Process Optimization and In Vitro Antioxidant Activity of Flavonoids Extracted from Acanthopanax senticosus
-
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。 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. -
Key words:
- Acanthopanax senticosus /
- extraction process /
- response surface /
- antioxidation
-
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 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 −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 表 3 响应面试验结果
Table 3. Results of response surface experiment
试验组
GroupA:乙醇体积分数
Ethanol
Concentration
/ %B:液料比
Liquid-Solid
ratio/
mL·g−1C:提取时间
Extraction
time/
minD:提取温度
Extraction
temperature/
℃总黄酮
得率
Yield/
mg·g−11 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 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 squareF值
F valueP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).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 -
[1] 廖炎, 乐园. 氧化应激与器官损害 [J]. 中国现代医学杂志, 2013, 23(18):57−61. doi: 10.3969/j.issn.1005-8982.2013.18.011LIAO Y, LE Y. Oxidative stress and organ impairment [J]. China Journal of Modern Medicine, 2013, 23(18): 57−61.(in Chinese) doi: 10.3969/j.issn.1005-8982.2013.18.011 [2] 杨海峰, 何宏勇, 陈海峰, 等. 刺五加提取物对断奶仔猪生长性能、免疫功能和肠道菌群的影响 [J]. 中国饲料, 2019(2):45−48.YANG H F, HE H Y, CHEN H F, et al. Effects of Acanthopanax senticosus extract on growth performance, immune function and intestinal flora in weaned piglets [J]. China Feed, 2019(2): 45−48.(in Chinese) [3] JIANG Y, WANG M H. Different solvent fractions of Acanthopanax senticosus harms exert antioxidant and anti-inflammatory activities and inhibit the human Kv1.3 channel [J]. J Med Food., 2015, Apr; 18(4): 468−575. [4] 王彦博, 石燕, 袁毅君. 麦积山野生刺五加多酚与黄酮的超声辅助提取与体外抗氧化活性的研究 [J]. 天然产物研究与开发, 2019, 31(12):2153−2162.WANG Y B, SHI Y, YUAN Y J. Ultrasonic assisted extraction of polyphenols and flavonoids from Maijishan wild Acanthopanax and their antioxidant activities in vitro [J]. Natural Product Research and Development, 2019, 31(12): 2153−2162.(in Chinese) [5] 高腾, 丁婵, 苏建青. 响应面优化超声辅助提取刺五加茎皮黄酮工艺 [J]. 中国畜牧杂志, 2017, 53(8):114−118.GAO T, DING C, SU J Q. Optimization of ultrasonic assisted extraction of flavonoids from the stem bark of Acanthopanax senticosus [J]. Chinese Journal of Animal Science, 2017, 53(8): 114−118.(in Chinese) [6] 吴桐, 徐慧春, 郑春英, 等. 快速溶剂萃取法提取刺五加叶中的黄酮类成分 [J]. 中国食品学报, 2013, 13(7):59−65.WU T, XU H C, ZHENG C Y, et al. Extraction of flavanoids from Acanthopanax Senticosus(Rupr. et Maxim) leaves by pressurized solvent extraction [J]. Journal of Chinese Institute of Food Science and Technology, 2013, 13(7): 59−65.(in Chinese) [7] 张晶, 邢媛媛, 金晓, 等. 响应面法优化艾蒿水提物的提取工艺及其抗氧化活性分析 [J]. 中国农业大学学报, 2020, 25(11):99−108. doi: 10.11841/j.issn.1007-4333.2020.11.11ZHANG J, XING Y Y, JIN X, et al. Optimizing the aqueous extraction process of Artemisia argyi by response surface methodology and its antioxidant activity analysis [J]. Journal of China Agricultural University, 2020, 25(11): 99−108.(in Chinese) doi: 10.11841/j.issn.1007-4333.2020.11.11 [8] 常飞, 吴文能, 曹晖. 白补药总黄酮含量测定方法的建立 [J]. 天然产物研究与开发, 2016, 28(1):71−75, 82.CHANG F, WU W N, CAO H. Determination of total flavonoids in Salvia scapiformis hance [J]. Natural Product Research and Development, 2016, 28(1): 71−75, 82.