| ObjectiveRecently there has been an ever-increasing incidence of obesity, which has become a worldwide increasingly serious public health problem. The serious harm of obesity is not only affecting human health itself, but also causing many related metabolic disorders. Because of the complexity of etiology and pathogenesis of the obesity, there has been no safe and effective therapy for the treatment of obesity. With the superiority of multiple indregents, multiple targets and multi-level action, comprehensive and synergistic effect, and less adverse reactions for Traditional Chinese medicine (TCM), developing new anti-obesity drugs from natural medicines has become one of the hottest research topics. So the thoroughly illumination the mechanism of anti-obesity and its therapeutic basis for the natural medicine is meaningful for developing new anti-obesity drugs.The leaves of Lithocarpus polystachyus Rehd (Baise sweet tea) have been used for reducing blood glucose and lipid for a long time in folk of south China. This study was based on our previous research, aim to further verify the glucose and lipid metabolic intervention effect of L. polystachyus in different obese mice models, study the molecular mechanism of improving lipid and glucose metabolism, and search for genuine therapeutic basis of L. polystachyus. Methods and Results1. Effect of the water extract of L. polystachyus on lipid and glucose metabolism in C57BL/6J and db miceThe anti-obesity effects of the water extract of L. polystachyus were evaluated in C57BL/6J mice and db mice after oral administration for 14 and 15 weeks, respectively. The effects on body weight, abdominal perimeter, Lee’s index, glucose resistance, insulin resistance, fasting blood-glucose, plasma total cholesterol, low density lipoprotein cholesterin, triglyeride, high density lipoprotein cholesterol, free fatty acid, and the effect on viscera such as liver, pancreas and adipose tissue were measured. The results demonstrate that L. polystachyus significantly reduce the increase of body weight, abdominal perimeter, Lee’s index, blood glucose and cholesterol levels (TC, TG, LDL and FFA) in C57BL/6J and db mice, which also ameliorate the lesion of liver, pancreas and adipose tissue induced by obesity.2. Study on the mechanism of the water extract of L. polystachyus on lipid and glucose metabolism in C57BL/6J and db miceThe related protein expression pathways, and phosphorylation levels of p38MAPK/mTORCl/p70S6K in liver were measured by Western blot analysis. The results showed that the level of p-p38MAPK was up-regulated, while the levels of p-mTOR and p-p70S6K were down-regulated in both nutritional and genetic obesity mice3. Effect of the water extract of L. polystachyus and drug-containing serum on lipid and glucose metabolism in the fatty degeneration of hepatic cells induced by oleic acid.The cytotoxicity of L. polystachyus extract and its drug-containing serum were evaluated using a MTT assay. The effect of the L. polystachyus extract and its drug-containing serum on lipid and glucose metabolism on the fatty degeneration of hepatic cells induced by oleic acid was studied. The results showed that the decrease effect on lipid droplets size and number, TG, TC, ALT and AST was not obvious for L. polystachyus extract, but effective for its drug-containing serum.4. Characterization of chemical constituents in L. polystachyus by LC-MSA rapid, sensitive and selective UPLC/MS method using LTQ Orbitrap mass spectrometry was established for the analysis and characterization of the components and their metabolites in rat plasma, urine and feces following oral administration of L. polystachyus extract. The optimized LC conditions were as follows:Column:Agilent SB-C18,100 x 4.6 mm,1.8 μm particle size; Column temperature:30 ℃; Mobile phase: (A) water with 0.1% formic acid; (B) methanol; Flow rate:300 μL/min; Injection volume: 10 μL; Gradient:Linear gradient of 30%-60% A over 35 minutes. The ESI-MS was operated in negative ion mode. The key optimized electrospray parameters were as follows: source voltage:3.0 kV; sheath gas (nitrogen):45 arb; auxiliary gas flow:5 arb; capillary voltage:-35.0 V; capillary temperature:325.0 ℃; tube lens:-110.0 V. The ion injection time was 100 ms. Seventy-two constituents, including 57 flavonoids,11 phenolic acids, and 4 propenamide derivatives, were identified or tentatively characterized. Among these constituents, eight flavonoids and one propenamide were found to be representative constituents in L. polystachyus, and the major constituents were dihydrochalcone glycosides such as phloridzin and 3-hydroxyphlorizin, but none of them were detected in rat plasma. Instead, dihydrochalcone phase II metabolites derived from phloridzin and 3-hydroxyphlorizin were found to be the main metabolites in rat plasma. A metabolism study of 3-hydroxyphlorizin and phloridzin was also conducted, and conjugation with glucuronic acid was the main metabolic pathway of phloridzin, whereas conjugation with glucuronic acid+sulfuric acid was the main metabolic reaction of 3-hydroxy phlorizin. For the excretion study, the phase II metabolites of dihydrochalcone were mainly excreted in the urine, whereas they are mainly excreted as the aglycone forms (3-hydroxy phloretin and phloretin) in feces.ResultsThe water extract of L. polystachyus could significantly reduce the body weight of both nutritional and genetic obesity mice, reduce the plasma and hepatic triglyceride and cholesterol levels, and improve lipid and glucose metabolism related liver, pancreatic lesions, thus to ameliorate the lipid and glucose metabolism abnormality in both two models of obesity mice. The liver lipid and glucose metabolism regulating mechanism may be through the activation of p38 MAPK protein pathway, which inhibit the expression of mTORCl, thus inhibit phosphorylation of its downstream protein p70S6K. Our further study found that the drug-containing serum rather than the L. polystachyus extract, could obviously improve the degeneration of liver injury and regulate lipid and glucose metabolism. Comparing the main constituents between the L. polystachyus extract and its drug-containing serum by LC-MS, we found that they showed inconformity, and the main constituents in drug-containing serum were dihydrochalcone II phase metabolites, and these metabolites maybe genuine anti-obesity material basis. |
PDF Full Text Request