Study On Chemical Constituents From Lapemis Curtus

Lapemis curtus is a reptile of Lapemis in Elapidae, which has many functions, such as dispelling wind, eliminating dampness, relaxing the tendons, and has been recorded in Guangxi Chinese Medicine Standard. To find the active constituents, and provide reference for its resource-oriented utilization, the chemical compounds in dry Lapemis curtus was extracted, separated, purified and identified. In order to provide theoretical information for further exploitation and comprehensive utilization of hydrolysis animal protein, the optimum hydrolysis conditions by applying four proteases were investigated, respectively.The whole specimen was air-dried, powdered and extracted with75%EtOH for four times at room temperature. The extract was filtered and evaporated at reduced pressure to obtain a crude residue, which was then successively partitioned between petroleum ether and water, and between ethyl acetate and water, to obtain petroleum ether-, EtOAc-, and water-soluble fractions, respectively. The compounds were isolated by repeated column chromatography (CC) on silica gel, Sephadex LH-20, and recrystallization. The structures of these compounds were elucidated by means of the physico-chemical properties and spectroscopic methods including MS,1D-NMR (’H-NMR,13C-NMR and DEPT) and2D-NMR (’H-’H COSY, HMBC, HSQC). At the same time, Lapemis curtus was hydrolyzed by enzyme. The effects of temperature, enzyme amount, solid/liquid ratio, and hydrolysis time on hydrolysis were studied through combining orthogonal trials. The optimum hydrolysis conditions of the four kinds of proteases, such as papain, hay bacillus protease, trypsin and alkaline Protease, were investigated.Seventeen compounds from Lapemis curtus were identified as cholesterol (1),5,6β-epoxysitosteryl oleate (2),7a-methoxyl-β-sitosterol (3),3β-methoxy-5a-cholestane (4), stigmasta-5,24(28)-diene,3-methoxy-,(3p,24E)(5),5α-cholestan-3p-o1,5,6β-dibromo-,nonanoate (6), cholestane-3,5,6-trio1,3-nonanoate (7), cholest-5-ene-3β,7β-dioI (8), cholest-5-ene-3β,7α-diol (9),1-methylhydantoin (10), tetracosanamide,N-[(1S,2R,3E)-2-hydroxy-l-(hydroxylmethyl)-3,7-heptadecadien-l-yl](11), palmitic acid (12), octdecenoic acid methyl ester (13),9-octadecenoic acid (14),1,3-distearoyl-2-oleoylglycerol (15), a-palmityl glycerin ether (16), chimyl alcohol (17). All of the above compounds were isolated from the dry Lapemis curtus for the first time except compound (1) and compound (10).The enzyme-based hydrolysis conditions of protein from Lapemis curtus was studied by utilizing a9-run orthogonal array design. The optimum conditions were as follows:the temperature was50℃, the pH value was7.0, the solid/liquid ration was1:9 (g/mL), the hydrolysis time was7.0h, the best enzyme was hay bacillus protease and the amount was1000U/g. Under the optimized conditions, the yield of hydrolysis animal protein could reach42.0%, and the SC50was0.5580mg/mL.