改良1,9-二甲基亚甲基蓝法定量检测岩藻多糖

doi:10.19586/j.2095-2341.2023.0046

生物技术进展 ›› 2023, Vol. 13 ›› Issue (5): 779-784.DOI: 10.19586/j.2095-2341.2023.0046

改良1,9-二甲基亚甲基蓝法定量检测岩藻多糖

赵薇萍¹(), 齐艺惠¹, 李晶晶¹^,²(), 宋爽¹, 翟睿³, 杜茜茜¹

^1.大连工业大学食品学院，辽宁大连 116034
^2.北京市计量检测科学研究院，北京 100029
^3.中国计量科学研究院国家市场监管技术创新中心（质谱）前沿计量科学中心，北京 100029

收稿日期:2023-04-06 接受日期:2023-05-04 出版日期:2023-09-25 发布日期:2023-10-10
通讯作者: 李晶晶
作者简介:赵薇萍 E-mail: zweiping46@163.com；
基金资助:
国家重点研发计划项目(2019YFD0902005);国家市场监管技术创新中心（质谱）项目(AKYKF2205)

Quantitative Determination of Fucoidan by Improved 1,9-dimethyl-methylene Blue Method

Weiping ZHAO¹(), Yihui QI¹, Jingjing LI¹^,²(), Shuang SONG¹, Rui ZHAI³, Qianqian DU¹

^1.College of Food，Dalian Polytechnic University，Liaoning Dalian 116034，China
^2.Beijing Institute of Metrology，Beijing 100029，China
^3.Technology Innovation Center of Mass Spectrometry for State Market Regulation，Center for Advanced Measurement Science，National Institute of Metrology，Beijing 100029，China

Received:2023-04-06 Accepted:2023-05-04 Online:2023-09-25 Published:2023-10-10
Contact: Jingjing LI

摘要/Abstract

摘要：

岩藻多糖是一类硫酸化多糖，主要提取自海参、褐藻等，其具有抗凝血、抗肿瘤、抗血栓等多种生物活性，在医药和食品行业具有广阔的应用前景。目前，1,9-二甲基亚甲基蓝(1,9-dirnethyl-methylene blue，DMMB)比色法是岩藻多糖定量检测的常用方法之一，但存在灵敏度差、操作繁琐、耗时长等问题。优化了DMMB法的显色液浓度、pH、显色反应时间等条件，改良后检测方法的线性范围、精密度、回收率和定量限均有显著改善。改进后DMMB法检测岩藻多糖的最佳反应条件为：DMMB溶液浓度为42.8 mg·mL^-1、pH 3.3，避光反应在10~15 min之间进行检测。方法学考察结果表明，改良后的DMMB比色方法提高了岩藻多糖定量检测方法的灵敏度，将检测浓度范围拓宽至0.012 5~0.300 0 mg·mL^-1，这为岩藻多糖定量分析提供了快捷有效的手段，有利于岩藻多糖的开发与利用。

关键词: 岩藻多糖, 1,9-二甲基亚甲基蓝, 定量检测, 条件优化

Abstract:

As a kind of sulfated polysaccharide mainly extracted from sea cucumbers and brown algae， etc.， fucoidan has anticoagulant， antitumor， antithrombotic and other biological activities， and has broad application prospects in the pharmaceutical and food industries. Currently， 1，9-dimethylmethylene blue （DMMB） colorimetry is one of the common methods for quantitative detection of fucoidan， but there are some problems such as poor sensitivity， cumbersome operation and long time consumption. This study optimized the concentration， pH， and reaction time of the DMMB method， and significantly improved the linear range， precision， recovery rate and limit of quantification of the improved detection method. The optimal reaction conditions for the improved DMMB method to detect fucoidan were a DMMB solution concentration of 42.8 mg·mL^-1, pH 3.3, a dark reaction, and detection between 10~15 minutes. The methodological investigation results showed that the improved DMMB colorimetric method raised the sensitivity of the quantitative detection method for fucoidan， expanded the detection concentration range to 0.012 5~0.300 0 mg·mL^-1， and provided a fast and effective means for the quantitative analysis of fucoidan， which was conducive to the development and utilization of fucoidan.

