花叶万年青功能成分提取条件优化与活性探究

doi:10.3969/j.issn.1004-1524.2023.02.16

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (2): 383-393.DOI: 10.3969/j.issn.1004-1524.2023.02.16

花叶万年青功能成分提取条件优化与活性探究

马波¹(), 陶震², 周瑞², 王雪², 吕茜茜², 孙士红¹, 王寒², 高金秋², 张楚涵¹, 陈凤清²^,^*()

1.白城医学高等专科学校基础医学院,吉林白城 137000
2.白城师范学院生命科学学院,吉林白城 137000

收稿日期:2022-04-21 出版日期:2023-02-25 发布日期:2023-03-14
通讯作者: 陈凤清
作者简介:*陈凤清,E-mail:chenfq@163.com
马波(1971—),女,吉林白城人,教授,研究方向为医学生物学。E-mail:443891659@qq.com
基金资助:
国家级大学生创新创业训练计划(202010206017);吉林省中医药科技项目(2022184)

Study on optimization of extraction conditions and activity of functional components from Dieffenbachia picta

MA Bo¹(), TAO Zhen², ZHOU Rui², WANG Xue², LYU Qianqian², SUN Shihong¹, WANG Han², GAO Jinqiu², ZHANG Chuhan¹, CHEN Fengqing²^,^*()

1. School of Basic Medical Sciences, Baicheng Medical College, Baicheng 137000, Jilin, China
2. College of Life Science, Baicheng Normal University, Baicheng 137000, Jilin, China

Received:2022-04-21 Online:2023-02-25 Published:2023-03-14
Contact: CHEN Fengqing

摘要/Abstract

摘要：

为优化花叶万年青中多糖与生物碱类成分的提取工艺,并探究其多糖提取物的抗氧化活性,以及生物碱的杀虫和抑菌活性,以花叶万年青叶为材料,通过正交设计试验优化多糖与生物碱类成分的提取工艺。杀虫试验以虫体浸渍法,探究花叶万年青生物碱提取物对棉铃虫、小菜夜蛾、大麦虫的触杀效果;用滤纸片法探究花叶万年青生物碱提取物对大肠埃希菌、金黄色葡萄球菌、铜绿假单胞菌、粪肠球菌、枯草芽孢杆菌、痢疾志贺氏菌、伤寒沙门氏菌的抑菌作用;通过测定对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(·OH)清除能力和对Fe³⁺的还原能力,研究花叶万年青叶多糖的抗氧化活性。结果表明,花叶万年青叶多糖的最佳提取工艺为料液比1∶ 30(g·mL^-1),提取次数3次,提取时间1 h,提取温度90 ℃。花叶万年青多糖的抗氧化活性研究结果显示,当多糖质量浓度为5.0 mg·mL^-1时,对DPPH自由基、ABTS自由基、羟基自由基的清除率分别为51.78%、48.60%、40.86%,Fe³⁺还原力达到0.338 6,且随多糖质量浓度的不断增加,抗氧化活性也逐渐增强,但均低于维生素C。花叶万年青叶片生物碱的提取中各因素的影响大小为超声温度>超声时间>液料比>乙醇体积分数,最佳工艺为超声温度66 ℃,超声时间80 min,料液比1∶30(g·mL^-1),乙醇体积分数90%。杀虫试验结果表明,花叶万年青生物碱提取物对3种虫的触杀效果有显著差异,处理36 h后棉铃虫、小菜夜蛾、大麦虫的死亡率分别为92.22%、96.67%、96.67%。抑菌试验结果表明:12.5 mg·mL^-1生物碱提取物对不同菌种的抑制效果有所不同,对大肠埃希菌、铜绿假单胞菌抑菌活性最强,抑菌环直径可达10.95、10.65 mm;其次是粪肠球菌、金黄色葡萄球菌、枯草芽孢杆菌、痢疾志贺氏菌,抑菌环直径分别为8.00、8.20、8.85、8.25 mm;而伤寒沙门氏菌表现出不敏感或敏感度不明显。

关键词: 多糖, 生物碱, 抑菌活性, 杀虫活性, 抗氧化活性, 花叶万年青

Abstract:

