摘要
本课题主要研究了毛竹叶提取物的抑菌活性,抑菌活性成分最佳提取工艺条件,探讨了毛竹叶提取物稳定性以及对活性成分进行初步的分析、分离,并对活性成分进行了抑菌、美白、抗氧化性能的测试。
采用滤纸片法、稀释平板计数法、比浊度法,以金黄色葡萄球菌和大肠杆菌为指示菌,考察毛竹叶提取物的抑菌活性,实验结果证明毛竹叶提取物具有很强的抑菌活性。采用滤纸片法,以抑菌圈直径为评价标准,金黄色葡萄球菌、大肠杆菌为指示菌,优化提取工艺。采用单因素实验确定每个单因素的最佳提取条件分别为:提取温度60℃,乙醇体积分数60 %,液固比20 : 1(mL : g),提取时间2 h。采用正交试验得出最佳提取工艺条件为:提取温度60℃,乙醇体积分数60 %,液固比20 : 1(mL : g),提取时间2 h。对毛竹叶提取物做了抗菌谱的测定,结果表明具有较宽的抗菌谱。毛竹叶提取物对金黄色葡萄球菌、大肠杆菌的最低抑菌浓度分别为3.125 g/L、6.25 g/L。
对毛竹叶提取物进行稳定性测试,结果表明毛竹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,pH 5~7范围内抑菌效果稳定。
对抑菌活性成分进一步分析,用四种不同极性的溶剂对毛竹叶提取物水溶液进行萃取,得到五个萃取部分:石油醚相、三氯甲烷相、乙酸乙酯相、正丁醇相和水相。采用滤纸片法,测定毛竹叶提取物不同萃取部分的抑菌活性,并对抑菌活性进行比较,结果表明:乙酸乙酯相抑菌活性最强。还原显色反应、金属盐类的络合反应、紫外可见光下显色,初步确定乙酸乙酯相主要活性成分为黄酮类化合物。
以芦丁为标准样品,建立了芦丁标准工作曲线的回归方程,测定毛竹叶黄酮类化合物的含量,将毛竹叶提取物经过乙酸乙酯、AB-8大孔吸附树脂处理,黄酮类化合物的含量得到很大的提高。
乙酸乙酯相经硅胶柱层析、聚酰胺柱层析、AB-8大孔吸附树脂分离,得到三种组分,经过RP-HPLC分析,毛竹叶提取物成分复杂。
将毛竹叶提取物加入到化妆品中进行测试,结果表明毛竹叶提取物具有很强的抑菌活性、美黑、抗氧化性能,在化妆品中将会有很好的应用。
In this paper,extract of Phyllostachys pubescens leaves was tested for its antimicrobial actives, the extraction technology of active components from P. pubescens leaves and their antimicrobial actives was studied. The stability to universal factors of the extracts was studied. At the same time, the primary isolation and purification of the antimicrobial components of P. pubescens leaves were also studied.
Using disc diffusion method, plate dilution method,photometric assay method investigate the antimicrobial actives of the extracts from P. pubescens leaves,with Staphylococcus aureus and Escherichia coli as indicative bacteria.The results showed that the extracts of P. pubescens leaves has the antimicrobial actives.
The extraction technology was optimized with disc diffusion method,use the diameter of bacteriostatic circle as assessing index, S. aureus and E. coli as indicative bacteria. The optimum extraction conditions of every factor for leaching active substances determined by the single factor experiment method were as follows: extraction temperature 60 oC, 60 %(volume fraction)ethanol, liquid:solid ratio 20:1(mL:g), extraction time 2 h. To With the orthogonal design test, the results showed that the optimum extracting condition was: extraction temperature 60 oC, 60 %(volume fraction)ethanol, liquid:solid ratio 20:1(mL:g), extraction time 2 h.The extracts of P. pubescens leaves has wide Antibacterial spectrum.The MIC of the extracts from P. pubescens leaves was 3.125mg/mL against S. aureus, which was 6.25mg/mL against E. coli.
The stability to temperature,ultraviolet and pH value of the extracts of P. pubescens leaves were also studied. The result showed the extract was very stable to temperature,ultraviolet and pH 6.5~7.5.
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 antimicrobial activities.The results showed the extraction of ethyl acetate had a strong capacity on S. aureus and E. coli.The colour reaction of reduction,metallic salts complexing and under ultraviolet and visible light showed that the main actives of the extraction of ethyl acetate is flavonoids.
With Rutin being taken as the standard sample,the regression equation of rutin standard working curve was established. Total flavonoids content was determined by Spectrophotometry.The treatment of the extracts from P. pubescens leaves with ethyl acetate and AB-8 macroporous adsorption resin made flavonoids content increased.
The extracts of ethyl acetate of P. pubescens leaves was seperated by silica gel column chromatography,polyamide columnchromatography, AB-8 macroporous adsorption resin.Three components were obtained.But the results of RP-HPLC showed that the extrats of P. pubescens leaves is complex.
The extract of P. pubescens leaves was applied in cosmetics. All the results confirmed that extract of P. pubescens leaves has the antimicrobial effect, the skin-lightening effect and the antioxidative effect. So it can be good used in the cosmetics.
引文
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