Optimization of Deep Eutectic Solvent Extraction Process of Polysaccharides from Dendrobium officinale
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摘要: 为提高铁皮石斛的综合利用率,建立一种绿色高效的铁皮石斛多糖提取方法。本研究以铁皮石斛多糖提取率为指标,通过单因素考察了低共熔溶剂浓度、提取温度及液料比对铁皮石斛多糖提取率的影响,采用响应面设计优化铁皮石斛多糖的提取工艺,并对纯化后的多糖进行结构分析。结果表明:响应面优化后得到最佳工艺为低共熔溶剂(deep eutectic solvent,DES)浓度40%、提取温度80 ℃、液料比110:1(mL/g),在此条件下实际提取率为33.2%±0.28%,与预测值33.5%接近,多糖的纯度为56.95%±1.2%。多糖经阴离子交换柱及葡聚糖凝胶柱纯化后,纯度可达90.8%,其单糖主要由葡萄糖和甘露糖构成,质量比约为43:37,此外还含有少量的木糖、鼠李糖、核糖等,结构中同时含有α糖苷键和β糖苷键。本研究提供了一种低共熔溶剂提取铁皮石斛多糖的高效绿色提取方案,具有多糖提取率高及绿色的特点,为后续铁皮石斛多糖的开发提供了借鉴。Abstract: In order to increase the comprehensive utilization of Dendrobium officinale, a green and efficient method for extracting of Dendrobium officinale polysaccharides (DOPs) was established. This research used the extraction rate of DOPs as the indicator for the effects of three factors. These included the concentration of deep eutectic solvent (DES), extraction temperature, and liquid-to-material ratio on the efficacy of polysaccharides from Dendrobium officinale, using single-factor tests. Response surface methodology (RSM) was employed to optimize the best extraction process of DOPs and the structure of the purified polysaccharides was analyzed. The experimental results showed that the optimal process obtained after RSM was a 40% concentration of DES, an extraction temperature of 80 ℃, and a liquid-to-material ratio of 110:1 (mL/g). Under these conditions, the actual extraction rate was 33.2%±0.28%, which was close to the predicted value of 33.5%, and the purity of polysaccharide was 56.95%±1.2%. After purification by anion-exchange column and glucose gel column, the purity could reach 90.8%, and its monosaccharides were mainly composed of glucose and mannose, with a mass ratio of 43:37. It also contained a small amount of xylose, rhamnose, and ribose, and the structure