【作者】 付宇新；

【导师】 罗丽萍；

【作者基本信息】 南昌大学 ， 植物学， 2010， 硕士

【摘要】 蜂胶是蜜蜂从植物的幼芽或树干上采集的树脂、挥发油等分泌物,经过蜜蜂反复咀嚼加工,与自身腺体分泌物和一定比例的蜂蜡、花粉混合转化而成的一种具有芳香气味和黏性的天然固体胶状物。它是一类重要药理功能的天然产物,一直作为民间的传统医药来使用。研究表明,蜂胶具有抗菌、抗病毒、抗氧化、消炎及抗肿瘤等活性。蜂胶的化学组成多样,目前从蜂胶中鉴定到的化合物就有300多种,包括酚类、萜烯类、多糖、维生素等多种化合物。蜂胶挥发油约占原胶总组成的10%,且具有丰富的生物活性,但是目前对于蜂胶挥发油研究并不多。中国地理气候分布类型多样,胶源植物也十分广泛,为了更好的了解到蜂胶挥发油这一重要组分的特点,本文以中国不同地区收集到的蜂胶为对象,对蜂胶挥发油化学成分、生物活性(抗氧化、抑菌)方面做以下方面的研究。1.以内蒙古蜂胶为实验材料,采用传统水蒸气蒸馏法(HD)、同时蒸馏萃取法(SDE)和动态顶空进样(DHS)提取制备挥发性成分,结合GC-MS检测结果,了解内蒙古蜂胶的挥发性化学成分组成并获得其优化提取方法。对获得的蜂胶挥发性成分进行了分析,从HD所得样品中检测出了12种化合物,主要是3-甲基-2-丁烯-1-醇(26.81%),苯乙醇(17.16%)、桉叶油醇(14.53%),2-甲氧基-4-乙烯基苯酚(9.47%),α-没药醇(4.29%)等。从SDE法所得样品中检测出了40种化合物,主要是α-没药醇(20.19%),2-甲基-3-丁烯-2-醇(10.76%),3-甲基-2-丁烯-1-醇(8.28%),莫(5.21%)等。从DHS法所得样品中检测出了70种化合物,主要是十七烷(6.96%),菲(3.99%),芳姜黄酮(3.80%),1-(1-,5-二甲基-4-己烯基)-4-甲基-苯(3.41%),十八烷(3.23%),1-甲氧基-4-(1-丙烯基)-苯(2.59%),十六烷(2.47%)等。研究表明：DHS得到的挥发性物质种类最多,SDE次之,HD最少。DHS是用于蜂胶挥发物检测的最优方法,但不能获得供进一步生物活性检测的样品；如需获得生物活性检测样品,SDE是一个更好的方法。2.采用DHS结合GC-MS方法分析中国23个不同地理来源蜂胶挥发油化学成分,结果表明,不同地区蜂胶挥发油化学成分组成和含量上存在差异。DHS-GC-MS分析中,总共鉴定得到84种化学成分,倍半萜13个,单萜2个,酚醇19个,醛酮类12个,酸酯类12个,烃类12个,其他类14个。蜂胶挥发性成分主要为α-雪松烯(0.08-8.12μg/μL),γ-桉叶油醇(0.04-4.33μg/μL),3-甲基-3-丁烯-1-醇(0.42-24.74μg/μL),α-萜品醇(0.02-1.31μg/μL),乙酸(2.23-21.99μg/μL),苄醛(0.13-10.23μg/μL),苯甲醇(0.01-7.65μg/μL),雪松醇(0.03-0.90μg/μL),乙酸乙酯(0.10-4.64μg/μL),γ-杜松烯(0.05-2.58μg/μL),苯乙醇(0.02-5.60μg/μL),苯乙烯(0.06-5.81μg/μL)等。聚类和主成分分析,所有样品根据相似系数的异同程度,蜂胶可以大致分为三类,其中温带蜂胶和亚热带蜂胶被分在了不同的两种类别中,不同气候蜂胶挥发油的化学成分具有差异性,而不同颜色蜂胶之间的差异不明显。3.采用SDE-GC-MS从来自不同地区的25个蜂胶样本共鉴定出406种成分。其中单萜类30种,倍半萜类94种,酚醇类31种,醛酮类56种,酸酯类90种,烃类81种,其他24种,其中主要的成分为愈创木醇(0.56-13.61%),苯甲醛(0.07-8%),苯甲醇(0.08-17.67%),雪松醇(0.48-37.46%),γ-桉叶油醇(1.81-21.48%)，苯乙醇(0.03-14.43%),2-甲氧基-4-乙烯基苯酚(0.31-14.06%),姜黄烯(0.23-5.9%),苯甲酸苄酯(0.06-6.59%),苯乙酮(0.08-2.05%),3,4-二甲氧基苯乙烯(0.31-14.06%),壬醛(0.1-1.48%),α-雪松烯(0.05-1.98%),β-桉叶油醇(6.1-27.89%)等。聚类和主成分分析结果表明,来自于亚热带、热带地区和温带地区的蜂胶之间有一定的差别。而从蜂胶的颜色上来看,不能显示出各样品之间的差异。4.采用还原力测试方法和清除DPPH自由基活性对挥发油的抗氧化能力进行了分析。不同地区蜂胶挥发油还原力测定中,以山东蜂胶抗氧化能力最强(K=0.0649),广东、云南蜂胶较弱(分别为K=0.0086,K=0.0118和0.0102)。清除DPPH自由基活性中,不同地区蜂胶的抗氧化表现出不同的活性大小,其中山东蜂胶清除DPPH自由基活性最强,其清除自由基指数AAI为2.533,最弱的湖南永州蜂胶,AAI只有0.19。蜂胶地理气候类型和蜂胶颜色对蜂胶挥发油的抗氧化活性有密切的关系,并且以温带地区蜂胶和黄色蜂胶的还原力和清除自由基活性较强。5.采用滤纸片扩散法和连续稀释法,以大肠杆菌、金黄色葡萄球菌、枯草杆菌、产气杆菌、变形杆菌、青霉、曲霉、毛霉及根霉等九种菌作为受试菌,分别测定蜂胶挥发油抗菌活性和最小抑菌浓度(MIC),结果表明,蜂胶挥发油对细菌和真菌都有一定程度的抑制作用,但抑制活性强弱不同。各地蜂胶挥发油的抑菌活性存在不同水平的差异。通过对蜂胶挥发油的化学成分、抗氧化活性和抗菌活性研究结果来看,不同蜂胶样品之间都或多或少存在着差异,这主要与蜂胶提取分析方法、气候和胶源植物不同有关。实验证明了中国蜂胶具有较为丰富的挥发性成分,并且有一定程度的抗氧化和抑菌活性,可以考虑作为一种天然的抗氧化和抑菌剂,推广应用到医药化工和食品工业等领域中。更多还原

【Abstract】 Propolis (bee glue) is a resinous or sometimes wax-like bee hive product collected by honeybees from buds and leaves of plants, mixed with pollen as well as enzymes secreted by bees. It has been used by man since ancient for its pharmaceutical properties. It is still used as remedy in folk medicine. Research shows it possesses antiseptic, antibacterial, antifungal, antiinflammatory, hepatoprotective and antitumor et al. properties. The chemical constituent of propolis is very complex, more than 300 compounds had been detected in it, such as polyphenols, terpenoids, steroids, sugars and amino acids, et al.. Among them, the volatile compounds are great important due to their