Process Optimization and in vitro Hypoglycemic Effect of Polyphenols Extracted from Jaboticaba Leaves
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摘要:目的 优化嘉宝果叶片总多酚的提取工艺，检测优化提取后的嘉宝果叶片提取物对α-葡萄糖苷酶的抑制作用。方法 利用超声波辅助与乙醇提取相结合的方法，以总多酚提取量为考察指标，采用单因素及正交试验，研究功率、时间、乙醇体积分数、料液比和温度对嘉宝果叶片总多酚提取量的影响，并采用酶动力学及紫外可见吸收光谱法探讨叶片提取物体外抑制α-葡萄糖苷酶的活性。结果 嘉宝果叶片总多酚的最佳提取工艺为：功率120 W，时间10 min，乙醇体积分数40%，料液比1:160（g:mL），温度40℃，在此条件下，嘉宝果叶片总多酚质量分数可达33.84%。优化提取后的嘉宝果叶片提取物对α-葡萄糖苷酶具有较强的抑制作用，半数抑制浓度IC50为0.003 1 mg·mL-1，阿卡波糖对α-葡萄糖苷酶的半数抑制浓度IC50为8.110 8 mg·mL-1；Lineweaver-Burk双倒数作图显示嘉宝果叶片多酚对α-葡萄糖苷酶的抑制类型为反竞争性抑制。结论 正交实验法优化嘉宝果叶片总多酚提取具有可行性，嘉宝果叶片总多酚对α-葡萄糖苷酶具有较强的抑制作用，可作为降血糖功能因子进行开发利用。Abstract:Objective To optimize the polyphenol extraction from leaves of jaboticaba and determine the α-glucosidase inhibition by the extract.Method The ethanol extraction with ultrasound application was optimized by an orthogonal experiment. Effects of the power and time of ultrasonic application as well as the ethanol volume fraction, material-solvent ratio, and processing temperature on the total polyphenols extracted from the leaves were analyzed. The inhibition on α-glucosidase activity by the extract was determined in vitro by an enzyme kinetic study and uv-vis absorption spectrometry.Result The optimal extraction conditions included the use of 120 W ultrasonic wave for 10 min, 40% ethanol volume fraction, 1:160(g:mL) material-solvent ratio, and 40℃ processing temperature to achieve a polyphenol extraction rate of 33.84%. The α-glucosidase activity was significantly inhibited by the extract with a low IC50 of 0.0031 mg·mL-1, as compared to IC50 of 8.110 8 mg·mL-1 on acarbose. The kinetic study by the Lineweaver-Burk plot showed that the polyphenol extract was an anti-competitive inhibitor of α-glucosidase.Conclusion The polyphenol extract from jaboticaba leaves significantly inhibited α-glucosidase and could be explored for use as a hypoglycemic agent.
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Keywords:
- Jaboticaba /
- polyphenol /
- α-glucosidase /
- orthogonal test
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致谢: 特别感谢福建龙海市惠昌树葡萄专业合作社，在采样过程中承担了主要的采集工作。
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图 2 时间对总多酚提取量的影响
Figure 2. Effect of ultrasonic wave application duration on polyphenol extraction
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图 3 乙醇体积分数对总多酚提取量的影响
Figure 3. Effect of ethanol volume fraction on polyphenol extraction
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图 6 嘉宝果叶片总多酚提取物(A)及阿卡波糖(B)对α-葡萄糖苷酶的抑制作用
Figure 6. Effects of polyphenol extract (A) and acarbose (B) on α-glucosidase
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图 7 嘉宝果叶片总多酚提取物对α-葡萄糖苷酶抑制作用类型
Figure 7. Inhibition type of polyphenol extract on α-glucosidase
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图 8 嘉宝果叶片总多酚提取物对α-葡萄糖苷酶抑制作用的Lineweaver-Burk曲线
Figure 8. Lineweaver-Burk plot on inhibition of polyphenol extract on α-glucosidase
表 1 正交试验因素水平
Table 1 Factors and levels of orthogonal experiment
水平
Level乙醇体积分数
Ethanol volume fraction/%料液比
Material-solvent ratio/(g:mL)温度
Temperature/℃1 20 1:120 20 2 40 1:160 30 3 60 1:200 40 
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表 2 正交试验设计与结果
Table 2 Orthogonal design, results and range analysis
试验号
Numbers因素 总多酚提取量
Total polyphenol/%A/% B/(g:mL) C/℃ D 1 1(20) 1(1:120) 1(20) 1 30.13 2 1 2(1:160) 2(30) 2 31.91 3 1 3(1:200) 3(40) 3 31.59 4 2(40) 1 2 3 32.67 5 2 2 3 1 34.52 6 2 3 1 2 34.06 7 3(60) 1 3 2 32.22 8 3 2 1 3 33.14 9 3 3 2 1 33.20 k1 93.63 95.02 97.33 97.85 k2 101.25 99.57 97.78 98.19 k3 98.56 98.85 98.33 97.40 R 7.62 4.55 1.00 0.79 注：表中因素A为乙醇体积分数，B为液料比，C为温度，D为空列。表 3同。
Note：A.Ethanol volume fraction，B.Material-solvent ratio，C.Temperature，D.Vacant column.The same as table 3.
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表 3 正交试验结果方差分析
Table 3 Analysis of variance on results of orthogonal experiment
变异来源
Variations偏差平方和
Sum of square自由度
FreedomF值
F valueF临界值
F critical valueP值
P valueA 9.956 2 94.819 19.000 ＜0.05 B 3.988 2 37.981 19.000 ＜0.05 C 0.167 2 1.590 19.000 >0.05 D 0.105 2 1.000 19.000 >0.05 误差Errors 0.105 2
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表 4 嘉宝果总多酚提取物对α-葡萄糖苷酶抑制作用的Lineweaver-Burk曲线方程
Table 4 Lineweaver-Burk plot equation for inhibition of polyphenol extract on α-glucosidase
项目
Items质量浓度
Mass concentration/(mg·mL-1)线性回归方程Linear regression equation R2 Vmax/(OD·min-1) Km/(mmol·L-1) 斜率Slope 截距Intercept 对照组Contrast 0 5.7203 8.0232 0.9976 0.1246 0.7130 嘉宝果总多酚
Total polyphenol0.003 5.8844 22.6842 0.9974 0.0441 0.2594 0.006 5.8105 61.6144 0.9976 0.0162 0.0943
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