雪松松针多糖的醇沉、脱蛋白工艺及抗氧化活性研究

doi:10.3969/j.issn.0253-2417.2023.05.011

摘要/Abstract

摘要：

对雪松松针多糖的醇沉、脱蛋白工艺及其体外抗氧化活性进行研究。在单因素试验基础上, 利用Box-Behnken模型对提取液浓缩比、乙醇体积分数和醇沉时间进行优化, 比较多糖得率确定最佳雪松松针多糖醇沉工艺条件。以蛋白质清除率和多糖损失率为指标, 比较了Sevage法和三氯醋酸(TCA)法的脱蛋白效果, 并对试剂用量、脱蛋白次数进行了考察，然后利用DPPH和ABTS自由基法对纯化后多糖的抗氧化活性进行评价。研究结果表明：优化的雪松松针多糖一级醇沉条件为粗提液体积和松针干质量的比例(浓缩比)为3∶1(mL∶g，下同), 乙醇体积分数为71%, 醇沉温度为40 ℃, 醇沉时间为10 h, 在此条件下, 雪松松针多糖得率为3.81%, 结果与预测值接近。收集多糖后的上清液再按浓缩比1∶1浓缩, 筛选得到二级醇沉的乙醇体积分数为80%。Sevage法脱蛋白效率优于TCA法, 其最佳条件为Sevage试剂与多糖溶液的体积比为1∶1, 脱除蛋白次数为3次，经UV法测得脱蛋白后的一级、二级醇沉多糖(以葡萄糖折算)质量分数分别为35.36%和36.62%。清除DPPH与ABTS自由基能力依次为Vc＞二级醇沉多糖＞BHT＞一级醇沉多糖, 纯化后的雪松松针80%醇沉多糖表现出良好的抗氧化活性。

关键词: 雪松松针, 多糖, 醇沉工艺, 脱蛋白, 抗氧化活性

Abstract:

The ethanol precipitation, deproteinization process and anti-oxidant activity of polysaccharides from pine needles of Cedrus deodara were studied. On the basis of single factor experiment, the concentration ratio of extraction solution, volume fraction of ethanol, and alcohol settling time were optimized by Box-Behnken design model. The optimum conditions of alcohol precipitation of C. deodara pine needles were determined by comparing the yield of polysaccharides. With the protein removal rate and polysaccharide loss rate as the index, the deproteinization effect of Sevage method and three chloroacetic acid method on polysaccharide of pine needles were compared, and the amount of reagent and the times of deproteinization were investigated. The anti-oxidant ability of the purified polysaccharides was evaluated by DPPH and ABTS free radical methods. The optimum alcohol precipitation conditions for the polysaccharide of pine needles were the concentration ratio(volume of crude extract to dry weight of pine needles) of 3:1(mL: g), the alcohol volume fraction of first order alcohol precipitation of 71%, the temperature of alcohol precipitation of 40℃, and the time of alcohol precipitation of 10 h. Under these conditions, the yield of pine needles polysaccharide was 3.81%, which was close to the predicted value. The supernatant after collecting polysaccharides was concentrated according to the ratio of solution to pine needle dry mass of 1:1(mL: g), and the volume fraction of alcohol precipitate at two level was 80%. The protein removal efficiency of Sevage method was superior to three chloroacetic acid method. Its optimum condition was that the volume ratio of Sevage reagent to polysaccharide solution was 1:1, and the number of protein removal was 3 times. After purification, the contents of primary and secondary alcohol precipitate polysaccharides were calculated by UV method with glucose conversions of 35.36% and 36.62%, respectively. The scavenging activities on DPPH and ABTS radicals were as follows: vitamin C(Vc)>secondary alcohol precipitate polysaccharides>butylated hydroxytoluene(BHT)>primary alcohol precipitates polysaccharides. The purified 80% alcohol precipitation polysaccharides of C. deodara pine needles showed good anti-oxidant activity.

Key words: pine needles of Cedrus deodara, polysaccharides, ethanol precipitation technology, deproteinization, anti-oxidant activity

中图分类号:

TQ35

马趣环, 王信, 石晓峰, 沈薇, 范彬, 王新娣. 雪松松针多糖的醇沉、脱蛋白工艺及抗氧化活性研究[J]. 林产化学与工业, 2023, 43(5): 81-88.