contained α-glycosidic and β-glycosidic bonds. The present research provides an efficient and green extraction scheme for extracting DOPS with DES, which has the characteristics of high polysaccharide extraction rate and greenness, providing a reference for the subsequent development of DOPs.
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铁皮石斛是兰科属草本植物,被称为“九大仙草之首”,含有多种活性物质,如多糖、氨基酸类和生物碱等[1]。其中,铁皮石斛多糖是铁皮石斛的主要功能活性成分,具有抗氧化、抗衰老、免疫调节、抗肿瘤和降血糖等作用[2−5]。因此,建立高效、绿色的铁皮石斛多糖提取工艺有利于铁皮石斛的药食两用价值的开发。邱现创等[6]通过超声辅助法提取铁皮石斛多糖,提取率可达25.39%。廖霞等[7]利用微波辅助结合酶解提取法,铁皮石斛多糖提取率可达29.40%。李贝贝等[8]研究了闪式提取法提取铁皮石斛多糖,在最佳优化工艺条件下浸泡30 min后,常温下提取3次,提取率达到31.45%。Kui等[9]从石斛中提取出了3种水溶性多糖,其主要由甘露糖和葡萄糖以不同的比例组成。高云霄等[10]用热水浸提法从铁皮石斛中提取得到一种以1,4-链接为主,存在少量1,2,4-、1,3,4-、1,4,6-分支结构和端基结构的O-乙酰化葡甘露聚糖。铁皮石斛多糖的结构是其呈现生物活性的基础,选择不同的提取、纯化分离方法,所得到的多糖在单糖组成及多糖结构上会有显著不同,而这往往会直接影响多糖的生物活性。
低共熔溶剂(deep eutectic solvent,DES)由氢键受体和氢键供体混合而成[11]。DES已被证明是一种新型绿色溶剂,具有成本低、易合成、无毒和可生物降解等优异性能[12−14]。其现已被用于功能成分的提取,如提取槲皮素[15−16]、提取甜菜碱[17−18]及提取姜黄素[19−20],目前,已有部分研究将DES的绿色提取工艺用于铁皮石斛总黄酮的提取[21],但其在铁皮石斛多糖的提取中应用较少。
DES具有安全性高、价格低廉及绿色等特点,课题组前期对近40种不同比例和原料的DES进行了筛选,发现基于氯化胆碱及乳酸的DES产生的氢键数最多[22]。因此,本研究选择氯化胆碱与乳酸作为制作DES的原料,利用响应面法优化铁皮石斛多糖的提取工艺。在铁皮石斛多糖提取后,进一步研究其单糖组成及可能的分子结构,以期为铁皮石斛多糖的高效提取提供理论依据。
1. 材料与方法
1.1 材料与仪器
铁皮石斛 购自云山斛皮石斛基地;苯酚、浓硫酸、乳酸、鼠李糖(纯度98%)、半乳糖(98%)、核糖(纯度>99.5%)、木糖(纯度99%) 国药集团化学试剂有限公司;氯化胆碱、阿拉伯糖(纯度98%) 杭州吉工生物科技有限公司;无水硫酸钠 杭州双木化工有限公司;DEAE-52纤维素柱填料、Sephadex G-100 上海源叶生物科技有限公司;葡萄糖(纯度99.7%)、乙酸酐 杭州邦易化工有限公司;无水乙醇、甘露糖(纯度99%) 杭州方平化工有限公司;D2O 安徽泽生科技有限公司;三氟乙酸 德国Merck公司;盐酸羟胺、吡啶 上海泰坦科技股份有限公司;所用试剂均为分析纯;实验室用水均为超纯水。
HWS-12型数显恒温水浴锅 常州智博瑞仪器制造有限公司;P7型紫外分光光度计 北京高能科迪科技有限公司;RE-52型旋蒸仪 上海锦赋实验仪器设备有限公司;DDSJ-308A型电导率测定仪 雷滋仪电科学仪器股份有限公司;Bruker AM-600型核磁共振 Bruker Physik-AG公司;Finnigan Trace Ultra-DSQ II单四级杆气相色谱-质谱联用仪器 Thermo公司。
1.2 实验方法
1.2.1 DES试剂的制备
参考DES的制作方法[23−25]并稍作修改,将氯化胆碱与乳酸以摩尔比4:1称量20 g氯化胆碱,50 g乳酸,置于蓝盖瓶中,后加入21 mL的去离子水混合,置于80 ℃的数显恒温水浴锅内加热至大部分试剂熔化,再转移至磁力加热搅拌器上,在80 ℃下不断搅拌获得均一透明的液体,即为DES,然后按照添加水稀释到不同浓度的DES,贮藏备用。
