山莓叶有效成分分离、解析及其生物活性研究
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摘要
山莓(Rubus corchorifolius L.f.)系蔷薇科(Rosaceae)蔷薇亚科悬钩子属植物(Rubus.L)中的一种落叶灌木,野生资源蕴藏量大。山莓作为传统的药用植物,已有悠久的应用历史,但其药效成分和药理作用基础并不清楚。本文以山莓叶为研究对象,在其活性成分分离分析鉴定及生物活性等方面做了系统而深入的研究。实验结果为开发山莓这一药用植物资源奠定了理论基础。其主要研究结果如下：
1黄酮类化合物是山莓的有效成分之一,建立亚硝酸钠-硝酸铝显色法测定山莓叶中总黄酮的含量,正交试验优化得到山莓叶总黄酮的最佳提取条件为乙醇浓度为60%、水浴温度80℃、提取时间1.5h。测定不同月份山莓叶黄酮的含量为0.134%～0.346%。采用溶剂提取、柱色谱分离等方法分离得到槲皮素1；槲皮素-3-O-芸香糖苷(这个化合物即为芦丁)2；山奈酚-3-O-β-D-(6”-对羟基桂皮酰基)-葡萄糖苷3。其中化合物1,2为首次从该植物中得到。
2研究了山莓叶黄酮单体化合物的抑菌活性及抗氧化活性。当槲皮素浓度为5mg/mL时,对大肠杆菌、金黄色葡萄球菌、痢疾志贺氏菌、苏云金杆菌大肠杆菌、痢疾志贺氏菌抑菌作用明显,抑菌效果优于中药黄连素。山莓叶黄酮单体化合物对Fenton体系产生的羟自由基清除具有良好的量效关系,为山莓抗菌消炎、抗腹泻作用提供理论依据。
3采用水蒸气蒸馏、同时蒸馏萃取、微波辅助蒸馏三种方法比较提取山莓叶挥发油,优化提取条件。建立3-10月份山莓挥发油的GC指纹图谱,发现3-10月份的山莓叶挥发油共了8个共有峰,以期为综合评价山莓及其挥发油的质量提供依据。首次采用刃天青显色法研究发现山莓叶挥发油对大肠杆菌、沙门氏菌、金黄色葡萄球菌、痢疾志贺氏菌、苏云金杆菌最低抑菌浓度(MIC)分别为62.5ug/mL.62.5ug/mL、125ug/mL、31.25ug/mL、15.6ug/mL,对痢疾志贺氏杆菌的最低抑菌浓度低于卡拉霉素标准品的最低抑菌浓度。首次采用DPPH法对山莓叶挥发油进行抗氧化活性初步研究,山莓叶挥发油、芦丁、Vc半清除率EC5o分别为121.945μg/mL、6.326μg/mL、4.817μg/mL,发现山莓叶挥发油具有一定抗氧化能力。
4采用溶剂萃取、硅胶柱色谱、高速逆流色谱及重结晶等方法从山莓叶中分离得到化合物五个对映贝壳杉烷型二萜类化合物,综合运用UV、IR、MS、1D-NMR、2D-NMR方法鉴定其结构分别为：对映-3α,16β,17,19-四羟基-贝壳杉烷-19-乙酸酯4；对映-3α,16β,17,19-四羟基-贝壳杉烷-19-乙酸酯-17-O-β-D-吡喃葡萄糖苷5：对映-3β,16β,17-贝壳杉-三醇6；对映-3p,16p,17,19-贝壳杉-四醇7；对映-2-羰基-16β-羟基-贝壳杉-17-O-β-D-葡萄糖苷8；其中化合物4、5为新化合物,化合物6首次从该植物中得到。
5利用高效液相色谱建立了山莓叶新二萜含量的检测方法,其线性回归方程为y=5×106x-10147,r=0.9942。表明二萜含量在0.139～0.695mg/mL范围内与吸光值线性关系良好,测定了不同月份山莓叶二萜的含量为0.656%-1.571%。K-B滤纸片测定发现新二萜单体化合物对金黄色葡萄球菌、痢疾志贺氏菌、大肠杆菌无明显抑制作用。MTT初探山莓新二萜化合物对HepG2肝癌细胞有一定的抑制作用,药品浓度与抑制率呈正相关,但其抑制率比5-FU低,可用于制备抗肿瘤保健品。
The Rubus corchorifolius L.f is a wild deciduous shrub of Rubus L. in Rosaceae, which is abundant in the wild. It has been used as traditional herb plants for a long time in the world, but we know little about its medical components and pharmacological action basis.The aauther takes the leaves of Rubus corchorifolius L.f as the research object, studies systematically on isolation, analysis and identification its active ingredient. The experiment results lay a theoretical foundation for exploring the herb resource of Rubus corchorifolius L.f. The main results are as follow:
1Flavone is one of the effective components of Rubus corchorifolius L.f. The optimum extraction condition are obtained through orthogonal experiment:60%ethanol as extracting solvent, temperature80℃, time1.5h. The flavone content is between0.134%-0.346%in Rubus corchorifolius L.f. leaves in different month byNaNO2-Al(NO)3coloration test. Then we obtain the quercetin(1), rutin(2)and kaenpferol-3-O-β-D-(6"-p-hydroxygalloy)-glucopyranoside(3)from crude extracts with solvent extraction and column chromtagraphy. It is the first time to obtain compounds1,2from this plant.
