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中国精品科技期刊2020

响应面法优化龙须菜多糖快速溶剂萃取提取工艺及其抗炎活性研究

王慧颖, 刘燕飞, 张敬远, 杜彬, 杨越冬

王慧颖,刘燕飞,张敬远,等. 响应面法优化龙须菜多糖快速溶剂萃取提取工艺及其抗炎活性研究[J]. 食品工业科技,2023,44(23):110−117. doi: 10.13386/j.issn1002-0306.2023030309.
引用本文: 王慧颖,刘燕飞,张敬远,等. 响应面法优化龙须菜多糖快速溶剂萃取提取工艺及其抗炎活性研究[J]. 食品工业科技,2023,44(23):110−117. doi: 10.13386/j.issn1002-0306.2023030309.
WANG Huiying, LIU Yanfei, ZHANG Jingyuan, et al. Optimization of Accelerated Solvent Extraction of Polysaccharides from Gracilaria lemaneiformis Using Response Surface Methodology and Anti-inflammatory Activity[J]. Science and Technology of Food Industry, 2023, 44(23): 110−117. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023030309.
Citation: WANG Huiying, LIU Yanfei, ZHANG Jingyuan, et al. Optimization of Accelerated Solvent Extraction of Polysaccharides from Gracilaria lemaneiformis Using Response Surface Methodology and Anti-inflammatory Activity[J]. Science and Technology of Food Industry, 2023, 44(23): 110−117. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023030309.

响应面法优化龙须菜多糖快速溶剂萃取提取工艺及其抗炎活性研究

基金项目: 中央引导地方科技发展资金项目(216Z3201G)。
详细信息
    作者简介:

    王慧颖(1996−),女,硕士,研究方向:天然产物化学,E-mail:WangHuiyingHYW@163.com

    通讯作者:

    杜彬(1980−),男,博士,研究员,研究方向:天然产物化学,E-mail:bindufood@aliyun.com

    杨越冬(1965−),女,博士,教授,研究方向:天然产物化学,E-mail:kycyyd@126.com

  • 中图分类号: TS255.3

Optimization of Accelerated Solvent Extraction of Polysaccharides from Gracilaria lemaneiformis Using Response Surface Methodology and Anti-inflammatory Activity

  • 摘要: 为了建立快速溶剂萃取技术(ASE)提取龙须菜多糖的新方法,本文以秦皇岛龙须菜为原料,利用ASE提取龙须菜粗多糖(GLP-K),以多糖得率为指标,采用单因素实验结合响应面试验法优化提取工艺条件。通过傅里叶红外光谱和高效液相色谱对多糖进行结构表征;探索GLP-K在脂多糖(LPS)诱导的RAW264.7巨噬细胞中的抗炎作用。结果表明,快速溶剂萃取法用于龙须菜多糖提取的最佳工艺参数为提取温度70 ℃,提取时间8.5 min,循环4次,在此条件下,多糖实验得率为9.58%±0.31%;红外光谱证实该多糖含有糖醛酸,重均分子量在4.4~747.1 kDa 之间;GLP-K在浓度1000 μg/mL及以下时对RAW264.7细胞增殖也无影响(P<0.001);与模型组相比,GLP-K给药组(50、100、200、300、400、500 μg/mL)NO的释放量显著降低43.76%~69.47%(P<0.001)。本文丰富了快速溶剂萃取技术在多糖提取方面的研究,为龙须菜多糖的开发和利用提供实验依据。
    Abstract: The aim of this work was to apply response surface methodology (RSM) to model and optimize the accelerated solvent extraction (ASE) technique for extracting the crude polysaccharides (GLP-K) from Gracilaria lemaneiformis. In terms of the yield of polysaccharide, single factor tests and Box-Behnken design response surface method were developed. The structure of the prepared polysaccharides was characterized by Fourier transform infrared (FT-IR) and high performance liquid chromatography (HPLC). And the anti-inflammatory potential of GLP-K in lipopolysaccharide (LPS)-induced RAW264.7 macrophages was also explored. Briefly, the optimal extraction conditions for GLP-K were as follows: 70 °C of extraction temperature, 8.5 min of extraction time, and 4 extraction cycles. Under the condition, the experimental yield of polysaccharide was 9.58%±0.31%. FT-IR showed that the polysaccharide contained uronic acid, and the weight-average molecular weight ranged from 4.4 to 747.1 kDa. GLP-K had no significant cytotoxic effects at or below the concentrations of 1000 μg/mL (P<0.001). Compared with the model group, GLP-K administration group (50, 100, 200, 300, 400, 500 μg/mL) displayed remarkable inhibitory effects on the release level of NO (P<0.001), which decreased by 43.76%~69.47%. This paper enriched the research on the extraction of polysaccharides by accelerated solvent extraction technology, and provided experimental basis for the development and utilization of Gracilaria lemaneiformis polysaccharides.
  • 龙须菜 (Gracilaria lemaneiformis) 又名江蓠、海发菜,属江蓠科红藻界天然海洋植物,其藻体直立,线形分支。其广泛分布在中国沿海地区,利于维持海洋生态环境,作为琼脂的主要来源,具有很高经济价值[1]。龙须菜富含多糖、膳食纤维、蛋白质、氨基酸、矿物质、多酚等可供人体吸收和利用的营养物质,其中多糖含量超过干重的30%[2]。研究表明,龙须菜多糖具有免疫调节[3]、降血糖[4]、抗氧化[5]、抗肿瘤[6]等优异活性,在功能食品、化妆品、医用材料和生物肥料领域实现高值化应用。

