纤维素酶法提取决明子粗多糖的工艺优化及其抗氧化活性

蒋德旗; 许焯彬; 罗云; 李子强; 肖家宝; 莫烨; 李盈盈; 蒋丽林; 陈晓白

doi:10.13386/j.issn1002-0306.2022060262

纤维素酶法提取决明子粗多糖的工艺优化及其抗氧化活性

doi: 10.13386/j.issn1002-0306.2022060262

玉林师范学院生物与制药学院，广西高校亚热带生物资源保护与利用重点实验室，广西农产资源化学与生物技术重点实验室，广西玉林 537000

基金项目: 玉林市科学研究与技术开发计划项目（玉市科能20194302）；广西自然科学基金面上项目（2021GXNSFAA220001）；国家级大学生创新创业训练计划项目（202210606028）。

详细信息

作者简介:
蒋德旗（1986−），男，博士，教授，研究方向：天然产物活性成分开发与利用，E-mail：jiangdq8@sina.com

通讯作者:
蒋丽林（1986−），女，本科，助理研究员，研究方向：天然产物活性成分开发，E-mail：jianglilin55@126.com

中图分类号: R284.2
计量
- 文章访问数: 13
- HTML全文浏览量: 66
- PDF下载量: 0
- 被引次数: 0
出版历程
- 收稿日期: 2022-06-28
- 刊出日期: 2023-05-01

Optimization of Cellulase Extraction Process of Crude Polysaccharide from Semen Cassiae and Study on Its Antioxidant Activity

College of Biology and Pharmacy, Guangxi Education Department Key Laboratory for Conservation and Utilization of Subtropical Bio-resources, Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology, Yulin Normal University, Yulin 537000, China

摘要

摘要: 目的：优化纤维素酶法提取决明子粗多糖的工艺，并研究决明子粗多糖的体外抗氧化活性。方法：在单因素实验的基础上，以酶解时间、酶解温度、酶用量、液料比及酶解pH为自变量，多糖得率为响应值，利用Box-Behnken响应面法进行工艺优化。以对DPPH自由基和羟自由基清除率的大小为指标考察决明子粗多糖的体外抗氧化活性。结果：纤维素酶法提取决明子粗多糖最佳工艺为酶用量1.4%、酶解时间50 min、液料比24:1 mL/g、酶解pH5.4、酶解温度48 ℃，此条件下决明子多糖得率为11.67%，与回归模型的理论预测值11.91%误差小于5%。决明子粗多糖对DPPH自由基和羟自由基均具有较强的清除作用，半数抑制浓度分别为1.025 mg/mL和0.894 mg/mL。结论：纤维素酶法可显著提高决明子粗多糖得率，工艺简便可行，获得的决明子粗多糖具有体外抗氧化活性。
- 决明子 /
- 粗多糖 /
- 纤维素酶 /
- 抗氧化 /
- 响应面法
Abstract: Objective: To optimize the cellulase extraction process of crude polysaccharide from Semen Cassiae and study its antioxidant activity in vitro. Methods: On the basis of single factor test results, Box-Behnken response surface methodology was used to optimize the extraction process of crude polysaccharide from Semen Cassiae with enzymolysis time, enzymolysis temperature, enzyme dosage, liquid-to-material ratio and enzymolysis pH as independent variables, and the yield of polysaccharide as response value. DPPH radical and hydroxyl radical scavenging rates were used to investigate the antioxidant activities of crude polysaccharides from Semen Cassiae in vitro. Results: The optimal cellulase extraction conditions of crude polysaccharide from Semen Cassiae were as follows: Enzyme dosage was 1.4%, enzymolysis time was 50 min, liquid-to-material ratio was 24:1 mL/g, enzymolysis pH was 5.4, and enzymolysis temperature was 48 ℃. Under these conditions, the yield of polysaccharide from Semen Cassiae was 11.67%, and the theoretical prediction value of regression model was 11.91%, the error between the two conditions was less than 5%. Crude polysaccharide from Semen Cassiae had strong scavenging effects on DPPH free radical and hydroxyl free radical, with median inhibitory concentrations of 1.025 mg/mL and 0.894 mg/mL, respectively. Conclusion: Cellulase enzymatic method can significantly improve the yield of crude polysaccharide from Semen Cassiae, the process is simple and feasible, and the crude polysaccharide obtained from Semen Cassiae has antioxidant activity in vitro.
- Semen Cassia /
- crude polysaccharide /
- cellulose /
- antioxidant activity /
- response surface methodology

