The Preclinical Pharmaceutical Research Of Dingchuanfang Keli

Objective:Under the guidance of traditional Chinese medicine theory and modern pharmaceutical technology,Dingchuanfang Keli for the treatment of asthma and chronic bronchitis was developed.In accordance with the requirements of the Regulations on Drug Registration,a series of proclinical pharmacy studies were carried out on Dingchuanfang Keli,such as quality inspection of Yinpian,optimization of preparation technology and quality standards.Meanwhile,the fingerprint and multi-component content determination methods of Dingchuanfang were established by UPLC,which laid a foundation for the study of new drugs of Dingchuanfang.Method:1.Study on preparation process of Dingchuanfang Keli:With the transfer rate of ephedrine hydrochloride and baicalin hydrochloride and the extraction rate as indexes,the extraction and purification process was optimized by single factor test and orthogonal test.Dingchuanfang Keli were prepared by one-step granulation,and the types of excipients and adhesives were investigated with the granulation condition and granulation rate as indexes.2.Study on quality standard of Dingchuanfang Keli:Through TLC identification,inspection and content determination of three batches of granules,the quality standard draft of Dingchuanfang Keli was preliminarily established.3.Study on stability of Dingchuanfang Keli:The stability of Dingchuanfang Keli was studied by investigating the Influencing factors test,long-term stability test and accelerated stability test of three batches of granules.4.Study on Fingerprint of Dingchuanfang Keli:The fingerprint of Dingchuanfang Keli was studied by ultra-high-performance liquid chromatography.The fingerprint of concentrated solution,extract,granule and 70%ethanol reflux samples of Chuan Fang were determined.The similarity model and common peak model of fingerprints were used to evaluate the differences between the fingerprints of each sample.5.Study on multi-component content determination of Dingchuanfang Keli:The ultra-high-performance liquid chromatography was used to determine the content of 12 components in Dingchuanfang Keli.The contents of each component in Dingchuanfang concentrate,extract and granule were determined,and the changes of chemical components in the preparation of Dingchuanfang granule were investigated.Result:1.According to the Chinese Pharmacopoeia 2015 edition,7 kinds of medicinal materials in Dingchuanfang,such as Ephedra sinica Stapf and Scutellaria baicalensis Georgi and so on,were inspected and the results were in accordance with the regulations.The optimal extraction process of Dingchuanfang Keli was plus 10 times the amount of water,soaking for 0.5 hours（adding Scutellaria baicalensis before decocting）,decocting twice（1 hour each time）.Dingchuanfang Keli were prepared by one-step granulation with dextrin after decompress concentration,vacuum drying（60°C）and comminution.2.The quality standard research:TLC method was used to establish the qualitative identification methods of six medicinal materials in the preparation.A method for the content determination of baicalin transfer rate in preparations by HPLC was established.Regression equation of puerarin is Y=36.871X-60.518（r=0.9997）.Puerarin has a good linear relationship in the range of 3.254μg/ml～325.4μg/ml.Average recovery rate is101.3%and RSD is 2.2%.A method for the content determination of ephedrine hydrochloride transfer rate in preparations by HPLC was established.Regression equation of puerarin is Y=3.9976X+0.5623（r=0.9999）.Puerarin has a good linear relationship in the range of 25.16μg/ml～1258μg/ml.Average recovery rate is 97.21%and RSD is 1.9%.The amount of Baicalin(C₂₁H₁₈O₁₁)in each bag should not be less than 265 mg,and that of ephedrine hydrochloride(C₁₀H₁₅NO?HCl)should not be less than 19 mg.3.Stability study:The experimental results of the factors affecting the stability of Dingchuanfang Keli showed that the high temperature（40 C）,strong light and high humidity conditions had no effect on the quality of the preparation,indicating that the temperature of the preparation should be avoided during storage and transportation.The results of long-term stability test and accelerated stability test for 3 months show that the particle has good stability.4.Study on Fingerprint of Dingchuanfang:The fingerprint of Dingchuanfang Keli was successfully established by UPLC.The analysis time of the fingerprint was 35 minutes,and210 nm was selected as the detection wavelength.There are 18 common peaks in the fingerprint of Dingchuanfang Keli,and the total area is more than 80%of the total peak area.By comparing with standard products,8 characteristic peaks of 18 common peaks were identified（1:Gallic acid,2:Ephedrine Hydrochloride,3:Pseudoephedrine Hydrochloride,11:Liquiritin,13:Baicalin,16:Wogonoside,17:Baicalein,18:Wogonin）.The fingerprints of concentrated liquid,dry extract and granule samples were determined,and the similarity was greater than 0.99.The RSD of the relative retention time of each common peak was less than 0.3%,the total peak area was 5.2-15.5%,and the relative peak area was 0.0-6.4%.The results showed that the fingerprints of water extracts,extracts and granules in the preparation process of Dingchuanfang Keli had high similarity,and the chemical composition was similar.At the same time,the fingerprint technology was also used to compare the chemical constituents of water extract and 70%ethanol reflux samples of Dingchuanfang.The results showed that there were significant differences between the common peaks 1,4,5,7,8,9,10 and 12 in the fingerprint of water extract and ethanol extract samples;the similarity was lower than 0.95;the common peak area RSD was 5.9-88.4%,and the relative peak area RSD was 0.0-95.8%.The fingerprint technology can be used for the quality control of the preparation process of Dingchuanfang Keli.5.Study on multi-component content determination of Dingchuanfang:A method for the simultaneous determination of 12 components in Dingchuanfang was established.The detection wavelength was 210,249,271,275,278,280 and 287 nm,respectively.The results of determination of Dingchuanfang concentrated liquid,dry extract and granule samples showed that the contents of each component in the concentrated liquid sample were0.01-0.24 mg/ml,0.15-6.29%in the dry extract sample and 0.08-3.25%in the granule sample.Compared with the concentrate samples,the transfer rates of components in the dry extract samples and granular samples were 79.53-89.32%and 76.75-85.92%.The results showed that the constituent transfer rate of the preparation process was high,and the method could be used for the quality control of the preparation.Conclusion:The preparation process of Dingchuanfang Keli is simple and suitable for industrial production;the established quality standard is simple and feasible;the quality of granules is controllable and stable,but attention should be paid to the storage and transportation process temperature below 40°C.In addition,the method of multi-component determination and fingerprint analysis of Dingchuanfang was successfully established,which laid a foundation for the preparation process and quality standard improvement of Dingchuanfang Keli.