黑木耳凝集素的提取优化及体外抗肿瘤活性的研究

doi:10.11924/j.issn.1000-6850.casb2021-0445

摘要/Abstract

摘要：

为了获得黑木耳凝集素的最优提取条件以及研究其对乳腺癌细胞MCF-7的抑制作用,采用单因素试验和响应面的方法,对提取时间、提取温度及料液比3个条件进行优化;采用CCK-8试剂法、形态学观察、划痕法来探究黑木耳凝集素对MCF-7的增殖、形态学变化和迁移率的影响来研究其抗肿瘤活性。结果表明：在浸提时间为8.47 h,料液比为1:51.71,(NH₄)₂SO₄浓度为50.23%时,粗提液中的凝集活性最高,可得最大理论值为5.4795 HU;黑木耳凝集素处理细胞后,细胞的存活率降低,细胞数量减少,细胞形态发生变化,划痕闭合速度减慢。且与凝集素浓度具有依赖性,浓度越高细胞活性越低,细胞数量越少,对细胞的迁移率抑制性越强。该实验获得了黑木耳凝集素的最优提取条件,以及对MCF-7细胞的抑制效果,这对凝集素的提取方法及其抗肿瘤活性的研究提供了参考。

关键词: 黑木耳, 凝集素, 提取优化, 响应面, 抗肿瘤

Abstract:

The aims are to obtain the optimal extraction condition of Auricularia auricula lectin and study its inhibitory effect on breast cancer cells MCF-7. Single factor test and response surface method were used to optimize the extraction time, extraction temperature and material-to-liquid ratio; CCK-8 reagent method, morphological observation and scratch method were used to explore the effects of Auricularia auricula lectin on the proliferation, morphological change and migration rate of MCF-7 to study the anti-tumor activity. The results indicated that: when the extraction time was 8.47 h, the material-to-liquid ratio was 1:51.71, and the (NH₄)₂SO₄ concentration was 50.23%, the agglutination activity in the crude extract was the highest, and the maximum theoretical value was 5.4795HU. After Auricularia auricula lectin treated the cells, the survival rate of the cells was decreased, the number of cells was reduced, the cell morphology was changed, and the scratch closure speed slowed down. And the change of cells was dependent on the concentration of lectin. The higher the concentration, the lower the cell viability and the smaller the number of cells, the stronger the inhibition on cell migration. This experiment obtained the optimal extraction condition of Auricularia auricula lectin and clarified its inhibitory effect on MCF-7 cells, which could provide certain reference for studying lectin extraction methods and its anti-tumor activity.

Key words: Auricularia auricula, lectin, extraction optimization, response surface method, anti-tumor

中图分类号:

S646.6

苏林贺, 黄东, 曾伟民, 张彦龙. 黑木耳凝集素的提取优化及体外抗肿瘤活性的研究[J]. 中国农学通报, 2022, 38(9): 143-150.

SU Linhe, HUANG Dong, ZENG Weimin, ZHANG Yanlong. Extraction Optimization of Auricularia auricula Lectin and Study on Its Anti-tumor Activity in Vitro[J]. Chinese Agricultural Science Bulletin, 2022, 38(9): 143-150.

