Study On The Chemical Constituents Of Feiyangchangweiyan Capsule And The Mechanism Of Its Main Component Of Gallic Acid On Experimental Gastric Ulcer

1BackgroundGastric ulcer（GU）is one of common gastrointestinal disease that seriously affects the life quality of people in China.It belongs to the category of"stomach pain"in traditional Chinese medicine theory.Our traditional Chinese medicine has rich theoretical foundation and clinical experience for treatment on epigastric pain syndrome.Feiyangchangweiyan Capsule（FY Capsule）is prepared from medicinal plants such as Feiyangcao,Huotanmu and Jiubibing through extraction and other processes and has the functions of purging fire,detoxifying,removing dampness and relieving pain.It has been approved by the National Medical Products Administration and produced by Shaanxi Junbisa Pharmaceutical Co.,Ltd.It is mainly used in the treatment of bacterial dysentery,acute and chronic gastroenteritis.Like the challenges faced by most traditional Chinese medicine prescriptions,FY Capsule contains complex chemical components,lacks the means of overall evaluation of drug quality,and its efficacy components and mechanism have not yet been clarified,which seriously limit the scientific application and wide recognition of FY Capsule.Therefore,it is of great practical significance to analyze the main chemical components of FY Capsule,clarify its functional components and mechanism of action,so as to improve the quality control standard of FY Capsule and provide better basic research data for clinical application.In this study,we analyzed the main chemical components of FY Capsule,established its fingerprint,and evaluated the pharmacokinetic characteristics of FY Capsule and its main component gallic acid（GA）,and their pharmacodynamic effect and mechanism in the treatment of ethanol-induced gastric ulcer.2 Methods2.1 Analysis of main chemical components and fingerprint of FY Capsule:An analytical method was established for the HPLC fingerprint of FY Capsule and simultaneous determination the contents of components in FY Capsule.Chromatographic conditions:Agilent ZORBAX SB-C₁₈（250×4.6 mm 5μm,Agilent）column,column temperature:40°C,mobile phase:methanol（A）-2%glacial acetic acid（B）,gradient elution method,time program:5%A（0-10 min）5-20%A（10-30 min）,20-40%A（30-50 min）,40-50%A（50-65 min）,flow rate:1 m L·min^-1,injection volume:20μL,detection wavelength:254 nm.On this basis,the fingerprint of FY Capsule was analyzed,and characteristic peaks were selected.Each characteristic peak was identified as the source of the material,and the chemical components corresponding to the characteristic peaks were identified and determined in combination with standard samples.2.2 Pharmacokinetics of FY Capsule and its main component GA:HPLC method for determination of gallic acid in rat plasma was established.Chromatographic conditions:Agilent ZORBAX SB-C₁₈（250×4.6 mm 5μm,Agilent）.Column,column temperature:40°C;mobile phase:methanol（A）-2%glacial acetic acid（B）,gradient elution method,time program:5%A（0-10 min）,5-20%A（10-20 min）,injection volume:20μL,detection wavelength:254 nm.Acquisition time:20 min.The content of GA in plasma was determined by the established method after administration of GA monomer,Feiyangcaoplus Huotanmu and FY Capsule,and the changes of main pharmacokinetic parameters were compared among different administration groups.2.3 Study the mechanism of FY Capsule and its main component of GA on experimental gastric ulcer:Rats were continuously administered with lansoprazole(30mg·kg^-1)and different doses of GA(10 mg·kg^-1,30 mg·kg^-1 and 50 mg·kg^-1)and FY Capsule(0.4 g·kg^-1).It was administered once a day for 7 consecutive days.On the last day,one hour after the administration an experimental model of gastric ulcer in rats was established by intragastric administration of absolute ethanol(5 m L·kg^-1).After intraperitoneal anesthesia with pentobarbital sodium(35 mg·kg^-1),serum and gastric tissue were separated,and pathological indicators,mucosal protective factors（PEG-2 and NO）,inflammatory factors（IL-1β,IL-6 and TNF-α）,oxidative stress-related factors（TBARS,GSH,SOD and CAT）,as well as oxidative stress and apoptosis-related pathway protein expression were detected.2.4 Statistical analysis:The experimental data were analyzed statistically using Graphpad Prism software version 5.01.GWM,p