蜂胶挥发油提取与抑蜜蜂球囊菌的研究
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摘要
本文主要对河南蜂胶挥发油的提取、成分鉴定及理化性质、清除DPPH自由基能力、抑蜜蜂球囊菌活性进行了研究,结果如下:
(1)采用有机溶剂浸提法提取蜂胶挥发油,比较了石油醚、正己烷、乙醚、四氯化碳四种溶剂的提取率,确定石油醚为最佳溶剂。采用正交试验优化微波-超声波辅助技术提取蜂胶挥发油的工艺,其最佳工艺条件为:料液比1:20,浸提温度40℃,微波炉选择中档功率,提取时间15 min。该条件下的提取率高达12.32%,说明微波-超声波协同辅助这种新技术具有很强的可行性。
(2)采用GC-MS技术对石油醚、正己烷、乙醚、四氯化碳四种溶剂浸提物(分别为PEP、HEP、TEP、DEP)的成分进行鉴定分析。其中PEP鉴定出60种化合物,HEP 49种化合物,TEP 46种化合物,DEP 47种化合物,其相对百分含量分别为34.18%,30.03%,47.66%,35.14%。主要成分有烷烃、烯、醇、酸、醛、酮、酯类等化合物,但各成分在含量上存在很大的差异,表明溶剂的极性会影响其对蜂胶挥发性成分的选择性。
(3)参考GB/T 14454,GB/T 14455等国家标准对蜂胶挥发油PEP的9项理化指标进行了初步的检测,结果显示:PEP为黄色透明油状液体,有浓郁香味;在25℃时,PEP的相对密度为0.936;折光率为1.43505;熔程为25℃～47℃;凝点为24℃;PEP在乙醇中的溶混度表示为1 mL的PEP能完全溶解于17.6 mL的90%乙醇溶液中;PEP的酸值与酯值分别为14.8和16.7;羰基化合物含量和含酚量分别为11.8 %,60.4 %。
(4)以DPPH自由基清除能力评价了TEP、PEP、HEP及DEP的抗氧化活性。Duncan多重比较分析结果显示:TEP、PEP、HEP及DEP之间的抗氧化活性差异极显著(P<0.01),抗氧化活由强到弱的顺序为TEP>PEP>HEP>DEP;TEP和蜂胶的抗氧化活性在5%水平上差异不显著,但均表现出很强的清除DPPH自由基能力。TEP、PEP、HEP及DEP的抗氧化活性都呈现量效关系,即DPPH自由基清除率随着浓度增大而升高;清除自由基的IC50分别约为5μg/mL,10μg/mL,50μg/mL,200μg/mL。TEP具有很强抗氧化活性与其含大量的高良姜素、松属素等黄酮类物质有关;而DEP的抗氧化活性很弱可能与DEP中含有具有抗氧化活性的酯类和酚类物质非常少有关。
(5)采用生长速率法和试管连续稀释法分析蜂胶与蜂胶挥发油PEP抑蜜蜂球囊菌活性,并与制霉菌素的抑菌效果作对比分析。生长速率法试验结果显示:蜂胶的抑菌活性强于蜂胶挥发油,蜂胶挥发油PEP在高浓度时对球囊菌有一定的抑制作用;蜂胶与蜂胶挥发油的抑菌效果不及制霉菌素效果好。试管稀释法测定蜂胶、PEP、制霉菌素的最低抑制浓度(MIC)分别为:0.078 mg/mL,0.156 mg/mL,0.156 mg/mL,蜂胶和PEP的MIC比制霉菌素的要低或相等,试管稀释法结果表明蜂胶与蜂胶挥发油对球囊菌均具有一定的抑菌活性。
In this paper, the extraction of volatile oil from propolis, composition identificat- ion, physical and chemical properties, DPPH radical-scavenging activities, and antifu- ngal activity against Ascosphaera apis were studied. The results were summarized as follows:
(1) Petroleumether, hexane, diethyl ether, tetrachloromethane, respectively, were used to extract volatile oil from propolis. The yeild of volatile oil were compared to choose the optimum solvent, and petroleumether was regarded as the optimum solvent. The orthogonal experiment was carried out to determine the optimum condition of extraction assisted by microwave–ultrasound technique, and the optimum conditions turned out to be solid-liquid ratio at 1:20, the extraction temperature at 40℃, micro- wave power at mid-range power, extraction time of 15 min. in this case, the yeild of volatile oil reached at as high as 12.32%, which proved the feasibility of this new technology.
