Study On Quality Standard Of Pulsatilla Total Saponins And Its Colon Targeting Capsules

The Radix Pulsatillae is the dry root of Pulsatilla chinensis（Bge.）Regel,which belongs to Ranunculaceae plants.And it is the common drug of Chinese medical treatment,with the function of clearing away heat,detoxicating,removing heat from the blood and arresting dysentery,and also has the reputation in dysentery treatment as an important medicine.Pulsatilla total saponins as the medicinal active site of Pulsatilla has the activities including anti-cancer,anti-ulcerative colitis,anti-bacterial,anti-pathogen,improving immunity and so on,which exists a great medicinal value.Moreover,Pulsatilla total saponins has a significant effect on resisting ulcerative colitis.When it is treated with its common preparation,because of its own defects,such as poor water solubility,dissolution difficulties,low bioavailability and so on,it is easy to be absorbed and damaged before the drug reached in the colon,which leads to a low drug concentration and making the effect reduced.However,when its colon targeting drug delivery system was used,it can make the concentration of drugs increased at the colon site and give full play to efficacy and improve efficacy due to the characteristics of drug releasing in the colon site.The quality standard of traditional Chinese medicine is the basic criterion and legal basis of traditional Chinese medicine on the quality evaluation and application,but the quality standard of Pulsatilla total saponins has not been determined.Therefore,it is very important to find and develop its quality standard.In this paper,the initial quality criteria for Pulsatilla total saponins and its colon targeting capsules will be determined by conducting the following works:（1）Taking the Pulsatilla Saponins D as the reference substance,using the UV-Visible spectrophotometry to determine the content of Pulsatilla total saponins,and the single factor test and orthogonal test were used to optimize the experimental conditions.The results were analyzed by intuitive analysis and variance analysis with the absorbance as the index.And the optimum reaction conditions were as follows:0.2 mL of 5%vanillin-glacial acetic acid and 0.8 mL of perchloric acid were added and reacted in a constant temperature water bath at 90℃for 40 min.The methodological investigation was carried out with the precision of the instrument,the stability of the sample,the repeatability and the sample recovery rate.Overall,the results showed that the method was sensitive and reproducible,and it could be used for the determination of Pulsatilla total saponins.（2）College Liquid Chromatography-Diode Array Detector（HPLC-DAD）method and the liquid chromatography-evaporative light scattering（HPLC-ELSD）method were analyzed and compared.And by changing the mobile phase ratio to adjust the polarity,changing the column temperature,detection wavelength and flow rate and so on to preferred the best chromatographic conditions.The optimum conditions were as follows:methanol-acetonitrile-water（14:36:50）as the mobile phase,the flow rate was 1.0 mL·min^-1,the column temperature was 35℃and the detection wavelength was 203 nm.Taking Pulsatilla saponin D,Pulsatilla saponin A,oleanolic acid 3-O-α-L-rhamnopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→4）]-α-L-arabinopyranoside and oleanolic acid 3-O-α-L-rhamnopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→4）-β-D-glucopyranosyl-（1→3）]-α-L-arabinopyranoside as the reference substance,and HPLC was established to determine the contents of the four saponins in the Pulsatilla total saponins.Besides,a methodological validation experiment was conducted.And the results showed that the four saponins of Pulsatilla total saponins have the good linear ranges at 1.92¹9.20、0.43⁴.32、1.54¹5.36、0.83⁸.28μg,respectively;The average recoveries of the four saponins were between 98.96%and 100.40%,and RSD values were less than 1.5%.It is indicated that the method in this paper was simple,accurate,and reproducible,which could provide the basis for the quality control of Pulsatilla total saponins.（3）The quality standards of Pulsatilla total saponins was established based on the systematic research from identification,examination and determination of the content.Qualitative identification was carried out by thin layer chromatography.using Pulsatilla saponin D,Pulsatilla saponin A,oleanolic acid 3-O-α-L-rhamnopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→4）]-α-L-arabinopyranoside and oleanolic acid 3-O-α-L-rhamnopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→4）-β-D-glucopyranosyl-（1→3）]-α-L-arabinopyranoside as the reference substance to identify the Pulsatilla total saponins.The results showed that the corresponding spots were clearly visible,and the blank control without interference.The items of examination included particle size and the dry weight loss with the appropriate results according to the standards.Qualitative studies were used for fingerprints of five batches of extracts.And the main components of the four saponins in the Pulsatilla total saponins were determined by a multi-evaluation method.The method was simple and accurate,and the results were accurate and reliable,which provided new ideas for the establishment and quality control of the Pulsatilla total saponins.（4）The solid dispersions of Pulsatilla total saponins were prepared with Eudragit S100 as the material and the drug loading of 20%according to the results of single factor study.And the Pulsatilla total saponins colon targeting capsules were prepared by No.3 capsules.The prepared capsules have compared with the extracts on qualitative studies of fingerprints as well.The quality standards of the prepared Pulsatilla total saponins colon targeting capsules were established based on the invention project of capsule from the"Chinese Pharmacopoeia"of the 2015 version including three aspects about identification,inspection,and content determination.The results show that the prepared capsules in this experiment have reached the purpose of drug releasing on colon,and it is of great significance to the application of Pulsatilla preparation for the treatment of ulcerative colitis.Instrument analysis is one of the main analysis methods in analysis chemistry,and it has broad application prospects.In this study,the quality standard of Pulsatilla total saponins and its colon targeting capsules were studied by the modern instrumental analysis method,such as UV,HPLC,HPLC-ELSD and so on.These studies will provide reference for the development,utilization and quality evaluation of the Pulsatilla total saponins.