The Synthesis Of Dihydromyricetin Esters By Enzymatic Acylation In Non-aqueous Solvent

Dihydromyricetin is the main active substances in the Chinese herbal tea Teng-cha, which has antioxidant and anti-tumor, regulating blood glucose, blood lipid and other physiological functions. But its low solubility limits its application in the fields of health food, cosmetics, pharmaceuticals and so on. Acylation of dihydromyricetin to improve its solubility can broaden the range of application. At present, chemical catalys is widely used in Acylation of dihydromyricetin. Which lack of specificity, also difficult to separate the product, And the low yield of the target product. Lipase catalyzed method was adopted in this research,which has the advantages of mild reaction conditions, simple process, low energy consumption, product environmental protection and so on. In this paper, Dihydromyricetin-3-acetate was achieved with lipase-catalyzed synthesis. The main conclusions include the five aspects in the following(1)Through single factor experiment the optimum synthesis reaction system was determined: Rhizopus niveus lipase is the best catalystin in acetonitrile. The initial water activity of 0.07 to 0.11 is appropriate; The optimum substrate molar ratio(Dihydromyricetin : vinyl acetate) 1:15 ~ 1:20; optimum reaction temperature is 45 ?. Considering the reaction rate, substrate conversion rate and cost factors, the amount of enzyme 500 U is appropriate; the reaction time is better controlled in 24-36 h. Under the optimal reaction conditions of DMY conversion rate could reach 87.6%.(2) Effect of hydrophobic [bmim][PF6] ionic liquid and hydrophilic [BF4] [emim] ionic liquid on enzymatic esterification was discuss.The experimental results showed that the ionic liquids are able to increase the initial reaction rate, but inhibit the reaction Conversion rate.(3)The methods to separate dihydromyricetin monoester by preparative high performance liquid chromatography and thin-layer chromatography was studied respectively. Preparative high performance liquid chromatography separation conditions: Mobile phase A was methanol and mobile phase B was 0.1% aqueous acetic acid; Gradient elution program: 0-12 min, A 28%- 28%; 12 to 20 min, A 30%-30%; 20 to 25 min, A 80%- 80%; Flow rate: 1 ml/min; injection volume 0.2ml; Thin-layer chromatography separation condition: developing solvent(chloroform: acetone: acetic acid = 60: 38: 2, by volume), purity of Dihydromyricetin-3- acetate can reach up to 95.17%.(4) The kinetics about The synthesis of dihydromyricetin esters by enzymatic acylation in non-aqueous solvent was established.The reaction competitive inhibition does not exist, The reaction containedTwo steps. The first step is that vinyl acetate combine to lipase to form an acylated enzyme and generating a first acetaldehyde. The second step is DMY combine to acylated enzyme, generating a second product Dihydromyricetin-3- acetate and the releasing the lipase.(5)The measurement of Dihydromyricetin-3- acetate performance, including oil soluble and hydrophobic constants, antioxidant activity, DPPH free radical scavenging. The appearance of Dihydromyricetin-3-acetate is light yellow crystal. Dihydromyricetin-3-acetate of oil soluble is 0.53 g / 100 g, Constant hydrophobic and anti-lipid peroxidation inhibition have increased two-fold. The period for lipid oxidation induction is 21.8h; DPPH radical scavenging rate was 45.8; A slight decrease compared with DMY; Antioxidant in lard system increased obviously.