棘豆波丝菌发酵产物化学成分及药理作用的研究
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摘要
植物内生真菌是指那些在其生活史中的某一段时期生活在植物组织内,对植物组织没有引起明显病害症状的真菌。是一类具有丰富多样性的微生物,他们能够产生具有生物活性的次级代谢产物。因此,植物内生真菌具有作为新型药物来源的巨大潜力而备受关注。本文选取甘肃棘豆的一种内生真—棘豆波丝菌(Undifilum oxytropis)作为研究材料,通过液体深层发酵培养获得的发酵产物,并对其活性成分的提取、药理活性及化学成分进行了初步的研究。实验结果如下：
1.本实验以ITS1和ITS4为引物,对研究的实验菌种菌丝体rDNA进行了PCR扩增,并对扩增产物进行测序,结果表明：该菌与Undifilum oxytropis同源相似性达到99%,在显微镜观察下,新生的菌落、菌丝、孢子的形态与文献报道一致,所以认为所转接到新的培养基上生长的菌种为波丝菌属真菌,即为棘豆波丝菌(Undifilum oxytropis)
2.本实验对棘豆波丝菌发酵产物化学成分进行了系统的预实验,初步确定了发酵产物菌丝体中主要含有生物碱、黄酮、蒽醌、多糖、皂苷、挥发油与酚类等物质,发酵液中含有多糖、生物碱、黄酮物质,与宿主植物甘肃棘豆所含成分基本相同,说明内生菌具有产生和宿主植物相同或相似的化学成分。
3.本文以总还原力、羟自由基抑制活性、超氧阴离子自由的清除、亚硝酸根离子的清除数据为评价指标,探明棘豆波丝菌次级代谢产物多糖的抗氧化作用。研究结果表明：在一定的浓度范围内,棘豆波丝菌多糖(胞内、胞外)对总还原力和羟自由基、超氧阴离子、亚硝酸根离子的清除率均随着浓度的增加而增大,呈现一种量效关系。其中,胞内多糖在抗氧化指标中表现最好。在总还原力的测定中,当浓度达到300ug/ml时,胞内多糖产生的光吸收值达到最大为1.594。浓度达到1.6mg/ml时,对羟自由基、超氧阴离子、亚硝酸根离子清除率分别为86.3%、86.2%、81.2%,与Vc清除率相比略低。
4.本实验对棘豆波丝菌发酵产物乙醇提取物极性部位的抗氧化作用进行了初步研究。结果表明,在一定的供试浓度范围内,棘豆波丝菌发酵产物乙醇提取物极性部位对总还原力的影响、对羟基自由基·OH、超氧阴离子O2·和亚硝酸根离子NO2-的清除均随着浓度的增加而增大,并表现出一定的抗氧化活性。其中,在羟基自由基！OH和亚硝酸根离子NO2-的清除过程中,菌丝体各极性萃取部位强于发酵液,当c=1.6mg/ml时,菌丝体氯仿萃取部位对羟基自由基·OH和亚硝酸根离子N02-清除率最大为86.7%、68.7%；在超氧阴离子O2-清除过程中,发酵液各极性萃取部位强于菌丝体,浓度为1.6mg/ml时,发酵液乙酸乙酯萃取部位清除率最大为74.1%。
5.本实验对棘豆波丝菌发酵产物乙醇提取物各极性进行了抑菌活性部位的筛选,选取了菌丝体和发酵液乙醇提取物氯仿、乙酸乙酯、正丁醇萃取部分作为供试品,以金黄色葡萄球菌、肠炎沙门氏菌、大肠杆菌、鼠伤寒沙门氏菌四种细菌为指示菌,以硫酸链霉素作为阳性对照,采用滤纸片扩散法进行体外抑菌实验。研究结果表明：菌丝体和发酵液氯仿萃取物、菌丝体乙酸乙酯萃取物表现出了一定的抑制作用。其中,发酵液氯仿萃取物对大肠杆菌和金黄色葡萄球菌的抑制作用较好,抑菌直径可达1.62cm、1.73cm,抑菌率分别为68.2%,61.4%；菌丝体氯仿萃取物对金黄色葡萄球菌、肠炎沙门氏菌、鼠伤寒沙门氏菌均有较好的抑制作用,抑菌率分别为65.4%、59.5%、60.3%；菌丝体乙酸乙酯萃取物表现最好,对4种指示菌均有较高的抑制活性,其中,对大肠杆菌抑菌效果最佳,抑菌率高达为89.6%。
6.本试验研究了棘豆波丝菌胞内多糖和乙醇提取物对小白鼠肉瘤S180的抑制作用,小白鼠皮下接种S180肉瘤后,经胃灌服胞内多糖和乙醇提取物,连续10天,试验结果表明：胞内多糖高、中、低剂量组对S180抑制率分别为43.91%、53.12%、35.42%,乙醇提取物高、中、低剂量组对S180抑制率分别为53.87%、48.90%、33.42%,与阳性药对照相比具有一定的显著性差异。
7.本实验以硅胶柱层析、薄层色谱和GC-MS分析为手段,对棘豆波丝菌乙醇提取石油醚萃取物进行了化学成分的初步研究,通过GC-MS分析,显示了22种化合物,确认了8种化合物,其中,烷烃4种,烯酸2种,酯2种。
Endophytic fungi are living in plants and have no significantly negative effects on the host plants. They have rich diversity of microorganism groups, and they can produce secondary metabolites with biological activity. Therefore, endophytic fungi are concerned as a source of new medicines due to their tremendous potential.In this study we selected Undifilum oxytropis (Q. Wang, Nagao & Kakish) as the research material, and obtained fermented products through the liquid deep fermentation. Then we had preliminary study the extractions, pharmacological activities and chemical constitutions were preliminarily studied.. The results were as follows:
1. In this study, ITS1 and ITS4 were selected as primers.The rDNA form Mycelium of Undifilum oxytropis were PCR amplified and the amplified products were sequenced. The result was as followed:The similarity of homologous of Undifilum oxytropis, with Undifilum oxytropis almost reached 99%. Under the microscope, the form of colonies, mycelium, spores of Undifilum oxytropis was Consistent With the preliminary reported. Therefore the bacteria growthed on the new medium was Undifilum oxytropis.
