诃子抗氧化活性的研究
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摘要
氧化损伤可以导致衰老、癌症、免疫力下降等多种病症,外源性抗氧化剂可调整机体的氧化平衡、预防或抑制氧化损伤。本研究从卫生部颁布的用作保健食品的原料中筛选出抗氧化活性最强的原料—诃子,采用多体系评价方法和生物活性追踪法对其抗氧化活性进行了研究,并初步分析了其抗氧化活性的物质基础。
选取19种氧化活性较强的原料并采用FRAP法和DPPH清除率对其进行分析评价,结果发现诃子提取液具有最强抗氧化活性,其FRAP值(以FeSO4·7H2O当量计)为(4422.33±123.56)mmol·g-1, DPPH自由基清率为(90.123±1.388)%,总多酚含量为(211.23±8.24)mg·g-1,总黄酮含量为(49.51±0.00)mg·g-1。原料FRAP法抗氧化能力和总多酚含量之间有较高的相关性,相关系数R2值为0.7779。
通过单因素和正交试验确定诃子抗氧化活性物质最佳提取工艺为50%乙醇、料液比1：20(w·v-1)、60℃恒温振荡水浴提取7min、重复提取4次。此时活性物质提取率最大,FeSO4·7H2O相当当量达(8958.07±145.05)mmol·g-1。
采用龙井茶和银杏叶为对照对诃子抗氧化活性的比较研究表明,诃子抗氧化活性显著高于龙井茶和银杏叶。诃子提取液FRAP法抗氧化能力(以FeSO4·7H2O当量计)是龙井茶的1.43倍、银杏叶的10.81倍；DPPH自由基清除率是龙井茶的1.27倍、银杏叶的1.99倍。
本研究选择总还原力、FRAP法抗氧化能力、DPPH自由基清除能力、总抗氧化力、亚油酸脂质过氧化抑制能力及超氧阴离子清除能力等六种评价方法分析诃子粗提物的抗氧化活性。结果表明,诃子粗提物具备与分析纯阳性对照BHT相当的总还原力和FRAP法抗氧化能力；DPPH自由基清除能力显著高于同质量浓度BHT(P<0.001)，超氧阴离子清除能力显著高于同质量浓度的Vc(P<0.001),具备较强的总抗氧化力和亚油酸脂质过氧化抑制能力。
首次采用生物活性追踪法并结合多方法评价体系研究诃子抗氧化活性及其物质基础。选用极性逐渐增大的溶剂正已烷、乙酸乙酯、正丁醇和水对诃子粗提物依次进行萃取,真空干燥后冷冻干燥获得各极性干粉。测定结果表明,在诃子不同极性部位中弱极性乙酸乙酯相抗氧化活性最强。25、50μg/mL乙酸乙酯部位总还原力是阳性对照BHT的1.16、1.30倍,FRAP法抗氧化力分别是阳性对照BHT的5.19、3.36倍,DPPH自由清除率分别是阳性对照BHT的1.45、1.19倍；100、200μg·mL-1乙酸乙酯部位总抗氧化力与阳性对照BHT相当(P>0.001)；100μg·mL-1乙酸乙酯相亚油酸脂质过氧化抑制率在120h内持续上升至最高值(86.18±0.26)%,该浓度抑制率与同质量浓度BHT相当(P>0.001)；乙酸乙酯相超氧阴离子清除率显著高于同质量浓度阳性对照Vc和其它各极性相的清除率,500μg·mL-1乙酸乙酯部位超氧阴离子清除率最高(89.52±1.27)%,是同质量浓度阳性对照Vc的5.44倍(P<0.001)。活性成分含量和功能活性评价结果相关关系表明,诃子强抗氧化活性主要由其高含量总多酚类化合物所贡献。
研究结果表明,诃子具有较全面的强抗氧化活性,可用作抗氧化活性成分添加到食品中,以制作具有抗氧化活性的高附加值功能性食品。
The oxidation stress is being implicated in many diseases such as aging, cancer, and immunity deficiency etc, which can be prevented by xenobiolic antioxidant. Terminalia chebula Retz.was proven to be of the strongest antioxidant activity among 19 stuffs as food additives permitted by Ministry of Health of China for the manufacture of functional foods..
The antioxidant activities of 19 stuffs were studied based on the FRAP values and the data of Reducing power. The results showed that the extract solution from Terminalia chebula Retz has highest antioxidant activity among 19 stuffs tested. The FRAP value and the percentage of scavenging DPPH radicals from it were (4422.33±123.56) mmol·g-1 and (90.123±1.388)%, respectively. The contents of total flavonoids and phenols were (211.23±8.24) mg·g-1 and (49.51±0.00) mg·g-1, respectively. The FRAP value of different stuffs has high correlation with the total phenols contents with the correlation coefficient up to 0.7779.
