紫皮豇豆总多酚提取工艺研究及组分分析

吴琼; 王明月; 葛会林; 马晨; 吕岱竹

doi:10.19303/j.issn.1008-0384.2020.06.014

紫皮豇豆总多酚提取工艺研究及组分分析

doi: 10.19303/j.issn.1008-0384.2020.06.014

中国热带农业科学院分析测试中心/农业农村部热作产品质量安全风险评估实验室（海口）/海南省热带果蔬产品质量安全重点实验室，海南　海口　571101

基金项目: 海南省自然科学基金（No.318MS089）；国家农产品质量安全风险评估项目（GJFP201900605）

详细信息

作者简介:
吴琼（1984−），女，助理研究员，研究方向：农产品营养品质研究（E-mal：joan627@126.com）

通讯作者:
吕岱竹（1972−），女，研究员，研究方向：农产品营养品质研究（E-mail：ldz162000000@126.com）

中图分类号: TS 201.4
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- 被引次数: 0
出版历程
- 收稿日期: 2020-01-19
- 修回日期: 2020-06-11
- 刊出日期: 2020-08-10

Extraction and Composition of Polyphenols from Purple Cowpeas

Analysis & Testing Center, Chinese Academy of Tropical Agricultural Sciences/Agricultural Ministry Laboratory for Tropical Crop Product Quality and Safety Risk Assessment/Hainan Provincial Key Laboratory of Quality and Safety for Tropical Fruits and Vegetables, Haikou, Hainan　571101, China

摘要

摘要: 目的采用溶剂萃取法提取紫皮豇豆中的总多酚，多酚提取物经大孔树脂分离纯化，再利用高效液相色谱（HPLC）分析其多酚类化合物的组成及含量，为紫皮豇豆中多酚类物质的提取、分析提供理论依据。方法以总多酚提取量为考察指标，采用单因素和正交试验，分析提取溶剂、料液比、温度和pH对紫皮豇豆总多酚提取量的影响，再通过Sephadex LH-20大孔树脂对粗提物进行分离纯化，并建立紫皮豇豆中6种多酚类组分的HPLC分析方法，采用外标法定量。结果紫皮豇豆中总多酚的最佳提取工艺为：经料液比1 ∶ 5（m ∶ V）的80%丙酮水溶液，在4℃、pH 6条件下，提取3次，每次提取5 h时，所得的总酚含量最高为614 mg（GAE）·100 g⁻¹。在优化的试验条件下，没食子酸、没食子儿茶素、绿原酸、芦丁、p-香豆酸和阿魏酸等6种多酚单体在相应的浓度范围内具有良好的线性关系，相关系数（r）为0.995 9～0.999 9，方法检出限和定量限分别为0.1～1 mg·100 g⁻¹和0.2～2.5 mg·100 g⁻¹；加标试验平均回收率74.3～107%，相对标准偏差0.4～8.6%。经HPLC分析，紫皮豇豆中6种多酚单体均有检出，含量在0.46～6.5 mg·100 g⁻¹，其中含量最高的是芦丁。结论采用80%丙酮水溶液提取紫皮豇豆总多酚，并利用HPLC法测定紫皮豇豆中几种单体酚的方法提取率高、准确性好，适用于紫皮豇豆中多酚类化合物的提取及定性定量分析。
- 紫皮豇豆 /
- 多酚 /
- 提取纯化 /
- 成分分析
Abstract: Objective Polyphenol compounds in purple cowpeas were solvent-extracted, purified, and then chemically analyzed by HPLC. Method Single factor and orthogonal experiments were conducted to optimize the polyphenol extraction from purple cowpeas. Using polyphenol yield as the criterion, effects of solvent, solid-liquid ratio, temperature, and pH applied for the process were evaluated for optimization. The crude extract was purified using a Sephadex LH-20 macroporous resin. HPLC with external standards was employed for the chemical determination on the purified product. Result The optimized process applied a peas-to-solvent ratio of 1 ∶ 5 (m ∶ V) with an 80% acetone solution to extract 3 times at 4^oC and pH 6 for 5 h per cycle to reach a maximum total polyphenols content of the gallic acid equivalent of 614 mg·100 g⁻¹. The correlation coefficients on the linear functions of the 6 polyphenols against their respective standards ranged from 0.995 9 to 0.999 9 with a limit of detection (LOD) of 0.1～1.0 mg·100 g⁻¹ and that of quantitation 0.2～2.5 mg·100 g⁻¹ on the HPLC test. The average recoveries on these compounds ranged from 74.3% to 107% with a relative standard deviation (RSDs) of 0.4～8.6%. The 6 polyphenols in the purple cowpeas thus determined averaged between 0.46 mg·100 g⁻¹ and 6.5 mg·100 g⁻¹, with rutin being the most abundant.ing the most abundant. Conclusion The polyphenol extraction from purple cowpeas was optimized to deliver high yields on 6 monomer phenols, which could be accurately determined quantitatively by HPLC.
- Purple cowpea /
- total polyphenols /
- extraction and purification /
- composition analysis