Quhuan MA, Xin WANG, Xiaofeng SHI, Wei SHEN, Bin FAN, Xindi WANG. Ethanol Precipitation, Deproteinization and Anti-oxidant Activity of Polysaccharides from Pine Needles of Cedrus deodara[J]. Chemistry and Industry of Forest Products, 2023, 43(5): 81-88.

图/表 7

图1

表1

表2

图2

图3

图4

图5

参考文献 17

1	李时珍. 本草纲目[M]. 北京: 人民卫生出版社, 1982: 1917.
	LI S Z . Compendium of Materia Medica[M]. Beijing: People's Medical Publishing House, 1982: 1917.
2	ZENG W C , JIA L R , ZHANG Y , et al. Antibrowning and antimicrobial activities of the water-soluble extract from pine needles of Cedrus deodara[J]. Journal of Food Science, 2011, 76 (2): 318- 323.
3	郑万钧, 傅立国. 中国植物志: 第七卷[M]. 北京: 科学出版社, 1978: 167- 203.
	ZHENG W J , FU L G . Flora of China: Vol.7[M]. Beijing: Science Press, 1978: 167- 203.
4	白朝辉, 石晓峰, 刘东彦, 等. 雪松松针的化学成分及药理作用研究概述[J]. 中国药师, 2012, 15 (12): 1791- 1793.
	BAI Z H , SHI X F , LIU D Y , et al. Chemical constituents and pharmacological effects of Cedrus Trew pine needles[J]. China Pharmacist, 2012, 15 (12): 1791- 1793.
5	葛霞, 王文君, 欧阳克蕙, 等. 水提法提取雪松松针多糖[J]. 食品研究与开发, 2010, 31 (7): 20- 23.
	GE X , WANG W J , OUYANG K H , et al. Extract polysaccharide from Cedar pine needles with the method of boiling water-extraction[J]. Food Research and Development, 2010, 31 (7): 20- 23.
6	范彬, 石晓峰, 沈薇, 等. 正交试验法优选雪松松针中总黄酮提取工艺[J]. 中国药师, 2008, 11 (8): 938- 940.
	FAN B , SHI X F , SHEN W , et al. Study on extraction technics of total flavonoids in pine needles of Cedrus deodara by orthogonal test[J]. China Pharmacist, 2008, 11 (8): 938- 940.
7	隋玉荣. 多糖类药物的研究进展[J]. 天津药学, 2013, 25 (2): 41- 43.
	SUI Y R . Research progress of polysaccharide drugs[J]. Tianjin Pharmacy, 2013, 25 (2): 41- 43.
8	时潇丽, 姚春霞, 林晓, 等. 多糖药物应用与研究进展[J]. 中国新药杂志, 2014, 23 (9): 1057- 1062.
	SHI X L , YAO C X , LIN X , et al. The applications and research progresses of polysaccharide drugs[J]. Chinese Journal of New Drugs, 2014, 23 (9): 1057- 1062.
9	刘东彦, 张莉霞, 石晓峰, 等. 雪松松针中莽草酸和多糖的提取工艺研究[J]. 中药材, 2017, 40 (8): 1915- 1917.
	LIU D Y , ZHANG L X , SHI X F , et al. Study on extraction technology of shikimic acid and polysaccharide from Cedrus deodara pine needles[J]. Journal of Chinese Medicinal Materials, 2017, 40 (8): 1915- 1917.
10	杨青青, 龚吉军. 响应面法优化超声辅助葛根浸提工艺及浸提液抗氧化活性研究[J]. 食品安全质量检测学报, 2021, 12 (13): 5409- 5417.
