Characterization of Theabrownins Prepared From Tea Polyphenols by Enzymatic and Chemical Oxidation and Their Inhibitory Effect on Colon Cancer Cells

Abstract

Theabrownins (TBs) are prepared from dark tea and contain a large number of complex heterogeneous components, such as carbohydrates, proteins, and flavonoids, which are difficult to remove. In addition, some toxic and harmful extraction solvents are used to purify TBs. These obstacles hinder the utilization and industrialization of TBs. In this study, tea polyphenols were used as substrates and polyphenol oxidase and sodium bicarbonate (NaHCO₃) were used successively to prepare theabrownins (TBs-E). The UV-visible characteristic absorption peaks of the TBs-E were located at 203 and 270 nm and Fourier-transform IR analysis showed that they were polymerized phenolic substances containing the hydroxy and carboxyl groups. The TBs-E aqueous solution was negatively charged and the absolute values of the zeta potential increased with increasing pH. A storage experiment showed that TBs-E were more stable at pH 7.0 and in low-temperature environments around 25°C. HT-29 human colon cancer cells were used to evaluate the biological activity of TBs-E through 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT), H&E staining, propidium iodide immunofluorescent staining, flow cytometry, and real-time PCR assays. The TBs-E significantly inhibited cell growth and caused late apoptosis, particularly at the dose of 500 μg/ml. The TBs-E markedly reduced the expression of antioxidant enzyme genes and increased the generation of reactive oxygen species to break the redox balance, which may have led to cell damage and death. These results will promote research and industrialization of TBs.

Keywords: characterization; colon cancer cell; enzyme; inhibition; theabrownins.

Figure 1

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to detect PPO in 2.00 mg/ml theabrownins (TBs-E) (5.00% concentrated gel, 12.00% separation gel, and 70 V constant-voltage electrophoresis). Among the lanes, lane 1 refers to polyphenol oxidase (PPO), lanes 2 and 3 refer to the TBs-E, and lane 4 refers to the marker proteins.

Figure 2

UV-vis spectra of 10.00 μg/ml TBS-E and 10.00 μg/ml tea polyphenols in the 200–700 nm scanning region (A), and the Fourier-transform infrared spectrum of TBs-E in the 500–4,000 cm⁻¹ scanning region (B).

Figure 3

Zeta potential curves of a 0.5 mg/ml TBs-E aqueous solution as a function of pH 3.0–9.0 adjusted using HCl and NaOH at an interval of 1.0 and a temperature of 25°C.

Figure 4

Scanning electron microscopy (SEM) micrographs of TBs-E; (A) zoom in 1,000X (10 μm), (B) zoom in 500X (50 μm), (C) zoom in 300X (50 μm), and (D) zoom in 100X (100 μm).

Figure 5

Effects of different pHs and temperatures on the stability of TBs-E. (A) Change in the TBs-E absorbance curves in pH 3.0–9.0 solutions and a purified aqueous solution (AS) after 5 days of storage, (B) the color of newly prepared TBs-E in different pH and ultrapure aqueous solutions, and (C) change in the absorbance curves of TBs-E aqueous solutions stored at 25, 50, and 90°C for 8 h.

Figure 6

The inhibitory effect on HT-29 cell proliferation.

Figure 7

Effect of TBs-E on HT-29 cell morphology (200X); (A) control (24 h), (B) control (48 h), (C) control (72 h), (D) TBs-E treatment (24 h), (E) TBs-E treatment (48 h), and (F) TBs-E treatment (72 h).

Figure 8

Effect of TBs-E on cell apoptosis (200X); (A) control, (B) TBs-E treatment (24 h), (C) TBs-E treatment (48 h), and (D) TBs-E treatment (72 h).

Figure 9

Determination of the apoptotic rate by flow cytometry; (A) PI-annexin V flow cytometry to detect cell apoptosis and (B) statistical analysis of cell apoptosis.

Figure 10

Effects of TBs-E on the expression of the antioxidant enzyme genes in HT-29 cells and immunofluorescence assay to determine ROS in HT-29 cells after the TBs-E treatment (200X). HT-29 cells were treated with 500 μg/ml of TBs-E for 24, 48, or 72 h. After extracting the DNA and synthesizing the cDNA, the final PCR products were subjected to agarose gel electrophoresis (A). The density of the bands was scanned and analyzed by Quantity One software (B–D). *P < 0.05, **P < 0.01, compared to the control group. (E) Control, (F) TBs-E treatment (48 h).