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  热带亚热带植物学报  2021, Vol. 29 Issue (3): 293-300  DOI: 10.11926/jtsb.4282
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引用本文  

王丽丽, 林清霞, 宋振硕, 等. 白茶对弹性蛋白酶活性的抑制研究[J]. 热带亚热带植物学报, 2021, 29(3): 293-300. DOI: 10.11926/jtsb.4282.
WANG Li-li, LIN Qing-xia, SONG Zhen-shuo, et al. Studies on Anti-elastase Activity of White Tea[J]. Journal of Tropical and Subtropical Botany, 2021, 29(3): 293-300. DOI: 10.11926/jtsb.4282.

基金项目

福建省属公益类科研院所专项(2020R1029006,2019R1029-3);福建省自然科学基金项目(2020J011367)资助

通信作者

陈林, E-mail: chenlin_xy@163.com

作者简介

王丽丽(1985~), 女, 助理研究员, 从事茶叶生物化学及天然产物分析研究。E-mail: 875720551@qq.com

文章历史

收稿日期:2020-07-02
接受日期:2020-11-18
白茶对弹性蛋白酶活性的抑制研究
王丽丽 , 林清霞 , 宋振硕 , 陈林     
福建省农业科学院茶叶研究所, 福州 350013
摘要:为了解白茶对弹性蛋白酶活性的作用,测定了6个白茶样品对猪胰弹性蛋白酶(PPE)活性的抑制作用。结果表明,6个白茶样品中CB4对PPE抑制活性最强,为69.73%;且随体积分数增加,酶抑制活性增强,呈剂量依赖性抑制。白茶水提液的4个萃取层中乙酸乙酯层(WEA)的抑制活性最强,达到82.58%。CB4的多酚总量(TPs)较高,且表没食子儿茶素没食子酸酯(EGCG)、表儿茶素没食子酸酯(ECG)和咖啡碱(CAF)含量最高。WEA萃取层中TPs、EGCG、ECG和ECG含量均最高,CAF含量极低。PPE活性抑制率与TPs、EGCG和CAF含量呈极显著正相关,相关系数均大于0.90。0.1~1.0 mg/mL EGCG和0.05~2.0 mg/mL CAF对PPE无抑制活性,0.05~2.0 mg/mL没食子酸(GA)对PPE活性具有促进作用,且GA的促进作用与其浓度呈正相关。可见,白茶具有潜在抗衰老活性,但对PPE起抑制作用的活性成分不是EGCG、GA和CAF。
关键词白茶    乙酸乙酯萃取层    猪胰弹性蛋白酶    抑制活性    
Studies on Anti-elastase Activity of White Tea
WANG Li-li , LIN Qing-xia , SONG Zhen-shuo , CHEN Lin     
Tea Research Institution, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China
Foundation item: his work was supported by the Special Project for Public Welfare Research Institutes in Fujian (Grant No. 2020R1029006, 2019R1029-3), and the Natural Science Foundation in Fujian (Grant No. 2020J011367)
Abstract: In order to understand the anti-elastase effect of white tea, the inhibitory effect of aqueous extract from six white tea samples (CB1-CB6) on porcine pancreatic elastase (PPE) were measured. The results showed that CB4 had the strongest inhibition on PPE among six white tea samples with inhibition rate of 69.73%. In addition, elastase inhibition activity of CB4 increased with the concentration, showing a dose-dependent manner. Among the four extraction layers, ethyl acetate (WEA) layer had the strongest inhibitory activity, reaching 82.58%. The total polyphenol (TPs) content in CB4 was high, and the contents of epigallocatechin gallate (EGCG), epicatechin gallate (ECG), and caffeine (CAF) were the highest among six samples. Among 4 extraction layers, the contents of EGCG, ECG, ECG, and total polyphenols in WEA layer were the highest and that of CAF was very low. There were significant positive correlation between elastase inhibition rate and the contents of TPs, EGCG and CAF with correlation coefficient above 0.90. However, neither 0.1-1.0 mg/mL EGCG nor 0.05-2.0 mg/mL CAF had inhibitory effects on PPE activity. Conversely, 0.05-2.0 mg/mL GA showed a promotion effect on PPE activity, and the promoting effect of GA was positively correlated with its concentration. Therefore, it was suggested that white tea exhibited potentially anti-aging activity, but the active ingredients that inhibited PPE were not EGCG, GA and CAF.
Key words: White tea    Ethyl acetate extraction layer    Porcine pancreatic elastase    Inhibitory activity    

