Studies On Fluoroscopy Analysis And Interaction Between Protein And Piperine Derivatives

Pipeline was one of the active components that extracted from Long Pepper. Piperine derivatives were synthesized from Piperine. Long Pepper was the grown ear-fruits of Piper Longum L., which frequently used as Chinese medicine and Mongolian medicine. Clinical practice indicated that Long Pepper had significant antilipidic effect. Piperine and its derivatives with similar structure were extracted from Long Pepper. Our research group found that Piperine and piperlonguminine had significant antilipidic effect. According to the structures of Piperine and its derivatives, a series of derivatives such as ethyl piperate (GBA) were synthesized using piperine as starting materials. In these derivatives, GBA was the most effective one with low toxicity. GBA was very valuable to do study for developing it into new kind of anti-hyperlipidemic medicine. Therefore, it is important to study and get a quick and convenient method for detecting and analyzing these kinds of compounds for our study on developing the compounds into new drugs in the future. The coalescence of these compounds and bovine serum albumin (BSA) should be studied.The fluorescence characteristics of piperine in micellar system with cation were studied. The influences of experimental condition on fluorescence intensity also were studied. New sensitive and stable fluorescence methods for analyzing the derivatives of GBA and GBB were developed. The experiments indicated that cationic surfactant-cetyltrimethylammonium bromide was able to enhance the fluorescence intensity of Mn(II)-piperine. There was a linear relationship between the enhancement of fluorescence intensity and the concentration of cationic surfactant-cetyltrimethylammonium bromide. The linear range of concentration of piperine was 2.02-10.1μg·mL^-1with the detection limit of 0.0602μg·mL^-1. The relative standard deviation (RSD) was 1.10%. The proposed method has been successfully applied to the quantitative determination of piperine in Long Pepper used as Mongolian medicine. The results were very satisfactory.The experiments indicated that non-ionic surfactant OP-10 enhanced the fluorescence intensity of piperlonguminine. There was a linear relationship between the enhancement of fluorescence intensity and the concentration of non-ionic surfactant OP-10. Based on this result, a sensitive method was developed for the quantitative determination of piperlonguminine. The linear range of concentration of piperlonguminine was 2.0-16.0μg·mL^-1with the detection limit of 0.0651μg·mL^-1. The relative standard deviation (RSD) was 1.40%. The proposed method was successfully applied to the quantitative determination of piperine, of Long Pepper. The results were good.The interaction between piperine, piperlonguminine, GBA and bovine serum albumin (BSA) was studied by fluoroscopy and UV spectroscopy. The experimental results showed that the molecules of piperine, piperlonguminine, GBA quenched the intrinsic fluorescence of BSA by forming complex. The mechanism of fluorescence quenching was confirmed combining by both static quenching and non-radiation energy transferring. The apparent binding constants (K_A) were calculated, were it 1.02×10⁷ L·mol^-1(25℃) and 1.11×10⁷L·mol^-1(37℃); 2.40×10⁵ L·mol^-1(25℃) and 1.01×10⁶ L·mol^-1 (37℃); 9.48×10⁵ L·mol^-1' (25℃) and 1.36×10⁶ L·mol^-1 (37℃). The value of binding sites (n) were calculated, were it 1.45(25℃) and 1.46(37℃); 1.10(25℃) and 1.24(37℃); 1.18(25℃) and 1.24(37℃). According to the Forster theory of non-radiation energy transfer, the binding distances (r) were obtained, were it 3.28 nm (25℃) and 3.30 nm (25℃); 3.12 ran (25℃) and 3.15 nm (37℃); 2.68 nm (25℃) and 2.81 nm (37℃). The process of binding was a spontaneous molecular interaction in which entropy increased and Gibbs free energy decreased, indicating that the interaction of between piperine, piperlonguminine, GBA and BSA was driven mainly by hydrophobic force. The effect of piperine on the conformation of BSA was also analyzed by synchronous fluoroscopy. The influences of ordinary ions on the interaction of piperine derivatives with BSA were discussed. The relations of structure, pharmacology and combination characteristics of piperine derivatives were also discussed.The interactions between piperine, piperlonguminine, GBA and trypsin were investigated by fluoroscopy and UV spectroscopy. The experimental results showed that the piperine, piperlonguminine and GBA quenched the intrinsic fluorescence of trypsin by forming complex. The result of fluorescence quenching might be caused by static quenching and non radiative energy transferring. The apparent binding constants (K_A) were calculated, were it 9.29×10⁵ L·mol^-1 (25℃) and 1.44×10⁶ L·mol^-1 (37℃); 1.19×10⁶ L·mol^-1 (25℃) and 9.57×10⁵ L·mol^-1 (37℃); 1.66×10⁶ L·mol^-1 (25℃) and 5.05×10⁵ L·mol^-1 (37℃). According to the Forster theory of non-radiation energy transfer, the binding distances (r) were obtained, were it 2.79 run (25℃) and 3.02 nm (37℃); 3.08 nm(25℃) and 2.93 nm(37℃); 3.04 nm(25℃) and 2.99 nm(37℃). According to the relationship of parameters of thermodynamics and the interactions force, determined the inter force between the pipeline derivatives and trypsin. The relationships of the structure, pharmacology and the combination of piperine derivatives were also discussed.