茶多糖的抗氧化活性及对细胞氧化损伤的保护机制

doi:10.11983/CBB22088

植物学报 ›› 2022, Vol. 57 ›› Issue (4): 444-456.DOI: 10.11983/CBB22088

茶多糖的抗氧化活性及对细胞氧化损伤的保护机制

张明珠¹, 秦华光¹, 穆丹¹, 杨龙宇¹, 李虎¹, 岂泽华¹, 王玉玺¹, 张永成¹, 叶利利¹, 殷文晶², 王树元¹, 饶玉春²^,^*(), 吴彦¹^,^*()

¹安庆师范大学生命科学学院, 皖西南生物多样性研究与生态保护安徽省重点实验室, 安庆 246133
²浙江师范大学化学与生命科学学院, 金华 321004

收稿日期:2022-04-26 修回日期:2022-06-28 出版日期:2022-07-01 发布日期:2022-07-14
通讯作者: 饶玉春,吴彦
作者简介:sportman821@sina.com
* E-mail: ryc@zjnu.cn;
第一联系人：^† 共同第一作者
基金资助:
国家自然科学基金(31800316);安徽省高校优秀青年人才支持计划(gxyq2018034);皖西南生物多样性研究与生态保护安徽省重点实验室开放基金(Wz2021002);皖西南生物多样性研究与生态保护安徽省重点实验室开放基金(Wz2021003);皖西南生物多样性研究与生态保护安徽省重点实验室开放基金(Wsz202206);皖西南生物多样性研究与生态保护安徽省重点实验室开放基金(Wsz202207);皖西南生物多样性研究与生态保护安徽省重点实验室开放基金(Wy202206);安徽省大学生创新创业训练计划(S202110372108);安徽省大学生创新创业训练计划(S2021-10372104X);安徽省大学生创新创业训练计划(S202110372107);安庆师范大学研究生教育教学改革项目(2021aqnujyxm64);安徽省教学示范课昆虫学(1545);安庆师范大学“敬敷育英”创新创业引领计划和安庆师范大学教研项目(2019aqnujyzc127)

Antioxidant Activity of Tea Polysaccharide and Its Protective Mechanism Against Oxidative Damage

Zhang Mingzhu¹, Qin Huaguang¹, Mu Dan¹, Yang Longyu¹, Li Hu¹, Qi Zehua¹, Wang Yuxi¹, Zhang Yongcheng¹, Ye Lili¹, Yin Wenjing², Wang Shuyuan¹, Rao Yuchun²^,^*(), Wu Yan¹^,^*()

¹The Province Key Laboratory of the Biodiversity Study and Ecology Conservation in Southwest Anhui, School of Life Sciences, Anqing Normal University, Anqing 246133, China
²School of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China

Received:2022-04-26 Revised:2022-06-28 Online:2022-07-01 Published:2022-07-14
Contact: Rao Yuchun,Wu Yan
About author:First author contact:^† These authors contributed equally to this paper

摘要/Abstract

摘要： 茶多糖是一种从茶叶中提取的酸性糖蛋白, 具有良好的抗氧化活性。以自由基清除率为指标, 分析皖西南地区夏秋茶多糖的抗氧化活性, 基于H₂O₂和EDTA-Fe²⁺建立的外源性羟基自由基(·OH)损伤细胞模型和PMA诱导内源性羟基自由基损伤模型, 进一步探讨茶多糖对自由基损伤的修复作用机制。结果表明, 茶多糖具有良好的体外抗氧化活性, 对DPPH·和·OH均具有较强的清除效果, EC₅₀值分别为209.5和535.2 µg∙mL^-1, 最大清除效率与Vc相当。细胞增殖实验表明, 外源性和内源性自由基氧化损伤模型中细胞存活率均随着茶多糖浓度的增加而升高, 在茶多糖浓度为800 µg∙mL^-1时细胞存活率分别高达87.41%和85.84%, 且显著高于模型组(47.67%和48.03%)。在修复机制上, 利用激光共聚焦显微镜显影细胞内活性氧(ROS)分布以及荧光强度, 分析结果显示, 与模型组相比, 茶多糖对于细胞模型中外源和内源性ROS均具有明显的清除效果, 与体外抗氧化实验结果一致。茶多糖在体外表现出良好的自由基清除效率, 可在细胞水平上改善自由基损伤。该研究在细胞水平上揭示了茶多糖清除自由基的抗氧化损伤机制, 为后续进一步阐明茶多糖抗衰老作用奠定了基础。

