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OBJECTIVE：To i nvestigate the metabolism stabilities of novel hypoglycemic compound LSM- 13 in rat liver microsomes，and to analyze the possible metabolites. METHODS ：LSM-13 was dissolved in rat liver microsome incubation system initiated by reduced nicotinamide adenine dinucleotide phosphate ，and was incubated in water at 37 ℃. The reaction was terminated with acetonitrile at 0，5，10，15，30，45 and 60 min，respectively. Using indomethacin as internal standard ，the concentration of LSM-13 in incubation system was determined by HPLC. The residual percentage and enzyme kinetic parameters of LSM- 13 were calculated at different incubation time points with the concentration of LSM- 13 incubated for 0 min as reference. UPLC-Q-TOF/MS was used to analyze and speculate the metabolites of LSM- 13 in rat liver microsomes. RESULTS ：After 60 min incubation ，the remaining percentage of LSM- 13 was（56.07±0.95）％，the half-life was 42.78 min，and the intrinsic clearance was 0.032 4 mL/（min·mg）. Compared with total ion flow diagram of rat liver microsome blank samples ，three chromatographic peaks were added in the samples incubated for 60 min；the corresponding molecular ion peaks were m/z 505.133 8，417.102 4，293.111 7 [M+H]+；the possible metabolites may be dehydrogenation ，O-debentylation and hydrolysis products of LSM- 13. CONCLUSIONS ： The compound LSM- 13 has moderate stability in rat liver microsomes ，and may undergo dehydrogenation ，O-debentylation and hydrolysis.
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AIM To study the interactions of component (group) compatibility of Sanwu Huangqin Decoction in incubation model of rat liver microsomes.METHODS With maackiain and ephedrine as internal standards,HPLC was adopted in the simultaneous content determination of total tlavonoids (baicalin,wogonoside,baicalein,wogonin and oroxylin A) from Scutellariae Radix and total alkaloids (oxymatrine,oxysophocarpine and matrine)from Sophorae flavescentis Radix in whole prescription component (group) compatibility and single medicinal material part (group) at seven time points (0,15,30,45,60,90 and 120 min),followed by the calculation of their in vitro metabolic rates.RESULTS In whole prescription component (group) compatibility,the contents of baicalin and wogonoside were first increased (0-0.5 h) and then decreased (0.5-2.0 h),those of baicalein,wogonin and oroxylin A showed increasing trends (more obvious within 0.5 h).The stable and gende metabolisms of various alkaloids reached balance within 20 min.CONCLUSION The bioavailability improvement and efficacy enhancement of total flavonoids from Scutellariae Radix may attribute to the compatibility with total alkaloids from Sophorae flavescentis Radix and total polysaccharides from Rehmanniae Radix in Sanwu Huangqin Decoction
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AIM To study the interactions of component (group) compatibility of Sanwu Huangqin Decoction in incubation model of rat liver microsomes.METHODS With maackiain and ephedrine as internal standards,HPLC was adopted in the simultaneous content determination of total tlavonoids (baicalin,wogonoside,baicalein,wogonin and oroxylin A) from Scutellariae Radix and total alkaloids (oxymatrine,oxysophocarpine and matrine)from Sophorae flavescentis Radix in whole prescription component (group) compatibility and single medicinal material part (group) at seven time points (0,15,30,45,60,90 and 120 min),followed by the calculation of their in vitro metabolic rates.RESULTS In whole prescription component (group) compatibility,the contents of baicalin and wogonoside were first increased (0-0.5 h) and then decreased (0.5-2.0 h),those of baicalein,wogonin and oroxylin A showed increasing trends (more obvious within 0.5 h).The stable and gende metabolisms of various alkaloids reached balance within 20 min.CONCLUSION The bioavailability improvement and efficacy enhancement of total flavonoids from Scutellariae Radix may attribute to the compatibility with total alkaloids from Sophorae flavescentis Radix and total polysaccharides from Rehmanniae Radix in Sanwu Huangqin Decoction
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Objective Re Du Ning Injection (RDN), a Chinese materia medica injection, is made from the extracts of Lonicerae Japonicae Flos, Gardeniae Fructus, and Artemisiae Annuae Herba. Since last decade, RDN has been widely used in China for the treatment of viral infection, fever, and inflammation. To assess the potential interacting of RDN with co-administered drugs, the inhibitory effects of RDN on the enzyme activities (CYP1A1, CYP1A2, CYP2C11, CYP2D1, and CYP3A1/2) of rat liver microsomes were investigated by a cocktail method. Methods A sensitive and specific LC-MS method capable of simultaneous quantification of five metabolites in rat liver microsomes was developed and validated. Then RDN (0.625%–1.0%) was incubated with rat liver microsomes and specific substrates. The enzyme activities were expressed as the formation rate of the specific metabolites of the substrates (pmol · mg · protein−1 · min−1). Results RDN competitively inhibited the activities of CYP1A2 and CYP2C11, with inhibition constant (Ki) values determined to be 0.18% and 0.63%, respectively. RDN exhibited the mixed inhibition on the activity of CYP2D1, with a Ki value of 0.15%. The activities of CYP1A1 and CYP3A1/2 were not markedly inhibited even by 1.0% RDN. Conclusion RDN could inhibit the rat enzyme activities of CYP1A2, 2C11, and 2D1 in vitro with different inhibition modes, which is worthy of promoting safety and efficacy of RDN.