    目前,多糖提取[7]的传统方法有微波辅助提取法[8]、溶剂提取法[9]和酶提取法[10]等。此外,快速溶剂萃取技术[11]是一种新兴的高压自动萃取技术,通过改变温度和压力影响多糖的提取率。该方法具有工序简单、自动化提取、效率高、成本低等优点,在食品[12]、化工[13]和环境[14]领域有广泛的应用。王国明等[15]研究发现快速溶剂萃取法提取人参粗多糖得率高于传统水提法;金建华等[16]探究了快速溶剂萃取法提取唐古特白刺果实多糖的最佳工艺为温度102 ℃、时间10 min、压力10 MPa、循环提取2次;王宇晴等[17]探究了快速溶剂萃取法提取灵芝多糖的最佳工艺参数为提取温度110 ℃,提取压力90 Pa,提取时间20 min,循环2次。然而,快速溶剂萃取技术提取龙须菜多糖尚未报道。

    本文将探究快速溶剂萃取法提取龙须菜多糖的提取工艺参数(提取温度、提取时间、循环次数),以获得龙须菜多糖的最高得率。系统分析提取工艺参数对龙须菜多糖得率的影响及其相互作用,并对其结构和抗炎活性进行实验,有望为龙须菜多糖提取方法和生物活性提供一定参考价值。

    龙须菜 2021年采自河北省秦皇岛海域,经除杂、清洗和晾晒后,得到的龙须菜在50 ℃恒温干燥箱中干燥至恒重,粉碎过40目筛,得到龙须菜粉末并置于−20 ℃冰箱内储存备用;3500 Da透析袋 上海源叶有限公司;RAW264.7(小鼠单核巨噬细胞白血病细胞) 武汉普诺赛生命科技有限公司;DMEM高糖培养基 上海富衡生物科技有限公司;胎牛血清 上海源培生物科技股份有限公司;CCK-8试剂盒 上海七纯生物科技有限公司;一氧化氮(nitric oxide,NO)检测试剂盒 上海碧云天生物技术有限公司;LPS(0111:B4) 美国Sigma公司;硫酸铵、叔丁醇、无水乙醇等 分析纯,天津欧博凯化工有限公司。

    APLE-3000全自动加压溶剂萃取仪(配有100 mL萃取池) 北京吉天仪器有限公司;ALpha2-4L D plus冷冻干燥机 德国CHRIST公司;TENSOR27傅立叶变换红外光谱仪 德国Bruker公司;HPLC1100高效液相色谱仪 美国Agilent公司;1300Ⅱ级A2型生物安全柜、3111型CO2细胞培养箱、MultifugeX1R型高速冷冻离心机 美国Thermo Fisher Scientific公司;XSZ-D2普通倒置显微镜 德国ZEISS公司;SPECTRA MAX 190型酶标仪 美谷分子仪器(上海)有限公司。

