箭根草总黄酮抗氧化抗动脉粥样硬化作用研究
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摘要
箭根草是豆科植物蔓性箭根草Flemingia philippinensis(Merr.et Rolfe)的根,其富含酚类、黄酮、鞣质、糖类、甾醇、生物碱和氨基酸类等成分,其中以黄酮类成分含量较高。
在单因素试验的基础上,以乙醇为提取溶剂,以总黄酮含量为指标,采用正交试验方法,考察溶剂浓度、提取温度、提取时间和溶剂用量对超声提取箭根草总黄酮得率的综合影响,优化箭根草总黄酮的提取工艺。
通过抗氧化活性的测定,检验箭根草总黄酮提取物对羟自由基、超氧阴离子自由基、过氧化氢的清除活性及还原能力。采用诱导和非诱导两种体系,通过测定肝匀浆中过氧化终产物MDA的含量,检验箭根草总黄酮在体外对肝细胞脂质过氧化和由Fe2+、H2O2诱导氧化的保护作用;通过检测红细胞溶血率和抑制率,考察箭根草总黄酮是否能够抑制红细胞因自氧化和由H2O2诱导氧化而导致的溶血作用。
建立实验性高脂血症大鼠模型,观察箭根草总黄酮提取物对大鼠血脂和肝脂含量的影响及抗氧化性物质的调节作用,通过病理学及免疫组化检测箭根草总黄酮对血管内皮细胞及肝脏的保护作用。
实验结果表明:采用超声波提取箭根草总黄酮的最佳条件为:溶剂为50%乙醇,提取温度40℃,提取时间30min,料液比1︰20,黄酮提取率为1.41%。急性毒性实验提示该提取物为低毒化合物。
箭根草总黄酮提取物在体外能有效地清除O 2~(-·)、·OH自由基和H2O2,具有较强的还原力,且具有良好的剂量效应关系。
箭根草总黄酮提取物能明显降低实验性高脂血症大鼠总胆固醇、甘油三酯、低密度脂蛋白胆固醇和动脉硬化指数值,提高高密度脂蛋白胆固醇、谷胱甘肽、一氧化氮含量,明显降低血清和肝组织中丙二醛含量,提高超氧化物歧化酶、一氧化氮合酶活性。
组织病理学及免疫组化结果显示,箭根草总黄酮提取物能够减轻高血脂对血管内皮细胞和肝脏的损伤作用。
箭根草总黄酮提取物通过清除多种自由基,提高抗氧化酶的活性,降低自由基及过氧化产物对机体的损伤,预防动脉粥样硬化的发生及发展。
Flemingia philippinensis is the root of bean plant Flemingia philippinensis (Merr.et Rolfe) with the characteristics of sweet, sour and gentle action. It can remove humidness, improve circulation, build strong bones and kill pains etc and has therapeutic effects on arthritis, boneaches, torn waist muscles, cruises, swelling, side paralysis etc. So far around 40 species of Flemingia philippinensis have been identified, 16 species and one variant can be found in China, mainly located in southwestern, midshouthern and southeastern provinces of our country. Flemingia philippinensis abounds in phenol, flavone, tannin, saccharide, sterol, alkaloid and amino acid etc, among them the content of flavone is relatively higher. Flemingia philippinensis has been used in patients for many years with definite pharmaceutic actions. Since it is added to the phamaceutical complex with its original root, its phamaceutical actions are not significant.In order to clarify the active component of Flemingia philippinensis and expand its application in pharmacology, the present study is to optimize the extraction conditions for obtaining the total flavone through Flemingia philippinensis coarse powder. Since the solubility of flavone varies with the different structure and existing state, so it is necessary to determine the type of solvents and then take the solvent concentration, extracting temperature, time and amount into account. Fix the proper range of the above factors through single factor experiment and through orthogonal experiment, based on the harvesting content of total flavone, to find out the effects of solvent concentration, extracting temperature, extracting time and volume of solvents on the total flavone harvest to determine the best extraction techniques.
Flavones contain multiple molecules of phenolic hydroxyl group which confer them the antioxidative abilities. The present study is to test the scavenging and reducing abilities of total flavones to hydroxyl free radicals and superoxide anion via H2O2/Fe2+ system in which hydroxyl free radicals and superoxide were generated via Fenton reaction and pyrogallol auto-oxidation method; Through induction and non-induction two systems, the MDA level test was utilized to determine the protective effects of total flavones against oxidative damage induced by Fe2+ and H2O2 in vitro; Haematolysis rate of red blood cells was tested to verify that if total flavone from Flemingia philippinensis could inhibit the red blood cell haematolysis due to autooxidation of red blood cell and oxidative damage caused by Fe2+.
