桑叶的抑菌作用及其活性物质的提取分离研究
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摘要
本课题主要对桑叶提取物的抑菌活性进行了研究,包括抑菌谱及最低抑菌浓度的确定,优化抑菌活性物质的提取工艺,探讨了抑菌活性物质的稳定性并进一步对抑菌活性物质进行分离纯化和成分粗测。最后对桑叶提取物作了一些应用研究。
将桑叶用水和70%乙醇分别提取后,用滤纸片法和平板计数法进行抑菌实验,得到抗菌谱。采用线性回归法快速准确的得出它们对大肠杆菌和金黄色葡萄球菌的最低抑菌浓度。乙醇提取物对金黄色葡萄球菌的MIC:0.43%,对大肠杆菌的MIC:0.055%;桑叶水提取物对金黄色葡萄球菌的MIC:0.86%;对大肠杆菌的MIC:1.54%。可见桑叶乙醇提取物对两种代表性细菌的抑菌效果优于水提取物。
采用单因素实验确定桑叶抑菌活性物质提取最佳条件为:乙醇浓度70%(V/V)、提取温度70°C、提取时间4h、液料比(W/V)1:30。采用正交实验,得出最佳工艺条件为乙醇浓度70%,提取温度70°C,提取时间6h,液料比1:40。此条件下,桑叶提取物对金黄色葡萄球菌的抑菌圈直径为8.81mm。
对桑叶提取物抑菌活性做了稳定性测试,结果表明桑叶提取物的抑菌活性具有较好的热稳定性,对紫外光照射稳定。对菌体的抑制能力随pH值的升高而有所下降,其中酸性条件下抑菌活性较强,在pH6-7之间,缓冲液对提取物的抑菌活性没有影响,并且提取物的抑菌效果好。
对活性物质进一步分析,首先将桑叶提取物分为石油醚部、二氯甲烷部、乙酸乙酯部、正丁醇部和水部等不同极性溶剂萃取物,经抑菌活性比较,确定乙酸乙酯部的抑菌活性最强,正丁醇部次之。乙酸乙酯部经薄层层析,展开后,有多个斑点,用1%的AlCl3显色,多个点显示黄色,紫外灯光下显黄绿色和棕色。推断抑菌组分属于黄酮类化合物。乙酸乙酯部分上聚酰胺和硅胶柱分离,得到抑菌效果最好的组分,薄层层析,发现三个清晰的斑点,Rf值分别0.20,0.27,0.72。桑黄素的Rf值为0.27,通过HPLC验证桑黄素是桑叶抑菌有效成分之一。
把桑叶提取物加入到化妆品中作测试,结果表明桑叶提取物具有很好的防腐、美白、保湿效果,可以在化妆品中广泛应用。
In this paper, extract of Mulberry leaves was tested for its antimicrobial activities, including investigation of its antimicrobial spectrum, MIC value. The processing conditions of leaching active substances from Mulberry leaves were optimized with the orthogonal design test. The sensitivity to universal factors of the extract was studied. At the same time, the isolation and purification of the antimicrobial components of Mulberry leaves were also studied .
The antimicrobial effect by water system extracting and alcohol- water system extracting in different concentrations were studied. We use D-value method to fast determine the efficacy. The results showed that both the extract by water system and alcohol- water system can effectively inhibit the growth of bacteria thus kill large amounts of the bacteria. The MIC of extraction from Mulberry leaves by alcohol- water system was 0.43% against Staphylococcus aureus,which was 0.055% against Escherichia coli. As to the extract by water system, the data was 0.86% against Staphylococcus aureus and 1.54% against Escherichia coli. Apparently, the effect by alcohol- water system was better.
The optimum extraction conditions of leaching active substances determined by the single factor experiment method were as follows: the concentration of ethanol was 70%; temperature for extracting was 70°C with 4h, the ratio of material and solvent was l:30. with the orthogonal design test, the results showed that the optimum extracting condition was the concentration of ethanol was 70%; temperature for extracting was 70°C with 6h, the ratio of material and solvent was l:40. Under this condition, the diameter of antibacterial of the extract was up to 8.81mm.
The stability to temperature, acid, alkali and ultraviolet of the extract of Mulberry leaves were also studied. The result showed the extract was very stable to temperature and ultraviolet. Antibacterial activities were better when pH< 7.
All extracts obtained in different fractions by using different polarities of solvents such as petroleum ether, chloroform, ethyl acetate, normal butyl alcohol and water were compared through the testing of their antibacterial activities. The results showed the antibacterial activities of ethyl acetate were strongest and normal butyl alcohol took second place. To track antimicrobial components in the extracts of ethyl acetate in which many different antibacterial points appeared after diffusion. The qualitative analysis of extracting sample is conducted by the specific reaction of total flavonoids with AlCl3 and so on, It is confirmed that the extraction material is flavonoids.
the extracts of ethyl acetate of mulberry leaves by polyamide columnchromatography and silica gel columnchromatography had three antibacterial components at least. The Rf value were 0.20, 0.27, 0.72. Antibacterial component was determined by HPLC. The results showed that one of antibacterial components was morin.
