荔枝草提取物的体外抗氧化活性研究
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摘要
人工合成的抗氧化剂如二叔丁基羟基甲苯(butylated hydroxy toluene,BHT)和叔丁基羟基茴香醚(butylated hydroxy anisole,BHA)等抗氧化活性较好,但其安全性受到怀疑,研究开发高效安全的天然抗氧化剂,已成为当今抗氧化剂研究领域中的热点之一。我国植物资源丰富,很多植物中都含有抗氧化物质。荔枝草(Salvia plebeia R.Br.)属于唇形科,是从719种中草药中筛选出来的一种抗氧化活性较强,分布较广,具有潜在开发前景的植物。目前已有研究表明荔枝草具有止咳平喘、祛痰、抗组胺、抑菌及抗油脂氧化的作用,但对其清除自由基和抗线粒体损伤的性能尚未见报道。
本实验采用体外化学模拟法对提取物的体外抗氧化活性以及对线粒体的保护进行了研究,为探索荔枝草药理作用的机制提供依据。首先,采用70%乙醇回流提取,重复3次,再用等体积的石油醚和乙酸乙酯萃取,弃醚相,将水相和乙酸乙酯相部分分别减压浓缩后烘干,得到两种荔枝草提取物(Salvia plebeia R.Br.extract,SE),分别为SE1和SE2。然后通过一系列的体外分析体系,包括DPPH·/O_2~-·/·OH自由基清除体系,还原能力分析体系,Fe~(2+)诱导的脂质过氧化反应和β-胡萝卜素/亚油酸自氧化体系,研究SE的体外抗氧化活性。最后用蔗糖浓度差迷离心法制备大鼠肝线粒体,利用Fe~(2+)-Vc系统产生·OH,诱导大鼠肝线粒体损伤,再用荔枝草提取物加以保护,通过对肝线粒体脂质过氧化水平、肿胀程度、蛋白质羰基水平、ATP酶的活性以及生成超氧阴离子的多少来评价SE抗线粒体氧化损伤的作用。
清除自由基及其抗氧化实验结果显示,SE能够有效清除DPPH·、O_2~-·和·OH自由基,具有较强的还原能力,可以有效抑制Fe~(2+)诱导的脂质过氧化反应和β-胡萝卜素/亚油酸的自氧化,且呈剂量依赖关系。其中,SE2对DPPH·和O_2~-·的半数清除浓度IC_(50)分别为0.79mg/mL和0.26mg/mL,清除能力高于SE1(IC_(50)分别为1.40mg/mL和0.39mg/mL);SE1清除·OH的能力明显强于SE2和D-甘露醇,在1.0mg/mL时,SE1的清除率可达62.27%,是SE2(17.23%)的3.62倍,D-甘露醇(39.15%)的1.59倍;在所检测浓度范围内,SE2的还原能力远远超过了SE1;SE1和SE2皆对Fe~(2+)诱导的脂质过氧化反应表现出较好的抑制作用,在1.0mg/mL时,其最大抑制率分别可达55.55%和66.36%;二者对β-胡萝卜素/亚油酸自氧化体系均有一定的抑制作用,且呈较好的量效关系,在0.05mg/mL时,抑制率分别为3.47%和6.69%,在0.20mg/kmL时,抑制率分别为10.96%、14.14%。
线粒体抗氧化实验结果表明,SE可以有效地抑制线粒体损伤时的脂质过氧化反应和线粒体膨胀,降低蛋白质羰基的含量,恢复ATP酶的活性,清除线粒体中产生的O_2~-·,并且呈现良好的量效关系。相比较而言,SE2和SE1抑制脂质过氧化反应和线粒体膨胀的能力均随着浓度的增大而增强,SE2的作用稍强于SE1,在0.50mg/kmL时,二者对脂质过氧化抑制率分别为96.25%和95.12%;在所检测浓度范围内,SE2对受损线粒体中蛋白质羰基含量和ATP酶活性的影响比SE1要显著的多;SE1和SE2对线粒体中产生的O_2~-·都有很强的清除作用,其半数清除浓度分别为0.36mg/mL和0.29mg/kmL。
以上实验结果表明,SE具有较强的抗氧化能力,可以有效清除DPPH·、O_2~-·和·OH自由基,具有较强的还原能力,并且可以有效抑制Fe~(2+)诱导的脂质过氧化反应、β-胡萝卜素/亚油酸的自氧化、线粒体损伤时的脂质过氧化反应以及线粒体膨胀程度,能够明显降低蛋白质羰基的含量,恢复ATP酶的活性,清除线粒体中产生的O_2~-·。其中SE2的效果较好,可能是该提取物中黄酮含量较高所致。此外,SE是否有较好的体内抗氧化活性,还有待进一步的实验研究。
The synthetic antioxidants such as butylated hydroxyltoluene(BHT) and butylated hydroxyanisole (BHA) have antioxidant activities.However,the synthetic antioxidants have the problem with their mutagenic activity.The development of effective and safe natural antioxidants becomes one of the main topics in the field of food chemistry.There are many Chinese herbs available in China and it has been verified that many of them are full of natural antioxidants.Salvia plebeia R.Br.which was screened out of 719 species of Chinese herbs grows indigenously in wet places almost throughout China and India,belonging to the Labiateae.It was traditionally used in folk medicine for its effects such as relieving cough and asthma,antihistamine,antimicrobial property and antioxidant activity on lipid peroxidation.But the reports about its free radical scavenging activity and antioxidant effect in mitochondria are even less.