关键词: 茶多糖, 抗氧化, 细胞氧化损伤, 修复机制

Abstract: Wan tea polysaccharides (WTPs) is a kind of acid glycoprotein extracted from tea, which has good antioxidant activity. The antioxidant activity of WTPs in Southwest Anhui was analyzed using the scavenging rate of free radicals as an index, and the repair effect of WTPs on free radical damage was further explored based on the exogenous ·OH injury cell model established by H₂O₂ and EDTA-Fe²⁺ and the endogenous hydroxyl radical injury model induced by PMA. The results showed that WTPs had good antioxidant activity against DPPH· and ·OH, with EC₅₀ values of 209.5 and 535.2 µg∙mL^-1 on scavenging efficiency, respectively, which was similar with Vc. The survival rate of cells in both exogenous and endogenous free radical oxidative damage models increased with the increase of WTPs concentration by cell proliferation assay, which could reach 87.41% and 85.84% when WTPs concentration was 800 µg∙mL^-1, which was significantly higher than that in model group (47.67% and 48.03%). In terms of protective mechanism, laser confocal imaging of reactive oxygen species (ROS) distribution in cells and fluorescence intensity analysis showed that compared with the model group, WTPs had significant scavenging effect on exogenous and endogenous ROS in cell models, which was consistent with the results of antioxidant test in vitro. WTPs showed good free radical scavenging efficiency in vitro and its improvement effect on free radical damage at the cellular level. The study revealed antioxidant damage mechanism of WTPs in scavenging free radicals at the cellular level, which laid a foundation for further research on the anti-aging effect of tea polysaccharides on the body aging caused by free radicals.

Key words: tea polysaccharides, antioxidant, cell oxidative damage, repair mechanism

张明珠, 秦华光, 穆丹, 杨龙宇, 李虎, 岂泽华, 王玉玺, 张永成, 叶利利, 殷文晶, 王树元, 饶玉春, 吴彦. 茶多糖的抗氧化活性及对细胞氧化损伤的保护机制. 植物学报, 2022, 57(4): 444-456.

Zhang Mingzhu, Qin Huaguang, Mu Dan, Yang Longyu, Li Hu, Qi Zehua, Wang Yuxi, Zhang Yongcheng, Ye Lili, Yin Wenjing, Wang Shuyuan, Rao Yuchun, Wu Yan. Antioxidant Activity of Tea Polysaccharide and Its Protective Mechanism Against Oxidative Damage. Chinese Bulletin of Botany, 2022, 57(4): 444-456.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22088

https://www.chinbullbotany.com/CN/Y2022/V57/I4/444

图/表 8

图1 不同浓度茶多糖(WTPs)对自由基的清除能力 (A) DPPH自由基; (B) 羟基自由基; (C) 超氧自由基

Figure 1 Free radical scavenging ability of different concentrations of Wan tea polysaccharides (WTPs) (A) DPPH·; (B)·OH; (C) O2-

图2 不同浓度茶多糖(WTPs)对HepG-2细胞(A)及RAW264.7巨噬细胞(B)存活率的影响

Figure 2 Effects of Wan tea polysaccharides (WTPs) concentration on the survival rate of HepG-2 cells (A) and RAW264.7 cells (B)

图3 茶多糖(WTPs)对细胞生理状态的影响 (A) 对照细胞线粒体形态; (B) 不同处理细胞选定区域的放大显微照片及强度分析; (C) WTPs处理细胞线粒体形态; (D) 不同处理细胞选定区域的放大显微照片及强度分析。DIC: 微分干涉称镜检术。Bars=20 μm

Figure 3 Effects of Wan tea polysaccharides (WTPs) on cell physiological state (A) Morphology of mitochondria in control cells; (B) Zoom in micrograph from selected regions from different treated cells and their intensity analysis; (C) Morphology of mitochondria in WTPs incubated cells; (D) Zoom in micrograph from selected regions from different treated cells and their intensity analysis. DIC: Differential interference contrast. Bars=20 μm

图4 茶多糖(WTPs)对损伤细胞的保护作用 (A) 不同浓度外源性羟基自由基对HepG-2细胞存活率的影响; (B) 不同浓度茶多糖对羟基自由基诱导的HepG-2细胞存活率的影响。与对照组相比, *P<0.05, **P<0.01; 与模型组相比, #P<0.05, ##P<0.01