2. The chemical constitutions of the fermented products from Embellisia oxytropis were performed as systematic pre-experiment,and the fermented products was preliminarily confirmed that mainly contained Alkaloids, Flavonoids, Anthraquinones, Polysaccharides, Saponins, Essential oil and Phenols and so on. In addition, fermenting liquor contained Polysaccharides, Alkaloids, Flavonoids. Those were similar to the host plants, Oxytropis kansuensis (Leguminosae; Oxytropis DC.). It was showed that endophytic fungi could generate chemical constitutions that were same or similar to the host plants.
3. The antioxidant activities of the secondary metabolites of Undifilum oxytropis were investigated by indexes of the total reduction power; inhibition activity of·OH; elimination of O2- and NO2-. The results showed that, in certain concentration range, eliminate ability of polysaccharides from Undifilum oxytropis (intracellular and exocellular) to the total reduction power;·OH; O2- and NO2- increased with the increasing concentrations and showed dose-effect relationship. The antioxidant capacity of intracellular polysaccharide was the best. When the concentration of intracellular polysaccharide was 300μg/ml, the highest light absorption value was 1.594 during the determination of total reducing power. When the concentration was 1.6mg/ml, the eliminate rates for·OH; O2- and NO2- were 86.3%,86.2% and 81.2%, respectively. They were a little lower than those of Vc. The Index of Evaluation in this study were otal reduction power; inhibition activity of·OH; elimination of O2-and NO2-.
4. We also preliminarily did the selection studying on the antioxidant activities of each part of ethanol extracts form fermented products by the indexes of the total reduction power; inhibition activity of·OH; elimination of O2- and NO2-. The results showed that, in certain concentration range, the eliminate ability of each polarity part which was extracted by ethanol from secondary metabolites increased with increasing concentrations by the indexes of the total reduction power;·OH; O2-and NO2-. And it also showed a certain antioxidant activities. During the experience the total reduction power; elimination of·OH and NO2-,the ability of mycelia were better than fermenting liquor. When concentration was 1.6mg/ml, the eliminate rate of mycelia chloroform extracts to·OH and NO2- were good, (86.7% and 68.7%, respectively). During the experience on eliminating of O2-, the ability of fermenting liquor was better than mycelia. When concentration was 1.6mg/ml, eliminate rate of fermented n-butanol part was the best, (74.1%).
