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中国精品科技期刊2020

菊苣苦味素提取工艺优化及其体外抗氧化活性分析

韩舒晨, 何天枫, 曹荣安, 李朝阳, 李良玉

韩舒晨,何天枫,曹荣安,等. 菊苣苦味素提取工艺优化及其体外抗氧化活性分析[J]. 食品工业科技,2024,45(18):165−174. doi: 10.13386/j.issn1002-0306.2023090104.
引用本文: 韩舒晨,何天枫,曹荣安,等. 菊苣苦味素提取工艺优化及其体外抗氧化活性分析[J]. 食品工业科技,2024,45(18):165−174. doi: 10.13386/j.issn1002-0306.2023090104.
HAN Shuchen, HE Tianfeng, CAO Rong'an, et al. Optimization of the Extraction Process for Chicory Bitter Principles and Analysis of Its in Vitro Antioxidant Activity[J]. Science and Technology of Food Industry, 2024, 45(18): 165−174. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023090104.
Citation: HAN Shuchen, HE Tianfeng, CAO Rong'an, et al. Optimization of the Extraction Process for Chicory Bitter Principles and Analysis of Its in Vitro Antioxidant Activity[J]. Science and Technology of Food Industry, 2024, 45(18): 165−174. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023090104.

菊苣苦味素提取工艺优化及其体外抗氧化活性分析

基金项目: 黑龙江省教育厅黑龙江省“双一流”学科协同创新成果项目(LJGXCG2023-043);中央引导地方科技发展资金 项目(ZY23QY16)。
详细信息
    作者简介:

    韩舒晨(1995−),男,硕士研究生,研究方向:天然活性物质提纯与应用,E-mail:969210023@qq.com

    通讯作者:

    李良玉(1982−),男,博士,副研究员,研究方向:天然活性物质提纯与应用,E-mail:Liliangyu6@163.com

  • 中图分类号: TS201.1

Optimization of the Extraction Process for Chicory Bitter Principles and Analysis of Its in Vitro Antioxidant Activity

  • 摘要: 为充分利用菊苣资源,探究菊苣生产菊粉加工副产品中菊苣苦味素利用价值,本文在单因素实验的基础上,采用超声波结合响应面法对菊苣根苦味素进行提取与参数优化,以获得最佳提取参数,以液质分析对菊苣苦味素进行了成分分析,最后对菊苣苦味素的体外抗氧化活性进行了研究。结果表明:在乙醇浓度80%、超声温度35 ℃、超声时间24 min、超声功率450 W,液料比33:1 mL/g条件下,菊苣苦味素的得率为1.18%±0.015%。液质分析结果显示,菊苣苦味素主要含有7种化合物,分别为地胆草丁、秦皮甲素、秦皮乙素、绿原酸、莴苣酸、山莴苣素及山莴苣苦素。体外抗氧化活性实验结果表明,菊苣苦味素对DPPH·、ABTS+·、羟自由基清除能力的半数清除浓度(IC50)分别为0.271、0.0734和0.130 mg/mL,且清除能力与浓度呈现浓度依赖性;菊苣苦味素浓度为1.0 mg/mL时,菊苣苦味素的总还原能力达到了1.108,研究为菊苣苦味素的利用提供了理论和实践基础。
    Abstract: To fully utilize chicory resources and explore the value of chicory bitterness in by-products from chicory powder processing, this study based on single-factor experiments, used ultrasonic technology combined with response surface methodology to extract chicory root bitters and optimize the parameters, aiming to obtain the optimal extraction parameters. Meanwhile, liquid chromatography-mass spectrometry (LC-MS) was used to analyze the components of chicory bitterness. Finally, the in vitro antioxidant activity of chicory bitters was investigated. The results showed that under the conditions of 80% ethanol concentration, ultrasonic temperature of 35 ℃, ultrasonic time of 24 min, ultrasonic power of 450 W, and liquid-to-material ratio of 33:1 mL/g, the yield of chicory bitterness was 1.18%±0.015%. Liquid chromatography-mass spectrometry analysis revealed seven main compounds in chicory bitterness, including andrographidine, quinpirole A, quinpirole B, chlorogenic acid, lactucinic acid, lactucin, and lactucopicrin. The partial in vitro antioxidant activity analysis of chicory bitterness indicated IC50 values of 0.271, 0.0734, and 0.130 mg/mL for DPPH·, ABTS+·, and hydroxyl radicals, respectively, showing a concentration-dependent clearance ability. Additionally, at a concentration of 1.0 mg/mL, chicory bitterness exhibited a total reducing power of 1.108. This study provides a theoretical and practical basis for the utilization of chicory bitterness.
  • 菊苣(Cichorium intybus L.)是一种多年生植物,其起源可以追溯到古代欧洲,在我国的东北、华北及西北等地分布广泛[12]。嫩叶、叶球和叶芽是其主要的食用部分,因独特的苦味和丰富的营养价值而作为美食被广泛食用[34]。在我国,菊苣主要用于生产具有多种功能特性菊粉[59]。此外,菊苣根还含有许多其他活性成分,如酚类、糖类、黄酮类、萜内酯类和香豆素类等[1013],但这些物质在菊粉加工过程中并未得到充分利用。尤其是苦味素,制备菊粉时常采用洗脱苦味素的工艺技术,这一过程使得苦味素随着其他废弃物被一同丢弃[1416],导致了许多有效物质的浪费。