H,IL-1β,IL-6,TNF-α,PGE-2,NO,GSH,SOD,TBARS,CAT,Nrf2,HO-1,Bax,Caspase-3 and Bcl-2 were compared by one-way ANOVA（One-way ANOVA）.UI has been expressed as nonparametric rank with Kruskal-Wallis analysis of variance analysis and Mann-Whitney U test wasconducted for comparison between groups.P<0.05 indicated statistical difference.3 Results3.1 The main chemical components and fingerprint of FY Capsule:There are 28common chromatographic peaks in the fingerprint of FY Capsule,and the peaks 1,3,7,8,9,20,21 and 24 belong to Feiyangcao;peaks 1,2,3,4,5,14,16,17,18,22,23,26 and 28are from Huotanmu;peaks 10,11,12,13,15,25 and 27 are from Jiubiying;the remaining characteristic peaks include 6,19 which failed to be assigned.It is also determined that the peaks 3,5,9,10,11,13,14,21,22,23,24,25,26,and 28 respectively represent gallic acid,protocatechuic acid,methyl gallate,chlorogenic acid,caffeic acid,syringin,syringicacid,myricetin,isoquercitrin,ellagic acid,lithospermic acid,rosmarinic acid,quercitrin,quercetinanddetected their contents,of which the content of GA is the highest,accounting for more than 50%of the remaining known ingredients,and it is the common chemical composition of Feiyangcao and Huotanmu.3.2 Pharmacokinetics of FY Capsule and its main component GA:Compared with GA monomer administration,the mean retention time(MRT_0-∞)and half-life(t_1/2)of GA in extract of Feiyangcao plus Huotanmuand FY Capsulewere significantly prolonged（P<0.05 or P<0.01）,the area under the concentration-time curve(AUC_0-∞)of gallic acid was significantly decreased（P<0.05 or P<0.01）and the apparent volume of distribution（V_d）was significantly increased（P<0.001）,indicating that the elimination rate of GA in compound prescription was decreased and the tissue distribution was wider.3.3 The protective effect and mechanism of FY Capsule and its main component GA on experimental gastric ulcer:Compared with the model group,the histopathological morphology were significantly improved,the ulcer index,the levels of serum inflammatory factors（IL-1β,IL-6 and TNF-α）,the level of TBARS and the expression of apoptosis related proteins（Bax and Caspase-3）were significantly decreased in the GA 50group and FY Capsule group（P<0.001）;however,the protective index,p H,GWM,gastric mucosal protective factors（PEG-2 and NO）,gastric tissue antioxidant indexes（GSH,SOD and CAT）,antioxidant related proteins（Nrf2 and HO-1）and anti-apoptosis related protein Bcl-2 were significantly increased in the GA 50 group and FY Capsule group（P<0.001）.And the improvement of FY Capsule on the indexes of gastric ulcer rats was significantly better than that of the low and middle dosage groups of GA（P<0.05 or P<0.001）.4 Conclusions4.1 The main chemical components in FY Capsule were attributed:In this study,the HPLC fingerprint of FY Capsule was established.The 28 common chromatographic peaks were attributed for the first time,and 8 were derived from Feiyangcao,13 from Huotanmu and 7 from Jiubiying.Among them the chromatographic peak No.3,that is GA,is a common component of Feiyangcao and Huotanmu.4.2 The highest content of the compents consisted in FY Capsule was found:14 of the chromatographic peaks were identified and determined.The content of GA in FY Capsule was the highest,reaching 11.68±0.59 mg/capsule,accounting for more than 50%of the remaining known ingredients.4.3 Pharmacokinetic characteristics of GA after monomer and compound administrationwere revealed:Pharmacokinetic studies have revealed that compared with monomer administration,the elimination rate of GA is slower and the tissue distribution is wider after compound administration.4.4 Confirmed the therapeutic effect of gallic acid on gastric ulcer and it was speculated that it was one of the effective component of FY Capsule:The ethanol-induced gastric ulcer model of rats confirms that GA and FY Capsule have gastric mucosal protective effect,and its mechanism may be related to activating the anti-oxidative stress pathway and regulating the expression of the expression of apoptosis related proteins,indicating that GA may be the main functional component of FY Capsule for gastroenteritis.The above research provided data support for revealing the in-depth study of the prescription mechanism and efficacy mechanism of FY Capsule against ethanol-induced gastritis.However,the mechanism of its effect on enteritis will be the focus of subsequent research.