Optimization of Extraction Process of Flavonoids from Fagopyrum esculentum Moench Leaves and Its Antioxidant Properties
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摘要: 为了对荞麦叶黄酮的提取和抗氧化性进行研究，采用响应面法对超声波法辅助提取荞麦叶黄酮的工艺进行了优化，高效液相色谱（HPLC）法分析了荞麦叶黄酮成分，并对荞麦叶黄酮的抗氧化性进行了测定。结果表明：当超声频率45 kHz、超声功率100 W、液料比30:1 mL:g、超声时间22 min，超声温度为28 ℃，乙醇体积分数为51%时，荞麦叶黄酮的提取量为80.311 mg/g，与预测值81.414 mg/g相对误差为1.35%，表明该模型预测值与实际值拟合效果良好。HPLC检测表明，荞麦叶黄酮的主要成分为芦丁和槲皮素，其含量分别为66.5%和13.9%。抗氧化试验表明，荞麦叶黄酮对DPPH自由基（DPPH·）、ABTS自由基（ABTS+·）和羟基自由基（·OH）的半清除浓度（IC50）分别为0.012、0.044、0.344 mg/mL，表明其具有较强的抗氧化能力。本试验为荞麦叶的综合利用提供了理论依据。Abstract: In order to study the extraction process and antioxidant properties of flavonoid in Fagopyrum esculentum Moench leaves. The ultrasonic-assisted extraction process of flavonoids from Fagopyrum esculentum Moench leaves was optimized by response surface method, the contents of flavonoids was analyzed by high performance liquid chromatography (HPLC), and the antioxidant activity was explored.The results showed that when the ultrasonic frequency was 45 kHz, the ultrasonic power was 100 W, the liquid -solid ratio was 30:1(mL:g), the ultrasonic time was 22 min, the ultrasonic temperature was 28 ℃, and the volume fraction of ethanol was 51%, the yield of flavonoids in Fagopyrum esculentum Moench leaves was 80.311mg/g. The relative error was 1.35% compared with the predicted value of 81.414 mg/g, which proved that the theoretical predicted value of the model well fitted with the actual value. The result of HPLC showed that rutin and quercetin were the main components of flavonoid in Fagopyrum esculentum Moench leaves, and their contents were 66.5% and 13.9%, respectively. The antioxidant activity test showed that the half maximal inhibitory concentration (IC50) of the flavonoids to DPPH·, ABTS+· and ·OH was 0.012, 0.044, 0.344 mg/mL, respectively, indicated that the flavonoids had strong antioxidant capacity. This experiment provided a theoretical basis for the comprehensive utilization of Fagopyrum esculentum Moench leaves.
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图 1 提取因素对黄酮提取量的影响
注：图中小写字母不同表示差异显著（P< 0.05)。
Figure 1. Effects of extraction factors on extraction amout of flavonoids
图 2 各因素交互作用对荞麦叶黄酮提取量影响的响应面和等高线
Figure 2. Response surface and contour plots showing the effects of pairwise interactions among various process conditions on extraction amout of flavonoids
图 3 荞麦叶黄酮液相色谱图
注：1：芦丁，2：槲皮素；3～5： 未知成分。
Figure 3. HPLC chromatogram of Fagopyrum esculentum Moench leaves flavonoids
图 4 荞麦叶黄酮对DPPH·、ABTS+·和·OH清除能力
Figure 4. Scavenging effect of Fagopyrum esculentum Moench leaves flavonoids on DPPH·(A), ABTS+·(B) and·OH (C)
表 1 响应面试验因素水平表
Table 1. Factors and levels table of response surface experiment
水平 因素 A超声时间（min） B乙醇体积分数（%） C超声温度（℃） −1 15 45 25 0 20 50 30 1 25 55 35 
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表 2 响应面试验设计及结果
Table 2. Design and results of response surface experiment
试验号 A超声时间
（min）B提取剂乙醇
体积分数（%）C超声温度
（℃）黄酮提取量
（mg/g）1 0 0 0 80.79 2 0 0 0 80.68 3 1 1 0 79.15 4 −1 −1 0 72.54 5 −1 0 −1 72.01 6 −1 0 1 71.67 7 −1 1 0 74.92 8 0 0 0 80.12 9 1 0 −1 78.85 10 0 −1 −1 76.97 11 1 −1 0 76.27 12 0 1 1 74.71 13 1 0 1 72.67 14 0 −1 1 74.23 15 0 1 −1 77.99 16 0 0 0 80.84 17 0 0 0 80.96
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表 3 二次响应面回归模型方差分析及显著性检验结果
Table 3. ANOVA for quadratic response surface model and significance of difference
差异来源 平方和 自由度 均方 F值 P值 显著性 模型 183.81 9 20.42 64.11 <0.0001 ** A 31.21 1 31.21 97.96 <0.0001 ** B 5.71 1 5.71 17.93 0.0039 ** C 19.66 1 19.66 61.70 0.0001 ** AB 0.063 1 0.063 0.20 0.6712 AC 8.53 1 8.53 26.77 0.0013 ** BC 0.073 1 0.073 0.23 0.6470 A2 53.56 1 53.56 168.12 <0.0001 ** B2 8.15 1 8.15 25.59 0.0015 ** C2 46.17 1 46.17 144.94 <0.0001 ** 残差 2.23 7 0.32 失拟项 1.80 3 0.60 5.59 0.0650 误差项 0.43 4 0.11 总离差 186.04 16 R2=0.988 R2Adj=0.9726 R2Pred=0.8416 精密度=20.7 CV（%）=0.74 注：**:差异极显著（P<0.01）;*:差异显著（P<0.05）。
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