    苦味素(Bitter principles),指一类富含苦味的化合物,包括了一萜类、倍半萜类、二萜类和三萜类等多种不同类型的化合物[17]。菊苣含有的苦味素与其具有的清肝利胆、清热解毒、利尿消肿、降血脂血糖等功效有关[1821],包括马栗树皮素(Esculetin)、马栗树皮苷(Esculin)、山莴苣素(Lactucin)、山莴苣苦素以及野莴苣苷(Cichoriin)等成分[22]。其中,山莴苣苦素属倍半萜内酯类,对恶性疟原虫具有良好的抗疟功能[23]。另外,菊苣中的倍半萜内酯具有抗炎作用,这为开发新的保健品提供了新的方向[2425]。因此,对菊苣深入探究其提取方法并进行改进,并评估其在体外的抗氧化性能,充分利用其含有的苦味素,是非常必要且有潜力的[2629]

    本研究采用超声波辅助提取法,用以提取菊苣中的苦味素。响应面法对提取工艺进行优化,为菊苣苦味素的工业化生产和应用提供参考。同时也对菊苣苦味素的抗氧化活性进行了评估,为开发天然抗氧化剂提供理论支持,也促进了我国菊苣产业的健康发展和菊苣资源的利用最大化[3031]

    菊苣根 2022年采自毫州市口袋医生生物科技有限公司,产地吉林长白山地区;香草醛、无水乙醇、高氯酸、乙酸乙酯、冰醋酸 天津市科密欧化学试剂有限公司;DPPH、ABTS 分析纯,Sigma公司。

    T6紫外可见光分光光度计 北京普析通用仪器有限责任公司;JY99-IIDN超声波细胞粉碎机 宁波新芝生物科技股份有限公司;UPLC-Triple-TOF 5600+超高效液相色谱串联飞行时间质谱 美国AB SCIEX公司; ACQUITY UPLC HSS T3柱(1.8 μm,2.1 mm×150 mm) 沃特世科技(上海)有限公司。

    在50 ℃的条件下,将菊苣根置于烘箱中烘干,随后进行粉碎并过100目筛,最终获得菊苣粉。取20 g上述菊苣粉于烧杯中,根据实验设计加入不同浓度的乙醇溶液,溶解并摇匀,随后进行超声提取。超声完成后,以4000 r/min的速度离心15min,后将上清液倒入锥形瓶中。对于剩余的残渣,按照之前的步骤再次操作一次,将其与第一次获得的上清液合并并备用。

    探究不同浓度乙醇提取液(50%、60%、70%、80%、90%)对菊苣苦味素得率的影响。在固定其他工艺参数的前提下,设定超声时间为25 min、超声功率500 W、超声温度35 ℃、液料比20:1 mL/g。