The extracting technology of bioactive components of Terminalia chebula Retz. were studied. The optimal parameters of the extracting technology were that the concentration of ethanol was 50%, the ratio of sample to extracting solution was 1:20 (m·v-1), the extracting temperature was 60℃, the extracting times was 4 and the extracting time was 7 min. The recovery ratio of bioactive compounds of Terminalia chebula Retz. was (8958.07±145.05) mmol·g-1 by using this optimum technology.
In order to further prove the strongest antioxidant activity of Terminalia chebula Retz., comparison tests of antioxidant activities were conducted taking Longjing tea and Ginkgo biloba leaf as references which are conventionally accepted as strong antioxidant. The results showed that the FRAP value of Terminalia chebula Retz. was 1.43 times more than that of Longjing tea, and 10.81 times more than that of Ginkgo biloba leaf. The percentage of scavenging DPPH radicals of Terminalia chebula Retz. was 1.27 times more than that of Longjing tea, and 1.99 times more than that of Ginkgo biloba leaf.
The antioxidant activity of crude extracts from Terminalia chebula Retz.was proved to have strong antioxidant activity by using six evaluation indexs including the reducing power assay, the FRAP values, the DPPH radicals scavenging percentage, the total antioxidant activity, the inhibition of linoleic acid peroxidation and superoxide anion radicals scavenging percentage. It was found by the bioassay-guided method combing with multilateral method analysis system that the bioactive antioxidants of Terminalia chebula Retz. were mainly lay in the ethyl acetate fraction and the fraction was proved to has strongest antioxidant activities among all subfractions. As for the ethyl acetate fraction, the OD values at 700 nm (reducing power) of with the concentrations of 25 and 50μg·mL-1 were 1.16,1.30 times, and the FRAP values (OD593 nm) were 5.19,3.36 times, and the percentage of scavenging DPPH radicals were 1.45,1.19 times, more than that of BHT respectively. The total antioxidant activity at 695nm with concentrations of 100 and 200μg·mL-1 was a bit equivalent to that of BHT. Inhibition of linoleic peroxidation of the same fraction with concentration of 100μg·mL-1 was being increased up to (86.18±0.26)% during the culture of 120h, which was almost equal to that of BHT. The scavenging percentages for superoxide anion radical with concentration of 500μg·mL-1 was highest among all fractions, up to (89.52±1.27)%, which was 5.44 times more than that of Vc. The correlation of different subfractions between the active component contents and the various methods showed that the strong antioxidant activity mainly resultes from the high contents of the total phenols and total flavonoids.
It comes to conclusion that Terminalia chebula Retz., with high antioxidant activity can be used as functional ingredient for prodution of functional foods with high added value.
选取19种氧化活性较强的原料并采用FRAP法和DPPH清除率对其进行分析评价,结果发现诃子提取液具有最强抗氧化活性,其FRAP值(以FeSO4·7H2O当量计)为(4422.33±123.56)mmol·g-1, DPPH自由基清率为(90.123±1.388)%,总多酚含量为(211.23±8.24)mg·g-1,总黄酮含量为(49.51±0.00)mg·g-1。原料FRAP法抗氧化能力和总多酚含量之间有较高的相关性,相关系数R2值为0.7779。
通过单因素和正交试验确定诃子抗氧化活性物质最佳提取工艺为50%乙醇、料液比1：20(w·v-1)、60℃恒温振荡水浴提取7min、重复提取4次。此时活性物质提取率最大,FeSO4·7H2O相当当量达(8958.07±145.05)mmol·g-1。
采用龙井茶和银杏叶为对照对诃子抗氧化活性的比较研究表明,诃子抗氧化活性显著高于龙井茶和银杏叶。诃子提取液FRAP法抗氧化能力(以FeSO4·7H2O当量计)是龙井茶的1.43倍、银杏叶的10.81倍；DPPH自由基清除率是龙井茶的1.27倍、银杏叶的1.99倍。
本研究选择总还原力、FRAP法抗氧化能力、DPPH自由基清除能力、总抗氧化力、亚油酸脂质过氧化抑制能力及超氧阴离子清除能力等六种评价方法分析诃子粗提物的抗氧化活性。结果表明,诃子粗提物具备与分析纯阳性对照BHT相当的总还原力和FRAP法抗氧化能力；DPPH自由基清除能力显著高于同质量浓度BHT(P<0.001)，超氧阴离子清除能力显著高于同质量浓度的Vc(P<0.001),具备较强的总抗氧化力和亚油酸脂质过氧化抑制能力。
首次采用生物活性追踪法并结合多方法评价体系研究诃子抗氧化活性及其物质基础。选用极性逐渐增大的溶剂正已烷、乙酸乙酯、正丁醇和水对诃子粗提物依次进行萃取,真空干燥后冷冻干燥获得各极性干粉。测定结果表明,在诃子不同极性部位中弱极性乙酸乙酯相抗氧化活性最强。25、50μg/mL乙酸乙酯部位总还原力是阳性对照BHT的1.16、1.30倍,FRAP法抗氧化力分别是阳性对照BHT的5.19、3.36倍,DPPH自由清除率分别是阳性对照BHT的1.45、1.19倍；100、200μg·mL-1乙酸乙酯部位总抗氧化力与阳性对照BHT相当(P>0.001)；100μg·mL-1乙酸乙酯相亚油酸脂质过氧化抑制率在120h内持续上升至最高值(86.18±0.26)%,该浓度抑制率与同质量浓度BHT相当(P>0.001)；乙酸乙酯相超氧阴离子清除率显著高于同质量浓度阳性对照Vc和其它各极性相的清除率,500μg·mL-1乙酸乙酯部位超氧阴离子清除率最高(89.52±1.27)%,是同质量浓度阳性对照Vc的5.44倍(P<0.001)。活性成分含量和功能活性评价结果相关关系表明,诃子强抗氧化活性主要由其高含量总多酚类化合物所贡献。
