| Objective: The toxicological experimental methods were used to investigate the toxic dose,toxic target organs and mechanisms of Liu Wei An Xiao San,to calculate the kinetic parameters of Liu Wei An Xiao San of Mongolian medicine by pharmacokinetics,to determine the content of toxic components of Liu Wei An Xiao San,and to provide scientific basis for the safe and rational clinical application of Liu Wei An Xiao San.Methods:In this study,we used the method of splitting the formula,divided into single flavor rhubarb,de-rhubarb formula and de-hubarb formula and Liouwei Anxiexian as the drugs shown,and used rats as the study subjects to evaluate the subacute toxicity and toxicity mechanism of Liouwei Anxiexian,analyze the in vivo kinetic characteristics of rhubarbin,and determine the content of the four toxic components.1.Subacute toxicity test of Liu Wei An Dissemination: The rats were divided into 9 groups,including Liu Wei An Dissemination high dose group(AG),Liu Wei An Dissemination medium dose group(AZ),Liu Wei An Dissemination low dose group(AD),Da Huang high dose group(DG),Da Huang medium dose group(DZ),Da Huang low dose group(DD),De Da Huang group(QD),De Hao Zi group(Qh)normal group(Z).The corresponding drug solution was administered once daily by gavage in groups other than the normal group and given for 28 d.The relevant indexes of serum liver and kidney functions were detected;liver and kidney organ indices were calculated;and histopathological examination of liver and kidney histology was performed.To evaluate the poisoning dose of Liu Wei An Xiao San and the target organs of poisoning.2.Preliminary investigation on the mechanism of subacute toxicity of hexamethonium based on 16 S sequencing of intestinal flora: Combining bioinformatics analysis and statistical methods,we analyzed the species composition components of each group,screened the phylum,just,order,family,genus and species level,and initially clarified the differential flora and differential pathways related to the toxicity of hexamethonium.3.Pharmacokinetic experiments: The rats were fasted for 12 h before the experiments,and after the gavage of Liu Wei An Xiao San,the rats were anesthetized with 10% chloral hydrate,and blood samples were collected from the orbits at 0,0.25,0.5,1,1.5,2,4,6,12 and 24 h after the gavage.Preliminary evaluation of absorption,distribution,metabolism and excretion pattern was performed.4.Determination of dissipative toxic components of Liuwei an xiao san:Determination of the content of Aloe vera rhodopsin,rhodolic acid,rhodopsin and rhodopsin methyl ether on a ZORBAX SB-C18(46×250mm,5um)column with the mobile phase of methanol-0.1% phosphoric acid solution(70:30),detection wavelength of 254 nm,flow rate of 1m L/min,column temperature of 25℃.Results:1.The results of subacute toxicity experiments showed that:(1)the effects on liver and kidney function indexes: compared with the normal group,the ALT content was significantly increased in the Liu Wei An Dissemination High,Liu Wei An Dissemination Low,Da Huang Zhong,Da Huang Low,lack of Chebul,lack of Da Huang group(p < 0.05,p < 0.01);the AST content was significantly increased in the Liu Wei An Dissemination High,Da Huang High,lack of Chebul,lack of Da Huang group(p < 0.05,p < 0.01);UREAL content was increased in the Liu Wei An Dissemination High,Rhubarb High,Rhubarb Medium,Chebulus Deficient,and Rhubarb Deficient groups(p < 0.05,p < 0.01);CREP content was increased in the Rhubarb High,Chebulus Deficient,and Rhubarb Deficient groups(p < 0.05,p <0.01).(2)Effects on organ indices: compared with the normal group,the group liver indices of rats in the rhubarb high dose,rhubarb deficiency and rhubarb deficiency groups were significantly lower(p < 0.05,);the kidney indices of the Liu Wei An Xiao San high dose,rhubarb high dose and rhubarb deficiency groups were significantly higher(p < 0.05,p < 0.01).This shows that Liu Wei An Xiao San has some sexual toxicity to both liver and kidney of rats.(3)Observation on the histological changes of liver and kidney pathology:(1)Liu Wei An Xiao San group: the structure of liver tissue was moderately abnormal,some hepatocytes were edematous,cytoplasm was lightly stained,some hepatocytes were fatty degeneration,a few vacuoles were visible in the cytoplasm,a few hepatocytes were edematous in the field of view,a few inflammatory cells were infiltrated in the fine liver parenchyma.The kidney tissue structure was moderately abnormal,some of the glomeruli were lobulated and atrophied,the number of thylakoid cells was reduced,some of the renal tubules were cellular tubular,a few cells were visible in the lumen,some of the tubular brush border was damaged,some of the tubular epithelial cells were edematous,the cells were swollen,the cytoplasm was lightly stained,and no obvious inflammatory cell infiltration was seen in the tissue.