响应面优化刺五加总黄酮提取工艺及体外抗氧化研究

阚琦缤; 刘瑞雪; 王晓娅; 苏建青; 褚秀玲

doi:10.19303/j.issn.1008-0384.2021.03.015

响应面优化刺五加总黄酮提取工艺及体外抗氧化研究

doi: 10.19303/j.issn.1008-0384.2021.03.015

聊城大学农学院，山东　聊城　252000

基金项目: 国家自然科学基金（31872515）

详细信息

作者简介:
阚琦缤（1995−），男，硕士研究生，研究方向：天然药物提取与机理研究（E-mail：kanqibin@163.com）

通讯作者:
褚秀玲（1969−），女，博士，副教授，硕士生导师，研究方向：中兽医药机理研究（E-mail：chuxiul@163.com）

中图分类号: TS 254.1
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出版历程
- 收稿日期: 2020-07-10
- 修回日期: 2021-03-05
- 网络出版日期: 2021-04-20
- 刊出日期: 2021-03-31

Process Optimization and In Vitro Antioxidant Activity of Flavonoids Extracted from Acanthopanax senticosus

College of Agriculture, Liaocheng University, Liaocheng, Shandong　252000, China

摘要

摘要: 目的采用响应面优化刺五加总黄酮的提取工艺，并探究其抗氧化能力，为刺五加的高效利用提供参考。方法在单因素试验的基础上，采用Box-Benhnken Design法优化总黄酮提取工艺，并通过DPPH自由基、ABTS自由基、羟基自由基、总还原能力以及对巨噬细胞RAW264.7氧化应激的保护作用评价刺五加总黄酮的抗氧化能力。结果通过响应面分析确定最佳提取工艺为：乙醇质量分数55%，提取时间73 min，液料比45 g·mL⁻¹，提取温度72 ℃，在此条件下刺五加总黄酮提取率为（24.11±0.17 ）mg·g⁻¹；刺五加总黄酮对DPPH自由基、ABTS自由基和羟基自由基的半数抑制浓度（IC₅₀）分别为28.38 μg·mL⁻¹、103.77 μg·mL⁻¹和228.70 μg·mL⁻¹，并具有较强的总还原能力。刺五加总黄酮对H₂O₂刺激的RAW264.7细胞氧化损伤具有明显的保护作用。结论通过响应面法优化了刺五加总黄酮的提取工艺，并发现刺五加总黄酮具有较好的抗氧化能力，为其临床开发和应用奠定了基础。
- 刺五加总黄酮 /
- 提取工艺 /
- 响应面 /
- 抗氧化
Abstract: Objective Extraction process of flavonoids from roots of Acanthopanax senticosus plants was optimized by response surface method, and in vitro antioxidant activity of the extract determined. Methods A single factor Box-Benhnken design experiment was conducted to optimize the flavonoid extraction. The antioxidant activity of the extracted flavonoids was measured against DPPH, ABTS, and hydroxyl free radicals as well as the total reducing capacity and protection of macrophages against oxidative stress. Results The optimized process by a response surface experiment with 4 factors and 3 levels used 55% ethanol at a liquid-solid ratio of 45:1 to extract at 72 ℃ for 73 m. It resulted in a yield of total flavonoids of 24.11±0.17 mg·g⁻¹. The IC₅₀ of the extract on DPPH, ABTS, and hydroxyl free radicals were 28.38 μg·mL⁻¹, 103.77 μg·mL⁻¹, and 228.70 μg·mL⁻¹, respectively. The extract exhibited a capacity in raising the proliferation of RAW264.7 cells and in protecting against the oxidative damage induced by H₂O₂. Conclusion The flavonoids extracted from the roots of A. senticosus exhibited significant in vitro antioxidant activities.
- Acanthopanax senticosus /
- extraction process /
- response surface /
- antioxidation

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图 1 芦丁标准曲线

Figure 1. Standard curve of rutin

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图 2 乙醇体积分数对刺五加总黄酮得率的影响

Figure 2. Effect of ethanol concentration on extraction yield of total flavonoids

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图 3 液料比对刺五加总黄酮得率的影响

Figure 3. Effect of liquid-solid ratio on extraction yield of total flavonoids

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图 4 提取时间对刺五加总黄酮得率的影响

Figure 4. Effect of extraction time on yield of total flavonoids

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图 5 提取温度刺五加总黄酮得率的影响

Figure 5. Effect of extraction temperature on yield of total flavonoids

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图 6 两因素间交互作用对刺五加总黄酮得率的影响

Figure 6. Effect of interaction between two factors on extraction of total flavonoids

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图 7 DPPH自由基清除活性

Figure 7. DPPH free radical scavenging ability of extracted flavonoids

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图 8 ABTS⁺自由基清除活性

Figure 8. ABTS⁺ free radical scavenging ability of extracted flavonoids

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图 9 羟基自由基清除活性

Figure 9. Hydroxyl free radical scavenging ability of extracted flavonoids

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图 10 刺五加总黄酮还原能力

Figure 10. Reducing power of flavonoids extract from A. senticosus

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图 11 H₂O₂对RAW264.7细胞增殖的影响

注：不同字母标号代表组别间差异性显著（P＜0.05）。

Figure 11. Effects of H₂O₂ on proliferation of RAW264.7 cells

Note: Different letters indicate significant difference at P＜0.05.

