Enzymatic synthesis and purification of Vitamin A docosahexaenoate
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摘要: 维生素A具有维持正常视力、改善皮肤、抗衰老等多种生理活性，但极不稳定，很容易在光、热、氧的作用下发生分解。选择二十二碳六烯酸乙酯 (Ethyl docosahexaenoate, EDHE) 与维生素A醋酸酯 (Vitamin A acetate, VAAE) 作为底物，在脂肪酶Novozyme435的催化下发生转酯反应，从而合成一种新型的维生素A-DHA酯 (Vitamin A docosahexaenoate, VADHE)。利用液相及质谱对目的产物进行鉴定，在此基础上，利用硅胶柱层析 (C18填料) 对反应体系中的VADHE进行纯化。经液相色谱检测，VADHE的纯度达到90%以上。为了进一步提高目的化合物得率，分别在有机溶剂体系和无溶剂体系中对转酯反应进行了优化。在有机溶剂体系中，反应6 h转化率可达40.61%。在无溶剂体系中，用100 mg脂肪酶催化反应4 h，转化率可达56.39%。该研究首次设计并合成了新型脂质VADHE，实现了该物质的纯化制备，为后期的活性检测实验奠定了基础。Abstract: Vitamin A shows a variety of physiological activities, such as maintaining normal vision, improving skin and anti-aging activity. However, it is extremely unstable and easily decomposed under the action of light, heat and oxygen. In this study, Novozyme435 was used to catalyze the transesterification reaction of Vitamin A acetate (VAAE) and ethyl docosahexaenoate (EDHE). The target product (Vitamin A docosahexaenoate, VADHE) was identified by high performance liquid chromatography (HPLC) and mass spectrometry (MS). On this basis, VADHE in the reaction system was purified by silica gel column chromatography (C18 filler). The result of HPLC shows that the purity of VADHE reached 90%. In order to increase the yield of VADHE, the transesterification reaction was optimized in organic solvent system and solvent-free system, respectively. In the organic solvent system, the conversion rate reached 40.61% after 6 h. In the solvent-free system, when 100 mg Novozyme was used to catalyze the reaction for 4 h, the conversion rate reached 56.39%. VADHE was firstly designed and synthesized in this study, and the purified VADHE can be further used in the physiological activity experiment.
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图 1 维生素A-DHA酯 (VADHE) 的液相 (a) 和质谱 (b) 鉴定
Figure 1. Identification of vitamin A docosahexaenoate (VADHE) by HPLC (a) and MS (b)
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图 2 VADHE的纯化洗脱曲线 (a) 、全波长扫描 (b) 和核磁共振碳谱 (c) 结果
Figure 2. Elution curve (a), full wavelength scanning (b) and 13C NMR (c) of VADHE
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图 3 VADHE在有机溶剂体系中合成反应条件的优化
Figure 3. Optimization of synthetic reaction conditions in organic solvent systems of VADHE
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