昆布多糖不同提取工艺优化及其理化性质和抗肿瘤活性比较
|
摘要
在单因素试验的基础上通过正交试验对昆布多糖的提取条件进行优化,并对比分析热水浸提法、超声辅助提取法和复合酶法3种方法提取的昆布多糖的理化性质和抗肿瘤活性。结果表明,优化后昆布多糖的提取条件为,热水浸提法:提取温度85℃、提取时间5 h、料液比1∶80(g/mL),此条件下多糖平均提取率为5.97%;超声辅助提取法:超声功率500 W、超声时间10 min、料液比1∶90(g/mL),此条件下多糖平均提取率为5.81%;复合酶法:木瓜蛋白酶75 U/g、果胶酶15 000 U/g、纤维素酶108 000 U/g、pH 5、温度50℃、料液比1∶70(g/mL)、时间3 h,此条件下多糖平均提取率为14.29%。不同提取方法对昆布多糖的成分有显著影响,复合酶法提取的昆布多糖的溶解性显著优于水浴和超声提取。提取的昆布多糖中均具有少量蛋白质或多肽存在,并且具有三股螺旋结构。噻唑蓝(MTT)实验表明,不同提取方法得到的昆布多糖具有不同的抗肿瘤活性,其中超声辅助提取多糖对人肺癌细胞A549的抑制作用最强,而复合酶法所得多糖对人乳腺癌细胞MCF-7的抑制效果最好。
Three extraction methods, including hot water extraction(HWE), ultrasonic-assisted extraction(UAE) and multiple enzymatic extraction(MEE), were used to extract polysaccharides from Laminaria japonica. Extraction conditions were optimized using one-factor-at-a-time and orthogonal array design methods, and the physicochemical properties and antitumor activity of the polysaccharides were comparatively analyzed. The results showed that the optimized HWE conditions were as follows: temperature 85 ℃, extraction time 5 h, and solid to solvent ratio 1:80(g/m L); the optimized UAE conditions were ultrasonic power 500 W, extraction time, 10 min, and solid to solvent ratio 1:90(g/mL); the optimized MEE conditions were papain 75 U/g, pectinase 15 000 U/g, cellulase 108 000 U/g, pH 5, temperature 50 ℃, solid to solvent ratio1:70(g/m L), and extraction time 3 h. Under the above conditions, the average yield of polysaccharides was 5.97%, 5.81%and 14.29%, respectively. There were signi?cant effects of different extraction methods on the polysaccharide composition.The solubility of the polysaccharides obtained by MEE was signi?cantly better than those obtained with HWE and UAE. All the polysaccharides contained a small amount of protein or polypeptide, and had three-strand helical structures. MTT assay showed that the three polysaccharides exhibited different antitumor activities, among which the polysaccharides obtained by UAE showed the strongest inhibitory effect on human lung cancer cell A549 while the polysaccharides extracted by CEE displayed the strongest inhibitory effect on human breast cancer cell line MCF-7.
Three extraction methods, including hot water extraction(HWE), ultrasonic-assisted extraction(UAE) and multiple enzymatic extraction(MEE), were used to extract polysaccharides from Laminaria japonica. Extraction conditions were optimized using one-factor-at-a-time and orthogonal array design methods, and the physicochemical properties and antitumor activity of the polysaccharides were comparatively analyzed. The results showed that the optimized HWE conditions were as follows: temperature 85 ℃, extraction time 5 h, and solid to solvent ratio 1:80(g/m L); the optimized UAE conditions were ultrasonic power 500 W, extraction time, 10 min, and solid to solvent ratio 1:90(g/mL); the optimized MEE conditions were papain 75 U/g, pectinase 15 000 U/g, cellulase 108 000 U/g, pH 5, temperature 50 ℃, solid to solvent ratio1:70(g/m L), and extraction time 3 h. Under the above conditions, the average yield of polysaccharides was 5.97%, 5.81%and 14.29%, respectively. There were signi?cant effects of different extraction methods on the polysaccharide composition.The solubility of the polysaccharides obtained by MEE was signi?cantly better than those obtained with HWE and UAE. All the polysaccharides contained a small amount of protein or polypeptide, and had three-strand helical structures. MTT assay showed that the three polysaccharides exhibited different antitumor activities, among which the polysaccharides obtained by UAE showed the strongest inhibitory effect on human lung cancer cell A549 while the polysaccharides extracted by CEE displayed the strongest inhibitory effect on human breast cancer cell line MCF-7.