Key words: fucoidan, 1，9-dimethylmethylene blue, quantitative detection, condition optimization

中图分类号:

Q539

赵薇萍, 齐艺惠, 李晶晶, 宋爽, 翟睿, 杜茜茜. 改良1,9-二甲基亚甲基蓝法定量检测岩藻多糖[J]. 生物技术进展, 2023, 13(5): 779-784.

Weiping ZHAO, Yihui QI, Jingjing LI, Shuang SONG, Rui ZHAI, Qianqian DU. Quantitative Determination of Fucoidan by Improved 1,9-dimethyl-methylene Blue Method[J]. Current Biotechnology, 2023, 13(5): 779-784.

图/表 6

图1 DMMB显色液浓度对标准曲线的影响

Fig. 1 The effect of DMMB concentration on standard curve

图2 DMMB溶液pH对标准曲线的影响

Fig. 2 The effect of DMMB pH on standard curve

图3 DMMB-岩藻多糖溶液的紫外可见光谱图

Fig. 3 UV-vis spectra of DMMB-fucoidan solution

图4 显色液的吸光度变化

Fig. 4 Absorbance change of chromogenic solution

表1 回收率和精密度考察结果

Table 1 The results of recovery and precision

样品	RSD	回收率	加标水平/(mg·mL^-1)
样品1	0.8%	95.8%	0.04
样品1	0.8%	99.8%	0.10
样品2	1.0%	104.1%	0.06
样品2	1.0%	95.1%	0.14

图5 杂质对DMMB法检测岩藻多糖的影响注：不同小写字母表示样品间在P<0.05水平上具有统计学意义。

Fig. 5 The effects of impurities on DMMB method for determination of fucoidan

参考文献 22

1	HOAGLAND D R, LIEB L L. The complex carbohydrates and forms of sulphur in marine algae of the Pacific Coast[J]. J. Biol. Chem., 1915, 23(1): 287-297.
2	KANG S M, KIM K N, LEE S H, et al.. Anti-inflammatory activity of polysaccharide purified from AMG-assistant extract of Ecklonia cava in LPS-stimulated RAW 264.7 macrophages[J]. Carbohydr. Polym., 2011, 85(1): 80-85.
3	NARAYANI S S, SARAVANAN S, RAVINDRAN J, et al.. In vitro anticancer activity of fucoidan extracted from Sargassum cinereum against Caco-2 cells[J]. Int. J. Biol. Macromol., 2019,138: 618-628.
4	SHANG Q, SHAN X, CAI C, et al.. Dietary fucoidan modulates the gut microbiota in mice by increasing the abundance of Lactobacillus and Ruminococcaceae [J]. Food Funct., 2016, 7(7): 3224-3232.
5	HIFNEY A F, FAWZY M A, ABDEL-GAWAD K M, et al.. Industrial optimization of fucoidan extraction from Sargassum sp. and its potential antioxidant and emulsifying activities[J]. Food Hydrocoll., 2016, 54: 77-88.
6	SINGDEVSACHAN S K, AUROSHREE P, MISHRA J, et al.. Mushroom polysaccharides as potential prebiotics with their antitumor and immunomodulating properties: a review[J]. Bioactive Carbo. hydr. Diet. Fibre, 2016, 7(1): 1-14.
7	ZHANG Z, TILL S, KNAPPE S, et al.. Screening of complex fucoidans from four brown algae species as procoagulant agents[J]. Carbohydr. Polym., 2015, 115: 677-685.
8	HEMMINGSON J A, FALSHAW R, FURNEAUX R H, et al.. Structure and antiviral activity of the galactofucan sulfates extracted from Undaria pinnatifida (phaeophyta)[J]. J. Appl. Phycol., 2006, 18(2): 185-193.
9	李宝花.黄山石耳多糖的提取纯化及其体外模拟消化的研究[D].南京:南京农业大学, 2020.
10	李桂芝,刘永明,秦华伟.亚甲基蓝褪色分光光度法测定藻酸双酯钠[J].分析化学,2005,33(9):1324-1326.
11	王泽文,冷凯良,翟毓秀,等.亚甲基蓝比色法测定海参不同组织酸性黏多糖含量[J].海洋科学,2011,35(3):77-82.
12	DE LIMA C R, BACCARIN R Y A, MICHELACCI Y M. Reliability of 1, 9-dimethylmethylene blue tests in comparison to agarose gel electrophoresis for quantification of urinary glycosaminoglycans[J]. Clin. Chim. Acta, 2007, 378(1-2): 206-215.
13	国家药典委员会.2010中华人民共和国药典(第3部)[M]. 北京: 中国医药科技出版社, 2010.
14	吴泽,魏琦峰,邱庆庆,等.褐藻多糖的分离提取及生理活性研究进展[J].生物技术进展,2015,5(3):201-206.
15	刘红英,薛长湖,李兆杰,等.海带岩藻聚糖硫酸酯测定方法的研究[J].青岛海洋大学学报(自然科学版),2002,32(2):236-240.
16	范丽萍,刘志伟,钟瑜萍,等.pH值对壳寡糖-柠檬酸盐-果胶自组装的影响[J].食品科技,2022,47(8):248-255.
17	陶红平,张觅,游长江,等. IL-13抑制剂在损伤大鼠尾椎间盘退变中的作用研究[J].生物技术进展,2021,11(3): 369-377.
18	PTAK S H, SANCHEZ L, FRETTÉ X, et al.. Complementarity of raman and infrared spectroscopy for rapid characterization of fucoidan extracts[J/OL]. Plant Meth., 2021, 17(1): 130[2023-07-18]. .
19	RICHARDSON J C, DETTMAR P W, HAMPSON F C, et al.. A simple, high throughput method for the quantification of sodium alginates on oesophageal mucosa[J]. Eur. J. Pharm. Biopharm., 2004, 57(2): 299-305.
20	李玉芹,陈赛兰,贾淑婷,等.岩藻多糖氧化降解工艺优化及其对多糖结构和抗氧化活性的影响[J].湘潭大学学报(自然科学版), 2023,45(1):13-26.
21	张海毅,丛大鹏,李广生,等.岩藻多糖原料药多糖含量测定方法的研究[J].生物化工,2021,7(4):69-73+77.
22	FARNDALE R W, BUTTLE D J, BARRETT A J. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue[J]. Biochim. Biophys. Acta, 1986, 883(2): 173-177.

