| Lipid metabolism disorder of the body refers to the abnormality of lipids in plasma.It is often accompanied by metabolic dysfunction such as obesity,hypertension,hyperlipidemia and type 2 diabetes.Poria cocos(Schw.)Wolf has the effects of inducing excreting dampness,strengthening spleen,and sedative properties.As a traditional Chinese herbal medicine,PC also known as Fuling and enjoyed the reputation of “nine Fuling in ten prescriptions”.In the research,PC is rich in a variety of chemical components.Such as Triterpenoids,polysaccharides,sterols,volatile oils,et al.Among them,triterpenoids and polysaccharides are the major bioactive components of PC.Modern pharmacological research indicates that the components in PC,have been widely used in clinical treatment including diuretic,anti-oxidant,antitumor,anti-inflammatory,anti-bacterial,hepatoprotective effects.However,there are few reports on the correlation analysis between the fingerprint of serum samples and therapeutic effects.There are also no reports on the efficacy quality markers of PC in regulating lipid metabolism disorders.ObjectThe spectrum-effect relationship was used to discover the quality markers of PC in regulating lipid metabolism disorder.Besides,the pharmacodynamic activity of PC quality markers were explored through in vitro and in vivo experiments.MethodFirst,preliminary evaluation of the differences in chemical composition of PC from different origins: To establish the quantitative fingerprint of 16 batches of PC from different origins.Combined fingerprint with quantitative analysis of multiple components using a single marker(QAMS).To qualitative and quantitative analysis of PC from different origins.Methodological investigation and calculation of relative correction factor were also conducted.Subsequently,three types of the major bioactive chemical groups in 16 batches of PC from different places of origin were measured by UV–Vis spectrophotometry.Second,the efficacy of PC total triterpenoids in regulating lipid metabolism disorder in rats was evaluated: The total triterpenoid content extracted with PC was high dose group(168.9 mg/kg;PC-H),low dose group(56.3 mg/kg;PC-L)and fenofibrate(20 mg/kg;FC)as a positive drug in rat animal experiments.Serum and liver TG,TC,AST,ALT,HDL-C and LDL-C levels were measured after the experiment.Determining liver organ index;and oil red O staining was used to observe the changes of lipid droplets in liver cells.Furthermore,the spectrum-effect correlation analysis was performed to screen potential quality markers: Serum samples containing PC high dose group were analyzed by UPLC/Q-TOF-MS.Selected the common peak of rat serum containing drug in negative ion mode.A total of 28 peaks were obtained,and the fingerprint of serum containing drug was established.Serum TG,TC,AST,ALT,HDL-C and HDL-C levels of each rat in PC high dose group were taken as the pharmacodynamic part.SPSS software was used to normalize the common peak area and the related efficacy indicators.The spectrum-effect analysis was performed by SIMCA-P software.The PLS model was established to analyze the correlation between 28 common peaks(predictor variables)and six indicates(response variable)of hypolipidemic activity.the main active compounds were screened according to their VIP values and correlation coefficients.Then,carrying out cell experiment on the screened potential PC quality markers:Three concentrations of potential PC quality markers,low dose group(25 μmol/L),medium dose group(50 μmol/L)and high dose group(100 μmol/L),were applied to hepatic L-02 cells cultured with 5% medical fat emulsion for 24 h to replicate the L-02 steatosis cell model,and cell experiments were conducted with fenofibrate(150 μmol/L)as the positive control.The levels of intracellular TG,TC,AST and ALT were detected after the experiment.Finally,the screened two potential PC quality markers are subjected to animal experiments: The C57BL/6J male mice were fed with high-fat diet for 4 weeks to model.The mice were treated with pachymic acid low dose group(20 mg/kg/d;PA-L)and pachymic acid high dose(40 mg/kg/d;PA-H);dehydropachymic acid low dose group(20 mg/kg/d;DPA-L)and dehydropachymic acid high dose group(40 mg/kg/d;DPAH);fenofibrate(14 mg/kg/d)as the positive control was administered by intraperitoneal injection for 4 weeks.Serum and liver TG,TC,AST,ALT,HDL-C and LDL-C levels were measured after the experiment.Determining of organ index(liver,spleen,kidney);and