乌饭树树叶水提物功能性的研究
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摘要
本论文主要研究了乌饭树树叶(VBTL)水提物的功能性。首先研究了VBTL水提物对STZ诱导的糖尿病小鼠的作用。动物实验结果表明VBTL水提物对STZ诱导的糖尿病小鼠有改善作用。灌胃四周后水提物组糖尿病小鼠的体重增加了61%,血糖降低了14.18%,胰岛素含量明显增加,血脂含量明显降低且水提物对小鼠的内脏器官没有影响。
采用二倍稀释法和管碟法对VBTL水提物进行体外抑菌试验,研究了VBTL水提物的抑菌作用及其最小抑菌浓度(MIC)。结果发现,VBTL水提物对金黄色葡萄球菌、大肠杆菌和枯草芽孢杆菌的抑菌圈大小分别为12.6±0.6mm,9.9±0.1mm和8.1±0.6mm,但对霉菌和酵母的抑制效果不明显。VBTL水提物对金黄色葡萄球菌的MIC为7.65 mg/mL,对大肠杆菌和枯草芽孢杆菌的MIC均为15.30mg/mL。
根据VBTL水提物的成分及一些抑菌和糖尿病方面的相关研究,推测抑菌作用的有效成分为黄酮,对糖尿病作用的有效成分为多糖。通过Box-Benhnken中心组合实验和响应面分析法,得到了VBTL水溶性黄酮提取的最佳工艺条件:料水比1:40,提取时间2h,提取温度50℃,提取次数2次,此时其得率为16.82mg/g。
采用二倍稀释法和管碟法对VBTL水溶性黄酮进行体外抑菌试验,主要研究了VBTL水溶性黄酮的抑菌作用及其最小抑菌浓度。结果表明VBTL水溶性黄酮提取物对金黄色葡萄球菌、大肠杆菌和枯草芽孢杆菌的抑菌圈大小分别为24.6±0.6mm,22.9±0.5mm和18.5±0.9mm,但对霉菌和酵母的抑制效果不明显。VBTL水溶性黄酮提取液对金黄色葡萄球菌、大肠杆菌和枯草芽孢杆菌的MIC分别为1.03mg/mL、2.06mg/mL和4.12mg/mL。将VBTL水溶性黄酮提取物的抑菌效果和山梨酸钾和苯甲酸钠的抑菌效果进行了比较,发现其抑菌效果要好于山梨酸钾和苯甲酸钠的抑菌效果。
利用管碟法研究了不同处理温度对VBTL水溶性黄酮提取物的抑菌作用的影响以及VBTL水溶性黄酮提取物和葡萄糖或NaCl的协同抑菌作用。实验结果表明随温度的升高黄酮提取物的抑菌效果先增强后减弱。在抑菌作用上黄酮提取物和葡萄糖或NaCl有较强的协同抑菌作用。对VBTL水溶性黄酮提取物的杀菌时间和黄酮提取物的抑菌机理进行了研究。杀菌时间实验结果表明浓度为1×MIC,3×MIC黄酮提取液对金黄色葡萄球菌的杀菌时间都为12h,而浓度为5×MIC黄酮提取液对金黄色葡萄球菌的杀菌时间为7h。浓度为3×MIC,5×MIC黄酮提取液对枯草芽孢杆菌和大肠杆菌的杀菌时间都为12h。抑菌机理的实验结果表明VBTL水溶性黄酮提取物的抑菌机理之一为破坏了细胞膜的结构,影响了细胞的生理活性从而起到了抑菌作用。
通过Box-Benhnken中心组合实验和响应面分析法,得出VBTL水溶性糖提取的最佳工艺条件:提取温度65℃,提取时间2.5h,pH值9.5,料水比1:45,此时糖的得率为89.80mg/g。再利用单因素实验确定乙醇醇沉VBTL水溶性多糖的最佳工艺条件为:提取液和乙醇的体积比1:5,乙醇体积分数90%,乙醇醇沉静置时间6h,醇沉2次,此时多糖得率为36.55mg/mL。
通过对四种大孔树脂的脱色效果进行研究,选择AB-8型大孔树脂对VBTL水溶性多糖进行脱色。通过对AB-8型大孔树脂脱色的静态吸附条件进行研究,确定了VBTL水溶性多糖脱色的最佳静态吸附条件为:树脂添加量8%,溶液pH2,作用温度35℃,作用18h,此时多糖保留率为82.03%、脱色率为88.11%、蛋白脱除率为78.35%。利用DEAE-Sepharose FF柱对脱色后的多糖进行进一步纯化,结果表明经过DEAE-Sepharose FF柱分离后得到两种多糖成分。
This paper mainly studied the functions of water extract from Vaccinium bracteatum Tbunb. leaves(VBTL). Animal experiment was used to study the effect of the water extract on STZ-induced diabetic mice. Animal experiments results showed that water extract from VBTL can improve the condition of diabetic mice. After 4 weeks, the body weights of diabetic mice treated with water extract from VBTL were increased 61%, the blood glucose levels were reduced 14.18%, insulin level significantly increased. Water extract from VBTL possess a potential hypolipidemic effect in diabetic mice, but have no effect on organ.
