Extraction and activity of flavonoids from Morus alba leaves by ultrasonic-semi-bionic method
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摘要:
目的 对桑叶黄酮的提取工艺及其自由基清除能力，对α-葡萄糖苷酶和α-淀粉酶的活性抑制作用进行研究，旨在获得充分保持桑叶黄酮活性的新型制备方法，为桑叶资源开发利用提供理论依据。 方法 采用超声波–半仿生法提取桑叶黄酮，考察液料比、超声时间、超声温度、超声功率4个因素对桑叶黄酮得率和DPPH·以及OH·的平均清除率的影响，通过响应面试验优化桑叶黄酮提取工艺，评价桑叶黄酮对α-葡萄糖苷酶和α-淀粉酶的抑制作用。 结果 桑叶黄酮最佳提取工艺为液料比30 mL/g、提取总时间97 min（3个阶段的时间比例为1∶2∶2）、超声温度49 ℃、超声功率400 W，黄酮得率为（38.23 ± 0.42） mg/g，自由基平均清除率为（57.04 ± 0.97）%。桑叶黄酮对α-葡萄糖苷酶和α-淀粉酶活性抑制的IC50值为（1.081 ± 0.130） g/L和（1.204 ± 0.190） g/L。超声波–半仿生法比单一超声波法提取桑叶黄酮的自由基清除能力强，比单一半仿生法提取桑叶黄酮的得率高。 结论 采用超声波辅助半仿生法提取的桑叶黄酮具有良好的自由基清除能力，且对α-葡萄糖苷酶和α-淀粉酶有抑制作用，与单一提取法相比，超声波–半仿生法提取桑叶黄酮具备更高的生物活性和得率。桑叶黄酮可开发为高糖人群调节血糖的产品，超声波–半仿生法可为工业制备桑叶黄酮提供良好的技术储备。 Abstract:Objective This project investigates the extraction process of Morus alba leaves flavonoids and their free radical scavenging ability, inhibition of α-glucosidase and α-amylase activity, aiming at obtaining a novel preparation method that fully maintains the activity of Morus alba leaves flavonoids, and providing a theoretical basis for the development and utilization of Morus alba leaves resources. Method The ultrasonic-semi-bionic method was used to extract the flavonoids from the Morus alba leaves. We investigated the effects of four factors (liquid-to-material ratio, ultrasonic time, ultrasonic temperature, and ultrasonic power) on the yield of Morus alba leaves flavonoids and the average scavenging of DPPH· and OH· radicals. The extracting process was optimized using the response surface test to evaluate the inhibitory effects of Morus alba leaves flavonoids on α-glucosidase and α-amylase. Result The results showed that optimal extraction conditions of Morus alba leaves flavonoids were as follows: ratio of liquid to solid 30 mL/g, ultrasonic time 97 min (the time ratio of the three stages 1∶2∶2), ultrasonic temperature was 49 ℃, ultrasonic power was 400 W. Under conditions, the flavonoid yield up to (38.23 ± 0.42) mg/g was obtained with an average clearance rate of (57.04 ± 0.97) %. Under optimal conditions, the IC50 value for α-glucosidase inhibition by Morus alba leaves flavonoids was (1.081 ± 0.130) g/L, and that for α-amylase inhibition was (1.204 ± 0.190) g/L. The free radical scavenging ability of Morus alba leaves flavonoids extracted by ultrasonic-semi-bionic method was stronger than that of the ultrasonic method, and the yield of Morus alba leaves flavonoids extracted by ultrasonic-semi-bionic method was higher than that of the semi-bionic method. Conclusion Morus alba leaves flavonoids extracted by ultrasonic-semi-bionic method have good free radical scavenging ability and inhibitory effect on α-glucosidase and α-amylase. Compared with the single extraction method, the Morus alba leaves flavonoids extracted by ultrasonic-semi-bionic method have higher bioactivity and yield. Morus alba leaves flavonoids can be developed as blood glucose regulating products for people with high glucose levels, and the ultrasonic-semi-bionic method can provide a better technical reserve for the industrial preparation of Morus alba leaves flavonoids. -
Key words:
- Morus alba leaves /
- flavonoids extraction /
- semi-bionic method /
- response surface
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图 1 各因素对桑叶黄酮得率、DPPH·和OH·平均清除率的影响
图中的不同小写字母代表显著性差异（P < 0.05）。下同。Different lowercase letters in the figure indicate that there are significant differences (P < 0.05). The same below.