oil red O staining was used to observe the changes of lipid droplets in liver cells.ResultsFirst,in this study,16 PC samples from different origins were collected,and 15 common peaks were obtained from the HPLC fingerprint.The similarity of the 16 batches ranged from 0.880 to 0.999.The precision,repeatability and stability of the method requirements of HPLC fingerprint.Calculation of the content of five triterpenoid by QAMS.There was no difference between the results determined by QAMS and the external standard method(ESM).In this study,established the quantitative fingerprint of PC,could reflect the fingerprint characteristics and chemical characteristics more comprehensively.The system of vanillin-glacial acetic acidphenol-concentrated sulfuric acid was used to determine the triterpenoids(TS),watersoluble polysaccharides(WSP),and acidic polysaccharides(AP).The method has good stability,precision,repeatability and recovery.The total triterpenoids content is 1.47%–5.87%,the water-soluble polysaccharide content is 0.48%–2.60%,and the acidic polysaccharide content is 50.81%–88.78%.Second,in animal experiments,the rat model with lipid metabolism disorder was successfully induced by high-fat diet feeding.After PC extract was given,the liver index of the rat was decreased,and the accumulation of lipid droplets in liver cells was decreased.PC-L/H group could significantly reduce the serum TC,TG,LDL-C and AST levels(P < 0.05).In addition,PC-L/H group showed a trend of increasing serum HDL-C levels.PC-L/H group could significantly reduce the liver TC and TG levels(P< 0.05).In the PC-L group,HDL-C level in the liver was significantly increased(P <0.05),and AST content was decreased(P<0.05).Third,after the spectrum-effect correlation analysis.Following these results,the17 peaks were considered as potentially active compounds for PC effect on lipid metabolism disorder,and eight were inferred.They are: No.4 peak dehydropachymic acid,No.8 peak 3-epidehydrotumulosic acid,No.9 peak tumulosic acid,No.11 peak polyporenic acid C,No.19 peak dehydrotumulosic acid,No.20 peak pachymic acid,No.21 peak dehydrotrametenolic acid,No.22 peak dehydroeburicoic acid.Fourth,the cell experiment of potential quality markers of PC showed that after the liver L-02 fatty degeneration cell model was given with the potential quality markers of PC,the levels of TC,TG,AST and ALT in cells were significantly reduced(P < 0.01,P < 0.05,P < 0.001).Fifth,animal experiments of the two screened PC quality markers showed that:after eight weeks of high-fat diet treatment,the mice with successfully induced lipid metabolism disorder were administrated with pachymic acid and dehydropachymic acid,respectively.And the results showed that the hepatomegaly and whitening of the mice with lipid metabolism disorder were appropriately alleviated.In addition,pachymic acid and dehydropachymic acid also reduced liver and serum levels of related lipid metabolism indexes: TC,TG,LDL-C,AST and ALT(P < 0.01,P < 0.05,P <0.001),and increased liver and serum HDL-C levels(P < 0.01,P < 0.05,P < 0.001).ConclusionsIn this study,the quantitative fingerprints of PC from different origins and the determination methods of three chemical groups(total triterpenoids,water-soluble polysaccharides and acidic polysaccharides)were established.Besides,the methods were preliminarily evaluated from multiple perspectives to the determination of the content,and the analytical method in the quality standard for PC was improved.Second,the study showed that PC total triterpenoids could effectively regulate lipid metabolism disorders by regulating lipid metabolism in liver and serum,and protecting liver injury.Furthermore,based on the research strategy of spectrum-effect correlation analysis,eight quality markers for regulating lipid metabolism disorder of PC were screened and identified,and the pharmacodynamic activity was evaluated by combining cell and animal experiments.Studies confirmed that the eight components can effectively improve liver L-02 cell steatosis.Among them,pachymic acid and dehydropachymic acid were found in vivo to be effective in regulating the lipid levels and transaminases contents in serum and liver,and reducing the lipid metabolism disorder in mice induced by a high-fat diet.The results will provide an important scientific basis for establishing a more scientific quality evaluation method of PC. |
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