| 英文摘要 |
Objective: Tangzhiqing formula, a traditional Chinese medicine, was composed of five crude drugs: Nelumbo nucifera Gaertn., Morus alba L., Paeonia lactiflora Pall, or Paeonia veitchii Lynch, Salvia miltiorrhiza Bge. and Crataegus pinnatifida Bge. var. major N.E.Br. or Grataegus pinnatifida Bge. Tangzhiqing formula could improve the clinical symptoms of patients with prediabetes, had the effect of reducing fasting and postprandial blood glucose, regulating blood lipids. Hence, it was suitable for patients with disorder of glycolipid metabolism. Furthermore, Tangzhiqing formula has obtained approval documents of clinical trials in 2010. Lotus leaf was the dry leaves of Nelumbo nucifera Gaertn. Lotus leaf was the main component of the Tangzhiqing formula, and it was also a representative drug of bitterness that could reduce blood glucose and adjust lipid metabolism. Modern research indicated that lotus leaf alkaloids were the main active ingredients. Therefore, this study took the lotus leaf as the research object, and discussed the hypothesis that “the lotus leaf alkaloids exhibits the hypoglycemic and lipid-lowering effect in the form of metabolites in vivo”, and in order to explain the association of the “active components-in vivo form-mechanism” of the lotus leaf. Moreover, this study could understand material basis and mechanism of action of Tangzhiqing formula. And provide scientific basis for its rational application. Method: (1) Separation of chemical constituents from lotus leaves: The lotus leaves were extracted by heating with Et0H/H₂O, and evaporated of the solvent and partitioned with different polar solvents to obtain alkaloids and flavonoids. They were separated and purified by column chromatography and recrystallization to prepare compounds. The structure was identified by MS, NMR, etc., in combination with its physical and chemical properties. (2)Studyon metabolites of nuciferine in normal rats: The rats were administered with nuciferine, and plasma, urine, feces and bile samples were collected at different time points. The UPLC-Q-TOF/MS method was developed to characterize the metabolites in rat biological samples through the retention time and MS fragmentation behavior of nuciferine. (3)Studyon pharmacokinetics and pharmacodynamics of lotus leaf alkaloids in rats with disorder of glycolipid metabolism: ZDF rats were divided into blank group, model group, low and high drug groups. Rats of the drug groups were given lotus leaf total alkaloids for 4 weeks. Then plasma samples were collected at different time points and level of glucose, triglycerides and cholesterol was measured. Nuciferine, O-nomuciferine and N-nomuciferine were used as analytical indicators to establish an UPLC-MS/MS method to detect plasma drug concentration. In addition, the correlation of pharmacokinetics and pharmacodynamics was analyzed. (4)Studyon the mechanism of glucose lipid metabolism in 3T3-L1 adipocytes by lotus leaf alkaloids: Nuciferine and its major metabolites (N-demethylolaline and O-demethylolaine) were considered to affect the mechanism of glycolipid metabolism through 3T3-L1 adipocytes. Firstly, three alkaloids were tested for influence on cell proliferation via MTT. Secondly, insulin resistant 3T3-L1 adipocytes were established. The optimum incubation time and concentration of the alkaloids were measured by intracellular glucose uptake. And the alkaloids were detected for their effects on intracellular triglyceride levels at this concentration and time. Finally, the effects of alkaloids on related gene and protein expression were analyzed by qRT-PCR and Western blotting. (5)Studyon metabolites of Tangzhiqing formula in normal rats: The rats were administered with Tangzhiqing formula, and plasma, urine and feces samples were collected at different time points. Compared chromatography and MS with blank samples, prototype compounds and metabolites were identified, combining existing data and control information. (6)Studyon metabolites of Tangzhiqing formula in patients with disorder of glycolipid metabolism: Eligible patients with disorder of glycolipid metabolism were selected. After oral administration of Tangzhiqing formula, plasma samples were collected for analysis. The detection method was the same as that of Tangzhiqing formula in normal rats. The same and different metabolic pathways in patients were identified by comparison with the metabolites of Tangzhiqing formula in rats. Result: (1) Separation of chemical constituents from lotus leaves: A total of 12 compounds were isolated from the ethanol extract of lotus leaf. They were nuciferine (C1), O-nomuciferine (C2), N-nomuciferine (C3), dehydronuciferine (C4), rutin (C5), hyperoside (C6), isoquercitrin (C7), astragalin (C8), quercetin (C9), kaempferol (C10), isorhamnetin (C11) and luteolin (C12). (2)Studyon metabolites of nuciferine in normal rats: Compared with blank samples, 15 compounds (7 phase I and 8 phase II metabolites) considered to be potential metabolites of nuciferine were screened from biological samples of rats, and their structures were tentatively characterized based on retention