1.2.2 铁皮石斛粗多糖的提取
称取约1.00 g铁皮石斛粉末,按照一定的液料比加入不同浓度的DES,设定提取温度及提取时间,提取完成后离心(4000 r/min、20 min),取上清液,加入其4倍体积的无水乙醇进行沉淀,并常温静置过夜,第二天进行离心去上清液后复溶铁皮石斛粗多糖,利用旋蒸去除乙醇,得到铁皮石斛粗多糖。
1.2.3 单因素实验
以铁皮石斛多糖提取率为指标,参照李佳等[2]选取2 h为本试验的提取时间,温度设置为70 ℃,液料比为90:1 mL/g,考察DES浓度(20%、30%、40%、50%、60%);DES浓度为30%,液料比为90:1 mL/g,考察提取温度(50、60、70、80、90 ℃);温度设置为70 ℃,DES浓度为30%,考察液料比(50:1、70:1、90:1、110:1、130:1 mL/g)对铁皮石斛多糖提取率的影响。
1.2.4 响应面试验
依据单因素实验结果,运用 Design-Expert 8.0.6软件进行 Box-Behnken 试验,采用三因素三水平(A:DES浓度、B:提取温度、C:液料比)进行响应面试验设计,考察铁皮石斛多糖提取率变化,因素水平设计见表1。
表 1 响应面设计因素与水平Table 1. Factors and levels in response surface design水平 因素 A:DES浓度(%) B:提取温度(℃) C:液料比(mL/g) −1 30 70 90:1 0 40 80 110:1 1 50 90 130:1 1.2.5 阴离子交换柱纯化多糖
将所得粗铁皮石斛多糖粉末溶解,用微孔滤膜去除杂质后,经压力泵上样注入DEAE-52阴离子交换柱,采用 0、0.05、0.1、0.3、0.5 mol/L的氯化钠溶液进行梯度洗脱[13]。利用自动收集器收集洗脱液,每管收集体积为10 mL。采用苯酚-硫酸法测定每管洗脱液的多糖含量,并测定其在490 nm下的紫外分光光度,以管数为横坐标,多糖吸光度为纵坐标,绘制洗脱曲线。合并相同组分洗脱液浓缩冻干,得到纯多糖。
1.2.6 葡聚糖凝胶柱纯化多糖
对经阴离子交换柱纯化后所得的铁皮石斛多糖洗脱组分进行葡聚糖凝胶柱纯化[14],称取铁皮石斛多糖溶于蒸馏水中,配制成浓度为10 mg/mL的溶液,定容后用微孔滤膜过滤,经Sephadex G-100凝胶色谱柱进行纯化,用蒸馏水作为洗脱液,用自动收集器收集,每管收集体积8 mL。采用苯酚-硫酸法测定多糖含量,以管数为横坐标,多糖吸光度为纵坐标,绘制洗脱曲线。收集所得到的多糖溶液进行冻干得到铁皮石斛精多糖。
1.2.7 多糖提取率及纯度测定方法
葡萄糖标准曲线制备:采用苯酚-硫酸法[26]制作标准曲线,以吸光度为纵坐标,浓度为横坐标,绘制标准曲线。回归方程为y=0.9229x−0.0144,R2=0.9989。按照1.2.2的方法提取操作重复3次,用苯酚-硫酸法比对标准曲线测定总糖含量。参照贾夏等[27]的3,5−二硝基水杨酸(DNS)比色法测定还原糖的浓度,并计算多糖的纯度。
式中:M总,总糖的质量,g;M还,还原糖的质量,g;M,粗多糖质量,g。
按下列公式计算多糖提取率:
式中:M多糖,粗多糖中多糖的质量,g;m,称取的铁皮石斛粉的质量,g。
1.2.8 铁皮石斛多糖的单糖组成测定
参照Anna等[28]并稍作修改,精确称取10.0 mg铁皮石斛多糖至具塞试管,加入6 mL 2 mol/L三氟乙酸,密封置于110 ℃烘箱中水解2 h,冷却后加入2 mL甲醇旋转蒸发,再依次加入盐酸羟胺10 mg、吡啶2.5 mL,90 ℃水浴加热30 min,冷却后加入乙酸酐2.5 mL,90 ℃水浴加热30 min,冷却,即得多糖衍生化物。其余各单糖标准品的制备方法同上。最后进行GC-MS测定[19]。
1.2.9 铁皮石斛多糖的核磁共振测试
参照Xi等[29]并作修改,精确称取100.0 mg干燥的铁皮石斛多糖用D2O溶解后,冷冻干燥,如此反复3次,将溶解后的多糖置于核磁管中在Bruker AM-600型核磁共振仪上进行均一多糖的核磁谱图测定,扫描时间为8 h,对其进行1H-NMR和13C-NMR分析,得到核磁共振图谱。
1.3 数据处理