The antibacterial effect and the minimal inhibitory concentration (MIC) of water extract from VBTL were determined by the cylinder plate method and two-fold dilution method. The antibacterial experiment results showed that water extract from VBTL had obvious inhibition effects on bacteria, but had no obvious inhibition effects on mold and yeast. The MIC experiment results showed that the MIC to Staphylococcus aureus was 7.65 mg/mL, the MIC to Escherichia coli and Bacillus subtilis were 15.30 mg/mL.
After analysising the component of water extract from VBTL, we speculate that the effective element of antibacterial effect was flavonoids, and the effective element of the effect on STZ-induced diabetic mice was polysaccharides.
We used Box-Benhnken central group experiment and response surface analysis to obtain the optimum conditions for the extract technique of water-soluble flavonoids. The results showed that the optimum conditions were: extraction temperature 50℃, extraction time 2h, ratio of water to material 40 and extract twice. In these conditions, the yield of water-soluble flavonoids from VBTL was 16.82mg/g.
The antibacterial effect and the MIC of water-soluble flavonoids from VBTL was determined by the cylinder plate method and two-fold dilution method. The antibacterial experiment results showed that water extract of VBTL leaves had obvious inhibition effects on bacteria, but had no obvious inhibition effects on mold and yeast. The MIC experiment results showed that the MIC to Staphylococcus aureus was 1.03 mg/mL, the MIC to Escherichia coli was 2.06 mg/mL, and the MIC to Bacillus subtilis was 4.12 mg/mL. The antibacterial experiment results showed that the antibacterial effect of water-soluble flavonoids is better than potassium sorbate and sodium benzoate.
Some other factors which influenced the antibacterial effect of water-soluble flavonoids from VBTL and the mechanism of antibacterial were preliminarily studied. The result showed that addition of glucose or NaCl can improve the antibacterial activity of water-soluble flavonoids from VBTL. The time-kill result showed that at 3×MIC and 5×MIC concentrations, the growth of Escherichia coli and Bacillus subtilis were completely wiped out within 12h, at 1×MIC and 3×MIC concentrations, the growth of Staphylococcus aureus were completely wiped out within 12h. At 5×MIC concentrations, the growth of Staphylococcus aureus were completely wiped out within 7h. The result of mechanism showed that flavonoids changed the permeability of the cell membrane, in this way the growth of bacterial was restrained.
We used Box-Benhnken central group experiment and response surface analysis to obtain the optimum extraction conditions for water-soluble sugars from VBTL. The results showed that the optimum conditions were: extraction temperature 65℃, extraction time 2.5h, pH value of solution 9.5, solid to liquid ratio 1:45, the yield of water-soluble sugars from VBTL was 89.80mg/g. Then single factor experiments were used to determine the optimum conditions for ethanol precipitated water-soluble polysaccharides from VBTL. The optimum conditions were: the ratio of extract volume and ethanol volume 1:5, ethanol concentration 90%, standing time 6h, and ethanol precipitated twice. In these conditions, the yield of water-soluble polysaccharides was 36.55mg/mL.
Four kinds of macroporous resins were screened to decolorize. After comparing macroporous resin AB-8 was chose to decolorize. According to the results optimum static adsorption conditions for AB-8 were: resin content 8%, pH value of solution 2, temperature 35℃, and reaction time 18h. In these conditions, polysaccharide retention rate was 82.03%, the decolorization rate was 88.11% and protein removal rate was 78.35%. Finally, DEAE-Sepharose FF column was used for further purification, the results showed that two polysaccharide components were isolated from water-soluble polysaccharides .
