葡萄酒抗氧化活性及其检测方法的研究
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摘要
流行性病学研究结果显示：适量的消费葡萄酒可以降低心血管疾病、动脉粥样硬化、血小板聚集和癌症等多种疾病的发病率。葡萄酒尤其是红葡萄酒中含有大量的酚类物质，葡萄酒的各种保健功效被认为与这些物质的抗氧化能力有关。我国葡萄酒工业以及葡萄酒市场正在不断地发展，并且这一趋势将会不断扩长。但目前为止，系统地研究我国葡萄酒的主要抗氧化成分和抗氧化能力的研究非常少。对于葡萄酒抗氧化能力的研究可以很好的评价葡萄酒的质量，为消费者提供参考，并且为葡萄酒的工艺措施改革提供理论依据。然而，由于抗氧化自身的复杂性和反应机制的多重性，使得目前没有一种标准方法可以代替和概括其它测定方法。因此，本研究利用多种抗氧化检测方法对葡萄酒以及葡萄酒副产物一葡萄籽微粉的‘抗氧化轮廓’进行综合评价。
本实验通过对各种方法的比较，最终选择并且优化、发展两种快速、精确可以日常操作的抗氧化测定方法(CUPRAC和ORAC)。铜离子还原能力测定(CUPRAC)最佳检测条件为：铜离子浓度5 mM，新亚铜试剂浓度3．75 mM，醋酸铵缓冲液(pH=7)1M，反应温度37℃，反应时间30 min，检测波长450 nm；葡萄酒最佳稀释倍数为：红葡萄酒12．5-25倍，白葡萄酒1．25-5倍(分光光度计)；红葡萄酒62．5-125倍，白葡萄酒6．25-25倍(酶标法)。氧自由基吸收能力(ORAC)最佳检测条件为：荧光素钠溶液为4×10~(-3)μM，AAPH浓度为160 mM；红葡萄酒测定最佳稀释倍数为500-1000倍，白葡萄酒最佳稀释倍数为200-500倍。
使用DPPH自由基清除能力(DPPH)、ABTS自由基清除能力(ABTS)、铜离子还原能力(CUPRAC)、氧自由基清除能力(ORAC)、羟自由基清除能力(HRSA)、超氧自由基清除能力(SRSA)、脂质过氧化抑制力(TBARS)、金属离子鳌合能力(MC)共八种不同机制的方法测定葡萄酒的抗氧化能力。虽然各种方法的测定结果在数值上各不相同，但抗氧化能力呈现一致性：红葡萄酒＞桃红葡萄酒＞白葡萄酒。同时，比较了相同葡萄园、相同栽培管理条件和相同工艺条件的不同品种葡萄酒之间的差异，结果显示：红色品种中赤霞珠葡萄酒的抗氧化能力最强；白色品种中贵人香葡萄酒的抗氧化能力最弱。
葡萄酒中的总酚、总类黄酮、总黄烷醇和总花色苷含量被调查。与抗氧化能力测定结果一致，红葡萄酒＞桃红葡萄酒＞白葡萄酒。红葡萄酒不同品种的总酚、总类黄酮和黄烷醇含量比较，赤霞珠葡萄酒＞梅鹿辄葡萄酒＞蛇龙珠葡萄酒；总花色苷含量比较，蛇龙珠葡萄酒＞赤霞珠葡萄酒＞梅鹿辄葡萄酒；白葡萄酒不同品种的总酚和总类黄酮含量比较，雷司令葡萄酒的各项含量为最高，贵人香为最低。酚类物质与抗氧化能力的相关性研究结果显示：葡萄酒的总酚、总类黄酮和黄烷醇物质与抗氧化能力有很强的相关性，而总花色苷含量与抗氧化能力相关性弱。
通过大量的文献数据统计和本实验中葡萄酒的抗氧化测定结果，综合分析了九种最常用抗氧化测定方法间的相关性。在各方法相关性的研究中，TP、ORAC、ABTS、DPPH、CUPRAC、SRSA和HRSA方法之间表现出显著性；TBARS和MC法与其它各方法的相关性较差。结合文献统计数据，分析认为ORAC、ABTS、DPPH和CUPRAC法之间相关性达到极显著水平，其中任何一种可以代替其它三种方法测定葡萄酒。此外，不同的方法对于不同的材料适应性不一样。综合考虑，在目前情况下，抗氧化能力测定需要使用不同机制的方法同时进行测定，包括生理相关性高的方法和其它机理的方法。
葡萄籽微粉不同溶剂提取液均有比较高的总酚含量，表现出强的抗氧化能力。不同提取溶剂之间的比较，葡萄籽微粉70％丙酮提取液的各项指标最高，而葡萄籽微粉水提物的各项指标最低。
模拟胃肠消化环境处理的体外抗氧化能力测定方法，是一种快速、廉价、重复性高可以日常操作的方法。模拟胃肠环境的体外检测方法包括：胃环境处理、肠环境处理以及利用透析膜模拟的小肠被动吸收，其中涉及了酸性环境的酸解、胃蛋白酶和胰蛋白酶酶解等多种生理反应。与体外方法相比，模拟生理方法更加接近真实的生理环境。与体内方法相比，模拟生理方法更加简单、快速，并且可以克服生物体存在的多种不可预知的变异发生。葡萄酒和葡萄籽微粉模拟胃肠环境处理后，在总酚成分和抗氧化能力的测定中均表现出比较高的水平。在模拟小肠吸收的实验中，不论葡萄酒还是葡萄籽微粉均为透析液测定值低于未透析液测定值，其比例为3：7到1：1。
Epidemiological evidence indicates that the moderate consumption of wines reduces theincident of coronary heart disease,artherosclerosis,platelet aggregation and Cancer.Thisgreater protection may be due to the phenolic components of wines,which are particularlyabundant in the red wine,since they behave as reactive oxygene species scavengers and metalchelators.The wine industry is growing and the wine market has a wider space to develop inChina,it will be even more prosperous in future.To date,there has been no publishedresearch on the chemical quality and antioxidant