20g of the millet grains were cleaned and washed with potable water. 100ml of potable water was added and cooked well. Cooked meal was cooled to room temperature and 150ml of water was added. The mixture was fermented for overnight and the gruel was analyzed for propionic acid content by high performance thin layer chromatography (HPTLC) technique. The 1.0-ml fermented gruels were centrifuged for 5 min at 5000g. The resulting supernatants were then transferred to an eppendorf tube to perform the identification process of acetic, propionic, lactic and butyric acids. Prior to use, the samples were filtered through 0.45μm filter.

A Camag HPTLC system equipped with an automatic TLC sampler, TLC scanner 3 and integrated software WINCATS version 1.4.1 was used for the analysis. Chromatrography was performed on 10 cm × 10 cm HPTLC plates coated with silica gel 60F 254 (E-Merck) of 200 μm layer thickness for the quantification of propionic acid. Standard and samples were applied to the plates as 8mm long bands, 8mm apart by use of a Camag Linomat(V) sample applicator equipped with a 10 µl microsyringe and an automatic TLC sampler under a flow of Nitrogen gas.

The linear ascending development was carried out in a Camag glass twin through chamber (20 cm × 10 cm) previously saturated with 20 mL mobile phase containing acetone: water: chloroform: ethanol: ammonium hydroxide (60:2:6:10:22) mixture (Lee et al., 2001) for 15 min at room temperature 25°C. Plates were developed to a distance of 80 mm. Subsequent to the development; the TLC plate was dried in a current of air with an air dryer. The TLC plates were then removed from the chamber and allowed to dry in air. The dried TLC plates were sprayed with the indicator solution, and the color was developed by brief heating (1–3 min.) in a hot dry oven 165°C. Quantitative evaluation of the plate was performed in absorbance-reflectance mode at λ_max =210 nm, using a slit width 6 × 0.4 mm, data resolution 100 mm Step - 1, scanning speed 20 mms^-1 and baseline correction was used.

The millets were cooked and cooled to room temperature. The cooked millets were added with 100 ml of potable water and fermented for overnight. The fermented gruels were extracted with Petroleum ether and Diethyl ether (1:1) and the solvents are evaporated and dried. The residues were reconstituted in DMSO for the in vitro anticancer assay.

MCF-7 human breast carcinoma cell line was purchased from National Center for Cell Sciences (NCCS), Pune. The cell lines were grown at 37 °C in a 5% CO₂, 95% air humidified atmosphere, in DMEM supplemented with 10% heat inactivated Fetal Bovine Serum (FBS) to which penicillin (100 U/mL) and streptomycin (100 µg g/ml) had been added.µ/mL) and amphotericin B (5 The growth-inhibitory effect of L-glutaminase (freeze dried dialysed fractions) on the cancer cell lines was determined using a colorimetric test, MTT assay.

Cells were seeded in 96-well plates at a density of 5x10³ cells per well in 100 µl of medium with FBS then cultured in a CO₂ incubator at 37 °C for 24 h. After which, 10 μl of fermented millet gruels (or 20 µl of DMEM medium with FBS for the control) was added to each well and the plate was incubated for 96 h. After incubation, 20 μl MTT stock solution (5 mg/ml in phosphate buffered saline or PBS, pH 7.5, filtered through 0.22-μm cellulose acetate filter; Sigma, St. Louis) was added to each well, incubated for 4 h at 37 °C then the solution was decanted. To stop succinate tetrazolium reductase activity and solubilise formazan crystals, 100 µL of propanol was then added to each well. Absorbance was read on a plate reader (VERS Amax, Molecular Devices, Saint Gregoire, France) at 540 nm. The percentage growth inhibition was calculated using the following formula and the concentration of enzyme extract needed to inhibit cell growth by 50% (CTC50) values is generated from the dose response curves for each cell line.

The fermented millet gruels showed different concentrations of short chain fatty acids such as acetic, propionic, butyric and lactic acids (Table 1). P. miliare fermented gruel showed the acetic acid in the levels of 0.89 µM, lactic acid as 1.76 µM, propionic acid in the levels of 1.82 µM and butyric acids in the levels of 10.58 µM. The fermented S.italica gruel showed acetic, lactic and butyric acid levels of 1.12 µM, 2.41 µM and 42.85 µM respectively. The fermented P. scrobiculatum gruel showed the acetic acid of 1.04 µM, lactic acid of 0.89 µM, proponic acid of 2.26 µM and butyric acid of 3.09 µM. The fermented E.frumantacea also showed high levels of propionic acid (8.93 µM) and 2.7 µM of acetic acid.

The effect of extracellular and intracellular extracts on the viability of cancer cells was determined by MTT (3[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay on HeLa cells. The most active extracts were further screened on MCF-7 cells by MTT assay. The viability of the MCF-7 cells were determined by exposing cells to various concentrations of the fermented millet gruel (0.31 mg /mL, 0.62 mg /mL, 1.25 mg/mL and 2.5 mg/mL) for 72 h. The viability % was defined as the concentration of drug at which there was 50% less growth compared to control cells. Each experiment was performed in triplicate and is shown in the Tab (Fig. 1 to 4).

Results indicated that the extracts of Setaria italica fermented gruel and Panicum miliare gruels had promising anticancer activity against Breast cancer (MCF-7) cell lines. The S.italica gruel at a concentration of 2.5 mg/mL showed only 15.54% of growth compared to control on MCF-7 cells by the MTT method. The S. itailica extract showed 55.08 % growth of MCF-7 cells at the initial concentration of 0.31mg/mL and it gradually reduced to 15.54% growth which is more potential concentration of the drug against the MCF-7 breast cancer cell lines.

Panicum miliare extract showed the inhibition of the growth of breast cancer cells at the concentration of 21.34% at the maximum of dosage of 2.5 mg/mL and the maximum viability of the cancer cells were observed at the concentration of 0.31mg/mL concentration of the millet gruel extract. The P. scrobiculatum showed only 67.69% of inhibition of the MCF-7 cell lines at the concentration of 0.31g/mL and it showed 37.28% growth at the maximum concentration of 2.5 g/mL, which is comparatively lower than the P. miliare and S. italica fermented gruel extracts.

The E. frumentace fermented gruel extract has not shown significant reduction in the growth of breast cancer cell line MCF-7 at the concentrations from 0.31mg/mL to 2.5 mg/mL. It showed the growth of the cancer cells from 59.32% to 60.31% which were significantly lower than the other fermented millet gruels. The S. italica showed more anticancer efficacy compared to other fermented gruels. It showed more butyric acid content of 42.85 µM. The high anticancer activity of S. italica fermented gruel may be the high contribution of butyric acid content.

In vivo studies have associated butyrate levels with a decreased incidence of colon cancer (Mcintyre et al. 1993; Okata et al. 1989), and butyrate instilled into the colonic lumen reduced tumor production in a chemical model of colon carcinogenesis (Medina et al. 1988). Although its precise mechanisms of action are not well understood, butyrate inhibits histone deacetylase (HDAC), resulting in a relative hyperacetylation of core histone proteins (H3 and H4) (Sealy & Chalkley, 1978).