| Background:The coronary heart disease is a common and frequently-occurring disease, which severely threatens human health and affects the life quality of patients in varying degrees. Shuangshentongguan Formula (Corydalis Rhizoma total alkaloids, Ginseng total saponins and total salvianolic acid composition) is a component compatibility compound Chinese medicine, which are researched and developed by our research group. So far, a lot of prescription screening, pharmacodynamics, and pharmacokinetics studies have been carried out. However, few druggability prediction have been released. Especially, metabolic drug-drug interactions should be also explored for investigational drug safety in the druggability analysis.Objectives:1.In order to obtain a sufficient amount candidate compound, the target alkaloids were isolated and purified from constituents of Corydalis Rhizoma, and the target ginsenosides were isolated and purified from Ginseng Caulis et Folium.2.According to the FDA’s Guidance for Industry (draft)—Drug Interaction Studies. CYP1A2, CYP2C9. CYP2C19, CYP2D6, and CYP3A4isoenzymes were selected to study inhibitory effects of Corydalis Rhizoma alkaloids and ginsenosides entered the systemic circulation in Shuangshentongguan Formula on the CYP isoenzymes activities.3.To study biotransformation of corydaline by liver microsomes.4.To study inhibitory effects of Corydalis Rhizoma alkaloids and ginsenosides entered the systemic circulation in Shuangshentongguan Formula on the nitric oxide (NO) production using lipopolysaccaride (LPS)-activited BALB/c murine macrophage-like RAW264.7cells model.Methods:1.The alkaloids in Corydalis Rhizoma and the ginsenosides in Ginseng Caulis et Folium were isolated and purified by the methods of solvent extraction and different types of column chromatography. Their structures were elucidated on the basis of their physico-chemical properties and spectral data.2.The assay compounds (alkaloids in Corydalis Rhizoma and the ginsenosides in Ginseng) at a series of concentrations were incubated with human liver microsome in the presence of NADPH and a group of CYP probe substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6and CYP3A4. CYP isoform-specific inhibitors were assayed in parallel as positive controls. The relative activities of CYP isoforms were determined by analyzing the formation o f the substrate metabolites using LC-MS techniches. The half maximal inhibitory concentration (IC50) value was subsequently calculated for each CYP isoforms assayed. The inhibitory potencies of assayed compounds on CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4were then evaluated.3.A typical1000mL biotransformation incubation mixture consisted of100mL of rats liver microsomes (21.39mg protein/mL),100mM PBS (pH7.4) and300mg of corydaline as substrate. After preincubation for5min, the reaction was initiated by adding an NADPH generating system (1.0mM NADP,0.5mM NADH,10mM glucose-6-phosphate,1000IU/mL glucose-6-phosphate dehydrogenase and4.0mM MgCl2). Incubations were carried out at37℃for2h with continuous shaking in a Dubnoff incubator. Reactions were terminated by adding1000mL of ice-cold EtOAc, followed by extraction. The organic layer was collected and the residual water layer was extracted by adding1000mL of EtOAc for seven times. The EtOAc extract was combined and then were evaporated in vaccum, ollowed by dryness under gentle N? gas stream to give residues2.8g. The residues2.8g were separated and purified by silica gel column chromatography and prepared HPLC to afford biotransformation product. The chemical structure of the product was determined on the basis of their NMR and MS data analyses.For the analytical biotransformation of corydaline by rat or human liver microsomes assay, a typical500uL of incubation mixture in PBS (pH7.4) consisted of5μL of rat human liver microsome (21.39mg protein/mL) or human liver microsome (0.2mg protein/mL),100mM PBS (pH7.4), and0.1μ.M of corydaline as substrate. After5min of pre-incubation at37℃, the reaction was initiated by adding NADPH-generating system, and shaken gently for another5,10and30min, respectively. The reactions were terminated by adding100μL of ice-cold methanol-acetonitrile (1:4, v/v), which contained caffeine as an internal standard, followed by centrifugation for10min at16000×g and4℃. The supernatant was evaporated to dryness under gentle N2gas stream to give residues. The residues were resuspended in100μL of10%acetonitrile aqueous with vortexing, followed by filtration through a0.45μm pore size membrane filter. A10μL aliquot was injected into the LC-MS system for analyses. All the in vitro incubations were performed in duplicate.4.In BALB/c murine macrophage RAW264.7cells, LPS stimulation alone has been demonstrated to increase NO production. This cell system is an excellent model for drug screening