Hyperlipemia is characterized by lipid metabolism disorder and lipoprotein superoxidization, which are the primary cause for atherosclerosis. AS is the incentive for cardiovascular diseas. Therefore, the key measure to prevent the development of AS is to control hyperlipemia effectively. There are researches showed that free radicals is closely related to the development and progression of AS, very low density lipoprotein can be oxidized easily by free radicals to form oxidative low density lipoprotein and result in AS and cardiovascular diseases. Hyperlipemia rat models were given Flemingia philippinensis total flavone at the dose of 10 mg/kg, 20 mg/kg, 40 mg/kg to explore the effects of total flavones on the level of serum and liver total cholesterol(TC), triglyceride(TG), high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholestero(lLDL-c), superoxide dismutase(SOD), malonaldehyde(MDA), glutathion(GSH), hydrogen peroxide(H2O2), nitric oxide(NO), nitric oxide synthase(NOS)and find out if the flavone can reverse the lipid metabolism disorders induced by experimental hyperlipemia and inhibit the secondary damages caused by hyperlipemia. The above test were to demonstrate that if the total flavone could remove free radicals and protect the expeimental rats from oxidative stress and prevent the development of AS. The morphology of endothelial cells and liver as well as the expression of iNOS and VCAM-1 in aorta were examined to find out if the flavone could protect the them from hyperlipemia.
The best extracting technique for Flemingia philippinensis total flavone based on the experiment results and statistics analyses was the following: 50% ethanol as solvent, the ratio of Flemingia philippinensis to solvent1:20, temperature 50℃,the harvesting rate was 1.41%. Acute toxicity test showed that the flavone extract low grade chemical. The preliminary quantitation test indicated that the extract contained flavoid, flavonol and flavonone.
In vitro antioxidation experiment, Flemingia philippinensis flavone could scavenge O2-,·Oh , H2O2,reduce Fe2+ in a dose-effect manner, decrease the MDA level induced by autooxidation and oxidative stress derived from Fe2+ and H2O2, inhibit the haemolysis of red blood cells, which indicated that Flemingia philippinensis flavone could act as a natural antioxidative agent.
Hyperlipemia rat model was successfully established by feeding the rats with the high level of lipid diet, the model rats were classified into high lipid diet group, flavone treated group which was further into high, medium and low dose subgroups. Normal rats were used as control. There was a significant decrease in the serum and liver level of TC, TG, LDL-c, MDA and a significant increase in the level of HDL-c, SOD, T-AOC, NO, NOS compared to high lipid diet group, which indicated that the flavone could prevent the development and progression of AS.
There was a significant increase in the expression of iNOS in Flemingia philippinensis flavone high dose subgroup in a dose-effect manner, the iNOS was located in the macrophages and foam cells which was the center of atherosclerosis plaque by aorta immunohistochemistry staining.
There was a significant decrease in the expression of VCAM-1 in vessel wall and monocytes in rats from Flemingia philippinensis flavone group compared to high lipid diet group, which indicated that the action mechanisms of how Flemingia philippinensis flavone preventing the development of AS was to upregulate the expression of key enzyme in the synthesis of NO and downregulate the expression of VCAM-1.
在单因素试验的基础上,以乙醇为提取溶剂,以总黄酮含量为指标,采用正交试验方法,考察溶剂浓度、提取温度、提取时间和溶剂用量对超声提取箭根草总黄酮得率的综合影响,优化箭根草总黄酮的提取工艺。
通过抗氧化活性的测定,检验箭根草总黄酮提取物对羟自由基、超氧阴离子自由基、过氧化氢的清除活性及还原能力。采用诱导和非诱导两种体系,通过测定肝匀浆中过氧化终产物MDA的含量,检验箭根草总黄酮在体外对肝细胞脂质过氧化和由Fe2+、H2O2诱导氧化的保护作用;通过检测红细胞溶血率和抑制率,考察箭根草总黄酮是否能够抑制红细胞因自氧化和由H2O2诱导氧化而导致的溶血作用。
建立实验性高脂血症大鼠模型,观察箭根草总黄酮提取物对大鼠血脂和肝脂含量的影响及抗氧化性物质的调节作用,通过病理学及免疫组化检测箭根草总黄酮对血管内皮细胞及肝脏的保护作用。
实验结果表明:采用超声波提取箭根草总黄酮的最佳条件为:溶剂为50%乙醇,提取温度40℃,提取时间30min,料液比1︰20,黄酮提取率为1.41%。急性毒性实验提示该提取物为低毒化合物。
箭根草总黄酮提取物在体外能有效地清除O 2~(-·)、·OH自由基和H2O2,具有较强的还原力,且具有良好的剂量效应关系。
箭根草总黄酮提取物能明显降低实验性高脂血症大鼠总胆固醇、甘油三酯、低密度脂蛋白胆固醇和动脉硬化指数值,提高高密度脂蛋白胆固醇、谷胱甘肽、一氧化氮含量,明显降低血清和肝组织中丙二醛含量,提高超氧化物歧化酶、一氧化氮合酶活性。
组织病理学及免疫组化结果显示,箭根草总黄酮提取物能够减轻高血脂对血管内皮细胞和肝脏的损伤作用。
箭根草总黄酮提取物通过清除多种自由基,提高抗氧化酶的活性,降低自由基及过氧化产物对机体的损伤,预防动脉粥样硬化的发生及发展。
Flemingia philippinensis is the root of bean plant Flemingia philippinensis (Merr.et Rolfe) with the characteristics of sweet, sour and gentle action. It can remove humidness, improve circulation, build strong bones and kill pains etc and has therapeutic effects on arthritis, boneaches, torn waist muscles, cruises, swelling, side paralysis etc. So far around 40 species of Flemingia philippinensis have been identified, 16 species and one variant can be found in China, mainly located in southwestern, midshouthern and southeastern provinces of our country. Flemingia philippinensis abounds in phenol, flavone, tannin, saccharide, sterol, alkaloid and amino acid etc, among them the content of flavone is relatively higher. Flemingia philippinensis has been used in patients for many years with definite pharmaceutic actions. Since it is added to the phamaceutical complex with its original root, its phamaceutical actions are not significant.In order to clarify the active component of Flemingia philippinensis and expand its application in pharmacology, the present study is to optimize the extraction conditions for obtaining the total flavone through Flemingia philippinensis coarse powder. Since the solubility of flavone varies with the different structure and existing state, so it is necessary to determine the type of solvents and then take the solvent concentration, extracting temperature, time and amount into account. Fix the proper range of the above factors through single factor experiment and through orthogonal experiment, based on the harvesting content of total flavone, to find out the effects of solvent concentration, extracting temperature, extracting time and volume of solvents on the total flavone harvest to determine the best extraction techniques.