2The antibacterial activity of flavone compound is equivalent to that of berberine.When the content of quercetin is5mg/mL, it can inhibite the growth of Escherichia coli, Staphylococcus aureus, Shigella dysenteroae, Salmonella, Bacillusthuringiensis. the flavone compounds of Rubus corchorifolius L.f. have better dose-effect relationship in hydroxyl radical-scavenging, so as to provide theoretical evidence for antisepsis and anti-inflammation and anti-diarrhea of Rubus corchorifolius L.f.
3Contrasting extraction method of the volatile oil of Rubus corchorifolius, L.f. and optimizing extraction condition, establishing GC fingerprint of volatile oil from March to October, the result shows that there are8common peaks in total, so as to build up the quality evaluation for volatile oil of Rubus corchorifolius L.f. It was first time determined by resazurim color test, Escherichia coli, Staphylococcus aureus, Shigella dysenteroae, Salmonella, Bacillusthuringiensis were used as microbial strains to test the anti-bacterial activity of volatile oil and the minimum inhibitory concentration were62.5μg/mL,125μg/mL,15.6μg/mL,62.5μg/mL,31.25μg/mL. The antioxidant activity of volatile oil was first studied preliminarily by DPPH, the half DPPH radical-scavenging rate of volatile oil, rutin and Vc were respectively121.945μg/mL,6.326μg/mL,4.817μg/mL, the result shows that the volatile oil of Rubus corchorifolius L.f. has capacity of DPPH scavenging, which is lower than that of Vc and rutin.
4Using solvent extraction, silica gel column chromatography and crystallization methods to obtain five compounds:3,16,17-trihydroxykauran-18-yl acetate4;17-[(β-D-glucopyranosyl)oxy]-3,16-dihydroxykauran-18-yl acetate5, Ent-kaur-3β,16(3,17-triol6; ent-kauran-3β,16βM7,19-tetrol7; ent-2-carbonyl-16β-hydroxy-kauran-17-P-D-gluc8; It is the first time that purifying compound (5) from chloroform extracts by high speed counter current chromatography. The compounds (4,5) are new. In addition, the compounds (6) were first found from this plant.
5Established content determination of new diterpenoid of Rubus corchorifolius L.f. by HPLC and linear equation y=5×106×-10147, r=0.9942, concentration range of0.139-0.695mg/mL. diterpenoids contents in different month are betweenO.656%-1.571%. Through K-B paper disc method, the new diterpenoids (compound4,5) were discovered no antibacterial activity to Escherichia coli, Staphylococcus aureus, Shigella dysenteroae, Salmonella, Bacillus thuringiensis. The new diterpernoids can obviously restrained the HepG2cell lines proliferation in vary degree by MTT, the sample concentration correlated positively with suppression rate, but suppression rate are lower than that of5-FU, therefore the new diterpenoids of Rubus corchorifolius L.f can be used in anti-cancer health products.
