脱脂沙苑子乙醇提取物的体外抗氧化活性研究
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摘要
合成抗氧化剂(如BHA和BHT)大多数为芳胺或苯酚类,大多通过阻断链式反应机理发挥抗氧化作用,因其具有一定的毒性和致癌作用,限制了这些物质的使用。因此,开发廉价、高效、安全无毒的天然抗氧化剂成为众多研究者关注的热点之一。沙苑子为豆科植物扁茎黄芪(Astragalus complanatus)的干燥成熟种子,是一味补益肝肾的传统中药,广泛分布在东北、华北和西北。沙苑子黄酮是沙苑子的主要活性成分,具有抗衰老、降脂保肝、抑制血小板聚集、增强免疫功能及调节中枢神经系统等作用。目前已有一系列沙苑子保健食品上市。尽管已有报道证明沙苑子有清除超氧阴离子能力,但至今尚无沙苑子体外抗氧化活性的系统研究,因此本文首次系统研究沙苑子提取物的体外抗氧化活性,以期进一步揭示沙苑子的药理作用机制,促进沙苑子中药资源的开发利用。
本研究主要分三大部分：
第一步,制备脱脂沙苑子乙醇提取物(the defatted ethanolic extract from Semen Astragali Complanati, ESA),并采用分光光度法测定主要化学成分总糖、总黄酮、总多酚的含量。
第二步,通过构建体外化学体系,包括β-胡萝卜素/亚油酸自氧化体系、还原能力测定、DPPH·/O2-·/·OH三种自由基清除体系及Fe2+诱导的脂质过氧化反应,研究ESA的体外抗氧化活性。
第三步,研究ESA对·OH诱导的线粒体氧化损伤的保护活性。研究按照以下操作进行：采用0.25 M蔗糖溶液提取、差速离心法制备大鼠肝线粒体,利用Fe2+/Vc或Fe2+/H2O2系统产生·OH诱导大鼠肝线粒体损伤,测定ESA对肝线粒体肿胀程度、脂质过氧化和蛋白质羰基生成水平的抑制作用,特别是对ATP酶活性以及超氧阴离子的生成的影响。
研究结果表明：
1、干燥的沙苑子经提取得到ESA,得率为6.73%,ESA中主要成分为糖类、黄酮类、多酚类物质,其含量分别为44.92%、7.47%和14.24%。
2、ESA具有很强的清除自由基的能力,均呈现剂量依赖性效应,但在不同的体系中机制不同。其清除DPPH·、O2-·、·OH三种自由基的IC50依次为419.1μg/mL、117.9μg/mL和224.4μg/mL,其清除超氧阴离子的作用最强、羟基次之、DPPH·最差。
3、ESA有较强的还原力,且与浓度呈正相关。4.2 mg/mL的ESA相当于160μg/mL的Vc和300μg/mL的BHT的还原力。对β-胡萝卜素褪色有明显的抑制作用,150μg/mL、600μg/mL、1200μg/mL的ESA强于50μg/mL的Vc的总抗氧化力。能较好的抑制Fe2+诱导的不饱和脂质的过氧化反应,IC50为1.7 mg／mL。
4、ESA可以有效地抑制·OH诱导的线粒体肿胀、脂质过氧化和蛋白质羰基的生成,通过减少超氧阴离子自由基的生成和提高ATP酶活性,保护线粒体免受羟基诱导的氧化损伤。
5、ESA的抗氧化活性可能是沙苑子黄酮、多酚和多糖类综合作用的结果。
总之,ESA具有很强的体外抗氧化活性。其可能的作用机制为通过直接清除自由基或者供氢、供电子作用消除活性自由基而中断自由基链式反应,或保护线粒体免受氧化损伤。沙苑子提取物具有比Vc更好的稳定性,与比合成抗氧化剂更大的安全性,说明其具有可开发为天然抗氧化剂的潜在优势。
Synthetic antioxidants, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) exert their effects by different mechanisms, like breaking chain reaction and scavenging reactive oxygen species. However, use of such compounds has been restricted because of their toxicity and the carcinogenic effect. Therefore, there is an increasing interest in natural antioxidants for their high-performance, low-cost and wide availability. Semen Astragali Complanati is the ripe seed of the Astragalus Complanatus R. Br, family Leguminosae. In China pharmacopeia (PRC Pharmacopeia,1990), it is indicated that the traditional Chinese herb increases Yang, acts primarily on kidney and liver channels and improves function of sexual performance. It is widely distributed in northeast, northwest and north China. Flavonoids of Semen Astragali Complanali (FAC) is pharmacologically active, and it has been demonstrated to resist aging, protect liver, inhibit platelet aggregation and enhance the central nervous system and so on. A series of functional food made from Semen Astragali Complanati has been available in the market. It has been reported that it possessed superoxide scavenging activity, but, up to now, its antioxidant activities in vitro have never been systematically studied. The present study aimed to interpret its antioxidant mechanism and facilitate utilities of Semen Astragali Complanati.