弹性蛋白酶具有降解胶原蛋白、弹性蛋白等多种蛋白质的能力。皮肤组织中的弹性蛋白被弹性蛋白酶降解与皮肤衰老发生过程密切相关,因此对抗弹性蛋白酶对弹性蛋白的降解,恢复皮肤弹性是延缓皮肤衰老的重要途径之一[1]。研究表明,很多植物提取物如芦丁、黄芩素、高良姜素(又名3, 5, 7-三羟基黄酮)、虎杖苷等是天然弹性蛋白酶抑制剂[25]。白茶是我国六大茶类之一,富含多酚类物质,具有清凉、退热、降火、祛暑等功效。近年来,随着人们对健康的追求日益高涨,白茶逐渐步入大众视野,其独特的风味品质和保健功效引起了国内外消费者的关注和喜爱。有研究表明,白茶水提取物对弹性蛋白酶的抑制率高达89%[6],且当其作用于体外培养的人皮肤成纤维细胞时,可有效缓解H2O2诱导造成的细胞凋亡损伤[7], 但对于白茶提取物中可能起作用的有效成分尚未见报道。本研究测定了6个白茶样品水提液及其不同萃取层对猪胰弹性蛋白酶(PPE, porcine pancreatic elastase)的抑制活性,并分析其化学组成,同时测定了没食子酸(GA, gallic acid)、表没食子儿茶素没食子酸酯(EGCG, epigallocatechin gallate)和咖啡碱(CAF, caffeine)的酶活抑制率,推测可能起作用的有效成分,以期为白茶延缓皮肤衰老研究提供理论依据, 为其在女性抗皮肤衰老化妆品领域的应用提供数据支撑。

1 材料和方法 1.1 试剂和材料

刚果红-弹性蛋白(德国Ruibio)与PPE (德国Ruibio, 活性≥30 U/mg)购自合肥博美生物科技有限责任公司;0.2 mol/L硼酸盐缓冲液(pH 8.8); 0.5 mol/L磷酸盐缓冲液(pH 6.0);GA (上海生工生物, 纯度 > 98%)、EGCG与CAF (上海阿拉丁,纯度 > 99%)标准品;白茶样品6份(表 1),市售,系白牡丹等级,分别记作CB1~CB6。

表 1 茶样信息 Table 1 Information of white tea samples
1.2 仪器

SKY-200B摇床(上海苏坤)、VARIOSKAN LUX多功能酶标仪(美国Thermo scientific)、ACD-0502-U超纯水机(重庆艾科浦)、PB-10酸度计(德国Sarto- rius)、MS 3 basic旋涡混匀器(德国IKA)、5418R小型台式离心机(德国Eppendorf)、CVE-3000型离心浓缩仪(日本EYELA)。

1.3 白茶水提液及其萃取层制备

称取白茶样品4 g,加100 mL沸水,沸水浴提取15 min,滤纸抽滤制得白茶水提液,当日使用。参照杨子银等[8]的方法制备CB4萃取层,依次得到氯仿层(WEC)、乙酸乙酯层(WEA)、正丁醇层(WEB)和剩余水层(WER)。各萃取层浓缩至干(呈浸膏状),–20℃冷冻保存备用。

1.4 对PPE抑制率的测定

参照姚亚红等[3]的方法,稍作修改。准确称取20 mg刚果红-弹性蛋白至50 mL离心管内,加入5 mL 37℃预热的0.2 mol/L硼酸盐缓冲液(pH 8.8),充分涡旋混匀,水浴20 min,加入2 mL经37℃预热的用硼酸盐缓冲液配制的PPE溶液(浓度为0.5、1、2、4、6 mg/mL),充分涡旋混匀,在37℃、400 r/min摇床振荡20 min,立即加入pH 6.0的0.5 mol/L磷酸盐缓冲液5 mL,涡旋混匀,取混匀液适量至2 mL离心管内, 在9 391×g下离心10 min,精密吸取上清液200 μL至96孔板内,在波长495 nm处测定吸光度。以PPE浓度为横坐标,吸光度为纵坐标,