| Objective:liparis nervosa is a folk medicine commonly used in southwestern China,it was first published in "Plans and Realities",it has bitter coldness,has the effects of cooling blood to stop bleeding,clearing heat and detoxifying.At present,the quality standards for liparis nervosa are only included in the "Sichuan Province Chinese Medicinal Materials Standards",which are mainly routine tests such as traits and microscopic identification,and lack of relevant quantitative detection methods.At the same time,a small amount of literature reports that they have good hemostatic and coagulation effects,but the material basis of its efficacy is not clear.Therefore,in order to further improve the quality standards of liparis nervosa and extracts,and to analyze the spectrum-effect relationships of different polar parts of liparis nervosa,this study intends to use high-performance liquid chromatography(HPLC)to establish fingerprints for liparis nervosa of different origins.Based on the similarity evaluation,combined with cluster analysis(HCA)and principal component analysis(PCA),systematically evaluate the 10 batches quality and their common peaks of fingerprints;at the same time,on the basis of the above research,HPLC fingerprints of different polar parts of liparis nervosa were constructed,and a series of pharmacodynamic tests were conducted.Modern mathematical analysis models such as gray correlation analysis and bivariate correlation analysis were used to detect different polar parts of liparis nervosa.Spectrum analysis of fingerprints and different medicinal effects,to explore the correspondence between the optimal medicinal effect and fingerprints,with a view to improving the quality control of s liparis nervosa,revealing the basis of medicinal materials,and providing resources for the development of liparis nervosa Scientific basis.Methods:1.Study on fingerprints of serum medicinal materials from different origins:High-performance liquid chromatography(HPLC)was used to establish fingerprints of liparis nervosa from different origins in 10 batches,and the common peak chromatogram information was obtained,the common peak material was confirmed by comparison with the reference substance;the software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Version A" was used to evaluate the similarity of fingerprints;on the basis of this,software SPSS 23 and Simca 14.1 were used to perform cluster analysis and master analysis on the above 10 batches of liparis nervosa.Composition analysis,to complete the research and quality evaluation of serum composition differences in different regions.2.The spectrum-effect relationship of serum extracts in different polar parts:(1)The establishment of fingerprints of different polar parts of liparis nervosa.The liparis nervosa were extracted by refluxing with 80% ethanol,and the alcohol extract was gradient-extracted with petroleum ether,ethyl sites,and n-butanol in order to obtain extracts of different polar parts of the liparis nervosa;HPLC was used to establish different polar parts of the liparis nervosa fingerprints,to identify the common peaks.(2)Pharmacodynamic study of different polar parts of liparis nervosa.Hemostatic coagulation of different polar parts of liparis nervosa was observed by tail-cutting method,glass slide method,and capillary method;the analgesic effects of the abovementioned parts were observed by twisting method,tail flick method,formalin method,and hot plate method;carrageen foot swelling method and agar granulomatous method were used to analyze its anti-inflammatory effect,and its related inflammatory factors were detected.(3)Spectrum-effect relationship analysis of different polar parts of liparis nervosa.Combined with the above research results,using modern mathematical analysis models such as gray correlation analysis and bivariate correlation analysis to systematically analyze the spectrum-effect relationship between common peaks in different polar parts of serum and different drug effects,identify the "effect map" of liparis nervosa.Results:1.Study on fingerprints of liparis nervosa from different origins:The study established 10 batches of HPLC fingerprints of liparis nervosa from different origins,and identified 40 common peaks,identifying peak 2 as protocatechuic acid,peak 4 as vanillic acid,peak 6 is p-hydroxycinnamic acid and peak 29 is linolein.The software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Version A" was used for similarity evaluation.Except for the two batches of S3 and S6,the similarity of each batch was above 0.9,which met the requirements of fingerprints.The software SPSS 23.0 was used to