Study On The Fingerprint Of Clematidis Radix Et Rhizoma

The Clematidis Radix et Rhizoma was derived from the dried roots and rhizomes of Clematis chinensis Osbeck,Clematis hexapctala Pall,Clematis manshurica Rupr.At present,Clematidis Radix et Rhizoma was quality-controlled by the Chinese Pharmacopoeia through cross-section microscopic identification and determination of the content of oleanolic acid after hydrolysis.Since the efficacy component of Clematidis Radix et Rhizoma has not yet been clarified,there were some problems of intrinsic quality control in the existing standards for Clematidis Radix et Rhizoma,which were needed further research and improvement.In order to provide research basis for the improvement of the quality standards of Clematidis Radix et Rhizoma,this paper carried out the fingerprint research of Clematidis Radix et Rhizoma on the scientific and technical problems existing in the current quality standards of Clematidis Radix et Rhizoma.The main research contents and conclusions of this paper were as follows:1.Study on the identification of the origins of Clematidis Radix et Rhizoma.A total of 13 batches of Clematidis Radix et Rhizoma purchased from the market were prepared by cross section and powder microscopic respectively.The preparation methods were investigated and optimized.The results showed that the microscopic features of the outer cortex cell arrangement from the cross section were remarkable,and the microscopic features of the wood fiber,bast fiber and stone cell from the powder were remarkable.In terms of the pharmacopoeia standards,Clematis chinensis Osbeck can be identified according to the direction of extension of the outer cortical cells in the cross section,Clematis hexapetala Pall and Clematis manshurica Rupr can be identified according to the features of stone cell and bast fiber in the powders.As the result,two of the 13 batches of Clematidis Radix et Rhizoma were identified as Clematis chinensis Osbeck,while2 batches as Clematis hexapetala Pall and 9 batches as Clematis manshurica Rupr.Therefore,powder microscopy can effectively make up the deficiency of cross-section microscopy identification.The combination of powder and cross-section microscopy could quickly and efficiently identify the origin of Clematidis Radix et Rhizoma,which was a powerful supplement to the present microscopic identification method of pharmacopoeia.2.Study on the extraction method of Clematidis Radix et Rhizoma.By comparing the effects of heating reflux and ultrasonic on the extraction of Clematidis Radix et Rhizoma,it was found that the overall composition was similar of the HPLC chromatogram by the two methods,but under the condition of heating reflux,the peak was larger and a peak was added.The structure was identified by NMR after purification,and combined with standard for HPLC analysis,the peak was confirmed to be 5-hydroxymethylfurfural.According to literature research,this substance was produced by the degradation of carbohydrate in Clematidis Radix et Rhizoma during heating.Ultrasonic extraction of water was determined as the method of extraction of Clematidis Radix et Rhizoma for the characteristics of high efficiency,short time,low temperature and easy operation.3.Study on HPLC fingerprint of Clematidis Radix et Rhizoma.The HPLC analysis method of the water extract of Clematidis Radix et Rhizoma was optimized:mobile phase was acetonitrile-0.5%aqueous acetic acid solution?V/V=5.5:94.5?;gradient elution was?0⁵ min,5%B;5²5 min,5%B¹8%B;30⁴5 min,18%B²7%B?;flow rate was 1.0 mL·min^-1;injection volume was 20?L;detection wavelength was 280 nm.The optimal ultrasonic extraction process was obtained by single factor and orthogonal design:drug particle size was 80-100mesh;material to liquid ratio was 1:12;extraction time was 30 min;extraction times was 3 times.The comparison fingerprint was established based on the HPLC chromatogram of 13 batches of Clematidis Radix et Rhizoma,and 16 fingerprint peaks were indicated,and similarity evaluation was performed.It was indicated that the water extracts of Clematidis Radix et Rhizoma from different places of production and origins were roughly similar,but there were some differences in the number and content of the components.The similarity of the seven batches of Clematidis Radix et Rhizoma was above 0.9,and the similarity of the other batches was between 0.63-0.80.There was no correlation between similarity and factors such as place of production and origin.In 13 batches of Clematidis Radix et Rhizoma,the similarity of the five batches purchased from pharmacies were above0.9,which indicated that the quality of Clematidis Radix et Rhizoma from pharmacies was relatively controllable.The fingerprint quality control method of Clematidis Radix et Rhizoma had certain feasibility,but it remained to be further studied after adding credible samples.