Figure 4 Protective effect of Wan tea polysaccharides (WTPs) on demaged cells (A) Effects of different concentrations of exogenous ·OH on the survival rate of HepG-2 cells; (B) Effects of different concentrations of WTPs on HepG-2 cell survival rate induced by ·OH. Compared with control group, *P<0.05, **P<0.01; compared with model group, #P<0.05, ##P<0.01

图5 茶多糖(WTPs)对PMA诱导的RAW264.7巨噬细胞活力的影响 (A) 不同浓度PMA诱导内源性羟基自由基对RAW264.7巨噬细胞存活率的影响; (B) 不同浓度PMA对RAW264.7巨噬细胞形态的影响(bars=20 μm); (C) 不同浓度茶多糖对内源性羟基自由基诱导的RAW264.7巨噬细胞存活率的影响。与对照组相比, *P<0.05, **P<0.01; 与模型组相比, #P<0.05, ##P<0.01

Figure 5 Effect of Wan tea polysaccharides (WTPs) on the viability of RAW264.7 cells induced by PMA (A) Effects of endogenous ·OH induced by different concentration of PMA on the survival rate of RAW264.7 cells; (B) The effect of different concentration of PMA on morphological changes of RAW264.7 cells (bars=20 μm); (C) Effects of different concentration of WTPs on RAW264.7 cells survival rate induced by endogenous ·OH. Compared with control group, *P<0.05, **P<0.01; compared with model group, #P<0.05, ##P<0.01

表1 茶多糖(WTPs)对RAW264.7氧化损伤细胞中谷胱甘肽还原酶(GSH-PX)和过氧化氢酶(CAT)含量的影响(平均值±标准误)

Table 1 Effects of Wan tea polysaccharides (WTPs) on the content of glutathione reductase (GSH-PX) and catalase (CAT) in oxidation-damaged RAW264.7 cells (means±SD)

Group	GSH-PX	CAT
Group	(U∙mL^-1)	(U∙mL^-1)
Control	0.45±0.08	1.08±0.12
Model	0.13±0.04**	0.59±0.04**
100 µg∙mL^-1 WTPs	0.16±0.04**	0.57±0.06**
200 µg∙mL^-1 WTPs	0.15±0.04**	0.60±0.07**
400 µg∙mL^-1 WTPs	0.12±0.03**	0.62±0.05**
800 µg∙mL^-1 WTPs	0.13±0.02**	0.57±0.08**

图6 茶多糖(WTPs)对HepG-2细胞氧化损伤的修复作用 (A) 不同处理组细胞中羟基自由基含量的共聚焦显影图(bars=20 μm); (B) 不同处理组细胞中羟基自由基荧光强度对比。与对照组相比, *P<0.05, **P<0.01; 与模型组相比, #P<0.05, ##P<0.01。

Figure 6 Repair effect of Wan tea polysaccharides (WTPs) on oxidative damage of HepG-2 cells (A) Confocal imaging of ·OH content in cells of different treatment groups (bars=20 μm); (B) Comparison of fluorescence intensity of ·OH in cells of different treatment groups. Compared with control group, *P<0.05, **P<0.01; compared with model group, #P<0.05, ##P<0.01.

图7 茶多糖(WTPs)对PMA诱导的RAW264.7巨噬细胞氧化损伤的影响 (A) 不同处理组细胞中羟基自由基含量的共聚焦显影图(bars=20 μm); (B) 不同处理组细胞中羟基自由基荧光强度对比。与对照组相比, *P<0.05, **P<0.01; 与模型组相比, #P<0.05, ##P<0.01

Figure 7 Effect of Wan tea polysaccharides (WTPs) on PMA-induced oxidative damage of RAW264.7 macrophages (A) Confocal imaging of ·OH content in cells of different treatment groups (bars=20 μm); (B) Comparison of fluorescence intensity of ·OH in cells of different treatment groups. Compared with control group, *P<0.05, **P<0.01; compared with model group, #P<0.05, ##P<0.01

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茶多糖的抗氧化活性及对细胞氧化损伤的保护机制

Antioxidant Activity of Tea Polysaccharide and Its Protective Mechanism Against Oxidative Damage

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