activity of wines produced in China.Withestablishing an“antioxidant profile”this paper will help to better understand the quality ofcurrent wines and stimulate the development of enological techniques for their enrichment.Because multiple reaction characteristics and mechanisms are usually involved,no singleassay will accurately reflect all antioxidants in a mixed or complex system.Thus,to fullyelucidate a full profile of antioxidant capacity,different antioxidant capacity assays may beneeded.
The purpose of the study was to develop a simple,accurate and rapid antioxidantcapacity analysis method for measuring antioxidant capacity in table wine.The developedmethod could have the application in routine quantific ation of antioxidant capacity forChinese table wine.The parameters of the CUPRAC (cupric reducing antioxidant capacity)method were optimized.It involves mixing the wine with solutions of 5mM CuCl_2,3.75 mMneocuproine,and ammonium acetate at pH 7,and measuring the absorbance at 450 nm after30 min.Optimum dilutions for white and red wines were set up as a function of their totalphenolic content (for spectrophotometric measurements,red wine,dilution 12.5 to 25,whitewine,dilution 1.25 to 5;for microplate reader,red wine,dilution 62.5 to 125,white wine,dilution 6.25 to 25).The parameters of the ORAC (oxygen radical absorbance capacity)method were optimized.It involves mixing the wine with solutions of 4×10~(-3)μM sodiumfluorescein,160 mM AAPH,and phosphate buffer at pH 7.4.Optimum dilutions for whiteand red wines were set up as a function of their total phenolic content (red wine,dilution 500to 1000;white wine,dilution 200 to 500).
The antioxidant activity of wines was measured by different analytical methods:oxygen radical absorbance capacity (ORAC),reducing power (CUPRAC),2,2-azino-di-(3-ethylbenzothialozine-sulphonic acid) (ABTS),2,2-diphenyl-1-picrylhydrazyl(DPPH),hydroxyl radical scavenger activity (HRSA),superoxide radical scavenger activity(SRSA),lipid peroxidation (TBARS) and chelating capacity (MC).As expected,the redwines had much higher antioxidant capacity than ros(?) wines and white wines.In order tocompare antioxidant capacity of different grape variety,we chose several wines.These wineshad same situations including same age,vintage and winemaking techniques.Amongst the redwines,Cabernet Sauvignon represented the wine with the highest antioxidant capacity.Amongst the white wines,Italian Riesling represented the one with lowest antioxidantcapacity.
