Study On The Remediation Of Clematis Varieties Based On DNA Barcode - Morphology

Clematis Radix et Rhizoma is a wildly used Chinese Medicine for its effects of expelling wind-damp and tongjinluo in clinical, and used as treating rheumatism palsy, numbness of limb, nconvenient flexing and stretching. And it is mainly sourced from Clematis chinensis Osbeck、Clematis hexapetala Pall and Clematis manshurica Rupr. based on Chinese Pharmacopoeia. Then according to the literature review, we find that there is many medicinal plants which used as Clematis Radix et Rhizoma since the ancient times after reading the herbal medicine works, and there is a difference in medicinal species of Clematis Radix et Rhizoma in different area and in different period. The phenomenon of synonyms and homonym is seriously. Now, there are many adulterants in medicinal market, in addition to three kinds of authentic medicinal materials. But the adulterants’ chemical composition and clinical effects are different from the authentic medicinal materials. It influences the development of the medicinal market, the safety of clinical drug use, the research of the medicine and the drug’s production. Therefore, it is very necessary and urgent to identify Clematis Radix et Rhizoma and its adulterants.Nowadays, the traditional identify method including origin identification, macroscopical identification, microscopic and physicochemical identification, and the DNA-Barcoding method which is wildspread used in Chinese herb identification. DNA-Barcoding method is very popular in pharmacognostic study for it is convenient, accurate, efficient. The identify method of DNA-Barcoding is similarity searching and building a N-J tree after analyzing plenty of gene sequence. But this method has been hampered by imcomplete sequence database and varying compare sequence. Herein, the leaves of Clematis manshuria and Clematis hexapetala and the sequence of Clematis chinensis and its adulterants from GenBank were used as materials. We built the DNA-Barcoding datebase of Clematis manshuria and Clematis hexapetal and used similarity threshold, specific mutation sites and N-J tree to identify Clematis Radix et Rhizoma and its adulterants.A new molecular identification method was successfully established to identify Clematis Radix et Rhizoma firstly quickly and accurately.The following results were drawn in this paper:(1)Build the DNA-Barcoding datebase of Clematis manshuria ITS and psbA-trnH sequence.We have built two DNA-Barcoding datebase, there are the ITS sequence datebase of Clematis manshuria, the psbA-trnH sequence datebase of Clematis manshuria. And the haplotype number of two haplotype datebase is 787 and 38, the percentage of main haplotype is 11.36% and 37.18%, respectively. We used the computer language to build the DNA-Barcoding database searching system of Clematis manshuria and Clematis hexapetal based on the sequences we obtained. In this searching system, the system will show the result containing species name, sequence name, similarity when user seach the sequence. And the administrator can increase, seach, edit, delete and restore the sequence in order tokeep the searching system running.(2) The similarity threshold is used as the authenticate target.The similarity threshold is used as the the authenticate target after we analysed the target including similarity threshold, specific mutation sites and N-J tree. Finally, the similarity threshold between the DNA-Barcoding datebase of Clematis manshuria and samples was 100%. If the similarity between the sample and haplotype in database was 100%, the sample is Clematis manshurica, but if it was less than 100%, we should use DNA-Barcoding method to identify it. The computing method of similarity threshold was convenient and accurately.(3) Use the authenticate target to systematize the variety of Clematis Radix et Rhizoma and its adulterants in medicinal market.Establish a new identify method for Clematis Radix et Rhizoma and its adulterants. We used the mehod which we established to identify 26 samples of Clematis Radix et Rhizoma collected from the medicinal market. The results show that 11 samlpes is Clematis manshuria, 2 samples are Clematis hexapetala, one sample is Clematis chinensis, and 11 samples are adulterants, which is Ardisia japonica. Ardisia japonica is folk commonly used Chinese herbal medicine and ornamental plants, which has the activating blood stasis, antitussive expectorant, detoxification effect. The identify results indicate that Clematis manshuria Rupr. is the main specie of Clematis Radix et Rhizoma, and there are so many adulterants of Clematis Radix et Rhizoma in the market. And the results also show this method can identify Clematis Radix et Rhizoma quickly and accurately. And it is necessary to renovate the the medicinal market.