Affinity Screening Of Pancreatic Lipase Inhibitors In Polygonum Cuspidatum And Prunella Vulgaris L

Obesity is a serious threat to human health,it directly or indirectly leads to a variety of diseases.Various weight loss drugs and alternative therapies are being developed.Among them,the study of pancreatic lipase inhibitors,has become the hotspots in the prevention and treatment of obesity.Orlistat was received international recognition,but there are still many side effects.Therefore,it has become the main task to develop an effective,safe and long-term drug that can inhibit pancreatic lipase activity.Natural plant compounds possess diverse structure,wide range of sources and relatively small toxic side effects,screening for safe and effective pancreatic lipase inhibitors from natural plants has become a major research target.Simultaneously,the traditional drug screening with long duration,low accuracy and other deficiencies,therefore,it is essential to choose a highly efficient,highly targeted and simplye separative means.Our team used magnetic beads immobilized enzyme to screen for pancreatic lipase inhibitor before,but its synthesis conditions were not optimized.In this paper,the synthesis conditions of immobilized pancreatic lipase were firstly optimized and verified.Second,8 natural plant extracts with good inhibitive ability to pancreatic lipase were selected by investigatinga lot of literature,their crude extracts and corresponding subfractions were prepared,their inhibitory activity was determined.Third,the subfractions with good inhibitory activity from Polygonum cuspidatum and Prunella vulgaris L were screened with immobilized enzyme,meanwhile the inhibitory enzyme activity and inhibitory type were determined.Finally,the interaction between different pancreatic lipase inhibitors was studied by co-index method?Chou-Talala?,it laid a foundation for preparation of active ingredents and understand of inhibitive mechanism.The main contents and research results were listed as follows:1.Optimization of synthesis conditions and model validation of immobilized enzymes.Firstly,the multiple regression equation of immobilized enzyme synthesis was established by single factor experiment and response surface analysis,the synthesis conditions of immobilized enzyme were optimized.Then,its structure was characterized by scanning electron microscope and infrared absorption spectrum.At last,the stability,specificity and reproducibility of the optimized immobilized enzyme were verified by the use of hesperidin?positive inhibitor?and schisandrin A?negative inhibitor?.The results showed that the optimal synthetic conditions were p H 7.48,PL 20.32 mg/mL and 35.40 ?.Pancreatic lipase hads been successfully immobilized on magnetic nanoparticles.And after two weeks,the enzyme activity still remained over 80%,which could specifically screen out positive inhibitors.In addtion,the enzyme activity kept over 50% after it had been reused for five times.This experiment had successfully laid a foundation for the following experiments about inhibitor screening.2.Determination of inhibitory enzyme activity of several natural plants.First of all,the inhibitory effects of eight natural plants on PL were determined.Then,four kinds of plants with good inhibitory activity were selected,and further separated.Finally,the inhibitory effect of each subfraction on pancreatic lipase was determined.The results showed that two subfractions of Polygonum cuspidatum?80% alcohol part?and Prunella vulgaris L?40% alcohol part?with high inhibitory activity,their IC₅₀ was 90 ?g/mL?80 ?g/mL respectively,could be used to following immobilized enzyme screening.3.Inhibitor screening in the highest inhibitory activity subfraction of Prunella vulgaris L and Polygonum cuspidatum.First,two subfractions were screened by immobilized enzyme,then,the inhibitory activity and inhibition type were determined.The results showed that four pancreatic lipase inhibitors had been screened out from Polygonum cuspidatum and Prunella vulgaris L respectively.They included one terpenoid?ursolic acid?,two polyphenols?resveratrol,coffee acid?,two flavonoids?rutin,hesperidin?and three anthraquinones?physcion,emodin-8-O-?-D-glucose,emodin?.Among these inhibitors,hesperidin had the best inhibitory effect(IC₅₀=31.5±4.4 ?g/mL).4.Study on synergistic effects between inhibitors of pancreatic lipase.Nine different types of inhibitors were selected,including noncompetitive inhibitors,competitive inhibitors and uncompetitive inhibition.The combination effect between different inhibitory types and the same inhibitory types were studied by Chou-Talalay method.The results showed that different uncompetitive inhibitors had antagonistic effect,and the rest of the inhibitors showed synergistic effect.