【作者】 赵强；

【导师】 张彬；

【作者基本信息】 南昌大学 ， 粮食、油脂与植物蛋白工程， 2007， 硕士

【摘要】 蜂胶（propolis）是由蜜蜂的工蜂将采自杨树、柳树、松树等植物的幼芽和愈伤组织分泌的树脂状物质与自身的消化腺和蜡腺等腺体的分泌物混合之后形成的一种具有黏性的天然混合物。一般来说,蜂胶的基本成分为:50%的树脂,30%的蜂蜡,10%的挥发物和芳香油,5%的花粉,以及5%的其它物质。近年来,研究发现蜂胶具有多种较强的药理活性,如抗菌、消炎、抗真菌、抗病毒、抗原虫、抗滤过性病原体、增强免疫调节、抗肿瘤、抗增殖、抗氧化和清除自由基、抗辐射、对心脏的保护作用、局部麻醉镇痛作用、降血脂等。蜂胶挥发油,是蜂胶的重要有效活性组份之一,约为蜂胶的百分之十。目前,研究已发现蜂胶挥发油具有抗菌和抗脂质过氧化等功能。本研究主要对蜂胶挥发油的提取、分离、理化性质及成分鉴定、抗氧化活性、抗菌活性进行了研究,其研究结果可为蜂胶挥发油作为化妆品或是药物的开发与利用提供一定的实验数据。本文主要研究结果归纳如下。采用传统与现代提取技术对蜂胶挥发油的提取进行了研究,并采用GC/MS对相应蜂胶挥发油的成分进行分析。通过比较水蒸汽蒸馏法、常温溶剂法、超声波辅助提取法、微波辅助提取法、超临界CO₂流体萃取法的提取工艺,发现各类方法对蜂胶挥发油的提取率由高到低依次为:微波辅助提取法>超声辅助提取法>常温溶剂法>超临界CO₂萃取法>水蒸汽蒸馏法,微波辅助萃取法最佳工艺条件为:萃取温度30℃,液料比为11,萃取时间32min,蜂胶挥发油的得率达13.83%,其提取率是溶剂提取法的1.51倍,并且时间大大缩短为其2/45。比较萜烯类及其酯类成分的相对含量可以看出,水蒸汽蒸馏法提取的蜂胶挥发油萜烯类及其酯类有效成分的相对含量最高,达到80.1%,主要成分为α-桉叶油醇（30.28%）、γ-桉叶油醇（10.99%）、异麦芽酚（9.25%）、正二十烷（8.76%）、α-没药醇（7.77%）、愈创木醇（6.15%）、顺-14-二十九烯（3.98%）桉叶烷-3,7（11）-二烯（3.45%）、二十九烷（3.21%）、长叶醛（1.90%）、菖蒲烯（1.74%）、姜黄烯（1.68%）、雪松醇（1.51%）、匙叶桉油烯醇（斯巴醇）（1.51%）等;其次是超临界CO₂提取法提取的蜂胶挥发油,萜烯类及其酯类成分相对含量为26.43%,其中主要的活性成分有α-桉叶油醇（6.89%）、羊毛甾醇醋酸酯（5.31%）、γ-桉叶油醇（2.97%）、羽扇豆醇（2.55%）。采用乙醇溶解法和系统溶剂法对蜂胶渣中的挥发油组分进行了分离。乙醇溶解法使萜烯类及其酯类成分的相对含量由1.18%提高到9.57%;系统溶剂法可以有效地分离得到相对含量为38.71%的挥发油,同时得到相对含量为45.26%的含有四种黄酮成分的组分,还分离出相对含量为51.02%的油酸。采用国标和药典的方法对蜂胶挥发油13项理化指标的进行了初步鉴定。采用DPPH法和化学发光法对蜂胶挥发油的抗氧化活性进行了研究,并采用三氯化铝分光光度法和福林酚法分别测定了蜂胶挥发油的总黄酮含量、总酚含量,探讨了蜂胶挥发油抗氧化的作用机理。结果表明,五种方法提取的蜂胶挥发油都具有清除DPPH自由基、抑制超氧自由基和羟基自由基的的抗氧化活性,表现为中等强度抗氧化活性,总体较蜂胶醇提物、水提物、超临界CO₂萃取物弱;超临界CO₂萃取的蜂胶挥发油具有很强的抗氧化活性,清除DPPH自由基IC₅₀约为50μg/mL,清除超氧阴离子自由基的IC₅₀约为0.75mg/mL,清除羟基自由基的IC₅₀约为1.3mg/mL;蜂胶清除DPPH的作用机理与其中的成分有关,具有抗氧化活性的物质可以分为超临界CO₂萃取的蜂胶挥发油中的萜烯类、蜂胶乙醇提取物中的黄酮及总酚、蜂胶水提物中的几种含量较低的水溶性成分四类;蜂胶挥发油清除·O₂^-和·OH的作用机制,可能主要是与其中的黄酮类成分有关。采用滤纸片法和试管连续稀释法对蜂胶挥发油的抗菌活性进行了比较研究,并对其作用机理做了探讨。结果表明,超临界CO₂萃取的蜂胶挥发油的抗菌活性与蜂胶超临界CO₂萃取物相当,两者均强于蜂胶醇提物;常温溶剂法、超声波辅助提取法及超临界CO₂流体萃取法提取的蜂胶挥发油对黑曲霉有抑制作用,并且其效果均强于蜂胶醇提物;蜂胶挥发油样品对大肠埃希氏杆菌均有抑制作用,水蒸汽蒸馏法和超临界CO₂流体萃取法提取的蜂胶挥发油对大肠埃希氏杆菌、枯草芽孢杆菌、金黄色葡萄球菌、普通变形杆菌MIC为0.0391～0.625mg/mL;超临界CO₂提取的蜂胶挥发油对大肠杆菌、金黄色葡萄球菌的作用机制可能分为以下三种情形,分别为提取方式的影响、酚类物质主要是黄酮类化合物与萜烯类化合物的协同作用、挥发油对细菌及真菌的作用方式。更多还原

【Abstract】 Propolis is a resinous product collected by honeybees （Apis mellifera） from leaf buds and cracks of various plant sources, mainly from poplar（Populus）genus and, to a lesser extent, beech, horsechestnut, birch and conifer trees, mixed by bees with beeswax to form a sealing material in their honeycombs, smooth out the internal walls, and protect the entrance against intruders. In general, it is composed of 50% resin and vegetable balsam, 30% wax, 10% essential and aromatic oils, 5% pollen, and 5% various other substances, including organic debris. Recently, Some of the considerable biological activities of propolis extracts were reported, such as antibacterial, anti-inflammatory, antifungal, antitoxin, antiprotozoan, antiviral, Imunostimulant, anticancer, antiproliferative, antioxidative, scavenging