5. We measured the antibacterial activity of ethanol extracts of fermented products from Undifilum oxytropis. The antibacterial experiments used filter paper diffusion method, while selected chloroform, ethyl acetate and n-butyl alcohol parts of ethanol extracts from mycelia and fermenting liquor as test samples, Staphylococcus aureus, Enteritis salmonella, Escherichia coli and Rat typhoid salmonella bacteria were selected as indicator bacteria, and Streptomycin sulfate was selected as positive control. The results showed that, either chloroform extracts of mycelia and fermented liquid or ethyl acetate extracts of mycelia all showed a little antibacterial activity. The antibacterial activities of chloroform extracts from fermenting liquor to Escherichia coli and Staphylococcus aureus were better, the antibacterial diameters were 1.62cm and 1.73cm, the antibacterial rates were 68.2% and 61.4%, respectively. The antibacterial activities of chloroform extracts of mycelia to Staphylococcus aureus, Enteritis salmonella and Rat typhoid salmonella bacteria also were better, the antibacterial rates were 65.4%,59.5% and 60.3%, respectively. The antibacterial activities of ethyl acetate extracts from mycelia was the best, it showed all of the four indicator bacteria have well antibacterial activities.especially on Escherichia coli, the antibacterial rate was 89.6%.
6. In this paper, the antitumor activity of polysaccharide and ethanol extract of Undifilum oxytropis was studied. The inhibition rate of high, medium and low dose group of intracellular polysaccharide was 43.91%. 53.12%、35.42%, respectivily, and the inhibition rate of high, medium and low dose group of ethanol extract was 53.87%、48.90%、33.42%, respectivily.
7. Chemical constitutions of ethanol and petroleum ether extracts from Embellisia oxytropis were preliminarily investigated through the means of silica gel column chromatography, thin layer chromatography and GC-MS analysis. It showed 22 compounds by GC-MS analysis, and eight kinds of compounds were confirmed which included four alkanes, two olefine acids and two esters.
1.本实验以ITS1和ITS4为引物,对研究的实验菌种菌丝体rDNA进行了PCR扩增,并对扩增产物进行测序,结果表明：该菌与Undifilum oxytropis同源相似性达到99%,在显微镜观察下,新生的菌落、菌丝、孢子的形态与文献报道一致,所以认为所转接到新的培养基上生长的菌种为波丝菌属真菌,即为棘豆波丝菌(Undifilum oxytropis)
2.本实验对棘豆波丝菌发酵产物化学成分进行了系统的预实验,初步确定了发酵产物菌丝体中主要含有生物碱、黄酮、蒽醌、多糖、皂苷、挥发油与酚类等物质,发酵液中含有多糖、生物碱、黄酮物质,与宿主植物甘肃棘豆所含成分基本相同,说明内生菌具有产生和宿主植物相同或相似的化学成分。
