| With the classical small prescription "Si-Ni-San" as research object, its therapeutic basis for anti-liver injury and compatibility mechanism were investigated based on the theory of traditional Chinese medicine by using the whole prescription, drug-pairs,each single drugs and pure chemical compounds.1. Study on the chemical constituents of Si-Ni-SanThe chemical constituent chromatograms of the whole prescription, drug-pairs and each single drugs were established. LC-MS method was developed to identify the chemical constituents in Si-Ni-San. There were 31 chromatographic peaks detected. Comparing with reference substances, paeoniflorin, naringin, hesperidin, neohesperidin, glycyrrhizic acid,naringenin, saikosaponin a and glycyrrhetinic acid were affirmed. The other chemical constituents were identified based on molecular weight information, MS/MS data and literature.Comparing the chemical constituent fingerprints in different groups, no new peak was found.However,the contents of paeoniflorin, naringin, hesperidin, neohesperidin, glycyrrhizic acid,naringenin and saikosaponin a in Si-Ni-San were significantly higher than those in each single drugs. The contents of above 7 chemical constituents in drug-pairs were higher than those in each single drugs to varying degrees.2. Study on the dynamic changes of the constituents of Si-Ni-San migrating into bloodDrug-containing plasma chromatograms of Si-Ni-San were established to monitor the changes of the constituents migrating into blood. A UPLC-MS/MS method was developed to analyse the components and metabolites in plasma. Comparing with reference substances and data in literature, 23 components were detected in rat plasma after oral administration of Si-Ni-San decoction. Paeoniflorin, liquiritin, isonaringin, naringin, hesperidin, neohesperidin,glycyrrhizic acid, ononin, naringenin, saikosaponin a, nobiletin and glycyrrhetinic acid are prototype components in Si-Ni-San. Ten metabolites are sulfate conjugate of liquiritigenin,glucuronide and sulfate conjugate of naringenin, glucuronide and sulfate conjugate of hesperetin,glucuronide conjugate of naringenin, glucuronide conjugate of hesperetin, glucuronide conjugate of hesperetin, sulfate conjugate of liquiritigenin-dihydrochalcone, glucuronide conjugate of liquiritigenin and hydroxyglycyrrhetinic acid. The dynamic changes of the components and metabolites in different time were studied. The prototype components absorbed into the blood were supposed to be the therapeutic basis of Si-Ni-San.3. Study on the anti-liver injury effect of Si-Ni-SanThe rats were randomly divided into eight groups, which were control group, model group,silymarin group (30 mg/kg), Si-Ni-San group (40 g/kg), Chai-Shao group (20 g/kg), Chai-Zhi group (20 g/kg), Shao-Zhi group (20 g/kg) and Shao-Gan group (20 g/kg). Liver injury model was prepared with the intraperitoneal injection of 40% CCl4 (3 mL/kg) for one time after administration of drugs for 5 days. The blood and liver tissues were obtained 24 h after injection,the content of ALT, AST, TB, TP and ALB in the serum, MDA, SOD and GSH-Px in the liver were measured. The pathological observation of liver tissues was conducted. The administration of Si-Ni-San and its four drug-pairs decreased the content of serum transaminase, protect hepatic cell and liver function, and relieved liver tissues damage. Chai-Shao pair protected liver excretoryfunction. Shao-Gan pair improved liver synthetic ability and anti-radicals activity, and showed higher antioxidation ability. Si-Ni-San group performed the best effect on acute liver injury. The reasonability of the compatibility of Si-Ni-San was elucidated by pharmacodynamics.4. Study on the relationship of chromatogram and efficacy of Si-Ni-SanBy the correlation analysis and the stepwise regression between the chemical and pharmacologic data, it was found that paeoniflorin, naringin, saikosaponin a and glycyrrhetinic acid were defined as therapeutic basis for anti-liver injury of Si-Ni-San. Saikosaponins a,paeoniflorin, glycyrrhizic acid and glycyrrhetinic acid could decrease the content of serum transaminase, protect hepatic cell. Saikosaponin a could decrease the content of TB, improve liver excretive ability. Liquiritin, glycyrrhizic acid, ononin and glycyrrhetinic acid could increase the content of TB in the serum and SOD and GSH-Px in the liver, improve liver synthetic ability,strengthen the enzyme activity of clearing the radicals and improve liver cells antioxidant ability.The constituents in immature could decrease the content of MDA in the liver, depress the peroxidation action.5. Study on pharmacokinetics of active components in rats after oral administration of Si-Ni-San decoctionA UPLC-MS/MS method was developed for the simultaneous determination of paeoniflorin,naringin,naringenin and glycyrrhetinic acid in rat plasma. A Waters BEH C18 column was used with a gradient mobile phase system of methanol-water containing 2 mmol/L ammonium acetate.The analysis was performed on a positive ionization electrospray mass spectrometer via multiple reaction monitoring (MRM). One-step protein precipitation with acetonitrile was used to extract the analytes from plasma. The limits of quantification were 9.80 ng/mL for paeoniflorin, 5.10 ng/mL for naringin, 5.20 ng/mL for naringenin and 10.6 ng/mL for glycyrrhetinic acid,respectively. The selectivity, linearity, precision, accuracy, extraction recovery and stability of the method were satisfactory. The validated method was applied to a comparative pharmacokinetic study in rats after oral administration of pure chemical compounds, each single drugs and Si-Ni-San decoction. The results showed that the absorption of paeoniflorin and the transformation of paeoniflorin to its metabolites were increased after administration of radix paeoniae alba and Si-Ni-San decoction; the transformation of naringin to its metabolite naringenin was increased after administration of Si-Ni-San decoction, the absorption of naringin was accelerated after administration of fructus aurantii immaturus; the absorption of glycyrrhetinic acid and the transformation of glycyrrhizic acid to glycyrrhetinic acid were increased after administration of glycyrrhiza, the bioavailability and the transformation of glycyrrhizic acid to glycyrrhetinic acid were futher increased after administration of Si-Ni-San decoction. |
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