Study On The Intestinal Absorption Characteristics Of The Extract Of Leptochloa Crus-galli Before And After Creaming And Its Effect On Intestinal Flor

Objective:The aim of this study was to establish an ultrahigh-performance liquid chromatography method coupled with quadrupole time of flight mass spectrometry(UPLC-Q-TOF/MS)to analyze differences of chemical composition of clarify Semen Euphorbiae and Semen Euphorbiae Pulveratun.To investigate the differences of rat intestinal absorption trait of the main diterpenoid ester components Euphorbia Factor L1,Euphorbia Factor L2,Euphorbia Factor L3 in Semen Euphorbiae and Semen Euphorbiae Pulveratun extract in rats by in-situ single-pass perfusion model.To study what about the impact of Semen Euphorbiae and Semen Euphorbiae Pulveratun on the microflora in rats gastrointestinal From the aspect of the intestinal microecology.so as to provide reference for further reveaingl mechanism of the attenuation after processing of Semen Euphorbiae.Methods:1.The changes of the chemical constituents between Semen Euphorbiae and Semen Euphorbiae Pulveratun were investigated by ultrahigh-performance liquid chromatography method coupled with quadrupole time of flight mass spectrometry(UPLC-Q-TOF/MS).The data of chemical constituents between Semen Euphorbiae and Semen Euphorbiae Pulveratun were analyzed by SIMCA-P software.The analytical approach was Partial Least Squares Discriminant Analysis(PLS-DA)and Principal Component Analysis(PCA).2.The absorption of Euphorbia Factor L1,Euphorbia Factor L2,Euphorbia Factor L3 of Semen Euphorbiae and Semen Euphorbiae Pulveratun in small intestine(duodenum,jejunum and ileum)and colon of rats were investigated using in situ single-pass perfusion method.And the content was measured by HPLC.The quantity of the four kind of representative intestinal flora-Bacillus bifidus,Bacillus acidi lactici,enteric bacilli,enterococci in rat feces were investigated by the method of plate culture count.At stationary phase of growth curve of the four kind of representative intestinal flora,difference of the number of bacteria and the time to enter the logarithmic phase determinated by turbidimetric method.Results:There were the significant differences of the chemical constituents between Semen Euphorbiae and Semen Euphorbiae Pulveratun based on the analysis of unsupervised PCA and supervised Partial Least Squares Discriminant Analysis.This study also selected potential chemical markers that variable was Variable Importance for the Projection(VIP)>2,of which 14 compounds were found.The quantity of Euphorbia Factor L2,L3,L7a,L8,L9 and lathyrol showed a dramatic decline in Semen Euphorbiae Pulveratun in positive(ES+)electrospray ionization modes.The quantity of Aesculetin,euphorbetin,Ingenol,palmitic acid significantly increased and the quantity of Euphorbia Factor L1,L4,L5 significantly declined in negative(ES-)electrospray ionization modes.2.The absorption rate constants(Ka)and apparent permeability coefficient(Papp)of the main diterpenoid ester components in Semen Euphorbiae and Semen Euphorbiae Pulveratun were significantly different.Ka of Euphorbia Factor L1,Euphorbia Factor L2,Euphorbia Factor L3 in Semen Euphorbiae Pulveratun extract were at the range of 15.38±1.27～21.92±3.74,9.67±1.01～17.51±2.35,14.56±0.93～22.31±2.29;Ka of Euphorbia Factor L1,Euphorbia Factor L2,Euphorbia Factor L3 in Semen Euphorbiae extract were at the range of 19.81±0.24～26.89±1,01,15.79±1.70～24.03±2.60,19.37±2.14～26.66±1.33;and Papp of Euphorbia Factor L1,Euphorbia Factor L2,Euphorbia Factor L3 in Semen Euphorbiae Pulveratun extract were at the range of 1.74±0.18～2.55±0.51,0.99±0.22～1.58±0.14,1.62±0.20～2.15±0.14;Euphorbia Factor L1,Euphorbia Factor L2,Euphorbia Factor L3 in Semen Euphorbiae extract were at the range of 2.36±0.23～3.10±0.11,1.84±0.30～2.51±0.35,2.32±0.20～2.87±0.24;Ka and Papp of these three constituents in Semen Euphorbiae Pulveratun extract were less than Semen Euphorbiae extract.3.The quantity of the four kind of representative intestinal flora-Bacillus bifidus,Bacillus acidi lactici,enteric bacilli,enterococci in rat feces were different after a period of time by gavage with Semen Euphorbiae and Semen Euphorbiae Pulveratun extract.The degree of the intastinal flora disturbance caused by Semen Euphorbiae Pulveratun extract was mucn lower than Semen Euphorbiae.However,the quantity of enteric bacilli and enterococci reduced in low dose group.When extract of Semen Euphorbiae and Semen Euphorbiae Pulveratun were in the concentration of 0.002-0.02g/mL,stationary phase of growth curve of the four kind of representative intestinal flora were under the blank control group,and logarithmic phase lagged,bacteria number reduced as to the increase of drug concentration.stationary phase of growth curve of the four kind of representative intestinal flora in Semen Euphorbiae Pulveratun group were under Semen Euphorbiae group.Conclusion:1.UPLC-Q-TOF/MS had advantages of high sensitivity,high resolution,high efficiency on studying the influence of the frost-processing on the chemical composition of Semen Euphorbiae.Then scientific basis was provided that mechanism of the attenuation after processing of Semen Euphorbiae.2.These three constituents in raw and processed Semen Euphorbiae extract can be absorbed in the whole intestinal segments and they absorbed are less in Semen Euphorbiae Pulveratun extract.3.Degree of intastinal flora disturbance coused by Semen Euphorbiae Pulveratun was much lower was the same with previous research results.Meanwhile,Semen Euphorbiae maybe have the influence on regulating the balance of intestinal flora under the lower dose usage.The turbidimetric method experimenta result showed that with the dose of the extract of Semen Euphorbiae and Semen Euphorbiae Pulveratun had inhibitory effectes on the four representative of intestinal flora and the inhibitory effect increased with the increase of drug concentration.Compared to the extract of Semen Euphorbiae,the extract of frost-processing Semen Euphorbiae decreased the inhibition of the four intestinal flora.That the extract of Semen Euphorbiae and Semen Euphorbiae Pulveratun both affect the biological diversity of intestinal flora,the intestinal microecological balance effect.What was more,Semen Euphorbiae Pulveratun could decrease the four intestinal flora which caused by intestinal flora disturbance.This is consistent with the result of Semen Euphorbiae Pulveratun moderate study.