3.本文以总还原力、羟自由基抑制活性、超氧阴离子自由的清除、亚硝酸根离子的清除数据为评价指标,探明棘豆波丝菌次级代谢产物多糖的抗氧化作用。研究结果表明：在一定的浓度范围内,棘豆波丝菌多糖(胞内、胞外)对总还原力和羟自由基、超氧阴离子、亚硝酸根离子的清除率均随着浓度的增加而增大,呈现一种量效关系。其中,胞内多糖在抗氧化指标中表现最好。在总还原力的测定中,当浓度达到300ug/ml时,胞内多糖产生的光吸收值达到最大为1.594。浓度达到1.6mg/ml时,对羟自由基、超氧阴离子、亚硝酸根离子清除率分别为86.3%、86.2%、81.2%,与Vc清除率相比略低。
4.本实验对棘豆波丝菌发酵产物乙醇提取物极性部位的抗氧化作用进行了初步研究。结果表明,在一定的供试浓度范围内,棘豆波丝菌发酵产物乙醇提取物极性部位对总还原力的影响、对羟基自由基·OH、超氧阴离子O2·和亚硝酸根离子NO2-的清除均随着浓度的增加而增大,并表现出一定的抗氧化活性。其中,在羟基自由基！OH和亚硝酸根离子NO2-的清除过程中,菌丝体各极性萃取部位强于发酵液,当c=1.6mg/ml时,菌丝体氯仿萃取部位对羟基自由基·OH和亚硝酸根离子N02-清除率最大为86.7%、68.7%；在超氧阴离子O2-清除过程中,发酵液各极性萃取部位强于菌丝体,浓度为1.6mg/ml时,发酵液乙酸乙酯萃取部位清除率最大为74.1%。
5.本实验对棘豆波丝菌发酵产物乙醇提取物各极性进行了抑菌活性部位的筛选,选取了菌丝体和发酵液乙醇提取物氯仿、乙酸乙酯、正丁醇萃取部分作为供试品,以金黄色葡萄球菌、肠炎沙门氏菌、大肠杆菌、鼠伤寒沙门氏菌四种细菌为指示菌,以硫酸链霉素作为阳性对照,采用滤纸片扩散法进行体外抑菌实验。研究结果表明：菌丝体和发酵液氯仿萃取物、菌丝体乙酸乙酯萃取物表现出了一定的抑制作用。其中,发酵液氯仿萃取物对大肠杆菌和金黄色葡萄球菌的抑制作用较好,抑菌直径可达1.62cm、1.73cm,抑菌率分别为68.2%,61.4%；菌丝体氯仿萃取物对金黄色葡萄球菌、肠炎沙门氏菌、鼠伤寒沙门氏菌均有较好的抑制作用,抑菌率分别为65.4%、59.5%、60.3%；菌丝体乙酸乙酯萃取物表现最好,对4种指示菌均有较高的抑制活性,其中,对大肠杆菌抑菌效果最佳,抑菌率高达为89.6%。
6.本试验研究了棘豆波丝菌胞内多糖和乙醇提取物对小白鼠肉瘤S180的抑制作用,小白鼠皮下接种S180肉瘤后,经胃灌服胞内多糖和乙醇提取物,连续10天,试验结果表明：胞内多糖高、中、低剂量组对S180抑制率分别为43.91%、53.12%、35.42%,乙醇提取物高、中、低剂量组对S180抑制率分别为53.87%、48.90%、33.42%,与阳性药对照相比具有一定的显著性差异。
7.本实验以硅胶柱层析、薄层色谱和GC-MS分析为手段,对棘豆波丝菌乙醇提取石油醚萃取物进行了化学成分的初步研究,通过GC-MS分析,显示了22种化合物,确认了8种化合物,其中,烷烃4种,烯酸2种,酯2种。
Endophytic fungi are living in plants and have no significantly negative effects on the host plants. They have rich diversity of microorganism groups, and they can produce secondary metabolites with biological activity. Therefore, endophytic fungi are concerned as a source of new medicines due to their tremendous potential.In this study we selected Undifilum oxytropis (Q. Wang, Nagao & Kakish) as the research material, and obtained fermented products through the liquid deep fermentation. Then we had preliminary study the extractions, pharmacological activities and chemical constitutions were preliminarily studied.. The results were as follows:
1. In this study, ITS1 and ITS4 were selected as primers.The rDNA form Mycelium of Undifilum oxytropis were PCR amplified and the amplified products were sequenced. The result was as followed:The similarity of homologous of Undifilum oxytropis, with Undifilum oxytropis almost reached 99%. Under the microscope, the form of colonies, mycelium, spores of Undifilum oxytropis was Consistent With the preliminary reported. Therefore the bacteria growthed on the new medium was Undifilum oxytropis.