Total phenols,total flavonoids,total flavanols and total anthocyanins of wines weredetermined.As expected,the red wines had much higher phenolic content than ros(?) wines andwhite wines.The phenolic content of three red wines (Cabernet Sauvignon,CabernetGernischet and Merlot) and three white wines (Chardonnay,Italian Riesling and Riesling)were analyzed and compared.Amongst the red wines,the total phenols,the total flavonoidsand the total flavanols decrease in the order:Cabernet Sauvignon＞Merlot＞CabernetGernischet,while the total anthocyanins decrease in the order:Cabernet Gernischet＞Cabernet Sauvignon＞Merlot.Amongst the white wines,Chardonnay and Italian Riesling,respectively,represented the wine with the highest and lowest phenolic contents.The totalphenol,flavonoids and flavanol contents of wines exhibited the strongest correlation withantioxidant properties,while total anthocyanins exhibited weaker correlations.
This work analyzed the relativity about the most common nine methods that can measureantioxidant activity by studying many literatures.At the time,the result of wine antioxidantactivity in this paper was analyzed.Regarding different methods,the significant correlationbetween two methods was confirmed with seven methods (TP,ORAC,ABTS,DPPH,CUPRAC,SRSA and HRSA),while TBARS and MC exhibited weaker correlations withother methods.Taken together,a relatively tight coupling of these four parameters (ORAC,DPPH,ABTS and CUPRAC) indicates that every one of them can be considered as a relevantand reliable characteristic of the antioxidant capacity of wines.And also,there is muchchange of relativity in different methods for different materials.So,at the present time,weshould choose several methods which have highest relativity with physiology,and choose themethods which based on other mechanism at the same time.
The total phenolic content and antioxidant activity of grape seed powder extracted by invitro procedure.It has been found that grape seed powder has higher total phenolic contentand antioxidant capacity.As for solvents extracts,extraction with acetone:water (70:30) led to maximum phenol content and antioxidant capacity,while water gave the lowest phenolcontent and antioxidant capacity.
The in vitro digestion method is simple,cheap,and reproducible and can be used tostudy a wide range of experimental conditions.Therefore,the biological properties ofantioxidants may depend on their release from the food matrix during the digestion processand may be more useful for nutritional purposes than the values determined in chemicalprocedure.It has been found that both wine and grape seed powder have higher total phenoliccontent and antioxidant capacity by in vitro physiological procedure.As for digestiveenzymatic extracts,total phenolic content and antioxidant capacity of the dialysates of wineand grape seed powder were lower than those of the retentates (3:7-1:1).
