Study On The Preparation Process And Quality Standard Of Effective Part From Hedyotis Diffusa Willd

hedyotis diffusa is a dry herb that belongs to hedyotis L,Rubiaceae.It was included in Appendix Three of Chinese Pharmacopoeia,2010 version,which contains the medicinal materials and processed products that are not included in prescription preparation.It is a common clinical medicine because of its curative effect,which can be used for clearing heat,detoxification,diuresis and detumescence.Study on the material foundation shows that flavone and organic acid,included in hedyotis diffusa,are active ingredients for antitumor.But currently,there are few studies on preparation technology of the effective part of hedyotis diffusa.This thesis did a preliminary study on the quality standard of effective parts and midbody,try to establish best preparation process of the effective parts of hedyotis diffusae.Details are as follows:ObiectiveFurther studied antitumor medicine hedyotis diffusae,which is commonly used in clinical:Isolated and purified its active part(flavonoids and organic acids).Quantitative study on effective components and made fingerprints,preliminarily established the quality standards of effective fractiion intermediates.Method1.LEstablished determination method of total flavonoids,total triterpene acids and total phenylpropanoids organic acids inhedyotis diffusa by spectrophotometry.2.To Optimization extraction method by studying on the extraction effect of hedyotis diffusa effective components by methanol ultrasonic extraction,alkali water reflux extraction,ethyl acetatere flux extraction and ethanol reflux extraction respectively.Single factor investigated the effect of total flavonoids and total organic acids by the rate of extract effective Components.To study the effect of solvent concentration,solvent volume,extraction times and extraction time on Extraction of total Flavone and total organic acids in hedyotis diffusa By the L9(34)orthogonal experiment.therefore,the optimum process conditions were made.3.Investigated the static adsorption and resolution rate of the effective components by the purity ofhedyotis diffusa effective parts(total flavonoids and organic acids)as indicators,optimized the best resin that purify hedyotis diffusa effective parts;Single factor investigated related parameters that enrichment of effective parts by the resin respectively,therefore developed the best purification process.4.To determine the contents of total flavonoids and organic acids in hedyotis diffusa effective parts by UV spectrophotometry.Determination method of oleanolic acid,ursolic acid,p-coumaric acid and rutin were established by high performance liquid chromatography(HPLC).Quality analysis of five producing area hedyotis diffusa by established technology and methods of cotent determation.Single factor investgated the effect of column,the mobile phase systems,detection wavelength and column temperature on fingerprinting.Established fingerprint of the effective parts intermediates.Results:1.The establishment of determination method of the effective components in hedyotis diffusaeBy spectrophotometry,the determination methods of total flavone,three terpene acid,phenylpropanoids and other organic acid in hedyotis diffusae are established respectively.Specific methods are:(1)determination of content of total flavonoids:retrieve sample ethanol extract to 10 mL volumetric flask,fill in 70%ethanol to 5 mL,add 0.4 mL 4%sodium nitrite,let stand for 6min,then add 10%aluminum nitrate 0.4 mL,let stand for 6min,add 4 mL 4%sodiumhydroxide,set the volume at scale with 70%ethanol,let stand for 15 min.Take the same determination methods of 70%ethanol without specimen as blank control,assay the absorbance at 510 nm.(2)The determination of the content of triterpenes and organic acids:take sample liquid to 10 mL centrifuge tube with plug,valatilize and dry it with 70 ? water,add 0.2 mL new prepared 5%vanillina-glacial acetic acid,0.8 mL perchloric acid,seal up,then put it in 70 ? water bath and coloration for 15min,take out.Put it in ice-water bath to refrigerate for 5 min.then add 5 mLglacial acetic,take the same coloration methods of anhydrous methanol without specimen as blank control,put it in spectrophotometer and test the absorbance at 510 nm;(3)The determination of the content of phenylpropanoids and other organic acid:Take some samples to 10 mL volumetric flask,make it to scale with anhydrous methanol.Take anhydrous methanol as blank control,test the absorbance of the samples at 320 nm.2.Study on the extraction technology of effective parts of hedyotis diffusaTake the extraction rate of the effective components as index,using single factor investigation to inspect the causes of extraction the total flavonoids and total organic acid in hedyotis diffusa Winid.then use,9(34)orthogonal experiment to study these four factors,extraction of ethanol concentration,solvent volume,extraction frequency and extraction time,on the extraction of total flavonoids and total organic acid from herba hedyotis diffusae.The optimum process conditions were determined:take coarse powder from Oldenlandia diffusa,adding 20 times amount of 70%ethanol and reflux extraction 1.5h,filtration,add 10 times amount of 75%ethanol to the medicine residue and reflux extraction 1.5h,merge filtrate,decompress and retrieve ethanol.3.Study on the purification technology of effective parts of hedyotis diffusaTake the purity of the effective part of Oldenlandia diffusa(total flavonoids and total organic acids)as index,by studying on the static adsorption and desorption rate of effective components,select theh macroporous adsorption resin as the best resin to purify effective components ofherba hedyotis diffusae.Take h resin as the medium and single-factor study the related parameters when effective parts concentrated.The optimum purification conditions are determined as follows:(1)The preparation of sample solution:Acid-base Precipitation Centrifugal method.