1黄酮类化合物是山莓的有效成分之一,建立亚硝酸钠-硝酸铝显色法测定山莓叶中总黄酮的含量,正交试验优化得到山莓叶总黄酮的最佳提取条件为乙醇浓度为60%、水浴温度80℃、提取时间1.5h。测定不同月份山莓叶黄酮的含量为0.134%～0.346%。采用溶剂提取、柱色谱分离等方法分离得到槲皮素1；槲皮素-3-O-芸香糖苷(这个化合物即为芦丁)2；山奈酚-3-O-β-D-(6”-对羟基桂皮酰基)-葡萄糖苷3。其中化合物1,2为首次从该植物中得到。
2研究了山莓叶黄酮单体化合物的抑菌活性及抗氧化活性。当槲皮素浓度为5mg/mL时,对大肠杆菌、金黄色葡萄球菌、痢疾志贺氏菌、苏云金杆菌大肠杆菌、痢疾志贺氏菌抑菌作用明显,抑菌效果优于中药黄连素。山莓叶黄酮单体化合物对Fenton体系产生的羟自由基清除具有良好的量效关系,为山莓抗菌消炎、抗腹泻作用提供理论依据。
3采用水蒸气蒸馏、同时蒸馏萃取、微波辅助蒸馏三种方法比较提取山莓叶挥发油,优化提取条件。建立3-10月份山莓挥发油的GC指纹图谱,发现3-10月份的山莓叶挥发油共了8个共有峰,以期为综合评价山莓及其挥发油的质量提供依据。首次采用刃天青显色法研究发现山莓叶挥发油对大肠杆菌、沙门氏菌、金黄色葡萄球菌、痢疾志贺氏菌、苏云金杆菌最低抑菌浓度(MIC)分别为62.5ug/mL.62.5ug/mL、125ug/mL、31.25ug/mL、15.6ug/mL,对痢疾志贺氏杆菌的最低抑菌浓度低于卡拉霉素标准品的最低抑菌浓度。首次采用DPPH法对山莓叶挥发油进行抗氧化活性初步研究,山莓叶挥发油、芦丁、Vc半清除率EC5o分别为121.945μg/mL、6.326μg/mL、4.817μg/mL,发现山莓叶挥发油具有一定抗氧化能力。
4采用溶剂萃取、硅胶柱色谱、高速逆流色谱及重结晶等方法从山莓叶中分离得到化合物五个对映贝壳杉烷型二萜类化合物,综合运用UV、IR、MS、1D-NMR、2D-NMR方法鉴定其结构分别为：对映-3α,16β,17,19-四羟基-贝壳杉烷-19-乙酸酯4；对映-3α,16β,17,19-四羟基-贝壳杉烷-19-乙酸酯-17-O-β-D-吡喃葡萄糖苷5：对映-3β,16β,17-贝壳杉-三醇6；对映-3p,16p,17,19-贝壳杉-四醇7；对映-2-羰基-16β-羟基-贝壳杉-17-O-β-D-葡萄糖苷8；其中化合物4、5为新化合物,化合物6首次从该植物中得到。
5利用高效液相色谱建立了山莓叶新二萜含量的检测方法,其线性回归方程为y=5×106x-10147,r=0.9942。表明二萜含量在0.139～0.695mg/mL范围内与吸光值线性关系良好,测定了不同月份山莓叶二萜的含量为0.656%-1.571%。K-B滤纸片测定发现新二萜单体化合物对金黄色葡萄球菌、痢疾志贺氏菌、大肠杆菌无明显抑制作用。MTT初探山莓新二萜化合物对HepG2肝癌细胞有一定的抑制作用,药品浓度与抑制率呈正相关,但其抑制率比5-FU低,可用于制备抗肿瘤保健品。
The Rubus corchorifolius L.f is a wild deciduous shrub of Rubus L. in Rosaceae, which is abundant in the wild. It has been used as traditional herb plants for a long time in the world, but we know little about its medical components and pharmacological action basis.The aauther takes the leaves of Rubus corchorifolius L.f as the research object, studies systematically on isolation, analysis and identification its active ingredient. The experiment results lay a theoretical foundation for exploring the herb resource of Rubus corchorifolius L.f. The main results are as follow:
1Flavone is one of the effective components of Rubus corchorifolius L.f. The optimum extraction condition are obtained through orthogonal experiment:60%ethanol as extracting solvent, temperature80℃, time1.5h. The flavone content is between0.134%-0.346%in Rubus corchorifolius L.f. leaves in different month byNaNO2-Al(NO)3coloration test. Then we obtain the quercetin(1), rutin(2)and kaenpferol-3-O-β-D-(6"-p-hydroxygalloy)-glucopyranoside(3)from crude extracts with solvent extraction and column chromtagraphy. It is the first time to obtain compounds1,2from this plant.