引文
[1]王丽波,程龙,徐雅琴,等.南瓜籽多糖热水提取工艺优化及其抗氧化活性[J].农业工程学报,2016,32(9):284-290.DOI:10.11975/j.issn.1002-6819.2016.09.040.
[2]李奎,李雪玲.响应面法优化碱液提取百蕊草多糖的工艺研究[J].食品工业科技,2015,36(7):204-207;213.DOI:10.13386/j.issn1002-0306.2015.07.035.
[3]税丹,王立峰,袁建,等.酸法提取菜籽多糖的抗氧化活性研究[J].食品科学,2011,32(21):98-101.
[4]CHEN C,ZHANG B,HUANG Q,et al.Microwave-assisted extraction of polysaccharides from Moringa oleifera Lam.leaves:characterization and hypoglycemic activity[J].Industrial Crops and Products,2017,100:1-11.DOI:10.1016/j.indcrop.2017.01.042.
[5]邱现创,赵宁,李晨,等.铁皮石斛多糖提取工艺优化及对果蝇抗氧化能力的影响[J].食品科学,2018,39(2):273-280.DOI:10.7506/spkx1002-6630-201802043.
[6]赵亚红,王文侠,张显斌,等.高温高压提取甜菜废粕多糖的工艺及其抗氧化性研究[J].食品与发酵工业,2015,41(4):230-235.DOI:10.13995/j.cnki.11-1802/ts.201504043.
[7]BHOTMANGE U D,WALLENIUS H J,SINGHAL S R,et al.Enzymatic extraction and characterization of polysaccharide from Tuber aestivum[J].Bioactive Carbohydrates and Dietary Fibre,2017,10:1-9.DOI:10.1016/j.bcdf.2017.02.001.
[8]张帅,郑宝东,林良美,等.笋壳多糖的微波-超声波联合辅助提取工艺优化及其抗氧化活性[J].食品科学,2015,36(16):72-76.DOI:10.7506/spkx1002-6630-201516013.
[9]万宇俊,殷军艺,聂少平,等.微波提取对胡萝卜中的多糖基本结构特征及固体形貌的影响[J].食品科学,2017,38(7):1-5.DOI:10.7506/spkx1002-6630-201707001.
[10]SUN Y,HOU S,SONG S,et al.Impact of acidic,water and alkaline extraction on structural features,antioxidant activities of Laminaria japonica polysaccharides[J].International Journal of Biological Macromolecules,2018,112:985-995.DOI:10.1016/j.ijbiomac.2018.02.066.
[11]ZHU Z Y,DONG F,LIU X,et al.Effects of extraction methods on the yield,chemical structure and anti-tumor activity of polysaccharides from Cordyceps gunnii mycelia[J].Carbohydrate Polymers,2016,140:461-471.DOI:10.106/j.carbpol.2015.12.053.
[12]WANG J L,ZHANG J,WANG X F,et al.Comparison study on microwave-assisted extraction of Artemisia sphaerocephala polysaccharides with conventional method:molecule structure and antioxidant activities evaluation[J].International Journal of Biological Macromolecules,2009,45(5):483-492.DOI:10.1016/j.ijbiomac.2009.09.004.
[13]ZHAO C L,LI X,MIAO J,et al.The effect of different extraction techniques on property and bioactivity of polysaccharides from Dioscorea hemsleyi[J].International Journal of Biological M a c r o m o l e c u l e s,2 0 1 7,1 0 2:8 4 7-8 5 6.D O I:1 0.1 0 1 6/j.ijbiomac.2017.04.031.
