Enzymatic Synthesis And Anti-oxidative Activity Of Astragalin

Astragalin (AST), also known as kaempferol (KMF)-3-O-glucoside or KMF-3-O-β-D-glucopyranoside, is a natural flavonoid from many traditional herbs and medicinal plants. It has been reported that AST possesses a wide range of properties, such as anti-inflammatory, anti-oxidative, anti-viral, analgesic, anti-bacterial, anti-allergic, and anti-hepatotoxic activity. However, traditional methods for extraction of AST from plants consume a great quantity of organic solvents and raw materials, and the chemical methods for direct synthesis of this flavonoid are often coupled with rigor and complicated reaction condition and massive by-products, which greatly limit its industrial-scale production, whereas in vitro enzymatic synthesis provides a simpler, milder, more environment-friendly, and stereospecific method for production of medicinal compounds, which also indicates a bright perspect. This study focused on the development of a reaction system for the enzymatic synthesis of AST in vitro to replace the traditional methods.Firstly, a pathway for the enzymatic synthesis of AST was designed based on the general biosynthesis of flavonoids, which included 5 main enzymes encoding glycogen phosphorylase (GP), flavanone-3-hydroxylase (F3H), flavanols synthase (FLS1), glucose-1-phosphate uridylyltrans-ferase (GalU), and flavonoid-3-O-glycosyltransferase (UGT78K2). These 5 genes were cloned into plasmids pET-32a or pGEX-5X-1, respectively. The recombinant prokaryotic expression vectors were transformed into E.coli BL21(DE3) following sequence validation to induce the expression of fusion proteins by IPTG. The induced fusion proteins were purified by affinity chromatography using Ni-agarose or Glutathione--agarose resin. SDS-PAGE analysis demonstrated that all fusion proteins were successfully expressed in a soluble state and the purities were all more than 90%.Then, the enzyme activities of all fusion proteins were analyzed by setting up a reaction system with a fusion protein and its corresponding substrates in a specific buffer and detecting the reaction product by polyamide thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), with the product further confirmed by liquid chromatography-quantitative time of flight-electrospray ionization mass spectrometry (LC-QTOF-ESI/MS) analysis. The results showed that all fusion proteins trasformed its substrate into a predicted product. The specific activities were 59.50 IU/mg for GP,31426.77 IU/mg for GalU,309.61 IU/mg for F3H(At),659.21 IU/mg for F3H(Gm),4374.45 IU/mg for FLS1, and 271.81IU/mg for UGT78K2, respectively. Based on the reported reaction condition and further modification, a reaction system was developed, which included 5 purified fusion proteins in a buffered system and transformed raw materials naringenin and glycogen directly into AST in a single step. The synthesized AST was crudely extracted by ethyl acetateand purified and analyzed by Sephadex LH-20 column chromatography and HPLC. The purity of the final product AST was 94.11%.Finally, the inhibitory effects of synthesized AST on superoxide anion and 2,2-diphenyl-l-picrylhydrazyl (DPPH) free radical scavenging activity were analyzed and evaluated. The data showed that there was no significant difference in the anti-oxidative activity between synthesized and naturally-extracted AST (p>0.05).In conclusion, we developed a new method for the synthesis of AST and made a preliminary research on the anti-oxidative activity of synthesized AST. The reaction system we developed in this thesis provided an important tool for the production of AST and possessed a guiding significance for the development and utilization of other flavonoid compounds.