图/表 11

参考文献 27

[1]	张金霞, 谢宝贵. 食用菌菌种生产与管理手册[M]. 北京: 中国农业大学出版社, 2006:5-7.
[2]	XU S Q, XU X J, ZHANG L N. Branching structure and chain conformation of water-soluble glucan extracted from Auricularia auricula-judae[J]. Journal of agricultural and food chemistry, 2012, 60(13):3498-506. doi: 10.1021/jf300423z URL
[3]	CHEN G, LUO Y C, JI B P, et al. Effect of polysaccharide from Auricularia auricula on blood lipid metabolism and lipoprotein lipase activity of icr mice fed a cholesterol-enriched diet[J]. Journal of food science, 2008, 73(6):H103-H108. doi: 10.1111/jfds.2008.73.issue-6 URL
[4]	REGINALD C O, ESSIEN E B. Evaluation and characterization of nutritive properties of the jelly ear culinary-medicinal mushroom Auricularia auricula-judae (Agaricomycetes) from Nigeria[J]. International journal of medicinal mushrooms, 2017, 19(2):173-177. doi: 10.1615/IntJMedMushrooms.v19.i2 URL
[5]	NASCIMENTO K S, CUNHA A I, NASCIMENTO K S, et al. An overview of lectins purification strategies[J]. Journal of molecular recognition, 2012, 25(11):527-541. doi: 10.1002/jmr.v25.11 URL
[6]	VALENTINA E N, EKATERINA A L, ELENA P. Vetchinkina. Lectins from mycelia of basidiomycetes[J]. International journal of molecular sciences, 2017, 18(7):1334-1334. doi: 10.3390/ijms18071334 URL
[7]	MISHRA A, BEHURA A, KUMAR A, et al. Soybean lectin induces autophagy through P2RX7 dependent activation of NF-κB-ROS pathway to kill intracellular mycobacteria[J]. Biochimica et biophysica acta (bba) - general subjects, 2021, 1865(2):789-791.
[8]	陈雨秋, 周国华, 顾军. 半乳糖凝集素3与乳腺癌的研究进展[J]. 东南国防医药, 2020, 22(5):510-515.
[9]	李疆, 王颖, 郭巧珍. 菌类凝集素免疫调节作用的研究进展[J]. 食品安全质量检测学报, 2020, 11(21):7669-7673.
[10]	HAROLD R, HANS J G. Plant lectins: Occurrence, biochemistry, functions and applications[J]. Glycoconjugate journal, 2001, 18(8):589-613. doi: 10.1023/A:1020687518999 URL
[11]	RAM S S, RANJEETA B, HEMANT P K. Mushroom lectins: current status and future perspectives[J]. Critical reviews in biotechnology, 2010, 30(2):99-126. doi: 10.3109/07388550903365048 URL
[12]	BOVI M, CARRIZO M E, CAPALDI S, et al. Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms[J]. Glycobiology 2011, 21:1000-1009. doi: 10.1093/glycob/cwr012 URL
[13]	LIU Q, WANG H, NG TB. First report of a xylose-specific lectin with potent hemagglutinating, antiproliferative and anti-mitogenic activities from a wild ascomycete mushroom[J]. Biochim biophys acta, 2006, 1760(12):1914-1919.
[14]	ZHAO S, ZHAO Y C, LI S H, et al. A novel lectin with highly potent antiproliferative and HIV-1 reverse transcriptase inhibitory activities from the edible wild mushroom Russula delica[J]. Glycoconjugate journal, 2010, 27(2):259-265. doi: 10.1007/s10719-009-9274-5 URL
[15]	刘振东, 黄悦, 李鹏飞, 等. 西藏黑木耳中1种新的凝集素的分离纯化及鉴定[J]. 中草药, 2018, 49(20):4785-4790.
[16]	于洋, 马成瑶, 黄东, 等. 黑木耳凝集素对RAW264.7鼠巨噬细胞活化的影响[J]. 黑龙江大学自然科学学报, 2021, 38(1):86-92.
[17]	房佳敏, 马成瑶, 张彦龙. 黑木耳凝集素对RAW264.7细胞免疫调节作用的研究[J]. 中国农学通报, 2021, 37(9):125-130.
[18]	于雪慧, 童军茂, 张建. 奶花芸豆中凝集素的提取、纯化及鉴定[J]. 食品工业科技, 2018, 39(15):48-54.
[19]	张柏林, 刘宁, 王双双, 等. 大豆凝集素纯化及凝集活性测定的研究[J]. 江苏农业科学, 2019, 47(12):221-224.
[20]	LIANG R J. Optimization of extraction process of Glycyrrhiza glabra polysaccharides by response surface methodology[J]. Carbohydrate polymers, 2008, 74(4):858-861. doi: 10.1016/j.carbpol.2008.05.005 URL
[21]	敖丽, 王凯, 鲁晓岚, 等. 荧光显微镜联合CCK8法测定原花青素B2对胃癌细胞自噬和凋亡的影响[J]. 中国医疗设备, 2020, 35(1):82-84.
[22]	张舒, 王长远, 谭朝印, 等. 绿豆清蛋白Osborne分级提取工艺优化及亚基组成分析[J]. 食品与机械, 2019, 35(10):174-178.
[23]	肖俊琪, 翟爱华, 李嘉庆, 等. 蒸汽爆破预处理辅助提取米糠蛋白的工艺研究[J]. 中国食品添加剂, 2018(9):134-141.
[24]	肖俊琪, 翟爱华, 张东杰. 赤小豆凝集素的分离纯化及电泳分析[J]. 食品与机械, 2020, 36(7):161-166,200.
[25]	邓政东, 程爱芳, 蒲玉婷. 黑木耳凝集素的提取工艺[J]. 江苏农业科学, 2015, 43(3):259-260.
[26]	马成瑶, 曾伟民, 黄东, 等. 黑木耳凝集素的分离、纯化及抗肿瘤活性研究[J]. 黑龙江大学自然科学学报, 2019, 36(4):465-472.
[27]	GABIUS H J, SABINE A, JESÚS J B, et al. From lectin structure to functional glycomics: principles of the sugar code[J]. Trends in biochemical sciences, 2011, 36(6):298-313. doi: 10.1016/j.tibs.2011.01.005 URL

试验因素	试验水平
试验因素	-1	0	1
A：提取时间/h	7	8	9
B：料液比	1:40	1:50	1:60
C：(NH4)₂SO₄浓度/%	40	50	60

试验因素	试验水平
试验因素	-1	0	1
A：提取时间/h	7	8	9
B：料液比	1:40	1:50	1:60
C：(NH4)₂SO₄浓度/%	40	50	60

方差来源	平方和	自由度	均方	F值	P值	显著性
模型	13.84	9	1.54	28.56	0.0001	^**
A	6.13	1	6.13	113.73	<0.0001	^**
B	0.25	1	0.25	4.55	0.0704
C	0.00	1	0.00	0.00	1.00
AB	2.5E-0.03	1	2.5E-0.03	0.046	0.8356
AC	0.023	1	0.023	0.42	0.5387
BC	2.5E-0.03	1	2.5E-0.03	0.046	0.8356
A²	3.74	1	3.74	69.45	<0.0001	^**
B²	1.24	1	1.24	23.01	0.0020	^**
C²	1.74	1	1.74	32.27	0.0008	^**
残差	0.38	7	0.054
失拟项	0.34	3	0.11	14.37	0.131
纯误差	0.032	4	8.0E-0.03
总离差	14.22	16

方差来源	平方和	自由度	均方	F值	P值	显著性
模型	13.84	9	1.54	28.56	0.0001	^**
A	6.13	1	6.13	113.73	<0.0001	^**
B	0.25	1	0.25	4.55	0.0704
C	0.00	1	0.00	0.00	1.00
AB	2.5E-0.03	1	2.5E-0.03	0.046	0.8356
AC	0.023	1	0.023	0.42	0.5387
BC	2.5E-0.03	1	2.5E-0.03	0.046	0.8356
A²	3.74	1	3.74	69.45	<0.0001	^**
B²	1.24	1	1.24	23.01	0.0020	^**
C²	1.74	1	1.74	32.27	0.0008	^**
残差	0.38	7	0.054
失拟项	0.34	3	0.11	14.37	0.131
纯误差	0.032	4	8.0E-0.03
总离差	14.22	16