蜂胶中挥发性成分的提取方法、化学组成及生物学活性
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摘要
蜂胶是由蜜蜂采集树脂并混入蜂蜡、花粉及自身腺体分泌物所得到的一种天然产物,具有广泛的生物学活性。蜂胶成分复杂,包含大量的黄酮类化合物、酚酸类化合物及萜类化合物等生物学活性组分。目前对蜂胶中的挥发性成分的组成及生物学活性研究较少。由于蜂胶生物学活性的发挥被认为是蜂胶中多种组分共同作用的结果,因此对蜂胶挥发性成分的组成及生物学活性的研究可为蜂胶生物学活性的全面评价及蜂胶产品的开发利用提供基础。
本研究采用水蒸气蒸馏法、常温有机溶剂提取法、超声辅助提取法及微波辅助提取法对蜂胶中的挥发性成分进行了提取；采用均匀实验设计及正交设计优化了超声辅助提取及微波辅助提取蜂胶中挥发性成分的工艺参数；采用GC-MS对微波辅助法提取所得的中国蜂胶和巴西蜂胶中挥发性成分的化学组成进行了分析与比较；采用抑菌圈法及最小抑菌浓度法对中国蜂胶和巴西蜂胶中挥发性成分的抑菌活性进行了研究；采用DPPH自由基清除实验、抗氧化能力指数ORAC测定实验及H202诱导的红细胞氧化溶血试验评价了中国蜂胶和巴西蜂胶中挥发性成分的体外抗氧化活性；利用束缚所致的小鼠氧化应激评价了蜂胶挥发性成分对机体氧化应激的改善作用；利用动物实验评价了蜂胶挥发性成分对类焦虑样行为的影响。
研究结果发现：
1、超声辅助提取及微波辅助提取较适于蜂胶挥发性成分的提取,挥发性成分提取得率较高、性状较好。
2、优化的超声辅助提取蜂胶中挥发性成分的工艺参数：提取温度59℃、料液比1：15、提取时间62 min。在此条件下得到中国蜂胶和巴西蜂胶中的挥发性成分提取率分别为8.70%和8.75%。
3、优化的微波辅助提取蜂胶中挥发性成分的工艺参数：微波处理温度为35℃,微波处理15min,料液比为1：10,提取2次。在此条件下得到中国蜂胶和巴西蜂胶中的挥发性成分提取率分别为9.73%和11.26%。
4、鉴定出巴西蜂胶挥发性组分5D个,主要是倍半萜类及其含氧衍生物,长叶稀、桉叶醇、愈创木醇等。中国蜂胶挥发性组分47个,主要是芳香酸及萜类衍生物,肉桂酸、桉叶醇等。两地蜂胶挥发性成分中含有21个具有定香、抑菌、驱虫活性的共有组分。
5、中国蜂胶和巴西蜂胶中的挥发性成分具有抑菌活性,对革兰氏阳性细菌、酵母茵及霉菌抑菌活性强,对革兰氏阴性细菌抑菌活性较弱；中国蜂胶和巴西蜂胶中的挥发性成分具有DPPH自由基清除作用,其IC50值分别为178.3和309.5μg/ml; ORAC值分别为0.82和0.88μMTrolox eq/g;对H2O2诱导的兔红细胞氧化溶血具有保护作用,其IC50值分别为0.871和0.879 mg/ml.
6、蜂胶中挥发性成分可显著降低束缚应激小鼠血清中MDA含量,提高SOD活性；显著提高肝组织中SOD和CAT的活性,增加肝组织中GSH的含量,降低肝组织中MDA和NO含量,降低XOD活性。
7、蜂胶中挥发性成分可显著增加束缚应激小鼠在高架十字迷宫实验中进入开臂次数及进入开臂时间百分比,降低防卫性探头及防卫性伸展比例,且对自主活动次数无影响；显著降低束缚应激小鼠血清ACTH和CORT水平并能显著降低脑组织中MDA含量,提高脑组织中SOD活性及GSH含量。
总之,蜂胶挥发性成分是蜂胶生物学活性组分的重要组成部分,不同来源蜂胶挥发性成分化学组成不同；蜂胶挥发性成分具有抑菌及体外抗氧化活性；并对机体氧化应激具有改善作用；对焦虑症亦有一定改善作用。作为蜂胶一种新的产品形式,蜂胶挥发性成分值得研究与开发。
Propolis is a resinous material collected by honeybees (Apis mellifera L.) from leaf buds and cracks in the bark of the plants and mixed with pollen, wax and bee enzymes. It possesses a broad spectrum of biological activities. The chemical composition of propolis is very complicated, containing great amounts of bioactive compounds such as flavonoids, phenolic acids and terpenoids. Essential oil is an important part of propolis which contains great amounts of bioactive ingredients, and may contribute a lot for propolis'pharmacological activities. However, the researchs on propolis essential oil were far from sufficient. The studies on chemical composition and biological activities of propolis essential oil can provide foundations for more comprehensive evaluation of the biological activities of propolis and better use of propolis.