	YANG Q Q , GONG J J . Optimization of ultrasonic-assisted extraction technology of Pueraria lobata by response surface methodology and antioxidant activity of extracts[J]. Journal of Food Safety and Quality, 2021, 12 (13): 5409- 5417.
11	马趣环, 石晓峰, 沈薇, 等. 响应面分析法优化雪松松针多糖提取工艺研究[J]. 中华中医药杂志, 2018, 33 (3): 1106- 1110.
	MA Q H , SHI X F , SHEN W , et al. Optimization of extraction technique of polysaccharides from pine needles of Cedrus deodara via response surface methodology[J]. China Journal of Traditional Chinese Medicine and Pharmacy, 2018, 33 (3): 1106- 1110.
12	张心驰, 惠和平, 李晓东, 等. 响应面法优化败酱草多糖超声提取工艺及其体外抗氧化活性研究[J]. 化学研究与应用, 2022, 34 (9): 2074- 2081.
	ZHANG X C , HUI H P , LI X D , et al. Optimization of ultrasonic extraction technology and in vitro antioxidant activity of polysaccharide from Herba Patriniae using response surface methodology[J]. Chemical Research and Application, 2022, 34 (9): 2074- 2081.
13	蒋俊, 罗珍, 刘艳芳, 等. 醇沉条件对猴头菌多糖得率和品质的影响[J]. 食用菌学报, 2011, 18 (2): 57- 61.
	JIANG J , LUO Z , LIU Y F , et al. Effect of precipitation conditions on the yield and quality of Hericium erinaceus polysaccharides[J]. Acta Edulis Fungi, 2011, 18 (2): 57- 61.
14	李宏燕, 樊君. 大枣多糖的水提醇沉工艺研究[J]. 宁夏工程技术, 2005, 4 (3): 265- 267.
	LI H Y , FAN J . Research on extraction and isolation of jujube polysaccharide[J]. Ningxia Engineering Technology, 2005, 4 (3): 265- 267.
15	周静华, 万顺康. 大理百合多糖的醇沉分离工艺及脱蛋白方法[J]. 食品研究与开发, 2010, 31 (12): 7- 9.
	ZHOU J H , WAN S K . Study on ethanol precipitation process and deproteinization in extraction of polysaccharides from DaLi Lilium brownie[J]. Food Research and Development, 2010, 31 (12): 7- 9.
16	叶润, 蔡静, 李尽哲, 等. 油茶叶多糖的闪式提取工艺优化及其抗氧化活性[J]. 生物质化学工程, 2021, 55 (5): 42- 46.
	YE R , CAI J , LI J Z , et al. Optimization of flash extraction technology of polysaccharide from leaves of Camellia eleifera Able.and Its antioxidant activity[J]. Biomass Chemical Engineering, 2021, 55 (5): 42- 46.
17	杨力, 常寒, 王逍, 等. 响应曲面法优化栀子活性成分的提取工艺[J]. 华西药学杂志, 2021, 36 (5): 531- 534.
	YANG L , CHANG H , WANG X , et al. Optimization of extraction process of gardenia active ingredients by response surface methodology[J]. West China Journal of Pharmaceutical Sciences, 2021, 36 (5): 531- 534.