(2) The compositions of four kinds of volatile voils-PEP, HEP, TEP and DEP were analyzed by GC-MS. The results showed that 60 compounds were identified in PEP, 49 compounds in HEP, 46 compounds in TEP, 47 compounds in DEP, and the relative contents of volatile oils were 34.18%, 30.03%, 47.66%, 35.14% respectively. The major components of the volatile oils were alkanes, alkenes, alcohols, acids, aldehydes, esters etc, but differed greatly in content, which indicated that the polarity could affect the extract of volatile substance.
(3) Nine physical and chemical properties of PEP were detected according to National Standard: the oil was an yellow transparent oily liquid, with aromatic flavor; at 25℃, relative density of PEP was 0.936, refractive index was 1.43505, melting range was about 25℃~47℃, freezing point was 24℃, 1 mL of the volatile oil was insoluble in 17.6 mL of 90% alcohol solution; volatile oil acid value, ester value were 14.8, 16.7 respectively; content of carbonyl compounds and phenols were 11.8%, 60.4% respectively.
(4) DPPH radical-scavenging activity was tested to evaluate the antioxidant activity of propolis and four volatile oils—TEP, PEP, HEP and DEP. Duncan multiple comparison showed that there were extremely significant difference (P<0.01) among antioxidative activitiy of TEP, PEP, HEP and DEP. the order of antioxidative activitiy was: TEP>PEP>HEP>DEP. TEP and propolis had strong antioxidative activitiy, which showed no significant difference at the level of 5%, both of them showed significant scavenging activity to DPPH freeradical. All the activities had an dose-effect relationship, DPPH radical scavenging rate depended on the concentration, the higer the better. IC50 were approximately at 5μg/mL, 10μg/mL, 50μg/mL, 200μg/mL respectively,TEP showed strong antioxidant activity due to the high content of flavonoids such as Pinocembrin and galangin. DEP had a very weak antioxidant activity, which may be due to the lack of esters and phenols.
(5) Growth Rate method and Tube Dilution method were used to evaluate antifungal activity of propolis and volatile oil PEP against Ascosphaera apis, which effects were compared with that of nystatin, and the mechanism was discussed. Propolis showed a better antifungal activity than volatile oil, which may attribute to higher flavonoids content in PEP than in propolis. At high concentration, PEP showed a good activity, and the activity of nystatin was the best. The minimum inhibitory concentration of Propolis, PEP, nystatin were: 0.078 mg/mL, 0.156 mg/mL, 0.156 mg/ mL respectively, the MIC of EEP and PEP is lower than nystatin, which indicated that volatile oil and propolis had a good antifungal activity against Ascosphaera apis.
(1)采用有机溶剂浸提法提取蜂胶挥发油,比较了石油醚、正己烷、乙醚、四氯化碳四种溶剂的提取率,确定石油醚为最佳溶剂。采用正交试验优化微波-超声波辅助技术提取蜂胶挥发油的工艺,其最佳工艺条件为:料液比1:20,浸提温度40℃,微波炉选择中档功率,提取时间15 min。该条件下的提取率高达12.32%,说明微波-超声波协同辅助这种新技术具有很强的可行性。
(2)采用GC-MS技术对石油醚、正己烷、乙醚、四氯化碳四种溶剂浸提物(分别为PEP、HEP、TEP、DEP)的成分进行鉴定分析。其中PEP鉴定出60种化合物,HEP 49种化合物,TEP 46种化合物,DEP 47种化合物,其相对百分含量分别为34.18%,30.03%,47.66%,35.14%。主要成分有烷烃、烯、醇、酸、醛、酮、酯类等化合物,但各成分在含量上存在很大的差异,表明溶剂的极性会影响其对蜂胶挥发性成分的选择性。
(3)参考GB/T 14454,GB/T 14455等国家标准对蜂胶挥发油PEP的9项理化指标进行了初步的检测,结果显示:PEP为黄色透明油状液体,有浓郁香味;在25℃时,PEP的相对密度为0.936;折光率为1.43505;熔程为25℃～47℃;凝点为24℃;PEP在乙醇中的溶混度表示为1 mL的PEP能完全溶解于17.6 mL的90%乙醇溶液中;PEP的酸值与酯值分别为14.8和16.7;羰基化合物含量和含酚量分别为11.8 %,60.4 %。
(4)以DPPH自由基清除能力评价了TEP、PEP、HEP及DEP的抗氧化活性。Duncan多重比较分析结果显示:TEP、PEP、HEP及DEP之间的抗氧化活性差异极显著(P<0.01),抗氧化活由强到弱的顺序为TEP>PEP>HEP>DEP;TEP和蜂胶的抗氧化活性在5%水平上差异不显著,但均表现出很强的清除DPPH自由基能力。TEP、PEP、HEP及DEP的抗氧化活性都呈现量效关系,即DPPH自由基清除率随着浓度增大而升高;清除自由基的IC50分别约为5μg/mL,10μg/mL,50μg/mL,200μg/mL。TEP具有很强抗氧化活性与其含大量的高良姜素、松属素等黄酮类物质有关;而DEP的抗氧化活性很弱可能与DEP中含有具有抗氧化活性的酯类和酚类物质非常少有关。
(5)采用生长速率法和试管连续稀释法分析蜂胶与蜂胶挥发油PEP抑蜜蜂球囊菌活性,并与制霉菌素的抑菌效果作对比分析。生长速率法试验结果显示:蜂胶的抑菌活性强于蜂胶挥发油,蜂胶挥发油PEP在高浓度时对球囊菌有一定的抑制作用;蜂胶与蜂胶挥发油的抑菌效果不及制霉菌素效果好。试管稀释法测定蜂胶、PEP、制霉菌素的最低抑制浓度(MIC)分别为:0.078 mg/mL,0.156 mg/mL,0.156 mg/mL,蜂胶和PEP的MIC比制霉菌素的要低或相等,试管稀释法结果表明蜂胶与蜂胶挥发油对球囊菌均具有一定的抑菌活性。
