Screening Of Effective Lipid-lowering Ingredient Composition Of Jiangzhining And Study Of Its Mechanism Of Actio

Jiangzhining grains was composed of Radix Polygoni Multiflori Preparata,Fructus Crataegi,Folium Nelumbinis,and Semen Cassiae,which is known to reduce hematic fat levels and soften blood vessels,being clinically used for coronary circulation enhancement and arrhythmia/hyperlipidemia treatment.In the current quality standards,the qualitative identification of Jiangzhining grains is made by using ursolic acid and lotus leaf as control materials and the quantitative determination of it is made by using quercetin as content measurement index,while the curative effect of Chinese medicine compound is the whole manifestation of multiple effective components in Chinese medicine,it is impossible to control and evaluate the quality of Jiangzhining grains comprehensively and reasonably by using the index ingredients selected in the standards which has a poor correlation with antihyperlipidemic effect.Biological evaluation method which has unique advantages of related efficacy can be used to qualitatively or quantitatively evaluate the quality of tested drugs,and the establishment of this method relies on the study of pharmacological activity and action mechanism.Therefore,in this thesis,with antihyperlipidemic effect as the point of contact,the pharmacodynamics of the effective fraction of Jiangzhining are verified,and the qualitative and quantitative studies on chemical components and components in plasma of it are counducted to screen the effective ingredient combination of Jiangzhining.Furthersmore,the bioequivalence and mechanism of antihyperlipidemic effect of the effective ingredient combination of Jiangzhining are clarified which will lay a foundation for the establishment of biological evaluation methods.The main research contents and results are as follows:Part one:summary of the related literaturesThis chapter reviews the establishment of the biological evaluation method and its application in the field of traditional Chinese medicine.At the same time,and the future of traditional Chinese medicine quality control mode is prospected.Meanwhile,a total of 232 chemical components from Jiangzhining(JZN)and its component herbs are summarized.84 literatures are cited in this chapter.Part two:A chemical analysis of the effective fraction of Jiangzhining1.The antihyperlipidemic effect of the effective fraction of Jiangzhining was verified.Wistar rats were selected to establish hyperlipidemia model with high-fat diet.After succeeding,the effective fraction of Jiangzhining was administered by gavage for three weeks,and then the TC and TG were determined.Through statistical analysis,the TG and TC contents of model rats were increased significantly compared with the normal rats(P<0.01).Compared with model rats,the TG contents of different dose group of rats decreased significantly(P<0.05 or P<0.01)and no significant difference with positive control rats;the TC contents of medium,high dose group of rats decreased significantly(P<0.05)and no significant difference with positive control rats,which showed that the effective fraction of Jiangzhining has antihyperlipidemic effect.2.UPLC-ESI-Q-TOF-MSn was applied to investigate the chemical constituents of the effective fraction of Jiangzhining.A total of 45 components,including twelve alkaloids,four phenolic acids,thirteen flavonoids,five anthraquinones,two stilbene glucosides,three naphtha-pyrone glycosides,and six others(Vanillic acid-4-β-D-glucoside、Primulaverin or Primeverin、Cassitoroside、Lyoniresinol-3α-O-glucoside、Prupaside and Torachrysone 8-glucoside).And the composition of the effective ingredient combination of Jiangzhining was determined as follows:isoquercitrin,rutin,quercetin-3-O-glucuronide,(E)-2,3,5,4’-tetrahydroxystilbene-2-O-glucoside,armepavine,astragalin,hyperoside,emodin-8-O-glucoside,higenamine,nuciferine,N-nornuciferine,O-nornuciferine,aurantio-obtusin-6-O-glucoside,rubrofusarin-6-O-gentiobioside,cassiaside C,coclaurine and N-norarmepavine.3.The content determination method for the 17 compounds in the effective fraction of JZN by using UPLC-QqQ-MSn was established(seven alkaloids were in positive ion mode technology,the other compounds were in negative ion mode technology),and the validated method was used for the quantification of 17 representative compounds in 10 batches samples.The result of method validation showed that the established approach had a good linearity,precision,stability,repeatability and recovery.The determination results of 10 batches samples showed that the contents of the 17 ingredients differed greatly from each other.Quercetin-3-O-glucuronide was the most abundant(32.750-76.500 mg/g),being followed by(E)-2,3,5,4’-tetrahydroxystilbene-2-O-glucoside(20.950-28.200 mg/g),and nuciferine displayed the lowest amounts(0.017-0.096 mg/g).In addition,the contents of individual markers were broadly variable between different samples,which were probably caused by differences in the quality of medicinal materials and processing methods.On the one hand,the