Figure 1. Effects of various factors on the flavonoids yield and average clearance rate of DPPH· and OH· in Morus alba
图 2 各因素对桑叶黄酮得率的交互影响
Figure 2. Interactive effects of various Morus alba leaves factors on flavonoid yield
图 3 不同质量浓度的桑叶黄酮对酶抑制率的影响
Figure 3. Effect of different mass concentrations of Morus alba leaves flavonoid on enzyme inhibition rate
图 4 不同提取方法的桑叶黄酮得率与活性对比
Figure 4. Comparison of the yield and activity of Morus alba leaves flavonoids by different extraction methods
表 1 响应面试验设计及其结果
Table 1. Box-Behnken design and the results
试验号
NO.液料比
Liquid to material
ratio (A)/(mL·g−1)超声时间
Ultrasonic time
(B)/min超声温度
Ultrasonic temperature
(C)/℃超声功率
Ultrasonic power
(D)/W黄酮得率
Flavonoid
yield/(mL·g−1)平均清除率
Average clearance
rate/%1 30 60 50 420 35.76 ± 0.35 50.30 ± 1.52 2 30 90 50 360 38.26 ± 0.47 56.06 ± 1.23 3 30 90 50 360 38.75 ± 0.98 57.96 ± 0.97 4 30 60 60 360 34.22 ± 0.69 49.13 ± 0.68 5 25 90 60 360 32.76 ± 1.22 47.92 ± 1.09 6 30 90 40 300 35.15 ± 0.83 52.13 ± 1.32 7 30 60 40 360 33.71 ± 1.03 49.71 ± 0.59 8 25 90 40 360 33.32 ± 1.08 49.14 ± 0.88 9 25 120 50 360 34.44 ± 0.62 50.71 ± 1.24 10 30 90 50 360 38.46 ± 0.54 57.04 ± 0.63 11 30 90 50 360 38.35 ± 0.14 57.55 ± 1.07 12 25 90 50 420 35.83 ± 1.22 51.75 ± 0.92 13 30 120 50 300 35.63 ± 0.37 49.39 ± 1.41 14 30 60 50 300 34.06 ± 0.55 51.60 ± 1.05 15 30 120 50 420 37.41 ± 1.16 55.56 ± 1.53 16 35 90 50 420 35.76 ± 0.27 52.30 ± 0.97 17 35 90 60 360 33.85 ± 0.85 49.59 ± 1.34 18 30 90 40 420 37.15 ± 0.64 53.88 ± 1.22 19 30 90 50 360 38.06 ± 0.92 57.13 ± 1.02 20 30 90 60 300 35.02 ± 1.31 52.80 ± 0.96 21 35 60 50 360 34.98 ± 0.81 48.39 ± 1.25 22 25 90 50 300 33.89 ± 1.17 50.32 ± 1.16 23 35 90 50 300 34.68 ± 0.64 50.46 ± 0.73 24 30 120 60 360 34.86 ± 1.48 49.60 ± 1.64 25 30 120 40 360 35.66 ± 0.25 52.68 ± 2.01 26 35 120 50 360 34.10 ± 0.69 49.26 ± 1.38 27 25 60 50 360 32.52 ± 0.84 45.75 ± 0.56 28 30 90 60 420 36.36 ± 1.23 54.27 ± 1.14 29 35 90 40 360 32.89 ± 0.87 49.58 ± 1.36 注：平均清除率为DPPH·清除率与OH·清除率的均值。Note: average clearance rate is the average of DPPH· clearance rate and OH· clearance rate. 
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表 3 平均清除率方差分析表
Table 3. Average clearance rate ANOVA table
方差来源
Source平方和
Sum of squares自由度
Degrees of freedom均方
Mean squareF P 显著性
Significance模型 Model 280.21 14 20.01 36.17 < 0.000 1 ** A 1.33 1 1.33 2.40 0.143 8 B 12.65 1 12.65 22.86 0.000 3 ** C 1.21 1 1.21 2.19 0.161 4 D 10.75 1 10.75 19.44 0.000 6 ** AB 4.18 1 4.18 7.56 0.015 7 * AC 0.38 1 0.38 0.68 0.422 2 AD 0.042 1 0.042 0.076 0.786 9 BC 1.56 1 1.56 2.82 0.115 0 BD 13.95 1 13.95 25.21 0.000 2 ** CD 0.020 1 0.020 0.035 0.853 4 A2 152.80 1 152.80 276.16 < 0.000 1 ** B2 103.26 1 103.26 186.61 < 0.000 1 ** C2 56.30 1 56.30 101.75 < 0.000 1 ** D2 8.65 1 8.65 15.63 0.001 4 ** 残差 Residual 7.75 14 0.55 失拟项 Lack of fit 5.73 10 0.57 1.14 0.490 4 纯误差 Pure error 2.02 4 0.50 总和 Total 287.95 28
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表 2 黄酮得率方差分析表
Table 2. Flavonoid yield ANOVA table
方差来源
Source平方和
Sum of squares自由度
Degrees of freedom均方
Mean squareF P 显著性
Significance模型 Model 90.97 14 6.50 35.45 < 0.000 1 ** A 1.02 1 1.02 5.57 0.033 3 * B 3.91 1 3.91 21.33 0.000 4 ** C 0.055 1 0.055 0.30 0.593 6 D 8.07 1 8.07 44.02 < 0.000 1 ** AB 1.96 1 1.96 10.69 0.005 6 * AC 0.58 1 0.58 3.15 0.097 6 AD 0.18 1 0.18 1.01 0.332 3 BC 0.43 1 0.43 2.34 0.148 3 BD 1.600 × 10−3 1 1.600 × 10−3 8.728 × 10−3 0.926 9 CD 0.11 1 0.11 0.59 0.4537 A2 51.25 1 51.25 279.59 < 0.000 1 ** B2 20.31 1 20.31 110.82 < 0.000 1 ** C2 27.78 1 27.78 151.57 < 0.000 1 ** D2 2.34 1 2.34 12.78 0.003 0 * 残差 Residual 2.57 14 0.18 失拟项 Lack of fit 2.31 10 0.23 3.53 0.117 6 纯误差 Pure error 0.26 4 0.065 总和 Total 93.54 28 注：**为差异极显著（P < 0.01），*为差异显著（P < 0.05），下同。 Notes: ** means highly significance (P < 0.01), and * means significance (P < 0.05). The same below.
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