(2)Rhubarb group: the structure of liver tissue was moderately abnormal,some hepatocytes in the visual field were edematous with swollen cells,cytoplasm was lightly stained,a few vacuoles were visible in the cytoplasm,no expansion was seen,the hepatic cords were arranged neatly and closely;a small amount of inflammatory cells infiltration was seen in the liver parenchyma.The kidney tissue structure was moderately abnormal,some glomeruli were atrophied,the number of thylakoid cells was reduced,some glomeruli were lobulated,some renal tubules were cellular tubular,a few cells were seen in the tubular lumen,and no obvious inflammatory cell infiltration was seen in the tissue.(3)Lack of chelonia group: the liver tissue structure was moderately abnormal,the hepatocytes in the field of view were structurally intact,with rounded nuclei and abundant cytoplasm,no necrosis or degeneration was seen;the hepatic sinusoids were uniform in size and not dilated,the hepatic cords were neatly arranged and tight;a small amount of inflammatory cell infiltration was seen in the liver parenchyma.The kidney tissue structure was moderately abnormal,a large number of renal tubules showed cellular tubular type,a few cells were seen in the lumen,some of the tubular brush border was damaged,some of the tubular epithelial cells were edematous and cellular swelling,no obvious inflammatory cell infiltration was seen in the tissue.(4)The hepatic tissue structure was mildly abnormal,with intact hepatocytes in the visual field,rounded nuclei,abundant cytoplasm,and no necrosis or degeneration;the hepatic sinusoids were uniform in size,not dilated,and the hepatic cords were neatly and closely arranged.The kidney tissue structure was mildly abnormal,some of the glomeruli in the visual field were lobulated,the number of membrane cells was normal,some of the renal tubules showed cellular tubular type,a small number of cells were seen in the tubular lumen,and the tissue did not see obvious inflammatory cell infiltration.2.The experimental results of the subacute toxicity mechanism analysis of Liu Wei An Xiao San showed that there were 31 differential flora in normal group,31 differential flora in Qh group,29 differential flora in QD group and 24 differential flora in DG group.The main Prevotella,Helicobacter,Clostridium,Lactobacillus, Daniostoma,Bifidobacterium,Campylobacter,and Desulfovibrio affect the Fox O signaling pathway pathway.3.The results of pharmacokinetic experiments showed that the half-life(t1/2)of rhodopsin was 7.015 ± 2.730,and the highest concentration in plasma was reached after 1.017 ± 0.014,with a peak concentration(Cmax)of 1.2450 ± 0.04087 μg/L.The concentration then decreased rapidly,with an in vivo retention time of 9.256± 2.368 and an area under the blood concentration curve(AUC)was 7.048±1.034.4.The experimental results of content determination showed that the average recoveries of Liu Wei An Xiao San were 98.39%,98.84%,101.48% and 96.38%,and the average recoveries of Rhubarb were 96.67%,99.41%,99.03% and 97.37%.The contents of aloe-emodin,rhodolic acid,rhodopsin and rhodopsin methyl ether in Liu Wei An Xiao San were 0.90mg/g,1.79mg/g,0.70mg/g and 0.34mg/g,respectively,and the contents of aloe-emodin,rhodopsin,rhodopsin and rhodopsin methyl ether in Rhubarb were 4.58mg/g,7.79mg/g,3.12mg/g and 1.56mg/g,respectively.Conclusion:1.Liuwei An dissipate has no subacute toxicity at clinical equivalent dose,and the administration dose more than 4 times the clinical equivalent dose can produce subacute toxicity in liver and kidney.Liuwei An dissipative subacute toxicity characteristics are similar to Rhubarb.It is suggested that Liuwei An dissipates liver and kidney subacute toxicity mainly from Rhubarb.2.Liou Wei An Dissolve increased the biochemical indexes of ALT,AST,UREAL and CREP in rat serum related to the disorder of Prevotella,Helicobacter,Clostridium,Lactobacillus,Daniostoma,Bifidobacterium,Campylobacter and Desulfovibrio in the intestinal tract.The changes of intestinal flora in the liver and kidney subacute toxicity of Liu Wei An Xiao San were similar to those of Da Huang.3.The pharmacokinetics of emodin in Liuwei An were studied by LMS.The content rule of emodin in blood was explained,which provided methodological basis for the kinetic analysis of emodin,aloe emodin and emodin methyl ether in Liuwei an.4.Based on the determination of the contents of rhubarbin and rhubarbol in the2020 edition of the Chinese Pharmacopoeia,a method was established for the first time to determine the contents of rhubarbin,rhubarbin,rhubarbic acid and rhubarbin methyl ether in Liuwei Anxianxian by HPLC,which further provided a basis for the perfect quality control of Liuwei Anxianxianxian. |
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