Research On Quality Standard Of The Opisthoplatia Orientalis Burm.

ObjectiveThe quality standards of Opisthoplatia orientalis Burm.had been comprehensively and systematically studied by modern detection technology and analysis methods.And formulate quality standard draft of Opisthoplatia orientalis Burm.Methods1.Microscopic identification method will be performed to observe the powders of Opisthoplatia orientalis.Thin-layer chromatography（TLC）identification method will be used to optimize existing TLC identification methods,which make the method more suitable to the Opisthoplatia orientalis and its adulterants.2.Molecular biotechnology will be applied to analyse and compare the identify efficiency of COI、12SrRNA、16SrRNA、ITS2 of Opisthoplatia orientalis.And the DNA barcode of Opisthoplatia orientalis will be conformed.Based on the above molecular research,specific primer will be analyzed and designed.Using the specific primer to complete identification by specific site polymerase chain reaction（PCR）method.3.According to the existing standards,the water content,total ash,acid-insoluble ash and extractive of 24 batches of Opisthoplatia orientalis will be detected.And on the basis of this,high performance liquid chromatography（HPLC）,ultra high performance liquid chromatography-mass spectrometry（UHPLC-MS）,inductively coupled plasma mass spectrometry（ICP-MS）will be respectively used for aflatoxin,veterinary drug residues,and inorganic elements limited detection and analysis.4.Pre-column derivatization of phenyl isothio cyanate（PITC）-high performance liquid chromatography（HPLC）method will be applied to study the fingerprint of Opisthoplatia orientalis and its adulterants from different areas.The similarity evaluation and cluster analysis methods are carried out on the high performance liquid chromatography（HPLC）of each batch sample and the fingerprint will be established.On the foundation of above research,the contents of 6 amino acids in Opisthoplatia orientalis will be tested and limited.Results1.A large amount of fat droplets were found in the powder of Opisthoplatia orientalis.A thin-layer chromatography（TLC）method for the identification of Opisthoplatia orientalis and its adulterants was established.2.The PCR amplify conditions of 4 segment sequences of COI、12SrRNA、16SrRNA、ITS2 in Opisthoplatia orientalis were conformed.Genetic distance test,neighbor-2-joining tree（NJ）and maximum likehood tree（ML）analysis methods were applied to evaluate the identification efficiency of each sequence.In the end,the sequence of 12SrRNA can be used as the main sequence of DNA barcode in Opisthoplatia orientalis,which could successfully make a distinction between division of Opisthoplatia orientalis and its adulterants.It was found that the difference of ITS2 sequence between species was obvious.Therefore,a pair of specific primers were designed and a specific site PCR identification method was established in Opisthoplatia orientalis.3.There are 14 samples of 24 batches of Opisthoplatia orientalis complied with Opisthoplatia orientalis under the standard of Chinese medicinal materials in Guangdong Province（Volume II）.There are only 6 samples’the total contents of aflatoxin B₁（≦5μg/kg）and aflatoxin B₁,B₂,G₁and G₂（≦10μg/kg）of14 batches of Opisthoplatia orientalis mets the regulations.And there are only 6 samples’the tetracycline veterinary drug residues of 14 batches of Opisthoplatia orientalis in accordance with the provisions of Proclamation No.235 of the Chinese Agriculture Ministry（≦100μg/kg）.The 14 batches contained 23 kinds of inorganic elements,there were 16 kinds of common elements.4.The common pattern of amino acid HPLC fingerprint was established.And21 common peaks were comformed,and 13 amino acids were marked.The similarity calculation results of the HPLC fingerprints of 14 batches of Opisthoplatia orientalis showed that similarity of 14 samples were above 0.9,it showed that the quality of Opisthoplatia orientalis from different areas was stable.On the basis of this,a HPLC method for test the contents of glycine,arginine,threonine,valine,isoleucine,lysine was established and respectively stipulated that the contents should not be less than 0.12%、0.47%、0.72%、0.09%、0.13%、0.34%.ConclusionResearched the identification,inspection item,fingerprints and quantitative determination,the overall quality of Opisthoplatia orientalis was diversity analysed and detected.The quality standard draft of Opisthoplatia orientalis was drawn up,which provided experimental basis for quality control and clinical efficacy and safety,and improved the current standard.