Response Surface Optimization of Flavonoid Extraction and in Vitro Antioxidant Activity of Extract from Dendrobium Chrysotoxum Lindl. Flowers
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摘要:
目的 优化鼓槌石斛花总黄酮提取工艺条件，为鼓槌石斛花种质的开发提供技术参考。 方法 以鼓槌石斛花为原料，乙醇溶液为提取剂，在单因素的基础上，选择乙醇体积分数、液料比、超声温度、超声时间4个因素为影响因素，鼓槌石斛花总黄酮提取率为响应值，采用Box-Behnken试验设计方法，研究各自变量及其交互作用对鼓槌石斛花总黄酮提取率的影响。并通过鼓槌石斛花总黄酮对1，1-二苯基-2-三硝基苯肼（DPPH）自由基、羟自由基的清除作用评价其抗氧化活性。 结果 鼓槌石斛花总黄酮最佳提取工艺条件为：乙醇体积分数83%，液料比31 mL·g-1，超声温度59℃，超声时间44 min，在此条件下，得到的实际提取率9.71 mg·g-1，与理论值（9.60 mg·g-1）相对误差为1.15%。采用响应面法优化鼓槌石斛花总黄酮提取工艺切实可行，回归模型拟合度及重现性较好。鼓槌石斛花黄酮对DPPH自由基半清除浓度（IC50）值为11.30 μg·mL-1，其清除能力是L-抗坏血酸的0.51倍、2，6-二叔丁基对甲酚（BHT）的1.42倍；对羟自由基IC50值为137.26 μg·mL-1，其清除能力是L-抗坏血酸的0.70倍、BHT的1.95倍。体外抗氧化活性试验表明，相同浓度下其对两种自由基清除能力优于BHT，弱于L-抗坏血酸。 结论 采用响应面法优化得出的鼓槌石斛花总黄酮提取的最佳工艺条件，能有效提高总黄酮提取率。 Abstract:Objective Extraction process of flavonoids from the flowers of Dendrobium chrysotoxum Lindl. was optimized, and the antioxidative activity of the extract determined in vitro. Method Flowers of D. chrysotoxum was subjected to an ethanol extraction for flavonoids. The resulting yield was weighed against the processing conditions based on the single-factor tests and Box-Benhnken center composite experiment with four factors including ethanol volume fraction, solvent-to-substrate ratio, temperature, and time of ultrasonic treatment. The antioxidant capacity was estimated by the ability of the extract to scavenge 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl free radicals. Result The optimized processing conditions were determined to include ethanol volume fraction at 83%, solvent-to-substrate ratio at 31 mL·g-1, temperature at 59℃ and ultrasound application for 44m to obtain up to 9.71 mg·g-1 of total flavonoids in the extract. The yield exceeded the theoretically expected 9.60 mg·g-1 with a relative deviation of 1.15%. The in vitro tests showed an IC50 for DPPH to be 11.30 μg·mL-1, a scavenging ability 0.51-time of L-ascorbic acid or 1.42-time of BHT, while that for hydroxyl radicals, 137.26 μg·mL-1, a scavenging ability 0.70-time of L-ascorbic acid or 1.95-time of BHT. Conclusion The optimized flavonoid extraction paved the way for scale-up utilization of D. chrysotoxum flowers. -
图 1 各单因素对鼓槌石斛花总黄酮提取率的影响
Figure 1. Effect of various factors on yield of flavonoid extraction from D. chrysotoxum flowers
图 2 各因素交互作用对总黄酮提取率影响的响应面
Figure 2. Response surface of interactive of various factors on flavonoid yield
图 3 鼓槌石斛花总黄酮对DPPH自由基的清除效果
Figure 3. Scavenging capacity of flavonoid extract of D. chrysotoxum flowers on DPPH free radicals
图 4 鼓槌石斛花总黄酮对羟自由基的清除效果
Figure 4. Scavenging capacity of flavonoid extract of D. chrysotoxum flowers on hydroxyl free radicals
表 1 响应面分析因素及水平
Table 1. Factors and levels of response surface methodology
因素
Factor水平Levels -1 0 1 乙醇体积分数Ethanol concentration/% 70 80 90 液料比Liquid-solid ratio/(mL·g-1) 20 30 40 超声温度Ultrasonic temperature/℃ 50 60 70 超声时间Ultrasonic time/min 25 35 45 
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表 2 响应面设计方案及结果
Table 2. Design and results of response surface experiments
试验号
No.因素Factor 提取率
Yield/(mg·g-1)A B C D 1 80 30 60 35 9.34 2 70 30 60 25 7.11 3 90 20 60 35 7.42 4 80 30 60 35 9.56 5 80 20 60 45 8.91 6 70 20 60 35 6.97 7 90 30 60 25 8.55 8 70 30 50 35 7.03 9 80 40 60 45 8.96 10 90 40 60 35 8.25 11 80 30 60 35 9.44 12 80 30 70 45 8.47 13 80 30 50 45 8.36 14 90 30 70 35 8.38 15 90 30 50 35 8.48 16 80 20 50 35 7.35 17 80 40 70 35 8.29 18 80 20 60 25 7.22 19 80 30 50 25 7.11 20 80 30 60 35 9.34 21 80 30 60 35 9.01 22 70 30 60 45 8.40 23 80 20 70 35 7.53 24 80 30 70 25 9.06 25 70 30 70 35 8.23 26 80 40 60 25 7.48 27 80 40 50 35 7.59 28 90 30 60 45 9.18 29 70 40 60 35 6.70
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表 3 响应模型方差分析
Table 3. Variance analysis of response model
方差来源
Source of variance平方和
Sum of squares自由度
DOF均方
Mean squareF值
F valueP值
P value显著性
Significance模型Model 19.34 14 1.38 11.94 ＜0.0001 ** A 2.83 1 2.83 24.46 0.0002 ** B 0.29 1 0.29 2.51 0.1351 C 1.38 1 1.38 11.90 0.0039 ** D 2.74 1 2.74 23.70 0.0002 ** AB 0.30 1 0.30 2.62 0.1276 AC 0.43 1 0.43 3.73 0.0741 AD 0.11 1 0.11 0.95 0.3471 BC 0.07 1 0.07 0.59 0.4566 BD 0.01 1 0.01 0.09 0.7678 CD 0.85 1 0.85 7.32 0.0170 * A2 4.06 1 4.06 35.06 ＜0.0001 ** B2 7.08 1 7.08 61.22 ＜0.0001 ** C2 2.69 1 2.69 23.26 0.0003 ** D2 0.50 1 0.50 4.31 0.0567 残差Residual 1.62 14 0.12 失拟度Lack of fit 1.45 10 0.15 3.50 0.1191 绝对误差Pure error 0.17 4 0.04 总离差Cor total 20.96 28 注：*表示差异显著(P＜0.05)；**表示差异极显著(P＜0.01)。
Note：*, significant difference(P＜0.05); **, extreme significant difference(P＜0.01).
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