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
计量
- 文章访问数: 1429
- HTML全文浏览量: 655
- PDF下载量: 32
- 被引次数: 0
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

HTML全文

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.

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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.

下载: 全尺寸图片幻灯片

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.

下载: 导出CSV

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

下载: 导出CSV

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

下载: 导出CSV

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)．

下载: 导出CSV

参考文献(28)

[1]	CHEN X Y, GOU S H, SHI Z Q, et al. Spectrum-effect relationship between HPLC fingerprints and bioactive components of Radix Hedysari on increasing the peak bone mass of rat [J]. Journal of Pharmaceutical Analysis, 2019, 9(4): 266−273. doi: 10.1016/j.jpha.2018.10.004
[2]	SHEN C H, LIU C T, SONG X J, et al. Evaluation of analgesic and anti-inflammatory activities of Rubia cordifolia L. by spectrum-effect relationships [J]. Journal of Chromatography B, 2018, 1090: 73−80. doi: 10.1016/j.jchromb.2018.05.021
[3]	ZHAO Y, YOU X M, JIANG H, et al. Spectrum-effect relationships between high-performance liquid chromatography fingerprints and anti-inflammatory activities of Leontopodium leontopodioides (Willd.) Beauv [J]. Journal of Chromatography B, 2019, 1104: 11−17. doi: 10.1016/j.jchromb.2018.11.001
[4]	李秋月. 延胡索生物碱谱-效关系及相互作用研究[D]. 北京: 北京协和医学院, 2014.
[5]	吕邵娃, 董书羽, 郭玉岩, 等. 数据分析技术在中药谱效关系中的应用进展 [J]. 中国实验方剂学杂志, 2015, 21(15):226−230. LV S W, DONG S Y, GUO Y Y, et al. Advance in application of data analysis technique in spectrum-effect relationship of traditional Chinese medicines [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2015, 21(15): 226−230.(in Chinese)
[6]	曾令军, 林兵, 宋洪涛. 中药谱效关系研究进展及关键问题探讨 [J]. 中国中药杂志, 2015, 40(8):1425−1432. ZENG L J, LIN B, SONG H T. Progress in study of spectrum-effect relationship of traditional Chinese medicine and discussions [J]. China Journal of Chinese Materia Medica, 2015, 40(8): 1425−1432.(in Chinese)
[7]	徐晶晶, 刘斌. 基于DPPH、FRAP法的薄荷药材抗氧化谱效关系研究 [J]. 北京中医药大学学报, 2015, 38(6):405−410. doi: 10.3969/j.issn.1006-2157.2015.06.009 XU J J, LIU B. Spectrum-effect relation in antioxidant activity of Menthae haplocalycis Herba based on DPPH and FRAP assay [J]. Journal of Beijing University of Traditional Chinese Medicine, 2015, 38(6): 405−410.(in Chinese) doi: 10.3969/j.issn.1006-2157.2015.06.009
[8]	ZHU C S, ZHANG B, LIN Z J, et al. Relationship between high-performance liquid chromatography fingerprints and uric acid-lowering activities of Cichorium intybus L [J]. Molecules, 2015, 20(5): 9455−9467. doi: 10.3390/molecules20059455
[9]	谭庆龙, 欧筱争, 谢丽霞, 等. 藏方甲嘎松汤挥发油体外抗氧化活性的谱效关系研究 [J]. 中药新药与临床药理, 2015, 26(3):360−364. TAN Q L, OU X Z, XIE L X, et al. Study on spectrum-effect relationship between fingerprints and antioxidant activities of essential oil from Tibetan medicine jiagasong decoction in vitro [J]. Traditional Chinese Drug Research and Clinical Pharmacology, 2015, 26(3): 360−364.(in Chinese)
[10]	XU G L, XIE M, YANG X Y, et al. Spectrum-effect relationships as a systematic approach to traditional Chinese medicine research: current status and future perspectives [J]. Molecules, 2014, 19(11): 17897−17925. doi: 10.3390/molecules191117897
[11]	王媛媛, 马飞祥, 李凤英, 等. 在线色谱联用技术检测天然产物抗氧化活性的研究进展 [J]. 北方药学, 2016, 13(9):123−125. WANG Y Y, MA F X, LI F Y, et al. Progress in the determination of antioxidant activity of natural products by on-line chromatography [J]. Journal of North Pharmacy, 2016, 13(9): 123−125.(in Chinese)
[12]	韦献雅, 殷丽琴, 钟成, 等. DPPH法评价抗氧化活性研究进展 [J]. 食品科学, 2014, 35(9):317−322. doi: 10.7506/spkx1002-6630-201409062 WEI X Y, YIN L Q, ZHONG C, et al. Advances in the DPPH radical scavenging assay for antioxidant activity evaluation [J]. Food Science, 2014, 35(9): 317−322.(in Chinese) doi: 10.7506/spkx1002-6630-201409062
[13]	ZHANG C, SUEN CLAIREL C, YANG C, et al. Antioxidant capacity and major polyphenol composition of teas as affected by geographical location, plantation elevation and leaf grade [J]. Food Chemistry, 2018, 244: 109−119. doi: 10.1016/j.foodchem.2017.09.126
