Effect of Brewing Duration on the Antioxidant and Hepatoprotective Abilities of Tea Phenolic and Alkaloid Compounds in a t-BHP Oxidative Stress-Induced Rat Hepatocyte Model

Abstract

Tea is an interesting source of antioxidants capable of counteracting the oxidative stress implicated in liver diseases. We investigated the impact of antioxidant molecules provided by a mixture of teas' leaves (green, oolong, pu-erh) after different infusion durations in the prevention of oxidative stress in isolated rat hepatocytes, by comparison with pure epigallocatechin-3-gallate (EGCG), the main representative of tea catechins. Dried aqueous tea extracts (ATE) obtained after 5, 15 and 30 min infusion time were characterized for total polyphenols (gallic acid equivalent), catechins, gallic acid and caffeine (HPLC-DAD/ESI-MS) contents, and for scavenging ability against 2,2-diphenyl-1-picrylhydrazyl free radical. Hepatoprotection was evaluated through hepatocyte viability tests using tert-butyl hydroperoxide as a stress inducer, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, neutral red uptake, real-time cellular impedance) and mitochondrial function tests. We showed that a 5-min incubation time is sufficient for an optimal bioaccessibility of tea compounds with the highest antioxidative ability, which decreases for longer durations. A 4-h pretreatment of cells with ATE significantly prevented cell death by regulating reactive oxygen species production and maintaining mitochondrial integrity. Pure EGCG, at doses similar in ATE (5-12 µM), was inefficient, suggesting a plausible synergy of several water-soluble tea compounds to explain the ATE beneficial effects.

Keywords: Camellia sinensis; EGCG; ROS; antioxidant; bioaccessibility; hepatocytes; mitochondrial membrane integrity; polyphenols; tea.

Figure 1

HPLC-DAD/ESI-MS chromatogram of tea extracts ATE-5 (A), ATE-15 (B) and ATE-30 (C) at 280 nm (retention time/molecular weight). The numbered peaks are denoted in Table 2.

Figure 2

Antioxidant activity of tea extracts ATE-5, ATE-15 and ATE-30 using DPPH scavenging activity. The DPPH radical-scavenging activity was calculated as percent inhibition according to the following equation: % Inhibition = [(Absorbance_control − Absorbance_sample)]/(Absorbance_control)] × 100. Results are means ± SEM (n = 3). For a given concentration, means with different superscript letters (a, b) are significantly different (p < 0.05).

Figure 3

Effect of ATE or EGCG on the viability of rat hepatocytes. Cell viability was assessed by the MTT (A) and neutral red uptake (NRU) test (B) after 4 h pretreatment with 25, 100 and 500 µg/mL tea extracts or 5, 10 and 12 µM EGCG followed by 1 h exposure to 150 µM t-BHP. MTT and NRU results are presented as % viability over non-treated cells (mean ± SEM of three separate experiments; note: * p < 0.05 when compared to non-treated cells, ^# p < 0.05 when compared to non-pretreated + t-BHP treated cells; − indicates the absence of t-BHP or ATE, and + indicates the presence of t-BHP, in the culture medium). For cell impedance tests (C–F), cells were pretreated for 4 h with 25, 100 and 500 µg/mL of tea extracts or 5, 10 and 12 µM EGCG, and incubated for 44 h with t-BHP at 150 µM. Cell impedance was measured in real-time (RTCI) and cell index was normalized prior the addition of t-BHP. Results are means ± SEM for triplicates of one experiment and are representative of three independent experiments (note: * p < 0.05 when compared to non-treated cells, ^# p < 0.05 when compared to non-pretreated + t-BHP treated cells).

Figure 4

Effect of ATE or EGCG on mitochondrial superoxide anion (O₂⁻) production and on mitochondrial functionality. Cells were seeded onto 96-well E-plates, pretreated for 4 h with various concentrations of tea extracts (25, 100 and 500 µg/mL) or EGCG (5, 10 and 12 µM), then treated for 1 h with t-BHP at 150 µM. Cells were labeled with Hoechst 33342 and MitoSox (A–C) or TMRE (D,E) for 30 min before being read on the ArrayScanXTI. Mitochondrial superoxide (O₂⁻) content (A,B) was calculated from the MitoSox fluorescence intensity following the “compartmental analysis protocol”. Mitochondrial functionality (D) was calculated from the TMRE fluorescence intensity following the “compartmental analysis protocol”. Error bars indicate the mean ± SEM of triplicate determinations in three independent experiments (note: * p < 0.05 when compared to non-treated cells, ^# p < 0.05 when compared to non-pretreated + t-BHP treated cells). Objective magnification ×20. General note: − indicates the absence of t-BHP or ATE, and + indicates the presence of t-BHP, in the culture medium.