*3.6. Cytotoxicity Test of Kombucha Tea Against Caco-2 and NIH*/*3T3 Cells*

The inhibition of the kombucha in different types of tea leaves was investigated against Caco-2 cancer cells and NIH/3T3 cells by MTT assay (Table 3). Kombucha tea prepared from green, oolong and black teas at 15 days of fermentation inhibited Caco-2 cancer cells with 50% inhibitory concentrations (IC50) of 2.603%, 1.899%, and 6.077%, respectively. Moreover, Kombucha from green, oolong and black teas inhibited NIH/3T3 cells with 50% inhibitory concentrations (IC50) of 2.851%, 1.922%, and 6.697%, respectively.



<sup>a</sup> Results are presented as mean ± SD of three independent experiments. <sup>b</sup> Fermented tea: kombucha tea fermented at 15 days without any treatment. <sup>c</sup> Unfermented tea: broth culture contained only 1% of tea leaves. <sup>d</sup> Neutralized kombucha: kombucha tea neutralized with NaOH (1 M) at pH 7.0. <sup>e</sup> Heat-denatured kombucha M1: kombucha tea treated at 100 ◦C for 20 min. <sup>f</sup> Heat-denatured kombucha M2: kombucha tea treated at 121.5 ◦C for 15 min. <sup>g</sup> Acetic acid: acid prepared at the same total acidity content of kombucha for each type of tea leaf. \* The values were significantly different between the cytotoxicity of Caco-2 cells and NIH/3T3 cells (*p* < 0.05).

The effect of kombucha tea on cancer cells showed cytotoxicity values higher than cytotoxicity on normal cells. However, kombucha tea prepared from green tea and black tea significantly showed toxicity against cancer cells when compared to normal cells. Moreover, cytotoxicity of kombucha prepared from green tea and black tea after boiling (100 ◦C for 20 min) also showed significantly toxicity against cancer cells.

Moreover, all unfermented tea containing 1% (*w*/*v*) of tea leaf-infusion also displayed toxicity toward Caco-2 cancer cells. In contrast, kombucha that was neutralized with 1 M NaOH at a pH value of 7.0 revealed levels of toxicity on Caco-2 cells that were lower than those in the heat-denatured kombucha. Acetic acid was used for acidic activity control and was prepared with the same total acidity content of kombucha for each type of tea. After treatment of Caco-2 cancer cells with acetic acid, IC50 values of the kombucha prepared from green, oolong, and black teas were 33.155%, 12.610%, and 8.720%, respectively. Thus, the toxicity of the acetic acid on cancer cells was lower than in the fermented kombucha that had been prepared from each type of tea leaves.