perform cluster analysis(HCA)on 10 batches of liparis nervosa.Combining with the clustering tree diagram,it can be seen that there were cross-originations of the classification of liparis nervosa from different batches,that the content of chemical components in medicinal materials is not affected by the place of origin,and the results of cluster analysis are consistent with the results of principal component analysis(PCA).At the same time,the partial least squares discriminant analysis(PLS-DA)model found that peaks 1,2,4,5,6,8,12,15,20,21,21,23,27,30,35,37,38,and 39 are the main chromatographic peaks that cause differences between sample groups;the comprehensive quality scores of 10 batches were calculated,and the results showed that Leshan,Sichuan(S10)has the highest overall score(2.470)and the best quality.2.The spectrum-effect relationship of liparis nervosa extracts in different polar parts:(1)Establish HPLC fingerprints of different polar positions of liparis nervosa and determine 53 common peaks,including 49 peaks in ethanol,33 peaks in n-butanol,23 peaks in ethyl sites,and petroleum ether and 5 peaks in petroleum ether.(2)Relevant pharmacodynamic experiments:(1)The results of three coagulation and hemostasis experiments showed that the liparis nervosa petroleum ether and ethyl acetate sites both significantly promoted coagulation and enhanced hemostasis,especially the petroleum ether site.(2)Analgesic effect: The alcohol extraction site and n-butanol site of liparis nervosa show good peripheral analgesic effect in mice writhing experiment and formalin experiment II,and ethanol site is the best;meanwhile,The results of central analgesic experiments showed that the ethyl sites and ethanol sites showed a good analgesic effect on the hot plate test,tail-flick test,and formalin test of mice in phase I,especially acetic acid.Ethyl sites has the best effect.(3)Antiinflammatory effect: Both the ethanol and n-butanol sites can significantly inhibit the swelling of carrageen horn-induced foot swelling and agar-induced granulomatosis in mice,and reduce the levels of inflammatory factors TNF-α and IL-1β,especially the ethanol site The effect is the most obvious.(3)Using gray correlation analysis and bivariate correlation analysis to study the spectrum-effect relationship between the common peaks of fingerprints in different polar parts of liparis nervosa and the above-mentioned pharmacodynamic effects,it was found that:(1)In terms of hemostasis and coagulation,the correlation between peaks 46 and 49 was more Strong,and the correlation between peaks 20,22,and 24 is poor;(2)in the peripheral analgesia,the correlation between peaks 34 and 22 is strong,and the correlation between peaks 14 and 35 is poor;(3)In terms of central analgesia,the correlation between peaks 5 and 53 is strong,while the correlation between peaks 6 and 9 is poor;(4)In terms of anti-inflammatory effects,peaks 19,20,21,22,24,25,26,27 and 28 has a strong correlation with efficacy,while peak 14 and 35 have a poor correlation.The polar positions of the above-mentioned common peak distribution are consistent with the efficacy results.that the petroleum ether site is rich in peaks of hemostatic coagulation,the ethyl acetate site is rich in central analgesic peaks,and the ethanol site is rich in peripheral analgesic and anti-inflammatory peaks.Conclusion:1.Establish HPLC fingerprints of liparis nervosa.The retention time of the whole peaks in each batch are basically consistent with the control spectra.The quality of the liparis nervosa can be seen from the overall reaction.Based on the similarity evaluation,cluster analysis and principal component analysis were performed to evaluate the quality of 10 batches of liparis nervosa from different places of origin.The research method is simple and easy,and the data is reliable.It provides scientific basis for the quality control and evaluation of liparis nervosa and the further development of medicinal material resources.2.Establish HPLC fingerprints of different polar parts of liparis nervosa,and compare the pharmacological effects of hemostatic coagulation,analgesia,antiinflammatory and other "effects" on the above parts,and combine the spectrum analysis method to determine the serum "effect map ",That is,the correlation between the polar part that exerts the pharmacological effect and the peak group of the pharmacological substance.The research method is simple,accurate and feasible,and provides a scientific basis for revealing the serum "pharmacologically active component group" and the substance basis of the medicinal effect. |
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