蜂胶中抑制人白细胞弹性蛋白酶活性成分的研究
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摘要
蜂胶是蜜蜂从植物的芽和叶子中采集的树脂，混入花粉、蜂蜡和其他分泌物而形成的强粘性的天然物质。蜂胶的成分复杂，鉴定出的化合物超过300多种。蜂胶中含有大量的黄酮、酚酸及其酯类，因而具有广泛的药理活性。近年来，蜂胶抗炎活性受到广泛关注。本实验采用体外抑制人白细胞弹性蛋白酶（HLE）的筛选模型，对蜂胶不同组分以及从中分离得到的化合物的人白细胞弹性蛋白酶抑制活性进行评价，为蜂胶抗炎活性的开发提供参考。
（1）以蜂胶中存在的14种化合物为样品，研究了蜂胶样品的薄层色谱展开条件和显色方法。确定了较好的展开条件为氯仿：甲醇：甲酸92：4：9，较好的显色条件为1%～2%的三氯化铁乙醇溶液。
（2）对活性筛选方法的有效性进行了验证。以高效液相色谱法测定酶反应产物，以熊果酸作为阳性对照，证明了HLE活性筛选方法是有效的。
（3）通过不同浓度乙醇分级提取蜂胶得到4个提取物，比较了分级提取物的薄层色谱显色差异和HLE抑制活性差异。在质量浓度为100μg/mL时，40%乙醇水溶部分HLE抑制活性较低，为48.77%。而40%乙醇醇溶部分、70%乙醇提取物和95%乙醇提取物的薄层色谱显色斑点特征相似，HLE抑制活性较为接近，分别为75.68%、84.33%和79.90%，高于阳性对照品熊果酸（66.24%），统计学上存在极显著差异（p<0.01）。说明蜂胶醇溶组分具有很好的HLE抑制活性，是蜂胶中HLE抑制活性的物质基础所在。
（4）将40%乙醇醇溶部分、70%乙醇提取物和95%乙醇提取物合并后，采用溶剂分级萃取得到3种萃取物，比较了萃取物的薄层色谱显色差异和HLE抑制活性差异。石油醚萃取物的HLE抑制活性只有7.35%，远低于氯仿萃取物和乙酸乙酯萃取物。其薄层色谱显色与氯仿萃取物和乙酸乙酯萃取物差异极大。乙酸乙酯萃取物的HLE抑制活性（85.09%）高于氯仿萃取物（64.53%），是蜂胶中抑制HLE的主要活性组分。
（5）乙酸乙酯萃取物经硅胶柱层析分离，用石油醚-乙酸乙酯体系梯度洗脱，得到的3个洗脱组分。在质量浓度为100μg/mL时，三者的洗脱组分的活性分别介于44.77%～60.37%、21.38%～56.02%和85.85%～93.68%之间，HLE抑制活性存在极显著差异（p<0.01）。A9～A13的HLE抑制活性高于熊果酸（63.19%），是乙酸乙酯萃取物中对HLE具有高抑制活性的组分。A1～A4组分亦为活性组分，可从中分离得到活性化合物。
（6）活性组分A1～A4和A9～A13通过柱层析分离，结合薄层层析制备和液相色谱制备，共得到6个化合物，其中5个化合物经鉴定为球松素、高良姜素、白杨素、异鼠李素、槲皮素-5,7-二甲基醚，化合物VI尚未鉴定。在质量浓度为100μg/mL时，异鼠李素无HLE抑制活性，球松素、白杨素、槲皮素-5，7-二甲基醚和未知化合物VI的HLE抑制活性分别为26.22%、4.96%、50.63%和24.39%，具有一定的HLE抑制活性，但低于阳性对照熊果酸（70.85%）。高良姜素对HLE的抑制活性强于阳性药物熊果酸，其IC50为6.88μmol/L，是HLE的强抑制剂。
Propolis is a resinous, strongly adhesive natural substance, collected byhoneybees from buds and leaves of trees and plants, mixed with pollen, beeswax andother bee secretions. Propolis is a complex mixtrue of more than300constituents. Theactive components, flavonoids, phenolic acids, and their esters contribute to the widespectrum of its biological activities. Propolis has attracted much attention in recentyears for its anti-inflammatory properties. In order to offer some reference to thedevelopment of propolis with its anti-inflammatory properties, the in vitro model wasused to evaluate the human leukocyte elastase (HLE) inhibitory activity by differentfractions and compounds of propolis.
(1) The analitical TLC mobile phases and methods for visualisation of spots wereinvestigated using14chemical compounds of propolis as TLC sample. The mobilephase with chloroform-methnol-formic acid92:4:9and the1%～2%ferrictrichloride-ethanol solution for visualisation were selected as optimal TLC condition.
