Study On The Quality Control Of Rhubarb Based On Biological Activity

Research backgroundRhubarb is the dried root and rhizome of Rheum Palmatum L.,Rheum Tanguticum Maxim.Ex Balf.,or Rheum Officinale Baill.It is the most commonly used bulk Chinese medicinal material in China and mainly distributed in Gansu,Sichuan,Qinghai and other places.Rhubarb,which was first published in Shen nong bencao jing,has a medicinal history of more than 2,500 years in China.It has the functions of relieving accumulation,clearing away heat and fire,cooling blood and detoxification,and driving away blood stasis and passing through the scriptures.It is widely used in treatment of various diseases and syndromes of Internal,External,Gynecological and Pediatric Medicines.Modern researches have shown that rhubarb have the following effects of diarrhoea,antitumor,anti-inflammatory,analgesic,antioxidant,protecting liver,and reduding blood fat and so on.Furthermore,different constituents exert different effects,such as anthraquinone and sennoside are the main purgative ingredients,and polyphenols represented by catechin and gallic acid can expel blood statis.Moreover,it has been reported that rhubarb from different sources has different dominant activity.Previous studies have found that the chemical composition of rhubarb from different sources and different habitats is quite different,and its dominant activity also should be different theoretically.However,in no matter Chinese Pharmacopoeia or the world main herb pharmacopoeia,such as the European Pharmacopoeia,Hong Kong Pharmacopoeia and Japanese Pharmacopoeia,the quality standard of rhubarb only stipulate the anthraquinones or sennoside content without considering the fact that rhubarb has multiply effects and different constituents exert different actions.Therefore,we think that different quality control standards should be established on the different efficacy of rhubarb,so as to contribute to the precision medication in clinic and the full use ofrhubarb resources.PurposeThrough the study of fingerprint-activity relationship and constituent-activity relationship,the active components of Rhubarb that exert the purgative,anti-tumor,anti-inflammatory and antipyretic effects were identified,and then the quality standards for different biological activities of rhubarb were established based on these components,so as to promote the precise medication in clinic.Methods1.HPLC was used to establish the fingerprint chromatography of rhubarb,and the fingerprint data were statistically analyzed to screen out representative samples.2.The effects of methanol extrac of the typical rhubarb samples on the defecation,incidence of diarrhea and small intestine propulsion of ICR mice were investigated,and then the active components of rhubarb were identified by correlation analysis between the chemical components and the activities of the typical samples.3.MTT method was used to investigate the inhibitory effect of 28 batches of rhubarb methanol extracts on human liver cancer HepG 2 cells proliferation,and IC50 value was calculated.Through multivariate statistical analysis,correlation analysis was conducted between fingerprint data of 28 batches of rhubarb and IC50 value of each batch of crude drugs to identify their anti-tumor active components.4.The effects of 28 batches of rhubarb methanol extract on TNF-? secretion of monocyte macrophages(RAW264.7)stimulated by LPS were investigated,and the inhibition rate of TNF-? secretion was calculated.the fingerprint data of 28 batches of rhubarb were correlated with the inhibition rate of TNF-? ecretion by SPSS Pearson correlation analysis,and the anti-inflammatory active components were identified.5.28 batches of rhubarb samples were measured by the color difference instrument.The correlation analysis was conducted between the fingerprint data of 28 batches of rhubarb and the chromaticity values L*,a*and b*of the corresponding batches of crude drugs by the multivariate statistical analysis method.The correlation between the color of rhubarb and its chemical composition was discussed to provide scientific basis for the "color differentiation theory" of rhubarb.6.The antipyretic activity of rhubarb was measured by investigating the inhibitory effect of different polar parts of rhubarb on the increase of body temperature in pharmacological models of pyrexia induced by yeast in rats,UPLC-ESI-Orbitrap-MS/MS technology was used to analyze the chemical components of the active fractions and identify the antipyretic active components of rhubarb.7.A method for the simultaneous determination of 7 anthraquinone glycosides in rhubarb was established by HPLC,including Aloe-emodin-8-O-?-D-glucoside,Rhein-8-O-?-D-glucoside,Emodin-1-O-?-D-glucoside,Chrysophan-1-O-?-D-glucoside,Chrysopha nol-8-O-?-D-glucoside,Emodin-8-O-?-D-glucoside and Physcion-8-O-?