HTML全文

图 1 酶用量对决明子多糖得率的影响

注：不同小写字母表示差异显著（P<0.05），图2~图5同。

Figure 1. Effects of enzyme addition on polysaccharide yield of Semen Cassia

下载: 全尺寸图片幻灯片

图 2 酶解温度对决明子多糖得率的影响

Figure 2. Effects of enzymolysis temperature on polysaccharide yield of Semen Cassia

下载: 全尺寸图片幻灯片

图 3 酶解pH对决明子多糖得率的影响

Figure 3. Effects of enzymolysis pH on polysaccharide yield of Semen Cassia

下载: 全尺寸图片幻灯片

图 4 酶解时间对决明子多糖得率的影响

Figure 4. Effects of enzymolysis time on polysaccharide yield of Semen Cassia

下载: 全尺寸图片幻灯片

图 5 液料比对决明子多糖得率的影响

Figure 5. Effects of liquid to material ratio on polysaccharide yield of Semen Cassia

下载: 全尺寸图片幻灯片

图 6 因素间交互作用的响应面图

Figure 6. Response surface diagram of interaction between factors

下载: 全尺寸图片幻灯片

图 7 决明子粗多糖对DPPH自由基的清除作用

Figure 7. Scavenging effects of crude polysaccharide from Semen Cassia on DPPH free radical

下载: 全尺寸图片幻灯片

图 8 决明子粗多糖对羟自由基的清除作用

Figure 8. Scavenging effects of crude polysaccharide from Semen Cassia on hydroxyl radical

下载: 全尺寸图片幻灯片

表 1 响应面设计因素与水平

Table 1. Experimental design factors and levels of response surface methodology

水平	因素
水平	A 酶解时间（min）	B 酶用量（%）	C 液料比（mL/g）	D 酶解pH
−1	50	0.8	16:1	4.6
0	80	1.2	20:1	5.0
1	110	1.6	24:1	5.4

下载: 导出CSV

表 2 响应面试验设计与结果

Table 2. Design and results of response surface test

试验号	A	B	C	D	多糖得率（%）
1	−1	0	0	1	11.2
2	0	1	0	−1	9.4
3	0	0	1	1	11.2
4	0	−1	1	0	10.1
5	0	0	0	0	10.3
6	−1	0	0	−1	9.2
7	−1	1	0	0	10.8
8	1	0	1	0	9.9
9	0	1	−1	0	10.2
10	0	−1	−1	0	9.9
11	0	1	1	0	10.9
12	0	0	0	0	10.7
13	0	−1	0	−1	8.8
14	0	1	0	1	10.7
15	0	0	0	0	11.1
16	0	0	−1	1	9.9
17	0	0	0	0	10.9
18	1	0	0	−1	9.7
19	1	−1	0	0	10.3
20	−1	0	−1	0	10.2
21	1	1	0	0	10.9
22	−1	−1	0	0	11.0
23	0	0	0	0	10.4
24	0	0	−1	−1	8.9
25	1	0	−1	0	9.9
26	1	0	0	1	10.0
27	0	−1	0	1	10.2
28	0	0	1	−1	9.3
29	−1	0	1	0	11.3

