharmacologica mode of CCl₄-induced hepatic injury in newborn rats was employed. The results indicated that the hepatoprotective fraction of Phyllanthus urinaria L （PE） significantly blocked the elevations of ALT and AST, alleviated the swelling and inflammation of hepatocytes, reduced the production of MDA, improved the activity of SOD, increased the synthesis of ALB, raised the value of A/G. So, it is obvious that PE has hepatoprotective activity.3. The HPLC fingerprint of Phyllanthus urinaria L.from different habitats, different harvest time, different parts was firstly established in this study. The mutual HPLC fingerprint consisted of 18 mutual peaks and 4 mutual peaks was confirmed: peak No. 2 of gallic acid, peak No.3 of protocatechuic acid, peak No.6 of caffeic acid, peak No.8 of corilagin. The fingerprint had good attribute, stability, repeatability and able to reflect the difference of Phyllanthus urinaria L. from different habits, different harvest time, different part.The data from different habits were analyzed by computer-aided-similarity-evaluation software. The results indicated that the similarity was more than 0.9 except No.3 sample （Hangzhou City, Zhejiang Province） and No.8 sample（Kunming City, Yunnan Province）.The similarity of 7 samples from different harvest time was more than 0.94. The peak area of most peaks increased with the growth period, the highest in October 5, significantly reduced afterwards. So, it was suggested the best harvest time was on the beginning of October annually.The similarity of samples from different parts was analyzed and evaluated. Compared with the mutual fingerprint, the similarity of leaf and fruit was more than 0.94, and the similarity of root and stem was less than 0.9. The difference of content of constituents brought about the difference in similarity. The content of peaks in the leaf was the highest. Next is in the fruit, the stem, the lowest is in the root. Because the root accounts for less than 20% in proportion, also various ingredients content is very low, therefore, suggested only collecting the aerial parts.4. A HPLC method for the determination of corilagin in Phyllanthus urinaria L. was established. The calibration curve of the corilagin showed good linearity in the range of 0.458～4.580 mg/g, Y=302 172 X + 22 305 （r=0.995 9）. The average recovery of the corilagin was 97.98%. The contents of corilagin varied with different habits evidently, with the average of 2.288 mg/g. The content of the sample from Zhongshan City in Guangdong Province is the highest at 4.224 mg/g and from Hangzhou City in Zhejiang Province is the lowest at 0.982 mg/g. The highest content was 4.090 mg/g with the harvest time of October 5 in the samples from different harvest time. In the samples from different parts, leaf contained the highest content at 4.437 mg/g, followed by fruit and stem. The content in root was the lowest, with 1/5 of leaf.5. The chromatography fingerprint of Yexiazhu Capsules was established by HPLC. The mutual HPLC fingerprint consisted of 17 mutual peaks and 3 mutual peaks was confirmed: peak No.3 of gallic acid, peak No.4 of protocatechuic acid, peak No.8 of corilagin. The similarity of 10 samples was more than 0.9. The results indicated that 10 samples were of good consistency and quality. The correlation of HPLC fingerprint of Yexiazhu Capsules with reference herb of Phyllanthus urinaria L. was investigated. The chemical constituents of Yexiazhu Capsules were nearly consistent with that of reference herb of Phyllanthus urinaria L., but the peak area of some peaks varied very much because of processing of Yexiazhu Capsules.6. The HPLC method for the determination of corilagin in Yexiazhu Capsules was established. The calibration curve of the corilagin showed good linearity in the range of 8.400～42.000 mg/g, Y=199 850 X- 608 070 （r=0.999 8）. The average recovery of the corilagin was 98.51%. There exist small difference in content of corilagin which was from 17.19 mg/g to 21.84 mg/g, and the average content was 19.82 mg/g.更多还原