| Caulophyllum robustum Maxim,also known as "Hong Mao Qi" in Chinese,belong to the family of Berberidaceae Caulophyllum plants.The dry roots and rhizomes are the medicinal parts of the plant and these parts are used as a folk medicine in China to treat rheumatoid arthritis,bruises,stomach pain,menoxenia,etc.,especially in the treatment of rheumatoid aspects has significant curative effect.Because of the complexity of chemical composition,Looking for the effectiveness of specific chemical composition becomes hotspot and difficulty in research.In this paper,we established high performance liquid chromatogram(HPLC)fingerprint about 11 batches of Caulophyllum robustum Maxim effective parts,which come from different regions and different harvest time.Meanwhile,Collagen-induced arthritis(CIA)were used as pharmacodynamic model and characteristic efficacy index was studied.The spectrum-effect relationship was applied in studies on the Caulophyllum robustum Maxim treatment of rheumatoid arthritis by using correlation analysis and multiple regression analysis method.1 On the basis of the process screening,the research using(L934)orthogonal test method optimized the extraction conditions of Caulophyllum robustum Maxim in order to obtain more comprehensive fingerprint,in which the extraction rate to combine inflammation inhibition rate pharmacodynamic index of the effectively part were used as comprehensive measure.Optimum process were as follows:80%ethanol was extracted three times,each time with 6 times the amount of ethanol,extraction of 2.5 hours.2 The HPLC fingerprint establishment of 11 batches of Caulophyllum robustum Maxim.By the optimization of chromatographic conditions and the study system methodology,We using the "traditional Chinese medicine(TCM)chromatographic fingerprint similarity evaluation system for 2004 A version " software processing to generate mutual model refere nce map,in which 17 common peaks were determined and 9 peaks were identified.The peak 1:N-CH3 cytisine,retention time of 3.671;Peak2 Magnoflorine,retention time 17.720;Peak 8:Cauloside H,retention time 24.181;peak 9:Leonticin D,retention time 24.804;peak 10:Cauloside G,retention time 25.328;peak 11:Cauloside D,retention time 27.220;peak 13:CaulosideC,retention time 39.347;peak 15:oleanolic acid,retention time 43.940;peak 16:?-sitosterol,a retention time of 45.422;The rest characteristic peaks which not identifyied were gussed by ultraviolet spectrum:Peak 4,peak 6,peak 7 were alkaloids,peak 3,peak 5,peak 12,peak 14 and peak 17were saponins.The results of si milarity analysis and cluster analysis showed that the herbs similarity mostly meet the requirements which was in different regions,different harvest years,The similarity values were 0.977,0.935,0.985,0.945,0.984,0.965,0.979,0.987,0.985,0.976,0.971 respectively.However,The herbs similarity between different regions and different harvest years is somewhat different,indicating regions,harvesting years have different degrees of impact on medicines.The cluster analysis showed that 11 batches of herbs can be divided into two categories,categoryfor S1 and S2,and the other for S3-S11.Wherein S7 and S9,S8 and S10,S3,S 4 and S11;S5 and S6 can be divided into four small classes,thus S4,S11 and S6 belong to categories,but S6 and S4,S11 also have differences,The cluster analysis and similarity analysis results are almost inagreement.3 1mg/ml collagen-complete Freund's adjuvant emulsion was injected in the mouse base of the tail to establish the CIA model.Respectively inflammatory cytokines which contained TNF-?,IL-1?,IL-6,IL-17 content,PGE2 content and T lymphocyte subsets CD4+/CD8+ ratio were used as quantitative indicators to evaluat herbs anti-inflammatory immune function,meanwhile,the changes in body weight of mice,paw swelling,arthritis score and visceral index as a secondary measure.The results showed that anti-inflammatory immune function were different between different harvest regions and harvest years.Anti-inflammatory immune effect was 2014 in Heilongjiang>2013 in Heilongjiang>2014 in Sichuan>2014 in Hebei>2012 in Heilongjiang>2013 in Sichuan.In other words,In the same harvest years,Heilongjiang>Sichuan>Hebei;In the same harvest region,2014>2013>2012.4 The correlation analysis and multiple regression analysis method were respectively applied in the study of the spectrum-effect relationship about Caulophyllum robustum Maxim acting on rheumatoid arthritis.Comprehensive analysis as follows:17 fingerprint peaks in HPLC fingerprint,(1)With inflammatory factor TNF-? content as index,the no.2,6,11,17 peak combination play anti-inflammatory effect;(2)With inflammatory factor IL ?1?as indexes,the no 1,3,6,7,12,13,14,peak combination play anti-inflammatory effect;(3)With inflammatory factor IL-6 as indexes,the no 1,2,13,17 peak combination play anti-inflammatory effect;(4)With inflammatory factor IL-17 as indexes,the no.1,2,7,10,15 peak combination play anti-inflammatory effect;(5)the PGE2 content as indexes,the peak 1,2,10 combination play anti-inflammatory effect;(6)With the T lymphocytes CD4+/CD8 + ratio as indexes,the no.1,2,17 peak combination play immune effect.Based on the above results,not only a linear relationship between Caulophyllum robustum Maxim chemical composition and efficacy,the multi-component common play anti-inflammatory immune efficacy.The main characteristic peaks are no.1,2,3,6,7,10,12,13,14,15 and 17 peak,in which peak 1:N-CH3 cytisine;Peak 2:Magnoflorine;peak 10:Cauloside G;peak 12:Cauloside D;peak 13:CaulosideC;peak 15:oleanolic acid.In summary,this study take Caulophyllum robustum Maxim,as the research object,and the effect of the anti-rheumatoid arthritis as a pharmacodynamic evaluation index.Meanwhile the effect of using HPLC fingerprint to characterize its overall chemical characteristics,and the inflammatory index content of TNF-?,IL-1?,IL-6,IL-17 and PGE2;T lymphocyte CD4+/CD8+ ratio measures were respectively taken as anti-rheumatoid arthritis efficacy indicators.The integrated using of correlation analysis and multiple linear regression analysis established the medicinal materials correlation between "spectrum" and "efficiency" from the different regions,different harvest years.The results showed that the alkaloids(N-CH3 cytisine,Magnoflorine,peak 6 and peak 7),saponins(peak 3,Cauloside G,CaulosideC,peak 12 and peak 14 and peak 17)and saponins(oleanolic acid)together played an anti-inflammatory and immune efficacy.The study laid a solid foundation for effective substances identification in Caulophyllum robustum Maxim,at the same time provide the basis for its quality control. |
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