(2) The effectiveness of the sceening method of HLE inhibition was confirmed.High performance liquid chromatography was used to detect the product of theenzymatic reaction. Ursolic acid was used as positive control, and the HLE sceeningmethod was confirmed to be valid.
(3) Propolis was extracted by different concentrations of ethanol, and fourextracts were obtained. The differences of the thin layer chromatograph between thesefour extracts and their inhibition on HLE were compared. When the concentrationswere100μg/mL, the inhibition of the40%ethanol water fraction on HLE was48.77%,lower than that of the other three extracts. The thin layer chromatography spots andthe inhibition of the40%ethanol fraction,70%ethanol extract and95%ethanolextract were approximate. Their HLE inhibitory activity were75.68%,84.33%and79.90%, respectively, higer than that of ursolic acid (66.24%) with a statisticallysignificant difference (p<0.01). These facts demonstrated that the ethanol extracts ofpropolis were the material basis for the high HLE inhibitory activity they exerted.
(4) The active extracts were merged and then extracted by solvents with differentpolarities, and three extracts were abtained. The differences of the thin layerchromatograph between these three extracts and their inhibition on HLE werecompared. When the concentrations of the three ethanol extracts were100μg/mL, theinhibion of petroleum ether extract on HLE was only7.35%, much lower than that ofchloroform extract and ethyl acetate extract. Its TLC spots were quite different from that of the latter two. The inhibion of ethyl acetate extract on HLE was85.09%,higher than that of the chloroform extract (64.53%). The ethyl acetate extract was themain active extract of propolis.
(5) The ethyl acetate extract was separated by column chromatography method,with petroleum ether-ethyl acetate as mobile phase, and three fractions were obtained.When the concentrations of the three ethanol extracts were100μg/mL, the range ofinhibition of these eluents were44.77%～60.37%,21.38%～56.02%and85.85%～93.68%, repectively, with a statistically significant difference (p<0.01). The fractionA9～A13were highly active eluents of the ethyl acetate extract, their inhibion werehigher than that of ursolic acid (63.19%). The eluent A1～A4was also active eluentand can be separated and purified to obtain some active components.
(6)Active eluents were separated by the column chromatography methodcombined with preparative TLC and preparative HPLC methods. And six compoundswere isolated, and five of them were identified to be pinostrobin, galangin, chrysin,isorhamnetin, quercetin-5,7-dimethyl-ether and the compound VI hasn’t beenidentified. Isorhamnetin had no inhibition on HLE when its concentration was100μg/mL. Pinostrobin, chrysin, quercetin-5,7-dimethyl-ether and the unknowncompound VI were somehow active, their inhibion were26.22%,4.96%,50.63%and24.39%, respectively. The inhibitory activity of galangin was stronger thanpositive drug ursolic acid. Galangin was a strong inhibitor of HLE with an IC50of6.88μmol/mL.
（1）以蜂胶中存在的14种化合物为样品，研究了蜂胶样品的薄层色谱展开条件和显色方法。确定了较好的展开条件为氯仿：甲醇：甲酸92：4：9，较好的显色条件为1%～2%的三氯化铁乙醇溶液。
（2）对活性筛选方法的有效性进行了验证。以高效液相色谱法测定酶反应产物，以熊果酸作为阳性对照，证明了HLE活性筛选方法是有效的。
（3）通过不同浓度乙醇分级提取蜂胶得到4个提取物，比较了分级提取物的薄层色谱显色差异和HLE抑制活性差异。在质量浓度为100μg/mL时，40%乙醇水溶部分HLE抑制活性较低，为48.77%。而40%乙醇醇溶部分、70%乙醇提取物和95%乙醇提取物的薄层色谱显色斑点特征相似，HLE抑制活性较为接近，分别为75.68%、84.33%和79.90%，高于阳性对照品熊果酸（66.24%），统计学上存在极显著差异（p<0.01）。说明蜂胶醇溶组分具有很好的HLE抑制活性，是蜂胶中HLE抑制活性的物质基础所在。
（4）将40%乙醇醇溶部分、70%乙醇提取物和95%乙醇提取物合并后，采用溶剂分级萃取得到3种萃取物，比较了萃取物的薄层色谱显色差异和HLE抑制活性差异。石油醚萃取物的HLE抑制活性只有7.35%，远低于氯仿萃取物和乙酸乙酯萃取物。其薄层色谱显色与氯仿萃取物和乙酸乙酯萃取物差异极大。乙酸乙酯萃取物的HLE抑制活性（85.09%）高于氯仿萃取物（64.53%），是蜂胶中抑制HLE的主要活性组分。
（5）乙酸乙酯萃取物经硅胶柱层析分离，用石油醚-乙酸乙酯体系梯度洗脱，得到的3个洗脱组分。在质量浓度为100μg/mL时，三者的洗脱组分的活性分别介于44.77%～60.37%、21.38%～56.02%和85.85%～93.68%之间，HLE抑制活性存在极显著差异（p<0.01）。A9～A13的HLE抑制活性高于熊果酸（63.19%），是乙酸乙酯萃取物中对HLE具有高抑制活性的组分。A1～A4组分亦为活性组分，可从中分离得到活性化合物。
（6）活性组分A1～A4和A9～A13通过柱层析分离，结合薄层层析制备和液相色谱制备，共得到6个化合物，其中5个化合物经鉴定为球松素、高良姜素、白杨素、异鼠李素、槲皮素-5,7-二甲基醚，化合物VI尚未鉴定。在质量浓度为100μg/mL时，异鼠李素无HLE抑制活性，球松素、白杨素、槲皮素-5，7-二甲基醚和未知化合物VI的HLE抑制活性分别为26.22%、4.96%、50.63%和24.39%，具有一定的HLE抑制活性，但低于阳性对照熊果酸（70.85%）。高良姜素对HLE的抑制活性强于阳性药物熊果酸，其IC50为6.88μmol/L，是HLE的强抑制剂。
