| Objective:In this study,the “predict-verify” LC-MS analysis strategy was used to establish a target analysis method for the triterpenoid saponins of the kuding tea.The chemical structures of triterpenoid saponins were identified and characterized.Thus,the substantial foundation of the anti-hyperlipidemic effect of the triterpenoid saponins was explored.Then,target UHPLC-QTOF-MS/MS technology was used in qualitatively and quantitatively analyze the dynamic changes of lipid microenvironment on serum lipoprotein during the pathogenesis of hyperlipidemic golden hamster.At the same time,based on the differential expression levels of SR-related genes,the protective effect of triterpenoid saponins of the kuding tea on hyperlipidemic golden hamsters was preliminary ensured.Finally,based on the modern pharmaceutics theories and nano technologies,preparation process and prescription of chitosan-triterpenoid saponins nanosphere preparation was screened and characterized.In addition,its pharmacodynamic and pharmacokinetic evaluation were carried out.Therefore,our results provided the scientific basis for the clinical application of the anti-hyperlipidemic effect and further exploitation of the triterpenoid saponins of the kuding tea.Methods:Experiment 1 The study of identification and characterization of the triterpenoid saponins of the kuding tea1.The dried leaves of Ilex kudingcha were boiled to obtain the water extract of kuding tea.This water extract was further separated by HP20 SS Middle Chromatogram Isolated Gel to obtain the triterpenoid saponins' fraction of the kuding tea.2.According to literature survey,fragmentation pattern of the triterpenoid saponins of the kuding tea were summarized.Thus,the "prediction-verification" analysis strategy was used to predict the potential triterpenoid saponins and the compounds library of the triterpenoid saponins was established.1.The UHPLC-QTOF-MS/MS technology was used to identify and characterize the chemical construction of the triterpenoid saponins of the kuding tea.Experiment 2 Pharmacodynamics and mechanism study on the hypolipidemic effect of the triterpenoid saponins of the kuding tea1.The high-fat diet was used to establish a golden hamster model of hypercholesterolemia.After intragastric administration of the triterpenoid saponins of the kuding tea,the changes of its serum biochemical indexes were determined by reagent test kit.2.The UHPLC-QTOF-MS/MS analysis was used to establish the profile of serum lipoprotein Lyso-GPLs of hyperlipidemic golden hamsters.3.Using electrophoresis technology,the hyperlipidemic golden hamster' lipoproteins were extracted by agarose gel.Thus,the qualitative distribution and relatively quantitative changes of serum lipoproteins Lyso-GPLs in hyperlipidemic golden hamster were determined using above serum lipoprotein Lyso-GPLs' profile.4.In addition,the expression levels of SR-related genes were determined by real time RT-PCR.Based on the correlation analysis and multivariate statistics analysis,the lipidome analysis was explored and the Lyso-GPLs of hyperlipidemic golden hamster with strong relation to SR expression as a potential biomarker of anti-hyperlipidemia mechanism were screened for the triterpenoid saponins of the kuding tea.Experiment 3 Preparation of nano-microsphere of the triterpenoid saponins of the kuding tea,its pharmacodynamics and pharmacokinetics study1.With the single factor experiment and Box-Behnken design test,the preparation method of nano-microsphere was optimized.The optimal preparation process and prescription of nano-microsphere based on the chitosan and the triterpenoid saponins of the kuding tea was screened according to the above model fitting results.2.The physical and chemical property of prepared nano-microsphere were characterized.The pharmacodynamic evaluation of prepared nano-microsphere were investigated.3.The method for determination for the triterpenoid saponins of the kuding tea in vivo was established.The comparison on the pharmacokinetic study of prepared nano-microsphere with its prototype was investigated.Results:Experiment 1 The study of identification and characterization of the triterpenoid saponins of the kuding tea1.The triterpenoid saponins' fraction was obtained by eluting with 60%(V/V)aqueous ethanol solution.2.The the potential triterpenoid saponins of the kuding tea molecules were predicted and the 1881 compounds' library of the potential triterpenoid saponins established.3.Based on the auto-matching and manual verification,80 compounds of kuding tea TS were identified and characterized.In addition,11 of them seem to be novel TS from kuding tea(9 of them seem to be novel).Experiment 2 Pharmacodynamics and mechanism study on the hypolipidemic effect of the triterpenoid saponins of the kuding tea.1.The golden hamster model of hypercholesterolemia was established.The contents of serum TC and LDL-C of the gold hamster in model group were significantly increased than that of normal group.In addition,the