time, accurate mass measurement and fragment ions. They were 1-O-glucuronyl-N-nomuciferine or 2-O-glucuronyl-N-nornuciferine(HMl or HM4), 2-O-glucuronyl-1-methoxyaporphine or l-O-glucuronyl-2-methoxyaporphine (HM2 or HM5), 5-OH-O-nornuciferine sulfate (HM3), 1-O-sulphate-N-nomuciferine or 2-O-sulphate-N-nomuciferine (HM6), 2-O-sulphate-O-nomuciferine (HM7), asimilobine (HM8), O-nomuciferine (HM9), caaverine (HM10), 5-O-glucuronylnuciferine (HM11), N-oxygenic-1-demethylated nuciferine or N-oxygenic-2-demethylatednuciferine (HM12 or HM13), N-nomuciferine (HM14) and N-oxygenatednuciferine (HM15). Among them, 7 new metabolites were identified for the first time, including HM3, HM6, HM8 and HM10-HM13. By summing up, the results found that demethylation, oxidation, glucuronidation and sulfation were the predominant metabolic reactions of nuciferine in rats (3)Study on pharmacokinetics and pharmacodynamics of lotus leaf alkaloids in rats with disorder of glycolipid metabolis: An UPLC-MS/MS method was used to establish for simultaneous detection of nuciferine, O-nomuciferine and N-nomuciferine in rat plasma. The results showed that the C〓 and AUC of the three alkaloids increased along with dosage. Among them, O-nomuciferine had the largest V〓/F, indicating that his distribution was broader and had a stronger effect when entering the blood circulation. The lotus leaf total alkaloids had the effect of reducing glucose and regulating lipids, and the effect of the high drug group was significantly better than that of the low drug group, which was tested by pharmacodynamics. In addition, the dynamic process of lotus leaf alkaloids in vivo had a certain correlation with the content of glucose and total cholesterol, and the correlation with the content of and triglyceride was smaller. (4)Studyon the mechanism of glucose lipid metabolism in 3T3-L1 adipocytes by lotus leaf alkaloids: MTT results showed that nuciferine had no significant effect on cell growth within 50μg/mL, while N-nomuciferine and O-nomuciferine inhibited cell proliferation at concentration greater than 20μg/mL. By measuring the intracellular glucose uptake, the optimal action time and content of nuciferine and N-nomuciferine was 48h and 10μg/mL, while O-nomuciferine had no significant effect on intracellular glucose uptake. Moreover, at the above time and concentration, nuciferine and N-nomuciferine had an obvious inhibitory effect on triglyceride content. qRT-PCR results showed that nuciferine could stimulated the gene expression of PPAR-α and AMPK and inhibit the expression of TNF-α, while N-nomuciferine could only promote the gene expression of PPAR-α and had the same inhibitory effect on TNF-α. Western blotting results showed that nuciferine and N-nomuciferine could up-regulate the protein expression of PPAR-α and p-AMPK, and down-regulate the expression of TNF-α. (5)Studyon metabolites of Tangzhiqing formula in normal rats: Through the analytical method of UPLC-Q-TOF/MS, 38 prototype compounds and 48 related metabolites were identified in rat biological samples, including alkaloids, flavonoids, phenolic acids, diterpenoid quinones and monoterpenoids. Of these, 29 chemical compounds (11 prototypes and 18 metabolites) were detected in plasma, and 72 chemical compounds 25 prototypes and 47 metabolites) were detected in urine, and 41 chemical compounds (34 prototypes and 7 metabolites) were detected in feces. By summing up, the major metabolic reactions of Tangzhiqing formula were methylation, demethylation, oxidation, glucuronidation and sulfation. (6)Studyon metabolites of Tangzhiqing formula in patients with disorder of glycolipid metabolism: The plasma of patients with disorder of glycolipid metabolism was analyzed by the same UPLC-Q-TOF/MS method, and 11 prototype compounds and 15 metabolites were identified. These 26 compounds were also present in rats. Moreover, by analyzing the structure of metabolites, the main metabolic pathways of the Tangzhiqing formula in patients were glucuronidation and sulfation. Conclusion: The research results of chemical constituents, pharmacokinetics and cellular pharmacology confirmed that lotus leaf alkaloids and their related metabolites had certain hypoglycemic and lipid-lowering effects. Moreover, the study also explained that there was a correlation between “lotus leaf alkaloids-related metabolites-mechanism of regulating glucose and lipid metabolism”, which provided data reference for the in-depth research of the material basis and mechanism of regulating disorder of glycolipid metabolism of Tangzhiqing formula. Based on the above research, this study clarified the chemical basis of the regulation of disorder of glycolipid metabolism of Tangzhiqing formula by inferring the proposed metabolic pathways of drugs in normal rats and patients with disorder of glycolipid metabolism. This study provided valuable data for safety estimate of Tangzhiqing formula, which will be advantageous for clinical application. Key words: Lotus leaf; alkaloids; material basis; metabolites; mechanism of action; glycolipid metabolism; Tangzhiqing fomula
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