Optimization of Ethanol Extraction Process for Active Components from Broussonetia papyrifera Root Bark and Its Antioxidant Activity
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摘要: 以构树根皮为原料，通过单因素实验考察不同因素对构树根皮总黄酮和多酚提取量的影响。运用Design-Expert 11软件设计响应面法优化构树根皮乙醇回流提取工艺，并进行工艺验证。最后对提取得到的构树根皮乙醇提取物进行DPPH·、ABTS+·、羟自由基清除能力和总还原能力的测定，评价其抗氧化活性。响应面分析表明，构树根皮总黄酮和多酚的最佳提取工艺为提取温度75 ℃、提取时间117 min、料液比1:16 g/mL、乙醇浓度70%。此条件下，构树根皮总黄酮和多酚提取量分别为23.93±0.30 mg/g和14.69±0.56 mg/g，与预测理论值接近。抗氧化实验表明，构树根皮乙醇提取物对DPPH·、ABTS+·和羟自由基的半数清除浓度（IC50）分别为5.256 μg/mL、0.259 mg/mL和0.310 mg/mL，且清除能力与其浓度呈现一定的量效关系。当提取物浓度为1.0 mg/mL时，总还原能力达到1.484±0.062。此优化实验有效可行，构树根皮乙醇提取物具有较强的抗氧化活性。本研究为构树资源的综合利用提供了一定的理论依据。Abstract: In this study, single factor experiments were employed to determine the effects of various factors on yields of total flavonoids and polyphenols from Broussonetia papyrifera root bark. Then Box-Behnken design and response surface methodology were used to optimize the ethanol reflux extraction process using the Design-Expert 11 software. Moreover, the antioxidant activity of the ethanol extract of Broussonetia papyrifera root bark was evaluated by determining the scavenging capacity of DPPH·, ABTS+·, and the hydroxyl free radical, as well as the total reducing power. Results showed that the optimal conditions were as follows: Extraction temperature 75 ℃, extraction time 117 min, solid-liquid ratio 1:16 g/mL, and ethanol concentration 70%. Under these conditions, the experimental extraction yield values of total flavonoids and polyphenols from Broussonetia papyrifera root bark were 23.93±0.30 mg/g and 14.69±0.56 mg/g, respectively, which was not significantly different in comparison to predicted values. The IC50 values of scavenging rates on DPPH·, ABTS+·, and the hydroxyl free radical were 5.256 μg/mL, 0.259 mg/mL, and 0.310 mg/mL, respectively, and the scavenging rates showed a certain dose-effect relationship with the sample concentration. In addition, the total reducing power of 1.0 mg/mL ethanol extract of the root bark was 1.484±0.062. These results indicated that this optimization test was effective and feasible, and the ethanol extract of Broussonetia papyrifera root bark had good antioxidant activity in vitro. The present study provides supplement information for the comprehensive utilization of Broussonetia papyrifera in food and medicine ingredients.
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图 2 提取时间对构树根皮黄酮和多酚含量的影响
Figure 2. Effect of extraction time on flavonoids and polyphenols contents of Broussonetia papyrifera root bark
图 3 料液比对构树根皮黄酮和多酚含量的影响
Figure 3. Effect of solid-liquid ratio on flavonoids and polyphenols contents of Broussonetia papyrifera root bark
图 4 乙醇浓度对构树根皮黄酮和多酚含量的影响
Figure 4. Effect of ethanol concentration on flavonoids and polyphenols contents of Broussonetia papyrifera root bark
图 5 交互项对总黄酮提取量影响的响应面图
Figure 5. Response surface diagram of the effect of interactive items on the yield of total flavonoids
图 6 交互项对多酚提取量影响的响应面图
Figure 6. Response surface diagram of the effect of interactive items on the yield of polyphenols