采用二倍稀释法和管碟法对VBTL水提物进行体外抑菌试验,研究了VBTL水提物的抑菌作用及其最小抑菌浓度(MIC)。结果发现,VBTL水提物对金黄色葡萄球菌、大肠杆菌和枯草芽孢杆菌的抑菌圈大小分别为12.6±0.6mm,9.9±0.1mm和8.1±0.6mm,但对霉菌和酵母的抑制效果不明显。VBTL水提物对金黄色葡萄球菌的MIC为7.65 mg/mL,对大肠杆菌和枯草芽孢杆菌的MIC均为15.30mg/mL。
根据VBTL水提物的成分及一些抑菌和糖尿病方面的相关研究,推测抑菌作用的有效成分为黄酮,对糖尿病作用的有效成分为多糖。通过Box-Benhnken中心组合实验和响应面分析法,得到了VBTL水溶性黄酮提取的最佳工艺条件:料水比1:40,提取时间2h,提取温度50℃,提取次数2次,此时其得率为16.82mg/g。
采用二倍稀释法和管碟法对VBTL水溶性黄酮进行体外抑菌试验,主要研究了VBTL水溶性黄酮的抑菌作用及其最小抑菌浓度。结果表明VBTL水溶性黄酮提取物对金黄色葡萄球菌、大肠杆菌和枯草芽孢杆菌的抑菌圈大小分别为24.6±0.6mm,22.9±0.5mm和18.5±0.9mm,但对霉菌和酵母的抑制效果不明显。VBTL水溶性黄酮提取液对金黄色葡萄球菌、大肠杆菌和枯草芽孢杆菌的MIC分别为1.03mg/mL、2.06mg/mL和4.12mg/mL。将VBTL水溶性黄酮提取物的抑菌效果和山梨酸钾和苯甲酸钠的抑菌效果进行了比较,发现其抑菌效果要好于山梨酸钾和苯甲酸钠的抑菌效果。
利用管碟法研究了不同处理温度对VBTL水溶性黄酮提取物的抑菌作用的影响以及VBTL水溶性黄酮提取物和葡萄糖或NaCl的协同抑菌作用。实验结果表明随温度的升高黄酮提取物的抑菌效果先增强后减弱。在抑菌作用上黄酮提取物和葡萄糖或NaCl有较强的协同抑菌作用。对VBTL水溶性黄酮提取物的杀菌时间和黄酮提取物的抑菌机理进行了研究。杀菌时间实验结果表明浓度为1×MIC,3×MIC黄酮提取液对金黄色葡萄球菌的杀菌时间都为12h,而浓度为5×MIC黄酮提取液对金黄色葡萄球菌的杀菌时间为7h。浓度为3×MIC,5×MIC黄酮提取液对枯草芽孢杆菌和大肠杆菌的杀菌时间都为12h。抑菌机理的实验结果表明VBTL水溶性黄酮提取物的抑菌机理之一为破坏了细胞膜的结构,影响了细胞的生理活性从而起到了抑菌作用。
通过Box-Benhnken中心组合实验和响应面分析法,得出VBTL水溶性糖提取的最佳工艺条件:提取温度65℃,提取时间2.5h,pH值9.5,料水比1:45,此时糖的得率为89.80mg/g。再利用单因素实验确定乙醇醇沉VBTL水溶性多糖的最佳工艺条件为:提取液和乙醇的体积比1:5,乙醇体积分数90%,乙醇醇沉静置时间6h,醇沉2次,此时多糖得率为36.55mg/mL。
通过对四种大孔树脂的脱色效果进行研究,选择AB-8型大孔树脂对VBTL水溶性多糖进行脱色。通过对AB-8型大孔树脂脱色的静态吸附条件进行研究,确定了VBTL水溶性多糖脱色的最佳静态吸附条件为:树脂添加量8%,溶液pH2,作用温度35℃,作用18h,此时多糖保留率为82.03%、脱色率为88.11%、蛋白脱除率为78.35%。利用DEAE-Sepharose FF柱对脱色后的多糖进行进一步纯化,结果表明经过DEAE-Sepharose FF柱分离后得到两种多糖成分。
This paper mainly studied the functions of water extract from Vaccinium bracteatum Tbunb. leaves(VBTL). Animal experiment was used to study the effect of the water extract on STZ-induced diabetic mice. Animal experiments results showed that water extract from VBTL can improve the condition of diabetic mice. After 4 weeks, the body weights of diabetic mice treated with water extract from VBTL were increased 61%, the blood glucose levels were reduced 14.18%, insulin level significantly increased. Water extract from VBTL possess a potential hypolipidemic effect in diabetic mice, but have no effect on organ.
