Studies On Quality Evaluation Of Rhei Radix Et Rhizoma And Extraction And Purification Of Anthraquinones

Rhubarb(Rhei Radix et Rhizoma,RRR)is dry root and rhizome of Rheum palmatum L.,Rheum tanguticum Maxim.ex Balf.and Rheum officinale Baill.RRR is one of the most commonly used traditional Chinese medicine in our country.Modern research has shown that RRR play a part of protection and poisonousness in liver and kidney.Therefore,advantages and disadvantages of RRR quality is related to the medication safety and effectiveness.Currently,on the market,quality of RRR is uneven.And the quality evaluation method has certain limitations.Therefore,a comprehensive and scientific quality evaluation methods of RRR is very important to ensure clinical medication safety and effective.Chemical composition in RRR are complex,including anthraquinones(AQs),anthrones,Tannins,butyrophenones and Stilbenes,et al.Studies have shown that AQs play a major role of protection and poisonousness in liver and kidney.AQs in RRR is the material basis of protection and poisonousness in liver and kidney.According to the literature,At present,22 AQs were isolated from RRR the other constituents of up to 22,including 8 Free anthraquinones(FAs)and 14 Bound anthraquinones(BAs).But which one plays a role of protection?Which one plays a role of poisonousness?The relationship between them is unclear.Therefore,extraction and purification of FAs and BAs in rhubarb ingredients for follow-up studies on liver and kidney protection and toxic effect and the poison-effect relationship of substances in rhubarb This paper divided into two parts:the review and experimental research.In part 1,87 references were cited.The literature review in regard to rhubarb chemical components and quality evaluation and present situation on extraction and purification technology research of anthraquinones constituents separately,On this basis,the paper introduces the research ideas and design of the project briefly.In part 2,the experimental study.Include the following three aspects:1、Study on quality evaluation of RRR(1)Study on HPLC fingerprints of RRRTo determine the fingerprint of 22 RRR samples by using HPLC and establish control fingerprints under two wavelength of 280 nm and 430 nm.Under the 280 nm wavelength.There are 41 common peaks in the fingerprint common model.Under the 430 nm wavelength.There are 21 common peaks in the fingerprint common model.Compared with retention time and ultraviolet spectrogram of the reference substance,we have pointed out 20 peaks under the 280 nm.8 BAs(Aloe emodin-8-O-glucopyranoside,Rhein-8-O-glucopyranoside,Emodin-1-Oglucopyranoside,Chrysophanol-1-O-glucopyranoside,Chrysophanol-8-O-glucopyranoside,Emodin-8-O-glucopyranoside,Aloe emodin-3-CH2-O-glucopyranoside,Physcion-8-Oglucopyranoside),5 FAs(emodin,Rhein,Chrysophanol,Aloe emodin,Physcion),4 tannins(Gallic acid,Epigallocatechin gallate,Catechin,Epicatechin gallate),2 stilbenes(Resveratrol-4’-O-glu-copyranoside,Resveratrol-4’-O-β-D-(6"-O-galloyl)-glucopyranoside),1 butyrophenone:4-(4’-hydroxyphenyl)-2-butanone-4’-O-β-D-(2"-O-galloyl-6"-O-p-coumaroyl)glucopyranoside.All of 21 common peaks are anthraquinones constituents under 430 nm.We have pointed out 13 peaks,including 8 BA and 5 FA.(2)Assaying of 5FAs and TAs in RRR by HPLCBy using the statistical package of SPSS Statistics 20 to processe data,we have investigatedthat FA and TA extracted by several times(solid-liquid ratio 0.3:50 and 1:50)instead of one-time extraction(solid-liquid ratio 0.3:50),and we have changed the extraction method by reflux instead of ultrasonic method.The results show that the extraction rate of 5 FA was improved obviously(P<0.01),on the basis of the best material and liquid ratio of 0.3:50,It has no obvious difference between two extraction methods for 5 FA and TA(P>0.05).Five FA and TA of 22 batches of samples were determined and analyzed by the optimized method.(3)Assaying of 8 BAs in RRR by QAMSWe have established relative correction factor and location method of chromatographic peak of the component under test between 7 BA and Rhein-8-O-glucopyranoside,according to internal content of Rhein-8-O-glucopyranoside.On this basis of methodology examination.We have calculated the content in the samples by QAMS.We also have calculated the content of 8BA in 22 batches of samples by external standard method.We have compared difference between observed and calculated values,it is a verification of accuracy of QAMS.The repeatability of relative correction factor that we have established is very well.Chromatographic peaks relative retention values can be used for the positioning of the chromatographic peaks of components under test,There was no significant difference between measured and calculated values of Aloe emodin-8-O-glucopyranoside、Emodin-l-O-glucopyranoside、Chrysophanol-1-Oglucopyranoside、Chrysophanol-8-O-glucopyranoside、Aloe emodin-3-CH2-O-glucopyranoside、Physcion-8-O-glucopyranoside by RCF,the six FA can be used in QAMS with Rhein-8-O-glucopyranoside.There were greater differences between measured and calculated values in allusion to emodin-8-O-glucopyranoside.