Optimization of Extraction Process of Polysaccharides from Hovenia dulcis Fruit Pedicels and Its Antioxidant Activity
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摘要: 为获得枳椇果梗多糖，并进一步评价其自由基清除及抑制生物大分子（蛋白质、脂质、DNA）氧化的能力。以枳椇果梗为试验材料，在单因素实验的基础上，结合正交试验及方差分析优化枳椇果梗多糖热水提取工艺条件；对所提多糖清除DPPH、ABTS+自由基能力进行测定；并利用Cu2+/H2O2、FeSO4、APPH分别诱导牛血清蛋白、亚油酸、鲱鱼精子DNA氧化，构建体外蛋白质、脂质、DNA氧化模型，对所提多糖体外抑制生物大分子氧化能力进行评价。结果表明：醇沉体积分数对枳椇果梗多糖的得率有显著性（P<0.05）的影响，最佳的热水提取工艺条件为：料液比1:25 g/mL，提取温度85 ℃，提取时间1 h，醇沉体积分数80%，此时多糖得率为3.06%±0.181%；且随着浓度的增大，所提多糖对自由基的清除和生物大分子的氧化抑制效果也逐渐提高，对DPPH自由基、ABTS+自由基清除IC50分别为1.687、1.824 mg/mL，对牛血清蛋白羰基化、亚油酸过氧化和鲱鱼精子DNA氧化抑制IC50分别为：13.84、10.88、74.70 mg/mL。研究结果可为枳椇果梗多糖的提取及在功能性食品中的应用提供参考。Abstract: The aim of this study was to obtain polysaccharides from Hovenia dulcis fruit pedicels, and further evaluate its ability to scavenge free radicals and inhibit the oxidation of biological macromolecules (proteins, lipids, DNA). On the basis of a single-factor experiment, the hot water extraction process of polysaccharides from Hovenia dulcis fruit pedicels was optimized using orthogonal test combined with an analysis of variance. The ability of the extracted polysaccharides to scavenge DPPH and ABTS+ free radicals was determined. In addition, Cu2+/H2O2, FeSO4 and APPH were used to induce the oxidation of bovine serum albumin, linoleic acid and herring sperm DNA, respectively, to construct in vitro protein, lipid and DNA oxidation models and the ability of the extracted polysaccharides to inhibit biological macromolecule oxidation in vitro was evaluated. The results showed that the volume fraction of ethanol precipitation had a significant effect on the yield of polysaccharides from Hovenia dulcis fruit pedicels (P<0.05). The optimal hot water extraction conditions were as follows: solid-liquid ratio, 1:25 g/mL, extraction temperature 85 ℃, extraction time 1 h, ethanol precipitation volume fraction 80%, with the polysaccharide yield of 3.06%±0.181%. With the increase in the concentration, the effects of the extracted polysaccharides in scavenging free radicals and inhibiting the oxidation of biological macromolecules were gradually improved. The IC50 for scavenging DPPH and ABTS+ free radicals were 1.687 and 1.824 mg/mL, and the IC50 for inhibiting bovine serum albumin carbonylation, linoleic acid peroxidation and herring sperm DNA oxidation were 13.84, 10.88 and 74.70 mg/mL, respectively. The results can provide a reference for the extraction of polysaccharides from Hovenia dulcis fruit pedicels and its application in functional food.
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图 2 提取时间对多糖得率的影响
Figure 2. The effects of time on the extraction yield of polysaccharides
图 3 料液比对多糖得率的影响
Figure 3. The effects of solid liquid ratio on the extraction yield of polysaccharides
图 4 醇沉体积分数对多糖得率的影响
Figure 4. The effects of ethanol precipitation volume fraction on the extraction yield of polysaccharides
图 5 枳椇果梗多糖对DPPH自由基的清除作用
Figure 5. Effects of Hovenia dulcis fruit pedicels polysaccharides on DPPH free radical removal
图 6 枳椇果梗多糖对ABTS+自由基的清除作用
Figure 6. Effects of Hovenia dulcis fruit pedicels polysaccharides on ABTS+ free radical removal
图 7 枳椇果梗多糖对蛋白质羰基化的影响
Figure 7. Effects of Hovenia dulcis fruit pedicels polysaccharides on protein carbonylation
图 8 枳椇果梗多糖对脂质过氧化的影响
Figure 8. Effects of Hovenia dulcis fruit pedicels polysaccharides on lipid oxidation
图 9 枳椇果梗多糖对DNA氧化的影响
Figure 9. Effects of Hovenia dulcis fruit pedicels polysaccharides on DNA oxidation
表 1 正交试验因素水平表
Table 1. Orthogonal test factor level table
水平 A 提取温度（℃） B 提取时间（h） C 料液比（g/mL） D 醇沉体积分数（%） 1 85 1 1:25 60 2 90 1.5 1:30 70 3 95 2 1:35 80 
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表 2 正交试验设计与结果
Table 2. Design and results of orthogonal test
试验号 因素 多糖得率（%） A B C D 1 1 1 1 1 1.36 2 1 2 2 2 1.31 3 1 3 3 3 2.56 4 2 1 2 3 2.70 5 2 2 3 1 1.07 6 2 3 1 2 1.54 7 3 1 3 2 1.57 8 3 2 1 3 2.91 9 3 3 2 1 1.57 K1 1.743 1.877 1.937 1.333 K2 1.770 1.763 1.860 1.473 K3 2.017 1.890 1.733 2.723 极差R 0.274 0.127 0.204 1.390
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表 3 正交试验方差分析
Table 3. Variance analysis of orthogonal test
因素 偏差平方和 自由度 F比 F临界值 P值 显著性 A 0.136 2 4.690 19.000 0.183 B 0.029 2 1.000 19.000 0.039 C 0.063 2 2.172 19.000 0.085 D 3.514 2 121.172 19.000 0.008 * 误差 0.03 2 注：*表示P<0.05。
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