Folin-Ciocalteus's phenol reagent, sodium carbonate, gallic acid (GA), quercetin (QE), FeCl₃, NaNO₂, 1,1-Diphenyl-2-picrylhydrazyl (DPPH), ascorbic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Sodium nitro preside (SNP), α-napthyl-ethylenediamine, potassium ferricyanide, trichloroacetic acid (TCA), ammonium molybdate, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulphoxide (DMSO) were purchased from Merck Chemical Supplies (Damstadt, Germany). All the chemicals used including the solvents, were of analytical grade.

Seeds of Njavara variety were obtained from Kerala Agricultural University, Kerala, Vasumathi, Yamini and Jyothi varieties were obtained from Directorate of Rice Research, Hyderabad; Central Soil Salinity Research Institute, Karnal and National Seed Corporation, Warangal respectively.

Rice bran from the four varieties was obtained by milling rice grain in a local grinding mill, followed by sieving to separate grain from bran. Stabilization of rice bran was done by heating the bran in microwave oven at 850W and with 2450 MHz. The microwave chamber was preheated to 100% power for 3 min. The moisture content of the raw rice bran was adjusted to 21% by adding deionized water 19. The sample was heated for 3 min at 100% power. The temperature of the sample after heating in the microwave was 107 ± 2°C. The sample was allowed to cool to room temperature and stored in an ultralow freezer (-80°C) until further analysis.

Rice bran extract was prepared according to a modified method of Choi et al., 20. Rice bran powder (5 g) was extracted thrice with 30 ml methanol for 3 h in an electrical shaker at 40°C. The extracts were filtered through Whatman No.2 filter paper and evaporated under vaccum using a rotary evaporator (Heidolph, Germany). The residual crude methanolic rice bran extract was weighed and dissolved in dimethyl sulphoxide (DMSO), and filtered through a 0.45 μm of Nylon membrane filter and stored at -20°C until further analysis.

The total phenolic content of the bran extracts was determined using the Folin- Ciocalteu reagent 21. The reaction mixture contained: 200 μl of diluted rice bran extract, 800 μl of freshly prepared diluted Folin Ciocalteu reagent and 2 ml of 7.5% sodium carbonate. The final mixture was diluted to 7 ml with deionized water. Mixtures were kept in dark at ambient conditions for 2 h to complete the reaction. The absorbance at 765 nm was measured. Gallic acid was used as standard and the results were expressed as mg gallic acid (GAE)/g bran.

Total flavonoid content was determined using aluminium chloride (AlCl₃) according to a known method 22 using quercetin as a standard. The plant extract (0.1 ml) was added to 0.3 ml distilled water followed by 5% NaNO₂ (0.03 ml). After 5 min at 25°C, AlCl₃ (0.03 ml, 10%) was added. After further 5 min, the reaction mixture was treated with 0.2 ml of 1 mM NaOH. Finally, the reaction mixture was diluted to 1 ml with water and the absorbance was measured at 510 nm. The results were expressed as mg quercetin (QE)/g bran.

The reducing power of rice bran extract was measured according to the method described by Yen and Duh 23 with some modifications. Various concentrations (100, 200, 300, 400 and 500 μg) of rice bran extract were mixed with 2.5 ml of 0.2 M sodium phosphate buffer (pH 6.6). The dilute sample was then mixed with 5.0 ml of 1% potassium ferricyanide and the mixture was incubated at 50°C for 20 min. 5.0 ml of 10% trichloroacetic acid was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. 5.0 ml of the supernatant was mixed with 5.0 ml of distilled water and 1.0 ml of ferric chloride (1%). The absorbance was measured at 700 nm. Ascorbic acid was used for comparison.

For total antioxidant assay various concentrations (20, 40, 60, 80 and 100 μg) of rice bran extract were mixed with 1 ml of the reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The reaction mixture was incubated in a water bath at 95°C for 90 min. After cooling to room temperature, the absorbance was measured at 695 nm 24. Ascorbic acid was used for comparison.

Nitric oxide scavenging activity was determined according to Griess Illosvoy reaction 25. The reaction mixture contained: 10 mM SNP in 0.5 M phosphate buffer, pH 7.4, and various doses (50-250 μg/ml) of the test solution in a final volume of 3 ml. After incubation for 60 min at 37°C, Griess reagent (α-napthyl-ethylenediamine 0.1% in water and sulphanilic acid 1% in H₃PO₄ 5%) was added. The pink chromophore generated during diazotization of nitrite ions with sulphanilamide and subsequent coupling with α-napthyl-ethylenediamine was measured spectrophotometrically at 540 nm. Ascorbic acid was used as a positive control. Nitric oxide scavenging ability (%) was calculated by using the formula:

DPPH radical scavenging activity of rice bran extract was determined according to the method reported by Blois 26 with slight modifications. An aliquot of 0.5 ml of sample solution in methanol was mixed with 2.5 ml of 0.5 mM methanolic solution of DPPH. The mixture was shaken vigorously and incubated for 37 min in the dark at room temperature. The absorbance was measured at 517 nm using UV-vis spectrophotometer. Ascorbic acid was used as a positive control. DPPH free radical scavenging ability (%) was calculated by using the formula:

The cell cytotoxicity of methanolic rice bran extracts against the C6 glioma cells was determined by MTT assay 27. Cells were seeded into 96-well plate at 1 × 10⁴ cells/well density and treated with different concentrations of rice bran extracts for 48 h. After 48 h MTT was added to each well and solubilized the formazan crystals by DMSO. Then absorbance was measured at 570 nm in a microplate ELISA reader. All experiments were performed in triplicate.