    探究不同超声温度(20、25、30、35、40 ℃)对菊苣苦味素得率的影响。固定乙醇浓度70%,其余参数同1.2.2.1。

    探究不同超声时间(10、15、20、25、30 min)对菊苣苦味素得率的影响,其余参数同1.2.2.1。

    探究不同超声功率(100、200、300、400、500 W)对菊苣苦味素得率的影响,其余参数同1.2.2.1。

    探究不同液料比(10:1、15:1、20:1、25:1、30:1、35:1、40:1 mL/g)对菊苣苦味素得率的影响,其余参数同1.2.2.1。

    基于前述实验结果,遵循Box-Behnken原则,以菊苣苦味素得率为响应值,超声温度、超声时间、超声功率、液料比为响应面因素,具体试验设计如表1所示[32]

    表  1  响应面因素与水平设计
    Table  1.  Response surface factors and level design
    编码值 X1超声温度(℃) X2超声时间(min) X3超声功率(W) X4液料比(mL/g)
    −2 20 20 300 20:1
    −1 25 22.5 350 25:1
    0 30 25 400 30:1
    +1 35 27.5 450 35:1
    +2 40 30 500 40:1
    下载: 导出CSV 
    | 显示表格

    分别取(0.1、0.2、0.3、0.4、0.5 mL)10 mg/mL山莴苣苦素标准溶液于试管中,加热至挥发。分别加入0.4 mL 5%香草醛-冰醋酸和0.8 mL的高氯酸溶液,60 ℃水浴20 min。冷却至室温,用乙酸乙酯定容至5 mL[33]。山莴苣苦素浓度作为横坐标,于560 nm处测吸光度值为作为纵坐标,空白试剂作对照。经线性回归分析,得到标准曲线回归方程为Y=0.2718X+0.0039,决定系数R2为0.9991。

    按1.2.4.1步骤测得1 mL菊苣苦味素提取液吸光度,按标准曲线计算山莴苣苦素含量。按公式(1)计算山莴苣苦素得率。

    W(%)=c×vm×1000×100
    (1)

    式中,W:苦味素得率,%;c:苦味素含量,g/L;v:苦味素提取液体积,mL;m:菊苣根原料的质量,g。

    使用超高效液相色谱串联飞行时间质谱(UPLC-Q-TOF-MS)对菊苣苦味素提取液的化学成分进行定性分析。液相色谱条件:使用Waters ACQUITY UPLC HSS T3柱(1.8 μm,2.1 mm×150 mm);流动相A为水,流动相B为乙腈,采用线性梯度洗脱。0~3 min时A为98%,B为2%;3~20 min时A为95%,B为5%;20~30 min时A为65%,B为35%;30~32 min时A为5%,B为95%;流速为0.3 mL/min;柱温维持在50 ℃。

    质谱条件:负离子扫描模式下,质谱扫描范围为m/z 100~1500;雾化气(GS1)压力为55 psi;雾化气(GS2)压力为55 psi;气帘气(CUR)压力为35 psi;离子源温度(TEM)为550 ℃;离子源电压(IS)为−4500 V(负离子模式);一级扫描使用去簇电压(DP)为100 V和聚焦电压(CE)为10 V;二级扫描采用IDA模式,CID能量为40±20 eV。

    参考Cheung等[34]的方法,制备0.1、0.2、0.3、0.4、0.5 mg/mL浓度样品溶液与无水乙醇共同放置,各吸取2 mL,分别加入同体积 0.4 mmol DPPH溶液,避光30 min。在517 nm处测定加入DPPH后各样品吸光值Ai ,加入DPPH后无水乙醇吸光度为A0。取同浓度各样品加入同体积无水乙醇,后重复上述操作测吸光值为Aj。依据式(2)计算清除率。

    DPPH(%)=1AiAjA0×100
    (2)

    参考Soong 等[35]的方法,制备0.1、0.2、0.3、0.4、0.5 mg/mL浓度样品溶液,各取1 mL样品,随后分别混合入4 mL ABTS工作液中(为确保ABTS工作液在734 nm处的吸光值在0.70±0.02之间,需用无水乙醇进行调整),并充分混匀。避光静置6 min,734 nm 处测量吸光值Ai,分别用无水乙醇替代 ABTS和样品溶液,测其吸光值Aj及A0。依据式(3)计算清除率。

    ABTS(%)=1AiAjA0×100
    (3)

