【作者】 徐元君；

【导师】 罗丽萍；

【作者基本信息】 南昌大学 ， 植物学， 2010， 硕士

【摘要】 近年来由于蜂胶在医药学中的广泛应用而受到很高的关注,它具有抗氧化、抑菌、消炎、抗肿瘤等生物活性,而这些生物活性与其复杂的化学成分有密切关系,因此对其化学成分及生物活性的研究具有很高的科学价值。中国地域广大,跨越温带、亚热带、热带地区,各地蜂胶性状不同,而季节、地理位置对蜂胶品质的影响一直是蜂胶研究的重点,因此本实验研究了季节、地理位置对中国蜂胶醇提物的影响。本论文重点研究了中国不同季节的热带、亚热带和温带蜂胶醇提物(Ethanol Extraction of Propolis,以下简写为EEP)的化学成分、抗氧化活性及对9种常见菌的抑菌性,以期对中国蜂胶的影响因子做初步了解,为中国蜂胶的研究奠定基础。化学成分的研究采用分光光度法测定其色度、特定吸收率(E1%1cm)、总酚、总黄酮、黄酮-黄酮醇和黄烷酮的含量以及HPLC分析EEP的液相色谱图相似度。抗氧化活性的测定包括普鲁士兰法测定EEP总抗氧化能力；以1,1-二苯基-2-苦基肼(2,2-Diphenyl-l-picrylhydrazyl, DPPH)为清除对象,考查EEP清除自由基活性。抑菌性包括对3个革兰氏阳性菌：金黄色葡萄球菌(Staphilococcus aureus)、枯草杆菌(Bacillus subtilis)、产气杆菌(Clostridium perfringens);2个革兰氏阴性菌：大肠杆菌(Escherichia coli)、变形杆菌(proteusbacillus vulgaris);4个真菌：青霉(Penicillium glaucum)、曲霉(Aspergillus niger)、毛霉(Mucor racemosus)、根霉(Rhizopus nigricans)的抑菌圈以及最小抑菌浓度的研究。本论文还研究了中国不同地区29个蜂胶样本的EEP对9种常见菌的抑菌性,具体包括测定EEP对这些菌种的抑菌圈和最小抑菌浓度,不仅丰富了课题组的研究内容,也为较为全面的研究不同地区中国蜂胶提供了理论依据。研究结果如下。1.季节对中国EEP化学成分的影响较显著,总酚、总黄酮、黄酮-黄酮醇和黄烷酮的含量均是在7-8月达到峰值,而9月份各化学成分含量大多最低。其中不同月份间总酚和总黄酮含量差异极显著,总酚最大值为243.87±152.13 mg/g最小值为187.25±146.45 mg/g；总黄酮最大值为189.34±107.51 mg/g,最小值为122±81.02 mg/g；而黄酮-黄酮醇和黄烷酮的含量均值随月份变化不大。地理位置对中国EEP化学成分的影响显著,特别是热带地区EEP与亚热带和温带地区EEP总酚、总黄酮和黄酮-黄酮醇的含量差别巨大。其中总酚、总黄酮含量差异极显著,热带EEP的总酚和总黄酮含量是亚热带和温带地区EEP的1/6-1/4。不同地理位置EEP的E1%1cm值均显示与总黄酮含量相关性显著。2.通过测定中国不同季节不同地理位置EEP的总抗氧化能力和清除DPPH自由基活性发现,地理位置对中国EEP的抗氧化活性影响显著,不同地理位置EEP的抗氧化活性差异显著,热带EEP的总抗氧化能力最弱,在0.32～0.51之间,亚热带EEP的总抗氧化能力在1.33～1.48之间,温带EEP的总抗氧化能力最强,在2.28～2.62之间；地理位置对EEP清除DPPH自由基活性和对总抗氧化能力的影响趋势是一样的,热带EEP的AAI基本都小于0.5,其清除DPPH自由基活性最弱,亚热带EEP的AAI在1.87～2.47之间,其清除DPPH自由基活性强,温带EEP的AAI在4.61～6.29之间,其清除DPPH自由基活性非常强。说明中国蜂胶中温带蜂胶的抗氧化活性较好,亚热带蜂胶次之,而热带蜂胶的抗氧化活性最弱。季节对中国EEP抗氧化活性影响也显著,同时EEP的E1%1cm值与清除DPPH自由基活性和总酚或者总黄酮的含量相关性显著。3.通过测定中国不同季节不同地理位置EEP的抑菌圈和最小抑菌浓度来研究季节和地理位置对中国EEP抑菌性的影响。不同季节不同地理位置EEP对9个菌种的抑菌圈直径和最小抑菌浓度结果负相关显著,即抑菌圈越大,最小抑菌浓度越小,而代表的抑菌性也越大。同一地理位置不同季节EEP的不同月份抑菌圈直径进行显著性分析结果显示大多数并没有显著性差异,即抑菌性没有显著性差异,因此季节对中国EEP的抑菌性没有显著影响。地理位置对中国EEP对革兰氏阳性菌的抑菌性的影响显著,温带EEP抑菌性最强,亚热带EEP次之,而热带EEP最弱,这与EEP的化学成分含量的趋势相似,但是将抑菌性与EEP各主要化学成分和抗氧化活性进行相关性分析,并没有得到统一的规律。地理位置对中国EEP对革兰氏阴性菌和真菌的抑菌性的影响不显著,但是热带EEP对真菌的抑菌性明显好于其他菌种,与亚热带和温带EEP几乎没有区别。4.采用HPLC法对中国不同季节不同地理位置EEP的化学成分进行分析,色谱图的相似度结果表明热带EEP的化学成分与其他地区显著不同,而亚热带和温带EEP的化学成分种类类似,季节对热带EEP的化学成分的影响明显,但对其他地区EEP的化学成分影响不明显。5.为了解中国不同地区蜂胶的抑菌性,我们测定了中国不同地区29个EEP的抑菌圈和最小抑菌浓度,并根据中国的气候区域对蜂胶产地划分,结果显示中国不同地区EEP对革兰氏阳性菌、革兰氏阴性菌和真菌都具有较好的抑菌性,但气候区域对EEP的抑菌性没有显著影响。EEP对革兰氏阳性菌的平均抑菌圈直径为9.88±1.47 mm,对革兰氏阴性菌的平均抑菌圈直径8.75±1.56 mm,对真菌的平均抑菌圈直径为10.01±0.65 mm,可见中国不同地区EEP对真菌的抑菌性最强,其次为革兰氏阳性菌,对革兰氏阴性菌的抑菌性最弱。在这9个菌种中,对变形杆菌的抑菌性最差,对根霉的抑菌性最好。更多还原

【Abstract】 Propolis has been attracted much attention in recent years as an useful substance applied in medicine, it possessed various biological activities, such as antioxidant, antibacterial, antiinflammatory, antitumor and so on. And these activities were strongly related to the complex chemical composition, thus it had high value to investigate the chemical composition and biological activities of propolis to the academic field. On account of China comprehended large areas, which crossed from tropical and sub-tropical to temperate zones, the characteristics of propolis were extremely diverse. The influences of the season and geographic area to propolis were always the emphasis on propolis research, thus we worked on the influences of the season and geographic area to Chinese propolis.Our work was focused on the investigation of the Chemical composition, antioxidant activity and antimicrobial activity against 9 microbe strains