[1]	刘蕾, 周欣悦, 朱桢, 曹烨, 王文彬. 副溶血弧菌外膜铁蛋白受体pvuA基因的原核表达及产物的诱导条件优化[J]. 生物技术进展, 2022, 12(3): 396-404.
[2]	张文静, 佟晔, 杨锡文, 曹彦金, 魏计东. 高产中性蛋白酶菌株的筛选、优化及中试放大[J]. 生物技术进展, 2022, 12(1): 112-119.
[3]	缪青梅,赵杨,徐晓丽,徐俊锋,汪小福,陈笑芸. 转g10-epsps基因耐除草剂大豆ZUTS-33转化体特异性检测方法的建立[J]. 生物技术进展, 2020, 10(6): 696-703.
[4]	武文艳,刘新香,周秒依,刘金香,刘亚. 转基因玉米2A-5特异性PCR方法的建立[J]. 生物技术进展, 2020, 10(4): 363-370.
[5]	刘晓,朱鹏宇,景小艳,李志红,张永江,李想,付伟,. 双重数字PCR在转基因大豆检测中的应用[J]. 生物技术进展, 2020, 10(1): 60-66.
[6]	余爱丽,赵晋锋,张晏萌,张正,郭二虎. 猪SsBHMT基因的原核表达及条件优化[J]. 生物技术进展, 2015, 5(2): 132-136.
[7]	杨煌建,张祝兰,黄小珍,郑卫. 链霉菌FIM-0916产安福霉素的发酵条件优化[J]. 生物技术进展, 2014, 4(2): 118-123.

改良1,9-二甲基亚甲基蓝法定量检测岩藻多糖

Quantitative Determination of Fucoidan by Improved 1,9-dimethyl-methylene Blue Method

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 22

相关文章 7

编辑推荐

Metrics

本文评价