| Traditional Chinese medicine(TCM),regarded as the material carrier of disease prevention and treatment frequently priscribed by doctors,its quality directly relates to the safety and efficacy of clinical medication.Therefore,the quality control of TCM not only has been the hotspot and difficulty of TCM research,but also has been precondition and important foundation of TCM modernization.The choice of quality control components is the key of TCM quality control.Although extensively used in clinic,the active ingredients of TCM are very complicated and difference in content,so it is very important to choose the appropriate components and research strategies on TCM quality control study for a long time.Epimedium,as one of TCM,are belong to be rberidaceae Epimedium L.and derived from their dry leaf.The China Pharmacopoeia(2015 version)contains four varieties of Epimedium,which are Epimedium brevicornum Maxim.,Epimedium pubescens Maxim.,Epimedium koreanum Nakai.,and Epimedium sagittatum Maxim.,respectively.Epimedium has function of nourishing kidney-yang,strengthening the bones and muscles,and expelling wind-damp.Which mainly were used for the treatment of kidney-yang deficiency,impotence,spermatorrhea,bones and muscles impotent soft,rheumatism pain and numbness in clinical application.The quality control components of Epimedium are icariin and total flavonoids.This research mainly analyzed chemical compositions of Epimedium itself,the absorbed compositions from Epimedium in rat(blood,urine,feces)by UPLC-Q/TOF-MS.Meanwhile,we everted intestinal sac model which was utilized to research absorption process and the characteristics of main ingredients in Epimedium.Furthermore,the method of network pharmacology was used to predict activity of absorbed compounds.In addition,quality control indicators were screened out and the method of quality control based on these indicators was established.The method was used to evaluate the quality of 34 batches of Epimedium.All the results were used to provide references for screening of Epimedium efficacy material and guiding to establish quality control method under their pharmacological activities.The concrete research content is divided into the following several aspects:(1)UPLC-Q/TOF-MS was used to identify chemical compositions of Epimedium itself,to analyze the absorbed compositions from Epimedium in rat and to predicte activity of absorbed compounds by network pharmacology.UPLC-Q/TOF-MS was used to identify chemical compositions of Epimedium itself.Comparing the precise molecular ion weight(△ppm<10)of reference substances and analyzing their fragment ions,we summarized the pyrolysis rule of prenylated flavonoids.In totally,52 compounds were identified based on the pyrolysis rule of prenylated flavonoids,most of which are flavonoids.Furthermore,7 prototype compounds,13 prototype compounds,and 27 prototype compounds were identified in rats’plasma,urine,and feces,respectively.We choosed 13 compounds which were found in rats’ plasma and urine with similar structure.Then,enrichment analysis the pathway of candidate targets which were extracted from drugbank databases and their molecular interaction was carried out to find 35 signaling pathways(P<0.02)related to inflammation,osteoporosis,cancer and other biological function.Meanwhile,we established a network diagram about the relationship of absorption ingredients,targets and their corresponding channel,which revealed that this herb apply a multi-components and multi-targets approach in the treatment of diseases.All results provided a reference for further experimental verification and active ingredients selection.(2)Intestinal absorption ingredients in Epimedium folium and investigation of their absorption characteristics were studied by everted intestinal sac method.We choosed five main flavonoids ingredients,i.e.epimedin A,epimedin B,epimedin C,icariin,icariside Ⅱto study absorption ingredients in vivo through intestinal sac model.Firstly,we put forward the experimental methodology.Then,accumulative absorbed dose(Q)of five flavonoids were calculated,intestinal absorption kinetics of five flavonoids were studied and the proportions of five flavonoids between intestinal absorption liquid and epimedium extract were compared.The results indicated that five flavonoids have the same absorption selectivity in different intestinal segments,with the increase of drug concentration,and Q of five flavonoids reached maximum value inintestinal segments moved from front and middle jejunum segments to ileum segments.The linearly dependent coefficient(R2)of five flavonoids between Q and time were greater than 0.9,which was in consistent with zero order absorption rate.Absorption rate constant(Ka)of five flavonoids increased with increasing of concentration of Epimedium extract,which indicated that intestinal absorption mechanism of five flavonoids were passive diffusion.The proportions of five flavonoids in intestinal absorption liquid were different from those in Epimedium extract,which might be caused by enzyme hydrolysis in intestinal cells and the different absorption characteristics of five flavonoids.(3)The quality control on Epimedium was studied based on multi-ingredients quantification and chromatographic fingerprint.We choosed six main flavonoids ingredients from absorption ingredients of epimedin A,epimedin B,epimedin C,icariin,icariside I and icariside II.The quality control method was established based on multi-ingredient quantification and chromatographic fingerprint by HPLC.The extraction method,type of solvents,solvent volume and extracting time were investigated in our study.Meanwhile,the chromatographic column,mobile phase and other chromatographic conditions were optimized and the methodology of precision,stability,accuracy and repeatability were conformed to the requirement of the Pharmacopoeia.The content of six flavonoids in 34 batchs of Epimedium which involved five breeds were determined and their fingerprints were analyzed.The results showed that the content of icariin was the highest in all samples,and 26 batchs of 34 batches samples showed the content of epimedin C was higher than icariin.From the mean of content,the content of epimedin C was the highest in Epimedium brevicornum Maxim.,Epimedium pubescens Maxim.,Epimedium leptorrhizum Steam.and Epimedium acuminatum Franch.,the content of epimedin B was the highest in Epimedium koreanum Nakai.,and.the content of icariin was the highest in little samples of Epimedium brevicornum Maxim.,Epimedium pubescens Maxim.,Epimedium pubescens Maxim.Moreover,epimedin A,epimedin B,epimedin C,and icariin can be detected in five breeds,icarisid I only can be detected in small part of samples,and icarisid II can be detected in most of samples and its content were lower than those of epimedin A,epimedin B,epimedin C,icariin.The results of fingerprints analysis showed that the similarities of comparison sample for five specieswas in 0.569～0.917,the similarities of comparison sample between Epimedium pubescens Maxim.and Epimedium leptorrhizum Stearn.has the largest similarity of 0.569,and the similarities of comparison sample for Epimedium brevicornum Maxim.,Epimedium koreanum Nakai.and Epimedium pubescens Maxim.was in 0.665～0.910.Above the results showed that larger quality differences were existed in different varieties of Epimedium. |