    参考Li等[36]方法,制备0.1、0.2、0.3、0.4、0.5 mg/mL浓度样品溶液与无水乙醇共同放置。各取2 mL先后加入2 mL浓度为6 mmol/mL的FeSO4、H2O2与水杨酸溶液,其中加入H2O2溶液后静置10 min。加入水杨酸溶液后37 ℃ 水浴 30 min,3700 r/min 离心10 min。510 nm处测量各样品吸光值Ai,无水乙醇吸光度为A0。样品加入H2O2溶液后将水杨酸替换成蒸馏水,后重复上述操作测量吸光值Aj;按照公式(4)计算清除率。

    (%)=1AiAjA0×100
    (4)

    制备0.1、0.2、0.3、0.4、0.5 mg/mL的提取物溶液与无水乙醇共同放置。各取1 mL先后加入同体积的1%(w/v)K3Fe(CN)6溶液、pH6.6磷酸缓冲液和两倍体积的10%(w/v)的C2HCl3O2溶液,后取2 mL上清液添加同体积蒸馏水与0.4 mL浓度为0.1%(w/v)的FeCl3溶液。其中加入磷酸缓冲液后50 ℃水浴20 min,加入C2HCl3O2溶液后3000 r/min离心10 min。加入FeCl3溶液后50 ℃水浴 10 min,测样品组与无水乙醇组在700 nm处吸光值。上述实验均以相同浓度的VC溶液作为阳性对照组。

    实验数据取三次实验平均值。响应面设计与统计分别使用Design-Expert 10.0与SAS 8.2软件完成。

    图1可知,乙醇浓度在50%~80%时,菊苣苦味素得率与乙醇浓度成正相关,在80%浓度时达到巅峰得率0.91%。乙醇浓度超过80%后得率随浓度增加而降低。原因可能是现有乙醇量不能完全溶解菊苣苦味素,所以随着浓度增加苦味素溶解度增加。当达到80%乙醇浓度时,乙醇量可完全溶解菊苣苦味素。但当乙醇浓度过高时,溶解出菊苣中更多杂质反而使菊苣苦味素得率降低。因此,后续实验选择80%乙醇为溶剂浓度。

    图  1  乙醇浓度对菊苣苦味素得率的影响
    Figure  1.  Effect of ethanol concentration on the yield of chicory bitterness

    图2可知,随着超声温度的逐渐升高,菊苣苦味素的得率呈现先升高后下降的趋势。当超声温度为30 ℃时,苦味素的提取效率最高,得率达到1.03%。超声温度超过30 ℃后,苦味素的得率逐渐减少。其原因是超声温度升高与溶解能力在一定温度范围内正相关,温度升高加快了菊苣苦味素分子的运动速度。但温度过高时,高温会导致倍半萜类化合物分解转化为单萜类化合物[37],得率会降低。因此,本实验在最佳处理的超声温度为30 ℃,中心点为20~40 ℃时进行响应面试验。

    图  2  超声温度对菊苣苦味素得率的影响
    Figure  2.  Effect of ultrasonic temperature on the yield of chicory bitterness

    图3可知,随着超声时间的延长,菊苣苦味素的得率先增加后下降。当超声时间达到25 min时,得率最高,为1.01%。这是因为超声波会对细胞产生破坏,促进更多苦味素的溶解,因此产量增加。超声时间超过25 min后,苦味素的提取效率开始降低。其原因可能是样品长期处于超声波的作用下,苦味素被提取殆尽,导致苦味素得率下降。因此,为了优化超声处理时间,本实验选择以25 min为中心,范围设定为20~30 min。

    图  3  超声时间对菊苣苦味素得率的影响
    Figure  3.  Effect of ultrasonic temperature on the yield of chicory bitterness

    图4可知,超声功率在100~400 W区间,超声功率与苦味素得率成正相关。达到400 W时,菊苣苦味素在溶剂中的溶解最充分,得率最高。超过此范围后,可能因超声波过强得热效应,破坏了菊苣苦味素使苦味素得率下降。因此,为优化超声功率选以400 W为中心,范围300~500 W。

    图  4  超声功率对菊苣苦味素得率的影响
    Figure  4.  Effect of ultrasound power on the yield of chicory bitterness