content of serum HDL-C level was significantly decreased in model gold hamster.The above levels of the gold hamster in positive and triterpenoid saponins group showed callback.2.The profile for 338 Lyso-GPLs in the serum of hyperlipidemic golden hamsters was established.This profile including 189 Lyso-PCs,58 Lyso-Pes,22 Lyso-PSs,33 Lyso-Pas,28 Lyso-PIs and 8 Lyso-PGs.3.The aualitative and relative quantitative analysis of 251 Lyso-GPLs in the serum lipoproteins of hyperlipidemic golden hamsters was finished based above profile.The result is that the type and the abundance of Lyso-GPLs have changed significantly between normal group and model group.Compared with the total content of Lyso-GPLs in normal group,the total content of Lyso-GPLs in model group showed a significant increase.In addition,the Lyso-GPL contents of HDL was higher than that of LDL of normal group.On the contrary,the Lyso-GPL contents of HDL was lower than that of LDL of modern group.4.The expression levels of SR related genes(SR-A1,SR-B2 and CD36)in liver were determinded.The expression levels of SR-A1,CD36 and SR-B2 in the model group showed a significant increase than that of normal group.Furthermore,the expression levels of SR-A1 and CD36 in triterpenoid saponins group were significantly lower than that of the model group.In addition,four Lyso-GPLs(16:1/Lyso-PC,18:4/Lyso-PC,Lyso-PC/18:1,22:7/Lyso-PE)were screened as potential biomarkers for anti-hyperlipidemia mechanisms according to the correlation analysis and the multivariate statistical analysis on the relationship of the content of lipoprotein Lyso-GPLs and the expression levels of SR related genes.Experiment 3 Preparation of nano-microsphere of the triterpenoid saponins of the kuding tea,its pharmacodynamics and pharmacokinetics study1.Through single factor experiment and Box-Behnken design optimization experiment,the optimal preparation process and prescription of nanosphere preparation based on the chitosan and the triterpenoid saponins was screened according to the model fitting results.The best preparation process and prescription were as follow: concentration of CS,2.0 mg·m L-1;concentration of triterpenoid saponins,0.5 mg·m L-1;concentration of TPP,1.0 mg·m L-1;adding speed,4 m L·min-1;stirring rate,1200 r·min-1;shearing time,45 min;swelling time,6 h.2.The physical and chemical property of prepared nano-microsphere were characterized.The physical and chemical parameters were as follows: the Pd I was 0.192,the particle size was 193.6 nm,the Zeta potential was 26.3 m V,and the drug loading rate was 8.65%,the encapsulation efficiency is 72.07%,and the appearance of approximately spherical shape is observed by transmission electron microscope.The pharmacodynamic evaluation of prepared nanomicrosphere and prototype drug were compared and the anti-hyperlipidemic effect were investigated.3.The method for determination for the three saponins of kuding tea in vivo was established.The pharmacokinetics study of the prepared nano-microsphere in vivo was explored.By comparing the pharmacokinetic parameters,it showed that the time to peak(1.5h)of the prepared nano-microsphere was obviously faster than that of prototype drug(2.0h)after intragastric administration.In addition,the elimination half-life(t1/2)and AUC area(2.0 h and 6507 ng·h·m L-1)were larger than that of the prototype drug(1.2 h and 4242 ng·h·m L-1).These results indicating that the administration with the prepared nano-microsphere has higher bioavailability compared with the prototype drug.Conclusion:Using the “predict-verify” LC-MS analysis strategy,the identification and characterization on chemical composition of the triterpenoid saponins of the kuding tea was carried out.Then,the hyperlipidemic golden hamster model was established to test its lipid-lowering effect.In addition,the RT-q PCR technology was used to measure the expression levels of SR related genes among different group.By the established profile of Lyso-GPLs in serum lipoprotein of hyperlipidemic golden hamster,the biomarker of Lyso-GPLs' species were selected by their correlation of expression levels of SR related genes.Thus,the mechanism of lowering blood lipids for the triterpenoid saponins of the kuding tea was preliminarily clarified.Therefore,the preparation process of the nanomicrosphere based on the chitosan and the triterpenoid saponins of the kuding tea was determined.In addition,the pharmacodynamic and pharmacokinetic studies carried out confirmed that the nano-microsphere preparation could significantly increase the bioavailability and anti-hyperlipidemic activity.These research results would provide a new scientific basis of the clinical application and further utilization of the prevention on hyperlipidemia of the triterpenoid saponins of the kuding tea.It could provide a new scientific perspective for the prevention and treatment of hyperlipidemia by TCM on the regulation on serum lipoprotein lipid microenvironment. |
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