    参照何俊平等[18]的实验方法,并做修改。精确称取龙须菜粉末(5.0000±0.0003)g并与硅藻土(1:1,w/w)混合均匀,装入100 mL不锈钢萃取池中,用硅藻土填满萃取池剩余空间后放到加压溶剂萃取仪中,以水为溶剂,固定加载溶剂25 mL;固定10 MPa压力,在一定温度下静态萃取一定时间,循环一定次数,后将萃取液离心(5000 r/min,10 min),记录体积,加四倍体积无水乙醇4 ℃条件下醇沉过夜、离心取沉淀,将沉淀复溶;然后加入20% (w/v) 硫酸铵和1.5倍(v/v)叔丁醇,35 ℃反应30 min,过3500 Da透析膜,透析48 h,浓缩至四分之一,冷冻干燥得龙须菜粗多糖GLP-K。提取工艺流程见图1

    图  1  龙须菜多糖快速溶剂提取工艺流程图
    Figure  1.  Accelerated solvent extraction process flow chart of GLP-K

    以水为溶剂,固定投料量为5 g,提取压力为10 Mpa,提取时间2 min,循环次数1次,探究提取温度(40、50、60、70、80 ℃)对龙须菜多糖得率的影响。每个实验平行重复三次。

    以水为溶剂,固定投料量为5 g,提取压力为10 Mpa,提取温度60 ℃,循环次数1次,探究提取时间(2、4、6、8、10 min)对龙须菜多糖得率的影响。每个实验平行重复三次。

    以水为溶剂,固定投料量为5 g,提取压力为10 Mpa,提取温度60 ℃,提取时间2 min,探究循环次数(1、2、3、4、5次)对龙须菜多糖得率的影响。每个实验平行重复三次。

    基于Box-Benhnken试验设计原理,综合单因素实验结果,固定提取投料量为5 g,提取压力为10 MPa,以多糖得率为响应值,选择提取温度(A)、提取时间(B)和循环次数(C)为自变量,设计三因素三水平响应面优化试验,响应面试验设计表见下表1

    表  1  响应面试验设计因素与水平
    Table  1.  The design factors and levels of response surface test
    水平 A提取温度(℃) B提取时间(min) C循环次数(次)
    −1 60 6 3
    0 70 8 4
    1 80 10 5
    下载: 导出CSV 
    | 显示表格

    参考任鑫等[19]的方法,按公式(1)计算龙须菜多糖得率:

    Y(\%)=M1M2×100
    (1)

    式中:Y为龙须菜多糖得率,%;M1为龙须菜粗多糖质量,g;M2为龙须菜粉的质量,g。

    选用苯酚硫酸法测定多糖含量。标准曲线:先配制0.1 mg/mL葡萄糖标准液母液,再配成一系列浓度(0、4、8、12、16、20、24、28、32、36、40 μg/mL)的标准溶液,各取1 mL于试管中,加1 mL 6%苯酚和5 mL浓硫酸,静置20 min,在490 nm处测定吸光度值,作图得标准曲线。配制1 mg/mL的GLP-K溶液,按上述步骤加苯酚和浓硫酸,静置20 min,在490 nm处测吸光度值。

    将干燥多糖样品与干燥KBr于研钵中顺时针充分研磨至混匀,置于专用模具内手动压成透明薄片后测试。扣除KBr的空白背景,扫描范围:500~4000 cm−1,扫描间隔2 cm−1

    采用高效液相色谱法[20](HPLC)对龙须菜多糖进行测定。将样品溶解在蒸馏水中,过0.22 μm的微孔滤膜后上机检测。色谱条件:Agilent PL aquagel-OH MIXED-H凝胶色谱柱(300×7.5 mm,8 μm),流动相为蒸馏水,流速为0.8 mL/min,柱温为35 ℃;配制2 mg/mL 葡聚糖标准品及样品,测样体积为20 μL。实验平行重复三次。

    将RAW 264.7细胞株采用生长培养液(含89% DMEM培养基、10%胎牛血清和1%的青霉素、链霉素双抗混悬液)于37 ℃含5% CO2的培养箱中常规培养。将培养皿放在普通倒置显微镜下,放大不同倍数观察细胞密度和生长情况。