and the subsequent evaluation of potential inhibitors against NO production. Using this cell model, the inhibitory effects of the isolated alkaloids from Corydalis Rhizoma and ginsenosides from Ginseng Caulis et Folium against NO production were evaluated. Since NO is a very labile molecule with a very short halflife. NO production was assayed indirectly by measuring the accumulation of nitrite according to a published spectrophotometric method.Results:1.Nineteen alkaloids were isolated and purified from Corydalis Rhizoma and identified as tetrahydrocoptisine (1),tetrahydrocolumbamine (2),tetrahydropalmatine (3), corybulbine (4), isocorybulbine (5),8-trichloromethyl-7,8-dihydrocoptisine (6), corydaline (7),8-oxocoptisine (8),(-)-corypalmine (9), dehydrocorydaline (10),13-methylpalmatrubine (11), oxoglaucine (12), protopine (13), noroxyhydrastinine (14), tetrahydroberberine (15), didehydroglaucine (6a,7-didehydroglaucine,16), pontevedrine (17), coptisine (18), palmatine (19), and berberine (20), Eight saponins were isolated and purified from Ginseng Caulis et Folium and determined as20S"-ginsenoside Rg1(21), ginsenoside Rb1(22), ginsenoside Re (23),20S-ginsenoside Rh1(24),20R-ginsenoside Rh1(25),20S-ginsenoside Rg2(26),20R-ginsenoside Rg2(27), and ginsenoside Rd (28).2. In vitro assay, ginsenosides Rb1, Rd, Re, and Rg1exhibited no inhibition against human liver microsomes CYP1A2. CYP2C9, CYP2C19, CYP2D6. and CYP3A4activities with an IC50value of more than200μM. In alkaloids of Corydalis Rhizoma. corydaline showed selectively inhibitory effect against CYP2C9and CYP2C19, tetrahydropalmatine showed selectively inhibitory effect against CYP2D6, while didehydroglaucine showed the inhibitory effect against all of the five CYP subtypes with an IC50value of less than1μM.3.After corydaline was incubated with rat hepatic microsomes pretreated with sodium phenobarbital in an NADPH-generating system in vitro, seven biotransformation products were obtained and their structures were elucidated by NMR as9-O-desmethylcorydaline,9,10-di-O-desmethylcorydaline, yuanhunine,4-hydroxycorydaline,5-hydroxycorydaline, corybulbine, and isocorybulbine. The major biotransformation was9-O-desmethylcorydaline.4.Ginsenosides Rb1, Rd, Re, Rf, Rg1, Re5, and ginsenjilinol showed inhibition in a concentration-dependent manner against NO production in lipopolysaccaride-activated murine monocyte-macrophage RAW264.7with an IC50value of69.67±3.28.62.41±3.02.85.59±2.43,74.14±2.65,80.89±2.00,79.83±1.78, and70.96±2.05μM, respectively. Indomethacin was used as a positive control with an IC50value of63.75±3.33μM. The selected protoberberine-type alkaloid, tetrahydropalmatine,8-oxocoptisine, dehydrocorydaline, and oxoglaucine exhibited the moderate inhibitory activity against NO production with an IC50 value of218.02±1.50,161.37±7.88and133.29±4.81μM, respectively. Whereas aporphine-type alkaloid, oxoglaucine showed the potential inhibition with an IC50value of46.04±1.99μM, which the inhibitory activity was stronger more than that of indomethacin.Conclusions:1.The alkaloids in Corydalis Rhizoma were main divided into the two types of protoberberine and aporphine, which latter is a derivatives of morphine. A series of ID and2D NMR techniques were especially applied to the assignment of all proton and carbon signals of8-trichloromethyl-7,8-dihydrocoptisine and didehydroglaucine for the first time. And8-trichloromethyl-7,8-dihydrocoptisine,(-)-corypalmine and didehydroglaucine were isolated from Corydalis Rhizoma for the first time.2.For ingredients of Shuangshentongguan Formula entering systemic circulation, the no clinical significant metabolic interaction seems likely to occur between the CYP enzymes and ginsenosides (ginsenosides Rb1, Rd, Re, and Rg1) tested because these ginsenosides exhibited no inhibition against human liver microsomes CYP1A2, CYP2C9, CYP2C19. CYP2D6, and CYP3A4. Potential drug-drug interactions should be considered when drugs contained corydaline and tetrahydropalmatine are used, which corydaline showed the selectively inhibitory activity against CYP2C9and CYP2C19with an IC50value of36.81and25.54μM. respectively, and tetrahydropalmatine exhibited the selectively inhibitory activity against CYP2D6with an IC50value of11.26μM. Didehydroglaucine showed the inhibitory activity against all of the five CYP subtypes with an IC50value of less than1μM.3.The major biotransformation product of corydaline in rat hepatic microsomes in vitro was9-O-desmethylcorydaline. Corydaline was primarily metabolised by O-demethenylation and hydroxylation.4.The results from ginsenosides Rb1, Rd,Re, and Rg1showing inhibition against NO production indiceted that Shuangshentongguan Formula perhaps exerted beneficial anti-inflammatory effects and treatment of cardiovascular disease. |
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