In order to optimize the extraction process of polysaccharides and alkaloids from Dieffenbachia picta Lodd, and explore the antioxidant activity of its polysaccharide extract, and the insecticidal and bacteriostatic activity of alkaloids, Dieffenbachia picta was used as material, the extraction processes of polysaccharides and alkaloids were optimized by orthogonal design. Insecticidal test was carried out by the insecticidal impregnation method, to explore the contact killing effect of alkaloid extract of D. picta on Helicoverpa armigera, Plutella xylostella and Zophobas atratus; Filter paper method was used to explore the bacteriostatic effect of alkaloid extract of D. picta on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Bacillus subtilis, Shigella dysentery and Salmonella typhi. The antioxidant activity of polysaccharide from Dieffenbachia picta Lodd was studied by measuring the ability of scavenging DPPH, ABTS, hydroxyl radical (·OH) and reducing ability to Fe³⁺. The results showed that the optimal extraction process of polysaccharide from D. picta were the solid-liquid ratio 1∶30 (g·mL^-1), extraction for 3 times, extraction time 1 h, extraction temperature 90 ℃. The antioxidant activity of polysaccharide from D. picta show that when the polysaccharide concentration was 5.0 mg·mL^-1, the scavenging rates of DPPH radical, ABTS radical and hydroxyl (·OH) radical were 51.78%, 48.60% and 40.86%, respectively, and the reducing power of Fe³⁺ reached 0.338 6. With the continuous increase of polysaccharide concentration, the antioxidant activity also gradually increased, but they were lower than those in the control group. The influence of each factor on the extraction of alkaloids from the leaves of D. picta was ultrasonic temperature>ultrasonic time>solid-liquid ratio>ethanol volume fraction. The optimal extraction process of alkaloids from the leaves of D. picta were ultrasonic temperature 66 ℃, ultrasonic time 80 min, solid-liquid ratio 1∶ 30 (g·mL^-1), ethanol volume fraction 90%. The results of the insecticidal test showed that the contact toxicity of the alkaloids from D. picta leaves to the three insects was significantly different. After 36 h of treatment, the mortality rates of H. armigera, P. xylostella and Z. atratus worm were 92.22%, 96.67% and 96.67%, respectively. The results of bacteriostatic test showed that 12.5 mg·mL^-1 alkaloid extract had different inhibitory effects on different strains. It had the strongest antibacterial activity against E. coli and P. aeruginosa, and the diameter of bacteriostatic ring could reach 10.95 mm and 10.65 mm, followed by E. faecalis, S. aureus, B. subtilis and S. dysentery, the diameters of bacteriostatic rings were 8.00, 8.20, 8.85 and 8.25 mm, respectively. Salmonella typhi showed insensitive or insensitive.

Key words: polysaccharide, alkaloid, bacteriostatic acitivity, insecticidal activity, antioxidant activity, Dieffenbachia picta Lodd

中图分类号:

S567.239

马波, 陶震, 周瑞, 王雪, 吕茜茜, 孙士红, 王寒, 高金秋, 张楚涵, 陈凤清. 花叶万年青功能成分提取条件优化与活性探究[J]. 浙江农业学报, 2023, 35(2): 383-393.

MA Bo, TAO Zhen, ZHOU Rui, WANG Xue, LYU Qianqian, SUN Shihong, WANG Han, GAO Jinqiu, ZHANG Chuhan, CHEN Fengqing. Study on optimization of extraction conditions and activity of functional components from Dieffenbachia picta[J]. Acta Agriculturae Zhejiangensis, 2023, 35(2): 383-393.

图/表 15

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水平 Levels	因素Factors
水平 Levels	A提取温度 Extraction temperature/℃	B提取时间 Extraction time/h	C料液比 Solid-liquid ratio/(g·mL^-1)	D提取次数 Extraction times
1	85	1.0	1∶30	1
2	90	1.5	1∶35	2
3	95	2.0	1∶40	3

水平 Levels	因素Factors
水平 Levels	A提取温度 Extraction temperature/℃	B提取时间 Extraction time/h	C料液比 Solid-liquid ratio/(g·mL^-1)	D提取次数 Extraction times
1	85	1.0	1∶30	1
2	90	1.5	1∶35	2
3	95	2.0	1∶40	3

水平 Levels	因素Factors
水平 Levels	A料液比 Solid-liquid ratio/(g·mL^-1)	B超声时间 Ultrasonic time/min	C超声温度 Ultrasonic temperature/℃	D乙醇体积分数 Ethanol volume fraction/%
1	1∶30	40	58	85
2	1∶35	60	66	90
3	1∶40	80	74	95

水平 Levels	因素Factors
水平 Levels	A料液比 Solid-liquid ratio/(g·mL^-1)	B超声时间 Ultrasonic time/min	C超声温度 Ultrasonic temperature/℃	D乙醇体积分数 Ethanol volume fraction/%
1	1∶30	40	58	85
2	1∶35	60	66	90
3	1∶40	80	74	95

序号 Serial number	A提取温度 Extraction temperature	B提取时间 Extraction time	C料液比 Solid-liquid ratio	D提取次数 Extraction times	得率 Yield/(mg·g^-1)
1	1	1	1	1	72.89
2	1	2	2	2	31.43
3	1	3	3	3	72.53
4	2	1	2	3	72.89
5	2	2	3	1	49.41
6	2	3	1	2	71.79
7	3	1	3	2	56.58
8	3	2	1	3	76.37
9	3	3	2	1	43.12

花叶万年青功能成分提取条件优化与活性探究

Study on optimization of extraction conditions and activity of functional components from Dieffenbachia picta

