| 1 BackgroundGastritis is the most common clinical digestive system disease,which greatly affects people’s quality of life and life safety.At present,the treatment of acute gastritis mainly adopts clinical symptomatic treatment or surgical treatment,without exploring the cause of the disease;moreover,taking chemical drugs for a long time will further damage the gastric mucosa,leading to the development of chronic gastritis,gastrointestinal disorders and other recurrent diseases,and even induce gastric cancer.Traditional Chinese medicine pays attention to syndrome differentiation and treatment of acute gastritis,seeking the root of the disease,diagnosing the type of the disease,treating the disease according to the priority and development trend of the disease,repairing the gastric mucosa at the same time,fundamentally preventing the development of the disease and its reversal.Traditional Chinese medicine has obvious effect on acute and chronic gastritis,and its safety may be better.Thus,it has gradually become an important direction in the field of clinical digestive diseases to excavate more effective traditional Chinese medicine or compound medicine.Jiaolong capsule is an original class II Chinese patent medicine approved by the State Food and Drug Administration（SFDA）.It is composed of gentian glycosidess of Gentiana macrophylla.It has the effect of clearing liver heat,relieving heat and lowering stomach qi.It is mainly used to treat functional dyspepsia of liver and stomach stagnation heat syndrome.Clinical symptoms such as fullness of stomach,burning of stomach,dry mouth and bitter mouth can be seen.Although the listed drug is the gentian glycosides of single Chinese medicine"Gentiana macrophylla"However,it is not clear which the main components play a role in the treatment of functional dyspepsia.In view of this scientific problem,the main components of Jiaolong capsule were analyzed by means of fingerprint,serum pharmacochemistry,pharmacokinetics and other research methods.The distribution of Jiaolong capsule in various tissues/organs in vivo was further detected,and the effect of gentiopicroside on acute gastritis was preliminarily clarified,and the main effective components and mechanism of Jiaolong capsule in the treatment of experimental gastritis were preliminarily clarified.2 Methods2.1 Composition analysis and quality control of Jiaolong capsule:Using HPLC method to establish the fingerprint and multi-component content determination of Jiaolong capsule.Chromatographic conditions:Intersil C18column（250 mm×4.6mm,5μm）.The mobile phase was a mixture of acetonitrile（A）and 0.2%phosphoric acid solution（B）in gradient elution,the method of gradient elution time application as follow:B 95%→85%（0～10 min）;B 85%→82%（10～40 min）,82%→55%（40～80 min）.The flow rate was 0.8 m L/min,and the detection wavelength was 254 nm,and the column temperature was set at 25℃.The aim of this study was to determine the main alkaloids and their contents in Jiaolong capsule.2.2 Pharmacokinetics and tissue distribution of Jiaolong capsule in rats:We established HPLC method for the simultaneous determination of the plasma concentrations and pharmacokinetics parameters of the migrating components in rats,and the distribution characteristics of the tissues（heart,liver,spleen,lung,kidney,stomach,pancreas,brain,duodenum,colon,ileum）of the blood components in rats were analyzed.Chromatographic conditions:Akzo Nobel kromasil C18（250mm×4.6mm 5μm）;acetonitrile（A）-0.085%phosphoric acid solution（B）as mobile phase,gradient elution,flow rate:0.8m L/min;column temperature:25℃;detection wavelength:238nm;injection volume:20μL.The aim of this study was to clarify the possible effector organs or tissues and the distribution characteristics of the main chemical components in Jiaolong capsule.2.3 Effect and mechanism of gentiopicroside in Jiaolong capsule on acute gastritis:The protective effect of gentiopicroside on acute gastritis was observed by replicating the model of acute gastritis induced by alcohol in mice;The model of alcohol injury was replicated in GES-1 cells,and the protective effect of gentiopicroside on alcohol injury was observed by the changes of cell vitality,gene level of main inflammatory related factors and protein level;The changes of MMP-10 gene and protein level and ERK1/2 phosphorylation of gentiopicroside were detected on GES-1 cell model,so as to preliminarily explore the protective mechanism of Jiaolong capsule and gentiopicroside（the main effective component）on acute gastritis.3 Results3.1 Composition analysis and quality control of Jiaolong capsule:There were 23peaks in the fingerprint of Jiaolong capsule,among which 8 peaks were identified as:loganic acid,shanzhiside methyl ester,6′-O-β-D-glucosylgentiopicroside,swertiamarin,gentiopicroside,sweroside,isoorientin,isovitexin.We established a method for the simultaneous determination of 8 components in Jiaolong capsule.The specific contents were 104.337mg/g,7.170mg/g,49.291 mg/g,8.304 mg/g,483.760 mg/g,5.124 mg/g,0.885 mg/g,0.608 mg/g,respectively.The method is simple,reproducible and precise,which provides data and methodological support for the improvement and revision of the quality standard of the preparation.3.2 Pharmacokinetics and tissue distribution of Jiaolong capsule in rats:The five components of loganic acid,swertiamarin,6′-O-β-D-glucosylgentiopicroside,gentiopicroside and sweroside were simultaneously determined in the plasma of rats.The concentration of each active component reached a high level in the plasma and tissues at 60 min,and the content of each active component in the gastrointestinal digestive organs was high,among which gentiopicroside was the highest.However,the contents of the active components in brain and lung were lower.The results showed that the main effector organ of the main component of Jiaolong capsule may be gastric/intestinal tissue.3.3 Effect and mechanism of gentiopicroside in Jiaolong capsule on acute gastritis:Gentiopicroside have a very obvious protective effect on alcohol-induced acute gastritis,which can reduce the damage of gastric mucosa,the level of TNF-a,IL-1β,IL-8,and increase the level of IL-10 in the process of inflammation;It can also reduce the expression of MMP-10 gene and protein,ERK1/2 phosphorylation level in the process of inflammation,and this effect can be reversed by ERK pathway inhibitor U0126,in MMP-10 gene knock-out model also verified that MMP-10 plays an important role in inflammation.It is suggested that the protective mechanism of gentiopicroside on gastritis is related to down regulating the expression of MMP-10and blocking ERK signal transduction pathway.4 ConclusionsThe study established the HPLC fingerprint of Jiaolong capsule for the first time,in which confirmed 23 common chromatographic peaks and identified 8components.For the first time,five components of Jiaolong capsule were detected,which were loganic acid,swertiamarin,6′-O-β-D-glucosylgentiopicroside,gentiopicroside and sweroside,which revealed the high distribution of the five main components in stomach and intestine.It is confirmed that gentiopicroside is the main component of Jiaolong capsule,which has a significant therapeutic effect on acute alcoholic gastritis,and its mechanism is related to the regulation of MMP-10 and p ERK1/2 signaling pathway;meanwhile,gentiopicroside may be the main effective component or one of the effective components of Jiaolong capsule. |
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