In this study, four procedures for the extraction of essential oils from propolis were compared, including steam distillation extraction, solvent extraction, ultrasonic assisted extraction and microwave assisted extraction. The Uniform design was used to optimize ultrasonic assisted extraction process of propolis essential oil and the orthogonal design was used to optimize microwave assisted extraction process of propolis essential oil. The chemical components of essential oil from Brazilian and Chinese propolis were separated and identified by gas chromatography-mass spectrometry (GC-MS). Antimicrobial activities of propolis essential oil were measured by disc diffusion method and minimum inhibitory concentration (MIC) determination. Antioxidant properties of propolis essential oil were measured by both cell-free and cell-based experimental models:2,2-diphenyl-l-picrylhydrazyl (DPPH) assay, oxygen radical absorbance capacity (ORAC) assay and the assay to inhibit the H2O2-induced lysis of rabbit red blood cells (RBC). The effects on oxidative stress and anxiety-like behavior of propolis essential oil were evaluated with restraint stress animal model.
Results showed that:
1. Among the four procedures chosen for extraction, ultrasonic assisted extraction and microwave assisted extraction were suitable for the extraction of propolis essential oil. With these two methods, essential oil was of a high yield and quality.
2. The optimal extracting procedure for ultrasonic assisted extraction was as follows:at the extraction temperature of 59℃with 1:15 feed ratio and 62min of the extraction time, the essential oil yield reached 8.70% for Chinese propolis and 8.75% for Brazilian propolis.
3. The optimum technological parameters for microwave assisted extraction were obtained as:35℃of microwave extraction temperature,15min of microwave extraction time,1:10 of the solid to liquid ratio and 2 times of extraction. Under such optimal conditions, it was found that the essential oil content was 11.21% in Brazilian propolis and 9.73% in Chinese propolis.
4. Fifty-one components were identified from Brazilian propolis essential oil and the major components were terpenoids:Longifolene, a-eudesmol,β-eudesmol, P-Caryophyllene and Guaiol. Forty-seven components were identified from Chinese propolis essential oil and the major components were aromatic compound and terpenoids:Cinnamic acid,β-eudesmol, a-eudesmol, and y-eudesmol. Twenty-one compounds were found existing in both of Chinese and Brazilian propolis.
5. The essential oil from both Brazilian and Chinese propolis showed good to moderate antimicrobial activity against G-positive bacteria and fungi, while showed slight activity on G-negative bacteria. The essential oil from both Brazilian and Chinese propolis showed antioxidant activities in vitro. The IC50 of DPPH free radical were 178.3μg/ml and 309.5μg/ml respectively. The relative ORAC values of them were 0.82μMTrolox eq/g and 0.88μMTrolox eq/g, while the IC50 of H2O2 induced RBC hemolysis were 0.871 mg/ml and 0.879 mg/ml respectively.
6. Essential oil of propolis can significantly decrease MDA level and increase SOD activity in serum of restraint stress mice, ameliorate liver function by improving the oxidative stress status. It can also significantly increase SOD and CAT activity and the GSH level, and decrease MDA and NO level as well as XOD activity in liver tissue.
7. Propolis essential oil can significantly increase the Open Arm Entry percentage and Open Arm Time percentage, and decrease the protect Head-dipping percentage and protect rearing percentage of restraint stress mice in elevated plus-maze test, which showed no significant effects on the number of activities in spontaneous locomotor activity test. Propolis essential oil also depressed the serum level of CORT and ACTH, decreased the content of MDA and improved SOD and GSH in brain tissues of restraint stress mice.
