Optimization of Aqueous Two-phase Extraction Technology of Total Flavonoids from Lonicera japonica Thunb. Leaves and Its Antioxidant Activity
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摘要: 研究了乙醇-(NH4)2SO4双水相提取金银花叶总黄酮工艺，并探讨了总黄酮的抗氧化活性。以(NH4)2SO4质量分数、液料比、浸提温度和浸提时间等因素为自变量，以总黄酮得率为指标，采用正交试验对工艺参数进行了优化。通过对DPPH自由基、羟基自由基和超氧阴离子自由基清除率的测定，评价总黄酮的抗氧化性能。最佳实验条件为：(NH4)2SO4质量分数15%，液料比25:1 mL/g，浸提温度50 ℃，浸提时间30 min。在该条件下，总黄酮得率为4.98%。当金银花叶总黄酮浓度为0.48 mg/mL时，DPPH自由基的清除率为97.61%，浓度为0.72 mg/mL时，羟基自由基的清除率为86.53%，超氧阴离子自由基的清除率为75.29%，表现出良好的抗氧化性能。Abstract: The ethanol-ammonium sulfate aqueous two-phase extraction of total flavonoids from the leaves of Lonicera japonica Thunb. leaves was studied, and the antioxidant activity of the total flavonoids was further discussed. The extraction process was optimized by orthogonal test with the parameters including mass fraction of ammonium sulfate, liquid-to-material ratio, extraction temperature and extraction time as independent variables and the yield of the flavonoids as index. The antioxidant activity of the total flavonoids was evaluated by measuring the clearance rates of DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals. The optimum experimental conditions were as follows: The mass fraction of ammonium sulfate was 15%, the liquid-to-material ratio was 25:1 mL/g, the extraction temperature was 50 ℃, and the extraction time was 30 min. Below this term, the yield of flavonoids was 4.98%. The clearance rate of DPPH free radicals was 97.61% when the concentration of total flavonoids from the leaves of Lonicera japonica Thunb. was 0.48 mg/mL, and those of the hydroxyl free radicals and superoxide anion free radicals were 86.53% and 75.29%, respectively, when the concentration of flavonoids was 0.72 mg/mL, showing good antioxidant activity.
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图 1 硫酸铵质量分数对金银花叶总黄酮得率和萃取率的影响
Figure 1. Effects of mass content of ammonium sulfate on the yield and extraction rate of the total flavonoids from Lonicera japonica Thunb. leaves
图 2 液料比对金银花叶总黄酮得率和萃取率的影响
Figure 2. Effects of liquid-to-material ratio on the yield and extraction rate of the total flavonoids from Lonicera japonica Thunb. leaves
图 3 浸提温度对金银花叶总黄酮得率和萃取率的影响
Figure 3. Influence of extraction temperature on the yield and extraction rate of the total flavonoids from Lonicera japonica Thunb. leaves
图 4 浸提时间对金银花叶总黄酮得率和萃取率的影响
Figure 4. Effects of extraction time on the yield and extraction rate of the total flavonoids from Lonicera japonica Thunb. leaves
图 5 金银花叶总黄酮的HPLC图
Figure 5. HPLC chromatogram of the total flavonoids from the leaves of Lonicera japonica Thunb
注：（a）混合标准品溶液；（b）金银花叶总黄酮样品溶液；1：木犀草苷；2：槲皮素；3：木犀草素。
图 6 金银花叶总黄酮对DPPH自由基的清除率
Figure 6. DPPH free radical scavenging rate of the total flavonoids from Lonicera japonica Thunb. leaves
图 7 金银花叶总黄酮对羟基自由基的清除率
Figure 7. Hydroxyl radical scavenging rate of the total flavonoids from Lonicera japonica Thunb. leaves
图 8 金银花叶总黄酮对超氧阴离子自由基的清除率
Figure 8. Superoxide anion free radical scavenging rate of the total flavonoids from Lonicera japonica Thunb. leaves
表 1 正交试验因素和水平设计
Table 1. Design of factors and levels of orthogonal experiment
水平 A （NH4）2SO4
质量分数（%）B 液料比
（mL·g−1）C 浸提温度
（℃）D 浸提时间
（min）1 13 25:1 40 20 2 14 30:1 50 30 3 15 35:1 60 40 
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表 2 正交试验结果
Table 2. Results of orthogonal experiments
编号 A （NH4）2SO4
质量分数（%）B 液料比
（mL·g−1）C 浸提温度
（℃）D 浸提时间
（min）总黄酮得率
（%）1 13 25:1 40 20 3.21 2 13 30:1 50 30 3.81 3 13 35:1 60 40 2.99 4 14 25:1 50 40 4.91 5 14 30:1 60 20 4.70 6 14 35:1 40 30 4.05 7 15 25:1 60 30 4.92 8 15 30:1 40 40 4.52 9 15 35:1 50 20 4.71 k1 3.337 4.347 3.927 4.207 k2 4.553 4.343 4.477 4.260 k3 4.717 3.917 4.203 4.140 R 1.380 0.430 0.550 0.120
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