雁荡山铁皮石斛多糖的提取、结构表征与体外抗氧化活性

doi:10.3969/j.issn.1004-1524.20230543

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (8): 1898-1908.DOI: 10.3969/j.issn.1004-1524.20230543

雁荡山铁皮石斛多糖的提取、结构表征与体外抗氧化活性

赵小亮¹(), 鲁雲¹, 康兴兴¹, 龙则宇¹, 郑晓杰²^,^*()

1.兰州理工大学生命科学与工程学院,甘肃兰州 730050
2.温州市农业科学研究院食品科学研究所,浙江温州 325006

收稿日期:2023-04-27 出版日期:2024-08-25 发布日期:2024-09-06
作者简介:*郑晓杰,E-mail: 852117648@qq.com
赵小亮(1982—),男,甘肃灵台人,博士,副教授,研究方向为糖化学与糖药物。E-mail: zhxl819@163.com
通讯作者: 郑晓杰
基金资助:
国家自然科学基金(32260231);国家自然科学基金(31860250);甘肃省高校教师创新基金(2023A-020);浙江省农业农村厅市级农科院联盟区域示范性项目(2022SJLM06)

Extraction, structural characterization and antioxidant activity of polysaccharide from Dendrobium officinale in Yandang Mountain, China

ZHAO Xiaoliang¹(), LU Yun¹, KANG Xingxing¹, LONG Zeyu¹, ZHENG Xiaojie²^,^*()

1. School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China
2. Institute of Food Science, Wenzhou Academy of Agricultural Sciences, Wenzhou 325006, Zhejiang, China

Received:2023-04-27 Online:2024-08-25 Published:2024-09-06
Contact: ZHENG Xiaojie

摘要/Abstract

摘要：

以雁荡山铁皮石斛鲜茎为材料,选取液料比、提取温度、提取时间、提取次数为因子,以铁皮石斛多糖得率为响应值,分别基于单因素试验和响应面法试验优化铁皮石斛多糖的提取工艺,通过傅里叶红外光谱和高效液相色谱法分析对其进行初步的结构表征,并对其清除自由基的活性进行研究。结果表明:雁荡山铁皮石斛多糖提取的最佳工艺条件为液料比55∶1(mL·g^-1),提取温度80 ℃,提取时间130 min,提取次数2次。在此条件下,实际测得的多糖得率为31.02%。通过红外光谱分析,铁皮石斛多糖同时包含α和β型糖苷键,由甘露糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖和木糖组成,其物质的量之比为1.57∶0.49∶0.99∶2.08∶1.00。铁皮石斛多糖清除1,1-二苯基-2-三硝基苯肼(DPPH)自由基、2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)自由基、羟基自由基、超氧阴离子的半抑制浓度(IC₅₀)分别为2.46、4.47、5.60、2.66 mg·mL^-1,说明其具备一定的抗氧化活性。

关键词: 铁皮石斛, 多糖, 工艺优化, 结构表征, 抗氧化活性

Abstract:

In the present study, the fresh stem of Dendrobium officinale in Yandang Mountain was selected as the test object, the liquid-to-material ratio, extraction temperature, extration time and extraction times were selected as the key factors, as well as the yield of polysaccharide being response value, to optime the extraction process of polysaccharide from D. officinale in Yandang Mountain. The structure of the extracted polyssacharide was preliminarily explored by Fourier infrared spectroscopy and high performance liquid chromatography (HPLC). Besides, the in vitro activity of polysaccharides to scavenge free radicals was examined. The results showed that the optimal process conditions for the extraction of polysaccharide were liquid-to-material ratio of 55∶1 (mL·g^-1), extraction temperature of 80 ℃, extraction time of 130 min, and extraction times of 2 times. The actual polysaccharide yield under these conditions was 31.02%. By infrared spectroscopic analysis, it was shown that the polysaccharide contained both α-type and β-type glycosidic bonds, and the extracted polysaccharide was composed of mannose, glucuronic acid, galacturonic acid, glucose and xylose, and the amount of substance ratio of the above monosaccharides was 1.57∶0.49∶0.99∶2.08∶1.00. In vitro, the extracted polysaccharide could scavenge 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) radicals, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals, hydroxyl free radicals and superoxide anion, and the determined half maximal inhibitory concentrations (IC₅₀) were 2.46, 4.47, 5.60, 2.66 mg·mL^-1, respectively, indicating that the extracted polysaccharide had antioxidant activity.