下载: 导出CSV

表 3 方差分析结果

Table 3. Results of ANOVA analysis

来源	平方和	自由度	均方	F值	P值	显著性
模型	12.62	14	0.90	11.76	<0.0001	**
A	0.83	1	0.83	10.79	0.0054	**
B	0.53	1	0.53	6.85	0.0203	*
C	1.22	1	1.22	15.87	0.0014	**
D	5.12	1	5.12	66.86	<0.0001	**
AB	0.16	1	0.16	2.09	0.1704
AC	0.32	1	0.32	4.24	0.0586
AD	0.71	1	0.71	9.32	0.0086	**
BC	0.06	1	0.06	0.75	0.4005
BD	0.01	1	0.01	0.09	0.7633
CD	0.14	1	0.14	1.88	0.1914
A²	0.01	1	0.01	0.19	0.6660
B²	0.08	1	0.08	1.06	0.3199
C²	0.59	1	0.59	7.66	0.0151	*
D²	3.03	1	3.03	39.54	<0.0001	**
残差	1.07	14	0.08
失拟项	0.62	10	0.06	0.55	0.8009
纯误差	0.45	4	0.11
总和	13.69	28
注：P<0.05显著，*P<0.01极显著。

下载: 导出CSV

参考文献(32)

[1]	于凡, 孙乐, 许利嘉, 等. 决明子现代应用的研究进展[J]. 中国现代中药,2018,20(5):626−630. [YU F, SUN L, XU L J, et al. Research progress on modern application of Cassiae semen[J]. Modern Chinese Medicine,2018,20(5):626−630.
[2]	董玉洁, 蒋沅岐, 刘毅, 等. 决明子的化学成分、药理作用及质量标志物预测分析[J]. 中草药,2021,52(9):2719−2732. [DONG Y J, JIANG Y Q, LIU Y, et al. Research progress on chemical composition and pharmacological effects of Cassiae semen and predictive analysis on quality markers[J]. Chinese Traditional and Herbal Drugs,2021,52(9):2719−2732.
[3]	ZHANG M, LI X, LIANG H, et al. Semen Cassiae extract improves glucose metabolism by promoting GlUT4 translocation in the skeletal muscle of diabetic rats[J]. Frontiers in Pharmacology,2018,9:235. doi: 10.3389/fphar.2018.00235
[4]	LEE J Y, LIAO W L, LIU Y H, et al. Oral administration of processed Cassia obtusifolia L. seed powder may reduce body weight and cholesterol in overweight patients with schizophrenia: A 36-week randomized, double-blind, controlled trial of high and low doses[J]. Journal of Ethnopharmacology,2022,292:115111. doi: 10.1016/j.jep.2022.115111
[5]	朱周靓, 张世鑫, 郑云燕, 等. 决明子提取物对高脂血症大鼠血脂和肝肾功能的影响[J]. 预防医学,2021,33(12):1290−1294. [ZHU Z L, ZHANG S X, ZHENG Y Y, et al. Effects of extract from Cassiae semen on blood lipid and liver and kidney function in hyperlipidemia rats[J]. Preventive Medicine,2021,33(12):1290−1294.
[6]	高小敏, 赵勤. 应用网络药理学预测决明子治疗高血压的作用机制[J]. 大理大学学报,2021,6(8):11−15. [GAO X M, ZHAO Q. Application of network pharmacology to predict the mechanism of Semen cassiae in the treatment of hypertension[J]. Journal of Dali University,2021,6(8):11−15. doi: 10.3969/j.issn.2096-2266.2021.08.003
[7]	WANG Q Y, ZHOU J W, XIANG Z N, et al. Anti-diabetic and renoprotective effects of Cassiae semen extract in the streptozotocin-induced diabetic rats[J]. Journal of Ethnopharmacology,2019,239:111904. doi: 10.1016/j.jep.2019.111904
[8]	WANG Q Y, TONG A H, PAN Y Y, et al. The effect of Cassia seed extract on the regulation of the LKB1-AMPK-GLUT4 signaling pathway in the skeletal muscle of diabetic rats to improve the insulin sensitivity of the skeletal muscle[J]. Diabetology Metabolic Syndrome,2019,11(1):108. doi: 10.1186/s13098-019-0504-0
[9]	LUO H Y, WU H W, WANG L X, et al. Hepatoprotective effects of Cassiae semen on mice with non-alcoholic fatty liver disease based on gut microbiota[J]. Communications Biology,2021,4(1):1357. doi: 10.1038/s42003-021-02883-8