[14]YAN Y J,LI X,WAN M,CHEN J,et al.Effect of extraction methods on property and bioactivity of water-soluble polysaccharides from Amomum villosum[J].Carbohydrate Polymers,2015,117:632-635.DOI:10.1016/j.carbpol.2014.09.070.
[15]国家药典委员会编.药典:一部[S].北京:化学工业出版社,2010.
[16]XU H L,KITAJIMA C,ITO H,et al.Antidiabetic effect of polyphenols from brown alga Ecklonia kurome in genetically diabetic KK-A(y)mice[J].Pharmaceutical Biology,2011,50(3):393-400.DOI:10.3109/13880209.2011.60146.
[17]SIN S S,YOON M.The herbal composition GGEx18 from Laminaria japonica,Rheum palmatum,and Ephedra sinica inhibits highfat diet-induced hepatic steatosis via hepatic PPARαactivation[J].Pharmaceutical Biology,2012,50(10):1261-1268.DOI:10.3109/13880209.2012.666982.
[18]HUANG L,WEN K,GAO X,et al.Hypolipidemic effect of fucoidan from Laminaria japonica in hyperlipidemic rats[J].Pharmaceutical Biology,2010,48(4):422-426.DOI:10.3109/13880200903150435.
[19]CHOI J S,BAE H J,KIM S J,et al.In vitro antibacterial and antiinflammatory properties of seaweed extracts against acne inducing bacteria,Propionibacterium acnes[J].Journal of Environmental Biology,2011,32(3):313-318.
[20]季宇彬,娄艳华,高世勇.昆布多糖分离纯化及其抗肿瘤活性的研究[J].中草药杂志,2009(1):132-135.
[21]ZENG M,WU X,LI F,et al.Laminaria japonica polysaccharides effectively inhibited the growth of nasopharyngeal carcinoma cells in vivo and in vitro study[J].Experimental and Toxicologic Pathology,2017,69(7):527-532.
[22]万琴,萧伟,王振中,等.女贞子多糖除蛋白工艺的研究[J].中草药,2010,41(3):407-410.
[23]KONG L S,YU L,FENG T,et al.Physicochemical characterization of the polysaccharide from Bletilla striata:effect of drying methods[J].Carbohydrate Polymers,2015,125:1-8.DOI:10.1016/j.carbpol.2015.02.042.
[24]徐雅琴,刘菲,郭莹莹,等.黑穗醋栗果实超声波降解多糖的结构及抗糖基化活性[J].农业工程学报,2017,33(5):295-300.
[25]于曼曼,夏光华,李川,等.罗非鱼头长链碱的分离纯化及抗肿瘤活性[J].食品科学,2018,39(5):166-172.DOI:10.7506/spkx1002-6630-201805025.
[26]魏晓梅,吴丽芳,柏旭,等.云南松茸多糖提取方案优化及其测定[J].中国食用菌,2018(1):46-49.DOI:10.13629/j.cnki.53-1054.2018.01.012.
[27]朱振元,韩丹.滑子菇多糖提取工艺优化[J].食品研究与开发,2018,39(4):68-72.DOI:10.3969/j.issn.1005-6521.2018.04.012.
[28]王振斌,刘加友,马海乐,等.无花果多糖提取工艺优化及其超声波改性[J].农业工程学报,2014,30(10):262-269.DOI:10.3969/j.issn.1002-6819.2014.10.033.
[29]刘娜女,张静,李岱,等.利用傅立叶红外光谱和原子力显微镜研究超声提取对鸡腿菇多糖结构的影响[J].生物加工过程,2010(1):56-60.DOI:10.3969/j.issn.1672-3678.2010.01.011.
[30]MENG X,LIANG H,LUO L.Antitumor polysaccharides from mushrooms:a review on the structural characteristics,antitumor mechanisms and immunomodulating activities[J].Carbohydrate Research,2016,424:30-41.DOI:10.1016/j.carres.2016.02.008.
[31]丁振东,张玉影,张宇,等.五味子多糖对脑肿瘤干细胞的凋亡诱导及生长抑制作用[J].吉林大学学报(医学版),2018(2):305-309.DOI:10.13481/j.1671-587x.20180218.