采用软件Design-Expert.V8.0.6.1进行响应面分析,数据通过Graphpad Prism 8软件进行计算绘图。采用多重比较法进行显著性分析(P<0.05表示差异显著)。实验数据为三组数据平均值,以平均值±标准偏差()表示。
2. 结果与分析
2.1 单因素实验
2.1.1 DES浓度对铁皮石斛多糖提取率的影响
DES浓度的变化对铁皮石斛多糖提取率的影响如图1所示。由图1可知,铁皮石斛多糖的提取率呈先上升后逐渐下降的趋势,当DES的浓度从20%升到40%时多糖提取率从15.1%升到30.7%,原因可能是随着DES浓度的提高,DES中氢键受体和配体的数量逐渐增加,导致氢键数目会增加[22],让更多的多糖渗入到提取液中,从而提高了多糖提取率。当DES浓度继续提高,多糖的提取率逐渐下降,这是因为DES试剂是由氯化胆碱和乳酸配制而成,试剂内部离子力通常较强且黏度较大,不利于溶质的溶出,因此在使用过程中加入适量的蒸馏水来降低溶液的剪切力[30]。因此在40%DES浓度时更有利于铁皮石斛多糖的提取,并选取DES浓度为30%、40%、50%做响应面试验以确定最佳DES浓度。
2.1.2 温度对铁皮石斛多糖提取率的影响
温度变化对铁皮石斛多糖提取率的影响如图2所示。由图2可知,铁皮石斛多糖的提取率变化呈缓慢上升后下降的趋势,当温度从50 ℃升高到80 ℃时,多糖提取率最高,从14.2%升高到23.4%,是因为随着温度的升高,增加了物质的热运动,提高了多糖分子扩散效率,使多糖在整个体系中的溶解更迅速。当温度继续提升到90 ℃,多糖的提取率反而略有下降。一方面,可能是因温度继续升高时,铁皮石斛多糖中可能存在热敏性物质,在温度升高条件下会使得这些成分易变性降解[31],另一方面,较高温度导致蒸发,影响了溶剂平衡,从而降低了提取率。因此,选取提取温度为70、80、90 ℃做响应面试验以确定最佳提取温度。
2.1.3 液料比对铁皮石斛多糖提取率的影响
液料比对铁皮石斛多糖提取率的影响如图3所示,铁皮石斛多糖的提取率变化呈现先上升后下降的趋势,当液料比为110:1时提取率最高,从18.1%升高到32.2%,可能是由于DES为黏稠液体,在一定范围内,随着液料比提高,溶剂与铁皮石斛的接触面积增多,增加了多糖的溶出,使得多糖提取率增加;但溶剂加入过多时,溶剂用量较大,铁皮石斛粉与溶剂接触面积不断增大,溶出了其他杂质,导致多糖溶解被抑制[32]。因此,选取90:1、110:1、130:1(mL/g)做响应面试验以确定最佳液料比。采用DES不仅对铁皮石斛多糖实现了绿色提取,同时在提取率上也优于超声辅助、微波辅助结合酶解及闪式提取[6−8]。
2.2 响应面试验
2.2.1 响应面试验设计与结果
在铁皮石斛多糖提取的过程中,以DES浓度、提取温度和液料比为影响因素,多糖提取率为响应值。试验设计与结果见表2。
表 2 响应面优化试验设计及结果Table 2. Designs and results of Box-Behnken test试验号 A:DES浓度 B:温度 C:液料比 Y:提取率(%) 1 −1 1 0 19.5 2 0 0 0 33.9 3 −1 0 1 20.4 4 −1 0 −1 19.1 5 1 −1 0 22.3 6 −1 −1 0 18.3 7 0 0 0 33.6 8 1 1 0 21.8 9 0 −1 1 20.9 10 0 1 −1 20.6 11 0 0 0 33.2 12 1 0 −1 21.9 13 0 0 0 33.2 14 1 0 1 23.8 15 0 1 1 22.3 16 0 0 0 33.7 17 0 −1 −1 19.8 2.2.2 响应面二次回归模型的建立与方差分析
由表2的试验结果,用响应面分析软件操作,得出铁皮石斛多糖提取率为响应值 Y的回归方程:Y=33.55+1.56A+0.36B+0.75C−0.42AB+0.14AC+0.13BC−6.29A2−6.75B2−5.92C2。
由表3方差分析结果可知,该模型的F=681.25,回归模型项 P<0.0001,失拟项P>0.05,为不显著,说明试验误差小[33]。试验结果与模型拟合度好[34]。由F检验可知,影响铁皮石斛多糖提取率的主次因素为DES浓度>液料比>提取温度,方差分析显示,A、C 对 Y影响极显著(P<0.001),B对 Y影响显著(P<0.05),A2、B2、C2都是极显著的模型项(P<0.001)。
