Study On Mechanism Of Activity And Expression Regulation Of Lipoprotein Lipase By D-catechin From Red Skin Of Lotus Seeds

A large number of lotus seed red-skin powder are produced in the process of lotus seeds each year in China,and these by-products have not been in-depth utilization and mostly abandoned.At the same time,the incidence of obesity has increased dramatically all over the world.Lipoprotein lipase?LPL?is a rate limiting enzyme in the process of hydorolysis to triglycerides,because its activity determines whether free fatty acids were stored in fat cells or oxidated in skeletal muscle cells,therefore LPL is closely related with the occurrence of obesity.Many polyphenol extracts from plant have been reported to be effective in anti-obesity,but the relationship with LPL activity is not clear and the mechanisms are poorly reported.In the present study,the effect of D-catechin,which were separated and purificated from lotus seed red-skin powder,on LPL activity and expression in vitro and vivo were explored by using kit,Western blot and RT-PCR technologies.Meanwhile,the effect of anti-obesity of D-catechin was also evaluated in mice models fed a high fat diet.In addition,surface plasmon resonance?SPR?technology was applicated to explore the action mechanism between catechin and LPL.The results were summarized as follows:1.Separation and purification of D-catechin from lotus seed-red skin.Extracts of lotus seed red-skin was obtained by microwave method,and its contents were including 273.6mg/g of D-catechin,251.0mg/g of gallic acid,107.4mg/g of epicatechin,44.0mg/g of epigallocatechin and 13.4mg/g of epigallocatechin gallate according to the determination of HPLC.Then,D-catechin was separated from the extracts by polyamide resin,and its purity was 69.35%.The D-catechin was further purified by Toyopearl HW-40F column,and the optimal conditions for the purification process were chosen as follows:the sample volume 3.0 mL was loaded at a flow rate of 2.5 m L/min followed by elution with 95%ethanol.Under these conditions,the purity of D-catechin reached 93%.2.Effects of D-catechin on LPL activity in vitro.The primary culture models of skeletal muscle cell and adipocyte were successfully established.The data showed that the safe dose of D-catechin on skeletal muscle cells and adipocytes was below 80?g/m L.Following treatment with 80?g/mL of D-catechin,the LPL activity in skeletal muscle cells was increased by 121.98%,whereas that in adipocytes was decreased by 57.08%.3.Interaction between D-catechin and LPL.SPR analysis indicated that the affinity constant between D-catechin and LPL was 2.7×10³1/M,and the dissociation equilibrium constant was 4.82×10^-4M.Thus,the rate constant of D-catechin binding to LPL was much greater than the dissociation rate constant.In addition,the correlation coefficient was 6.50 and showed a good fitting relationship.Therefore,the regulation of D-catechins on LPL activity might be due to the affinity binding between them.4.Anti-obesity evaluation of D-catechin and its effect on LPL activity and expression in vivo.The mice were randomly divided into three groups:normal control group,obesity control group and D-catechin experimental group.The control group was fed with normal diet,both of obesity control group and D-catechin experimental group were fed a high fat diet;meanwhile,D-catechin experimental group was treated with intragastric administration of 500mg/kg bw/d D-catechins daily,and both of normal control group and obese control group were treated with equal volumes of stilled water.Kit method,real-time PCR and western blot technologies were used to analyze the activity and gene expression of LPL involved in lipid metabolism in skeletal muscle and epididymal fat of mice.At the 9th experimental week,the activity of LPL in skeletal muscle of experimental group was increased by 170%compared to the obesity control group,while that in epididymal fat was reduced by 89%compared to the obesity control group.Therefore,D-catechin could speciafically regulate LPL activity in different tissues in vivo.In the meantime,the levels of mRNA and protein of LPL were also improved in skeletal muscle and decreased in epididymal fat under the intervention of D-catechin.Consequently,we observed that the body weight,serum TC,TG,LDL-C and hepatic MDA levels in D-catechin experimental group were significantly lower than those in obesity control group,while hepatic SOD?GSH?T-OC and serum HDL-C in D-catechin experimental group were markedly higher than those in obesity control group.The results indicated that D-catechin could effectively reduce body weight and improve various biochemical indexes of mice fed a high fat diet.In summary,high-purity D-catechin could be obtained via polyamide resin combined with TSK-40F gel.Moreover,D-catechin played good anti-obesity effect on mice fed a high fat diet with speciafically regulating LPL activity and expression in skeletal muscle and epididymal fat tissue,and it might be duo to the interaction between D-catechins and LPL.