above content variation could be attributed to the different geographic locations of collected herbs,the corresponding environmental conditions,collection times,sources and storage conditions.On the other hand,some compounds could be damaged or transformed.during processing or exposure to high temperature.Finally,the proportion of the effective ingredient combination of Jiangzhining was determined as follows:cassiaside C:rubrofusarin-6-O-gentiobioside:aurantio-obtusin-6-O-glucoside:hyperoside:isoquercitrin:quercetin-3-O-glucuronide:(E)-2,3,5,4’-tetrahydroxystilbene-2-O-glucoside:rutin:emodin-8-O-glucoside:astragalin:armepavine:O-nornuciferine:coclaurine:N-nornuciferine:nuciferine:N-norarmepavine:higenamine was 3.30:16.06:9.15:23.94:98.40:417.45:189.68:8.62:1.28:5:3.51:14.57:1.06:1.35:1:5.64:6.06.Part three:Serum chemistry study of the effective fraction of Jiangzhining1.UPLC-MSn was applied to investigate components in plasma of the effective fraction of Jiangzhining.A total of 108 components was identified,a few of them wereprototype,while the rest were the demethylation,methylation,glucuronidation and sulfate metabolites.The metabolites identified above,most of them were isomers with different retention time,and it may be due to the combination of glucuronidation and sulfate with different positions of hydroxyl.2.The plasma sample content determination method for 14 ingredients absorbed into blood of the effective fraction of JZN using UPLC-MSn was established.Six alkloids were in positive ion mode technology with berberine selected as the internal and the plasma samples were treated with liquid-liquid extraction(ethyl acetate-acetone(10:1)).The other compounds were in negative ion mode technology with linarin selected as the internal standard and the samples were treated with solid phase extraction.The result of method validation showed that the established method had good method stability,short term stability,long term stability,freeze thaw stability,matrix effect and extraction recovery with high precision and accuracy,which accordance with the detecting requirements of the biological sample.The plasma concentration of rats with different time for administration(3,7 and 24 d)is detected by the established method,and the trend to change was analysed.The trend to change of plasma concentration of compounds may be related to its absorption,metabolism,accumulation,excretion,mutual transformation and enterohepatic circulation.Part four:A preliminary study on the bioequivalence and mechanism of the effective ingredient combination of Jiangzhining1.The effects of Jiangzhining granules,the effective fraction of Jiangzhining,the effective ingredient combination of Jiangzhining,simvastatin and oleic acid on the activity of HepG2 cell were investigated by CCK-8 assay.And the maximum non-toxic dose of Jiangzhining granules,the effective fraction of Jiangzhining,the effective ingredient combination of Jiangzhining,simvastatin and oleic acid were 100 μg/mL、40 μg/mL、40μg/mL,3 μmol/L and 0.25 mmol/L,respectively..2.Using OA-induced HepG2 cell,this paper studied the effects of different concentrations of Jiangzhining granules,the effective fraction of Jiangzhining,the effective ingredient combination of Jiangzhining on the accumulation of TC and TG in cells.The results showed that,compared with the normal control group,OA increased significantly the levels of TC and TG;treating Jiangzhining granules,the effective fraction of Jiangzhining,the effective ingredient combination of Jiangzhining,the accumulation of TG and TC in HepG2 cells induced by oleic acid was inhibited and dose dependent,and the trends of them were consistent.In addition,there was no significant difference between the three groups in the corresponding dose group,indicating that there was certain equivalence in the pharmacodynamic effect.3.The effects of the effective ingredient combination of Jiangzhining(4,12 and 40μg/mL)on the mRNA expression of LDL-R、HMG-CoA、CYP7A1、SREBP-1c and AMPKα1 mRNA were studied by qRT-PCR.The results showed that,compared with the normal control group,OA(3 μmol/L)decreased the expression levels of LDL-R、CYP7A1 and AMPKα1 mRNA,increased the expression levels of HMG-CoA and SREBP-1c mRNA;treating the different concentrations of the effective ingredient combination of Jiangzhining,expression levels of L LDL-R、CYP7A1 and AMPKal mRNA were increased and the expression levels of HMG-CoA and SREBP-1c mRNA was reduced,and dose dependent was presented.These results exhibited that the effective ingredient combination of Jiangzhining exerted an antihyperlipidemic effect by regulating expression levels of LDLR、HMG-CoA、CYP7A1、SREBP-1c and AMPKαl mRNA.Innovative:1.UPLC-MSn was employed for first-time chemical profiling of the effective fraction of Jiangzhining and its components in plasma.2.UPLC-QqQ-MSn was used for first-time quantification of the effective fraction of Jiangzhining and its components in plasma.3.The effective ingredient combination of Jiangzhining was selected and determinate first-time and its bioequivalence and action mechanism was clarified.