    图5可知,随着液料比的增加,菊苣苦味素的得率呈上升趋势。少量的溶剂会使苦味素迅速溶解至饱和,得率随溶剂量逐渐提高。液料比达到30:1 mL/g时得率最高。超过此料液比后得率出现下降趋势。这可能是因为当液料比高于30:1 mL/g时,样品中的苦味素已经基本完全溶解,因此导致苦味素的得率下降。因此,选择液料比为20:1~40:1 mL/g的范围进行响应面优化,以30:1 mL/g作为中心值。

    图  5  液料比对菊苣苦味素得率的影响
    Figure  5.  Effect of ultrasonic temperature on the yield of chicory bitterness

    基于单因素实验结果最佳条件设计31组响应面试验与结果见表2

    表  2  响应面试验设计与结果
    Table  2.  Response surface methodology experimental design and results
    实验号 X1超声温度 X2超声时间 X3超声功率 X4液料比 得率
    (%)
    1 1 1 1 1 1.01
    2 1 1 1 −1 0.85
    3 1 1 −1 1 0.92
    4 1 1 −1 −1 0.78
    5 1 −1 1 1 1.13
    6 1 −1 1 −1 1.08
    7 1 −1 −1 1 1.04
    8 1 −1 −1 −1 0.82
    9 −1 1 1 1 1.09
    10 −1 1 1 −1 0.92
    11 −1 1 −1 1 0.88
    12 −1 1 −1 −1 0.83
    13 −1 −1 1 1 0.88
    14 −1 −1 1 −1 0.72
    15 −1 −1 −1 1 0.73
    16 −1 −1 −1 −1 0.67
    17 2 0 0 0 0.93
    18 −2 0 0 0 0.71
    19 0 2 0 0 0.93
    20 0 −2 0 0 0.91
    21 0 0 2 0 1.09
    22 0 0 −2 0 0.82
    23 0 0 0 2 1.01
    24 0 0 0 −2 0.66
    25 0 0 0 0 1.11
    26 0 0 0 0 1.13
    27 0 0 0 0 1.04
    28 0 0 0 0 1.08
    29 0 0 0 0 1.07
    30 0 0 0 0 1.14
    31 0 0 0 0 1.11
    下载: 导出CSV 
    | 显示表格

    根据表3的数据分析可得,二次回归模型的F值为24.20,其P值<0.0001,高于0.0001水平上的F值,失拟项的F值为1.84,P值为0.2344>0.05,说明对菊苣的苦味素产量影响显著且该模型拟合度良好。一次项X1、X3、X4以及交互项X1X2和各因素的平方项对结果菊苣的苦味素产量的影响程度均达到极显著水平(P<0.01)其它项对结果的影响程度并不显著(P>0.05)。根据F检验结果显示,各因素对菊苣的苦味素产量的影响顺序为X4>X3>X1>X2,即液料比>超声功率>超声温度>超声时间。综上所述,响应面二次回归方程的拟合度较好,该模型可用于对菊苣的苦味素提取工艺条件预测和分析。

    表  3  二次回归模型方差分析结果
    Table  3.  Analysis of variance results for the second-order regression model
    方差来源平方和自由度均方FP显著性
    模型0.6505140.046524.20<0.0001**
    X10.075910.075939.55<0.0001**
    X20.002610.00261.360.2612
    X30.100110.100152.14<0.0001**
    X40.121810.121863.46<0.0001**
    X1X20.094610.094649.25<0.0001**
    X1X36.250E-0616.250E-060.00330.9552
    X1X40.001110.00110.55020.4690
    X2X30.000510.00050.26370.6146
    X2X40.000110.00010.02930.8662
    X3X40.000310.00030.15950.6949
    X120.127910.127966.64<0.0001**
    X220.050210.050226.140.0001**
    X320.031410.031416.360.0009**
    X420.114010.114059.38<0.0001**
    残差0.0307160.0019
    失拟项0.0232100.00231.840.2344
    纯误差0.007560.0013
    总变异0.681230
    注:*表示因素对响应值的影响显著,P<0.05;**为极显著,P<0.01。
    下载: 导出CSV 
    | 显示表格

    以菊苣苦味素的得率为Y值,各因素为X值得回归方程为:

