摘要
阿魏酸(Ferulic acid, (E)-3-甲氧基-4-羟基肉桂酸)是植物界普遍存在的一种酚酸,在植物中主要与多糖和木质素交联构成细胞壁的一部分,是阿魏、当归、川芎、升麻等重要的有效成分之一。尽管阿魏酸在某些方面具有确切的药理活性,但其分子中含有双键,烷烃基较短,亲水性较强,难以透过生物膜脂质分子层,这在很大程度上限制了阿魏酸应用,因此在不改变其生物活性的情况下增加其脂溶性成为亟待解决的问题。通过化学方法将长烷烃链引入阿魏酸进行修饰,已有报道,但是化学改性很大程度限制和破坏了阿魏酸生理活性的发挥。通过生物催化技术改性可以很好的解决这一难题,酶催化反应的特点在于所需要的反应条件温和、选择性高、催化剂可以重复利用,这可以在保证不改变阿魏酸生理活性前提下赋予其更广泛的功能性,选择性的合成不同结构的阿魏酸结构脂更是引起了广大研究学者的极大关注。
本文以非水相为反应介质,建立了脂肪酶催化合成阿魏酸三油酸甘油酯的一种新的合成方法,即通过化学方法对底物阿魏酸进行修饰,制备了阿魏酸乙酯和阿魏酸乙烯酯,然后在脂肪酶的催化下一锅法制备阿魏酸三油酸甘油酯,并比较阿魏酸乙酯和阿魏酸乙烯酯在相同条件下作为底物合成阿魏酸三油酸甘油酯的合成效率；研究了阿魏酸三油酸甘油酯的体外清除自由基的生理活性；采用静电纺丝的方法,制备速溶阿魏酸及阿魏酸三油酸甘油酯聚乙烯吡咯烷酮纤维膜,并对其速溶性进行测试。
通过化学方法对阿魏酸单体进行修饰,制备了阿魏酸乙酯和阿魏酸乙烯酯从而提高酶促反应的效率。在有机相甲苯中探讨了酶促合成阿魏酸三油酸甘油酯的研究。考察了不同反应条件,包括底物比、反应时间、反应温度、水活度,酶用量对转化率的影响,从而确定酶促合成阿魏酸三油酸甘油酯的最佳反应条件,另外对酶在本体系中的稳定性做了探讨。与此同时,在相同的条件下比较阿魏酸乙酯和阿魏酸乙烯酯分别作为反应底物时的合成效率,确定最佳底物。通过当实验可以得出阿魏酸乙烯酯做为底物时具有更高的转化率。阿魏酸乙烯酯为底物酶促合成阿魏酸油酸甘油酯的最佳条件：有机介质3.OmmL甲苯中底物阿魏酸乙烯酯与三油酸甘油酯的底物比为1：3,Novozyn 435脂肪酶60mg,550C下反应62h,摇床转速210r/min,水活度0.07条件下转化率可达90.2%,此时阿魏酸单油酸甘油酯和阿魏酸双油酸甘油酯含量分别为32.5%,57.6%。批试反应进行13批后相对酶活还保持在75%以上。阿魏酸乙酯作为底物酶促合成阿魏酸油酸甘油酯的最佳条件：有机介质3.0mL甲苯中底物阿魏酸乙烯酯与三油酸甘油酯的底物比为1:3, Novozyn 435脂肪酶100mg,50℃下反应96h,摇床转速210 r/min,水活度0.07条件下最大转化率为61.9%,此时阿魏酸单油酸甘油酯和阿魏酸双油酸甘油酯含量分别为30.4%,31.4%。
研究了阿魏酸单油酸甘油酯(FMOG)、阿魏酸双油酸甘油酯(FDOG)清除二苯代苦味肼基自由基(DPPH·)、羟自由基(·OH)和超氧阴离子自由基(02·)的能力,并与VE作比较。实验结果表明：阿魏酸三油酸甘油酯清除自由基的能力随着浓度的增大而增大。清除DPPH·自由基的能力依次为：VE>FMOG>FDOG;清除·OH自由基的能力依次为：VE>FMOG>FDOG;清除O2-·自由基的能力依次为：VE>FMOG>FDOG。
采用易溶于水的高聚物聚乙烯吡咯烷酮(PVP),利用静电纺丝法制备阿魏酸及阿魏酸三油酸甘油酯PVP速溶超细纤维膜,探讨了浓度和电压对纤维膜表面形态的影响,从而确定最佳电纺条件。并对两种纳米纤维膜的速溶性质做了测试。实验表明,制备阿魏酸PVP速溶超细纤维的最佳制备条件为：聚乙烯吡咯烷酮40(w/v)%,阿魏酸5(w/w)%,溶剂为无水乙醇,喷丝口流速为0.8 mL/h,两级间屏距为12cm,电压12 kV,所制得的纤维直径分布为800—1000nm,在水中完全溶解用时为3.4±1.5s；制备阿魏酸三油酸甘油酯PVP速溶超细纤维的最佳制备条件为：聚乙烯吡咯烷酮5(w/v)%,阿魏酸三油酸甘油酯25(w/w)%,三氯甲烷和乙醇的混合溶剂(V (CHCl3): V(C2H5OH)=4:1),喷丝口流速为0.8 mL/h,两级间屏距为12cm,电压为14 kV,所制得的纤维直径分布在800—1000nm,在水中完全溶解用时为2.0.±1.5s。
Ferulic acid (4-hydroxy-3-methoxy cinnamic acid, FA), is a phenolic compound that is abundant in plant cell walls. It has been shown to have many physiological functions, including antioxidant, antimicrobial, anti-inflammatory, anti-allergic, antiviral, anti-carcinogenic, free radical scavenging, and UV filter properties, which made FA widely used in the food, cosmetics, and pharmaceutical industries. However a major obstacle in the application of FA in oil-based food processing and other corresponding industries is its low solubility and stability in hydrophobic media. To overcome this limitation the modification of FA through esterification with aliphatic alcohols or transesterification with triacylglycerols has been widely reported. Chemical synthesis of feruloylated lipids is difficult due to heat-sensitivity and susceptibility of ferulic acid to oxidation at high temperature and under certain pH conditions. The advantages associated with enzymatic synthesis include mild-operating reaction conditions, high specificity, and easy recovery of the end-product.