Key words: Dendrobium officinale, polysaccharide, process optimization, structural characterization, antioxidant activity

中图分类号:

TQ281
S567

赵小亮, 鲁雲, 康兴兴, 龙则宇, 郑晓杰. 雁荡山铁皮石斛多糖的提取、结构表征与体外抗氧化活性[J]. 浙江农业学报, 2024, 36(8): 1898-1908.

ZHAO Xiaoliang, LU Yun, KANG Xingxing, LONG Zeyu, ZHENG Xiaojie. Extraction, structural characterization and antioxidant activity of polysaccharide from Dendrobium officinale in Yandang Mountain, China[J]. Acta Agriculturae Zhejiangensis, 2024, 36(8): 1898-1908.

图/表 12

表1 响应面分析的因素和水平

Table 1 Factors and levels in response surface analysis

编码水平 Coding level	各因素的设定值Setting values for factors
编码水平 Coding level	LTM/(mL·g^-1)	ET/min	ETP/℃	ETS
-1	30	60	50	1
0	45	120	70	2
1	60	180	90	3

图1 液料比、提取时间、提取温度和提取次数对铁皮石斛多糖得率的影响

Fig.1 Effect of liquid-to-material ratio, extraction time, extraction temperature and extraction times on the yield of polysaccharides from Dendrobium of ficinale

表2 响应面试验设计与结果

Table 2 Design and results of response surface experiment

编号 No.	各因素的编码水平Coding levels of factor				Y/%
编号 No.	X₁	X₂	X₃	X₄	Y/%
1	-1	0	0	1	28.21
2	0	-1	0	1	26.82
3	0	1	-1	0	25.31
4	0	0	-1	-1	24.63
5	0	1	1	0	28.26
6	-1	0	1	0	27.86
7	-1	0	-1	0	25.32
8	0	0	-1	1	25.15
9	0	-1	1	0	28.56
10	0	0	1	1	29.92
11	0	0	0	0	31.05
12	0	-1	0	-1	26.96
13	-1	-1	0	0	26.13
14	1	1	0	0	27.29
15	0	0	0	0	30.76
16	0	0	0	0	31.02
17	0	1	0	1	26.86
18	1	0	1	0	31.13
19	0	0	0	0	30.45
20	0	0	1	-1	29.80
21	0	-1	-1	0	24.09
22	0	0	0	0	30.86
23	0	1	0	-1	26.96
24	1	-1	0	0	27.21
25	1	0	-1	0	24.65
26	1	0	0	-1	28.16
27	-1	0	0	-1	26.36
28	-1	1	0	0	27.15
29	1	0	0	1	27.86

