基于DPPH法的茶叶儿茶素类抗氧化谱效关系研究

林清霞; 王丽丽; 杨军国; 宋振硕; 陈林

doi:10.19303/j.issn.1008-0384.2020.02.012

基于DPPH法的茶叶儿茶素类抗氧化谱效关系研究

doi: 10.19303/j.issn.1008-0384.2020.02.012

1.
福建省农业科学院茶叶研究所，福建　福州　350013
2.
闽南师范大学生物科学与技术学院，福建　漳州　363000

基金项目: 福建省科技计划公益类专项（2019R1029-3）；福建省农业科学院青年人才创新基金项目（YC2018-7）

详细信息

作者简介:
林清霞（1989−），女，硕士，研究实习员，研究方向：茶叶精深加工（E-mail：735801309@qq.com）

通讯作者:
陈林（1975−），男，博士，研究员，研究方向：茶叶加工、茶叶生物化学及综合利用研究（E-mail：chenlin_xy@163.com）

中图分类号: TS 272
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出版历程
- 收稿日期: 2019-10-23
- 修回日期: 2019-12-26
- 刊出日期: 2020-02-01

Spectrum-effect Relationship between HPLC Fingerprints and DPPH-scavenging Activities of Tea Catechins

1.
Tea Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China
2.
School of Biological Science and Biotechnology, Minnan Normal University, Zhangzhou, Fujian　363000, China

摘要

摘要: 目的研究茶叶儿茶素HPLC指纹图谱与其自由基清除活性的关系。方法采用70%甲醇对绿茶、白茶、闽北乌龙、闽南乌龙、红茶5类共25批次样本进行提取；以福林酚法测定总酚含量；以1,1-二苯基-2-三硝基苯肼（DPPH）法研究自由基清除活性；同时通过高效液相色谱法（HPLC）获取指纹图谱数据并对所获取的指纹图谱数据进行主成分分析；采用偏最小二乘回归分析研究HPLC指纹图谱与DPPH清除活性的谱效关系，结合皮尔逊（Pearson）相关性分析对偏最小二乘回归模型进行验证。结果获取25批次样本的HPLC指纹图谱，确定了8个儿茶素类化合物，成功构建了偏最小二乘回归方程，其决定系数R²=0.900 9。结果表明表没食子儿茶素没食子酸酯（EGCG）、表没食子儿茶素（EGC）、表儿茶素没食子酸酯（ECG）与DPPH清除力相关性最强，均呈极显著正相关；没食子酸（GA）与DPPH清除力呈负相关。Pearson相关性分析结果与偏最小二乘回归模型结果基本一致。结论本研究通过HPLC指纹图谱信息可预测抗氧化活性。
- 儿茶素 /
- 抗氧化 /
- 高效液相色谱（HPLC） /
- 谱效关系
Abstract: Objective Correlation between the high-performance liquid chromatography (HPLC) fingerprints and the DPPH radical scavenging activities of catechins from varieties of teas was studied. Method From 5 categories of teas including green tea, white tea, northern Fujian oolong, southern Fujian oolong, and black tea, 25 specimens were collected and extracted with 70% methanol. The extracts were used to determine polyphenols content by the Folin-Ciocalteu method, the free radical scavenging activity by [1,1-diphenyl-2-picrylhydrazylradical2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl, DPPH] assay, and the fingerprints by HPLC for a principal component analysis. A correlation model between the HPLC fingerprints and DPPH radical scavenging activity was established by a partial least squares regression analysis and validated by the Pearson analysis. Result The HPLC fingerprints of the 25 specimens had 8 common compounds. The established regression model showed a coefficient of determination of R²=0.900 9. The 3 compounds, epigallocatechin gallate (EGCG), epigallocatechin (EGC), and epicatechin gallate (ECG), exhibited the strongest and significant correlations with DPPH radical scavenging activity. On the other hand, gallic acid (GA) correlated inversely with the scavenging activity. The Pearson analysis showed a result consistent with what the partial least squares regression test did. Conclusion It appeared that the antioxidant activity of catechins in the various teas could be satisfactorily predicted by HPLC fingerprints.
- catechins /
- antioxidant activities /
- high-performance liquid chromatography (HPLC) /
- spectrum-effect relationship