[32]CHEN YJ,JIANG X,XIE H Q,et al.Structural characterization and antitumor activity of a polysaccharide from Ramulus mori[J].Carbohydrate Polymers,2018,190:232-239.DOI:10.1016/j.carbpol.2018.02.036.
[2]李奎,李雪玲.响应面法优化碱液提取百蕊草多糖的工艺研究[J].食品工业科技,2015,36(7):204-207;213.DOI:10.13386/j.issn1002-0306.2015.07.035.
[3]税丹,王立峰,袁建,等.酸法提取菜籽多糖的抗氧化活性研究[J].食品科学,2011,32(21):98-101.
[4]CHEN C,ZHANG B,HUANG Q,et al.Microwave-assisted extraction of polysaccharides from Moringa oleifera Lam.leaves:characterization and hypoglycemic activity[J].Industrial Crops and Products,2017,100:1-11.DOI:10.1016/j.indcrop.2017.01.042.
[5]邱现创,赵宁,李晨,等.铁皮石斛多糖提取工艺优化及对果蝇抗氧化能力的影响[J].食品科学,2018,39(2):273-280.DOI:10.7506/spkx1002-6630-201802043.
[6]赵亚红,王文侠,张显斌,等.高温高压提取甜菜废粕多糖的工艺及其抗氧化性研究[J].食品与发酵工业,2015,41(4):230-235.DOI:10.13995/j.cnki.11-1802/ts.201504043.
[7]BHOTMANGE U D,WALLENIUS H J,SINGHAL S R,et al.Enzymatic extraction and characterization of polysaccharide from Tuber aestivum[J].Bioactive Carbohydrates and Dietary Fibre,2017,10:1-9.DOI:10.1016/j.bcdf.2017.02.001.
[8]张帅,郑宝东,林良美,等.笋壳多糖的微波-超声波联合辅助提取工艺优化及其抗氧化活性[J].食品科学,2015,36(16):72-76.DOI:10.7506/spkx1002-6630-201516013.
[9]万宇俊,殷军艺,聂少平,等.微波提取对胡萝卜中的多糖基本结构特征及固体形貌的影响[J].食品科学,2017,38(7):1-5.DOI:10.7506/spkx1002-6630-201707001.
[10]SUN Y,HOU S,SONG S,et al.Impact of acidic,water and alkaline extraction on structural features,antioxidant activities of Laminaria japonica polysaccharides[J].International Journal of Biological Macromolecules,2018,112:985-995.DOI:10.1016/j.ijbiomac.2018.02.066.
[11]ZHU Z Y,DONG F,LIU X,et al.Effects of extraction methods on the yield,chemical structure and anti-tumor activity of polysaccharides from Cordyceps gunnii mycelia[J].Carbohydrate Polymers,2016,140:461-471.DOI:10.106/j.carbpol.2015.12.053.
[12]WANG J L,ZHANG J,WANG X F,et al.Comparison study on microwave-assisted extraction of Artemisia sphaerocephala polysaccharides with conventional method:molecule structure and antioxidant activities evaluation[J].International Journal of Biological Macromolecules,2009,45(5):483-492.DOI:10.1016/j.ijbiomac.2009.09.004.
[13]ZHAO C L,LI X,MIAO J,et al.The effect of different extraction techniques on property and bioactivity of polysaccharides from Dioscorea hemsleyi[J].International Journal of Biological M a c r o m o l e c u l e s,2 0 1 7,1 0 2:8 4 7-8 5 6.D O I:1 0.1 0 1 6/j.ijbiomac.2017.04.031.
[14]YAN Y J,LI X,WAN M,CHEN J,et al.Effect of extraction methods on property and bioactivity of water-soluble polysaccharides from Amomum villosum[J].Carbohydrate Polymers,2015,117:632-635.DOI:10.1016/j.carbpol.2014.09.070.
[15]国家药典委员会编.药典:一部[S].北京:化学工业出版社,2010.
