| Rhubarb is derived from the dry roots and rhizomes of Rheum palmatum L.,Rheum taguticum Maxim.ex Balf and Rheum officinale Baill.Raw piece of rhubarb is bitter and cold,showing the strong effect of purgation,and used for treatment of constipation in clinic.Compared with raw piece of rhubarb,steamed piece of rhubarb presents weak purgative effect and is applied for promoting blood circulation and removing blood stasis in clinic.The different in pharmacological effects between raw and steamed pieces of rhubarb may cause the changes in chemical compounds between them.In recent years,many scholars have carried out research on the properties theory of Traditional Chinese medicine and processing related to medicinal properties.In the previous study,the basic law of the change in purgative effect with bitter cold has been proved from the macroscopic point in the view of medicine functional property.There was not published about medicinal orientation property of rhubarb.Orientation property is also one of the theories of properties theory of Traditional Chinese medicine,including viscera,main and collateral channels and so on.Rhubarb went into qifen,used for treating stagnation of the circulation of vital energy and focused on intestinal tract.Therefore,rhubarb was mainly used to attack,treat constipation in clinic.Rhubarb went into xuefen,removed blood stasis and blood-heat,decompressed of solid heat in stomach and removed stagnant blood in the body.Steaded rhubarb was used to traet blood stasis disease.In order to determine the basic theory of the change in medicinal orientation property between raw and steamed rhubarb,and the relationship with qifen xuefen.The study was supported by National Natural Science Foundation of China.Based on the results of study on the material basis and different biological activity of rhubarb,the differences of pharmacokinatics behavior between them are explored in the constipation and blood stasis models by pharmacokinetic method.By analyzing the differences in the absorption and distribution of components in raw and cooked rhubarb,it revealed the scientific connotation of medicinal orientation property of raw and steamed rhubarb,explore the relationship between medicinal orientation property and qifen.The study provides a scientific basis for revealing the difference in efficacy of raw and steamed rhubarb,broaden the research ideas of science of processing Chinese materia medica.1.Chemical composition characterization of raw and steamed pieces of rhubarbQualitative analysis of the chemical constituents between raw and steamed rhubarb were identified by pattern recognition technology of RRLC-QTOF/MS.It was rapidly used to extract the information of all the compounds directly from the total ion chromatorgraphy(TIC)by the molecular feature extraction method for the collected the qualitative spectral data of raw and steamed pieces of rhubarb.Furthermore,73 marker compounds are identified that caused the difference in chemical composition between raw and steamed rhubarb by one-way analysis of variance method with the peak area of component as variable.By analyzing the fragment rule of the reference substance,the characteristic fragment ions of various components were summarized and identified and 73 chemical component markers were identified,including 27 anthraquinones,11 phenylbutanones,16 stilbenes and 19 other compounds.The result indicated that there are obvious differences between raw and steamed rhubarb.Meanwhile,anthraquinones play an important role in the indentified compounds.The pharmacokinetic parameters of rhubarb were analyzed scientifically and accurately.In this study,UPLC-DAD method was established for the quantitative analysis of chemical constituents in raw and steamed rhubarb.For the established method,the recovery,specificity,repeatability,accuracy and precision of the method are accord with requirements of determination.The contents of ethyl gallate(1),aloe-emodin-8-O-?-D-glucophyranoside(2),lindleyin(3),rhein-8-O-?-D-glucophyranoside(4),isolindleyin(5),emodin-8-O-?-D-glucophyranoside(6),4-(4'-hydroxyphenyl-2-butanone-4'-O-?