表3 回归模型的方差分析结果

Table 3 Analysis of variance of regression model

来源 Source	平方和 Sum of squares	均方 Mean square	F值 F value	P值 P value
模型Model	127.03	9.07	66.30	<0.000 1
X₁	2.31	2.31	16.91	0.001 1
X₂	0.35	0.35	2.58	0.130 3
X₃	57.99	57.99	423.77	<0.000 1
X₄	0.32	0.32	2.32	0.150 4
X₁X₂	0.22	0.22	1.61	0.224 6
X₁X₃	3.88	3.88	28.36	0.000 1
X₁X₄	1.16	1.16	8.44	0.011 5
X₂X₃	0.58	0.58	4.22	0.059 1
X₂X₄	0.01	0.01	0.0029	0.957 6
X₃X₄	0.04	0.04	0.2923	0.597 3
$X_{1}^{2}$	16.78	16.78	122.58	<0.000 1
$X_{2}^{2}$	35.05	35.05	256.09	<0.000 1
$X_{3}^{2}$	24.40	24.40	178.28	<0.000 1
$X_{4}^{2}$	15.85	15.85	115.82	<0.000 1
残差Residual	1.92	0.14	—	—
失拟项Lack of Fit	1.68	0.17	2.87	0.161 0
纯误差Pure error	0.23	0.06	—	—

表3 回归模型的方差分析结果

Table 3 Analysis of variance of regression model

来源 Source	平方和 Sum of squares	均方 Mean square	F值 F value	P值 P value
模型Model	127.03	9.07	66.30	<0.000 1
X₁	2.31	2.31	16.91	0.001 1
X₂	0.35	0.35	2.58	0.130 3
X₃	57.99	57.99	423.77	<0.000 1
X₄	0.32	0.32	2.32	0.150 4
X₁X₂	0.22	0.22	1.61	0.224 6
X₁X₃	3.88	3.88	28.36	0.000 1
X₁X₄	1.16	1.16	8.44	0.011 5
X₂X₃	0.58	0.58	4.22	0.059 1
X₂X₄	0.01	0.01	0.0029	0.957 6
X₃X₄	0.04	0.04	0.2923	0.597 3
$X_{1}^{2}$	16.78	16.78	122.58	<0.000 1
$X_{2}^{2}$	35.05	35.05	256.09	<0.000 1
$X_{3}^{2}$	24.40	24.40	178.28	<0.000 1
$X_{4}^{2}$	15.85	15.85	115.82	<0.000 1
残差Residual	1.92	0.14	—	—
失拟项Lack of Fit	1.68	0.17	2.87	0.161 0
纯误差Pure error	0.23	0.06	—	—

图2 各因素相互作用对铁皮石斛多糖得率的响应面图和等高线图

Fig.2 Response surface and contour plots of the interaction of various factors to the polysaccharide yield of Dendrobium officinale

图3 铁皮石斛多糖的紫外光谱图

Fig.3 Ultraviolet spectrum of polysaccharide extracted from Dendrobium officinale

图4 铁皮石斛多糖的红外光谱图

Fig.4 Infrared spectroscopy of polysaccharide from Dendrobium officinale

图5 单糖混合标准溶液(左)与铁皮石斛多糖水解液(右)的液相色谱图 1~9分别代表甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖、岩藻糖。

Fig.5 Liquid chromatograms of the mixed standard solution of monosaccharides and the polysaccharide hydrolysate of Dendrobium officinale 1-9 represent mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose, respectively.

图6 铁皮石斛多糖(PDO)和维生素C对DPPH自由基的清除作用

Fig.6 Scavenging effects of polysaccharide from Dendrobium officinale (PDO) and vitamin C on DPPH free radicals

图7 铁皮石斛多糖(PDO)和维生素C对ABTS自由基的清除作用

Fig.7 Scavenging effects of polysaccharide from Dendrobium officinale (PDO) and vitamin C on ABTS free radicals

图8 铁皮石斛多糖(PDO)和维生素C对羟基自由基的清除作用

Fig.8 Scavenging effects of polysaccharide from Dendrobium officinale (PDO) and vitamin C on hydroxyl free radicals

图9 铁皮石斛多糖(PDO)和维生素C对超氧阴离子的清除作用

Fig.9 Scavenging effects of polysaccharide from Dendrobium officinale (PDO) and vitamin C on superoxide anion

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雁荡山铁皮石斛多糖的提取、结构表征与体外抗氧化活性

Extraction, structural characterization and antioxidant activity of polysaccharide from Dendrobium officinale in Yandang Mountain, China

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