free radicals, antiradiation, cardioprotective effect, anaesthetic, and antilipemic, et al. 10% Essential oils of Propolis, which is one of the mostly effective active components of propolis, it had occasionally been reported to possess antimicrobial activity and antilipid peroxidative action. The thesis focused on the extraction, separation, identification of physical and chemical properties and chemical compositions, and, antimicrobial activity and antioxidant activity were investigated, which will provide experimental datas for developing and applying essential oils of propolis as a resource of cosmetic or medicine. The main results and conclusions are as follows:The extract methods of essential oils from propolis were studied with the traditional and modern technologies, and chemical compositions were assessed by GC/MS. The results of different methods, such as hydrodistillation extraction （H）, petroleum ether solvent extraction （P）, ultrasound-assisted extraction （U）, microwave-assisted extraction （M）, and supercritical CO₂ extraction （S） showed that the order of extract rates was as follow: M>U>P>S>H. The optimal conditions for essential oils by microwave-assisted extraction can be concluded as follows: 32 min at 30℃, the ratio of solvent to material is 11 by using petroleum ether as the solvent, and the yield of extract was 13.83%, at these conditions, compared with petroleum ether solvent extraction, microwave-assisted extraction needs only 2/45 of the time, and achieves 1.51 times yield for extraction of essential oils from propolis. The relative content of terpene and ester in essential oils of propolis extracted by hydrodistillation （HDE） is the highest, the relative content of terpene and ester reaches 80.1%, and the main constituents of HDE areα-Eudesmol（30.28%）,γ-Eudesmol（10.99%）, Isomaltol（9.25%）, Eicosane（8.76%）,α-Bisabolol（7.77%）, Guaiol （6.15%）, Z-14-Nonacosane （3.98%）, Eudesma-3,7（11）-diene （3.45%）, Nonacosane （3.21%）, Longifolenaldehyde （1.90%）, （-）-Calamenene （1.74%）, Curcumene （1.68%）, Cedrol （1.51%）, 2H-2, 4a-Ethanonaphthalen-8（5H）-one, hexahydro-2, 5, 5-trimethyl-（1.51%）, etc. Then, the relative content of terpene and ester in essential oils of propolis extracted by supercritical CO₂ extraction（SE） achieves 26.43%, the main active constituents of SE areα-Eudesmol（6.89%）, Lanosta-8,24-dien-3.beta.-ol, acetate （5.31%）,γ-Eudesmol（2.97%）, Lupeol（2.55%）, etc.The ethanol dissolution method and systematic solvent extraction method were adopted to separate the active compositions of essential oils from propolis residue. The results of dissolution method showed that the main active compositions such as terpene and ester were accumulated with a relative content from 1.18% to 9.57%; the results of systematic solvent extraction