of Chinese different season propolis from tropical, sub-tropical and temperate zones, for the purpose of getting the message of the influence factors on Chinese propolis and establishing foundation of Chinese propolis researches. Spectrophotography was used to determine color intensity, E1cm 1% , total phenol, total flavonoids, flavone-flavonol and flavanone of ethanolic extract of propolis (EEP), HPLC method was used and then we analyzed the similarity of EEP; prussian blue spectrophotometry was used to examine total antioxidant activity, and DPPH was utilized to evalue its free radical scavenging activity. Antimicrobial activity was tested against three Gram positive bacterias (Staphilococcus aureu, Bacillus subtilis, Clostridium perfringens), two Gram negative bacterias (Escherichia col, proteusbacillus vulgaris) and four fungus cultures (Penicillium glaucum, Aspergillus niger, Mucor racemosu, Rhizopus nigricans), and was investigated by the determination of inhibition zones and MIC of EEP.Antimicrobial activities of EEP of 29 propolis from 20 provinces of China were also researched by the means of determining the inhibition zones and MIC of EEP. It would helpful for deeply and widely analyzing Chinese propolis.Results were as followed.1. There was significant difference with regards to the seasonal effect on the chemical components of EEP, the contents of total phenol, total flavonoids, flavone-flavonol and flavanone reached maximum percentage in June and July, minimum in September, moreover, the disparity in the contents of total phenol and total flavonoids of EEP from different months was strongly significant. The maximum value of total phenol and total flavonoids contents were 243.87±152.13 mg/g and 189.34±107.51 mg/g respectively, the minimum of them were 187.25±146.45 mg/g and 122±81.02 mg/g respectively. The content of flavone-flavonol and flavanone seemed to be change a little with the change of season.The geographic area significantly influenced the chemical components of EEP, too. The contents of total phenol, total flavonoids and flavone-flavonol of tropical zone propolis were significant different with the contents of sub-tropical and temperate zones, especially total phenol and total flavonoids contents, the contents of them in tropical zone propolis were 1/6～1/4 of them in sub-tropical and temperate zones. The correlation between E1cm 1% and total flavonoids was significant.2. Prussian blue spectrophotometry was utilized to examine the total antioxidant activity of EEP from different seasons and areas, and DPPH was used to determine the free radical scavenging ability. The results showed that the geographic area significantly influenced the antioxidant activity. The total antioxidant activity of tropical zone propolis was the weakest, it ranged from 0.32～0.51, sub-tropical zone propolis had strong total antioxidant activity, and ranged from 1.33～1.48, and temperate zones propolis had the best total antioxidant activity, ranged from 2.28～2.62, the free radical scavenging activity of EEP had the same tendency with the total antioxidant activity, tropical zone propolis had the weakest free radical scavenging activity, AAI values were less than 0.5, and AAI value of sub-tropical zone propolis ranged from 1.87～2.47, and temperate zones propolis had the best free radical scavenging activity, AAI value ranged from 4.61-6.29. In a word, that the propolis