Essential oil is an important component of propolis which contains great amounts of bioactive ingredients and possesses various biological activities. The present work provides additional data about the propolis essential oil obtained by microwave extration and reveals the interesting character of its chemical composition, antimicrobial activity and antioxidant acitvity in vitro, ameliorative effects on oxdative stress and anxiety. As propolis essential oil is a complex mixture of chemicals which may differ by plant sources, more investigations and further studies are necessary to elucidate its components and the exact site to achieve their potential anxiolytic effects.
本研究采用水蒸气蒸馏法、常温有机溶剂提取法、超声辅助提取法及微波辅助提取法对蜂胶中的挥发性成分进行了提取；采用均匀实验设计及正交设计优化了超声辅助提取及微波辅助提取蜂胶中挥发性成分的工艺参数；采用GC-MS对微波辅助法提取所得的中国蜂胶和巴西蜂胶中挥发性成分的化学组成进行了分析与比较；采用抑菌圈法及最小抑菌浓度法对中国蜂胶和巴西蜂胶中挥发性成分的抑菌活性进行了研究；采用DPPH自由基清除实验、抗氧化能力指数ORAC测定实验及H202诱导的红细胞氧化溶血试验评价了中国蜂胶和巴西蜂胶中挥发性成分的体外抗氧化活性；利用束缚所致的小鼠氧化应激评价了蜂胶挥发性成分对机体氧化应激的改善作用；利用动物实验评价了蜂胶挥发性成分对类焦虑样行为的影响。
研究结果发现：
1、超声辅助提取及微波辅助提取较适于蜂胶挥发性成分的提取,挥发性成分提取得率较高、性状较好。
2、优化的超声辅助提取蜂胶中挥发性成分的工艺参数：提取温度59℃、料液比1：15、提取时间62 min。在此条件下得到中国蜂胶和巴西蜂胶中的挥发性成分提取率分别为8.70%和8.75%。
3、优化的微波辅助提取蜂胶中挥发性成分的工艺参数：微波处理温度为35℃,微波处理15min,料液比为1：10,提取2次。在此条件下得到中国蜂胶和巴西蜂胶中的挥发性成分提取率分别为9.73%和11.26%。
4、鉴定出巴西蜂胶挥发性组分5D个,主要是倍半萜类及其含氧衍生物,长叶稀、桉叶醇、愈创木醇等。中国蜂胶挥发性组分47个,主要是芳香酸及萜类衍生物,肉桂酸、桉叶醇等。两地蜂胶挥发性成分中含有21个具有定香、抑菌、驱虫活性的共有组分。
5、中国蜂胶和巴西蜂胶中的挥发性成分具有抑菌活性,对革兰氏阳性细菌、酵母茵及霉菌抑菌活性强,对革兰氏阴性细菌抑菌活性较弱；中国蜂胶和巴西蜂胶中的挥发性成分具有DPPH自由基清除作用,其IC50值分别为178.3和309.5μg/ml; ORAC值分别为0.82和0.88μMTrolox eq/g;对H2O2诱导的兔红细胞氧化溶血具有保护作用,其IC50值分别为0.871和0.879 mg/ml.
6、蜂胶中挥发性成分可显著降低束缚应激小鼠血清中MDA含量,提高SOD活性；显著提高肝组织中SOD和CAT的活性,增加肝组织中GSH的含量,降低肝组织中MDA和NO含量,降低XOD活性。
7、蜂胶中挥发性成分可显著增加束缚应激小鼠在高架十字迷宫实验中进入开臂次数及进入开臂时间百分比,降低防卫性探头及防卫性伸展比例,且对自主活动次数无影响；显著降低束缚应激小鼠血清ACTH和CORT水平并能显著降低脑组织中MDA含量,提高脑组织中SOD活性及GSH含量。
总之,蜂胶挥发性成分是蜂胶生物学活性组分的重要组成部分,不同来源蜂胶挥发性成分化学组成不同；蜂胶挥发性成分具有抑菌及体外抗氧化活性；并对机体氧化应激具有改善作用；对焦虑症亦有一定改善作用。作为蜂胶一种新的产品形式,蜂胶挥发性成分值得研究与开发。
Propolis is a resinous material collected by honeybees (Apis mellifera L.) from leaf buds and cracks in the bark of the plants and mixed with pollen, wax and bee enzymes. It possesses a broad spectrum of biological activities. The chemical composition of propolis is very complicated, containing great amounts of bioactive compounds such as flavonoids, phenolic acids and terpenoids. Essential oil is an important part of propolis which contains great amounts of bioactive ingredients, and may contribute a lot for propolis'pharmacological activities. However, the researchs on propolis essential oil were far from sufficient. The studies on chemical composition and biological activities of propolis essential oil can provide foundations for more comprehensive evaluation of the biological activities of propolis and better use of propolis.