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图 12 刺五加总黄酮对RAW264.7细胞氧化损伤保护作用

注：不同字母标号代表组别间差异性显著（P＜0.05）。

Figure 12. Effect of extracted flavonoids in protecting oxidative damage on RAW264.7 cells

Note: Different letters indicate significant difference at P＜0.05.

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表 1 单因素试验水平

Table 1. Level of single factor experiment

单因素变量 Single Factor	固定因素 Fixed Factors
乙醇体积分数 Ethanol concentration（30%、40%、50%、60%、70%、80%）	液料比（Liquid- Solid ration）mL·g⁻¹ ，提取时间（Extraction time）55 min，提取温度（ Extraction temperature）50 ℃，提取功率（ Extraction power）300 W
液料比 Liquid- Solid ration（10、20、30、40、50、60 mL·g⁻¹）	乙醇体积分数（Ethanol concentration）50%，提取时间（Extraction time）55 min，提取温度（Extraction temperature）50 ℃，提取功率（Extraction power）300 W
提取时间 Extraction time（25、40、55、70、85、100 min）	乙醇体积分数（Ethanol concentration）50%，液料比（ Liquid- Solid ration）30 mL·g⁻¹ ，提取温度（Extraction temperature）50 ℃，提取功率（Extraction power）300 W
提取温度 Extraction temperature（40℃、50℃、60℃、70℃、80 ℃）	乙醇体积分数（Ethanol concentration）50%，液料比（Liquid- Solid ration）30 mL·g⁻¹ ，提取时间（Extraction time）55 min，提取功率（ Extraction power）300 W

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表 2 四因素三水平响应面试验

Table 2. Response surface experiment of 4 factors and 3 levels

因素 Factor	水平Level
因素 Factor	−1	0	1
A：乙醇体积分数 Ethanol concentration/ %	40	50	60
B：液料比 liquid- Solid ration/ mL·g⁻¹	30	40	50
C：提取时间 Extraction time/ min	60	70	80
D：提取温度 Extraction temperatur/ ℃	60	70	80

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表 3 响应面试验结果

Table 3. Results of response surface experiment

试验组 Group	A:乙醇体积分数 Ethanol Concentration / %	B:液料比 Liquid-Solid ratio/ mL·g⁻¹	C:提取时间 Extraction time/ min	D:提取温度 Extraction temperature/ ℃	总黄酮得率 Yield/ mg·g⁻¹
1	40	40	80	70	21.13
2	50	30	70	60	21.26
3	50	30	70	80	21.69
4	50	50	60	70	22.26
5	40	40	60	70	21.76
6	50	40	80	80	22.51
7	40	40	70	60	20.94
8	40	40	70	80	22.51
9	50	50	80	70	23.03
10	60	40	80	70	23.18
11	60	40	60	70	22.41
12	50	40	60	60	21.18
13	50	40	70	70	23.51
14	50	40	80	60	21.99
15	50	50	70	60	22.32
16	50	40	70	70	23.59
17	50	30	80	70	21.43
18	60	50	70	70	23.34
19	50	40	60	80	22.34
20	50	40	70	70	23.59
21	50	40	70	70	23.43
22	60	40	70	60	22.76
23	40	50	70	70	21.93
24	40	30	70	70	20.82
25	50	40	70	70	23.43
26	50	50	70	80	23.24
27	60	40	70	80	23.01
28	60	30	70	70	22.01
29	50	30	60	70	21.57

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表 4 响应面试验方差分析

Table 4. Variance analysis of response surface experiment

方差来源　　 Source	平方和 Sum of squares	自由度 df	均方 Mean square	F值 F value	P值 P value	显著性 Significanc
模型 Model	21.08	14	1.51	70.44	＜0.000 1	**
A	4.84	1	4.84	226.37	＜0.000 1	**
B	4.49	1	4.49	210.04	＜0.000 1	**
C	0.26	1	0.26	11.94	0.003 9	**
D	1.96	1	1.96	91.70	＜0.000 1	**
AB	0.01	1	0.01	0.57	0.464 3	—
AC	0.49	1	0.49	22.92	0.000 3	**
AD	0.44	1	0.44	20.38	0.000 5	**
BC	0.21	1	0.21	9.69	0.007 6	**
BD	0.06	1	0.06	2.81	0.116 0	—
CD	0.10	1	0.10	4.79	0.046 1	*
A²	2.65	1	2.65	123.97	＜0.000 1	**
B²	3.66	1	3.66	171.45	＜0.000 1	**
C²	3.80	1	3.80	177.78	＜0.000 1	**
D²	2.70	1	2.70	126.41	＜0.000 1	**
残差 Residual	0.30	14	0.02	—	—	—
失拟项 Lack of fit	0.27	10	0.03	4.28	0.087 0	—
误差项 Pure error	0.03	4	0.01	—	—	—
总回归 Cor total	21.28	28	—	—	—	—
注：：差异性显著（P＜0.05）；：差异性极显著（P＜0.01）。 Note: significant difference (P＜0.05); **extremely significant difference (P＜0.01).

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表 5 刺五加总黄酮最佳提取条件

Table 5. Optimized conditions for flavonoid extraction from A. senticosus

因素 Factor	水平值 Level value
乙醇体积分数 Ethanol concentration	55%
液料比 Liquid-Solid ration	45 mL·g⁻¹
提取时间 Extraction time	73 min
提取温度 Extraction temperature	72 ℃
预测得率 Predicted value of extraction yield	23.89 mg·g⁻¹
实际得率 Actual value of extraction yield	24.11±0.17 mg·g⁻¹

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