    取生长状态良好的RAW 264.7制备细胞悬液,将细胞按1×104/孔接种到96孔板中,每孔100 μL培养基,贴壁培养24 h后弃培养基,空白组(无细胞)和对照组分别加入100 μL培养基,实验组加入100 μL含有不同浓度(7.8125、15.625、31.25、62.5、125、250、500、1000 μg/mL)龙须菜多糖的培养基干预24 h后,弃培养基,再将100 μL含有10% CCK-8试剂的细胞培养基加入空白组、对照组和实验组中,37 ℃避光孵育1 h。用酶标仪在450 nm处测量每个孔的吸光度(A450),计算细胞存活率。每组设三个复孔。存活率计算见公式(2)。

    (\%)=AsAbAcAb×100
    (2)

    式中:As为实验组吸光度;Ac为对照组吸光度;Ab为空白组吸光度。

    取生长状态良好的RAW 264.7制备细胞悬液,将细胞按2×105/孔接种到48孔板中,每孔200 mL培养基,贴壁培养24 h后弃培养基,实验组加入200 μL含有不同浓度(50、100、200、300、400、500 μg/mL)龙须菜多糖的培养基,LPS组和对照组加200 μL新鲜培养基,预处理2 h后,LPS组和实验组加入22.2 μL含10 μg/mL LPS的培养基,对照组加入22.2 μL新鲜培养基,刺激22 h后,根据NO试剂盒说明书步骤检测培养基上清中NO含量。

    通过Excel软件处理三次平行实验的实验结果作平均值和标准差分析,响应面试验数据采用Design-Expert8.0.6.1软件进行分析。采用Origin 9软件进行结构作图,GraphPad prism8.0.1软件进行活性作图分析。P<0.05表示差异显著,P<0.01表示差异极显著,P<0.001表示差异高度显著。

    图2所示,随着反应温度的升高,多糖的得率随之提高,当温度达到70 ℃时,多糖得率达到最大,继续升高温度时,得率略有下降,可能是温度的上升有利于溶剂与细胞壁的相互作用,使胞内物质溶出,但在10 MPa的压力加持下,高温导致多糖发生了热降解。因此后继试验中提取温度取值范围为60~80 ℃。

    图  2  提取温度对龙须菜多糖得率的影响
    Figure  2.  Effect of extracting temperature on the yield of GLP-K

    图3 所示,在2~8 min内,龙须菜多糖的得率随反应时间增加而提高,提取时间为8 min 时得率达到最大值,为5.59%;继续延长时间后,得率下降,分析可能是在较短时间范围内,随着提取时间的延长,细胞组织遭到持续性破坏,多糖溶出,得率增加;多糖在长时间[2122]的高压作用下发生水解或氧化。因此后继试验中提取时间取值范围为6~10 min。

    图  3  提取时间对龙须菜多糖得率的影响
    Figure  3.  Effect of extracting time on the yield of GLP-K

    图4所示,多糖得率随着循环次数的增加而提高,当循环次数为4时得率最高,继续增加循环次数,得率没有增加,分析是多糖在水中溶解度是一定的,达到平衡后不再溶出,更换新溶剂后多糖会继续溶出至再次达到平衡,但随着提取次数的增加,其他水溶性杂质会占据多糖的位置,因此后继试验中循环次数取值范围为3~5次。循环次数对多糖得率的影响结果与Du等[21]在青稞中β-葡聚糖的快速溶剂萃取试验结果一致。

    图  4  循环次数对龙须菜多糖得率的影响
    Figure  4.  Effect of cycle times on the yield of GLP-K

    以龙须菜多糖的得率为响应值,采用三因素三水平的响应面试验设计得到最佳提取工艺参数,实验结果见表2,方差分析结果见表3,龙须菜多糖得率在7.38%~9.75%之间,对表2中的响应面试验数据结果进行多元回归方程拟合,并获得以龙须菜多糖为响应值的多元回归方程:

    表  2  龙须菜多糖提取工艺条件响应面优化方案及结果
    Table  2.  Response surface optimization scheme and results of extraction process conditions of GLP-K
    实验号 A提取温度 B提取时间 C循环次数 Y龙须菜多糖得率(%)
    1 −1 −1 0 7.38
    2 1 −1 0 8.36
    3 −1 1 0 7.79
    4 1 1 0 8.20
    5 −1 0 −1 7.38
    6 1 0 −1 7.83
    7 −1 0 1 8.42
    8 1 0 1 9.16
    9 0 −1 −1 8.15
    10 0 1 −1 8.35
    11 0 −1 1 8.99
    12 0 1 1 9.13
    13 0 0 0 9.68
    14 0 0 0 9.73
    15 0 0 0 9.75
    16 0 0 0 9.69
    17 0 0 0 9.59
    下载: 导出CSV 
    | 显示表格
    表  3  响应面回归方程模型方差分析
    Table  3.  Analysis of variance of response surface regression equation model
    来源 偏差平方和 自由度 均方 F P 显著性
    模型 11.24 9 1.25 99.36 <0.0001 **
    A 0.83 1 0.83 66.13 <0.0001 **
    B 0.044 1 0.044 3.53 0.1022
    C 1.99 1 1.99 158.61 <0.0001 **
    AB 0.081 1 0.081 6.45 0.0387 *
    AC 0.022 1 0.022 1.73 0.2298
    BC 0.001 1 0.001 0.070 0.7989
    A2 5.18 1 5.18 411.70 <0.0001 **
    B2 1.77 1 1.77 140.97 <0.0001 **
    C2 0.62 1 0.62 49.36 0.0002 **
    残差 0.088 7 0.013
    失拟项 0.073 3 0.024 6.47 0.0515 不显著
    纯误差 0.015 4 0.001
    总和 11.33 16
    注:R2=0.9922,R2adj=0.9822;*表示有显著性差异(P<0.05);**表示有极显著性差异(P<0.01)。
    下载: 导出CSV 
    | 显示表格

    Y=9.69+0.32A+0.075B+0.50C−0.14AB+0.074AC−0.015BC−1.11A2−0.65B2−0.38C2

    结合表3可知,F值越高,P值越低,显著性则越高[23]。回归模型P<0.01,失拟项P=0.0515,A和C对响应值影响极显著(P<0.01),B对响应值影响不显著(P>0.05);交互项AB对响应值影响显著(P<0.05),AC、BC对响应值影响不显著(P>0.05);二次项对响应值影响均极显著(P<0.01);表明优化实验结果具有较高的相关性和真实性,三个因素显著性影响大小分别为循环次数>提取温度>提取时间。

    图5展示了三因素(提取温度、提取时间和循环次数)两两交互的响应曲面和等高线图。如图所示,相对比较而言,多糖得率在提取温度较低(60~70 ℃)时随提取时间的增大呈递增趋势,在提取温度较高时(70~80 ℃),呈递减趋势。提取温度和提取时间的响应面图的曲面较陡,表明其交互作用显著(P<0.05);多糖得率在提取温度较低(60~70 ℃)时随循环次数的增大呈递增趋势,在提取温度较高时(70~80 ℃),呈缓慢递减趋势(P>0.05);多糖得率在提取时间较低(6~8 min)时随循环次数的增大呈递增趋势,在提取时间较高时(8~10 min),呈缓慢递减趋势(P>0.05)。综合分析可得,温度和时间交互作用最强烈,温度和循环次数交互作用次之,时间和循环次数交互作用最弱,和表3方差分析结果相符。

    图  5  AB、AC、BC对龙须菜多糖得率影响的响应面图和等高线图
    Figure  5.  Response surface plots and contour plots of the effects of AB, AC and BC on the yield of GLP-K

    通过响应面软件对最佳提取工艺条件进行预测,得到龙须菜多糖快速溶剂萃取的最佳工艺条件为:蒸馏水为溶剂,萃取压力10 MPa,提取温度70.41 ℃,提取时间8.53 min,循环次数4.24次,预测得率为9.82%。基于实际情况,将最优条件调整为温度70 ℃,时间8.5 min,循环次数4次,在此条件下进行三次平行水平验证实验,龙须菜多糖得率为9.58%±0.31%,与多元回归方程得到的预测值相近,表明该模型的优化参数基本可靠,具有可行性。

    通过苯酚-硫酸法对葡萄糖标准溶液作图,得葡萄糖标准曲线方程:Y=0.006X+0.002,R2=0.998,代入方程计算GLP-K中总糖含量为75.28%。

    图6所示,在3386 cm−1处的宽而强的吸收峰是-OH伸缩振动特征吸收峰;在2916 cm−1处有一个-CH伸缩振动引起的弱而小的吸收峰,表明该样品类属于多糖类化合物[24];在1643 cm−1和1444 cm−1处的吸收峰分别对应C=O和C-O的拉伸振动,表明此多糖存在糖醛酸[25];1239 cm−1处的峰代表S=O的拉伸振动,表明含有硫酸基;1152 cm−1和1071 cm−1 的吸收表示半乳糖和葡萄糖的存在[26]。综上表明GLP-K是一种含有硫酸基和糖醛酸的粗多糖。