(2)The technological conditions of resin purification:take sample solution,adjust pH to 5.0,add sample as per 0.6g.mL-1?let stand for 1h,wash it with deionized water until the effluent is clear and pure,adjust ph to7 with ammonia and elute by 70%ethanol.Collect the eluant according to the ribbon,till the yellow ribbons were eluted.The specific purification parameters are as follows.The ph of sample solution is 5,the sample volume is not more than 0.6g resin/mL,the sample concentration is 0.4g crude drug/ML,the sample flow rate is 1.5-2BV/h,elution flow rate is 1.5-2BV/h.And adjust the Ph of eluent to 7 with ammonia water,then elute by 70%.alcohol.4.Preliminary study on the quality standard of effective parts of Oldenlandia diffusaUsing ultraviolet spectrophotometry to determine the content of total flavonoids and total organic acid in effective parts of herba hedyotis diffusae,and the total flavone and total organic acid is tentative fixed at not less than 50%;Then content determination methods for oleanolic acid and ursolic acid,p-coumaric acid,rutin,were established respectively by using high performance liquid chromatography(HPLC).(1)The content determination methods for rutin by HPLC:the krosmial C18 is chromatographic column,the detection wavelength is 320 nm,column temperature(T)is 30 ?,the injection volume is 10? L,flow rate is 0.6 mL/min,take methanol(A):0.5%acetic acid(B)as mobile phase,gradient elution is 0-20min:45-55%(A)?20-22min?55-45%(A);(2)The content determination methods for p-coumaric acid by HPLC:the krosmial C18 is chromatographic column,the detection wavelength is 322 nm,column temperature(T)is 30?,the injection volume is 10? L,take acetonitrile(A):0.1%glacial acetic acid(B)as mobile phase,the gradient elution is 0-5min:20-24%(A)?flow rate is 0.8-0.6 mL/min,5-20 min:24-26%(A),flow rate is 0.6-0.6 mL/min.20-22 min:26-20%(A),flow rate is 0.6-0.8 mL/min.The content determination methods are simple,accurate,reproducible,providing a quantitative basis for the comprehensive evaluation for quality of effective parts of hedyotis diffusa Willd.5.Study on the fingerprint of effective parts of hedyotis diffusaAfter studying the influence of chromatographic column,the mobile phase system,detection wavelength and the column temperature on fingerprint separately,the optimum chromatographic conditions are:the krosmial C18 is chromatographic column,the detection wavelength is 320 nm,flow rate is 0.7 mL/min,column temperature(T)is 30?,the injection volume is 10?l.Elute with acetonitrile(A)and 0.5%acetic acid(B)in different ratio:0-40min:15-25%(A)?40-60min:25-35%(A),60-62min:35-15%(A).Under this condition,many peaks of the effective parts of herba hedyotis diffusae appear,the resolution of adjacent peak is good,the analysis time is not long,and it is rapid and convenient.On the analysis of ten batches of different sources of hedyotis diffusa effective parts by the conditions above,a total of 14 obvious characteristic peaks are received.Take p-coumaric acid(6th peaks)as the reference peak,calculate the similarity of intermediate of ten batches hedyotis diffusa by chromatographic fingerprint evaluation system based on 2004A software.The result shows that the fingerprint of ten batches of intermediates of effective parts of hedyotis diffusae is of much similarity,the similarity of each batch were higher than 0.8.It indicates that the method of fingerprint is feasible,the prepared samples are of good quality,providing reference for the quality of intermediate of effective.ConlusionTo study preparation technology and quality standard of the antitumor active part in hedyotis diffusa by total flavonoids and total organic acid.,developed the best preparation technology of effective parts in hedyotis diffusa.The content of effective part prepared by the technology of the were no less than 50%,the technology is simple,stable,economic and reasonable,which is well suited for large scale production.Established determination methods of index component in effective parts intermediate,methodology study showed that the establishment of the method uite for the determination of the sample content.Established fingerprint of effective part intermediate,whch provided important reference for quality evaluate of the effective parts intermediate.The research has high academic value and application development,which has great significance on the development of new effective preparation and its industrialization of hedyotis diffusa.