| Objective:To design and develop a near-infrared fluorescence probe for the detection of lipase.Using the fluorescent probe to realize the visual identification of lipase functional fungi,which provides a reference for the biological function investigation of intestinal microbiota and the early diagnosis of metabolic diseases（such as dyslipidemia,obesity and hyperglycemia）.Using the developed fluorescent probe,a high-throughput screening system was constructed for the identification of lipase inhibitors.The effect of lipase inhibitors on weight loss and lipid reduction would be evaluated by the fluorescent probe,which provides an efficient technical method for the development of weight-loss and lipid-reducting drugs.Methods:（1）According to the metabolic characteristics of lipase and the near-infrared fluorophore,the enzymatic activatable fluorescence probe was synthesized by chemical reaction,and its chemical structure was determined by means of various spectroscopic data.（2）For the enzymatic reaction of lipase and fluorescent probe,the specificity and kinetic characteristics of the substrate were systematically evaluated,and the activity determination system of lipase was established.（3）The lipase functional fungus in intestinal microorganisms was visually identified by laser confocal and other fluorescence detection methods.（4）Based on the model of nutritional obesity mice,the effects of lipase functional fungi on nutritional obesity mice were evaluated by the determination of several aspects,including body weight,body fat,blood glucose,blood lipids and related inflammatory factors.（5）Utilizing high-throughput visualization screening,potential lipase inhibitors were obtained from traditional Chinese herbal medicines and identificated by chromatography and spectroscopy.（6）Based on the obesity mice induced by high-fat diet,the effects of bioactive compounds from Chinese herbal medicines on regulating lipid metabolism and reducing weight were evaluated by investigating body weight,body fat,blood sugar,blood lipids,liver lipid levels,etc.Results:（1）According to the functional characteristics of lipase to hydrolyze fatty acid,the fluorescent probe DDAO-C6 was synthesized using the near-infrared fluorophore DDAO.With 600 nm as excitation wavelength,DDAO produces the strongest near-infrared fluorescence at the emission wavelength of 658 nm,while DDAO-C6 has no significant fluorescence.Therefore,DDAO-C6 can be used as an OFF-ON fluorescent probe of lipase.（2）Through multi-factor investigation,an enzymatic reaction system for lipase activity assay was established by DDAO-C6.At different enzyme concentrations（lipase:0-50μg/m L）and different reaction time（0-45min）,the fluorescence intensity has a good linear relationship between enzyme concentration and reaction time.DDAO-C6 exhibits high specificity and is not affected by other biological enzymes,amino acids and ions.The enzymatic reaction about DDAO-C6 and lipase followed Michaelis-Menten kinetic,and the relevant kinetic parameters are as follows:Vmax=3.539 nmol/min/mg,Km=10.82μM.（3）Using the fluorescence probe DDAO-C6,the lipase in the fungus and colonies was visually detected by a variety of fluorescence detection instruments.A fungus strain with good lipase activity was screened and isolated from human feces,which identified as Trichosporon asahii Y2.Based on the obesity mice induced by high-fat diet,it is proved that the lipase produced by Y2 strain has the biological effect of promoting obesity.（4）According to the discernible changes of fluorescence emission caused by lipase-mediated hydrolysis of DDAO-C6,a high-throughput screening system for lipase inhibitors was constructed.It was found that Mume Fructus and Schisandrae Chinensis Fructus had strong lipase inhibition effects from 250 common Chinese herbal medicines.The main inhibitory compounds were further separated and identified from Mume Fructus and Schisandrae Chinensis Fructus(citric acid IC50 8.87μM,malic acid IC509.72μM).（5）For the experiment of obesity mice induced by high-fat diet,combined with the determination of body weight,blood sugar and lipid level of mice,it is shown that the Chinese herbal medicine Mume Fructus,Schisandrae Chinensis Fructus and their bioactive compounds,citric acid and malic acid,as lipase inhibitors,have effects on reducing weight and lipids in nutritional obesity mice.Conclusion:（1）Based on the mechanism of ICT,the OFF-ON fluorescent probe DDAO-C6 was designed and synthesized for detecting lipase activity.Its hydrolysate DDAO has excellent near-infrared fluorescence property.（2）For lipase enzymatic reaction,DDAO-C6 exhibits high selectivity and detection sensitivity,and can be used for the determination of endogenous lipase activity.（3）DDAO-C6 was successfully used for the visual detection of lipase in fungus and fungus imaging.In addition,DDAO-C6 can also be used for visual identification of lipase functional fungi in the complex intestinal microorganisms,which is an effective method.（4）Based on the obesity mice induced by high-fat diet,Trichosporon asahii Y2,a fungus strain with lipase activity,was found for the first time to promote obesity,giving a new insight into the role of gut microbiota in lipid metabolism.（5）A visual high-throughput system can be used to rapidly screen the inhibitory activity of Chinese hebal medicines against lipase,and guide the efficient tracing and sepration of main bioactive componenets,providing a novel method for the discovery of lipase inhibitors.（6）Based on the obesity mice induced by high-fat diet,it is obvious that Mume Fructus,Schisandrae Chinensis Fructus and their major bioactive substance,citric acid and malic acid,have modulatory effects on weight loss and lipid reduction,providing reference for the prevention and treatment of lipid metabolic diseases. |