    图  6  龙须菜多糖红外光谱图
    Figure  6.  FT-IR of GLP-K

    在最佳工艺参数下得到龙须菜多糖。图7给出了龙须菜多糖的HPLC洗脱曲线以及葡聚糖标准品[27](重均分子量Mw分别为180、667、47100、344000、708000 Da)的标准矫正曲线。得到标准曲线的线性方程lgMw=−0.9822t+14.9794,其中R2=0.994,呈良好的线性关系[28]。由表4可知,此方法得到的多糖分子量范围较宽,在4.4~747.1 kDa,响应信号最大值对应的Mw为29787 Da。

    图  7  HPLC的标准矫正曲线和龙须菜多糖的HPLC洗脱曲线
    Figure  7.  Standard calibrationcurve of HPLC and the HPLC elution profile of GLP-K
    表  4  保留峰时间和分子量
    Table  4.  Retention peak time and molecular weight
    样品保留时间(min)Mn(Da)Mw(Da)D(Mw/Mn
    龙须菜多糖110.69±0.034419±452747122±7508169.07±25.84
    下载: 导出CSV 
    | 显示表格

    图8所示,本实验探究在7.8125~1000 μg/mL浓度的GLP-K的细胞存活率[2931]。结果显示,与对照组相比,7.8125~1000 μg/mL浓度范围内的GLP-K组对巨噬细胞的活力分别增加了10.3%~46.7%,表明快速溶剂法萃取龙须菜多糖对细胞没有毒性作用。

    图  8  GLP-K对细胞活力的影响
    注:与对照组相比,***(P<0.001)。
    Figure  8.  Effects of GLP-K on cells viability

    NO作为一种有毒气体存在于空气中,但它在生物生理方面充当重要的信号分子作用,参与生物体免疫调节或抗炎活动。在炎症细胞模型中,可以通过NO释放量减少来判断炎症反应是否得到改善[3234]。由图9可知,与对照组相比,LPS组NO产生量有提升了4倍(P<0.001),证明炎症模型诱导成功;与模型组相比,不同浓度的实验组NO释放量降低,出现显著性差异(P<0.001),随着GLP-K实验组浓度的升高,NO抑制率呈正相关。在50 μg/mL时NO抑制率为43.76%,500 μg/mL时NO抑制率为69.47%。结果表明,GLP-K可以改善炎症反应。朱彦彬等[31]采用体外细胞实验,研究了桦褐孔菌醇抑制LPS诱导RAW264.7细胞炎症反应,结果表明,该提取物能有效抑制LPS诱导RAW264.7细胞中促炎因子和过氧化物的产生,提高细胞抗炎和抗氧化能力,从而保护细胞免受损伤。

    图  9  不同浓度GLP-K 对 RAW264.7 细胞NO分泌水平的影响
    注:与对照组相比用“*”表示,***(P<0.001);与LPS组相比用“#”表示,###(P<0.001)。
    Figure  9.  Effect of different concentrations of GLP-K on the NO secretion of RAW264.7 cells

    本研究使用快速溶剂萃取法提取龙须菜多糖,首先利用单因素实验获得提取温度、提取时间和循环次数对龙须菜多糖得率影响的最佳条件,再利用响应面法对快速溶剂法龙须菜多糖提取工艺进行优化,得到最佳提取工艺参数:温度70 ℃,时间8.5 min,循环次数4次,龙须菜多糖得率为9.58%±0.31%,与多元回归方程得到的预测值相近,表明该模型的优化参数基本可靠,具有可行性。红外光谱证实该多糖含有糖醛酸,重均分子量在4.4~747.1 kDa 之间;GLP-K在浓度1000 μg/mL及以下时对RAW264.7细胞增殖也无影响(P<0.001);与模型组相比,GLP-K给药组(50、100、200、300、400、500 μg/mL)NO的释放量显著降低43.76%~69.47%(P<0.001)。本文首次快速溶剂萃取法应用于龙须菜多糖的提取,并初步证实其抗炎活性,其抗炎机理还需进一步研究。