实验号 No.	A 浓缩比(mL∶g) concentration ratio	B 乙醇体积分数/% alcohol volume fraction	C 醇沉时间/h alcohol precipitation time	多糖得率(Y)/% polysaccharide yield
1	3∶1	70	8	3.82
2	4∶1	80	8	3.51
3	4∶1	70	12	3.59
4	3∶1	80	4	3.66
5	2∶1	70	4	3.33
6	2∶1	80	8	3.25
7	3∶1	60	12	3.64
8	3∶1	80	12	3.69
9	4∶1	70	4	3.58
10	3∶1	70	8	3.86
11	2∶1	70	12	3.41
12	3∶1	70	8	3.84
13	2∶1	60	8	3.13
14	3∶1	70	8	3.79
15	3∶1	70	8	3.85
16	3∶1	60	4	3.48
17	4∶1	60	8	3.45

方差来源 source	平方和 sum of squares	自由度 df	均方 mean square	F	P	显著性 significance
模型model	0.77	9	0.086	80.33	< 0.000 1	***
A	0.13	1	0.130	119.73	< 0.000 1	***
B	0.021	1	0.021	19.73	0.003 0	**
C	9.800×10^-3	1	9.800×10^-3	9.20	0.019 0	*
AB	9.000×10^-4	1	9.000×10^-4	0.85	0.388 5
AC	1.225×10^-3	1	1.225×10^-3	1.15	0.319 1
BC	4.225×10^-3	1	4.225×10^-3	3.97	0.086 7
A²	0.43	1	0.43	401.06	< 0.000 1	***
B²	0.13	1	0.13	125.97	< 0.000 1	***
C²	5.457×10^-3	1	5.457×10^-3	5.12	0.058 0
残差residual	7.455×10^-3	7	1.065×10^-3
失拟项lack of fit	4.375×10^-3	3	1.458×10^-3	1.89	0.271 8
纯误差pure error	3.080×10^-3	4	7.700×10^-4
总和cor total	0.78	16

[1]	胡泽坤, 晏婷婷, 李改云, 秦嘉惠, 吴新文, 陈媛. 奇楠与传统沉香特征成分的差异性分析及其生物活性研究[J]. 林产化学与工业, 2023, 43(5): 63-72.
[2]	徐梦娟, 卜庆, 陈宏杨, 张敏, 吴磊, 梁林富. 日本蛇根草化学成分的提取分离及其抗氧化活性研究[J]. 林产化学与工业, 2023, 43(4): 67-73.
[3]	詹梦茹, 王彦斌, 康淑荷, 罗兴平. 党参多糖的大孔树脂纯化及其美白、吸湿保湿性能[J]. 林产化学与工业, 2023, 43(3): 79-88.
[4]	安容苗, 袁婷, 郭雪峰. 簕竹属竹叶中黄酮类成分分析及其抗氧化活性[J]. 林产化学与工业, 2023, 43(1): 97-103.
[5]	雷响, 张闽峰, 胡可欣, 柯宇, 郑德勇. 1, 2, 3, 5, 6, 7-六羟基-9, 10-蒽醌的合成及其抗氧化活性研究[J]. 林产化学与工业, 2022, 42(6): 64-68.
[6]	何旭华, 石志娇, 彭小伟, 阚欢, 赵平, 刘云. 云南金花茶叶不同极性提取物的抗氧化活性分析[J]. 林产化学与工业, 2022, 42(5): 61-68.
[7]	李胜男, 程贤, 毕良武, 曾维星, 陈玉湘, 赵振东. 肉桂多糖的结构分析及抗氧化活性研究[J]. 林产化学与工业, 2022, 42(3): 34-40.
[8]	路露, 束成杰, 葛翎, 潘洁尘, 朱凯, 石宝俊. 肉桂精油和肉桂醛的抑菌、抗氧化和酪氨酸酶抑制活性研究[J]. 林产化学与工业, 2022, 42(3): 105-110.
[9]	黄维, 郭梦云, 王玉启, 林金垒, 张宇翔, 邹双全. 圆齿野鸦椿果皮多糖的提取及其抗炎活性研究[J]. 林产化学与工业, 2021, 41(3): 77-84.
[10]	程贤, 毕良武, 李胜男, 陈玉湘, 赵振东, 莫开林. 春秋两季油樟叶的油细胞形态、精油成分及其抗氧化活性研究[J]. 林产化学与工业, 2021, 41(1): 38-44.
[11]	蒋建新, 朱莉伟, 张伟, 李先平. 预处理温度对圆皂荚多糖胶制备与性能的影响[J]. 林产化学与工业, 2020, 40(6): 1-7.
[12]	程贤,毕良武,曾维星,赵振东,陈玉湘,张笮晦. 肉桂蒸油剩余物中活性成分的提取、纯化及其抗氧化活性研究[J]. 林产化学与工业, 2020, 40(2): 109-116.
[13]	李子江,刘慰,吴磊,王丹,司传领. 偃松松塔多糖的单糖组成分析及活性研究[J]. 林产化学与工业, 2020, 40(1): 37-44.
[14]	李嘉欣,孟斌斌,朱凯. 樟树叶精油组成分析及抗氧化活性研究[J]. 林产化学与工业, 2020, 40(1): 84-90.
[15]	王洁,路娇娇,杜涵,张萌,刘盼盼,陈小强. 五味子果肉和种子精油的化学成分与生物活性对比研究[J]. 林产化学与工业, 2019, 39(6): 59-67.