potent biological activities. In general, crude propolis contains 10% volatile oils and their chemical constituents are relatively complex, too. But there is little literature about volatile of Chinese propolis.China has diverse vegetation and original plants of propolis. So the chemical constituents of Chinese propolis are complex and different with propolis from other place. To understand the properties of Chinese propolis and analysis their chemical compositions, antioxidant and antimicrobial activity systematically, it is meaningful to collected propolis from different areas of China and study them.1 To study the volatile components in propolis collected in Inner Mongolia and get the optimized sampling method, we extracted the volatile components in the propolis by three methods, traditional hydrodistillation (HD), simultaneous distillation extraction (SDE) and dynamic headspace sampling (DHS), and analyzed the components by GC/MS. By searching the database, we got high similarity results with the reported compounds. The relative content of the components were calculated by peak area normalization method.12 kinds of volatile components can be measured by HD-GC/MS. The major components are 3-methyl-2-buten-l-ol (26.81%), phenylethyl alcohol (17.06%), eudesmol (14.53%),2-methoxy-4-vinylphenol (9.47%), a-bisabolol (4.29%).40 kinds of volatile components are identified by SDE-GC/MS. The main of them are a-bisabolol (20.19%), 2-methyl-3-buten-2-ol(10.76%),3-methyl-2-buten-l-ol (8.28%), azulene (5.21).70 kinds of volatile components are identified by DHS-GC/MS. The main of them are heptadecane (6.96%), phennathrene (3.99%), ar-tumerone (3.80%), 1-(1,5-dimethyl-4-hexenyl)-4-methyl-benzene (3.14%), octadecane (3.23%), 1-(1,5-dimethyl-4-hexenyl)-4-methyl-benzene (3.14%), hexadecane (2.47%). Among the three methods, the most volatile components can be detected by DHS. It is the best way to analytic, but the volatile components can not be obtained for further biological activity experiment. For that reason, SDE is the best way.2. The volatile components of 23 propolis samples from 17 provinces of China were analyzed by dynamic headspace sampling with gas chromatography and mass spectrometry (DHS-GC-MS). Eighty-four volatile components were identified from the propolis samples, includes sesquiterpenoids (13), monoterpenoids (2), phenolic and alcohols (19), aldehydes and ketones (12), organic acids and esters (12), hydrocarbons(12) and various other substances(14). The major components wereα-cedrene (0.08-8.12μg/μL),γ-eudesmol (0.04-4.33μg/μL),3-methyl-3-buten-l-ol (0.42-24.74μg/μL),α-terpineol (0.02-1.31μg/μL), acetic acid (2.23-21.99μg/μL), benzyl dehyde(0.13-10.23μg/μL), benzyl alcohol (0.01-7.65μg/μL), cedrol (0.03-0.90μg/μL), butanoic acid, ethyl ester (0.10-4.64μg/μL), y-cadinene (0.05-2.58μg/μL), phenylethyl alcohol (0.02-5.60μg/μL) and styrene (0.06-5.81μg/μL), et al.. The samples were subjected to principal component analysis (PCA) and hierarchical cluster analysis (CA) according to the composition corresponding essential oils in order to model their pattern. Different significant relationship