(4)Assaying of other 7 components in RRRwe have established methods of determination by HPLC that we have determined simultaneous of 7 element,including 4 tannins(Gallic acid,Catechin,Epigallocatechin gallate,Epicatechin gallate),2 stilbenes(Resveratrol 4’-O-glucopyranoside,Resveratrol 4’-O-β-D-(6"-O-galloyl)-glucopyranoside),1 Butyrophenone:4-(4’-hydroxyphenyl)-2-butanone 4’-O-β-D-(2"-O-galloyl-6"-O-p-coumaroyl)glucopyranoside.We have used the methods to get determination of components in 22 batches.The results showed that the other seven ingredients of rhubarb in 22 batch samples has large differences.2、Study on extraction technology of anthraquinones in RRRWe have conducted a test by using the orthogonal table L9(34),there are four investigation factors,such as ethanol concentration,dosage of ethanol,circumfluence times and reflux time.And each factor has three levels.Based on extraction amount of TAs and FAs,we carry on statistical and variance analysis,optimization of process(1)for five times the amount of 75%ethanol,circumfluence extraction,5 times and 30 min each time.Based on extraction amount of BAs,we carry on statistical and variance analysis,optimization of process(2)was 5 times 95%ethanol,circumfluence extraction,5 times and 60 min each time.The results of verification test(1)is that the average extraction yield of prototype anthraquinones and TAs was close to 90%.However,part of BAs had been hydrolyzed to FAs,the average extraction yield of BAs on the right side of 60%,the average extraction yield of FAs is 160%.The results of verification test(2)is that the hydrolysis of BAs has decreased obviously.It shows that the average extraction rate of BAs increased to 84.09%on basis of the average extraction rate of prototype anthraquinones was closed to 90%.And the average extraction rate of FAs had decreased to 101.07%.The two processes of optimization are both stable and feasible.3、Study on purification technology of anthraquinones in RRR(1)Study on separation of FAs and BAs and purification of FAsWe have used HPLC to investigate the result of separation between free anthraquinone and bound anthraquinones in rhubarb by method of alcohol extracting-water precipitating.According to chromatographs in two wavelength(280 nm and 430 nm),we can konow that most of the bound anthraquinones were shifted to supernatant,most of the free anthraquinones were moved to precipitation,Most tannins were shifted to supernatant,such as Gallic acid、Catechin、Epicatechin gallate,all of Stilbenes were transferred to supernatant,such as Resveratrol 4’-O-glucopyranoside.For purification of precipitation,we have used the method that named dynamic two-way extraction.The amount of 20%H2S04 and chloroform is three times and four times amount to medicinal materials.The results show that the purity of free anthraquinone is 92.63%after purification.(2)Study on purification of BAs by modified gelatin method and alcohol solution PH methodWe have purificated BAs by method of modified gelatin and by using alcohol solution PH.For the quantitative and qualitative analysis we have used HPLC then we know that the removal rate of four impurity,such as Gallic acid,Resveratrol-4’-O-glucopyranoside and so on,is 97.91%.However,the removal rate of impurity processed by method of modified gelatin is 6.94%.Comparison of retention rate of BA processed by method of modified gelatin and by using alcohol solution PH is 80.64%and 105,18%.Therefore,by using alcohol solution PH to purificate BAs is superior to method of modified gelatin.But the chromatogram of using alcohol solution adjust PH has an obvious impurity peak.(3)Study on purification of BAs by macroporous resinWe have compared the results of static adsorption and static desorption,dynamic adsorption and desorption test of three kinds of macroporous resin(D101,XAD1180,X-5),with the research object of 8 BAs.The results show that macroporous resin D101 is superior to other ones.we have investigated technological parameter of purification and verification test by use macroporous resin D101.The best concentration、flow rate、dosage of samples are 2 g/mL(equivalent to the original medicinal materials)、2 BV/h and 21 BV.The best concentration and dosage of eluent are 40%ethanol and 45 BV.Results of validation test showed that purity of bound anthraquinones by method of macroreticular resin D101 is 30.04%,content of BA increased more than 10 times.But the chromatogram of thick product has an obvious impurity peak.(4)Study on purification of BAs by joint method of alcohol solution PH and macroporous resinAfter dealing with the method of using alcohol solution adjust PH,we have conducted supernatant by macroreticular resin D101.The purity of BA is 47.56%after purification.Both impurity peak after the individual treatment of macroporous resin and using alcohol solution adjust PH disappear respectively.what we can know is that BA were more pure,those were consistented with the results of content determination.