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This study aimed to investigate the effects of tannins extracted from Tibetan medicine Phyllanthi Fructus on cytochrome P450 enzyme of liver microsomes in rats.Cocktail probe substrates were incubated with liver microsomes in vitro.Metabolic elimination percentages of the six probe substrates,including dapsone,dextromethorphan hydrobromide,coumarin,phenacetin,chlorzoxazone and tolbutamide,were determined by HPLC.The effects of tannins extracted from Tibetan medicine Phyllanthi Fructus on the enzymatic activity of rat liver microsomal P450s was evaluated.It was found that tannins extracted from Phyllanthi Fructus did not impact P450 enzymes.The IC50 values of CYP3A4 and CYP1A2 were over 200 μg·mL-1,while the IC50 value of CYP2C9 was superior to 500 tg·mL-1.In conclusion,Tannins extracted from Tibetan medicine Phyllanthi Fructus did not significantly affect cytochrome P450 enzymes.
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This study was aimed to investigate the induction effect ofRe-Du-Ning (RDN) Injection on rat liver microsome CYP450 enzymes. SD rats were randomly divided into the solvent control group, positive control group as well as the low, middle and high dose group of RDN (1, 2, 4 mL·kg-1·d-1). After drugs were administrated continuously for 7 days, the rats were sacrificed. The liver was weighed and prepared to microsomes. Meanwhile, the liver coefficients of rats were calculated. And the protein content was detected by BCA method. Finally, activities of five important subtypes of CYP450 enzymes such as CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A1/2 were measured by the“cocktail” method. The results showed that the levels of liver coefficients, microsome yield rate and activities of CYP450 subtypes increased significantly in the positive control group compared with the solvent control group (P < 0.01). There was no significant difference on the levels of liver coefficients, microsome yield and protein content between the low and middle dose group of RDN. However, there was significant difference on the levels of liver coefficients and microsome yield in the high dose group (P < 0.05). In terms of the influence on enzyme activity, RDN Injection can significantly induce the activities of CYP1A2 with dose dependence. It can induce the activities of CYP2C9 and CYP2C19 at the middle and high dose. However, there was no obvious influence on the activities of CYP3A1/2 and CYP2D6. It was concluded that the positive control group can obviously induce activities of CYP450, which can be used in the evaluation of induction experiments. RDN Injection had induction effect on CYP1A2, CYP2C9 and CYP2C19. But it had no influence on the activities of CYP3A1/2 and CYP2D6.