The present study was performed to evaluate the antioxidant activities of Semen Astragalus Complanati in three parts as follows:
The first part was to obtain the defatted ethanolic extracts from Semen Astragali Complanati (ESA), and to determine the content of active component using spectrophotometry.
Second, in vitro antioxidant activities of ESA was determined in different chemical systems, includingβ-carotene/linoleic acid assay, reducing power assay, determination of scavenging activities on OPPH·/O2-/·OH, and Fe2+induced lipid peroxidation assay.
Third, Hydrogen radical was used as pro-oxidant to investigate the mitochondrial protective effects of ESA against oxidative damage. This study was conducted in parallel as follows: preparation of rat liver mitochondrial using differential centrifugation in 0.25 M sucrose buffer, Fe2+/Vc or Fe2+/H2O2-induced isolated rat liver mitochondrial damage, evaluation the inhibition of mitochondrial swelling degree, the lipid peroxidation and the protein carbonyl formation by ESA, notably, to study the influence of ESA on ATPase activity and the generation of superoxide anion.
The main results of this paper showed that:
First, the rate of yields of ESA was 6.73%, and its main active components were sugar, ployphenols and flavonoids,44.92%,14.24% and 7.47% respectively.
Second, ESA displayed strong scavenging activities on free radical in dose-dependent manner but various mechanisms in different systems. The IC50 of scavenging activities on DPPH·, O2-·,·OH were 419.1μg/mL,117.9μg/mL and 224.4μg/mL respectively. The scavenging activities of ESA on the·OH was inferior to O2-·,but better than DPPH·.
Third, ESA possessed certain reducing power, which was positively correlated with concentration. As far as reducing power was concerned,4.2 mg/mL of ESA was equal to 160μg/mL of Vc or 300μg/mL of BHT. ESA significantly inhibited theβ-carotene bleaching, and the inhibition activity of ESA at 150μg/mL,600μg/mL,1200μg/mL was better than 50μg/mL of Vc in theβ-carotene/linoleic acid assay. ESA can well block the Fe2+induced lipid peroxidation chain reaction of polyunsaturated fatty acid from egg yolk, and the IC50 were 1.7 mg/mL.
Forth, ESA remarkably protected mitochondrial from·OH induced oxidative damage. The possible machanisms were inhibition of·OH induced mitochondrial swelling, lipid peroxidation and the protein carbonyl formation, reduction of superoxide anion generation, as well as increasing the ATPase activity.
Fifth, in vitro antioxidant activities of ESA could be comprehensive effects of flavonoids, sugar and ployphenols from ESA.
In conclusion, ESA showed effective antioxidant activities. It terminated free radical chain reactions by scavenging reactive oxygen species, and counteracting active oxygen by supplying electrons and hydrogen, or by protecting mitochondrial from oxidative damage. Better stability than vitamin C and better security than BHT implied it can be developed into natural antioxidant.
本研究主要分三大部分：
第一步,制备脱脂沙苑子乙醇提取物(the defatted ethanolic extract from Semen Astragali Complanati, ESA),并采用分光光度法测定主要化学成分总糖、总黄酮、总多酚的含量。
第二步,通过构建体外化学体系,包括β-胡萝卜素/亚油酸自氧化体系、还原能力测定、DPPH·/O2-·/·OH三种自由基清除体系及Fe2+诱导的脂质过氧化反应,研究ESA的体外抗氧化活性。
第三步,研究ESA对·OH诱导的线粒体氧化损伤的保护活性。研究按照以下操作进行：采用0.25 M蔗糖溶液提取、差速离心法制备大鼠肝线粒体,利用Fe2+/Vc或Fe2+/H2O2系统产生·OH诱导大鼠肝线粒体损伤,测定ESA对肝线粒体肿胀程度、脂质过氧化和蛋白质羰基生成水平的抑制作用,特别是对ATP酶活性以及超氧阴离子的生成的影响。
研究结果表明：
1、干燥的沙苑子经提取得到ESA,得率为6.73%,ESA中主要成分为糖类、黄酮类、多酚类物质,其含量分别为44.92%、7.47%和14.24%。
2、ESA具有很强的清除自由基的能力,均呈现剂量依赖性效应,但在不同的体系中机制不同。其清除DPPH·、O2-·、·OH三种自由基的IC50依次为419.1μg/mL、117.9μg/mL和224.4μg/mL,其清除超氧阴离子的作用最强、羟基次之、DPPH·最差。
3、ESA有较强的还原力,且与浓度呈正相关。4.2 mg/mL的ESA相当于160μg/mL的Vc和300μg/mL的BHT的还原力。对β-胡萝卜素褪色有明显的抑制作用,150μg/mL、600μg/mL、1200μg/mL的ESA强于50μg/mL的Vc的总抗氧化力。能较好的抑制Fe2+诱导的不饱和脂质的过氧化反应,IC50为1.7 mg／mL。
4、ESA可以有效地抑制·OH诱导的线粒体肿胀、脂质过氧化和蛋白质羰基的生成,通过减少超氧阴离子自由基的生成和提高ATP酶活性,保护线粒体免受羟基诱导的氧化损伤。
5、ESA的抗氧化活性可能是沙苑子黄酮、多酚和多糖类综合作用的结果。
总之,ESA具有很强的体外抗氧化活性。其可能的作用机制为通过直接清除自由基或者供氢、供电子作用消除活性自由基而中断自由基链式反应,或保护线粒体免受氧化损伤。沙苑子提取物具有比Vc更好的稳定性,与比合成抗氧化剂更大的安全性,说明其具有可开发为天然抗氧化剂的潜在优势。
