Study On The Isolation Of Hepatoprotective Components And HPLC Fingerprint Of Phyllanthus Urinaria L.

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.