# Thai Rice Vinegars: Production and Biological Properties
## 3 Results and Discussion
### Biological Properties of Rice Vinegars
#### 3.3.3 Cytotoxic Activity of Rice Vinegars  
The cytotoxic activities of rice vinegars against the human colon cancer cell line (HT-29) and mouse fibroblast normal cell line (L929) were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzolium bromide (MTT) assay. The MTT was reduced in mitochondria via succinate dehydrogenase to formazan products during incubation with the cell line [33]. The level of the reduced MTT form (formazan) was determined as the amount of cell line survival. The cytotoxicity was determined by the National Cancer Institute (NCI) as the 50% inhibitory concentration (IC\({}_{50}\)); an IC\({}_{50}\)\(\leq\) 30 \(\upmu\)g/mL represent an active level. However, [PERSON] et al., (1972) determined the level of cytotoxicity as IC\({}_{50}\)\(\leq\) 20 \(\upmu\)g/mL (highly active), IC\({}_{50}\) 21-200 \(\upmu\)g/mL (moderately active), IC\({}_{50}\) 201-500 \(\upmu\)g/mL (weakly active) and IC\({}_{50}\)\(>\) 501 \(\upmu\)g/mL (inactive) [34]. The cytotoxicities of the Thai rice vinegars are shown in Table 3. Black glutinous rice vinegar exhibited the highest cytotoxicity toward HT-29 cells with an IC\({}_{50}\) value of 74.02 \(\upmu\)g/mL. Weak activity of the vinegar against L929 cells was detected, showing an IC\({}_{50}\) value of 171.06 \(\upmu\)g/mL. Black fragment, glutinous and polished rice vinegar also showed cytotoxicity against HT-29 cells, with IC\({}_{50}\) values of 81.66, 90.17 and 110.92 \(\upmu\)g/mL, respectively. These results indicated that Thai rice vinegars contained cytotoxic activity against colon cancer cell lines and had a diminished effect on normal cell lines. The mouse fibroblast normal cell line (L929) cell line was used as a normal cell control, as recommended by the international standard for the testing of medical device, which demonstrates a more sensitive response than primary cell [18]. Several studies have reported the inhibitory effects of Kurosu (unpolished rice vinegar) extract on the growth of many cancer cell lines, such as colon and breast adenocarcinoma, lung, bladder and prostate carcinoma [35]. Previously, [PERSON] et al., (2003) reported that ethylacetate extract of Kurosu inhibited colonic aberrant crypt foci. Kurosu also showed an inhibitory effect toward colon adenocarcinoma growth induced by azoxymethane in male rats [36]. In addition, Izumi, the Japanese black vinegar made from unpolished rice, inhibited the proliferation of a human squamous cell carcinoma cell line [37]. These anticancer activities suggested that the phenolic compounds in rice bran and germ, such as ferulic acid and its derivatives contained in unpolished rice, are the major substances inhibiting cancer cell line growth [38]. [PERSON] et al. (2010) reported that an anthocyanin-rich extract from black rice exhibited cytotoxic effects on three human breast cancer cell lines, with IC\({}_{50}\) values of 374.7, 209.9 and 179.5 \(\upmu\)g/mL. They suggested that anthocyanin affected human breast cancer cells in vitro and in vivo by inducing apoptosis and suppressing angiogenesis [39]. Recently, sticky purple rice extract was found to prevent aberrant crypt focus (ACF) formation (the initial stage of colon carcinogenesis) in dimethylhydrazine (DMH)-induced rats [40]. Moreover, a red color strain of unpolished Thai rice (_Oryza sativa_ L.) extract was investigated for its anticancer activity against ACF formation in azoxymethane (AOM)-treated rats and efficacy for apoptotic induction and oxidative redox status in human colon cancer (Caco-2) cells. They found that feeding unpolished Thai rice extract might prevent ACF formation by inhibiting precancerous progression in AOM-treated rats. They also found that unpolished Thai rice extract significantly induced cancer cell apoptosis and increased cellular oxidants in human colon cancer cells, with an IC\({}_{50}\) of 5.87 mg/mL with the MTT assay at 24 h [41]. Therefore, phenolic compounds in rice grains such as phytochemicals and plant pigments are associated with anticancer activity.