Study On Lipase Inhibition And Intestinal Absorption

Objective:Lipase inhibition has become a relatively safe mechanism of weight loss. Not acting on the nerve, lipase inhibitors have become so far has good curative effect of medicine reducing weight, There have been some preliminary progress on the related research about Chinese material medicine ingredient currently, however, most studies before were focusing on only an in vitro model, which could not reflect the function characteristics of the lipase activity. In this study we choose lotus leaf as the traditional Chinese herbal medicine which has the exact effect of weight losing, two different types of models are also used to study lipase inhibition of lotus leaf. Characteristics of component absorption were also considered in to make further proof of possibility of weight loss on the inhibition for pancreatic lipase activity. A new model for the study of the activity of lipase in vitro was established to provide theoretical reference for the screening of the active components of active target before absorption like lipase.Methods:To establish effective analysis method of single component, multiple components in lotus leaf, orthogonal experimental design method were used to optimize extraction process of lotus leaf. And to establish two sets of lipase activity inhibition model with artificial substrate and natural substrate, study on the lipase inhibition of components of lotus leaf. To make the qualitative and quantitative analysis separately of composition absorption behavior of lotus leaf using everted-gut technique and rat single-pass intestinal perfusion technique.Results:(1) Orthogonal design was used to optimize the extracting process of total flavonoids in lotus leaves and nuciferine with the extraction rate as the evaluation index. The optimal extraction condition of total flavonoids in lotus leaves is 60% of alcohol,40:1 (ml/g) of liquid-material ratio, and two times with 60min per time. The optimal extraction condition of nuciferine in lotus leaves is 60% of alcohol,20:1 (ml/g) of liquid-material ratio, and two times with 60min per time. Comprehensive consideratd, the better optimal extraction condition of total flavonoids and nuciferine in lotus leaves as following:60% of alcohol,30:1 (ml/g) of liquid-material ratio, and two times with 60min per time. The total extract of lotus leaf was separated by D101 macroporous resin, which found that the various fractions of lotus leaves were separated to study the inhibition of lipase.(2) Two kinds of Lipase inhibition models were established and optimized. The inhibition of lipase from lotus leaves extracts increases with the increase of ethanol proportion with Artificial p-NPP as substrates. When compared at the same concentration of crude herbal dose,the inhibition of pancreatic lipase from lotus leaves extracts increases first and then decreases. The inhibition effects of 60% ethanol is optimize. It was found that the half inhibition concentrations (IC50) is 636 ug/ml. However, the inhibition of lipase from lotus leaves extracts is indistinctive with Natural olive oil as substrates hogonal test design; thermally induced phase separation;membrane preparation.(3) In this study, the total extract of lotus leaf was qualitatively analyzed by using the everted gut sac test, the possibility of absorption of ingredients could be determined, results found that in the lotus leaf extract, the vast majority of ingredients could be passed through the intestinal wall, the Papp levels of the known components were nuciferine (1×l0-5-1×10-6 cm·s-1), rutin (1×10-6-1×10-7 cms1), hyperoside (1×10-6 cm·s-1), isoquercitrin (1×10-6-1×10-7 cm·s-1) and quercetin (1×10-6-1×10-7 cm·s-1), respectively. Since the lotus leaf total extract content itself is relatively low, part of the components could not reach the limit of quantification in everted gut sac.For further quantitative analysis of intestinal permeability on lotus leaf components, Single-pass intestinal perfusion in vivo was experimented on the single component that had been determined of lotus leaf,it was found that the Peff of nuciferine, demethyla nuciferine, rutin, quercetin, kaempferol from lotus leaf were greater than 0.5 × 10-4 cm/s, In the BCS intestinal permeability property, these ingredients belong to high permeable components, while the hyperin belong to low permeable components. However, in the multi-component environment of the lotus leaf extract, component permeation is not static, semi quantitative analysis were done to the components which were still not clear in lotus leaf, four in seven components which content is relatively high Peff was less than 0.5× 10-4 cm/s. In the multi-component environment, among seven unknown components, three of them belong to high permeability, while other four were low permeability.Conclusion:Using the p-NPP artificial substrate, the inhibition effect of lipase of Lotus leaf extract has a great gap compared with the effect of control medicine-Orlistat. While the enzyme inhibition model, based on the natural substrates of olive oil shown that the components segments of Lotus leaf were not found lipase inhibition effect observably. Thus, it is inferred that Lipase inhibition might have substrate selectivity. Studis of intestinal absorption of ingredients showed that somekind of components were well absorbed, Lotus leaf was playing its role in lipid-lowering diet not only based on the mechanism of lipase inhibition, but also maybe some other mechanisms post-absorption target. Although components from Lotus Leaf lipase were negative, we set up a new research model, analyzing the pre-absorption target mechanism. In this study, we establish a new model, which use the artificial substrate p-NPP as a substrate of lipase inhibitory activity screening model, the natural substrate olive oil as the active verification model substrate, combined with the active ingredient intestinal permeability absorption of pre absorption target mechanism analysis.