Theanine Improves High-Dose Epigallocatechin-3-Gallate-Induced Lifespan Reduction in Caenorhabditis elegans

Abstract

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea. Our previous report showed that induced hormesis was a critical determinant for the promotion of a healthy lifespan in Caenorhabditis elegans. In the present study, we investigated the anti-aging effects of the main active ingredients in green tea. We found that galloylated catechins (EGCG and epicatechin gallate) could extend the lifespan of C. elegans, while their metabolites (gallic acid, epicatechin, and epigallocatechin) could not. Interestingly, the combination with theanine, not caffeine, could alleviate the adverse effects induced by high-dose EGCG, including the promotion of lifespan and locomotor ability. This was due to the attenuation of the excess production of reactive oxygen species and the activation of DAF-16. These findings will facilitate further studies on the health benefits of tea active components and their interactions.

Keywords: DAF-16; EGCG; Reactive Oxygen Species (ROS); hormesis; theanine.

Figure 1

Survival of wild-type C. elegans treated with polyphenols. (A) GA, (B) EGC, (C) EC, (D) ECG, and (E) EGCG tested at different concentrations (200, 500, and 1000 μM) for their ability to change the lifespan of the worms. (F) Molecular formulae of GA, EGC, EC, ECG, and EGCG. Survival rates were recorded every other day until all worms died (n = 90–105 worms/treatment). Statistical analysis using the log-rank (Mantel–Cox) test showed that the changes in the survival curves of 500 and 1000 μM ECG compared with the control group were significant (p < 0.05).

Figure 2

Survival of wild-type C. elegans treated with EGCG (200 and 1000 μM), caffeine (CAF) (5 and 10 m M), theanine (THA) (200, 500, and 1000 μM), and glutamine (Gln) (1000 μM). (A,B) Survival curves with EGCG and caffeine; (C,D) survival curves with EGCG and theanine; (E) survival curves with EGCG and glutamine. (F) Theanine was tested at different concentrations to evaluate its ability to change the lifespan of the worms. Survival was recorded every other day, until all worms died (n = 90–105 worms/treatment).

Figure 3

Effects of EGCG and theanine on body length and the physical exercise ability of C. elegans. (A) Nematode body length was measured on the sixth day of adulthood. The data are presented as the average of 3 trials with a total of 90–105 nematodes per concentration. (B) Worms were treated with EGCG (1000 μM) and theanine (200 μM) starting from adulthood (day 0). The thrashing of treated and untreated nematodes was measured on the sixth day to the fourteenth day of adulthood. All error bars represent Standard Error of Mean (SEM), and differences were considered significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 4

Changes in Reactive Oxygen Species (ROS) levels induced by high doses of EGCG were eliminated by theanine. (A) Relative formation of ROS throughout the life cycle after exposure to EGCG (1000 μM), N-acetylcysteine (NAC) (5 mM), and theanine (200 μM) in the JV1 worms. (B) Relative formation of ROS after 72 h of exposure to EGCG, NAC, and theanine in the JV1 worms. (C) Survival curves with EGCG, NAC, and theanine. (D) SOD-3 mutant strains were treated with EGCG and theanine starting from adulthood (day 0). Survival was recorded every two days, until all of the worms died (n = 90–105 worms/treatment). (E) SOD-3 expression after 72 h exposure to EGCG and theanine, representative images are shown (×200 magnification). All error bars represent SEM, and differences were considered significant at ** p < 0.01 and *** p < 0.001, ns—no significance.

Figure 5

DAF-16 mediates theanine regulation of high-dose EGCG-induced lifespan shortening. (A,B) DAF-16 and DAF-2 mutant strains were treated with EGCG (1000 μM) and theanine (200 μM) starting from adulthood (day 0). Survival was recorded every two days, until all worms died (n = 95–105 worms/treatment). (C,D) EGCG-induced nuclear accumulation of DAF-16::GFP in N2 worms treated with EGCG and theanine. Representative images and the quantitation result are shown; the localization of DAF-16::GFP from day 4 to day 12 was compared using the Fisher exact test. All error bars represent SEM, and differences were considered significant at ** p <0.01 and *** p < 0.001.