Revised Study On Quality Standards Of Granati Pericarpium And Granati Semen In The Chinese Pharmacopoeia

Granati pericarpium is dry skin of Punica granatum L., a medicine needing to be revised in the scientific research task of the Chinese pharmacopoeia 2015,and it was recorded in the Chinese pharmacopoeia 2010 previously, but the item of content determinatio in previous quality standard just provide that the total tannin content in Granati pericarpium shall not be less than 10.0%, lacking quantitative indicators of specific distinctive chemical components:Granati semen(Tibetan name "saizhu" (?)) is mature dry seeds of Punica granatum L.,a newly introduced medicine in the scientific research task of the Chinese pharmacopoeia 2015,and it was recorded in Tibetan medicine Standard (1995) enacted by The ministry of health previously,but the previous quality standard only included description of botanicalcharacter,leading that its production, circulating and clinical medication of the relevant compound preparation lack of basis for quality evaluation. So in this paper, the quality standards of Granati pericarpium and Granati semen were studied, providing scientific basis for the quality control of Granati pericarpium and Granati semen and their compound preparations.Purpose:1. Establish a new DNA barcodeing for identifying the origins of Granati pericarpium and Granati semen.2. Improve the quality standards of Granati pericarpium, and establish the quality standards of Granati semen, ensuring its clinical medication is availability and security.Methods:1. Using MEGA5.1 software, analysis the intraspecific variation of ITS2, psbA-trnH sequence of Punica granatum L.; According to K2P,calculate its genetic distance; Using NJ method, construct the system clustering tree of ITS2 and psbA-trnH sequence.2. Determine the content of ellagic acid in Granati pericarpium and Granati semen by HPLC.3. Identify the Granati semen by regular pharmacognosy method and TLC.4. Determine the content of moisture, total ash, acid-insoluble ash and extract in Granati semen according to the appendices of the Chinese pharmacopoeia(2010).Results:1. DNA barcodeing identification of Punica granatum L.:The similarity between the download GenBank sequences and the experimental ITS2, psbA-trnH sequences of 29 batch samples of Punica granatum L. amount to 99%;ITS2 sequence contains 244 bp,there is no intraspecific variation;PsbA-trnH sequence contains 405～409 bp and 2 haplotypes, genetic distance is 0-0.0025,the average of genetic distance is 0.0002; The Punica granatum L. and other plants can be clearly distinguished according to the system clustering tree of ITS2 and psbA-trnH sequences,and its monophyly is good.2. Quality standards of Granati pericarpium:Developed a content determination method by HPLC of ellagic acid in Granati pericarpium, and determined the limits of ellagic acid content, (calculated on dry product, ellagic acid≥ 0.45%), improved the quality standards of it.3. Quality standards of Granati semen:The description of botanical character and microscopic characteristics of Granati semen were determined; TLC method of Granati semen was established; Measured the content of moisture, total ash, acid-insoluble ash and extract in the 10 batches of Granati semen,and determined their limits (moisture≤16.0%、total ash≤4.0%、Acid insoluble ash≤1.0%、Extract≥ 42.0%); Developed a content determination method by HPLC of ellagic acid in Granati semen, and determined the limits of ellagic acid content, (calculated on dry product, ellagic acid≥0.07%), established the quality standard of Granati semen.Conclusion:1. ITS2, psbA-trnH sequence can be used as DNA barcodeing to identify the origins of Granati pericarpium and Granati semen,and this method is accurate, stable,simple operation and better repeatability.2. The quality standards of Granati pericarpium and Granati semen are scientific and comprehensive,and have specificity and controllability, improving the quality control level of Granati pericarpium and Granati semen and their compound preparations.