  • 图  1   龙须菜多糖快速溶剂提取工艺流程图

    Figure  1.   Accelerated solvent extraction process flow chart of GLP-K

    图  2   提取温度对龙须菜多糖得率的影响

    Figure  2.   Effect of extracting temperature on the yield of GLP-K

    图  3   提取时间对龙须菜多糖得率的影响

    Figure  3.   Effect of extracting time on the yield of GLP-K

    图  4   循环次数对龙须菜多糖得率的影响

    Figure  4.   Effect of cycle times on the yield of GLP-K

    图  5   AB、AC、BC对龙须菜多糖得率影响的响应面图和等高线图

    Figure  5.   Response surface plots and contour plots of the effects of AB, AC and BC on the yield of GLP-K

    图  6   龙须菜多糖红外光谱图

    Figure  6.   FT-IR of GLP-K

    图  7   HPLC的标准矫正曲线和龙须菜多糖的HPLC洗脱曲线

    Figure  7.   Standard calibrationcurve of HPLC and the HPLC elution profile of GLP-K

    图  8   GLP-K对细胞活力的影响

    注:与对照组相比,***(P<0.001)。

    Figure  8.   Effects of GLP-K on cells viability

    图  9   不同浓度GLP-K 对 RAW264.7 细胞NO分泌水平的影响

    注:与对照组相比用“*”表示,***(P<0.001);与LPS组相比用“#”表示,###(P<0.001)。

    Figure  9.   Effect of different concentrations of GLP-K on the NO secretion of RAW264.7 cells

    表  1   响应面试验设计因素与水平

    Table  1   The design factors and levels of response surface test

    水平 A提取温度(℃) B提取时间(min) C循环次数(次)
    −1 60 6 3
    0 70 8 4
    1 80 10 5
    下载: 导出CSV

    表  2   龙须菜多糖提取工艺条件响应面优化方案及结果

    Table  2   Response surface optimization scheme and results of extraction process conditions of GLP-K

    实验号 A提取温度 B提取时间 C循环次数 Y龙须菜多糖得率(%)
    1 −1 −1 0 7.38
    2 1 −1 0 8.36
    3 −1 1 0 7.79
    4 1 1 0 8.20
    5 −1 0 −1 7.38
    6 1 0 −1 7.83
    7 −1 0 1 8.42
    8 1 0 1 9.16
    9 0 −1 −1 8.15
    10 0 1 −1 8.35
    11 0 −1 1 8.99
    12 0 1 1 9.13
    13 0 0 0 9.68
    14 0 0 0 9.73
    15 0 0 0 9.75
    16 0 0 0 9.69
    17 0 0 0 9.59
    下载: 导出CSV

    表  3   响应面回归方程模型方差分析

    Table  3   Analysis of variance of response surface regression equation model

    来源 偏差平方和 自由度 均方 F P 显著性
    模型 11.24 9 1.25 99.36 <0.0001 **
    A 0.83 1 0.83 66.13 <0.0001 **
    B 0.044 1 0.044 3.53 0.1022
    C 1.99 1 1.99 158.61 <0.0001 **
    AB 0.081 1 0.081 6.45 0.0387 *
    AC 0.022 1 0.022 1.73 0.2298
    BC 0.001 1 0.001 0.070 0.7989
    A2 5.18 1 5.18 411.70 <0.0001 **
    B2 1.77 1 1.77 140.97 <0.0001 **
    C2 0.62 1 0.62 49.36 0.0002 **
    残差 0.088 7 0.013
    失拟项 0.073 3 0.024 6.47 0.0515 不显著
    纯误差 0.015 4 0.001
    总和 11.33 16
    注:R2=0.9922,R2adj=0.9822;*表示有显著性差异(P<0.05);**表示有极显著性差异(P<0.01)。
    下载: 导出CSV

    表  4   保留峰时间和分子量

    Table  4   Retention peak time and molecular weight

    样品保留时间(min)Mn(Da)Mw(Da)D(Mw/Mn
    龙须菜多糖110.69±0.034419±452747122±7508169.07±25.84
    下载: 导出CSV
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  • 收稿日期:  2023-03-28
  • 网络出版日期:  2023-10-08
  • 刊出日期:  2023-11-24

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