HTML全文

图 1 溶剂对总多酚提取率的影响

注：1-甲醇；2-异丙醇；3-二氯甲烷；4-氯仿；5-丙酮；6-乙酸乙酯；7-乙腈；8-乙醇

Figure 1. Effect of solvent on polyphenol extraction rate

Note: 1-Methanol; 2-Isopropanol; 3- Dichloromethane; 4-Trichloromethane; 5-Acetone; 6-Ethyl acetate; 7-Acetonitrile; 8-Ethanol

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图 2 温度对总多酚提取率的影响

注：a、b、c、d代表各水平之间的显著性差异

Figure 2. Effect of temperature on polyphenol extraction rate

Note: a,b,c and d represent the significant difference of each level

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图 3 料液比对总多酚提取率的影响

注：a、b、c、d、e代表各水平之间的显著性差异

Figure 3. Effect of solid-liquid ratio on polyphenol extraction rate

Note: a,b,c ,d and e represent the significant difference of each level

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图 4 pH对总多酚提取率的影响

注：a、b、c、d代表各水平之间的显著性差异

Figure 4. Effect of pH on polyphenol extraction rate

Note: a,b,c and d represent the significant difference of each level

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图 5 HPLC流动相优化色谱

注：a-流动相为水-乙腈；b-流动相为0.2%（V/V）甲酸水溶液-乙腈

Figure 5. HPLC chromatogram of mobile phase optimization

Note: a-Water-acetonitrile; b-0.2%(V/V) Formic acid in aqueous solution-acetonitrile

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图 6 纯化后紫皮豇豆中单体酚标准品与样品的HPLC色谱

注：1：没食子酸；2：没食子儿茶素；3：绿原酸；4：芦丁；5：p-香豆酸；6：阿魏酸；a：单体酚混合标准溶液（10 μg·mL⁻¹）HPLC 色谱图；b：紫皮豇豆样品 HPLC 色谱图。

Figure 6. HPLC chromatograms of 6 standards and polyphenols in purified purple cowpea extract

Note: 1: Gallic acid; 2: Gallocatechin; 3: Chlorogenic acid; 4: Rutin; 5: p-Coumalic acid; 6: Ferulic acid; a: HPLC chromatogram of polyphenols stanfards (10 μg·mL⁻¹); b: HPLC chromatogram of purple cowpea polyphenols.

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表 1 正交试验的因素及水平的选取

Table 1. Factors and levels of orthogonal test

水平 Level	A 溶剂 Solvent	B 温度 Temperature（℃）	C 料液比 Solid-liquid ratio[m (g)∶v (mL)]	D pH
1	80%丙酮 80% Acetone	4	1∶3	4
2	乙醇 Alcohol	25（室温RT）	1∶5	6
3	甲醇 Methanol	60	1∶7	8

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表 2 多酚的最佳吸收波长

Table 2. Analysis on orthogonal test results

多酚 Polrphenols	没食子酸 Gallic acid	没食子儿茶素 GC	绿原酸 Chlorogenicacid	芦丁 Rutin	p-香豆酸 p-Coumalicacid	阿魏酸 Ferulicacid
最佳吸收波长 Optimum absorption wavelength（nm）	273	278	327	254，355	308	323