研究结果表明,诃子具有较全面的强抗氧化活性,可用作抗氧化活性成分添加到食品中,以制作具有抗氧化活性的高附加值功能性食品。
The oxidation stress is being implicated in many diseases such as aging, cancer, and immunity deficiency etc, which can be prevented by xenobiolic antioxidant. Terminalia chebula Retz.was proven to be of the strongest antioxidant activity among 19 stuffs as food additives permitted by Ministry of Health of China for the manufacture of functional foods..
The antioxidant activities of 19 stuffs were studied based on the FRAP values and the data of Reducing power. The results showed that the extract solution from Terminalia chebula Retz has highest antioxidant activity among 19 stuffs tested. The FRAP value and the percentage of scavenging DPPH radicals from it were (4422.33±123.56) mmol·g-1 and (90.123±1.388)%, respectively. The contents of total flavonoids and phenols were (211.23±8.24) mg·g-1 and (49.51±0.00) mg·g-1, respectively. The FRAP value of different stuffs has high correlation with the total phenols contents with the correlation coefficient up to 0.7779.
The extracting technology of bioactive components of Terminalia chebula Retz. were studied. The optimal parameters of the extracting technology were that the concentration of ethanol was 50%, the ratio of sample to extracting solution was 1:20 (m·v-1), the extracting temperature was 60℃, the extracting times was 4 and the extracting time was 7 min. The recovery ratio of bioactive compounds of Terminalia chebula Retz. was (8958.07±145.05) mmol·g-1 by using this optimum technology.
In order to further prove the strongest antioxidant activity of Terminalia chebula Retz., comparison tests of antioxidant activities were conducted taking Longjing tea and Ginkgo biloba leaf as references which are conventionally accepted as strong antioxidant. The results showed that the FRAP value of Terminalia chebula Retz. was 1.43 times more than that of Longjing tea, and 10.81 times more than that of Ginkgo biloba leaf. The percentage of scavenging DPPH radicals of Terminalia chebula Retz. was 1.27 times more than that of Longjing tea, and 1.99 times more than that of Ginkgo biloba leaf.
The antioxidant activity of crude extracts from Terminalia chebula Retz.was proved to have strong antioxidant activity by using six evaluation indexs including the reducing power assay, the FRAP values, the DPPH radicals scavenging percentage, the total antioxidant activity, the inhibition of linoleic acid peroxidation and superoxide anion radicals scavenging percentage. It was found by the bioassay-guided method combing with multilateral method analysis system that the bioactive antioxidants of Terminalia chebula Retz. were mainly lay in the ethyl acetate fraction and the fraction was proved to has strongest antioxidant activities among all subfractions. As for the ethyl acetate fraction, the OD values at 700 nm (reducing power) of with the concentrations of 25 and 50μg·mL-1 were 1.16,1.30 times, and the FRAP values (OD593 nm) were 5.19,3.36 times, and the percentage of scavenging DPPH radicals were 1.45,1.19 times, more than that of BHT respectively. The total antioxidant activity at 695nm with concentrations of 100 and 200μg·mL-1 was a bit equivalent to that of BHT. Inhibition of linoleic peroxidation of the same fraction with concentration of 100μg·mL-1 was being increased up to (86.18±0.26)% during the culture of 120h, which was almost equal to that of BHT. The scavenging percentages for superoxide anion radical with concentration of 500μg·mL-1 was highest among all fractions, up to (89.52±1.27)%, which was 5.44 times more than that of Vc. The correlation of different subfractions between the active component contents and the various methods showed that the strong antioxidant activity mainly resultes from the high contents of the total phenols and total flavonoids.
It comes to conclusion that Terminalia chebula Retz., with high antioxidant activity can be used as functional ingredient for prodution of functional foods with high added value.
引文
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