    Y=−15.70+0.3148X1+0.5373X2+0.0124X3+0.1453X4−0.0027X12−0.0062X1X2+0.00001X1X3+0.0003X1X4−0.0067X22−0.0001X2X3+0.0001X2X4−0.0001X32+0.00001X3X4−0.0025X42

    图6中可以看出,超声温度与超声时间的等高线密集,响应面轮廓线呈弯曲的椭圆形,且响应面的斜率比较大,说明超声温度与超声时间之间有较强相互影响和制约[3839]。从轮廓线可以看出,超声温度变化的方向相对与沿超声时间方向轮廓线更为密集,表明超声温度对响应峰值的影响起主导作用[4041]。X1X2交互作用对响应值影响的响应面图坡度较为陡峭,跨度更大,较为显著,这与方差分析结果一致。

    图  6  超声温度和超声时间的交互作用对菊苣苦味素得率的影响
    Figure  6.  Effect of the interaction between ultrasound temperature and ultrasound time on the yield of chicory bitterness

    应用SAS8.2进行响应面典型分析,确证提取得率的最大值,获得最优提取条件[40]和得率。为乙醇提取液浓度为80%、超声温度为34.52 ℃,超声时间为23.9 min,超声功率为454.53 W,液料比为33.24:1 mL/g条件下,菊苣苦味素得率最大为1.177%。按照可操作性优化参数为乙醇提取液浓度为80%、超声温度为35 ℃,超声时间为24 min,超声功率为450 W,液料比为33:1 mL/g条件下条件进行三次重复实验,结果为1.18%±0.015%,实验值与模型的理论值相近,且相对偏差不超过5%,说明试验结果能够较好得重现,该模型能相对准确地的反映出超声波辅助提取菊苣苦味素的最佳条件。

    在上述最佳提取条件下制备菊苣苦味素提取液,并使用飞行时间质谱对苦味素进行定性分析,实验采用ESI离子源获得的基本都是准分子离子峰或者其他加和峰,很少直接得到分子离子峰,根据高分辨率质谱模拟的结果并结合相关研究结果分析,并使用Scifinder和Reaxy数据库检索和推测化合物,菊苣苦味素中主要含有7种化合物(图7)。化合物1,出峰时间为7.91,[M+NH3-H]为390.2,根据二级质谱m/z 231,114,化合物为地胆草丁,分子式为C20H22O7;化合物2,出峰时间为9.51,[M+nH2OH]为485.1,根据二级质谱m/z 485,439,421,259,215,197,119,化合物为秦皮甲素,分子式为C15H16O9;化合物3,出峰时间为10.7,[M+2H2O+H]为215.1,根据二级质谱m/z 277,215,200,197,化合物为秦皮乙素,分子式为C9H6O4;化合物4,出峰时间为11.42,[M+nH2O+H]为481.2,根据二级质谱m/z 481,257,215,200,197,化合物为绿原酸,分子式为C16H18O9;化合物5,出峰时间为16.73,[M-H]为475.2,根据二级质谱m/z475,261,217,199,化合物为莴苣酸,分子式为C22H18O12;化合物6,出峰时间为17.22,[M-2H2O-H]为243.1,根据二级质谱m/z 243,225,207,181,125,化合物为山莴苣素,分子式为C15H16O5;化合物7,出峰时间为20.66,[M+H]为411.2,根据二级质谱m/z 411,249,231,化合物为山莴苣苦素,分子式为C23H22O7图8)。

    图  7  菊苣苦味素总离子流图
    Figure  7.  Total ion chromatogram of chicory bitterness
    图  8  超高效液相色谱串联飞行时间质谱分析菊苣苦味素的一级质谱和二级质谱图
    Figure  8.  Primary mass spectrum and secondary mass spectrum analysis of chicory bitterness using ultra-high-performance liquid chromatography tandem time-of-flight mass spectrometry

    图9可以看出,菊苣苦味素和VC对DPPH自由基的清除能力均与质量浓度成正相关,菊苣苦味素对DPPH自由基的清除能力总体上低于VC。当浓度从0.2 g/L增加到0.3 g/L时,清除率迅速增加。当菊苣苦味素浓度为0.5 g/L时,其对DPPH自由基的清除率为97.57%,接近VC的清除能力。这表明,菊苣苦味素在抑制DPPH自由基方面能力出色。其IC50值为0.271 g/L,明显高于菊苣总酚(IC50=283.67mg/mL)的DPPH清除能力[42]