In this study, four procedures for the extraction of essential oils from propolis were compared, including steam distillation extraction, solvent extraction, ultrasonic assisted extraction and microwave assisted extraction. The Uniform design was used to optimize ultrasonic assisted extraction process of propolis essential oil and the orthogonal design was used to optimize microwave assisted extraction process of propolis essential oil. The chemical components of essential oil from Brazilian and Chinese propolis were separated and identified by gas chromatography-mass spectrometry (GC-MS). Antimicrobial activities of propolis essential oil were measured by disc diffusion method and minimum inhibitory concentration (MIC) determination. Antioxidant properties of propolis essential oil were measured by both cell-free and cell-based experimental models:2,2-diphenyl-l-picrylhydrazyl (DPPH) assay, oxygen radical absorbance capacity (ORAC) assay and the assay to inhibit the H2O2-induced lysis of rabbit red blood cells (RBC). The effects on oxidative stress and anxiety-like behavior of propolis essential oil were evaluated with restraint stress animal model.
Results showed that:
1. Among the four procedures chosen for extraction, ultrasonic assisted extraction and microwave assisted extraction were suitable for the extraction of propolis essential oil. With these two methods, essential oil was of a high yield and quality.
2. The optimal extracting procedure for ultrasonic assisted extraction was as follows:at the extraction temperature of 59℃with 1:15 feed ratio and 62min of the extraction time, the essential oil yield reached 8.70% for Chinese propolis and 8.75% for Brazilian propolis.
3. The optimum technological parameters for microwave assisted extraction were obtained as:35℃of microwave extraction temperature,15min of microwave extraction time,1:10 of the solid to liquid ratio and 2 times of extraction. Under such optimal conditions, it was found that the essential oil content was 11.21% in Brazilian propolis and 9.73% in Chinese propolis.
4. Fifty-one components were identified from Brazilian propolis essential oil and the major components were terpenoids:Longifolene, a-eudesmol,β-eudesmol, P-Caryophyllene and Guaiol. Forty-seven components were identified from Chinese propolis essential oil and the major components were aromatic compound and terpenoids:Cinnamic acid,β-eudesmol, a-eudesmol, and y-eudesmol. Twenty-one compounds were found existing in both of Chinese and Brazilian propolis.
5. The essential oil from both Brazilian and Chinese propolis showed good to moderate antimicrobial activity against G-positive bacteria and fungi, while showed slight activity on G-negative bacteria. The essential oil from both Brazilian and Chinese propolis showed antioxidant activities in vitro. The IC50 of DPPH free radical were 178.3μg/ml and 309.5μg/ml respectively. The relative ORAC values of them were 0.82μMTrolox eq/g and 0.88μMTrolox eq/g, while the IC50 of H2O2 induced RBC hemolysis were 0.871 mg/ml and 0.879 mg/ml respectively.
6. Essential oil of propolis can significantly decrease MDA level and increase SOD activity in serum of restraint stress mice, ameliorate liver function by improving the oxidative stress status. It can also significantly increase SOD and CAT activity and the GSH level, and decrease MDA and NO level as well as XOD activity in liver tissue.
7. Propolis essential oil can significantly increase the Open Arm Entry percentage and Open Arm Time percentage, and decrease the protect Head-dipping percentage and protect rearing percentage of restraint stress mice in elevated plus-maze test, which showed no significant effects on the number of activities in spontaneous locomotor activity test. Propolis essential oil also depressed the serum level of CORT and ACTH, decreased the content of MDA and improved SOD and GSH in brain tissues of restraint stress mice.
Essential oil is an important component of propolis which contains great amounts of bioactive ingredients and possesses various biological activities. The present work provides additional data about the propolis essential oil obtained by microwave extration and reveals the interesting character of its chemical composition, antimicrobial activity and antioxidant acitvity in vitro, ameliorative effects on oxdative stress and anxiety. As propolis essential oil is a complex mixture of chemicals which may differ by plant sources, more investigations and further studies are necessary to elucidate its components and the exact site to achieve their potential anxiolytic effects.
引文
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