图 7 构树根皮提取物对DPPH自由基的清除率
Figure 7. DPPH free radical scavenging rate of Broussonetia papyrifera root bark extract
图 8 构树根皮提取物对ABTS阳离子自由基的清除率
Figure 8. ABTS+ free radical scavenging rate of Broussonetia papyrifera root bark extract
图 9 构树根皮提取物对羟自由基的清除率
Figure 9. Hydroxyl free radical scavenging rate of Broussonetia papyrifera root bark extract
图 10 构树根皮提取物的总还原能力
Figure 10. Total reducing ability of Broussonetia papyrifera root bark extract
表 1 Box-Behnken试验因素和水平
Table 1. Test factors and levels of Box-Behnken
水平 因素 A温度（°C ） B时间（min） C料液比（g/mL） D乙醇浓度（%） −1 65 60 1:10 65 0 75 90 1:15 75 1 85 120 1:20 85 
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表 2 响应面试验设计与结果
Table 2. Design and results of response surface experiment
试验号 因素 总黄酮提取量（mg/g） 多酚提取量（mg/g） A温度 B时间 C料液比 D乙醇浓度 1 −1 −1 0 0 13.72 13.50 2 1 −1 0 0 17.00 12.86 3 −1 1 0 0 19.95 12.82 4 1 1 0 0 17.27 13.34 5 0 0 −1 −1 13.83 11.66 6 0 0 1 −1 19.63 12.29 7 0 0 −1 1 14.89 12.28 8 0 0 1 1 14.96 13.69 9 −1 0 0 −1 14.20 12.23 10 1 0 0 −1 19.24 12.85 11 −1 0 0 1 18.07 13.42 12 1 0 0 1 17.79 13.31 13 0 −1 −1 0 11.50 11.54 14 0 1 −1 0 17.22 12.77 15 0 −1 1 0 16.24 12.61 16 0 1 1 0 17.27 13.48 17 −1 0 −1 0 12.11 11.88 18 1 0 −1 0 11.96 11.34 19 −1 0 1 0 13.88 13.26 20 1 0 1 0 14.99 12.29 21 0 −1 0 −1 19.87 11.29 22 0 1 0 −1 23.55 14.64 23 0 −1 0 1 20.51 14.21 24 0 1 0 1 23.58 13.02 25 0 0 0 0 22.35 14.23 26 0 0 0 0 23.65 14.70 27 0 0 0 0 22.67 14.64 28 0 0 0 0 21.67 14.07 29 0 0 0 0 22.51 14.43
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表 3 总黄酮回归模型方差分析
Table 3. Analysis of variance of the regression model of flavonoids
方差来源 平方和 自由度 均方 F值 P值 显著性 模型 382.75 14 27.34 23.06 <0.0001 ** A温度 3.33 1 3.33 2.81 0.1158 B时间 33.33 1 33.33 28.12 0.0001 ** C料液比 19.88 1 19.88 16.77 0.0011 ** D乙醇浓度 0.0222 1 0.0222 0.0187 0.8931 AB 8.88 1 8.88 7.49 0.0160 * AC 0.4013 1 0.4013 0.3386 0.5699 AD 7.07 1 7.07 5.96 0.0285 * BC 5.48 1 5.48 4.62 0.0496 * BD 0.0929 1 0.0929 0.0783 0.7836 CD 8.22 1 8.22 6.93 0.0197 * A² 120.75 1 120.75 101.87 <0.001 ** B² 4.98 1 4.98 4.2 0.0597 C² 216.4 1 216.4 182.57 <0.001 ** D² 2.11 1 2.11 1.78 0.2033 残差 16.59 14 1.19 失拟项 14.55 10 1.46 2.85 0.1621 不显著 纯误差 2.04 4 0.5102 总方差 399.34 28 注：** P<0.01，表示差异性极显著；* P<0.05，表示差异性显著，表4同。
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表 4 多酚回归模型方差分析
Table 4. Analysis of variance of the regression model of polyphenols
方差来源 平方和 自由度 均方 F值 P值 显著性 模型 27.24 14 1.95 14.74 <0.0001 ** A温度 0.1051 1 0.1051 0.7962 0.3873 B时间 1.37 1 1.37 10.42 0.0061 ** C料液比 3.15 1 3.15 23.9 0.0002 ** D乙醇浓度 2.06 1 2.06 15.64 0.0014 ** AB 0.3381 1 0.3381 2.56 0.1318 AC 0.0466 1 0.0466 0.3529 0.5620 AD 0.1357 1 0.1357 1.03 0.3278 BC 0.0339 1 0.0339 0.257 0.6201 BD 5.17 1 5.17 39.21 <0.0001 ** CD 0.1542 1 0.1542 1.17 0.2981 A² 4.61 1 4.61 34.94 <0.0001 ** B² 1.44 1 1.44 10.92 0.0052 C² 11.77 1 11.77 89.14 <0.0001 ** D² 2.5 1 2.5 18.93 0.0007 ** 残差 1.85 14 0.132 失拟项 1.57 10 0.1567 2.23 0.2283 不显著 纯误差 0.2809 4 0.0702 总方差 29.09 28
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