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图/表 15

参考文献 24

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因素 Factor	得率Yield
因素 Factor	k₁/(mg·g^-1)	k₂/(mg·g^-1)	k₃/(mg·g^-1)	极差Range	最优水平Optimum level
A提取温度Extraction temperature	58.95	64.70	58.69	6.01	2
B提取时间Extraction time	67.45	52.41	62.48	15.05	1
C料液比Solid-liquid ratio	73.68	49.15	59.51	24.54	1
D提取次数Extraction times	55.14	53.27	73.93	20.66	3

评价指标 Evaluating indicator	样品号Number of samples						平均值 Average value	极差 Range	相对标准偏差 RSD/%
评价指标 Evaluating indicator	1	2	3	4	5	6	平均值 Average value	极差 Range	相对标准偏差 RSD/%
多糖得率	81.046	80.688	80.225	80.628	80.554	80.598	80.623	0.821	0.3
Polysaccharide yield/(mg·g^-1)
生物碱得率	2.215	2.215	2.161	2.223	2.223	2.206	2.207	0.017	1.1
Alkaloid yield/(mg·g^-1)

试验号 No.	试验设计方案Test design scheme				得率 Yield/(mg·g^-1)
试验号 No.	A料液比 Solid-liquid ratio	B超声时间 Ultrasonic time	C超声温度 Ultrasonic temperature	D乙醇体积分数 Ethanol volume fraction	得率 Yield/(mg·g^-1)
1	1	1	1	1	1.720 475
2	1	2	2	2	2.184 306
3	1	3	3	3	1.989 296
4	2	1	2	3	1.970 469
5	2	2	3	1	1.781 770
6	2	3	1	2	1.782 198
7	3	1	3	2	1.851 943
8	3	2	1	3	1.832 154
9	3	3	2	1	2.204 310

因素 Factors	得率Yield
因素 Factors	k₁/(mg·g^-1)	k₂/(mg·g^-1)	k₃/(mg·g^-1)	极差Range	最优水平Optimum level
A料液比Solid-liquid ratio	1.964 692	1.844 812	1.962 802	0.119 880	1
B超声时间Ultrasonic time	1.847 629	1.932 743	1.991 934	0.144 305	3
C超声温度Ultrasonic temperature	1.778 275	2.119 695	1.874 336	0.245 359	2
D乙醇体积分数Ethanol volume fraction	1.902 185	1.939 482	1.930 639	0.037 297	2

分组 Group	虫体死亡率Insect mortality/%
	棉铃虫Helicoverpa armigera			小菜夜蛾Plutella xylostella			大麦虫Zophobas atratus
	12 h	24 h	36 h	12 h	24 h	36 h	12 h	24 h	36 h
1	82.22 ±3.33 a	84.44 ±2.22 ab	90.00 ±0.01 a	84.44 ±2.22 b	87.78 ±3.33 b	87.78 ±3.33 b	42.22 ±2.32 d	61.11 ±5.32 d	64.44 ±1.32 f
2	82.22 ±3.33 a	86.67 ±3.33 a	92.22 ±2.22 a	92.22 ±2.22 a	94.44 ±1.23 ab	95.56 ±1.23 a	57.78 ±3.33 b	78.89 ±3.33 c	85.56 ±2.22 d
3	64.44 ±2.22 c	81.11 ±2.22 b	86.67 ±3.33 a	82.22 ±1.23 b	87.78 ±2.22 b	90.00 ±0.01 b	51.11 ±3.33 c	61.11 ±2.22 d	67.78 ±2.22 e
4	53.33 ±3.33 d	63.33 ±3.33 c	71.11 ±3.33 b	55.56 ±3.33 c	71.11 ±1.23 c	76.67 ±3.33 c	83.33 ±0.01 a	87.78 ±1.23 b	88.89 ±3.33 c
5	74.45 ±2.22 b	84.45 ±2.22 ab	87.78 ±3.33 a	54.44 ±1.23 c	63.33 ±3.33 c	72.22 ±3.33 c	80.00 ±3.33 a	88.89 ±2.22 ab	93.33 ±0.01 b
6	72.22 ±1.23 b	83.33 ±0.01 ab	88.89 ±1.23 a	88.89 ±1.23 a	96.67 ±0.01 a	96.67 ±0.01 a	78.78 ±3.33 a	93.33 ±0.01 a	96.67 ±0.01 a

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编号 No.	A浸渍次数 Impregnation times	B浸渍时间 Immersion time/s	C浸渍液生物碱浓度 Alkaloid concentration in impregnation solution/(mg·mL^-1)	D培养温度 Culture temperature/℃
1	1	4	24	65
2	2	8	20	80
3	3	12	16	60
4	4	2	12	75
5	5	6	8	55
6	6	10	4	70

编号 No.	A浸渍次数 Impregnation times	B浸渍时间 Immersion time/s	C浸渍液生物碱浓度 Alkaloid concentration in impregnation solution/(mg·mL^-1)	D培养温度 Culture temperature/℃
1	1	4	24	65
2	2	8	20	80
3	3	12	16	60
4	4	2	12	75
5	5	6	8	55
6	6	10	4	70