    图  9  菊苣苦味素对DPPH自由基的清除作用
    Figure  9.  Scavenging effect of chicory bitterness on DPPH radicals

    图10可知,菊苣苦味素对ABTS+·的清除率呈现一定的量效关系,随着菊苣苦味素提取物浓度增加,ABTS+·的清除率明显上升。清除率在浓度从0.1 g/L增加到0.2 g/L时,呈现最大的斜率,增长速度最快。当浓度为0.5 g/L 时,清除率达到 93.93%,接近VC的清除率。IC50值为0.0734 mg/mL,抗氧化能力显著。与同为倍半萜类的隐孔菌中倍半萜类化合物ABTS+ ·的清除率相近[43]

    图  10  菊苣苦味素对ABTS+自由基的清除作用
    Figure  10.  Scavenging effect of chicory bitterness on ABTS+ radicals

    图11可知,菊苣苦味素具有清除·OH的能力,其IC50值为 0.130 mg/mL,且具有良好的剂量效应关系。清除率从浓度从0.1 mg/mL增加到0.4 mg/mL时,持续上升,由48.07%增加到77.98%,没有明显的波动。当浓度为0.5 mg/mL 时,提取物的·OH清除率高达92.93%,显著高于VC的清除率(P<0.05),且高于同为菊苣生产的菊粉(IC50=4.38mg/mL)的羟基自由基清除能力[44],抗氧化能力显著。

    图  11  菊苣苦味素对羟基的清除作用
    Figure  11.  Scavenging effect of chicory bitterness on hydroxyl radicals

    图12可知,提取物的总还原能力随浓度增加呈现上升趋势且整体低于同浓度的VC溶液。当浓度为0.3 mg/mL后总还原力增加不显著,提取物的总还原能力接近VC;当浓度为0.5 mg/mL时提取物的总还原能力达到1.108。总还原能力强于同属菊科的艾叶挥发油[45],还原能力较佳。

    图  12  菊苣苦味素的总还原能力
    Figure  12.  Total reducing power of chicory bitterness

    从上述结果可以看出,菊苣苦味素有清除各种自由基(DPPH·、ABTS+·、·OH)的能力,其抗氧化活性和总还原能力均较好,具有应用于天然抗氧化剂的潜力。

    研究以菊苣根为原料,采用超声波法提取菊苣苦味素,通过响应面法优化了提取工艺,优化后的工艺参数为:乙醇浓度为80%、超声温度为34.52 ℃,超声时间为23.9 min,超声功率为454.53 W,液料比33.24:1 mL/g,菊苣苦味素得率可达到1.18%±0.015%。通过液质分析发现菊苣苦味素中主要含有:地胆草丁(C20H22O7);秦皮甲素(C15H16O9);秦皮乙素(C9H6O4);绿原酸(C16H18O9);莴苣酸(C22H18O12);山莴苣素(C15H16O5);山莴苣苦素(C23H22O7)等七种物质。通过体外部分抗氧化活性分析,发现菊苣苦味素对DPPH·、ABTS+·和·OH都具有出色的清除能力,展现出良好的抗氧化活性,同时还表现出强大的总还原能力。本研究为菊苣苦味素的综合开发及产业化利用奠定了理论及实践基础,对我国菊苣产业产业链延伸、利润提升具有一定的促进作用。

  • 图  1   乙醇浓度对菊苣苦味素得率的影响

    Figure  1.   Effect of ethanol concentration on the yield of chicory bitterness

    图  2   超声温度对菊苣苦味素得率的影响

    Figure  2.   Effect of ultrasonic temperature on the yield of chicory bitterness

    图  3   超声时间对菊苣苦味素得率的影响

    Figure  3.   Effect of ultrasonic temperature on the yield of chicory bitterness

    图  4   超声功率对菊苣苦味素得率的影响

    Figure  4.   Effect of ultrasound power on the yield of chicory bitterness

    图  5   液料比对菊苣苦味素得率的影响

    Figure  5.   Effect of ultrasonic temperature on the yield of chicory bitterness

    图  6   超声温度和超声时间的交互作用对菊苣苦味素得率的影响

    Figure  6.   Effect of the interaction between ultrasound temperature and ultrasound time on the yield of chicory bitterness