表 3 回归模型的方差分析结果Table 3. Regression model of variance analysis results来源 平方和 自由度 均方和 F P 模型 591.32 9 65.70 681.25 <0.0001** A 19.53 1 19.53 202.51 <0.0001** B 1.06 1 1.06 10.98 0.0129* C 4.52 1 4.52 46.82 0.0002** AB 0.70 1 0.70 7.23 0.0311* AC 0.076 1 0.076 0.78 0.4053 BC 0.063 1 0.063 0.65 0.4373 A2 166.80 1 166.80 1729.48 <0.0001** B2 191.64 1 191.64 1987.10 <0.0001** C2 147.64 1 147.64 1530.83 <0.0001** 残差 0.68 7 0.096 失拟项 0.31 3 0.10 1.11 0.4445 纯误差 0.37 4 0.092 总和 591.99 16 注:*(P<0.05)表示差异显著;**(P<0.01)表示差异极显著。 2.2.3 响应面曲面分析因素之间的交互作用
用Design-Expert 8.6 软件,对DES浓度、提取温度、液料比3个因素两两交互,做不同因素间响应面3D图,响应面图和等高线图可以反映各因素间的相互作用及最佳参数。三维图越陡峭[35],表明相应因素对多糖提取率影响较大。图4B等高线图为近圆形可知液料比和DES浓度的交互作用对响应值的影响不显著;由图4C可知,铁皮石斛多糖的提取率随液料比及提取温度的增加呈先上升后下降的趋势,等高线图为近圆形说明对铁皮石斛多糖提取率无显著影响。由图4知,两因素交互项AB>AC>BC,其中仅有AB的P<0.05,表明AB两因素间交互作用对响应值的影响显著,随着提取温度的升高,分子运动加快,提高了溶剂的渗透能力,提取率随之升高,但温度过高可能会对多糖分子结构产生破坏作用[36]。
2.2.4 响应面优化与验证实验
利由响应面软件分析显示预测最佳提取工艺条件为DES浓度39.5%、提取温度80.1 ℃、液料比112.4:1(mL/g),铁皮石斛多糖提取率为33.5%。根据实际情况调整提取参数为DES浓度40%、提取温度 80 ℃、液料比110:1(mL/g),在此条件下提取2 h,得出验证结果多糖提取率为 33.2%±0.28%,拟合度好,误差为1.7%,充分验证了模型的准确性,该工艺优化合理、有效。多糖的纯度为56.95%±1.2%。与传统的提取方法相比较,本文采用DES溶剂提取铁皮石斛多糖实现了较高的提取率,陈盛余等[37]采用微波辅助提取铁皮石斛多糖,其提取率为9.77%,而王琳等[38]采用热水浸提法提取铁皮石斛多糖,其提取率为30.83%,均较DES溶剂提取率低,此外本研究提取方法绿色、安全性高,可以为后续的提取提供一种高效绿色的方案。
2.3 阴离子交换柱纯化
如图5所示,DOP纯化后得到3个多糖洗脱组分,DOP1为蒸馏水洗脱得到的组分;DOP-2为0.05 mol/L NaCl溶液洗脱下的组分;DOP-3为0.1 mol/L NaCl溶液洗脱下的组分。由洗脱图谱可见三个组分均未出现拖尾现象,表明各组分间分离效果较好,与DOP-2和DOP-3相比较,DOP-1组分相对含量高,占比为72.6%。洗脱过程在260和280 nm波长处未出现特征性吸收峰,这表明其不包含游离的蛋白质和核苷酸[39],达到初步纯化的效果。
2.4 葡聚糖凝胶柱纯化
继续采用Sephadex G-100色谱柱对DOP-1进一步纯化,结果如图6所示。DOP-1获得一个集中的单峰,表明其纯度较高。收集8~30管DOP-1的洗脱液并进行冷冻干燥得到纯化多糖,测定其纯度为 90.8%,命名其为DOP-1-1。
2.5 铁皮石斛的单糖组成分析
采用GC-MS对DOP-1-1进行单糖组成分析,多糖的总离子流色谱图如图7所示。通过对比标准单糖(图7A)可知,铁皮石斛多糖组分主要由葡萄糖和甘露糖单糖组成,根据峰面积计算葡萄糖和甘露糖的总和占比80%,葡萄糖和甘露糖质量比约为43:37,此外还含有少量的木糖、鼠李糖、核糖等。表明DOP-1-1是一种葡甘露聚糖为主的杂多糖。
2.6 铁皮石斛多糖的核磁共振分析