Flavones contain multiple molecules of phenolic hydroxyl group which confer them the antioxidative abilities. The present study is to test the scavenging and reducing abilities of total flavones to hydroxyl free radicals and superoxide anion via H2O2/Fe2+ system in which hydroxyl free radicals and superoxide were generated via Fenton reaction and pyrogallol auto-oxidation method; Through induction and non-induction two systems, the MDA level test was utilized to determine the protective effects of total flavones against oxidative damage induced by Fe2+ and H2O2 in vitro; Haematolysis rate of red blood cells was tested to verify that if total flavone from Flemingia philippinensis could inhibit the red blood cell haematolysis due to autooxidation of red blood cell and oxidative damage caused by Fe2+.
Hyperlipemia is characterized by lipid metabolism disorder and lipoprotein superoxidization, which are the primary cause for atherosclerosis. AS is the incentive for cardiovascular diseas. Therefore, the key measure to prevent the development of AS is to control hyperlipemia effectively. There are researches showed that free radicals is closely related to the development and progression of AS, very low density lipoprotein can be oxidized easily by free radicals to form oxidative low density lipoprotein and result in AS and cardiovascular diseases. Hyperlipemia rat models were given Flemingia philippinensis total flavone at the dose of 10 mg/kg, 20 mg/kg, 40 mg/kg to explore the effects of total flavones on the level of serum and liver total cholesterol(TC), triglyceride(TG), high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholestero(lLDL-c), superoxide dismutase(SOD), malonaldehyde(MDA), glutathion(GSH), hydrogen peroxide(H2O2), nitric oxide(NO), nitric oxide synthase(NOS)and find out if the flavone can reverse the lipid metabolism disorders induced by experimental hyperlipemia and inhibit the secondary damages caused by hyperlipemia. The above test were to demonstrate that if the total flavone could remove free radicals and protect the expeimental rats from oxidative stress and prevent the development of AS. The morphology of endothelial cells and liver as well as the expression of iNOS and VCAM-1 in aorta were examined to find out if the flavone could protect the them from hyperlipemia.
The best extracting technique for Flemingia philippinensis total flavone based on the experiment results and statistics analyses was the following: 50% ethanol as solvent, the ratio of Flemingia philippinensis to solvent1:20, temperature 50℃,the harvesting rate was 1.41%. Acute toxicity test showed that the flavone extract low grade chemical. The preliminary quantitation test indicated that the extract contained flavoid, flavonol and flavonone.
In vitro antioxidation experiment, Flemingia philippinensis flavone could scavenge O2-,·Oh , H2O2,reduce Fe2+ in a dose-effect manner, decrease the MDA level induced by autooxidation and oxidative stress derived from Fe2+ and H2O2, inhibit the haemolysis of red blood cells, which indicated that Flemingia philippinensis flavone could act as a natural antioxidative agent.
Hyperlipemia rat model was successfully established by feeding the rats with the high level of lipid diet, the model rats were classified into high lipid diet group, flavone treated group which was further into high, medium and low dose subgroups. Normal rats were used as control. There was a significant decrease in the serum and liver level of TC, TG, LDL-c, MDA and a significant increase in the level of HDL-c, SOD, T-AOC, NO, NOS compared to high lipid diet group, which indicated that the flavone could prevent the development and progression of AS.
There was a significant increase in the expression of iNOS in Flemingia philippinensis flavone high dose subgroup in a dose-effect manner, the iNOS was located in the macrophages and foam cells which was the center of atherosclerosis plaque by aorta immunohistochemistry staining.
There was a significant decrease in the expression of VCAM-1 in vessel wall and monocytes in rats from Flemingia philippinensis flavone group compared to high lipid diet group, which indicated that the action mechanisms of how Flemingia philippinensis flavone preventing the development of AS was to upregulate the expression of key enzyme in the synthesis of NO and downregulate the expression of VCAM-1.
引文
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