Enzymatic Synthesis Of Fat-soluble Flavonoid Derivatives And Antioxidant Activity

Bioflavonoids widely distributed in nature not only content, but also has a variety of physiological activity. With superior antioxidant capacity, as early as in the1960s, has confirmed the role of flavonoids anti-lipid peroxidation; Since the1980s, research gradually discovered bioflavonoids in the free radical scavenging activity, anti-aging and the prevention and treatment of geriatric have a better effect. However, flavonoids nature exist in the form of a glycoside, the polarity of the glycoside having a hydrophilic, it is a poor compatibility with the grease, it is difficult to exist stably in fat, which limits the flavonoid compound in the oils and fats in the application. Flavonoids modified by esterification, the introduction of long hydrocarbon chain in the molecule can increase the fat-soluble, thus increasing the compatibility with the grease and fat oxidation resistance. Currently crest synthesis dioxide method mainly by chemical synthesis and enzymatic method. Chemical method has a higher yield, but vigorous reaction conditions, esterification sites is difficult to control, and easy to cause the unsaturated fatty acid oxidation; enzyme catalytic reaction under mild conditions can be achieved selectively esterified hydroxy rutin molecule, and the product is isolated The purification is simple.The study of lipase in the catalytic synthesis of flavonoid esters immaturity, lack of reaction kinetics of the status quo, enzymes of Novozym435fat, flavonoid esters enzymatic synthesis method based on optimization of process parameters by response surface methodology, lipase reaction kinetics to explore further separation and purification of the crude product of the flavonoid esters and analysis of its structure and physical and chemical characteristics, the main findings are as follows:1. Synthesis of rutin a-linolenate by Novozym435The lipase Novozym435for catalytic esterification of alpha-linolenic acid and rutin. By single factor and response surface experiments to explore the influencing factors of the reaction. The results show that the optional Novozym435of lipase as a catalyst, and acetone as the solvent, the acid to alcohol molar ratio of4:1, the substrate concentration of27.5mmol/L, the enzyme addition amount of15mg/mL, ADD Zeolite100mg/mL, the reaction temperature is55℃, reaction time60h reaction esterification rate of92.6%. Established by the response surface method immobilized lipase-catalyzed synthesis of alpha-linolenic acid plant sterol esters regression equation: Y=86.99+8.49A-3.77B+5.57C+1.33AB-2.58AC+0.043BC-2.97A2-2.30B2-4.89C2, esterification rate measure, the three factors on the size of the esterification rate as follows:the amount of immobilized enzyme Add> temperature> substrate concentration. Taking into account the factors of the process cost and energy consumption, to obtain the best experimental conditions:the amount of the immobilized enzyme and select the17.9g, substrate concentration of17.3mmol/L,55℃for the reaction60h, the esterification of sterol esters obtained was93.7%.2. Reaction kinetics of esterification by Novozym435Enzyme addition, rutin and alpha-linolenic acid concentration on the enzymatic impact of the initial rate, found that the reaction comply with the ping·pong mechanism, double substrates enzymatic esterification reaction kinetic model to calculate the kinetic parameters Vmax=2.865mmol/L·h, the Michaelis constants=1556.6mmol/L=220.5mmol/L, the kinetic equation: V=2.865[A][B]/1.556.6[B]+220.5[A]+[A][B]mmol/L-h3. Purification and structural characterization of flavonoid derivativesThe flavonoid ester products through the isolation and purification, pure purity more than95%.Then by infrared spectroscopy, mass spectrometry, nuclear magnetic resonance and other means of its structural identification.The results show that the esterification reaction sites esterified such as rhamnose C4hydroxy.High performance liquid chromatography monitoring esterification process, the first study of the molecular sieve Added esterification rate.The results show that the esterification rate and the reaction rate of the the molecular sieve esterification reaction is much larger than do not add to the molecular sieve is added. Zeolite added at different times, the esterification rate is also different.After reaction for8hours to add an esterification reaction of the molecular sieve can be obtained for the case of alpha-linolenic acid as the acyl donor, the maximum the esterification conversion rate of.In this study, for the first time rutin fatty acid ester UV-visible spectrum, rutin ester of fatty acid characteristic absorption at256nm and357nm.4. Flavonoid derivatives antioxidant activity evaluationThe flavonoid ester is a new class of anti-oxidants, flavonoids ester radical scavenging capacity and antioxidant activity is measured by the the Rancimat instrument induction period assay, DPPH method, beta carotene-linoleic acid method. The results showed that the oxidation induction period of up Naringin monooleate (10.81h), followed by the oleic Lo Dioxide (10.20h), naringin monooleate (10.08h) were higher than the natural vitamin E oxidation induction time (9.94h). the Lo-butyl ester of alpha-linolenic acid, linoleic acid, butyl Lo, oleic acid butyl Lo are the same students with the natural vitamin E radical scavenging capacity.