Separation And Purification Of Chemical Constituents From Opuntia Dillenii, Folium Mori, Vaccinium.spp And Study On Interaction Mechanism Of Drug With BSA

Objective: In order to investigate the chemical constituents of opuntia dillenii and folium mori and elucidate their structures, the Et OH extractions were studied through several column chromatography methods. With HSCCC and sephadex chromatography,the chemical components of vaccinium.spp fruits Et OH extractions were separated, the purity of these samples were determined and qualitatively identified. By combining fluoresecence and ultraviolet-visible spectroscopy, the interaction between benzocaine(BZC) and bovine serum albumin(BSA) was investigated under physiological condition(p H7.4 Tris-HCl buffer).Methods:(1) Opuntia dillenii and folium mori were extracted by ethanol-water purified and prepared through repeated silicagel column chromatography, sephadex column chromatography, preparative thin layer chromatography and PHPLC. Finally,the structures of the isolated compounds were elucidated based on the LC-MS, NMR data and chemical properties.(2) The dried vaccinium.spp fruits were extracted by ethanol-water(60%) with 0.1% HCl, and sephadex chromatography and high-speed countercurrent chromatography(HSCCC) were applied for further separation and purification. The purity of the compounds was analyzed by UPLC.(3) In close to human physiological environment(p H7.4 Tris-HCl buffer), the binding sites and binding distance were calculated through fluorescence quenching method and F?rster’s theory. The change of the conformation of BSA was also studied by synchronous fluorescence spectroscopy.Results:(1) Five compounds separated from opuntia dillenii were identified as:dibutyl phthalate, adaucosterol, 5-methoxy furan methanol, 2-(4-hydroxy-3-methoxyphenyl)-ethyl-O-β-D-glucopyra-noside, 5-hydroxymethylf- rfural.Two conpounds extracted from folium mori were identified as be: dihydrokaempfero,lupeol.(2) Two samples were separated from vaccinium.spp fruits, and the purity of samples was determinated by UPLC to be 65.0% and 90.0%.(3) The results showed that quenching constant(Kq) between benzocaine and BSA were 1.52×1012 L/(mol· s)(p H 7.4, 291 K) and 1.39 ×1012 L/(mol· s)(p H=7.4, T=310 K), respectively. The binding sites n was about 1 and the binding distance r was 2.02 nm.Conclusion:(1) Through four chromatography methods, five compounds was separated from opuntia dillenii, apart from compound Ⅱ, other compounds were isolated from opuntia dillenii for the first time. Through two chromatography methods,two compounds was separated from folium mori, compound Ⅱ were isolated firstly from the folium mori. This study will lay the foundation for further research on the chemical components and biological activity of opuntia dillenii and folium mori.(2)This study established the HSCCC and sephadex chromatography method to separate anthocyanidins from vaccinium.spp fruits, which showed that HSCCC is an efficient method for the samples purification of natural product. The study provides a basis for the research of vaccinium.spp fruits anthocyanidins.(3) The binding process between benzocaine and BSA is a static quenching process. And the presence of Benzocaine did not affect the microenvironment around the tyrosine residues and tryptophan residues.The study provides an important medical research proof for rational use of small drug molecules, and offers certain guiding significance and scientific basis in the body mechanism, drug synthesis, design and selection of new drugs. This article provide a useful attempt for further study on the interaction of natural product with macromolecules.