| Semen Cuscutae is a dried and mature seed of Cuscuta australis R.Br.or Cuscuta Cuscuta chinensis Lam..The flavor of the Semen Cuscutae is warm and sweet.It converges to liver,kidney and spleen channel.The effects of Semen Cuscutae are tonifying kidney and liver,miscarriage prevention.It have a long historical standing of dodder processing.The original processed product of Semen Cuscutae is wine-processed product(WPP),while the most popular used processed product in China is salt-processed product(SPP).After processing,the effect of nourishing liver whether is changed or not.As well as,the processing principle of dodder is still unknown.This study is based on National Natural Fund Project.The project of the study was to compare the liver-nourish effects of different processed products of Semen Cuscutae.We studied the hepatoprotective effect of three processed products of Semen Cuscutae based on pharmacodynamics and metabonomics.To investigate the relation between pharmacokinetics and pharmacodynamics of three processed products of Semen Cuscutae in rats with the combined PK-PD model.Objective:1.To compare the changes of flavonoids and polysaccharides in dodder after processed.2.To compare the hepatoprotective effect and mechanism of three processed products of Semen Cuscutae on the chemical liver injury rats.3.To compare the hepatoprotective effect and mechanism of three processed products of Semen Cuscutae on the alcoholic liver injury rats.4.To investigate the mechanism of action of hepatoprotective of Semen Cuscutae by a metabonomics model.5.To establish a UPLC-MS/MS method which used to determinate the chlorogenic acid,hyperoside and astragalin simultaneously in the plasma samples.6.To study the pharmacokinetics and pharmacodynamics of three processed products of semen dodder.To evaluate the pharmacokinetic properties and concentration-effect-time relation curve of three different processed products.Methods:1.The total polysaccharides and total flavonoids in three different processed peoducts were determinated by UV spectrophotometric.The use of HPLC method for simultaneous determination of three flavonoids monomer content in three different processed dodder.The monosaccharide components of three different processed product were analyzed by a precolumn derivatization with PMP of HPLC method.2.Studied on liver protective effect of three processed products of Semen Cuscutae by evaluated the content of serum and liver tissue biochemistries as well as pathological report against acute CCl4-hepatitis in rats.3.Studied on liver protective effect of three processed products of Semen Cuscutae by evaluated the content of serum and liver tissue biochemistries as well as pathological report against acute alcoholic-hepatitis in rats.4.The CCl4-hepatitis in rats were used to analyze the plasma metabolomics based on the UPLC-MS signal,and to find the related metabolites of the dodder’s liver protection by multivariate statistical analysis and medical statistics analysis,and analyze the pathway and principle of the liver protection action of Semen Cuscutae.5.A method for simultaneous determination of chlorogenic acid,hyperin and astragalin in rat plasma samples was established by UPLC-ESI-MS/MS.6.The pharmacokinetics of raw products(RP),salt processed products(SPP)and wine processed products(WPP)in rats with acute liver injury induced by CCl4 were studied by the established method and the pharmacokinetic parameters of three processed products of dodder were analyzed.7.A rat model of CCl4 acute liver injury was used to study the pharmacodynamics of three processed products of dodder and to establish a PK-PD model to analyze the concentration-effect-time relation curve of different processed products.Results:1.The quality of dodder is crisper and the color is darker after processing.The contents of total polysaccharides in RP,SPP and WPP were 5.74±0.19%,8.50±0.22%,10.37±0.38%,respectively.Total flavone contents were 3.20±0.07%,3.70±0.09%,3.69±0.10%,respectively.The contents of hyperoside were 3.86±0.09mg/g,4.16±0.11mg/g,5.03±0.03mg/g,respectively.The contents of astragulin were 0.56±0.01mg/g,0.83±0.00mg/g,0.78±O.Olmg/g,respectively.The contents of quercetin were0.05±0.00mg/g,0.09±0.00mg/g,0.08±0.00mg/g,respectively.The monosaccharide composition of the polysaccharide of Semen Cuscutae was basically unchanged.The main