-D-(2"-O-galloyl-6"-O-p-coumaroyl-glucophy ranoside(7),aloe-emodin(8),rhein(9),emodin(10),chrysophanol(11),physcion(12)were determined in raw and steamed rhubarb.The results of content were 0.99%,0.23%,5.46%,1.81%,7.73%,0.29%,1.69%,0.05%,0.66%,0.19%,0.12%,0.36%in raw rhubarb and 0.03%,0.18%,2.49%,1.76%,2.89%,0.14%,0.12%,0.13%,1.29%,0.23%,0.21%,0.55%in steamed rhubarb.After processing,the content of anthraquinone glycosides and stilbene glycosides are all decreased in steamed piece of rhubarb.Meanwhile,the content of anthraquinone aglycone is increased in steamed piece of rhubarb.This part of work is helpful for the subsequent comparison of pharmacokinetic differences between raw and steamed rhubarb.2.Pharmacokinetic study in normal rats of raw and steamed pieces of rhubarbBy UPLC-MS/MS method,the analysis method was estiblishend and determined the contents of aloe-emodin-8-O-?-D-glucophyranoside,emodin-8-O-?-D-glucophyranoside,rhein-8-O-?-D-glucophyranoside,rhein,aloe-emodin,emodin,chrysophanol and physcion in plasma and tissues of rats after administration of raw and steamed pieces of rhubarb.The methodological results show that the UPLS-MS/MS method has good specificity,high sensitivity,accuracy,precision,recovery and matrix effect.Meanwhile,the method are also suitable for the basic requirements of biological sample analysis methods in non-clinical pharmacokinetics.Raw and steamed pieces of rhubarb extracts are given by gavage in normal rats.The plasma and tissues are collected at different times in normal rats.The concentration of anthraquinones was determined after oral administration at different times.The concentration of anthraquinones was determined after oral administration at different times.According to the concentrations of components in the plasma sample,the pharmacokinetic parameters of each component were calculated by using the non-compartment model.The time to peak(Tmax)was faster in steamed rhubarb than in raw rhubarb.The plasma concentration(Cmax)and bioavailability(AUC0-24)of rhein-8-O-?-D-glucophyranoside were higher in raw piece of rhubarb than in the steamed piece of rhubarb.The lower clearance(CLz/F)indicated rhein-8-O-?-D-glucophyranoside showed higher absorption and bioavailability.As the same method,rhein and aloe-emodin were also higher bioavailability.It is speculated that it may be the potential medicinal substance basis of rhubarb.The in vivo retention time(MRT0-24)of chrysophanol,emodin and physcion was longer in steamed rhubarb.There were no significant differences between raw and steamed rhubarb for the concentration(Cmax)and area under the curve(A UC0-24)between raw and steamed rhubarb.The result indicated that there was no significant differences for the bioavailability of the three compounds between raw and steamed rhubarb or showed higher bioavailability in steamed rhubarb.The concentrations of components in the tissues were determined at three different time points(15min,60min,4h)after administration of raw and steamed rhubarb extracts with established method.The lower contents of rhein-8-O-?-D-glucophyranoside,rhein,aloe-emodin,emodin,chrysophanol,physcion and the higher contents of glucuronic acid conjugate indicated indicated the components were biotransformated in vivo.The compounds in raw and steamed rhubarb were mainly distributed in the stomach,large intestine and colon.The result of the distribution indicated the distribution of different components in the tissue targeted.This experiment provided the experimental basis for further study under different pathological models3 Pharmacokinetic study in constipation rats of raw and steamed pieces of rhubarbBy UPLC-MS/MS method,it is applied to determine the contents of anthraquinone aglycone,anthraquinone glycosides and glucuronic acid conjugation of anthraquinone aglycone in plasma and tissues in constipation rats after administration of rhubarb.To establish method for quantification of glucuronic acid conjugates of anthraquinone glycoelements in samples,the pretreatment steps of enzymatic hydrolysis of anthraquinone glucuronic acid metabolites was applied.The methodological results show that the UPLS-MS/MS method has good specificity,high sensitivity,accuracy,precision,recovery and matrix effect.Meanwhile,the method are also suitable for the basic requirements of biological sample analysis methods in non-clinical pharmacokinetics.By UPLC-MS/MS method,the concentration of anthraquinones was determined in constipation model rats after oral administration at different times.According to the concentrations of components in the plasma sample,the pharmacokinetic parameters of each component were calculated by using the non-compartment model.The higher clearance(CLz/F)in steamed rhubarb than in raw rhubarb indicated that the elimination rate of components in vivo is accelerated and accumulation time of components in vivo were reduced.Based on the result of contents in raw and steamed rhubarb,higher concentration(Cmax),area under the curve(AUC0-24)and lower clearance(CLz/F)in raw rhubarb indicated that rhein-8-O-?