Research On The Fingerprint Of Yinqiao Chai Gui Decoetion And Its Effect Against Influenza Virus In Vitro

Yinqiao Chai Gui Decoction (YQCGD)is a clinical prescription to the flu by the First Affiliated Hospital of Guangzhou University of TCM, which is composed by many kinds of medicine such as honeysuckle, forsythia, radix bupleuri and cassia twig. This prescription has the efficacy of regulating Ying-Wei, resolving superfecies and regulating activities of Qi. It is clinically used in the treatment of aversion to cold, fever, headache, muscle achesry, xerostomia, pharynx pain and other symptoms caused by influenza, which is effectual. Fingerprint analysis technology, combining anti-influenza virus experiment in vitro was adopted in the research to explore Yinqiao Chai Gui Decoction's pharmacodynamical material basis, providing beneficial reference for the development of its granules.Objective:To develop the assay methods of the index components and fingerprint of YQCGD. To detect the immanent quality and obtain the chemical composition information of the decoction from the qualitative and quantitative angle. To ascertain the chemical components and parts against influenza virus of YQCGD preliminary on the basis of the fingerprint combining the experiment against avian influenza H5N1 pseudovirus in vitro.Method:1,Using HPLC method to develop the content determination method of the index component (Chlorogenic acid, peony glucoside, forsythia ester glucoside and baicalin) in YQCGD.2. Adopting HPLC-DAD and Multipl-wavelength Fusion Technology to establish the fingerprint of YQCGD, and the fingerprint of Yinqiao Pulvis and Chaihu Guizhi Decoction,both of which are the related compounds, and using the Similarity Calculation Software of the Chromatographic Fingerprints of Traditional Chinese Medicine to evaluate the fingerprint.3. By the experiment in vitro, to detect the inhibitory activity of different combinations, different parts and the main chemical composition of Yinqiao Chai Gui Decoction on avian influenza H5N1 pseudovirus. The method is that constructing expression plasmid pHA-H5 and pNA-N1 based on pCDNA3.1 firstly, secondly use calcium phosphate method to make the two plasmids and HIV skeleton plasmid expressing luciferase but defecting in enveloped protein Rev genetic transfect 293T cells together,8 to 16 hours later changing fluid, continuing to cultivate 48 hours, getting supernatant fluid as avian influenza H5N1 pseudovirus. Placing MDCK cells in medicated culture medium, then adding H5N1 false virus in it, after 48 hours'incubation, cracking cells with cell lysis solution, and using Luciferase Assay kit to detect chemiluminesce value on the Multi-function Miroplate Luminometer, finally calculating the value of the drug's antiviral activity.Results:1 Using DAD detector in different wavelength to detect, this method had detected Yichlorogenic acid in 40 minutes simultaneously, peony glucoside and baicalin the three main ingredient 's content of YQCGD,realized detecting many kinds of multiple components at the same time, increased efficiency, of which the accuracy and precision was conforming to the requirements. The content of chlorogenic acid, baicalin, peony glucoside and forsythia esterA of 10 YQCGD was respectively (0.2532-0.3344 mg/ml, 0.2326-0.7958 mg/ml,0.1081-0.2226 mg/ml and 0.07986-0.1867 mg/ml), which had differencies in some degree among the three.2 YQCGD's fingerprint was established in the research,10 batches of YQCGD's fingerprint and Dual-Wavelength Fingerprint Chromatogram similarity was larger than 0.9, the method of which had better specificity, stability and repeatability.In the 90 minutes,the peaks in the fingerprints symbolize overall scattered distribution,with obvious characteristic. The obtained fingerprint had 22 common peaks, and five peaks' chemical composition was identified, number 4 peak of which was chlorogenic acid, number 7 peak was herbaceous peony, number 10 peak is licorice, number 14 was forsythia ester A,and number 17 peak was baicalin; In the same time the attribution of the common fingerprint peaks was analyzed,in which 7 peaks were from radix scutellariae, six peaks were from honeysuckle.Radix paeoniae alba had three peaks,both of forsythia and cassia twig had 2 characteristic peaks,and licorice had one characteristic peak. Number 1 peak was the superposition of the peaks of honeysuckle, forsythia and radix paeinoae alba.The Dual-Wavelength Fingerprint Chromatogram of Yinqiao Chai Gui Decoction made by the "integrating method",in comparison with the quondam fingerprint under a single wavelength of 230nm, characteristic peak number and the response strength increased significantly, of which the information was more abundant, and could more fully reflect the inner chemical composition treatmentresult characteristics.3 The fingerprint of Yinqiao Powder and Chaihu Guizhi Decoction was established in the research, of which the results showed that the similarity of the common medicinal herbs composing Yinqiao Chai Gui Decothion was higher,and these medicinal material could view fully the characteristics of the original medicinal prescription from the anger of the chemical composition, at the same time reflected that it had certain rationality that selecting these medicinal material to compose Yingiao Chai Gui Decotion from Yinqiao Powder and Chaihu Guizhi Decotion.4 The H5N1 flu virus inhibition in vitro experimental results showed that:Jun Medicine and Cheng Medicine had certain function of inhibiting flu viruses, of which the IC50 was 0.08854 mg/ml;Chlorogenic acid, Peony glucoside, Baicalin and Forsythia ester glucoside these four main chemical composition contained in the decoction had almost no inhibitory effEct on the avian influenza H5N1 pseudovirus.Conclusion:It can be concluded that the stability, precision requirements, and reproducibility of YQCGD's fingerprint and index component content determination method is up to the mustard. The chemical information of Jun medicine, Cheng medicine had the greatest contribution for the decoction's fingerprint, pharmacodynamic results showed that the both played the leading role in treating flu. Anti-influenza experimental results of different parts of the decoction showed that the weak polarity parts of Yinqiao, Chai Gui Decoction had stronger effect, which had good guidance meaning on the extraction method choice of Yinqiao Chai Gui granules preparation research process.The four main composition of YQCGD's pharmacodynamic experimental results showed that the characteristic components of the decoction's fingerprint was not the activated pharmaceutical ingredients of anti-influenza, which could only play the role of characterizing the chemical composition characteristics.