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图 1 茶样的总酚含量及其DPPH清除活性

注：G、W、N、S、B分别代表绿茶、白茶、闽北乌龙、闽南乌龙、红茶。图2同。

Figure 1. Polyphenol contents and DPPH radical scavenging activities of tea specimens

Note：G, W, N, S, and B represent samples of green tea, white tea, northern Fujian oolong, southern Fujian oolong, and black tea, respectively. The same for Fig.2.

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图 2 25个茶样共有峰的主成分分析

Figure 2. Scatter plot of PCA scores on common peaks of HPLC fingerprints from 25 samples

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图 3 25个样品的HPLC指纹图谱

注：图中各样品编号由上至下分别为W1、W2、W3、W4、W5、B1、B2、B3、B4、B5、G1、G2、G3、G4、G5、S1、S2、S3、S4、S5、N1、N2、N3、N4、N5。

Figure 3. HPLC fingerprints from 25 samples

Note：The sample numbers from the top to the bottom are: W1, W2, W3, W4, W5, B1, B2, B3, B4, B5, G1, G2, G3, G4, G5, S1, S2, S3, S4, S5, N1, N2, N3, N4, N5, respectively.

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表 1 25个样品HPLC指纹图谱共有峰峰面积

Table 1. Average areas of common peaks on HPLC fingerprints from 25 samples

样品 Samples	25个样品共有峰的峰面积 mAUS Average peak area of every common peak mAUS
样品 Samples	GA	GC	EGC	EGCG	EC	GCG	ECG	CG
G1	40.13±1.40	18.22±2.25	65.87±3.57	2 227.66±13.35	49.54±1.15	32.02±1.95	995.88±9..90	8.77±5.70
G2	25.03±1.54	8.54±0.57	74.24±0.11	1637.85±3.57	121.59±0.92	12.23±1.27	768.66±5.20	10.52±1.24
G3	21.94±0.46	17.81±0.64	80.63±0.67	1716.19±1.63	147.59±1.70	18.96±1.38	769.49±3.11	8.59±1.70
G4	21.11±1.15	14.70±3.50	79.25±3.20	1653.04±13.25	150.69±3.20	10.51±0.20	765.03±15.75	10.56±2.65
G5	13.18±1.15	13.06±0.45	91.82±4.33	1894.60±6.30	169.08±1.10	20.26±0.40	952.57±3.51	14.85±5.45
W1	41.36±0.90	4.55±3.05	111.84±5.55	2038.52±68.95	34.36±3.85	20.29±0.55	875.54±16.45	5.64±2.30
W2	43.63±1.10	10.71±1.70	89.49±2.95	1382.64±43.60	57.11±3.35	12.58±0.50	569.71±12.55	6.08±1.20
W3	41.22±0.35	5.80±0.55	91.56±0.45	1836.79±4.55	50.96±8.95	14.19±0.65	642.15±2.20	7.49±0.10
W4	27.76±1.10	8.32±0.80	95.98±5.95	1814.42±94.35	58.06±4.00	9.37±2.35	545.45±22.70	6.22±0.55
W5	47.33±1.30	7.55±0.30	70.44±0.15	1326.74±9.80	36.01±2.40	7.29±1.15	412.72±1.70	6.11±1.00
N1	106.43±0.50	6.64±0.45	64.66±0.30	965.38±67.50	79.93±11.40	16.89±4.35	321.53±39.65	16.39±1.05
N2	94.12±0.65	9.42±1.30	78.55±1.40	1037.62±2.00	91.03±1.5	22.86±0.75	313.46±5.20	15.66±1.30
N3	102.94±0.35	8.44±5.22	79.80±2.60	983.68±22.30	87.01±1.82	20.98±0.60	298.41±9.28	10.53±1.83
