Quality Control And "spectrum-effect" Relationship Of The Effective Parts Of Total Flavonoids From The Cultivar Of Guizhou (cut Red)

The research purposeThis subject aims to produce the Guizhou's Smilax glabra Roxb.(red of cross section)as the research object,establish the analysis method of quantitative analysis of multi-components by single marker(QAMS)in the medicine effective fraction of total flavonoids,through the relative correction factor(RCF)for other content of each component,both saving the cost analysis,and increase the multiple index of quality control,for the quality control of medicinal materials to provide an objective scientific basis for the rational use of medicinal materails.In the using condition of quantitative analysis of multi-components by single marker(QAMS),to establish the characteristic fingerprint of Guizhou's Smilax glabra Roxb.(red of cross section),and combine the characteristic fingerprint with quantitative analysis of multi-components by single marker(QAMS)method,the mutual benefit,in order to achieve the immanent quality of according to the control medicinal efficacy material base omni-directional.According to established characteristic fingerprint of total flavonoids fraction of the pharmacodynamics experiment and in vitro cell culture,application of hierarchical clustering analysis method(HCM),partial least-square regression analysis method(PLC)and stepwise regression analysis method(SRM),the characteristics fingerprint data and information and resistance to RA-HFLS cells(human rheumatoid arthritis into fiber kind of synovial cells)pharmacodynamics experiment results correlation research,find and resistance to RA-HFLS cells closely related compounds,to establish production Guizhou's Smilax glabra Roxb.(red of cross section)of the total flavonoids fraction spectrum-effect relationship,further defined the effective fraction in RA corresponding efficacy material base.The research methods1 The study of quantitative analysis of multi-components by single markerIn order to the astilbin for reference object of Guizhou's Smilax glabra Roxb.(red of cross section),using the HPLC analysis method,measure a relative correction factor for engeletin and 5-O-caffeoylshikimic acid to astilbin,through investigating the relative correction factor of durability and measuring the component chromatographic peak position,so as to establish the total flavonoids of medicinal materiais by quantitative analysis of multi-components by single marker method;through determinating the contents of engeletin and 5-O-caffeoylshikimic acid on the different origins by one point external standard method,external standard calibration curve method and quantitative analysis of multi-components by single marker method,and for analysis and comparison research,is proposed to evaluate the established quantitative analysis of multi-components by single marker method more than a test method of the accuracy and reliability.2 The study of the extraction and separation of total flavonoids fractionAccording to the single factor experiment and L9(34)orthogonal experiment,studied the concentration of extraction solvent,extraction time,extraction times and ratio of material to liquid has influenced on the extraction yield of total flavonoids,and process validation,in order to optimize the extraction process of total flavonoids in the medicinal,again according to the condition of extraction process,through the chemical system solvent method to remove total polysaccharides,total tannins and other categories of ingredients,further separation and purification of total flavonoids,tested by HPLC method,calculated by area normalization method content,in order to obtain high purity of the refined flavonoids.3 The study of characteristics of fingerprintBy optimizing the extraction technology of total flavonoids extraction and separation,and according to the application condition of quantitative analysis of multi-components by single marker of the total flavonoids analysis method,set up characteristic fingerprint of total flavonoids fraction in Guizhou's Smilax glabra Roxb.(red of cross section),by researching methodology and using similarity analysis,to generate the fingerprint of model,determine the feature of the fingerprint,and combine the quantitative analysis of multi-components by single marker analysis methods,further all-round and multi-level control the immanent quality of Smilax glabra(red of cross section)total flavonoids fraction.4 The study of spectrum-effect relationshipAccording to the condition of characteristics of fingerprint of Smilax glabra total flavonoids fraction,choose several representative samples,according to the extraction method for extraction and separation,put the RA-HFLS cells as an in vitro cell culture model,using the detection method by MTT,studied the groups of drug effects on RA-HFLS cell proliferation.Through the hierarchical clustering analysis method(HCM),partial least-square regression analysis method(PLS)and stepwise regression analysis method(SRM),to establish production Guizhou's Smilax glabra Roxb.