[14]	LV H P, ZHANG Y, SHI J, et al. Phytochemical profiles and antioxidant activities of Chinese dark teas obtained by different processing technologies [J]. Food Research International, 2017, 100: 486−493. doi: 10.1016/j.foodres.2016.10.024
[15]	林清霞, 项丽慧, 王丽丽, 等. 茶多酚高通量检测技术研究进展 [J]. 中国农学通报, 2019, 35(3):146−153. LIN Q X, XIANG L H, WANG L L, et al. High throughput test of tea polyphenols: a review [J]. Chinese Agricultural Science Bulletin, 2019, 35(3): 146−153.(in Chinese)
[16]	周卫龙, 徐建峰, 黄伙水. 茶叶中茶多酚和儿茶素类含量的检测方法: GB/T 8313-2018[S]. 北京: 中国标准出版社, 2018: 1-7.
[17]	王丽丽, 陈键, 宋振硕, 等. 茶叶中没食子酸、儿茶素类和生物碱的HPLC检测方法研究 [J]. 福建农业学报, 2014, 29(10):987−994. doi: 10.3969/j.issn.1008-0384.2014.10.011 WANG L L, CHEN J, SONG Z S, et al. Simultaneous HPLC determination of Gallic acid, catechins and alkaloids in tea [J]. Fujian Journal of Agricultural Sciences, 2014, 29(10): 987−994.(in Chinese) doi: 10.3969/j.issn.1008-0384.2014.10.011
[18]	王丽丽, 杨军国, 宋振硕, 等. 鲜叶、绿茶和白茶化学组分比较及清除DPPH自由基研究 [J]. 茶叶学报, 2015, 56(4):214−222. doi: 10.3969/j.issn.1007-4872.2015.04.004 WANG L L, YANG J G, SONG Z S, et al. Compositional differences and DPPH radical scavenging of fresh tea leaves, green tea and white tea [J]. Acta Tea Sinica, 2015, 56(4): 214−222.(in Chinese) doi: 10.3969/j.issn.1007-4872.2015.04.004
[19]	陈金娥, 丰慧君, 张海容. 红茶、绿茶、乌龙茶活性成分抗氧化性研究 [J]. 食品科学, 2009, 30(3):62−66. doi: 10.3321/j.issn:1002-6630.2009.03.013 CHEN J N, FENG H J, ZHANG H R. Effects of active ingredients in black tea, green tea and oolong tea on antioxidant capability [J]. Food Science, 2009, 30(3): 62−66.(in Chinese) doi: 10.3321/j.issn:1002-6630.2009.03.013
[20]	林玲, 龚志华, 袁冬寅, 等. 相同加工原料下的6类茶体外抗氧化性能比较 [J]. 中国农学通报, 2018, 34(2):107−112. doi: 10.11924/j.issn.1000-6850.casb16120045 LIN L, GONG Z H, YUAN D Y, et al. Comparison of antioxidative activity in vitro among six kinds of tea made from the same raw material [J]. Chinese Agricultural Science Bulletin, 2018, 34(2): 107−112.(in Chinese) doi: 10.11924/j.issn.1000-6850.casb16120045
[21]	ZHAO C N, TANG G Y, CAO S Y, et al. Phenolic profiles and antioxidant activities of 30 tea infusions from green, black, oolong, white, yellow and dark Teas [J]. antioxidants, 2019, 8: 215−228. doi: 10.3390/antiox8070215
[22]	杨伟丽, 肖文军, 邓克尼. 加工工艺对不同茶类主要生化成分的影响 [J]. 湖南农业大学学报(自然科学版), 2001, 27(5):384−386. YANG W L, XIAO W J, DENG K N. Effects of processing technology of different teas on the main biochemistry components [J]. Journal of Hunan Agricultural University(Natural Sciences Editon), 2001, 27(5): 384−386.(in Chinese)
[23]	龙海林, 李强, 司银楚, 等. 虎杖的功效成分组研究Ⅲ: 对血浆黏度影响的谱效分析研究 [J]. 现代医药卫生, 2012, 28(20):3080−3082. LONG H L, LI Q, SI Y C, et al. Spectrum-effect relationship study on influence of effective compositions research Ⅲ of polygoni Cuspidation plasma viscosity [J]. Journal of Modern Medicine & Health, 2012, 28(20): 3080−3082.(in Chinese)
[24]	肖遂. 基于谱效关系的中药铁苋菜抑菌物质辨识方法研究[D]. 北京: 中国农业科学院, 2013. XIAO S. A method for research of antibacterial constituent recognition of traditional Chinese medicine(Acalypha australis linn.) by spectrum-effect relationship[D]. Beijing: Chinese Academy of Agricultural Sciences, 2013. (in Chinese)
[25]	邓书鸿, 聂磊. 中药谱效关系的分析方法及数据处理技术研究进展 [J]. 中药材, 2010, 33(11):1819−1823. DENG S H, NIE L. Advances in analytical methods and data processing techniques for the relationship between the spectrum and effect of traditional Chinese medicine [J]. Journal of Chinese Medicinal Materials, 2010, 33(11): 1819−1823.(in Chinese)
[26]	DOU Q P. Recent advances on tea polyphenols [J]. Frontiers in Bioscience, 2012, E4(1): 111−131. doi: 10.2741/e363
[27]	卢怡雯, 李晓芬, 项朋志, 等. 没食子酸清除DPPH自由基的紫外-可见吸收光谱研究 [J]. 食品工业科技, 2014, 35(2):124−126, 130. LU Y W, LI X F, XIANG P Z, et al. Study on UV-Vis absorption spectrometric investigation of the gallic acid against DPPH free radicals [J]. Science and Technology of Food Industry, 2014, 35(2): 124−126, 130.(in Chinese)
[28]	马慧, 茹鑫, 王津, 等. 4种茶叶水提物及茶多酚的体外抗氧化性能研究 [J]. 食品研究与开发, 2019, 40(8):65−70. doi: 10.3969/j.issn.1005-6521.2019.08.011 MA H, RU X, WANG J, et al. Study on the antioxidant capacity of four tea water extracts and tea polyphenols in vitro [J]. Food Research and Development, 2019, 40(8): 65−70.(in Chinese) doi: 10.3969/j.issn.1005-6521.2019.08.011