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表 3 正交试验的结果及数据分析

Table 3. Optimal UV absorption wavelength for polyphenol detection

序号 Serial Numbers	A	B	C	D	总多酚含量（GAE） Total polyphenol （GAE）/ （mg·100 g⁻¹）
1	1	1	1	1	473
2	1	2	2	2	535
3	1	3	3	3	482
4	2	1	2	3	346
5	2	2	1	1	269
6	2	3	3	2	342
7	3	1	3	3	160
8	3	2	1	2	104
9	3	3	2	1	98
K₁	1 490	979	840	840
K₂	957	908	979	981
K₃	362	922	984	988
k₁	497	326	280	280
k₂	319	303	326	327
k₃	121	307	328	329
极差 R	376	24	48	49
因素主次顺序 Major factors	A＞D＞C＞B
优水平 Best level	A₁	B₁	C₂≈C₃	D₂
优组合 Best group	A₁B₁C₂D₂
注：K_i：表示任一列上水平号为i时所对应的收率试验结果之和；k_i=K_i·n⁻¹（n为该因素取第i个水平时所进行的试验次数）n=3；R= kimax- kimin（收率的正交分析结果的极差） Note: K_i: the sum of yield test results corresponding to the horizontal number i above any column; K_i =K_i·n⁻¹ (n is the number of tests performed when taking the i level of this factor) n =3; R= Kimax - Kimin (range of yield results from orthogonal analysis)

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表 4 紫皮豇豆中6种多酚组分检测的方法学验证

Table 4. Methodology validation for detecting 6 polyphenols in purple cowpeas

组分 Compound	回归方程 Regress equation	相关系数 Coefficient（r）	线性范围 Linear range/（μg·mL⁻¹）	检出限 LOD/（mg·100 g⁻¹）	定量限 LOQ/（mg·100 g⁻¹）
没食子酸 Galslicacid	Y=9.13e+003X−3.04e+002	0.997 6	0.025～1	0.2	0.50
没食子儿茶素 GC	Y=1.86e+004X−1.06e+001	0.999 9	0.150～1	1.0	2.50
绿原酸 Chlorogenic acid	Y=8.76e+003X+ 1.81e+002	0.995 9	0.025～1	0.1	0.25
芦丁 Rutin	Y=3.28e+004X+2.54e+002	0.999 2	0.100～1	0.5	1.00
p-香豆酸 p-Coumalic acidacid	Y=9.50e+003X+ 7.33e+001	0.999 8	0.025～1	0.1	0.20
阿魏酸 Ferulic acid	Y=8.50e+003X−1.74e+002	0.999 8	0.025～1	0.5	0.40

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表 5 紫皮豇豆中6种多酚组分的加标回收试验（n=6）

Table 5. Recovery rates of 6 polyphenols of purple cowpeas （n=6）

组分名称　　 Compound	本底值 Background （mg·100 ⁻¹）	加标浓度 Addition（mg·100 g⁻¹）	测量值 MeasuredValue （mg·100g⁻¹）	回收率 Recovery/%	相对标准偏差 RSD/%
没食子酸 Gallic acid	0.98±0.01	0.5	1.40±0.01	80.5±5.9	2.2
		10	12.40±0.19	85.1±6.3	6.0
		20	21.90±0.24	95.1±1.6	3.2
没食子儿茶素 GC	ND	2.5	1.90±0.05	76.8±4.3	5.6
		10	8.96±0.04	89.6±0.4	0.4
		20	18.50±0.03	92.7±0.7	0.8
绿原酸 Chlorogenic acid	0.51±0.04	0.5	0.96±0.02	89.7±3.1	3.4
		10	11.20±0.01	107.0±0.6	0.6
		20	21.20±0.13	104.0±2.3	2.2
芦丁 Rutin	1.00±0.06	1.0	1.80±0.04	78.3±5.8	7.4
		10	8.40±0.06	74.3±2.6	3.5
		20	16.40±0.29	76.8±4.3	5.6
p-香豆酸 p-Coumalic acid	1.54±0.07	0.5	1.97±0.02	85.7±3.8	4.4
		10	9.60±0.14	80.9±6.6	8.1
		20	18.89±0.34	86.8±5.8	6.7
阿魏酸 Ferulic acid	ND	0.5	0.39±0.06	77.3±2.5	3.1
		10	9.26±0.04	92.6±1.4	1.5
		20	20.00±0.34	100.0±8.6	8.6
注：ND是未检出。 Note：ND means not detected.

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表 6 纯化后紫皮豇豆多酚的单体酚种类及含量（n=10）

Table 6. Composition and contents of individual polyphenols in purified purple cowpea extract （n=10）

紫皮豇豆多酚类物质单体 Individual phenolic compounds in purple cowpea polyphenols	平均含量 Average Content/（mg·100 g⁻¹）
没食子酸 Gallic acid	0.462±0.019
没食子儿茶素 Gallocatechin	3.861±0.083
绿原酸 Chlorogenic acid	1.791±0.067
芦丁 Rutin	6.479±0.130
p-香豆酸 p-Coumalic acid	4.725±0.049
阿魏酸 Ferulic acid	2.627±0.025

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