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OBJECTIVE: To establish a highly effective HPLC method for analyzing 1-chlorine-2,4-dinitrobenzene (CDNB), and to determine the activity of GST enzyme in rat liver microsomes (RLM) by use of CDNB as probe and study its enzyme kinetics. METHODS: Welch Materials Ultimate™ XB C18 column (4.6 mm × 250 mm, 5 μm) was used, the mobile phase was acetonitrile-water(7:3), the flow rate was 0.8 mL · min-1, and the detection wavelength was 238 nm. CDNB was incubated with 0.02 mg · mL-1 protein in RLM at 37°C for 7 min and the reaction was terminated by cool acetonitrile. The reaction solution was centrifugated and the supernatant was filtered and analyzed by HPLC. The Vmax, Km and CLint were calculated by Sigma Plot. RESULTS: The retention time of CDNB was about 6 min without any interference. The lowest detection limit of CDNB was 1.0 μmol · L-1, and the calibration curve was linear from 2.5 to 100.0 μmol · L-1. The intra-day and inter-day relative standard deviations were less than 10% respectively. The method recoveries ranged from 99.38% to 108%. The reaction catalyzed by GST was terminated by acetonitrile and the incubation time was 7 min. The kinetic parameters, Vmax, Km, and CLint were 85.45 nmol · min-1 · (mg protein)-1, 15.09 μmol · L-1, and 5.73 mL · min-1 · (mg protein)-1, respectively. CONCLUSION: This method is reliable and the results can ac-curatelly indicate GST enzyme activity, which can be used for the kinetics study of GST in RLM.
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Objective: To study the enzymatic reaction kinetics of dl- tetrahydropalmatine (TET) in total alkaloid (TA) of Corydali Rhizoma in rat liver microsomes and to investigate the compatible effects of the effective components such as coumarin (Cou) and volatile oil (VO) in Angelicae Dahuricae Radix with TA in Corydali Rhizoma on enzymatic reaction kinetics of TET. Methods: Rat liver microsomes were prepared by ultracentrifugation and the TET concentration in incubation medium was determined by HPLC. Comparative study on the enzymatic reaction kinetics of TET in each group of TA, TA-Cou, TA-VO, and TA-Cou-VO to deduce the michaelis constant (Km) and maximum reaction rate (Vmax) of TET in each group and calculate the clearance rate (CLint) of TET in each group. Results: The Km, V max, and CLint in TA group were 0.12 μmol/ (L·min·mg), 5.40 μmol/L, and 0.022 L/(min·mg), respectively; In TA-Cou group they were 0.27 μmol/(L·min·mg), 40.18 μmol/L, and 0.006 L/(min·mg), respectively; In TA-VO group they were 0.57 μmol/(L·min·mg), 22.60 μmol/L, and 0.025 L/(min·mg), respectively; In TA-Cou-VO they were 0.84 μmol/(L·min·mg), 23.25 μmol/L, and 0.036 L/(min·mg), respectively. Conclusion: The effective components of TA in Corydali Rhizoma with Cou and VO in Angelicae Dahuricae Radix could decrease CLint of TET in TA of liver.
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Buddleja globosa Hope (matico) is a medicinal shrub native to Chile whose leaves have been traditionally used for wound and ulcer healing, pathologies associated to oxidative stress. Matico leaves display a high content of polyphenols, compounds with recognized antioxidant capacity, which may contribute to its therapeutic properties. Several factors, however, can modify the polyphenol content of matico leaf extracts, including plant material, production techniques, provenances, leaf age, harvest time, irrigation, and desiccation procedures. Thus, standardized leaf extracts prepared with plants from different provenances and harvest conditions were compared in terms of polyphenol content and their protecting antioxidant effects on rat liver microsomal lipids and thiol groups. All factors tested, but irrigation, changed both polyphenol content and antioxidant properties of matico extracts; water stress only affected their antioxidant properties without changing their polyphenol content. Correlation between polyphenol content and lipid peroxidation inhibition was only significant in the provenance study.
Buddleja globosa Hope (matico) es un arbusto medicinal nativo de Chile cuyas hojas han sido utilizadas en la medicina tradicional como cicatrizante en caso de patologías relacionadas con el estrés oxidativo. Las hojas tienen un alto contenido de polifenoles, compuestos con reconocidos efectos antioxidantes relacionados con la inhibición de la lipoperoxidación. Varios factores pueden afectar a su contenido, entre ellos el origen de la planta, edad de la hoja, momento de cosecha, riego y métodos de secado. En extractos estandarizados preparados de hojas de diferentes tratamientos se compararon el contenido de polifenoles y los efectos antioxidantes protectores de lipidos y grupos tioles microsomales. Todos los ensayos de cultivo y postcosecha mostraron diferencias significativas entre los tratamientos, excepto para la inhibición de lipoperoxidación en el tratamiento de riego. Plantas de diferente origen muestran que el contenido de polifenoles en las hojas es determinado genéticamente y sufre variaciones por efectos ambientales.