五倍子提取物的体外抗氧化作用研究

【作者】 代斌；

【导师】 杨建雄；

【作者基本信息】 陕西师范大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 由于机体内一些外源性自由基和内源性自由基的代谢过程,导致氧分子产生过量氧自由基及氧化应激。活性氧的介入引发细胞内外多种具有生物活性的化合物相互作用,最终导致机体产生病理变化。为了有效预防自由基对机体的损伤,维持正常的生命活动,已开发和研究了许多天然抗氧化剂和合成抗氧化剂。近年来,人工合成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）等的安全性受到质疑,而天然抗氧化剂以其来源广、抗氧化活性大、安全性高等特点倍受人们的亲睐。因此研究开发高效安全的天然抗氧化剂,成为当今抗氧化剂研究领域中的热点之一。五倍子（Galla chinensis）是一种天然无毒的传统中药,由绵蚜科昆虫在其寄主植物盐肤木叶总轴的翅叶上形成,具有潜在的开发前景。目前关于五倍子的研究多集中在抗菌,抗病毒,抑突变,加速血液凝固及收敛作用,对其清除自由基和抗线粒体氧化损伤的性能尚未见全面报道。本实验旨在研究五倍子提取物的抗氧化作用,为探索其药理机制提供实验依据。首先,采用4倍量的丙酮:水（2:1）于50℃浸提,再用乙醚和乙酸乙酯依次萃取,经减压浓缩后烘干,得到五倍子提取物GCE。然后通过建立体外化学模拟体系和鼠肝线粒体损伤模型,包括DPPH·/O₂^-·/·OH清除体系,还原能力分析体系,Fe²⁺诱导的脂质过氧化反应和β-胡萝卜素/亚油酸自氧化体系,Fe²⁺-Vc系统诱导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GCE总的抗氧化活性。体外抗氧化实验结果显示,五倍子提取物能够有效清除DPPH自由基、羟基自由基及超氧离子自由基,对脂质过氧化有明显的抑制作用,有较强的还原力和总抗氧化力,且呈现较好的剂量依赖效应。GCE对DPPH·和O₂^-·的半数清除浓度IC₅₀分别为13.75μg/mL和46.15μg/mL,而Vc分别为10.27μg/mL and 54.35μg/mL;清除·OH的能力明显强于D-甘露醇,在0.25 mg/mL时,GCE的清除率可达58.03%,是D-甘露醇（13.56%）的4.28倍;有较强的还原力和总抗氧化力;对Fe²⁺诱导的脂质过氧化反应表现出较好的抑制作用。线粒体抗氧化实验结果表明,五倍子提取物可以有效降低蛋白质羰基的含量,减缓线粒体的肿胀,恢复ATP酶的活性,保证了线粒体的膜内外渗透压;对线粒体脂质过氧化反应和O₂^-·产生有一定的抑制作用,对生物膜具有保护作用。综上所述,GCE具有显著的抗氧化活性,且能有效地保护线粒体的氧化损伤。但是否有较好的体内抗氧化活性,还有待进一步的实验研究。结果表明,五倍子可以作为一种有抗氧化活性的保健品或药物开发利用。更多还原

【Abstract】 The oxygen molecule might produce excessive oxygen free radicals and oxidative stress by some exogenous factors and endogenous metabolic processes in human body.Intervention of reactive oxygen species（ROS）leading to pathologic changes was a consequence of their interaction with various biological compounds inside and outside cells,In order to effectively prevent free radical damage to the body and to maintain normal life activities,numerous natural and synthetic ROS scavengers and antioxidants have been developed and studied.In recent years,synthetic antioxidants such as butylated hydroxyltoluene（BHT） and butylated hydroxyanisole（BHA） have been suspected in security,so nature antioxidant was getting people’s favor because of its extensive source,strong antioxidant activity and excellent security.Therefore,the development of effective and safe natural antioxidants becomes one of the main topics in the field of antioxidants.Galla chinensis（GAC）,a natural non-toxic traditional Chinese medicine with potential development prospects,is the gall produced by some parasitic aphids（family Pemphigidae） on the Rhus leaves of the family Anacardiaceae（mainly Rhus chinensis Mill）.Now,there are more researches on antibacterial,antiviral,suppression mutation and accelerating blood coagulation of GAC,but the reports about its free radical scavenging activity and antioxidant effect in mitochondria are even less.The experiment aims to evaluate the antioxidant activity of Galla chinensis extract （GCE）,in order to understand pharmacological mechanisms of this plant as a foodstuff as well as in medicine.First.GAC was macerated by water/acetone（1:2,v/v） with 4 times volume in a water bath at 50℃,then remove the acetone,the remaining aqueous solution was extracted with ether and ethyl acetate sequently,the extracts were evaporated to dryness and obtained GCE.Finally,we employed in vitro chemical assay systems,including 1,1-diphenyl-2-picrylhydrazyl（DPPH·）/ peroxide anion /hydroxyl radical-scavenging,reducing power assay system,peroxidation of polyunsaturated fatty acid from yolk lipoprotein induced by Fe²⁺ method,andβ-carotene/linoleic acid assay system,and in vitro mitochondrial damage models induced by Fe²⁺-Vc,including malondialdehyde（MDA） content,mitochondrial swelling.ATPase activity and protein carbonyl content along with superoxide anion generation,in order to evaluate total antioxidant activity of GCE.The results showed that GCE exposed strong antioxidant capability in a dose-dependent manner. IC₅₀ values in scavenging abilities on DPPH·and O₂^-·of GCE were 13.75 ug/mL和46.15ug/mL. whereas for Vc they were 10.27 ug/mL and 54.35 ug/mL.The hydroxyl radical scavenging ability of GCE is stronger than D-mannitol obviously,its scavenging ability（58.03%） is 4.28 times of D-mannitol（13.56%） at 0.25 mg/mL.GCE has preferable reducing power and excellent total antioxidant activity,and effectively inhibit lipid peroxidation induced by Fe²⁺.GCE had been shown to inhibit the rat liver mitochondrial swelling,oxidation of thiol groups and maintaining ATPase activity to ensure that the mitochondrial membrane osmotic pressure exterior and interior.It also could protect mitochondria membranes by inbibit lipid peroxidation and superoxide anions generation.To sum up,GCE has significant antioxidant activity and can effectively protect the oxidative damage of mitochondria.However,whether there is a better antioxidant activity in vivo,remains to be further studied.The results showed that Galla chinensis can be used as an antioxidant activity of the health care products or the use of drug development.更多还原