[16]XU H L,KITAJIMA C,ITO H,et al.Antidiabetic effect of polyphenols from brown alga Ecklonia kurome in genetically diabetic KK-A(y)mice[J].Pharmaceutical Biology,2011,50(3):393-400.DOI:10.3109/13880209.2011.60146.
[17]SIN S S,YOON M.The herbal composition GGEx18 from Laminaria japonica,Rheum palmatum,and Ephedra sinica inhibits highfat diet-induced hepatic steatosis via hepatic PPARαactivation[J].Pharmaceutical Biology,2012,50(10):1261-1268.DOI:10.3109/13880209.2012.666982.
[18]HUANG L,WEN K,GAO X,et al.Hypolipidemic effect of fucoidan from Laminaria japonica in hyperlipidemic rats[J].Pharmaceutical Biology,2010,48(4):422-426.DOI:10.3109/13880200903150435.
[19]CHOI J S,BAE H J,KIM S J,et al.In vitro antibacterial and antiinflammatory properties of seaweed extracts against acne inducing bacteria,Propionibacterium acnes[J].Journal of Environmental Biology,2011,32(3):313-318.
[20]季宇彬,娄艳华,高世勇.昆布多糖分离纯化及其抗肿瘤活性的研究[J].中草药杂志,2009(1):132-135.
[21]ZENG M,WU X,LI F,et al.Laminaria japonica polysaccharides effectively inhibited the growth of nasopharyngeal carcinoma cells in vivo and in vitro study[J].Experimental and Toxicologic Pathology,2017,69(7):527-532.
[22]万琴,萧伟,王振中,等.女贞子多糖除蛋白工艺的研究[J].中草药,2010,41(3):407-410.
[23]KONG L S,YU L,FENG T,et al.Physicochemical characterization of the polysaccharide from Bletilla striata:effect of drying methods[J].Carbohydrate Polymers,2015,125:1-8.DOI:10.1016/j.carbpol.2015.02.042.
[24]徐雅琴,刘菲,郭莹莹,等.黑穗醋栗果实超声波降解多糖的结构及抗糖基化活性[J].农业工程学报,2017,33(5):295-300.
[25]于曼曼,夏光华,李川,等.罗非鱼头长链碱的分离纯化及抗肿瘤活性[J].食品科学,2018,39(5):166-172.DOI:10.7506/spkx1002-6630-201805025.
[26]魏晓梅,吴丽芳,柏旭,等.云南松茸多糖提取方案优化及其测定[J].中国食用菌,2018(1):46-49.DOI:10.13629/j.cnki.53-1054.2018.01.012.
[27]朱振元,韩丹.滑子菇多糖提取工艺优化[J].食品研究与开发,2018,39(4):68-72.DOI:10.3969/j.issn.1005-6521.2018.04.012.
[28]王振斌,刘加友,马海乐,等.无花果多糖提取工艺优化及其超声波改性[J].农业工程学报,2014,30(10):262-269.DOI:10.3969/j.issn.1002-6819.2014.10.033.
[29]刘娜女,张静,李岱,等.利用傅立叶红外光谱和原子力显微镜研究超声提取对鸡腿菇多糖结构的影响[J].生物加工过程,2010(1):56-60.DOI:10.3969/j.issn.1672-3678.2010.01.011.
[30]MENG X,LIANG H,LUO L.Antitumor polysaccharides from mushrooms:a review on the structural characteristics,antitumor mechanisms and immunomodulating activities[J].Carbohydrate Research,2016,424:30-41.DOI:10.1016/j.carres.2016.02.008.
[31]丁振东,张玉影,张宇,等.五味子多糖对脑肿瘤干细胞的凋亡诱导及生长抑制作用[J].吉林大学学报(医学版),2018(2):305-309.DOI:10.13481/j.1671-587x.20180218.
[32]CHEN YJ,JIANG X,XIE H Q,et al.Structural characterization and antitumor activity of a polysaccharide from Ramulus mori[J].Carbohydrate Polymers,2018,190:232-239.DOI:10.1016/j.carbpol.2018.02.036.