The TFC of each sample was determined spectrophotometrically.
Briefly, 0.25 ml sample was mixed with 75 àl of 5% sodium nitrite(NaNO2) solution, 0.15 ml of freshly prepared 10% aluminium chloride (AlCl3) solution and 0.5 ml of 1 M sodium hydroxide solution (NaOH). The final volume of the mixture was adjusted to 2.5 ml with deionized water. The mixture was allowed to stand for 5 min at room temperature. The absorbance was measured at 510 nm. Catechin acid standards in the range 0 - 100 ppm were treated in a similar manner to generate a calibration curve (r2 = 0.9975) to calculate TFC. The TFC of the extracts were expressed as catechin acid equivalent (CE) g/100 g fresh weight and dry weight (Sun et al. 2007; Vale et al. 2014). All determinations were performed in triplicate (n = 3).
3.2.5. Determination of vitamin C contents
Vitamin C estimation was determined by the Folin–Ciocalteu reagent method by Jagota (Jagota and Dani 1982), with modifications. Samples (0.5 ml) were added to 0.8 ml of 10% trichloroacetic acid and vigorously shaken
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and the mixtures were kept on ice for 5 min and then centrifuged at 3000 g for 5 min. This extract (0.2 ml) was then diluted to 2 ml with distilled water.
Commercially prepared 2.0 M Flolin–Ciocalteu was diluted 10-fold with distilled water and 0.2 ml of this diluted reagent was added to the mixture and vigorously shaken. After 10 min at room temperature, the absorbance was measured at 760 nm. Ascorbic acid standards in the range 0 - 100 ppm were treated in a similar manner to generate a calibration curve (r2 = 0.9953) to calculate vitamins C content. The vitamins C content was expressed as ascorbic acid equivalent (AA) g/100 g fresh weight and dry weight (Anwar et al. 2013). All determinations were performed in triplicate (n = 3).
3.2.6. DPPH radical scavenging assay
DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging activity of broccoli samples were studied by the methods of Sharma (Sharma and Bhat 2009) and followed with modifications. During this experiment, the test samples of ethanol and water extracts of broccoli (5 mL) accompanied with standard compounds ascorbic acid or BHA methanolic solutions (5 mL) were separately mixed with freshly prepared DPPH methanolic solution (1 mM, 1 mL). The reaction mixture was then vortex for few minutes and allowed to stand at dark place at 25°C for 60 min. The absorbance of samples was read against a blank at 517 nm. The lower the absorbance, at 517 nm, the higher was DPPH scavenging activity. The percentage of the DPPH scavenging activity was expressed by the following formula [1 - (Abs. of sample – Abs.
of blank) / (Abs. of control)] x 100% (Vale et al. 2014). All determinations were performed in triplicate (n = 3).
3.2.7. ABTS radical cation decolorization assay
ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic) was dissolved in water to make a concentration of 7 mmol/l. ABTS.+ was produced by reacting the ABTS stock solution with 2.45 mmol/l potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. For the study of samples, the ABTS.+
stock solution was diluted with phosphate-buffered saline (PBS) 5 mmol/l, pH
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7.4 to an absorbance of 0.70 (±0.02) at 734 nmand equilibrated at 30°C for 30 min. After the addition of 1.0 ml of diluted ABTS.+ to 10 àl of sample, the absorbance reading was taken 5 min after the initial mixing. Decolorization of the assay was linear with increasing Trolox concentrations.Trolox standards in the range 0 - 100 ppm were treated in a similar manner to generate a calibration curve (r2 = 0.9958) to calculate vitamins C content. The activity is expressed as the Trolox-equivalent antioxidant capacity of the extract (àmol TE/100 g fresh weight and dry weight) (Re et al. 1999; Erel 2004). All determinations were performed in triplicate (n = 3).
3.2.8. Reducing power assay
The reducing powers of samples were determined following the methods developed by Oyaizu (1986) with modifications. Test samples of broccoli extracts (10 mL) accompanied with ascorbic acid or BHA methanolic solutions (10 mL) were spiked separately with phosphate buffer (2.5 mL, 0.2M, pH 6.6) and 1% potassium ferricyanide (2.5 mL). The mixture was then incubated at 50°C for 20 minutes, then cooled rapidly, again spiked with trichloroacetic acid (2.5 mL, 10%), and centrifuged at 3000 rpm for 10 minutes. The supernatant (5 mL) was then mixed with distilled water (5 mL) and ferric chloride (1 mL, 0.1%). The mixture was then allowed to stand for 10 minutes and the absorbance was recorded at 700 nm. A stronger absorbance indicates increased reducing power (Re et al. 1999; Jahan et al.
2010). All determinations were performed in triplicate (n = 3).
3.2.9. Cell lines and culture
The mouse liver normal cell line (FL83B) and the human lung tumor cell line (A549) grown in F-12 medium; the human liver tumor cell line (HepG2) cultured in DMEM medium, used in the present investigation. The mediums were supplemented with FBS (10%), penicillin (68 mg/l), streptomycin (100 mg/l), gentamicin (200 mg/l), NaHCO3 (2000 mg/l) and cells were maintained in CO2 incubator at 37oC with 90% humidity and 5%
CO2. Subcultures were performed with 1.5 mL of 1x trypsin in phosphate buffered saline (PBS). The morphology of cells was observed using an
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inverted microscope CK30-F100. The cells were treated with extracts dissolved in DMSO (2%) while the untreated control cultures received only DMSO. The sample extracts were dissolved in DMSO before being diluted with mediums and 10% FBS. The solution was then filtered and sterilised, using a 0.2 mM syringe filter, to prepare the working solution (Akanitapichat et al. 2010; Chaudhary et al. 2014).
3.2.10. Cell viability assay
The 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was performed following the well-described procedure with minor modifications (Mossman, 1983). Briefly, cell pellet was resuspended in complete growth medium to get 1 x 105 cells/ml and 100 àL of cell suspension per well was seeded in tissue culture plate. The cells were treated with different concentrations of test material and incubated for 24 h in CO2 incubator (37oC, 5% CO2 and 90% RH). Thereafter, 20 àL of freshly prepared MTT solution (5 mg/ml in PBS, sterile filtered) and 100 àL medium were added to each well and culture plates gently stirred at 150 rpm for 5 min, which further incubated for 4 h at 37oC to allow metabolization of MTT and MTT-formazan crystals (MTT metabolic product) were resuspended in 100 àl of DMSO. In order to dissolve formazan crystals plates were stir for 15 min and absorbance was measure at 570 nm. Cell viability was calculated as:
(absorbance of treatment/absorbance of control) × 100%. An IC50 value denoted the concentration of sample that show 50% inhibition of proliferation on any tested cell line, was calculated by CalcuSyn software (Akhlaghi and Bandy 2010; Dalar et al. 2014; Chaudhary et al. 2012). All determinations were performed in triplicate (n = 3).
3.2.11. Statistical analysis
For statistical studies SPSS 17.0 software (SPSS Inc. Chicago, IL) was used. Data were expressed as means ± SD of three independent replicates (n=3) and were studied using a one-way analysis of variance (ANOVA).
When ANOVA detected significant differences between mean values, means were compared using Duncan‟s test at value of p < 0.05 significance level.