Synthetic antioxidants, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) exert their effects by different mechanisms, like breaking chain reaction and scavenging reactive oxygen species. However, use of such compounds has been restricted because of their toxicity and the carcinogenic effect. Therefore, there is an increasing interest in natural antioxidants for their high-performance, low-cost and wide availability. Semen Astragali Complanati is the ripe seed of the Astragalus Complanatus R. Br, family Leguminosae. In China pharmacopeia (PRC Pharmacopeia,1990), it is indicated that the traditional Chinese herb increases Yang, acts primarily on kidney and liver channels and improves function of sexual performance. It is widely distributed in northeast, northwest and north China. Flavonoids of Semen Astragali Complanali (FAC) is pharmacologically active, and it has been demonstrated to resist aging, protect liver, inhibit platelet aggregation and enhance the central nervous system and so on. A series of functional food made from Semen Astragali Complanati has been available in the market. It has been reported that it possessed superoxide scavenging activity, but, up to now, its antioxidant activities in vitro have never been systematically studied. The present study aimed to interpret its antioxidant mechanism and facilitate utilities of Semen Astragali Complanati.
The present study was performed to evaluate the antioxidant activities of Semen Astragalus Complanati in three parts as follows:
The first part was to obtain the defatted ethanolic extracts from Semen Astragali Complanati (ESA), and to determine the content of active component using spectrophotometry.
Second, in vitro antioxidant activities of ESA was determined in different chemical systems, includingβ-carotene/linoleic acid assay, reducing power assay, determination of scavenging activities on OPPH·/O2-/·OH, and Fe2+induced lipid peroxidation assay.
Third, Hydrogen radical was used as pro-oxidant to investigate the mitochondrial protective effects of ESA against oxidative damage. This study was conducted in parallel as follows: preparation of rat liver mitochondrial using differential centrifugation in 0.25 M sucrose buffer, Fe2+/Vc or Fe2+/H2O2-induced isolated rat liver mitochondrial damage, evaluation the inhibition of mitochondrial swelling degree, the lipid peroxidation and the protein carbonyl formation by ESA, notably, to study the influence of ESA on ATPase activity and the generation of superoxide anion.
The main results of this paper showed that:
First, the rate of yields of ESA was 6.73%, and its main active components were sugar, ployphenols and flavonoids,44.92%,14.24% and 7.47% respectively.
Second, ESA displayed strong scavenging activities on free radical in dose-dependent manner but various mechanisms in different systems. The IC50 of scavenging activities on DPPH·, O2-·,·OH were 419.1μg/mL,117.9μg/mL and 224.4μg/mL respectively. The scavenging activities of ESA on the·OH was inferior to O2-·,but better than DPPH·.
Third, ESA possessed certain reducing power, which was positively correlated with concentration. As far as reducing power was concerned,4.2 mg/mL of ESA was equal to 160μg/mL of Vc or 300μg/mL of BHT. ESA significantly inhibited theβ-carotene bleaching, and the inhibition activity of ESA at 150μg/mL,600μg/mL,1200μg/mL was better than 50μg/mL of Vc in theβ-carotene/linoleic acid assay. ESA can well block the Fe2+induced lipid peroxidation chain reaction of polyunsaturated fatty acid from egg yolk, and the IC50 were 1.7 mg/mL.
Forth, ESA remarkably protected mitochondrial from·OH induced oxidative damage. The possible machanisms were inhibition of·OH induced mitochondrial swelling, lipid peroxidation and the protein carbonyl formation, reduction of superoxide anion generation, as well as increasing the ATPase activity.
Fifth, in vitro antioxidant activities of ESA could be comprehensive effects of flavonoids, sugar and ployphenols from ESA.
In conclusion, ESA showed effective antioxidant activities. It terminated free radical chain reactions by scavenging reactive oxygen species, and counteracting active oxygen by supplying electrons and hydrogen, or by protecting mitochondrial from oxidative damage. Better stability than vitamin C and better security than BHT implied it can be developed into natural antioxidant.
引文
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