This experiment aims to evaluate the antioxidant activity of Salvia plebeia R.Br extract(SE),in order to understand the usefulness of this plant as a foodstuff as well as in medicine.First,SE is extracted by 70%ethanol three times under reflux condition,then remove the ethanol,the remaining aqueous solution of the extraction was eluted with petroleum(v:v=1:1) and ethyl acetate(v:v=1:1), respectively.The aqueous solution fraction(SE1) and ethyl acetate solution fraction(SE2) were evaporated to dryness,giving for the next analyses.Second,we examined the antioxidant activity of SE,employing various in vitro assay systems,including DPPH·/O_2~-·/·OH radical-scavenging, reducing power assay system,peroxidation of polyunsaturated fatty acid from yolk lipoprotein induced by Fe~(2+) method,andβ-carotene/linoleic acid assay system.Third,we have investigated the effects of SE on damaged rat liver mitochondria induced by Fe~(2+)-Vc.Malondialdehyde(MDA), mitochondrial swelling,ATPase activity and protein carbonyl content along with superoxide anion generation were measured.
The results showed that both the extracts exposed strong antioxidant capability in a dose-dependent manner,but the results were different for each species depending on the applied test. IC_(50) values in scavenging abilities on DPPH·and O_2~- of SE2 were 0.79 mg/mL and 0.26 mg/mL, whereas for SE1 they were 1.40 mg/mL and 0.39 mg/mL.The hydroxyl radical scavenging ability of SE1 and SE2 were 62.27%and 17.23%at 1.0 mg/mL.Effectiveness in reducing powers was in a descending order of SE2>SE1.In lipid peroxidation assay,the maximum inhibition of SE1 and SE2 were 55.55%and 66.36%respectively.The total antioxidant activities of SE1 and SE2 were lower than BHT onβ-carotene/linoleic acid assay system.
SE2 has been shown to inhibit the rat liver mitochondrial swelling and the lipid peroxidation stronger than SE1.The two extracts were found to be effective in inhibiting the oxidation of thiol groups and maintaining ATPase activity.IC_(50) values in scavenging O_2~- of SE1 and SE2 were 0.36 mg/mL and 0.29 mg/mL respectively.