2The antibacterial activity of flavone compound is equivalent to that of berberine.When the content of quercetin is5mg/mL, it can inhibite the growth of Escherichia coli, Staphylococcus aureus, Shigella dysenteroae, Salmonella, Bacillusthuringiensis. the flavone compounds of Rubus corchorifolius L.f. have better dose-effect relationship in hydroxyl radical-scavenging, so as to provide theoretical evidence for antisepsis and anti-inflammation and anti-diarrhea of Rubus corchorifolius L.f.
3Contrasting extraction method of the volatile oil of Rubus corchorifolius, L.f. and optimizing extraction condition, establishing GC fingerprint of volatile oil from March to October, the result shows that there are8common peaks in total, so as to build up the quality evaluation for volatile oil of Rubus corchorifolius L.f. It was first time determined by resazurim color test, Escherichia coli, Staphylococcus aureus, Shigella dysenteroae, Salmonella, Bacillusthuringiensis were used as microbial strains to test the anti-bacterial activity of volatile oil and the minimum inhibitory concentration were62.5μg/mL,125μg/mL,15.6μg/mL,62.5μg/mL,31.25μg/mL. The antioxidant activity of volatile oil was first studied preliminarily by DPPH, the half DPPH radical-scavenging rate of volatile oil, rutin and Vc were respectively121.945μg/mL,6.326μg/mL,4.817μg/mL, the result shows that the volatile oil of Rubus corchorifolius L.f. has capacity of DPPH scavenging, which is lower than that of Vc and rutin.
4Using solvent extraction, silica gel column chromatography and crystallization methods to obtain five compounds:3,16,17-trihydroxykauran-18-yl acetate4;17-[(β-D-glucopyranosyl)oxy]-3,16-dihydroxykauran-18-yl acetate5, Ent-kaur-3β,16(3,17-triol6; ent-kauran-3β,16βM7,19-tetrol7; ent-2-carbonyl-16β-hydroxy-kauran-17-P-D-gluc8; It is the first time that purifying compound (5) from chloroform extracts by high speed counter current chromatography. The compounds (4,5) are new. In addition, the compounds (6) were first found from this plant.
5Established content determination of new diterpenoid of Rubus corchorifolius L.f. by HPLC and linear equation y=5×106×-10147, r=0.9942, concentration range of0.139-0.695mg/mL. diterpenoids contents in different month are betweenO.656%-1.571%. Through K-B paper disc method, the new diterpenoids (compound4,5) were discovered no antibacterial activity to Escherichia coli, Staphylococcus aureus, Shigella dysenteroae, Salmonella, Bacillus thuringiensis. The new diterpernoids can obviously restrained the HepG2cell lines proliferation in vary degree by MTT, the sample concentration correlated positively with suppression rate, but suppression rate are lower than that of5-FU, therefore the new diterpenoids of Rubus corchorifolius L.f can be used in anti-cancer health products.
引文
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