Study On The Molecular Identification Of Raw Materials In Chinese Patent Medicine

Objective To establish the new r the authentication of raw medicinal method fo materials in Chinese patent medicine by using molecular technology. Method(1) The genomic DNA was extracted from Lian Qiao Bai Du Wan through modified CTAB method. The psb A-trn H and rbc L sequences were gradient amplified, and PCR products were ligased with the p EASYTM-T5 vector and then transformed Trans1-T1 cells, respectively. Clones were selected randomly and sequenced. All sequences were analyzed by Blast N and the neighbor-joining(NJ) phylogenetic tree was constructed by MEGA 4.0.(2) The genomic DNA was isolated from 6 kinds of Chinese patent medicine through modified CTAB method, and was purified by the commercial DNA purification kits. The trn L-trn F and a pair of Fat B sequences were amplified. The trn L-trn F dilution samples were used as template, and were amplified by two pair of specific primers. The amplified products were sequenced in forward directions. All sequences were aligned and were analyzed by Blast N.(3) The DNA of Paeonia suffruticosa was used as template to amplify by five pair of FAM fluorescence labeling primers. Then, the amplified products were sequenced. The sequencing results were analyzed by Gene Marker V1.80 to screen the best fluorescence labeling primers. As a result, psb A-trn H fluorescence labeling primer was used to identify the raw materials of Liu Wei Di Huang Pill.(4) 13 samples of the different G. elegans materials and 58 samples of L. japonica, L. macranthoides and L. dasystyla were collected. The total DNA of the samples were extracted, and the DNA of G. elegans, L. japonica and L. macranthoides water extracts were extracted. Psb A-trn H sequence from G. elegans was amplified by PCR and sequenced unidirectionally, Clustul W has been used to align psb A-trn H sequences of the G. elegans and L. japonica and its close species from Gen Bank database. Results(1) The results showed that nine kinds of medicinal materials can be identified by psb A-trn H sequences, and six kinds of medicinal materials by rbc L sequences from Lian Qiao Bai Du Wan.(2) Lonicerae japonicae Flos can be identified by Lonicerae japonicae Flos primers from 6 kinds of Chinese patent medicine, and Lonicerae Flos medicinal materials can be identified by Lonicerae Flos primers from Jin Sang Kai Yin Wan. The results also indicated that Lonicerae japonicae Flos can be identified from 6 kinds of Chinese patent medicine, and Lonicerae Flos can be found in Niu Huang Qing Gong Wan and Jin Sang Kai Yin Wan by Fat B primers.(3) Three kinds of raw plant medicinal materials in Liu Wei Di Huang Pill can be correctly identified by psb A-trn H fluorescence labeling primer.(4) All samples were amplified by PCR with specific primers. DNA from G. elegans would be amplified 97 bp whereas PCR products from all of the adulterants have not bands. Conclusions Molecular marker technique can stably and accurately distinguish multi-origin medicinal materials in Chinese patent medicine.