The antibacterial effect and the minimal inhibitory concentration (MIC) of water extract from VBTL were determined by the cylinder plate method and two-fold dilution method. The antibacterial experiment results showed that water extract from VBTL had obvious inhibition effects on bacteria, but had no obvious inhibition effects on mold and yeast. The MIC experiment results showed that the MIC to Staphylococcus aureus was 7.65 mg/mL, the MIC to Escherichia coli and Bacillus subtilis were 15.30 mg/mL.
After analysising the component of water extract from VBTL, we speculate that the effective element of antibacterial effect was flavonoids, and the effective element of the effect on STZ-induced diabetic mice was polysaccharides.
We used Box-Benhnken central group experiment and response surface analysis to obtain the optimum conditions for the extract technique of water-soluble flavonoids. The results showed that the optimum conditions were: extraction temperature 50℃, extraction time 2h, ratio of water to material 40 and extract twice. In these conditions, the yield of water-soluble flavonoids from VBTL was 16.82mg/g.
The antibacterial effect and the MIC of water-soluble flavonoids from VBTL was determined by the cylinder plate method and two-fold dilution method. The antibacterial experiment results showed that water extract of VBTL leaves had obvious inhibition effects on bacteria, but had no obvious inhibition effects on mold and yeast. The MIC experiment results showed that the MIC to Staphylococcus aureus was 1.03 mg/mL, the MIC to Escherichia coli was 2.06 mg/mL, and the MIC to Bacillus subtilis was 4.12 mg/mL. The antibacterial experiment results showed that the antibacterial effect of water-soluble flavonoids is better than potassium sorbate and sodium benzoate.
Some other factors which influenced the antibacterial effect of water-soluble flavonoids from VBTL and the mechanism of antibacterial were preliminarily studied. The result showed that addition of glucose or NaCl can improve the antibacterial activity of water-soluble flavonoids from VBTL. The time-kill result showed that at 3×MIC and 5×MIC concentrations, the growth of Escherichia coli and Bacillus subtilis were completely wiped out within 12h, at 1×MIC and 3×MIC concentrations, the growth of Staphylococcus aureus were completely wiped out within 12h. At 5×MIC concentrations, the growth of Staphylococcus aureus were completely wiped out within 7h. The result of mechanism showed that flavonoids changed the permeability of the cell membrane, in this way the growth of bacterial was restrained.
We used Box-Benhnken central group experiment and response surface analysis to obtain the optimum extraction conditions for water-soluble sugars from VBTL. The results showed that the optimum conditions were: extraction temperature 65℃, extraction time 2.5h, pH value of solution 9.5, solid to liquid ratio 1:45, the yield of water-soluble sugars from VBTL was 89.80mg/g. Then single factor experiments were used to determine the optimum conditions for ethanol precipitated water-soluble polysaccharides from VBTL. The optimum conditions were: the ratio of extract volume and ethanol volume 1:5, ethanol concentration 90%, standing time 6h, and ethanol precipitated twice. In these conditions, the yield of water-soluble polysaccharides was 36.55mg/mL.
Four kinds of macroporous resins were screened to decolorize. After comparing macroporous resin AB-8 was chose to decolorize. According to the results optimum static adsorption conditions for AB-8 were: resin content 8%, pH value of solution 2, temperature 35℃, and reaction time 18h. In these conditions, polysaccharide retention rate was 82.03%, the decolorization rate was 88.11% and protein removal rate was 78.35%. Finally, DEAE-Sepharose FF column was used for further purification, the results showed that two polysaccharide components were isolated from water-soluble polysaccharides .
引文
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