Study On The Quality Control Of Triterpenoid Acids In Guizhi Fuling Capsule

Guizhi Fuling Capsule(GFC)is a traditional Chinese medicine preparation,which is made up of Ramulus Cinnamomi,Cortex MouTan,Persicae Semen,Radix Paeuniae Rubra and Poria cocos.It has the effect of activating blood,removing blood stasis and eliminating the disease.Modern pharmacological studies have shown that the triterpenoic acid is one of the most important active constituents in GFC,which has anti-inflammatory,immuno-modulatory and anti-tumor effects.Since the triterpenoic acid can affect the clinical efficacy of GFC,it is necessary to control the quality of the triterpenoic acid.Poria cocos is the sclerotiorum of Poria cocos(Schw.)WoIf(Polyporaceae)having the effects of excreting dampness and diuretic,as well as tonifying spleen and calming the mind.It is an edible and medicinal fungus that exists richly in our country.This study first separated different kinds of triterpenoic acid in Poria cocos via leveraging systematically chemic methodology,and then constructed the fingerprint of the triterpenoic acid inGFCby using the LC-MS technique.Moreover,this study investigated the method of quantitative analysis of the triterpenoic acid and finally determined the quality control method for the triterpenoic acid inGFCwith high sensitivity and selectivity.By utilizing chromatography and spectroscopy,the study successfully ex-tracted an d purified seven kinds of triterpenoic acid from the ethyl acetate distilled in the Poria cocos with 95%ethanol,including 3-epi-benzoyloxyl-dehydrotumulosic acid,poria aci d,dehydropachymic acid,dehydrotumulosic acid,dehydroeburicoic acid,polyporenic ac id C,and dehydrotraMetenolic aci-d.Then,these monomer compounds extracted above were further used for comparison in the quality control process of the triterpenoic aci d in GFC.Study on the quality control method of the triterpenoic acid in GFC:The first part is the study of fingerprints.It is the first time to use UPLC/Q-TOF MS for constructing the fingerprint of the triterpenoic acid in GFC.The analytical columns are Agilent RRHD SB-C18 chromatographic columns(2.1×100 mm,1.8 μm),together with acetonitrile-0.1%formic acid water as the fluxion phase for gradient elution,velocity of flow as 0.2 ml·min-1 and the column temperature as 25℃.The study established fingerprints for the triterpenoic acid coming from 10 different batches of GFC,which preserves a high precision,repeatability and stability.The similarity analysis was carried out by using the Similarity Evaluation System for Chromatographic Fingerprint of TCM,the similarity among the 10 batches of GFC varies from 0.978 to 0.998.According to the relevant technical requirements and the practical situations of fingerprint,26 common peaks were identified.Finally,by using UPLC/Q-TOF MS to analyze their structures and comparing with the reference substances,16 common peaks were identified totally,all of which are the triterpenoic acid.In addition,the correlation analysis on these common peaks of fingerprints showed that they all came from Poria cocos.The second part of this study is the quantitative analysis of the triterpenoic acid in GFC.This study,for the first time,established the method of measuring all six triterpenoid constituents in GFC at the same time by using UPLC-MS/MS with the Agilent Porosheell 120 SB-C18 column(4.6×150 mm,2.7 μm)and methanol-0.1%HCOOH aqueous solution for gradient elution.The velocity of flow of gradient elution is 0.3 ml·min-1,the column temperature is 40℃,and the injection volume is 5μl.Through the investigation of linear relation,precision,repeatability and stability,The results showed that the method is proved to be stable and reliable.the average recovery rate of six kinds of triterpenoic acid were 97.9%,100.2%,99.6%,106.7%,102.7%and 103.0%,respectively.Based on the study of the chemical constituents of Poria cocos,this paper obtained the monomers of the different kinds of triterpenoic acid from Poria cocos,which were applied to the quality control of the triterpenoid acid in GFC.This paper for the first time utilized the LC-MS technique,and proposed a method to determine the contents and construct their fingerprints of the triterpenoic acid in GFC,which was proved accurate,rapid,sensitive and good on selectivity.Moreover,it realized the qualitative and quantitative analysis of the triterpenoic acid in Poria cocos in the prescription preparation of Chinese medicine,which provides a reference method for the quality control of GFC.