表没食子儿茶素没食子酸酯（EGCG）酶法乙酰化分子修饰及其产物的抗氧化性能研究
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摘要
表没食子儿茶素没食子酸酯(EGCG)具有抗氧化、抗突变、防辐射、抗肿瘤、调节免疫和延缓衰老等卓越的生理活性。但是，其较高的亲水性和较低的亲脂性，极大的限制了EGCG在食品领域的应用，尤其是在油脂类食品中的应用。本文采用酶法分子修饰获得EGCG酰化产物以增加其脂溶性，通过分离纯化、结构表征，明确其酰化位点及最终产物结构，并对酰化产物的脂溶性和抗氧化性能进行评价。
首先，确立了酰化EGCG酶法合成路线为以乙烯酯作为酰基供体的酯交换反应。不同碳链乙烯酯作为酰基供体对EGCG的转化率影响较大，随着碳链的增加，EGCG的转化率逐渐减小，EGCG与乙酸乙烯酯之间的酯交换反应能达到最高的转化率。采用响应面分析法对酶法制备乙酰化EGCG反应条件进行优化，最优条件为：脂肪酶Lipozyme RM IM为酶催化剂，添加量为2.1%(w/w底物)；乙腈和异丙醇(质量比为1:1)为反应溶剂体系；40℃反应12h；EGCG与乙酸乙烯酯底物摩尔比为1.1。在该条件下，EGCG乙酰化转化率达到90.32%。采用液质联用选择离子分析明确了乙酰化EGCG反应产物为单-，二-和三取代乙酰化EGCG的混合物。在该反应体系中，脂肪酶LipozymeRM IM催化EGCG和乙酸乙烯酯的酯交换反应为动力学控制，在所研究的底物浓度范围内(EGCG浓度<15mmol/L，乙酸乙烯酯浓度<10mmol/L)，无底物抑制现象，反应遵循Michaelis-Menton方程，符合乒乓机制。
采用高速逆流色谱(HSCCC)与高效制备液相色谱对乙酰化EGCG粗产品进行分离纯化。确定HSCCC分离条件为:上相为固定相，下相为流动相，采用正己烷：乙酸乙酯：甲醇：水=1.5:5:1.5:5(v/v)为溶剂系统，分离温度为20℃，转速为700r/min，流动相以5mL/min的流速从首端进行洗脱。分离产物经过质谱和核磁共振鉴定分别为5″-O-乙酰基EGCG，3″,5″-2-O-乙酰基EGCG和5′,3″,5″-3-O-乙酰基-EGCG，确定了在脂肪酶Lipozyme RM IM催化作用下，EGCG发生酰化的位点是B环的5′位和D环的3″,5″位。
通过溶解度实验，透光率实验和油水分配系数(LogP)测定表明，乙酰化EGCG的脂溶性得到明显提高，30℃时在大豆油中溶解度为397mg/kg。色差实验表明在添加量为200mg/kg时，乙酰化EGCG对植物油色泽和亮度没有明显影响。采用过氧化值法和Rancimat仪方法考察并比较了乙酰化EGCG、未改性EGCG、2,6-二特丁基对甲酚(BHT)、叔丁基对苯二酚(TBHQ)在不同油脂油中的抗氧化性能。结果表明，乙酰化EGCG在油脂中具有良好的抗氧化性，在添加量相同时，乙酰化EGCG在油脂中的抗氧化活性要高于未改性EGCG、BHT，略低于TBHQ。乙酰化EGCG产物的溶剂残留符合欧洲药典标准，安全可靠。在添加乙酰化EGCG后，油脂的理化指标没有改变。
通过测定乙酰化EGCG对超氧阴离子自由基(O2·-)、羟基自由基(·OH)、DPPH自由基的清除能力，研究其体外抗氧化活性，结果表明，乙酰化EGCG对O2·-、·OH、DPPH·均具有较强的清除能力，半抑制率(IC50)分别为0.52mg/mL、0.43mg/mL和11.5mg/L。体外抗脂质过氧化实验表明当乙酰化EGCG浓度为320mg/L时，对H2O2和Fe2+诱导大鼠肝线粒体丙二醛(MDA)生成的抑制率分别为69.44%和74.77%，对H2O2诱导大鼠红细胞氧化溶血的抑制率达93.54%。乙酰化EGCG具有良好的体外抗氧化活性，其浓度与抗氧化活性呈现一定的量效关系。
(-)-Epigallocatechin-3-O-gallate (EGCG) is an active compound with the biologicalactivities of anti-oxidation, anti-mutation, radiation resistance, preventing cancer andcardiovascular diseases, modulating the immune system, anti-aging, etc.. However, with itslow solubility in lipophilic systems, the application of EGCG in food industry is limited. Inthis study, modification of EGCG by enzymatic acylation was carried out in order to improveits hydrophobic property. The acylated EGCG products were separated and purified, themolecular structure of the productes were determined and the acylation positions for EGCGwere obtained. And then the in vitro antioxidant properties and solubility for the acylatedEGCG products were further evaluated.