(in Chinese) [9] XIE Y, GUO Q S, WANG G S. Preparative separation and purification of the total flavonoids in Scorzonera austriaca with macroporous resins [J]. Molecules, 2016, 21(6): 768. doi: 10.3390/molecules21060768 [10] YE G H, TAO L Y, MA C L, et al. Influences of CCK-8 on expressions of apoptosis-related genes in prefrontal cortex neurons of morphine-relapse rats [J]. Neuroscience Letters, 2016, 631: 115−121. doi: 10.1016/j.neulet.2016.08.028 [11] SRIDHAR K, CHARLES A L. In vitro antioxidant activity of Kyoho grape extracts in DPPH and ABTS assays: Estimation methods for EC50 using advanced statistical programs [J]. Food Chemistry, 2019, 275: 41−49. doi: 10.1016/j.foodchem.2018.09.040 [12] ZHAO Z Y, ZHANG Q, LI Y F, et al. Optimization of ultrasound extraction of Alisma orientalis polysaccharides by response surface methodology and their antioxidant activities [J]. Carbohydrate Polymers, 2015, 119: 101−109. doi: 10.1016/j.carbpol.2014.11.052 [13] DAI C Y, LIU P F, LIAO P R, et al. Optimization of flavonoids extraction process in Panax notoginseng stem leaf and a study of antioxidant activity and its effects on mouse melanoma B16 cells [J]. Molecules, 2018, 23(9): 2219. doi: 10.3390/molecules23092219 [14] 徐金瑞, 张瑞芬, 潘国伟. 番石榴叶黄酮的微波提取及其抗氧化作用研究 [J]. 中国食品学报, 2010, 10(5):166−170. doi: 10.3969/j.issn.1009-7848.2010.05.024XU J R, ZHANG R F, PAN G W. Microwave extraction technology and antioxidation of flavonoid in guava leaf [J]. Journal of Chinese Institute of Food Science and Technology, 2010, 10(5): 166−170.(in Chinese) doi: 10.3969/j.issn.1009-7848.2010.05.024 [15] CHEN S S, ZENG Z, HU N, et al. Simultaneous optimization of the ultrasound-assisted extraction for phenolic compounds content and antioxidant activity of Lycium ruthenicum Murr. fruit using response surface methodology [J]. Food Chemistry, 2018, 242: 1−8. doi: 10.1016/j.foodchem.2017.08.105 [16] YU M, WANG B, QI Z, et al. Response surface method was used to optimize the ultrasonic assisted extraction of flavonoids from Crinum asiaticum [J]. Saudi Journal of Biological Sciences, 2019, 26(8): 2079−2084. doi: 10.1016/j.sjbs.2019.09.018 [17] ZHANG L, JIANG Y H, PANG X N, et al. Simultaneous optimization of ultrasound-assisted extraction for flavonoids and antioxidant activity of Angelica keiskei using response surface methodology (RSM) [J]. Molecules, 2019, 24(19): 3461. doi: 10.3390/molecules24193461 [18] ZHONG L J, LIU Y, XIONG B, et al. Optimization of ultrasound-assisted extraction of total flavonoids from Dendranthema indicum var. aromaticum by response surface methodology [J]. Journal of Analytical Methods in Chemistry, 2019, 2019: 1−10. [19] ZHU C P, ZHAI X C, LI L Q, et al. Response surface optimization of ultrasound-assisted polysaccharides extraction from pomegranate peel [J]. Food Chemistry, 2015, 177: 139−146. doi: 10.1016/j.foodchem.2015.01.022 [20] LIN X Y, BAI D P, WEI Z X, et al. Curcumin attenuates oxidative stress in RAW264.7 cells by increasing the activity of antioxidant enzymes and activating the Nrf2-Keap1 pathway [J]. PLoS One, 2019, 14(5): e0216711. doi: 10.1371/journal.pone.0216711 [21] ZHOU J, ZHANG L C, LI Q P, et al. Simultaneous optimization for ultrasound-assisted extraction and antioxidant activity of flavonoids from Sophora flavescens using response surface methodology [J]. Molecules (Basel, Switzerland), 2018, 24(1): 112−127. doi: 10.3390/molecules24010112