monosaccharides of the raw and two kinds of cannon products were mainly mannose,and more than 50%of the total amount of the sugar.2.After intraperitoneal injection of 40%CCl4 vegetable oil solution,the cellular swelling of the hepatocyte was moderate to severe,relative majority portal tracts are expand.The value of serum ALT and AST was significantly higher than that of the normal group.The liver damage were mitigated slightly in the raw group.The degree of liver cell edema and deformability is less than that of the model group.The liver structure of SPP group and the WPP group tended to be normal,with no obvious swelling and infiltration of inflammatory cells.The serum biochemical indexes showed that the content of ALT and AST in the serum of RP,SPP and WPP groups decreased significantly compared with that of the model group,indicating that the three products of dodder had a protective effect on the liver of CCl4 acute liver injury in rats.According to the antioxidant index of liver homogenate,there was no significant difference in the content of SOD and GSH from the model group(P>0.05),and the decrease of MDA content(P<0.01).The content of SOD and GSH caused by CCl4 decreased(P<0.01),and the content of MDA decreased significantly(P<0.01).From the level of liver homogenate cytokine,there was no significant difference in TNF-α and IL-1βlevels between the RP group and the model group(P>0.05).Compared with the model group,the level of TNF-α and IL-1β in liver homogenate was significantly lower than that in the model group(P<0.05).However,there was no significant difference between the two groups(P>0.05).3.After the rats were fed with 50%ethanol,the liver pathological sections showed that the liver cells were swelling obviously,and the liver cells with obvious fatty degeneration were visible,and the diffuse round lipid droplets appeared in the cytoplasm.The serum ALT and AST values in model group were significantly higher than those in normal group.The liver cell swelling and fatty change of the RP lightened,and the liver structure of dodder salt broiling group and dodder liquor group tended to be in the normal group,and the cells had no obvious swelling,and the fatty degeneration of liver cells in the liver lobule.The serum biochemical indexes showed that the content of ALT and AST in the serum of RP,SPP and WPP rats decreased significantly compared with that of the model group,which indicated that the three products of dodder had a protective effect on the liver injury of alcoholic liver injury in rats.According to the antioxidant index of liver tissue homogenate,there was no significant difference in GSH content between the SP and the model group(P>0.05),the decrease of SOD and MDA content(P<0.01),and the increase of CAT content(P<0.05).Compared with the model group,the content of SOD,GSH and CAT in SPP and WPP groups increased significantly(P<0.01)and MDA content decreased significantly(P<0.01).Compared with the model group,the levels of TNF-α and IL-1β in the liver tissue of the SPP and WPP rats were lower than those in the model group,which was statistically significant(P<0.05),but there was no statistical difference between the two groups(P>0.05).However,there was no significant difference in the level of TNF-α and IL-1β between the raw group and the model group(P>0.05).4.A UPLC-Q-TOF/MS analysis method was established for Metabonomics Study of CCl4 acute liver injury rats.The OPLS-DA model was used to analyze the samples.Under the positive and negative ion mode,the normal group and the model group could be clearly separated,proving that the CCl4 model was successful.The metabolic components of VIP>1.0 were analyzed by Mann Whitney U test and One-way ANOVA test,and the differential components of liver injury(P<0.05)were screened out.According to the fragment information and the exact mass number obtained by MSE,the selected components were identified,and 27 different compounds were screened by HMDB,KEGG,METLIN,Massbank and Chemspider.Statistical analysis was conducted on three processed products of Cuscuta chinensis,and potential biomarkers for liver protection were found.The pathway analysis and visual analysis of metabolites were carried out,and the main pathways related to the liver protection of dodder were mainly Sphingolipid metabolism,Taurine and hypotaurine metabolism,Glycerolipid