-D-glucoside.Usi ng Catechin as a control,The content of condensed tannin in rhubarb was determined by vanillin sulfuric acid method.Results1.HPLC was used to establish the fingerprint of 28 batches of rhubarb,and 28 common peaks were selected.The structure of 25 common peaks was speculated by LC-MS/MS,and 15 common peaks were identified by comparison of reference substances,including 7 anthraquinone glycosides,5 free anthraquinones,sensorineoside A,gallic acid and catechin.The contents of these 15 componets were determined under the same chromatographic condition.28 batches of samples were divided into two groupsaccording to the relative similarity index(RSI)between the fingerprint of each sample and the control fingerprint,with the RSI?0.88 for Group A and RSI<0.88 for Group B,The total content of glycosides(7 anthraquinone glycosides+senatosine A)in group A was significantly lower than that in group B.By PLS-DA analysis,typical samples of group A and Group B were selected as S10 and S22,respectively.The total anthraquinone content of S10 and S22 samples was 1.508%and 1.602%,respectively,by using 2015 edition pharmacopoeic method.However,the total anthraquinone glycosides content of S10 and S22 samples was far from each other,the former was 0.432%and the latter 1.229%.This result was consistent with the fingerprint analysis and content determination results determined by our own method.2.ICR mice were administered with 70%methanol extract from S10 and S22 samples.The results showed that both S10 and S22 showed laxative activity,but S22 had stronger laxative effect.As previously known,the difference mainly lies in the significantly higher content of anthraquinone glycosides in S22 sample than S10 sample,so anthraquinone glycosides are the active ingredients of rhubarb.3.Of the 28 batches of rhubarb,only 17 batches could calculate the ICso value,which ranged from 0.513 to 4.386 mg·mL-1.The statistical analysis results showed tha t there is a strong correlation between the antitumor activity of rhubarb is closely rela ted to and the Emodin-1-O-?-D-glucoside(16th peak),moderately related to seven co mponents including 1-O-galloyl-2-O-cinnamoyl-glucose/1-O-galloyl-6-O-cinnamoyl-glucos e(13th peak),1-O-galloyl-6-O-cinnamoyl-glucose/1-O-galloyl-2-O-cinnamoyl-glucose(15th peak),Chrysophanol-1-O-?-D-glucoside(17th peak),Chrysophanol-8-O-?-D-glucosi de(18th peak),Physcion-8-O-?-D-glucoside(21th peak)and Aloe-emodin(22th peak),a nd weakly correlated to ten components,including 3 tannins,2 anthraquinone glycosid es,1 acyl glycoside,1 unknown compound,and Senatosine A,Emodin and Physcion.4.ELISA results showed that only S1,S3 and S7 of 28 batches of rhubarb had inhibitory effect on LPS-induced RAW264.7 cell secretion of TNF-? to a certain extent,but the inhibitory activity was weak,with the maximum inhibitory rates of 20.8%,33.7%and 8.8%,respectively.The results showed that the anti-inflammatory activity of rhubarb was very weak under this model.5.The results showed that the best fit between the chemical composition and the color index a*(red and green)was obtained,and the curve fitting between the chemical composition and the color index b*,L*was not ideal.Therefore,the change of related components can be explained by the change of a*value of color index.Among them,Catechin,Procyanidin B-1,3-O-gallate,Procyanidin B-2,3,3'-di-O-gallate,(-)epicatechin-3-O-gallate,Rhein-8-O-?-D-glucoside and Physcion-8-O-?-D-glucoside with color index a*were fitted well,and the sig.Value of variance test was less than 0.05.The regression results were statistically significant,which could better reflect the relationship between them.According to Pearson correlation coefficient correlation analysis results,the color index a*(red and green)of rhubarb has a medium intensity correlation with the above six components,and all of them are positive correlation,indicating that the higher the content of these components is,the redder the color of rhubarb is.6.The results showed that the n-butanol extraction fraction and the remaing water fraction of rhubarb ethanol extract were the antipyretic active parts.By LC-MS/MS analysis,33 compounds were inferred from the n-butanolextract,including 10 tannins compounds(a gallic acid,six gallic acid glucoside,catechins,12 condensed tannins),15 anthraquinone glycosides,four anthrone glycosides,1 naphthalene glycoside,a phenolic glycoside,1 anthraquinone and 1 sucrose,among them 10 compounds were identified by comparison to the reference substances.A total of 12 compounds,including 5 gallic acids and their glycosides,6 anthraquinone glycosides and 1 sucrose,were deduced from the remaining water soluble fraction and all of which overlaps the compounds from n-butanol fraction.7.The total content of 7 anthraquinone glycosides in 28 batches of rhubarb is be tween 0.539%and 2.577%,of which the content range of Aloe-emodin-8-O-?-D-glucos ide is 0.036-0.403%and Rhein-8-O-?-D-glucoside 0.005-1.316%,Emodin-1-O-?-D-gluc oside 0.005-0.141%,Chrysophan-1-O-?-D-glucoside 0.050?0.355%,Chrysophan-8-O-?-D-glucoside 0.113-1.064%,Emodin-8-O-?-D-glucoside 0.165?0.436%,Physcion-8-O-?-D-glucoside is 0.038?0.243%.The content of condensed tannin in 28 batches of rhubar b medicinal materials ranged from 3.631%to 16.717%.Conclusion1.Anthraquinone glycosides are the main active ingredients for rhubarb to play a cathartic role.Anthraquinone glycosides and tannins are the main active components of rhubarb for its antitumor and antipyretic effects.The higher the content of anthraquinone glycosides and tannins,the darker the color of rhubarb.In terms of the cathartic effect,the reddish-brown rhubarb is better than that of yellow color.2.In the identification of Chinese medicine active ingredients by using fingerprint-activity relationship,the number of identified active ingredients largely depends on the number of common peaks in the fingerprint.Therefore,mass spectrographic fingerprint should be adopted so as to identify more active components.