(red of cross section)of the total flavono ids fraction spectrum-effect relationship,and further defined the correlation of resistance to RA-HFLS cell proliferation.The research results and analysis1 The study of quantitative analysis of multi-components by single markerPut the astilbin as a reference object,the relative correction factor(RCF)for engeletin and 5-O-caffeoylshikimic acid to astilbin,were the 1.2843 and 1.0605,through relative correction factor durability of the inspecting and positioning of the chromatographic peak of target component,the results RSD are less than 3%,suggest that the durability of relative correction factor and good reproducibility of the relative retention time ratio,conform to the research requirements of quantitative analysis of multi-components by single marker.Through the contents determination of engeletin and 5-O-caffeoylshikimic acid by one point external standard method,external standard calibration curve method and quantitative analysis of multi-components by single marker method.Results both the average percentage of 1.041%and 0.3361%,there was no significant among three different analysis methods,the relative deviation of 5-O-caffeoylshikimic acid were 1.76%to 4.29%and the engeletin were 2.64%to 4.74%,less than 5%;RSD were 0.65%to 3.63%and 1.09%to 2.61%,less than 4%.The results show that,by HPLC analysis method,the method of quantitative analysis of multi-components by single marker is accurate,reliable and repeatability,achieve the goal that lots of targets control the immanent quality of the medicinal herbs effective parts of total flavonoid by three active ingredients.2 The study of the extraction and separation of total flavonoids fractionThrough the study extraction process,found the best extraction technology conditions of total flavonoids in Guizhou' s Smilax glabra Roxb.(red of cross section),namely the concentration of ethanol is 70%,the extraction time is 1.5 hours,extraction for 3 times,and the ratio of material to liquid is 1:8(g/mL),and through the process validation.Results show that the selected parameters are reasonable of orthogonal experiment,higher extraction yield,the process is relatively stable.In major ingredient astilbin peak area of total flavonoids,its content accounted for 59.8%of the total,with the peak area of main components total flavonoids,its content accounted for 94.2%of the total,the results show that the fraction was the highly purified total flavonoids,can be used for pharmacodynamics experiment.3 The study of characteristics of fingerprintEstablished fingerprint of total flavonoids fraction and medicinal materials in Smilax glabra Roxb.(red of cross section)for 17 producing areas,and generate the characteristic fingerprint common model of medicinal materials and total flavonoids fraction,confirmed the 10 common peak of medicinal materials and 10 common peak of total flavonoids fraction;through the similarity analysis and relative retention time of the inspection,the similarity were greater than 0.900 of Smilax glabra Roxb medicinal materials and the total flavonoids for 17 producing areas,the RSD of relative retention time is less than 3%,accord with the requirement of characteristic fingerprint analysis.Through combining the characteristic fingerprint technology with quantitative analysis of multi-components by single marker method,with qualitative investigation by characteristic fingerprint technology,with quantitative analysis by quantitative analysis of multi-components by single marker method,mutual benefit,both the immanent quality of joint control effective fraction(total flavonoids fraction)of Guizhou's Smilax glabra Roxb.(red of cross section),meet the index component content determination and more reflect the overall Chinese medicine chemistry information request,a more comprehensive scientific evaluation Smiax glabra Roxb.effective fraction of the inner quality.4 The study of spectrum-effect relationshipAccording to the results of the characteristics fingerprint information and resistance to RA-HFLS pharmacodynamics experiment cell,found the most relevant efficacy phytochemical is mainly X2,X4,X5 and X8,established the spectrum-effect relationship of Guizhou's Smilax glabra Roxb.(red of cross section)total flavonoids fraction,according to the experimental results,further extraction and separation of the phytochemical,and efficacy of experimental verification,further clarify the RA corresponding efficacy material base,for the production resistance to RA new drug research of Guizhou's Smilax glabra Roxb.(red of cross section)and development of prototype laid the foundation research.According to the study rusults of spectrum-effect relationship of Guizhou's Smilax glabra Roxb.(red of cross section)total flavonoids fraction,in the most relevant to the four medicinal ingredients that resistance to RA-HFLS cell proliferation,there are three chemical composition is the major ingredients of quantitative analysis of multi-components by single marker method,the results further show that the quantitative analysis of multi-components by single marker method more than a effective test method,the method not only more index content determination of control the immanent quality of Guizhou's Smilax glabra Roxb.(red of cross section),but also it is the study of quality control that based on in pharmacodynamics experiment,achieve ther research goal that according to the efficacy material base,comprehensive control of Guizhou's Smilax glabra Roxb.(red of cross section)internal quality.