metabolism,Fructose and mannose metabolism.5.A UPLC-ESI-MS/MS method for simultaneous determination of chlorogenic acid,Hyperoside and milein in rat plasma samples was established.The retention time of the three components was 3.24min,4.06min and 4.32min,respectively,and the detection range was 0.5-300ng/mL.The precision of the two concentrations in the low,middle and high concentrations was<15%,the accuracy of the low,middle and high concentrations were all within the range of 80～120%,and the recovery and matrix effect of the three components were all in 85%～115%,and the stability of the samples was in accordance with the regulations.6.After administration of RP,SPP or WPP,chlorogenic acid,hyperin and astragalin in CCl4-hepatitis rats showed a two compartment model.It can be seen from the drug time curve that the absorption and elimination trend of the three components is similar,and that the maximum blood drug concentration is achieved within 2 hours,and then gradually eliminated.The Tmax of chlorogenic acid,hyperin and astragalin in RP group were 1.42±0.38h,1.17±0.26h,1.04±0.40h,respectively.The Cmax of chlorogenic acid,hyperin and astragalin in RP group were 81.34±15.22μg/L,48.44±12.54μg/L,12.76±4.10μg/L,respectively.The AUC(0-t)of chlorogenic acid,hyperin and astragalin in RP group were 477.97±30.32μg/L*h,159.85±28.71μg/L*h,49.65±4.85μg/L*h,respectively.The AUC(0-∞)of chlorogenic acid,hyperin and astragalin in RP group were 531.42±31.36μg/L*h,177.69±24.88μg/L*h,53.16±6.02μg/L*h,respectively.The Tmax of chlorogenic acid,hyperin and astragalin in SPP group were 1.17±0.52h,1.00±0.27h,0.54±0.29h,respectively.The Cmax of chlorogenic acid,hyperin and astragalin in SPP group were 20.41±4.06μg/L,14.54±3.12μg/L,3.53±1.69μg/L,respectively.The AUC(0-t)of chlorogenic acid,hyperin and astragalin in SPP group were 96.45±9.73μg/L*h,62.15±13.48μg/L*h,15.81±1.28μg/L*h,respectively.The AUC(0-∞)of chlorogenic acid,hyperin and astragalin in SPP group were 100.43±9.34μg/L*h,96.78±17.11μg/L*h,40.60±7.74μg/L*h,respectively.The Tmax of chlorogenic acid,hyperin and astragalin in WPP group were 0.83±0.20h,1.04±0.51h,0.83±0.13h,respectively.The Cmax of chlorogenic acid,hyperin and astragalin in WPP group were 66.40±13.55μg/L,22.32±1.50μg/L,13.11±1.09μg/L,respectively.The AUC(0-t)of chlorogenic acid,hyperin and astragalin in WPP group were 355.41±48.69μg/L*h,106.48±27.68μg/L*h,84.76±12.99μg/L*h,respectively.The AUC(0-∞)of chlorogenic acid,hyperin and astragalin in SPP group were 488.55±64.39μg/L*h,151.78±38.54μg/L*h,118.02±24.48μg/L*h,respectively.The relative absorptivity of chlorogenic acid was WPP>RP>SPP.The relative absorptivity of hyperin was RP>WPP>SPP.The relative absorptivity of astragalin was WPP>SPP>RP.7.Combined with the PK-PD model,the blood concentration of the three components of the three processed groups is higher,and the decrease of serum biochemical indexes in rats is more obvious.There is a more obvious correlation between the drug effect and the concentration of blood drug at about 1 hours.Conclusion:1.The content of flavonoids and polysaccharides were increased both in WPP and SPP.But the composition of main flavonoids and polysaccharides in three processed products were similar.2.All the RP,SPP and WPP have protective effect on the CCl4-hepatitis rat as well as alcholic-hepatitis rat.The effect of SPP and WPP was stronger than that of RP,while there was no significant difference between the SPP and WPP.The hepatoprotective effect of SPP and WPP may be related to the antioxidative effect and the regulation of cytokines.3.Three kinds of processed products of Semen Cuscutae have changed the potential biomarkers of chemical liver injury metabolism.The potential biomarkers of three products were similar,which were mainly lipid compounds,as well as amino acids,lysophosphatidylcholine and bile acids.Semen Cuscutae may play a protective role in liver by changing lipid metabolism and energy metabolism.4.Chlorogenic acid,hyperin and astragalin in CCl4-hepatitis rats showed a two compartment model.The pharmacokinetic parameters of three processed products of Semen Cuscutae were similar.The blood concentration of the three components is correlated with the efficacy of liver protection,which can be considered as an effective component of liver protection in Semen Cuscutae. |
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