The results of this text indicated that SE had high antioxidant capacities.It exhibited remarkable free radicals(DPPH·,O_2~- and·OH) scavenging abilities and effective reducing power.SE also showed powerful inhibitory effects on peroxidation of polyunsaturated fatty acid from yolk lipoprotein induced by Fe~(2+) and onβ-carotene/linoleic acid assay system.Furthermore,SE showed to inhibit the rat liver mitochondrial swelling and the oxidation of thiol groups.It also protected mitochondria membranes by scavenging superoxide anions and scavenging lipid free radicals as well as maintaining ATPase(one of membrane-bound enzymes) activity obviously.The higher efficiency of SE2,as compared to SE1,should be due to the presence of the more flavonoids in SE2.Whether there is more evident antioxidant activity of SE in vivo,it still needs further study.
本实验采用体外化学模拟法对提取物的体外抗氧化活性以及对线粒体的保护进行了研究,为探索荔枝草药理作用的机制提供依据。首先,采用70%乙醇回流提取,重复3次,再用等体积的石油醚和乙酸乙酯萃取,弃醚相,将水相和乙酸乙酯相部分分别减压浓缩后烘干,得到两种荔枝草提取物(Salvia plebeia R.Br.extract,SE),分别为SE1和SE2。然后通过一系列的体外分析体系,包括DPPH·/O_2~-·/·OH自由基清除体系,还原能力分析体系,Fe~(2+)诱导的脂质过氧化反应和β-胡萝卜素/亚油酸自氧化体系,研究SE的体外抗氧化活性。最后用蔗糖浓度差迷离心法制备大鼠肝线粒体,利用Fe~(2+)-Vc系统产生·OH,诱导大鼠肝线粒体损伤,再用荔枝草提取物加以保护,通过对肝线粒体脂质过氧化水平、肿胀程度、蛋白质羰基水平、ATP酶的活性以及生成超氧阴离子的多少来评价SE抗线粒体氧化损伤的作用。
清除自由基及其抗氧化实验结果显示,SE能够有效清除DPPH·、O_2~-·和·OH自由基,具有较强的还原能力,可以有效抑制Fe~(2+)诱导的脂质过氧化反应和β-胡萝卜素/亚油酸的自氧化,且呈剂量依赖关系。其中,SE2对DPPH·和O_2~-·的半数清除浓度IC_(50)分别为0.79mg/mL和0.26mg/mL,清除能力高于SE1(IC_(50)分别为1.40mg/mL和0.39mg/mL);SE1清除·OH的能力明显强于SE2和D-甘露醇,在1.0mg/mL时,SE1的清除率可达62.27%,是SE2(17.23%)的3.62倍,D-甘露醇(39.15%)的1.59倍;在所检测浓度范围内,SE2的还原能力远远超过了SE1;SE1和SE2皆对Fe~(2+)诱导的脂质过氧化反应表现出较好的抑制作用,在1.0mg/mL时,其最大抑制率分别可达55.55%和66.36%;二者对β-胡萝卜素/亚油酸自氧化体系均有一定的抑制作用,且呈较好的量效关系,在0.05mg/mL时,抑制率分别为3.47%和6.69%,在0.20mg/kmL时,抑制率分别为10.96%、14.14%。
线粒体抗氧化实验结果表明,SE可以有效地抑制线粒体损伤时的脂质过氧化反应和线粒体膨胀,降低蛋白质羰基的含量,恢复ATP酶的活性,清除线粒体中产生的O_2~-·,并且呈现良好的量效关系。相比较而言,SE2和SE1抑制脂质过氧化反应和线粒体膨胀的能力均随着浓度的增大而增强,SE2的作用稍强于SE1,在0.50mg/kmL时,二者对脂质过氧化抑制率分别为96.25%和95.12%;在所检测浓度范围内,SE2对受损线粒体中蛋白质羰基含量和ATP酶活性的影响比SE1要显著的多;SE1和SE2对线粒体中产生的O_2~-·都有很强的清除作用,其半数清除浓度分别为0.36mg/mL和0.29mg/kmL。
以上实验结果表明,SE具有较强的抗氧化能力,可以有效清除DPPH·、O_2~-·和·OH自由基,具有较强的还原能力,并且可以有效抑制Fe~(2+)诱导的脂质过氧化反应、β-胡萝卜素/亚油酸的自氧化、线粒体损伤时的脂质过氧化反应以及线粒体膨胀程度,能够明显降低蛋白质羰基的含量,恢复ATP酶的活性,清除线粒体中产生的O_2~-·。其中SE2的效果较好,可能是该提取物中黄酮含量较高所致。此外,SE是否有较好的体内抗氧化活性,还有待进一步的实验研究。
The synthetic antioxidants such as butylated hydroxyltoluene(BHT) and butylated hydroxyanisole (BHA) have antioxidant activities.However,the synthetic antioxidants have the problem with their mutagenic activity.The development of effective and safe natural antioxidants becomes one of the main topics in the field of food chemistry.There are many Chinese herbs available in China and it has been verified that many of them are full of natural antioxidants.Salvia plebeia R.Br.which was screened out of 719 species of Chinese herbs grows indigenously in wet places almost throughout China and India,belonging to the Labiateae.It was traditionally used in folk medicine for its effects such as relieving cough and asthma,antihistamine,antimicrobial property and antioxidant activity on lipid peroxidation.But the reports about its free radical scavenging activity and antioxidant effect in mitochondria are even less.
