Optimization of Flavonoids Extraction from Leaves of Helianthus tuberosus L. by Response Surface Methodology and Determination of Antimicrobial Activity of Resulting Extract
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摘要: 探索组培菊芋叶中总黄酮超声辅助提取最佳工艺条件，并对其抑菌活性进行评价。在单因素试验基础上，以总黄酮提取率为指标，应用响应面法优化其超声提取条件；并考察其总黄酮的抗菌活性。结果表明，响应面优化组培菊芋叶总黄酮提取的最佳工艺参数为：液料比1：40，提取时间20 min，乙醇含量（体积分数）40%，提取温度64℃，测得总黄酮提取率为1.47 mg·g-1。组培菊芋叶总黄酮对大肠杆菌的抑制效果良好，但对金黄色葡萄球菌和枯草芽孢杆菌的抑制效果不明显。结论：利用响应面法分析得到了组培菊芋叶总黄酮超声提取的最佳工艺条件，提取的总黄酮对大肠杆菌具有良好的抑菌活性，可为进一步研究开发组培菊芋奠定实验基础。Abstract: The ultrasound-assisted extraction of flavonoids from leaves of Helianthus tuberosus L. was optimized. The extract obtained was evaluated for its antimicrobial activity.Based on the results from single-factor tests, the extraction conditions were further optimized using the response surface methodology. It was found that when 40% ethanol was applied as the solvent in a substrate:solvent ratio of 1:40 to extract the leaves for 20 min at 64℃, the yield and efficiency of the process could be maximized to reach a flavonoid extraction rate of 1.47 mg·g-1. The flavonoids obtained showed an excellent antibacterial activity on Escherichiacoli, but not on Staphylococcus aureus or Bacillus subtilis.
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图 2 不同试验因素对总黄酮提取率的影响
Figure 2. Effects of different factors on the extration rate of flavonoids
图 3 液料比与提取时间交互影响总黄酮提取率的响应面与等值线
Figure 3. Response surface and contour plots on effects of extraction time and substrate:solvent ratio on yield of flavonoids
图 4 液料比与乙醇含量交互影响总黄酮提取率的响应面与等值线
Figure 4. Response surface and contour plots on effects of ethanol concentration and substrate:solvent ratio on yield of flavonoids
图 5 乙醇含量与提取时间交互影响总黄酮提取率的响应面与等值线
Figure 5. Response surface and contour plots on effects of ethanol concentration and extraction time on yield of flavonoids
图 6 提取温度与液料比交互影响总黄酮提取率的响应面与等值线
Figure 6. Response surface and contour plots on effects of substrate:solvent ratio and extraction temperature on yield of flavonoids
图 7 提取时间与提取温度交互影响总黄酮提取率的响应面与等值线
Figure 7. Response surface and contour plots on effects of extraction time and temperature on yield of flavonoids
图 8 乙醇含量与提取温度交互影响总黄酮提取率的响应面与等值线
Figure 8. Response surface and contour plots on effects of ethanol concentration and extraction temperature on yield of flavonoids
表 1 响应面分析的因素及水平
Table 1. Factors and levels of response surface analysis
水平 A液料比/
(mL·g-1)B提取时间
/minC提取液含量
/%D提取温度
/℃-1 20 20 40 50 0 30 30 50 60 1 40 40 60 70 
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表 2 响应面试验结果
Table 2. Experimental results of response surface analysis
试验号 A
液料比/
(mL·g-1)B
提取时间
/ minC
提取液含量
/%D
提取温度
/℃R
总黄酮提
取率/%1 30 40 40 60 1.31 2 30 30 40 50 1.29 3 30 30 50 60 1.43 4 40 40 50 60 1.43 5 40 30 50 70 1.41 6 20 30 50 70 1.36 7 20 40 50 60 1.37 8 40 30 50 50 1.37 9 30 30 40 70 1.32 10 30 40 50 50 1.37 11 20 30 50 50 1.31 12 30 20 60 60 1.30 13 20 20 50 60 1.37 14 40 30 60 60 1.43 15 30 30 50 60 1.44 16 30 20 50 70 1.39 17 30 30 50 60 1.44 18 20 30 60 60 1.39 19 30 30 60 50 1.30 20 30 20 50 50 1.30 21 30 30 50 60 1.43 22 30 40 60 60 1.38 23 30 30 50 60 1.42 24 20 30 40 60 1.37 25 30 40 50 70 1.29 26 30 20 40 60 1.44 27 40 30 40 60 1.46 28 40 20 50 60 1.44 29 30 30 60 70 1.30
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表 3 响应面试验方差分析
Table 3. Variance analysis of response surface test
方差来源 平方和 自由度 均方 F值 P值 模型 0.085 14 6.04×10-3 19.49 ＜0.0001 A(液料比) 0.011 1 0.011 36.74 ＜0.0001 B(提取时间) 5.74×10-4 1 5.74×10-4 1.85 0.195 C(乙醇含量) 6.28×10-4 1 6.28×10-4 2.03 0.1765 D(提取温度) 1.75×10-3 1 1.75×10-3 5.65 0.0322 AB 9.92×10-6 1 9.92×10-6 0.032 0.8605 AC 7.78×10-4 1 7.78×10-4 2.51 0.1353 AD 5.55×10-5 1 5.55×10-5 0.18 0.6786 BC 0.012 1 0.012 37.99 ＜0.0001 BD 6.80×10-3 1 6.80×10-3 21.94 0.0004 CD 3.46×10-4 1 3.46×10-4 1.12 0.3086 A2 5.88×10-4 1 5.88×10-4 1.9 0.1898 B2 5.61×10-3 1 5.61×10-3 18.09 0.0008 C2 0.011 1 0.011 34.63 ＜0.0001 D2 0.039 1 0.039 125.49 ＜0.0001 残差 4.34×10-3 14 3.10×10-4 - - 失拟项 4.05×10-3 10 4.05×10-4 5.55 0.0564 纯误差 2.91×10-4 4 7.29×10-5 - - 总和 0.089 28 - - -
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表 4 总黄酮对菌种的抑菌圈直径
Table 4. Diameters of inhibition zones on bacteria by flavonoid extract, mm
(单位/mm) 大肠杆菌 金黄色葡萄球菌 枯草芽孢杆菌 空白 菊芋总黄酮 18.48±0.69 8.34±0.29 8.53±0.34 - 18.21±0.90 8.53±0.35 8.56±0.34 18.05±0.88 8.57±0.27 8.43±0.23 阳性对照 19.00±0.22 18.18±0.28 17.00±0.25 - 18.76±0.31 18.07±0.25 16.94±0.25 18.81±0.45 18.11±0.16 16.98±0.22
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表 5 总黄酮对大肠杆菌的最低抑菌质量浓度(MIC)
Table 5. Minimal inhibitory concentration (MIC) of flavonoid extract against E.coli
抑菌液质量浓度
/(mg·mL-1)大肠杆菌 40 无长菌 20 无长菌 10 长菌 5 大量长菌 2.5 大量长菌 阳性对照 大量长菌 阴性对照 无长菌
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