[10]	HE S Q, MA X, MENG Q G, et al. Effects and mechanisms of water-soluble Semen Cassiae polysaccharide on retinitis pigmentosa in rats[J]. Food Science and Technology,2020,40(1):84−88. doi: 10.1590/fst.32718
[11]	杨冰, 任娟, 秦昆明, 等. 决明子药理作用及其机制研究进展[J]. 中药材,2018,41(5):1247−1251. [YANG B, REN J, QIN K M, et al. Research progress on pharmacological action and mechanism of Semen cassiae[J]. Journal of Chinese Medicinal Materials,2018,41(5):1247−1251.
[12]	LIU C, LIU Q, SUN J, et al. Extraction of water-soluble polysaccharide and the antioxidant activity from Semen Cassiae[J]. Journal of Food and Drug Analysis,2014,22(4):492−499. doi: 10.1016/j.jfda.2014.01.027
[13]	ZENG H, LIU Q, WANG M, et al. Target-guided separation of antioxidants from Semen Cassia via off-line two-dimensional high-speed counter-current chromatography combined with complexation and extrusion elution mode[J]. Journal of Chromatography B,2015,1001:58−65. doi: 10.1016/j.jchromb.2015.07.046
[14]	曲航, 高鑫, 伊娟娟, 等. 食源性天然产物对酒精性肝损伤的防护作用研究进展[J]. 食品科学,2020,41(17):283−290. [QU H, GAO X, YI J J, et al. Review on the protective effects of food-derived natural compounds on alcohol-induced liver injury[J]. Food Science,2020,41(17):283−290. doi: 10.7506/spkx1002-6630-20190920-262
[15]	肖莲, 杨道斌, 陈礼大, 等. 中药复方多成分多靶点协同增效药理药效评价[J]. 中医药研究前沿,2022,4(1):16−18. [XIAO L, YANG D B, CHEN L D, et al. Pharmacological efficacy evaluation of multi-component and multi-target synergistic synergy of compound prescription of TCM[J]. Frontiers of Chinese Medicine Research,2022,4(1):16−18. doi: 10.12238/fcmr.v4i1.5193
[16]	WU D T, LIU W, HAN Q H, et al. Extraction optimization, structural characterization, and antioxidant activities of polysaccharides from Cassia seed (Cassia obtusifolia)[J]. Molecules,2019,24(15):2817. doi: 10.3390/molecules24152817
[17]	KANG C Z, LIU Y N, CHI A P, et al. The anti-fatigue potential of water-soluble polysaccharides of Semen Cassiae on BALB/c mice[J]. Cellular and Molecular Biology,2021,67(2):148−154. doi: 10.14715/cmb/2021.67.2.23
[18]	SUN M M, HE N, LYU Z G. Polysaccharides extracted from Cassia seeds protect against high glucose-induced retinal endothelial cell injury[J]. International Ophthalmology,2021,41(7):2465−2472. doi: 10.1007/s10792-021-01801-5
[19]	FENG L, YIN J Y, NIE S P, et al. Fractionation, physicochemical property and immunological activity of polysaccharides from Cassia obtusifolia[J]. International Journal of Biological Macromolecules,2016,91:946−953. doi: 10.1016/j.ijbiomac.2016.05.030
[20]	王涛. 决明子多糖提取工艺研究[J]. 轻工标准与质量,2014,13(2):49−51. [WANG T. Study on extraction technology of polysaccharide from Semen Cassiae[J]. Standard & Quality of Light Industry,2014,13(2):49−51.
[21]	康佩姿, 梁磊, 黄清铧, 等. 响应面法优化决明子水溶性多糖提取条件研究[J]. 粮食与油脂,2015,28(11):63−67. [KANG P Z, LIANG L, HUANG Q H, et al. Optimization of ultrasonic extraction of polysaccharides from Cassia seeds[J]. Cereals & Oils,2015,28(11):63−67.