This experiment aims to evaluate the antioxidant activity of Salvia plebeia R.Br extract(SE),in order to understand the usefulness of this plant as a foodstuff as well as in medicine.First,SE is extracted by 70%ethanol three times under reflux condition,then remove the ethanol,the remaining aqueous solution of the extraction was eluted with petroleum(v:v=1:1) and ethyl acetate(v:v=1:1), respectively.The aqueous solution fraction(SE1) and ethyl acetate solution fraction(SE2) were evaporated to dryness,giving for the next analyses.Second,we examined the antioxidant activity of SE,employing various in vitro assay systems,including DPPH·/O_2~-·/·OH radical-scavenging, reducing power assay system,peroxidation of polyunsaturated fatty acid from yolk lipoprotein induced by Fe~(2+) method,andβ-carotene/linoleic acid assay system.Third,we have investigated the effects of SE on damaged rat liver mitochondria induced by Fe~(2+)-Vc.Malondialdehyde(MDA), mitochondrial swelling,ATPase activity and protein carbonyl content along with superoxide anion generation were measured.
The results showed that both the extracts exposed strong antioxidant capability in a dose-dependent manner,but the results were different for each species depending on the applied test. IC_(50) values in scavenging abilities on DPPH·and O_2~- of SE2 were 0.79 mg/mL and 0.26 mg/mL, whereas for SE1 they were 1.40 mg/mL and 0.39 mg/mL.The hydroxyl radical scavenging ability of SE1 and SE2 were 62.27%and 17.23%at 1.0 mg/mL.Effectiveness in reducing powers was in a descending order of SE2>SE1.In lipid peroxidation assay,the maximum inhibition of SE1 and SE2 were 55.55%and 66.36%respectively.The total antioxidant activities of SE1 and SE2 were lower than BHT onβ-carotene/linoleic acid assay system.
SE2 has been shown to inhibit the rat liver mitochondrial swelling and the lipid peroxidation stronger than SE1.The two extracts were found to be effective in inhibiting the oxidation of thiol groups and maintaining ATPase activity.IC_(50) values in scavenging O_2~- of SE1 and SE2 were 0.36 mg/mL and 0.29 mg/mL respectively.
The results of this text indicated that SE had high antioxidant capacities.It exhibited remarkable free radicals(DPPH·,O_2~- and·OH) scavenging abilities and effective reducing power.SE also showed powerful inhibitory effects on peroxidation of polyunsaturated fatty acid from yolk lipoprotein induced by Fe~(2+) and onβ-carotene/linoleic acid assay system.Furthermore,SE showed to inhibit the rat liver mitochondrial swelling and the oxidation of thiol groups.It also protected mitochondria membranes by scavenging superoxide anions and scavenging lipid free radicals as well as maintaining ATPase(one of membrane-bound enzymes) activity obviously.The higher efficiency of SE2,as compared to SE1,should be due to the presence of the more flavonoids in SE2.Whether there is more evident antioxidant activity of SE in vivo,it still needs further study.
引文
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