本实验通过对各种方法的比较，最终选择并且优化、发展两种快速、精确可以日常操作的抗氧化测定方法(CUPRAC和ORAC)。铜离子还原能力测定(CUPRAC)最佳检测条件为：铜离子浓度5 mM，新亚铜试剂浓度3．75 mM，醋酸铵缓冲液(pH=7)1M，反应温度37℃，反应时间30 min，检测波长450 nm；葡萄酒最佳稀释倍数为：红葡萄酒12．5-25倍，白葡萄酒1．25-5倍(分光光度计)；红葡萄酒62．5-125倍，白葡萄酒6．25-25倍(酶标法)。氧自由基吸收能力(ORAC)最佳检测条件为：荧光素钠溶液为4×10~(-3)μM，AAPH浓度为160 mM；红葡萄酒测定最佳稀释倍数为500-1000倍，白葡萄酒最佳稀释倍数为200-500倍。
使用DPPH自由基清除能力(DPPH)、ABTS自由基清除能力(ABTS)、铜离子还原能力(CUPRAC)、氧自由基清除能力(ORAC)、羟自由基清除能力(HRSA)、超氧自由基清除能力(SRSA)、脂质过氧化抑制力(TBARS)、金属离子鳌合能力(MC)共八种不同机制的方法测定葡萄酒的抗氧化能力。虽然各种方法的测定结果在数值上各不相同，但抗氧化能力呈现一致性：红葡萄酒＞桃红葡萄酒＞白葡萄酒。同时，比较了相同葡萄园、相同栽培管理条件和相同工艺条件的不同品种葡萄酒之间的差异，结果显示：红色品种中赤霞珠葡萄酒的抗氧化能力最强；白色品种中贵人香葡萄酒的抗氧化能力最弱。
葡萄酒中的总酚、总类黄酮、总黄烷醇和总花色苷含量被调查。与抗氧化能力测定结果一致，红葡萄酒＞桃红葡萄酒＞白葡萄酒。红葡萄酒不同品种的总酚、总类黄酮和黄烷醇含量比较，赤霞珠葡萄酒＞梅鹿辄葡萄酒＞蛇龙珠葡萄酒；总花色苷含量比较，蛇龙珠葡萄酒＞赤霞珠葡萄酒＞梅鹿辄葡萄酒；白葡萄酒不同品种的总酚和总类黄酮含量比较，雷司令葡萄酒的各项含量为最高，贵人香为最低。酚类物质与抗氧化能力的相关性研究结果显示：葡萄酒的总酚、总类黄酮和黄烷醇物质与抗氧化能力有很强的相关性，而总花色苷含量与抗氧化能力相关性弱。
通过大量的文献数据统计和本实验中葡萄酒的抗氧化测定结果，综合分析了九种最常用抗氧化测定方法间的相关性。在各方法相关性的研究中，TP、ORAC、ABTS、DPPH、CUPRAC、SRSA和HRSA方法之间表现出显著性；TBARS和MC法与其它各方法的相关性较差。结合文献统计数据，分析认为ORAC、ABTS、DPPH和CUPRAC法之间相关性达到极显著水平，其中任何一种可以代替其它三种方法测定葡萄酒。此外，不同的方法对于不同的材料适应性不一样。综合考虑，在目前情况下，抗氧化能力测定需要使用不同机制的方法同时进行测定，包括生理相关性高的方法和其它机理的方法。
葡萄籽微粉不同溶剂提取液均有比较高的总酚含量，表现出强的抗氧化能力。不同提取溶剂之间的比较，葡萄籽微粉70％丙酮提取液的各项指标最高，而葡萄籽微粉水提物的各项指标最低。
模拟胃肠消化环境处理的体外抗氧化能力测定方法，是一种快速、廉价、重复性高可以日常操作的方法。模拟胃肠环境的体外检测方法包括：胃环境处理、肠环境处理以及利用透析膜模拟的小肠被动吸收，其中涉及了酸性环境的酸解、胃蛋白酶和胰蛋白酶酶解等多种生理反应。与体外方法相比，模拟生理方法更加接近真实的生理环境。与体内方法相比，模拟生理方法更加简单、快速，并且可以克服生物体存在的多种不可预知的变异发生。葡萄酒和葡萄籽微粉模拟胃肠环境处理后，在总酚成分和抗氧化能力的测定中均表现出比较高的水平。在模拟小肠吸收的实验中，不论葡萄酒还是葡萄籽微粉均为透析液测定值低于未透析液测定值，其比例为3：7到1：1。
Epidemiological evidence indicates that the moderate consumption of wines reduces theincident of coronary heart disease,artherosclerosis,platelet aggregation and Cancer.Thisgreater protection may be due to the phenolic components of wines,which are particularlyabundant in the red wine,since they behave as reactive oxygene species scavengers and metalchelators.The wine industry is growing and the wine market has a wider space to develop inChina,it will be even more prosperous in future.To date,there has been no publishedresearch on the chemical quality and antioxidant activity of wines produced in China.Withestablishing an“antioxidant profile”this paper will help to better understand the quality ofcurrent wines and stimulate the development of enological techniques for their enrichment.Because multiple reaction characteristics and mechanisms are usually involved,no singleassay will accurately reflect all antioxidants in a mixed or complex system.Thus,to fullyelucidate a full profile of antioxidant capacity,different antioxidant capacity assays may beneeded.