was found between volatile composition and climatic characteristics and colors of the 23 populations, differences in the subtropical and temperate zones of samples from different samples, but nearly no significant relationship on volatile composition and propolis colors.3. The volatile components of 25 propolis samples from 18 provinces of China were analyzed by steam-distillation extraction with gas chromatography and mass spectrometry (SDE-GC-MS).406 volatile components were identified from the propolis samples, includes sesquiterpenoids (94), monoterpenoids (30), phenolic and alcohols (31), aldehydes and ketones (56), organic acids and esters (90), hydrocarbons (81) and various other substances (24). The major components were guaiol (0.56-13.61%), benzaldehyde (0.07-8%), benzyl alcohol (0.08-17.67%), cedrol (0.48-37.46%),γ-eudesmol (1.81-21.48%), benzeneethanol (0.03-14.43%), 2-methoxy-4-vinylphenol(0.31-14.06%), a-curcumene (0.23-5.9%), benzyl benzoate (0.06-6.59%), acetophenone (0.08-2.05%),4-ethenyl-1,2-dimethoxy-benzene (0.31-14.06%), nonanal (0.1-1.48%), a-cedrene (0.05-1.98%),β-eudesmol (6.1-27.89%). The samples were subjected to principal component analysis (PCA) and hierarchical cluster analysis (CA) according to the composition corresponding essential oils in order to model their pattern. The results obtanined, differences in the tropical, subtropical and temperate zones of samples from different samples, but maybe have no significant relationship about propolis colors.4. Reducing power was utilized to examined the total antioxidant activity of propolis extracts, and DPPH (1,1-diphenyl-2-picrylhydrazyl) was used to determined the free radical scavenging ability. Antioxidant activity varied with different samples. Reducing power of all samples are from 0.0102 to 0.0649. AAI (Antioxidant activity index) are from 0.19 to 2.533. Shandong propolis exhibited greatest reducing power and strongest free radical scavenging effect. All samples have been compared by the climatic characteristics and propolis colors, temperate zones or yellow propolis have stronger reducing power and free radical scavenging effect.5. The agar diffusion method using filter paper disks was employed. Antibacterial activity was measured by measure diameters of inhibition zones. The minimum inhibitory concentration (MIC) values obtained using the microdilution broth susceptibility assay. Propolis samples exhibited significant antibacterial and antifungal activities, affecting a wider apectrum of microorganisms and presenting lower or no activity against several bacterial and fungal.In contrast, the propolis samples are different in type and content of the chemical compositions. The differences manifest suggest to the volatile chemical composition in relation to different climates and original plants. The experiment demonstrated that the content of volatile oils in Chinese propolis was abundance, and it showed to process antioxidant activity and antimicrobial activity. It could been predicted that propolis is useful as a natural antioxidant and antimicrobial agents in medicine, chemical engineering and food industry.更多还原