铁皮石斛多糖的1H NMR(图8A)显示9个异头氢,其化学位移δ分别为1.85、2.13、3.29、3.50、3.76、4.07、4.70、5.35、5.45;13C NMR谱图(图8B)显示有7个信号峰,其化学位移δ分别为20.28、60.42、69.99、71.54、75.00、76.53、100.16。在4.070~4.741 ppm与4.036~4.703 ppm处的强烈异头质子信号峰是由D2O中的HDO所引起的[40]。1H NMR光谱中δ3.76 ppm和13C NMR光谱中δ71.54 ppm处的强烈信号提示其可能含有(1→3)糖苷键的连接方式[41]。1H NMR光谱中δ3.29 ppm和13C NMR光谱中δ69.99 ppm处的强烈信号提示多糖中存在(1→4)糖苷键[27]。在δ3.50 ppm处的信号峰提示提示可能存在(1→2,4)糖苷键[42]。1H NMR光谱中δ5.45 ppm的化学位移来自1,3-Galp的H1。此外1H NMR一般用于研究多糖糖苷键的结构特点[43],α糖苷键的质子信号通常集中在大于5 ppm的位置,而β糖苷键的化学位移一般低于5 ppm,表明其同时含有α糖苷键和β糖苷键。
3. 结论
本文通过DES溶剂提取铁皮石斛多糖的结果表明,响应面试验得到最佳提取工艺为DES浓度40%、提取温度 80 ℃、液料比110:1(mL/g),此条件下铁皮石斛多糖提取率达 33.2%±0.28%,多糖的纯度为56.95%±1.2%。在纤维素柱与凝胶柱纯化后,多糖的纯度可达90.8%,多糖组分主要由葡萄糖和甘露糖构成,其质量比约为43:37,还含有少量的木糖、鼠李糖、核糖等,结构中同时含有α糖苷键和β糖苷键,采用DES法提取多糖,可以有效提高多糖提取率。本研究建立了一套绿色、高效的铁皮石斛多糖提取方法,为后续的铁皮石斛多糖开发提供参考。
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表 1 响应面设计因素与水平
Table 1 Factors and levels in response surface design
水平 因素 A:DES浓度(%) B:提取温度(℃) C:液料比(mL/g) −1 30 70 90:1 0 40 80 110:1 1 50 90 130:1 表 2 响应面优化试验设计及结果
Table 2 Designs and results of Box-Behnken test
试验号 A:DES浓度 B:温度 C:液料比 Y:提取率(%) 1 −1 1 0 19.5 2 0 0 0 33.9 3 −1 0 1 20.4 4 −1 0 −1 19.1 5 1 −1 0 22.3 6 −1 −1 0 18.3 7 0 0 0 33.6 8 1 1 0 21.8 9 0 −1 1 20.9 10 0 1 −1 20.6 11 0 0 0 33.2 12 1 0 −1 21.9 13 0 0 0 33.2 14 1 0 1 23.8 15 0 1 1 22.3 16 0 0 0 33.7 17 0 −1 −1 19.8 表 3 回归模型的方差分析结果
Table 3 Regression model of variance analysis results
来源 平方和 自由度 均方和 F P 模型 591.32 9 65.70 681.25 <0.0001** A 19.53 1 19.53 202.51 <0.0001** B 1.06 1 1.06 10.98 0.0129* C 4.52 1 4.52 46.82 0.0002** AB 0.70 1 0.70 7.23 0.0311* AC 0.076 1 0.076 0.78 0.4053 BC 0.063 1 0.063 0.65 0.4373 A2 166.80 1 166.80 1729.48 <0.0001** B2 191.64 1 191.64 1987.10 <0.0001** C2 147.64 1 147.64 1530.83 <0.0001** 残差 0.68 7 0.096 失拟项 0.31 3 0.10 1.11 0.4445 纯误差 0.37 4 0.092 总和 591.99 16 注:*(P<0.05)表示差异显著;**(P<0.01)表示差异极显著。 -
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