The purpose of the study was to develop a simple,accurate and rapid antioxidantcapacity analysis method for measuring antioxidant capacity in table wine.The developedmethod could have the application in routine quantific ation of antioxidant capacity forChinese table wine.The parameters of the CUPRAC (cupric reducing antioxidant capacity)method were optimized.It involves mixing the wine with solutions of 5mM CuCl_2,3.75 mMneocuproine,and ammonium acetate at pH 7,and measuring the absorbance at 450 nm after30 min.Optimum dilutions for white and red wines were set up as a function of their totalphenolic content (for spectrophotometric measurements,red wine,dilution 12.5 to 25,whitewine,dilution 1.25 to 5;for microplate reader,red wine,dilution 62.5 to 125,white wine,dilution 6.25 to 25).The parameters of the ORAC (oxygen radical absorbance capacity)method were optimized.It involves mixing the wine with solutions of 4×10~(-3)μM sodiumfluorescein,160 mM AAPH,and phosphate buffer at pH 7.4.Optimum dilutions for whiteand red wines were set up as a function of their total phenolic content (red wine,dilution 500to 1000;white wine,dilution 200 to 500).
The antioxidant activity of wines was measured by different analytical methods:oxygen radical absorbance capacity (ORAC),reducing power (CUPRAC),2,2-azino-di-(3-ethylbenzothialozine-sulphonic acid) (ABTS),2,2-diphenyl-1-picrylhydrazyl(DPPH),hydroxyl radical scavenger activity (HRSA),superoxide radical scavenger activity(SRSA),lipid peroxidation (TBARS) and chelating capacity (MC).As expected,the redwines had much higher antioxidant capacity than ros(?) wines and white wines.In order tocompare antioxidant capacity of different grape variety,we chose several wines.These wineshad same situations including same age,vintage and winemaking techniques.Amongst the redwines,Cabernet Sauvignon represented the wine with the highest antioxidant capacity.Amongst the white wines,Italian Riesling represented the one with lowest antioxidantcapacity.
Total phenols,total flavonoids,total flavanols and total anthocyanins of wines weredetermined.As expected,the red wines had much higher phenolic content than ros(?) wines andwhite wines.The phenolic content of three red wines (Cabernet Sauvignon,CabernetGernischet and Merlot) and three white wines (Chardonnay,Italian Riesling and Riesling)were analyzed and compared.Amongst the red wines,the total phenols,the total flavonoidsand the total flavanols decrease in the order:Cabernet Sauvignon＞Merlot＞CabernetGernischet,while the total anthocyanins decrease in the order:Cabernet Gernischet＞Cabernet Sauvignon＞Merlot.Amongst the white wines,Chardonnay and Italian Riesling,respectively,represented the wine with the highest and lowest phenolic contents.The totalphenol,flavonoids and flavanol contents of wines exhibited the strongest correlation withantioxidant properties,while total anthocyanins exhibited weaker correlations.
This work analyzed the relativity about the most common nine methods that can measureantioxidant activity by studying many literatures.At the time,the result of wine antioxidantactivity in this paper was analyzed.Regarding different methods,the significant correlationbetween two methods was confirmed with seven methods (TP,ORAC,ABTS,DPPH,CUPRAC,SRSA and HRSA),while TBARS and MC exhibited weaker correlations withother methods.Taken together,a relatively tight coupling of these four parameters (ORAC,DPPH,ABTS and CUPRAC) indicates that every one of them can be considered as a relevantand reliable characteristic of the antioxidant capacity of wines.And also,there is muchchange of relativity in different methods for different materials.So,at the present time,weshould choose several methods which have highest relativity with physiology,and choose themethods which based on other mechanism at the same time.
The total phenolic content and antioxidant activity of grape seed powder extracted by invitro procedure.It has been found that grape seed powder has higher total phenolic contentand antioxidant capacity.As for solvents extracts,extraction with acetone:water (70:30) led to maximum phenol content and antioxidant capacity,while water gave the lowest phenolcontent and antioxidant capacity.
The in vitro digestion method is simple,cheap,and reproducible and can be used tostudy a wide range of experimental conditions.Therefore,the biological properties ofantioxidants may depend on their release from the food matrix during the digestion processand may be more useful for nutritional purposes than the values determined in chemicalprocedure.It has been found that both wine and grape seed powder have higher total phenoliccontent and antioxidant capacity by in vitro physiological procedure.As for digestiveenzymatic extracts,total phenolic content and antioxidant capacity of the dialysates of wineand grape seed powder were lower than those of the retentates (3:7-1:1).
引文
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