Sophora Alopecuroides Ameliorates Large Intestine Dampness-Heat Syndrome In Rats By Metabolomic Approach

The Chinese herbal Sophora alopecuroides is widely used to clear intestine and eliminate dampness,and it has good therapeutic effect on treating damp-heat diarrhea and dysentery,abdominal urgency and rectal heaviness,strangury disease with inhibited painful urination,etc.in clinical Traditional Chinese Veterinary Medicine?TCVM?.However,the mechanisms of treating diseases are not yet well understood.Thus,in this study,Sophora alopecuroides as object,the content of 5 alkaloids in Sophora alopecuroides was determined through the method of High performance liquid chromatography?HPLC?.On this basis,the model of large intestine dampness-heat syndrome?LIDHS?was built,and the water extract of Sophora alopecuroides was utilized to treat it.Meanwhile,the metabolomic approach was conducted to study the therapeutic mechanisms of Sophora alopecuroides on LIDHS from the level of endogenous metabolic small molecular in organism.The study provides the reference material and theoretical basis for Sophora alopecuroides in pharmacological function research and clinical practice in livestock.The main results were as follows:1.Determination of 5 alkaloids in Sophora alopecuroides by HPLC.The content of 5 alkaloids,including matrine,oxymatrine,sophocarpine,oxyophocrpine and sophoridine,was determined based on the method of HPLC and was extracted by the approach of NH₃·H₂O-CHCl₃.Separation was carried out on an Agilent ZORBAX SB-C₁₈?4.6×150 mm,5?m?under 35°C and at the detective wavelength of 205 nm.The mobile phase consisted of Methanol?A?and 0.2%Phosphoric acid?B?.The gradient elution of A was performed as follows:5%A at 0⁷ min,7%A at 7⁴5 min,and the flow rate was respectively 1 mL·min^-1 and 0.5 mL·min^-1.The methodological examination was evaluated through linear relationship,precision,stability,reproducibility and add sample collection experiments,etc.The results showed that the detection method was accurate and reliable.The ratio of the 5 alkaloids content in Sophora alopecuroides,including matrine,oxymatrine,sophocarpine,oxyophocrpine and sophoridine,was respectively 0.03%,0.03%,0.30%,0.18%and 0.26%.Specially,the ratio of sophoridine was maximum among them.2.Construction of LIDHS model and Sophora alopecuroide's therapeutic effect evaluation.32 Wistar rats were randomly divided into four groups with eight rats?half males and half females?in each group.Each group was designated as follows:control group?Control?,LIDHS model group?Model?,self-healing group of LIDHS model?ZY?and Sophora alopecuroides treatment group?KDC?.The LIDHS model was performed by the comprehensive factors,including high temperature and humidity environment,high-sugar and high-fat diet,and intraperitoneal injection of Escherichia coli.The general ethology,blood routine,serum inflammatory cytokine levels and intestinal histopathological changes of all of rats were detected and observed for evaluate the intervened effect of Sophora alopecuroides.The results showed that,in Model group,diarrhea symptom apparent,colon and ileum injury,white blood cell counts?WBC?,neutrophil counts?NE?,lymphocyte counts?LY?packed cell volume?PCV?,hemoglobin concentration?HGB?were significantly higher than those in the Control group?P<0.05?.Meanwhile,the content of inflammatory cytokines,including IL-1?,IL-2,IL-6 and TNF-?in vivo,were increased compared with control group?P<0.05?.Above results were in accordance with the evaluation criterion of LIDHS.However,after treatment with Sophora alopecuroides,the abnormality of blood routine indexes as well as inflammatory cytokines,including IL-1?,IL-2,IL-6 and TNF-?in vivo,tended to be normal,and the histopathological changes of intestine were improved,which indicated that Sophora alopecuroide has well therapeutic function for ameliorate LIDHS symptom,regulate immunoreaction and repair the intestinal injury.3.Exploration of the therapeutic mechanisms of Sophora alopecuroide on LIDHS by metabolomic approach.A serum and urinary metabolomic approach based on Ultra performance liquid chromatography coupled to quadrupole time-of-flight mass/mass spectrometry?UPLC-Q/TOF-MS/MS?was conducted to explore the changes in the endogenous metabolites and to assess the integral efficacy of Sophora alopecuroide on LIDHS model rats.Principal component analysis?PCA?and orthogonal partial least squares discriminate analysis?OPLS-DA?was used to find the potential metabolic markers and to screen the targeted metabolic pathways with respect to therapeutic effects of Sophora alopecuroides on LIDHS model rats.The results showed that 22 potential metabolic markers were screened from serum and urine?serum9,urine 13?,and may be related to therapeutic mechanisms of Sophora alopecuroides on LIDHS model rats.Compared with control group,the abnormal content change of these potential metabolic markers in LIDHS model rats was tended to normal after treatment by Sophora alopecuroides,and involved in taurine and hypotaurine metabolism,glycerolipid metabolism,tryptophan metabolism,Glutathione metabolism and Tryptophan metabolism,etc.Particularly,the therapeutic mechanisms of Sophora alopecuroides on LIDHS may be primarily through glycerophospholipid metabolism,primary bile acid biosynthesis and Melatonin biosynthesis,etc.through Pearson correlation analysis.