The extract of Mulberry leaves was applied in cosmetics. All the results confirmed that extract of Mulberry leaves has the antimicrobial effect, the skin-lightening effect and the moisturizing effect. So it can be widely used in cosmetics.
将桑叶用水和70%乙醇分别提取后,用滤纸片法和平板计数法进行抑菌实验,得到抗菌谱。采用线性回归法快速准确的得出它们对大肠杆菌和金黄色葡萄球菌的最低抑菌浓度。乙醇提取物对金黄色葡萄球菌的MIC:0.43%,对大肠杆菌的MIC:0.055%;桑叶水提取物对金黄色葡萄球菌的MIC:0.86%;对大肠杆菌的MIC:1.54%。可见桑叶乙醇提取物对两种代表性细菌的抑菌效果优于水提取物。
采用单因素实验确定桑叶抑菌活性物质提取最佳条件为:乙醇浓度70%(V/V)、提取温度70°C、提取时间4h、液料比(W/V)1:30。采用正交实验,得出最佳工艺条件为乙醇浓度70%,提取温度70°C,提取时间6h,液料比1:40。此条件下,桑叶提取物对金黄色葡萄球菌的抑菌圈直径为8.81mm。
对桑叶提取物抑菌活性做了稳定性测试,结果表明桑叶提取物的抑菌活性具有较好的热稳定性,对紫外光照射稳定。对菌体的抑制能力随pH值的升高而有所下降,其中酸性条件下抑菌活性较强,在pH6-7之间,缓冲液对提取物的抑菌活性没有影响,并且提取物的抑菌效果好。
对活性物质进一步分析,首先将桑叶提取物分为石油醚部、二氯甲烷部、乙酸乙酯部、正丁醇部和水部等不同极性溶剂萃取物,经抑菌活性比较,确定乙酸乙酯部的抑菌活性最强,正丁醇部次之。乙酸乙酯部经薄层层析,展开后,有多个斑点,用1%的AlCl3显色,多个点显示黄色,紫外灯光下显黄绿色和棕色。推断抑菌组分属于黄酮类化合物。乙酸乙酯部分上聚酰胺和硅胶柱分离,得到抑菌效果最好的组分,薄层层析,发现三个清晰的斑点,Rf值分别0.20,0.27,0.72。桑黄素的Rf值为0.27,通过HPLC验证桑黄素是桑叶抑菌有效成分之一。
把桑叶提取物加入到化妆品中作测试,结果表明桑叶提取物具有很好的防腐、美白、保湿效果,可以在化妆品中广泛应用。
In this paper, extract of Mulberry leaves was tested for its antimicrobial activities, including investigation of its antimicrobial spectrum, MIC value. The processing conditions of leaching active substances from Mulberry leaves were optimized with the orthogonal design test. The sensitivity to universal factors of the extract was studied. At the same time, the isolation and purification of the antimicrobial components of Mulberry leaves were also studied .
The antimicrobial effect by water system extracting and alcohol- water system extracting in different concentrations were studied. We use D-value method to fast determine the efficacy. The results showed that both the extract by water system and alcohol- water system can effectively inhibit the growth of bacteria thus kill large amounts of the bacteria. The MIC of extraction from Mulberry leaves by alcohol- water system was 0.43% against Staphylococcus aureus,which was 0.055% against Escherichia coli. As to the extract by water system, the data was 0.86% against Staphylococcus aureus and 1.54% against Escherichia coli. Apparently, the effect by alcohol- water system was better.
The optimum extraction conditions of leaching active substances determined by the single factor experiment method were as follows: the concentration of ethanol was 70%; temperature for extracting was 70°C with 4h, the ratio of material and solvent was l:30. with the orthogonal design test, the results showed that the optimum extracting condition was the concentration of ethanol was 70%; temperature for extracting was 70°C with 6h, the ratio of material and solvent was l:40. Under this condition, the diameter of antibacterial of the extract was up to 8.81mm.
The stability to temperature, acid, alkali and ultraviolet of the extract of Mulberry leaves were also studied. The result showed the extract was very stable to temperature and ultraviolet. Antibacterial activities were better when pH< 7.
All extracts obtained in different fractions by using different polarities of solvents such as petroleum ether, chloroform, ethyl acetate, normal butyl alcohol and water were compared through the testing of their antibacterial activities. The results showed the antibacterial activities of ethyl acetate were strongest and normal butyl alcohol took second place. To track antimicrobial components in the extracts of ethyl acetate in which many different antibacterial points appeared after diffusion. The qualitative analysis of extracting sample is conducted by the specific reaction of total flavonoids with AlCl3 and so on, It is confirmed that the extraction material is flavonoids.
the extracts of ethyl acetate of mulberry leaves by polyamide columnchromatography and silica gel columnchromatography had three antibacterial components at least. The Rf value were 0.20, 0.27, 0.72. Antibacterial component was determined by HPLC. The results showed that one of antibacterial components was morin.
The extract of Mulberry leaves was applied in cosmetics. All the results confirmed that extract of Mulberry leaves has the antimicrobial effect, the skin-lightening effect and the moisturizing effect. So it can be widely used in cosmetics.
引文
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