N4	70.68±0.85	10.03±1.83	69.03±0.70	955.95±2.85	93.20±1.65	22.93±3.45	303.44±0.60	14.65±0.99
N5	155.18±0.45	15.98±1.55	65.33±0.75	578.92±9.85	19.10±0.10	154.82±0.25	212.88±4.50	40.47±2.20
S1	4.20±0.90	16.01±3.05	94.03±5.55	1523.93±68.95	86.89±3.85	8.96±0.55	368.55±16.45	10.68±2.30
S2	5.38±1.10	12.29±1.70	70.83±2.95	1361.34±43.60	109.65±2.35	9.23±1.95	402.72±12.4	11.26±0.45
S3	6.53±0.35	11.52±0.50	65.74±0.45	1066.28±4.50	125.36±8.95	5.74±0.65	319.91±2.20	8.80±0.10
S4	4.93±1.10	11.48±0.80	70.38±5.95	1423.13±94.35	83.51±4.00	13.15±2.35	373.80±22.50	8.54±0.55
S5	6.43±1.30	15.79±0.11	65.66±0.15	1298.71±9.80	74.57±2.40	8.64±1.15	335.69±1.72	7.32±1.12
B1	203.94±17.35	0.48±0.07	45.49±2.35	11.04±0.39	2.85±0.09	3.24±0.12	71.26±7.35	8.22±1.84
B2	210.96±0.050	ND	50.50±1.25	110.06±3.46	3.21±0.12	3.16±0.32	119.59±3.52	15.38±3.10
B3	228.05±2.65	ND	70.50±4.55	434.56±3.45	15.89±2.80	5.83±2.35	343.13±4.00	12.55±2.65
B4	180.05±0.65	ND	61.54±3.55	221.03±0.90	15.74±0.43	1.63±0.020	233.35±13.25	8.37±0.86
B5	219.56±4.70	ND	63.60±7.00	155.15±4.15	9.92±0.30	4.38±1.25	261.40±7.30	19.73±4.05
注：ND指未检出。 Note：ND means undetected.

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表 2 偏最小二乘回归方程假设检验结果

Table 2. Hypothetic result of partial least squares regression equation

方差来源 Variance Source	自由度 df	方差 SS	均方差 MS	F	P
回归 Regress	8	1491	186	18	0
残差 Residual	16	164	10
总变异数 Total variation	24	1 655

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表 3 偏最小二乘回归方程预测结果

Table 3. Predicted result from partial least squares regression equation

模型检验 Model test	样品 Sample	预测值 Predictive	实测值 Measured	相对标准偏差 Relative standard deviation/%
内部验证 Internal test	G1	57.45	60.87	5.62
	B3	40.90	39.28	4.12
	N4	48.48	50.58	4.15
	W3	56.25	58.19	3.33
	S5	49.92	48.25	3.46
外部验证 External test	R1	47.41	53.25	10.97
	R2	62.15	58.34	6.53
	R3	58.83	61.46	4.28
	R4	47.93	45.16	6.13
	R5	46.17	43.05	7.25

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表 4 茶样中共同峰与其DPPH清除活性之间的关系

Table 4. Correlation coefficients between DPPH radical scavenging activities and common peaks

化合物 Composition	GA	GC	EGC	EGCG	EC	GCG	ECG	CG
相关系数 Correlation	−0.723^**	0.609^**	0.808^**	0.899^**	0.427^*	0.174	0.771^**	−0.170
注：*表示在0.01水平（双侧）上显著相关，表示在0.05水平（双侧）上显著相关。 Note: ** indicates a significant correlation at 0.01 level (double sides); * shows a significant correlation at 0.05 1evel (double sides)．

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