Lipase-catalyzed synthesis of functional feruloyl-oleyl-glycerol (FOG), consisted of 1(3)-feruloyl-monooleyl-glycerol (FMOG) and 1(3)-feruloyl-dioleyl-glycerol (FDOG), in toluene was investigated in this study. First, ferulic acid was chemically modified to ethyl ferulate and vinyl ferulate in order to make the substrate more soluble in the fatty alcohols. Second, feruloy-oleyl-glycerol was synthesis through transesterification between ethyl ferulate or vinyl ferulate and triolein using Novozym 435 as catalyst. The optimal reaction conditions were obtained by investigation of reaction factors, including substrate molar ratio, reaction time, temperature, water activity, enzyme content and enzyme stability. Meanwhile, under the same conditions, the synthetic efficiency of ethyl ferulate and vinyl ferulate was compared in this study.
The optimal conditions confirmed when vinyl ferulate was used as sustrate were:3.0 mL toluene,3 molar ration of vinyl ferulate to triolein, enzyme loading 60mg, reaction temperature 55℃, reaction time 62h, agitation speed 210 r/min, water activity 0.02. Under these optimal conditions, the highest reaction conversion achieved was 90.2%, which was composed of 1(3)-feruloyl-monooleyl-glycerol 32.5% and 1(3)-feruloyl-dioleyl-glycerol 57.6%. The optimal conditions confirmed when ethyl ferulate was used as sustrate were:3.0 mL toluene,3 molar ration of vinyl ferulate to triolein, enzyme loading 100mg, reaction temperature 50℃, reaction time 96h, agitation speed 210 r/min, water activity 0.07. Under these optimal conditions, the highest reaction conversion achieved was 61.9%, which was composed of 1(3)-feruloyl-monooleyl-glycerol 30.4% and 1(3)-feruloyl-dioleyl-glycerol 31.4%. The high synthetic efficiency of vinyl ferulate as substrate has been confirmed by the comparative studies. Furthermore, the residual activity of enzyme was keep about 75% after 13 runs when vinyl ferulate was used as substrate.
The radical scavenging properties of feruloyl-oleyl-glycerol were evaluated using several different tests, including hydroxyl radical scavenging, superoxide anion radical scavenging, 1,1-diphenyl-2-picrylhydrazyl (DPPH-) radical scavenging and using tocopherol as check sample. The results showed that feruloyl-oleyl-glycerol had strong radical scavenging ability and their abilities increased with the increase of their concentration. The experiment results indicated that the DPPH·scavenging properties of feruloyl-oleyl-glycerol in decreasing order was tocopherol> 1(3)-feruloyl-monooleyl-glycerol>1(3)-feruloyl-dioleyl-glycerol; for·OH radical the decreasing order was tocopherol> 1(3)-feruloyl-monooleyl-glycerol> 1(3)-feruloyl-dioleyl-glycerol; for O2·radical the decreasing order was tocopherol> 1(3)-feruloyl-monooleyl-glycerol> 1 (3)-feruloyl-dioleyl-glycerol.
Fast-dissolving ultrafine membranes (FDMs) were prepared with the water-soluble polymer polyvinylpyrrolidone (PVP) K90 as the filament-forming matrix and ferulic acid and feruloyl-oleyl-glycerol as a poorly water-soluble drug. The influence of PVP concentration and the applied voltage on the morphology of fibers were investigated. Wetting time tests were conducted to evaluate fast-dissolving properties. For preparation of ferulic acid-loaded PVP ultrafine membranes, the optimal conditions are:ferulic acid 5 (w/w)%, PVP 40 (w/v)%, solvent anhydrous ethanol, applied voltage 12 kV, distance from the syringe tip to aluminium foil 12cm, feed rate 0.8 mL/h. Under the optimal conditions, the fibers with the diameter ranging from 800 to 1000 nm were obtain, wetting time 3.4±1.5 s; For preparation of feruloyl-oleyl-glycerol-loaded PVP ultrafine membranes, the optimal conditions are:PVP 5(w/v)%, feruloyl-oleyl-glycerol 25 (w/w)%, mixed solvent V(CHCl3):V(C2H5OH)= 4:1, applied voltage 14 kV, distance from the syringe tip to aluminium foil 12 cm, feed rate 0.8 mL/h. Under the optimal conditions, the fibers with the diameter ranging from 800 to 1000 nm were obtain, wetting time 2.0±1.5 s
引文
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