-D-glucophyranoside showed higher bioavailability in raw rhubarb.Meanwhile,the higher in vivo retention time(MRT0-24)and area under the curve(AUC0.24)indicated rhein and aloe-emodin had higher bioavailability in raw rhubarb.Meanwhile,same conclusion were obtained for rhein,aloe-emodin,emodin,physcion glucuronic acid.As reported,rhein-8-O-?-D-glucophyranoside and aloe-emodin were related with purgative activity of rhubarb,indicated that rhein,aloe-emodin,rhein glucuronic acid and aloe-emodin glucuronic acid maybe pharmacodynamic material basis of purgative activity for rhubarb.The results of tissue distribution in the constipation model rats,there is still a high concentration of each component in the tissue after administration of rhubarb.Combined with the result of half time(t1/2),indicated the elimination of component was slower in vivo.The results of tissue distribution showed that rhein,emodin,aloe-emodin,chrysophanol,physcion and rhein-8-O-?-D-glucophyranoside are mainly distributed in colon and large intestine.Meanwhile,aloe emodin glucuronic acid is mainly distributed in the colon and rhein glucuronic acid is distributed in multiple tissue.The above results indicate that,in the constipation model,the distribution of rhubarb in tissues was mainly distributed in the colon.It is proved that the purgative activity of rhubarb was produced by distributing in the colon.4.Pharmacokinetic study in cold coagulation and blood stasis rats of raw and steamed pieces of rhubarbBy UPLC-MS/MS method,it is applied to determine the contents of anthraquinone aglycone,anthraquinone glycosides and glucuronic acid conjugation of anthraquinone aglycone in plasma and tissues in constipation rats after administration of rhubarb.Raw and steamed pieces of rhubarb extracts are given by gavage with cold coagulation and blood stasis model model in rats.The plasma and tissues are collected at different times in cold coagulation and blood stasis model model.In the model rats with cold coagulation and blood stasis,rhein,aloe-emodin,emodin,chrysophanol and corresponding glucuronic acid conjugation,physcion and rhein-8-O-?-D-glucophyranoside maybe the pharmacodynamic material basis of activating blood circulation and removing blood stasis for steamed rhubarb.The results of tissue distribution showed that all the compounds are distributed in the intestines.Aloe-emodin glucuronic acid conjugate and rhein-8-O-?-D-glycoside are mainly distributed in spleen.Rhein and rhein glucuronic acid conjugate are distributed in plasma.Aloe-emodin,emodin and emodin glucuronic acid conjugate are distributed in liver.Compared with the result in constipation model rats,the distribution in tissue of the above compounds was different in the model rats with cold coagulation and blood stasis.It is indicated the distribution in tissues of components is orientational,revealing that rhubarb can play the role of promoting blood circulation and removing blood stasis by distributing in targeted tissues.The results indicated that components in raw and steamed pieces of rhubarb mainly distributed in colon with constipation model in rats.Meanwhile,components in raw and steamed pieces of rhubarb mainly distributed in liver,spleen and plasma with cold coagulation blood stasis model in rats.Therefore,The distribution of compounds in raw and steamed pieces of rhubarb presented targeting in tissues with different pharmacological models,further led to the difference in medicinal orientation property of raw and steamed pieces of rhubarb.Rhubarb went into qifen,distributed in colon and showed the purgative activty.Rhubarb went into xuefen,distributed in liver,spleen and showed promoting blood circulation to remove blood stasis.The above result revealed the scientific connotation of differences in medicinal orientation property between raw and steamed pieces of rhubarb.The difference of orientation for raw and steamed rhubarb may be the important reason for the different efficacy.This study explores the scientific connotation of the processing principle from the perspective of medicinal orientation property,enriched the research methods of principle of medicine processing,provided a loan for the research on the processing of other Chinese medicine. |
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