method showed that the method was effective, terpene and ester, 4 kinds of flavanoids, and Oleic Acid with a relative content of 38.71%, 45.26%, 51.02% in three separated samples.The methods from national standard and prc codex for identification of physical and chemical properties of essential oils from propolis were adopted, and 13 kinds of index were determinated.The free radical scavenging activity of essential oils of propolis were examined by comparing to those of known antioxidants such as BHT, VE, and ethanol extract（EEP）, water extract（WEP）, supercritical CO₂ extract（SEP） of Propolis, using the method of DPPH and chemiluminescence; Total flavonoid contents in extract of propolis were determined by the method of aluminum chloride colorimetry, Total polyphenol contents in extract of propolis were determined by the Folin-Ciocalteau colorimetry method; Antioxidation mechanism of propolis also had been discussed. The results showed that five different extract of essential oils from propolis all had certain antioxidant activity, but weaker than EEP, WEP, and SEP, Generally speaking. The research also obtained a discovery that SE showed strong potential antioxidant activity, the 50% inhibitory concentration of SE to DPPH radical, super oxygen-anion radical and hydroxyl radical were 50μg/mL, 0.75mg/mL and 1.3mg/mL. propolis extracts had relatively strong DPPH radical scavenging activity were correlated with the 4 parts of chemical compositions, such as terpene and ester in essential oils of propolis, total polyphenol and flavonoid contents two part main in EEP, and several water-soluble chemical compositions with low content in WEP; Inhibition activity of propolis extract on super oxygen-anion radical and hydroxyl radical were mainly correlated with total flavonoid contents.The antimicrobial activity of essential oils of propolis was determinated by paper-disc method and tube dilution method, and it mechanism also had been discussed. The results showed that the essential oil displayed a broad antimicrobial spectrum, the antimicrobial activity of SE and SEP both were stronger than EEP, and essential oils of propolis extracted by P, U, and SE exerted a much stronger antimicrobial effect against Aspergillus niger than EEP. Unexpected or expected, all the essential oils of propolis had effect on Escherichia coli, and the MIC of HDE and SE for Escherichia coli, Bacillus subtilis, Proteus vulgaris, and Staphylococcus aureus indicated were 0.0391-0.625mg/mL. Aiming at Escherichia coli and Staphylococcus aureus, the strong antimicrobial activity of SE could be due to three aspects, which were exteaction method, synergistic action of polyphenol especially flavonoid and terpene and ester, the last one, mode of action, maybe. Further researchs of mechanism are expected.更多还原