from the tropical zone of China had the weakest antioxidant activity, the antioxidant activity of sub-tropical zone propolis of China was better than the tropical zone, and temperate zones propolis had the best antioxidant activity. There was also significant difference with regards to the seasonal effect on the antioxidant activity, moreover, the correlation among E1cm 1% , total flavonoids, flavone-flavonol and the free radical scavenging activity were significant.3. The antimicrobial activities against 9 microbe strains of EEP from different seasons and geographic areas were researched by the means of determining of inhibition zones and MIC of EEP. The results of the associativity between the inhibition zones and MIC of EEP showed strong negative correlation between them, which means the bigger inhibition zones, the smaller MIC, and betters the antimicrobial activity. Then we analyzed the influence of season on the antimicrobial activity, and found there had no different among the antimicrobial activity of different seasons propolis.The geographic area significantly influenced the antimicrobial activity against Gram positive, the antimicrobial activity against Gram positive bacteria of temperate zone EEP was the best, sub-tropical zone EEP was weaker than the temperate zone and tropical zones EEP had the weakest antimicrobial activity, it had the same tendency with the contents of chemical components, but the associativity between the antimicrobial activity and the chemical components contents is not good. The geographic area didn’t significantly influenced the antimicrobial activity against Gram negative bacteria and fungus culture, but fungus culture were shown to be more sensitive to tropical zone EEP than Gram positive bacteria and Gram negative bacteria, the antimicrobial activity of it was almost equal with the antimicrobial activity against fungus culture of sub-tropical zone and temperate zone EEP.4. The chemical composition of EEP from different seasons and geographic areas were detected by HPLC. The similarity was analyzed, and we found that the chemical composition of tropical zone EEP was much different with the other area EEP, the sub-tropical zone EEP had the similar chemical composition with temperate zone EEP, the season significantly influenced the chemical composition of tropical zone EEP, but not for the sub-tropical zone and temperate zone EEP.5. The antimicrobial activities of EEP of 29 propolis from 20 provinces of China were also researched by the means of determining the inhibition zones and MIC of EEP, we classified the propolis according to the climate areas, the results showed that fungus culture, Gram positive bacteria and Gram negative bacteria were all shown to be sensitive to Chinese EEP, but the climate area didn’t significantly influenced the antimicrobial activity. The average of the inhibition zones against Gram positive bacteria of EEP was 9.88±1.47 mm, The average of the inhibition zones against Gram negative bacteria was 8.75±1.56 mm, and the average of the inhibition zones against the fungus culture was 10.01±0.65 mm, so the antimicrobial activities of Chinese EEP against fungus culture was best, and weakest was against Gram negative bacteria. Moreover, EEP had the weakest antimicrobial activity against proteusbacillus vulgaris, and had the best antimicrobial activity against Rhizopus nigricans in 9 microbe strains.更多还原