Firstly, the enzymatic acylation process of EGCG was established. Different C chains ofvinyl ester had great effect on the conversion of EGCG. The conversion yield reduced withthe increasing of C chains. The highest conversion yield was obtained with the reactionbetween EGCG and vinyl acetate. By response surface methodology, the optimal reactioncondition was obtained under40°C, with the enzyme concentration of2.1%, the reactiontime was12h and EGCG/vinyl acetate mole ratio was1.1, respectively. The conversionyield of acylated EGCG products reached87.37%under the optimal conditions. The acylatedproducts were determined by liquid chromatography tandem mass spectrometry and infraredspectrometry. The presence of mono-, di-and tri-acetylated derivatives in acetylated EGCGwas confirmed by LC-MS-MS. The acylation reaction of EGCG catalyzed by lipase wascontrolled by the kinetic theory. There was no substrate inhibition with the concentrationrange of EGCG <15mmol/L and vinyl acetate <10mmol/L. The reaction was in accordancewith the Michaelis-Menton equation, which followed the ping-pong mechanism.
High speed counter current chromatography (HSCCC) and preparative high performanceliquid chromatography was used to separate and purify the acylated EGCG products. Theconditions for HSCCC were: rotation rate of700r/min under20℃at a flow rate of5mL/min.And two-phase solvent system contained n-hexane, ethyl acetate, methanol and water withthe ratio of1.5:5:1.5:5was selected.The structures of acylated EGCG products wereidentified as5″-O-acetyl-EGCG,3″,5″-2-O-acetyl-EGCG and5′,3″,5″-3-O-acetyl-EGCGby TOF MS/MS,1H NMR and13C NMR.
The light transmittance experiments and soluble properties of acylated EGCG showedthat the acetylation products had excellent lipid soluble property and high transparency inedible oil. The solubility in soybean oil was425mg/kg under30℃. There was little effect oncolor and brightness of soybean oil with the addition of200mg/kg of acylated EGCG asshowed by chromatic aberration experiments. Furthermore, the antioxidant effects ofacetylated EGCG, EGCG, butylated hydroxytoluene (BHT)，tert-butyl hydroquinone (TBHQ)on edible oils were evaluated and compared by POV values and Rancimat apparatus. Theresults indicated that the solubility of acetylated EGCG in edible oils was significantlyimproved by modification. And the antioxidant capabilities of acetylated EGCG in edible oilswere superior to that of EGCG and BHT, but slightly inferior to that of TBHQ. The residueof acylated EGCG products was under the level of the European pharmacopoeia. Thephysicochemical property of the oil was not changed after adding the acetylated EGCGproducts.
The in vitro antioxidant activity of acetylated EGCG products was evaluated bymeasuring its scavenging effect on superoixide anion (O2·-), hydroxyl radical (·OH) and1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical. The acetylated EGCG was found to havegood scavenging effects on O2·-,·OH and DPPH·and the50%inhibitory concentrations(IC50) were0.52mg/mL,0.43mg/mL and11.5mg/L, respectively. Anti lipid peroxidationexperiments showed that with acetylated EGCG concentration of320mg/L, the inhibitionrate of malonyldialdehyde (MDA) formation in rat liver mitochondrial induced by H2O2was61.11%and that of hemolysis for rats red blood cells induced by H2O2was93.54%. Itindicated that acetylated EGCG had strong in vitro antioxidant activity. The antioxidantactivity was dependent on the concentration of acetylated EGCG products.