[22]	杨黎燕. 决明子功效成分—蒽醌、多糖的提取及分析方法研究[D]. 西安: 西北大学, 2003: 1−8 YANG L Y. Study on the extraction and analysis methods of functional components of Cassia seeds-anthraquinone and polysaccharide[D]. Xi’an: Northwest University, 2003: 1−8.
[23]	邓泽元, 刘娟, 余迎利, 等. 决明子水溶性多糖提取的研究[J]. 食品科学,2002,23(1):72−75. [DENG Z Y, LIU J, YU Y L, et al. Study on extraction of water-soluble polysaccharide from Cassia seeds[J]. Food Science,2002,23(1):72−75. doi: 10.3321/j.issn:1002-6630.2002.01.016
[24]	郭晓强, 颜军, 邬晓勇, 等. 决明子水溶性多糖的纯化及抗氧化活性研究[J]. 食品科学,2007,28(8):205−208. [GUO X Q, YAN J, WU X Y, et al. Study on purification and antioxidation of water-soluble polysaccharide isolated from Semen Cassia[J]. Food Science,2007,28(8):205−208. doi: 10.3321/j.issn:1002-6630.2007.08.046
[25]	周晓红, 陈洁冰, 罗智, 等. 纤维素酶辅助提取茶多糖的工艺研究[J]. 湖南城市学院学报(自然科学版),2022,31(1):67−71. [ZHOU X H, CHEN J B, LUO Z, et al. Study on the extraction technology of tea polysaccharides assisted by cellulose[J]. Journal of Hunan City University (Natural Science),2022,31(1):67−71. doi: 10.3969/j.issn.1672-7304.2022.01.0011
[26]	LIU X X, GU L B, ZHANG G J, et al. Structural characterization and antioxidant activity of polysaccharides extracted from Chinese yam by a cellulase-assisted method[J]. Process Biochemistry,2022,121:178−187. doi: 10.1016/j.procbio.2022.06.023
[27]	GUO L, TAN D C, HUI F Y, et al. Optimization of the cellulase-ultrasonic synergistic extraction conditions of polysaccharides from Lenzites betulina[J]. Chemistry & Biodiversity,2019,16(11):e1900369.
[28]	蒋德旗, 柒善怀, 张兰熙, 等. 响应面法优化金果榄多糖提取工艺及抗氧化活性研究[J]. 中国中医药信息杂志,2019,26(4):85−90. [JIANG D Q, QI S H, ZHANG L X, et al. Optimization of extraction process of polysaccharide from Tinosporae radix by response surface method and study on its antioxidant activity[J]. Chinese Journal of Information on TCM,2019,26(4):85−90. doi: 10.3969/j.issn.1005-5304.2019.04.018
[29]	龚频, 王佩佩, 同美霖, 等. 红枣多糖的提取工艺及药理活性研究[J]. 食品工业科技,2022,43(13):198−207. [GONG P, WANG P P, TONG M L, et al. Study on extraction technology and pharmacological activities of polysaccharide from Ziziphus jujube[J]. Science and Technology of Food Industry,2022,43(13):198−207.
[30]	蒋德旗, 陈晓白, 农贵珍, 等. 鸡骨草多糖的酶法提取工艺优化及其抗氧化活性[J]. 食品工业科技,2019,40(3):153−158. [JIANG D Q, CHEN X B, NONG G Z, et al. Optimization of enzymatic extraction of polysaccharide from Abrus cantoniensis Hance and its antioxidant activity[J]. Science and Technology of Food Industry,2019,40(3):153−158.
[31]	叶兆伟, 叶润, 赫丁轩, 等. 息半夏多糖提取工艺优化及其抗氧化活性研究[J]. 中国食品添加剂,2022,33(1):90−98. [YE Z W, YE R, HE D X, et al. Optimization of polysaccharide extraction from Xi Pinellia ternate by response surface methodology and its antioxidant activity[J]. China Food Additives,2022,33(1):90−98.
[32]	陈红惠, 牛念拉姆. 底圩茶多糖的超声波辅助提取及其抗氧化活性[J]. 食品工业科技,2020,41(21):179−184. [CHEN H H, NIUNIAN L M. Ultrasonic extraction and antioxidant activity of polysaccharide from Dixu tea[J]. Science and Technology of Food Industry,2020,41(21):179−184.