2. The chemical constitutions of the fermented products from Embellisia oxytropis were performed as systematic pre-experiment,and the fermented products was preliminarily confirmed that mainly contained Alkaloids, Flavonoids, Anthraquinones, Polysaccharides, Saponins, Essential oil and Phenols and so on. In addition, fermenting liquor contained Polysaccharides, Alkaloids, Flavonoids. Those were similar to the host plants, Oxytropis kansuensis (Leguminosae; Oxytropis DC.). It was showed that endophytic fungi could generate chemical constitutions that were same or similar to the host plants.
3. The antioxidant activities of the secondary metabolites of Undifilum oxytropis were investigated by indexes of the total reduction power; inhibition activity of·OH; elimination of O2- and NO2-. The results showed that, in certain concentration range, eliminate ability of polysaccharides from Undifilum oxytropis (intracellular and exocellular) to the total reduction power;·OH; O2- and NO2- increased with the increasing concentrations and showed dose-effect relationship. The antioxidant capacity of intracellular polysaccharide was the best. When the concentration of intracellular polysaccharide was 300μg/ml, the highest light absorption value was 1.594 during the determination of total reducing power. When the concentration was 1.6mg/ml, the eliminate rates for·OH; O2- and NO2- were 86.3%,86.2% and 81.2%, respectively. They were a little lower than those of Vc. The Index of Evaluation in this study were otal reduction power; inhibition activity of·OH; elimination of O2-and NO2-.
4. We also preliminarily did the selection studying on the antioxidant activities of each part of ethanol extracts form fermented products by the indexes of the total reduction power; inhibition activity of·OH; elimination of O2- and NO2-. The results showed that, in certain concentration range, the eliminate ability of each polarity part which was extracted by ethanol from secondary metabolites increased with increasing concentrations by the indexes of the total reduction power;·OH; O2-and NO2-. And it also showed a certain antioxidant activities. During the experience the total reduction power; elimination of·OH and NO2-,the ability of mycelia were better than fermenting liquor. When concentration was 1.6mg/ml, the eliminate rate of mycelia chloroform extracts to·OH and NO2- were good, (86.7% and 68.7%, respectively). During the experience on eliminating of O2-, the ability of fermenting liquor was better than mycelia. When concentration was 1.6mg/ml, eliminate rate of fermented n-butanol part was the best, (74.1%).
5. We measured the antibacterial activity of ethanol extracts of fermented products from Undifilum oxytropis. The antibacterial experiments used filter paper diffusion method, while selected chloroform, ethyl acetate and n-butyl alcohol parts of ethanol extracts from mycelia and fermenting liquor as test samples, Staphylococcus aureus, Enteritis salmonella, Escherichia coli and Rat typhoid salmonella bacteria were selected as indicator bacteria, and Streptomycin sulfate was selected as positive control. The results showed that, either chloroform extracts of mycelia and fermented liquid or ethyl acetate extracts of mycelia all showed a little antibacterial activity. The antibacterial activities of chloroform extracts from fermenting liquor to Escherichia coli and Staphylococcus aureus were better, the antibacterial diameters were 1.62cm and 1.73cm, the antibacterial rates were 68.2% and 61.4%, respectively. The antibacterial activities of chloroform extracts of mycelia to Staphylococcus aureus, Enteritis salmonella and Rat typhoid salmonella bacteria also were better, the antibacterial rates were 65.4%,59.5% and 60.3%, respectively. The antibacterial activities of ethyl acetate extracts from mycelia was the best, it showed all of the four indicator bacteria have well antibacterial activities.especially on Escherichia coli, the antibacterial rate was 89.6%.
6. In this paper, the antitumor activity of polysaccharide and ethanol extract of Undifilum oxytropis was studied. The inhibition rate of high, medium and low dose group of intracellular polysaccharide was 43.91%. 53.12%、35.42%, respectivily, and the inhibition rate of high, medium and low dose group of ethanol extract was 53.87%、48.90%、33.42%, respectivily.
7. Chemical constitutions of ethanol and petroleum ether extracts from Embellisia oxytropis were preliminarily investigated through the means of silica gel column chromatography, thin layer chromatography and GC-MS analysis. It showed 22 compounds by GC-MS analysis, and eight kinds of compounds were confirmed which included four alkanes, two olefine acids and two esters.
引文
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