首先，确立了酰化EGCG酶法合成路线为以乙烯酯作为酰基供体的酯交换反应。不同碳链乙烯酯作为酰基供体对EGCG的转化率影响较大，随着碳链的增加，EGCG的转化率逐渐减小，EGCG与乙酸乙烯酯之间的酯交换反应能达到最高的转化率。采用响应面分析法对酶法制备乙酰化EGCG反应条件进行优化，最优条件为：脂肪酶Lipozyme RM IM为酶催化剂，添加量为2.1%(w/w底物)；乙腈和异丙醇(质量比为1:1)为反应溶剂体系；40℃反应12h；EGCG与乙酸乙烯酯底物摩尔比为1.1。在该条件下，EGCG乙酰化转化率达到90.32%。采用液质联用选择离子分析明确了乙酰化EGCG反应产物为单-，二-和三取代乙酰化EGCG的混合物。在该反应体系中，脂肪酶LipozymeRM IM催化EGCG和乙酸乙烯酯的酯交换反应为动力学控制，在所研究的底物浓度范围内(EGCG浓度<15mmol/L，乙酸乙烯酯浓度<10mmol/L)，无底物抑制现象，反应遵循Michaelis-Menton方程，符合乒乓机制。
采用高速逆流色谱(HSCCC)与高效制备液相色谱对乙酰化EGCG粗产品进行分离纯化。确定HSCCC分离条件为:上相为固定相，下相为流动相，采用正己烷：乙酸乙酯：甲醇：水=1.5:5:1.5:5(v/v)为溶剂系统，分离温度为20℃，转速为700r/min，流动相以5mL/min的流速从首端进行洗脱。分离产物经过质谱和核磁共振鉴定分别为5″-O-乙酰基EGCG，3″,5″-2-O-乙酰基EGCG和5′,3″,5″-3-O-乙酰基-EGCG，确定了在脂肪酶Lipozyme RM IM催化作用下，EGCG发生酰化的位点是B环的5′位和D环的3″,5″位。
通过溶解度实验，透光率实验和油水分配系数(LogP)测定表明，乙酰化EGCG的脂溶性得到明显提高，30℃时在大豆油中溶解度为397mg/kg。色差实验表明在添加量为200mg/kg时，乙酰化EGCG对植物油色泽和亮度没有明显影响。采用过氧化值法和Rancimat仪方法考察并比较了乙酰化EGCG、未改性EGCG、2,6-二特丁基对甲酚(BHT)、叔丁基对苯二酚(TBHQ)在不同油脂油中的抗氧化性能。结果表明，乙酰化EGCG在油脂中具有良好的抗氧化性，在添加量相同时，乙酰化EGCG在油脂中的抗氧化活性要高于未改性EGCG、BHT，略低于TBHQ。乙酰化EGCG产物的溶剂残留符合欧洲药典标准，安全可靠。在添加乙酰化EGCG后，油脂的理化指标没有改变。
通过测定乙酰化EGCG对超氧阴离子自由基(O2·-)、羟基自由基(·OH)、DPPH自由基的清除能力，研究其体外抗氧化活性，结果表明，乙酰化EGCG对O2·-、·OH、DPPH·均具有较强的清除能力，半抑制率(IC50)分别为0.52mg/mL、0.43mg/mL和11.5mg/L。体外抗脂质过氧化实验表明当乙酰化EGCG浓度为320mg/L时，对H2O2和Fe2+诱导大鼠肝线粒体丙二醛(MDA)生成的抑制率分别为69.44%和74.77%，对H2O2诱导大鼠红细胞氧化溶血的抑制率达93.54%。乙酰化EGCG具有良好的体外抗氧化活性，其浓度与抗氧化活性呈现一定的量效关系。
(-)-Epigallocatechin-3-O-gallate (EGCG) is an active compound with the biologicalactivities of anti-oxidation, anti-mutation, radiation resistance, preventing cancer andcardiovascular diseases, modulating the immune system, anti-aging, etc.. However, with itslow solubility in lipophilic systems, the application of EGCG in food industry is limited. Inthis study, modification of EGCG by enzymatic acylation was carried out in order to improveits hydrophobic property. The acylated EGCG products were separated and purified, themolecular structure of the productes were determined and the acylation positions for EGCGwere obtained. And then the in vitro antioxidant properties and solubility for the acylatedEGCG products were further evaluated.