In this paper, the extraction of volatile oil from propolis, composition identificat- ion, physical and chemical properties, DPPH radical-scavenging activities, and antifu- ngal activity against Ascosphaera apis were studied. The results were summarized as follows:
(1) Petroleumether, hexane, diethyl ether, tetrachloromethane, respectively, were used to extract volatile oil from propolis. The yeild of volatile oil were compared to choose the optimum solvent, and petroleumether was regarded as the optimum solvent. The orthogonal experiment was carried out to determine the optimum condition of extraction assisted by microwave–ultrasound technique, and the optimum conditions turned out to be solid-liquid ratio at 1:20, the extraction temperature at 40℃, micro- wave power at mid-range power, extraction time of 15 min. in this case, the yeild of volatile oil reached at as high as 12.32%, which proved the feasibility of this new technology.
(2) The compositions of four kinds of volatile voils-PEP, HEP, TEP and DEP were analyzed by GC-MS. The results showed that 60 compounds were identified in PEP, 49 compounds in HEP, 46 compounds in TEP, 47 compounds in DEP, and the relative contents of volatile oils were 34.18%, 30.03%, 47.66%, 35.14% respectively. The major components of the volatile oils were alkanes, alkenes, alcohols, acids, aldehydes, esters etc, but differed greatly in content, which indicated that the polarity could affect the extract of volatile substance.
(3) Nine physical and chemical properties of PEP were detected according to National Standard: the oil was an yellow transparent oily liquid, with aromatic flavor; at 25℃, relative density of PEP was 0.936, refractive index was 1.43505, melting range was about 25℃~47℃, freezing point was 24℃, 1 mL of the volatile oil was insoluble in 17.6 mL of 90% alcohol solution; volatile oil acid value, ester value were 14.8, 16.7 respectively; content of carbonyl compounds and phenols were 11.8%, 60.4% respectively.
(4) DPPH radical-scavenging activity was tested to evaluate the antioxidant activity of propolis and four volatile oils—TEP, PEP, HEP and DEP. Duncan multiple comparison showed that there were extremely significant difference (P<0.01) among antioxidative activitiy of TEP, PEP, HEP and DEP. the order of antioxidative activitiy was: TEP>PEP>HEP>DEP. TEP and propolis had strong antioxidative activitiy, which showed no significant difference at the level of 5%, both of them showed significant scavenging activity to DPPH freeradical. All the activities had an dose-effect relationship, DPPH radical scavenging rate depended on the concentration, the higer the better. IC50 were approximately at 5μg/mL, 10μg/mL, 50μg/mL, 200μg/mL respectively,TEP showed strong antioxidant activity due to the high content of flavonoids such as Pinocembrin and galangin. DEP had a very weak antioxidant activity, which may be due to the lack of esters and phenols.
(5) Growth Rate method and Tube Dilution method were used to evaluate antifungal activity of propolis and volatile oil PEP against Ascosphaera apis, which effects were compared with that of nystatin, and the mechanism was discussed. Propolis showed a better antifungal activity than volatile oil, which may attribute to higher flavonoids content in PEP than in propolis. At high concentration, PEP showed a good activity, and the activity of nystatin was the best. The minimum inhibitory concentration of Propolis, PEP, nystatin were: 0.078 mg/mL, 0.156 mg/mL, 0.156 mg/ mL respectively, the MIC of EEP and PEP is lower than nystatin, which indicated that volatile oil and propolis had a good antifungal activity against Ascosphaera apis.
引文
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