The data were subjected to correlation coefficient by using Sigmastat version 3.1 statistical analysis software. The correlation of the data was determined by Pearson's test and Stata statistical package was used for regression analysis. P < 0.05 was considered as statistically significant.

The total phenolic content (TPC) was expressed as gallic acid equivalents (Table 1). Significant differences were observed for TPC among the four rice varieties. TPC was in the range of 3.27-12.4 mg GAE/g bran. Highest TPC was observed in Njavara followed by Jyothi. Yamini and Vasumathi showed nearly equal TPC content which were in low quantities compared to Njavara.

Total flavonoid content of the methanolic extract was significantly high in Njavara compared to the other three varieties as recorded in quercetin equivalents (Table 1). TFC of Vasumathi, Yamini, Jyothi, and Njavara were 1.68, 2.57, 5.33 and 8.5 mg QEE/g bran respectively.

Total antioxidant activity of the rice bran extracts increased with the increasing concentration of the extracts and a significant change was observed at 0.02 to 0.1 mg/ml concentration of the extract (Figure 1). At 0.1 mg/ml of the methanolic rice bran extracts, the absorbance values of Vasumathi, Yamini, Jyothi and Njavara were 1.65, 2.21, 2.52 and 2.93 respectively. However, the total antioxidant activities of all the rice bran extracts were less than that of the positive control ascorbate (2.5-40 μg showed an absorbance 0.117-2.0).

The methanolic extracts from all the samples had shown considerable amount of reducing activity. The reducing power of the rice bran extracts increased with the increasing concentration and a significant change was observed at 0.1 to 0.5 mg/ml concentration of the rice extract (Figure 2). 0.5 mg/ml of the methanolic rice bran extracts showed absorbance values of 0.59, 1.04, 1.93 and 2.98 corresponding to Vasumathi, Yamini, Jyothi and Njavara were respectively. While 5-25 μg of the positive control ascorbate showed an absorbance 0.06-0.28.

Free radical scavenging activities of the rice bran methanolic extracts were assessed by the DPPH assay. Figure 3 illustrates a significant decrease in the concentration of DPPH radical due to scavenging ability of the rice bran. The results show that Njavara had the highest DPPH scavenging activity with an IC₅₀ value of 30.85 μg/ml. IC₅₀ values of the other three varieties were 48.88, 70.58 and 87.72 μg/ml for Jyothi, Yamini and Vasumathi respectively. IC₅₀ value of the positive control ascorbic acid was 3.2 μg/ml.

Njavara extract showed the highest nitric-oxide scavenging activity compared to the other three rice bran methanolic extracts in a moderate dose dependent inhibition of nitric oxide with an IC₅₀ value of 52.25 μg/ml. IC₅₀ values of Jyothi, Yamini and Vasumathi were 71.41, 107.18 and 102.48 μg/ml respectively (Figure 4). Ascorbic acid was used as a reference compound and its IC₅₀ value is 4.6 μg/ml.

The cytotoxic activity of the methanolic extracts from rice bran against C6 glioma cells were shown in figure 5. IC₅₀ values of the methanolic rice bran extracts of Vasumathi, Yamini, Jyothi, and Njavara were 57.78, 44.83, 25.52 and 17.53 μg/ml respectively.

Table 2 shows highly positive significant correlation coefficient values for phenolic content with DPPH and NO scavenging; TAA and RP. A negative significant correlation coefficient was observed for phenolic content with MTT in the four rice varieties. Table 3 shows the regression analysis for the four rice varieties. The slope of Vasumathi showed higher significant value for phenolic content with DPPH scavenging (6.44) and TAA (0.087) compared to the other varieties. It also showed a higher slope (3.39) with NO but less significant over Jyothi. Yamini had a higher significant slope (0.012) for phenolic content with RP. Njavara had higher negative significant slope (-0.017) for phenolic content with MTT.

Regression analysis of phenolic content with DPPH and NO scavenging, TAA and RP showed higher slope values in Vasumathi compared to the Njavara. Higher slope values in Vasumathi can be explained by the higher percentage of growth rate compared to Njavara. Minimum IC₅₀ values of Njavara has been predicted to be responsible for the lesser growth rate compared to Vasumathi resulting lesser slope values. Correlation coefficient and regression analysis showed that total phenolic content was responsible for antiradical efficiency in the rice bran extracts.