Firstly, the enzymatic acylation process of EGCG was established. Different C chains ofvinyl ester had great effect on the conversion of EGCG. The conversion yield reduced withthe increasing of C chains. The highest conversion yield was obtained with the reactionbetween EGCG and vinyl acetate. By response surface methodology, the optimal reactioncondition was obtained under40°C, with the enzyme concentration of2.1%, the reactiontime was12h and EGCG/vinyl acetate mole ratio was1.1, respectively. The conversionyield of acylated EGCG products reached87.37%under the optimal conditions. The acylatedproducts were determined by liquid chromatography tandem mass spectrometry and infraredspectrometry. The presence of mono-, di-and tri-acetylated derivatives in acetylated EGCGwas confirmed by LC-MS-MS. The acylation reaction of EGCG catalyzed by lipase wascontrolled by the kinetic theory. There was no substrate inhibition with the concentrationrange of EGCG <15mmol/L and vinyl acetate <10mmol/L. The reaction was in accordancewith the Michaelis-Menton equation, which followed the ping-pong mechanism.
High speed counter current chromatography (HSCCC) and preparative high performanceliquid chromatography was used to separate and purify the acylated EGCG products. Theconditions for HSCCC were: rotation rate of700r/min under20℃at a flow rate of5mL/min.And two-phase solvent system contained n-hexane, ethyl acetate, methanol and water withthe ratio of1.5:5:1.5:5was selected.The structures of acylated EGCG products wereidentified as5″-O-acetyl-EGCG,3″,5″-2-O-acetyl-EGCG and5′,3″,5″-3-O-acetyl-EGCGby TOF MS/MS,1H NMR and13C NMR.
The light transmittance experiments and soluble properties of acylated EGCG showedthat the acetylation products had excellent lipid soluble property and high transparency inedible oil. The solubility in soybean oil was425mg/kg under30℃. There was little effect oncolor and brightness of soybean oil with the addition of200mg/kg of acylated EGCG asshowed by chromatic aberration experiments. Furthermore, the antioxidant effects ofacetylated EGCG, EGCG, butylated hydroxytoluene (BHT)，tert-butyl hydroquinone (TBHQ)on edible oils were evaluated and compared by POV values and Rancimat apparatus. Theresults indicated that the solubility of acetylated EGCG in edible oils was significantlyimproved by modification. And the antioxidant capabilities of acetylated EGCG in edible oilswere superior to that of EGCG and BHT, but slightly inferior to that of TBHQ. The residueof acylated EGCG products was under the level of the European pharmacopoeia. Thephysicochemical property of the oil was not changed after adding the acetylated EGCGproducts.
The in vitro antioxidant activity of acetylated EGCG products was evaluated bymeasuring its scavenging effect on superoixide anion (O2·-), hydroxyl radical (·OH) and1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical. The acetylated EGCG was found to havegood scavenging effects on O2·-,·OH and DPPH·and the50%inhibitory concentrations(IC50) were0.52mg/mL,0.43mg/mL and11.5mg/L, respectively. Anti lipid peroxidationexperiments showed that with acetylated EGCG concentration of320mg/L, the inhibitionrate of malonyldialdehyde (MDA) formation in rat liver mitochondrial induced by H2O2was61.11%and that of hemolysis for rats red blood cells induced by H2O2was93.54%. Itindicated that acetylated EGCG had strong in vitro antioxidant activity. The antioxidantactivity was dependent on the concentration of acetylated EGCG products.
引文
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