| Cuscuta chinensis Lam.is a dry and mature seed of convolvulaceae,which is often used to nourish liver and kidney in clinic,and has the functions of tonifying yang and yin,strengthening kidney and preventing miscarriage.Previous studies by our research group showed that 95%ethanol extract of Cuscuta var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.chinensis var.Taking estrogen-deficient model rats as the research object,by constructing the model of "inherent components-in vivo metabolismbiological effects" of Cuscuta,the estrogen active components and in vivo direct acting substances in Cuscuta were found.However,it is not clear which inherent components metabolize and transform these in vivo direct acting substances,which seriously hindered the elucidation of the pharmacodynamic material basis of estrogen action of Cuscuta.Therefore,in this paper,the estrogenic parts and components of Cuscuta produced in Heilongjiang Province were taken as the research objects,and the metabolism of Cuscuta extract and its inherent components in intestinal flora and liver microsomes was studied,and the metabolic pathway of the directly acting substances in vivo was traced,and the inherent components related to the directly acting substances in vivo were found by reverse traceability analysis,which provided reference for further elucidating the material basis of Cuscuta playing estrogenic role.The main research methods and results are as follows:(1)using UPLC/Q-TOF-MS technology,taking quercetin and chlorogenic acid as metabolic substrates,the metabolic conditions were optimized by investigating the effects of different final concentration and incubation time on the number of metabolites.The results showed that the best metabolic reading concentration of liver microsome metabolism was 80μmol/L,and the best reaction time was 50min.(2)UPLC/Q-TOF-MS technique was used to analyze the metabolism of the inherent components of Cuscuta in the liver microsomes of female rats.Eight metabolites were detected in the samples incubated with quercetin liver microparticles.Two metabolites were detected in the samples incubated with quercitrin liver microparticles.Three metabolites were detected in the incubated samples of isoquercitrin liver microparticles.Three metabolites were detected in the samples incubated with hyperin liver microparticles.Seven metabolites were detected in the samples incubated with kaempferol liver microparticles.Three metabolites were detected in the incubated samples of Astragaloside liver microparticles.Three metabolites were detected in the samples incubated with apigenin liver microparticles.Three metabolites were detected in the incubated samples of isorhamnetin liver microparticles.Three metabolites were detected in the incubated samples of isorhamnetin-3-O-glucoside liver microparticles.Two metabolites were detected in the incubated samples of chlorogenic acid liver microparticles.Three metabolites were detected in the incubated samples of dicaffeoyl quinic acid liver microparticles.Glucuronic acid binding reaction is the main way of liver microsomal metabolism of Cuscuta chinensis.(3)UPLC/Q-TOF-MS technique was used to analyze the metabolism of estrogenic site of Cuscuta in intestinal flora of female rats.Eighteen prototype components were detected in 95%ethanol extract of Cuscuta for OH in positive and negative ion mode.They are quercetin-3-Ogalactose-(2→1)-apioside,quercetin-3-O-acetylgalactoside,kaempferol,isorhamnetin,caffeic acid,chlorogenic acid,quercetin-3-O-galactose-7-O-glucoside,hyperoside,isoquercitrin and Chinese milk vetch.Among them,quercetin-3-O-acetylgalactoside,unknown component 1 and unknown component 2 were first found in Cuscuta from Heilongjiang Province.Thirteen chemical components were found in the samples incubated for 48 hours in positive and negative ion mode,which were caffeic acid,quercetin-3-O-galactose-(2→1)-apigenin,hyperin,quercetin,cuscutoside A,kaempferol,isorhamnetin,p-hydroxybenzoic acid,dihydrocaffeic acid,dihydroferulic acid,3-hydroxyphenylpropionic acid and unknown components It contains 9 metabolites,among which caffeic acid,quercetin,kaempferol and isorhamnetin are both prototype components and metabolites.(4)Using UPLC/Q-TOF-MS technique,inherent components of Cuscuta in intestinal flora of female rats was analyzed.The results showed that one metabolite was detected in the warmincubated samples of quercitrin feces.One metabolite was detected in isoquercitrin fecal incubation samples.One metabolite was detected in hyperin fecal incubation samples.A metabolite was detected in the feces incubation samples of Astragaloside.A metabolite was detected in the fecal incubation samples of isorhamnetin-3-O-glucoside.Six metabolites were detected in chlorogenic acid fecal incubation samples.Seven metabolites were detected in the fecal incubation samples of dihydrocaffeic acid.Hydrolysis,hydrogenation,methylation and dehydroxylation are the main ways of intestinal flora metabolism of Cuscuta.(5)The previous research of our research group found that 9 components in the medicated serum of female ovariectomized rats were highly correlated with the estrogenic effect of Cuscuta chinensis.Combining the results of this experiment with the previous research results of our research group,we conducted a reverse traceability analysis,and preliminarily speculated that quercetin,kaempferol,apigenin,quercitrin,isoquercitrin,hyperin,and astragaloside were inherent components related to the estrogenic effect of Cuscuta chinensis.It provides a reference for elucidating the material basis of estrogenic action of Cuscuta chinensis. |
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