Propolis is a resinous, strongly adhesive natural substance, collected byhoneybees from buds and leaves of trees and plants, mixed with pollen, beeswax andother bee secretions. Propolis is a complex mixtrue of more than300constituents. Theactive components, flavonoids, phenolic acids, and their esters contribute to the widespectrum of its biological activities. Propolis has attracted much attention in recentyears for its anti-inflammatory properties. In order to offer some reference to thedevelopment of propolis with its anti-inflammatory properties, the in vitro model wasused to evaluate the human leukocyte elastase (HLE) inhibitory activity by differentfractions and compounds of propolis.
(1) The analitical TLC mobile phases and methods for visualisation of spots wereinvestigated using14chemical compounds of propolis as TLC sample. The mobilephase with chloroform-methnol-formic acid92:4:9and the1%～2%ferrictrichloride-ethanol solution for visualisation were selected as optimal TLC condition.
(2) The effectiveness of the sceening method of HLE inhibition was confirmed.High performance liquid chromatography was used to detect the product of theenzymatic reaction. Ursolic acid was used as positive control, and the HLE sceeningmethod was confirmed to be valid.
(3) Propolis was extracted by different concentrations of ethanol, and fourextracts were obtained. The differences of the thin layer chromatograph between thesefour extracts and their inhibition on HLE were compared. When the concentrationswere100μg/mL, the inhibition of the40%ethanol water fraction on HLE was48.77%,lower than that of the other three extracts. The thin layer chromatography spots andthe inhibition of the40%ethanol fraction,70%ethanol extract and95%ethanolextract were approximate. Their HLE inhibitory activity were75.68%,84.33%and79.90%, respectively, higer than that of ursolic acid (66.24%) with a statisticallysignificant difference (p<0.01). These facts demonstrated that the ethanol extracts ofpropolis were the material basis for the high HLE inhibitory activity they exerted.
(4) The active extracts were merged and then extracted by solvents with differentpolarities, and three extracts were abtained. The differences of the thin layerchromatograph between these three extracts and their inhibition on HLE werecompared. When the concentrations of the three ethanol extracts were100μg/mL, theinhibion of petroleum ether extract on HLE was only7.35%, much lower than that ofchloroform extract and ethyl acetate extract. Its TLC spots were quite different from that of the latter two. The inhibion of ethyl acetate extract on HLE was85.09%,higher than that of the chloroform extract (64.53%). The ethyl acetate extract was themain active extract of propolis.
(5) The ethyl acetate extract was separated by column chromatography method,with petroleum ether-ethyl acetate as mobile phase, and three fractions were obtained.When the concentrations of the three ethanol extracts were100μg/mL, the range ofinhibition of these eluents were44.77%～60.37%,21.38%～56.02%and85.85%～93.68%, repectively, with a statistically significant difference (p<0.01). The fractionA9～A13were highly active eluents of the ethyl acetate extract, their inhibion werehigher than that of ursolic acid (63.19%). The eluent A1～A4was also active eluentand can be separated and purified to obtain some active components.
(6)Active eluents were separated by the column chromatography methodcombined with preparative TLC and preparative HPLC methods. And six compoundswere isolated, and five of them were identified to be pinostrobin, galangin, chrysin,isorhamnetin, quercetin-5,7-dimethyl-ether and the compound VI hasn’t beenidentified. Isorhamnetin had no inhibition on HLE when its concentration was100μg/mL. Pinostrobin, chrysin, quercetin-5,7-dimethyl-ether and the unknowncompound VI were somehow active, their inhibion were26.22%,4.96%,50.63%and24.39%, respectively. The inhibitory activity of galangin was stronger thanpositive drug ursolic acid. Galangin was a strong inhibitor of HLE with an IC50of6.88μmol/mL.
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