    图  7   菊苣苦味素总离子流图

    Figure  7.   Total ion chromatogram of chicory bitterness

    图  8   超高效液相色谱串联飞行时间质谱分析菊苣苦味素的一级质谱和二级质谱图

    Figure  8.   Primary mass spectrum and secondary mass spectrum analysis of chicory bitterness using ultra-high-performance liquid chromatography tandem time-of-flight mass spectrometry

    图  9   菊苣苦味素对DPPH自由基的清除作用

    Figure  9.   Scavenging effect of chicory bitterness on DPPH radicals

    图  10   菊苣苦味素对ABTS+自由基的清除作用

    Figure  10.   Scavenging effect of chicory bitterness on ABTS+ radicals

    图  11   菊苣苦味素对羟基的清除作用

    Figure  11.   Scavenging effect of chicory bitterness on hydroxyl radicals

    图  12   菊苣苦味素的总还原能力

    Figure  12.   Total reducing power of chicory bitterness

    表  1   响应面因素与水平设计

    Table  1   Response surface factors and level design

    编码值 X1超声温度(℃) X2超声时间(min) X3超声功率(W) X4液料比(mL/g)
    −2 20 20 300 20:1
    −1 25 22.5 350 25:1
    0 30 25 400 30:1
    +1 35 27.5 450 35:1
    +2 40 30 500 40:1
    下载: 导出CSV

    表  2   响应面试验设计与结果

    Table  2   Response surface methodology experimental design and results

    实验号 X1超声温度 X2超声时间 X3超声功率 X4液料比 得率
    (%)
    1 1 1 1 1 1.01
    2 1 1 1 −1 0.85
    3 1 1 −1 1 0.92
    4 1 1 −1 −1 0.78
    5 1 −1 1 1 1.13
    6 1 −1 1 −1 1.08
    7 1 −1 −1 1 1.04
    8 1 −1 −1 −1 0.82
    9 −1 1 1 1 1.09
    10 −1 1 1 −1 0.92
    11 −1 1 −1 1 0.88
    12 −1 1 −1 −1 0.83
    13 −1 −1 1 1 0.88
    14 −1 −1 1 −1 0.72
    15 −1 −1 −1 1 0.73
    16 −1 −1 −1 −1 0.67
    17 2 0 0 0 0.93
    18 −2 0 0 0 0.71
    19 0 2 0 0 0.93
    20 0 −2 0 0 0.91
    21 0 0 2 0 1.09
    22 0 0 −2 0 0.82
    23 0 0 0 2 1.01
    24 0 0 0 −2 0.66
    25 0 0 0 0 1.11
    26 0 0 0 0 1.13
    27 0 0 0 0 1.04
    28 0 0 0 0 1.08
    29 0 0 0 0 1.07
    30 0 0 0 0 1.14
    31 0 0 0 0 1.11
    下载: 导出CSV

    表  3   二次回归模型方差分析结果

    Table  3   Analysis of variance results for the second-order regression model

    方差来源平方和自由度均方FP显著性
    模型0.6505140.046524.20<0.0001**
    X10.075910.075939.55<0.0001**
    X20.002610.00261.360.2612
    X30.100110.100152.14<0.0001**
    X40.121810.121863.46<0.0001**
    X1X20.094610.094649.25<0.0001**
    X1X36.250E-0616.250E-060.00330.9552
    X1X40.001110.00110.55020.4690
    X2X30.000510.00050.26370.6146
    X2X40.000110.00010.02930.8662
    X3X40.000310.00030.15950.6949
    X120.127910.127966.64<0.0001**
    X220.050210.050226.140.0001**
    X320.031410.031416.360.0009**
    X420.114010.114059.38<0.0001**
    残差0.0307160.0019
    失拟项0.0232100.00231.840.2344
    纯误差0.007560.0013
    总变异0.681230
    注:*表示因素对响应值的影响显著,P<0.05;**为极显著,P<0.01。
    下载: 导出CSV
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  • 收稿日期:  2023-09-10
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