Human neuroblastoma SH-SY5Y cell line was obtained from ATCC (Manassas, VA, USA). Minimum essential Eagle’s medium, Ham’s nutrient mixture F-12, fetal bovine serum, gentamicin, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and all other chemicals were obtained from Sigma (St. Louis, MO, USA).
3.2. Production of GBR
Brown rice (2 kg) was rinsed 5 times with tap water, out of which 500 g was soaked in sodium hypocloride (19 mL; 5.25%) solution for 30 min followed by washing with tap water for at least 1 min and soaking in H2O2 (20 mL; 25%) solution for 6 h at 35 °C in an incubator. After filtration, obtained BR sample was placed in a closed container (15 × 8 × 4 cm) at 35 °C for 18 h. About 10% of the total sample was removed for estimating the efficiency of germination. Rest of the GBR was transferred onto a tray and was kept in an oven at 50 °C, until the moisture content reached 8%–13%. Weight of dried GBR was recorded, and samples were packed in zipped bags and stored in chiller till further analyses.
3.3. Extraction of GBR and BR
Ground samples (100 g each of GBR and BR) were homogenized with ethanol (400 mL; 70%) for 30 min and centrifuged at 34,800 g for 20 min. The supernatant was filtered through Whattman No. 1 filter paper. The process was repeated twice further to ensure maximum extraction, supernatant of all the three batches was pooled and dried using rotatory evaporator (Rotavapor® R-210, BUCHI, Flawil, Switzerland) to concentrate the extract.
3.4. Determination of GABA Content in GBR and BR
GABA content in GBR and BR was determined using Liquid Chromatograph-Mass Spectrometer (LCMS) (Waters 2695, Wilford, MA, USA). Separation was carried out on C18 column (250 cm × 4.6 cm × 5 μm). Buffer A (0.1 M ammonium acetate; for 50 min) and buffer B (0.1 M ammonium acetate: acetonitrile: methanol) (44:46:10) were used as the mobile phases. Flow rate was maintained at 1.0 mL/min.
For analysis, GBR and BR extracts (50 μL each) were aliquoted into test tubes predried under vacuum. A 20 μL mixture of methanol: water: triethylamine (TEA) (2:2:1) was then added to these tubes, mixed well and dried under vacuum. Phenyl isothiocyanate (PITC) reagent (30 μL) composed of methanol, PITC, TEA and water mixed in a ratio of 7:1:1:1 was added to these tubes and contents were again dried under vacuum. Ammonium acetate buffer (500 μL; 0.1 M) was then added to each tube and resulting mixture was filtered into the vials using 0.45 μm syringe filter.
3.5. Cell Culture
The human neuroblastoma SH-SY5Y cells were grown in complete culture medium containing mixture (1:1) of Minimum essential Eagle’s medium and Ham’s nutrient mixture F-12, which was supplemented with 10% fetal bovine serum, 1% MEM non-essential amino acids and 50 μg/mL gentamicin. Cells were maintained at 37 °C under 5% CO2/95% air. Dimethyl sulfoxide (DMSO) concentration was maintained at 0.1% for all cell culture assays.
3.6. MTT Assay
The ability of GBR, BR and GABA to protect SH-SY5Y cells from H2O2 was determined by MTT assay, which is a potential indicator of cells viability. SH-SY5Y cells were seeded into 96-well culture plates at density of 1 × 105 cells/mL and were allowed to attach. After 24 h, cells were differentiated with retinoic acid (10 μM) for 6 days prior to treatment. To examine the possible toxic effects, the cells were treated with GBR, BR and GABA individually over a concentration range of 0.25–200 μg/mL for 24 h. Similarly, cells were treated with H2O2 over concentration range of 50–400 μM up to 24 h. For the determination of neuroprotective effects, cells were pretreated with GBR, BR and GABA diluted in serum-free medium for 24 h and then challenged with H2O2 for another 24 h. GBR, BR and GABA were dissolved in DMSO which was maintained at 0.1% as this concentration showed no toxicity to the cells. MTT (Sigma, St. Louis, MO, USA) was added to all the wells and allowed to incubate in dark at 37 °C for 4 h. The amount of MTT formazan product was determined by measuring absorbance at 540 nm using a Microplate reader (Opsys MR, Thermo Labsystems, Franklin, MA, USA). All the MTT assays were performed in triplicate.
3.7. Cell Cycle Analyses
SH-SY5Y cells were seeded in 96-well plates at density of 1 × 105 cells/mL. The cells were differentiated with 10 μM retinoic acid after 6 days. The cells were pretreated with each of the GBR, BR and GABA individually at 100 μg/mL for 24 h before exposing to 250 μM H2O2 for another 24 h. The cells were harvested using 0.1% trypsin-EDTA, fixed in 70% ethanol and kept at −20 °C overnight. After fixation, the pellets were washed with PBS to remove ethanol and mixed with 25 μL of RNAs, 50 μL of propidium iodide and 425 μL of PBS to make up the volume up to 500 μL. After 30 min of incubation in the dark, the DNA contents of the cells were analyzed using flow cytometer with Summit v4.3 software (Cyan ADP, Beckman Coulter, Brea, CA, USA).
3.8. Measurement of Mitochondrial Membrane Potential (MMP)
SH-SY5Y cells were seeded in 96-well plates at density of 1 × 105 cells/mL. The cells were differentiated with 10 μM retinoic acid for 6 days and then pretreated with GBR and GABA at 10, 50 and 100 μg/mL for 24 h before being exposed to 250 μM H2O2 for another 24 h. Mitochondrial membrane was monitored using the fluorescent dye Rhodamine 123, which preferentially partitions active mitochondria based on the highly negative MMP. Depolarization of MMP results in the loss of Rhodamine 123 from mitochondria and a decrease in intracellular fluorescence is observed. Rhodamine 123 (final concentration of 10 μM) was added to the harvested cells and analyzed using flow cytometer with Summit software (Version 4.3; CyAN ADP, Beckman Coulter: Brea, CA, USA).
3.9. Annexin V-FITC and Propidium Iodide Staining Assay
To detect the effects of GBR, BR and GABA on early and late apoptosis/necrosis induced by H2O2, SH-SY5Y cells were stained with FITC-conjugated Annexin V and propidium iodide. SH-SY5Y cells were seeded in 96-well plates at density of 1 × 105 cells/mL. The cells were differentiated with 10 μM retinoic acid for 6 days and pretreated with GBR at 1, 50 and 100 μg/mL for 24 h before being exposed to 250 μM H2O2 for another 24 h. The cells were then harvested using 0.1% trypsin-EDTA and cell pellets were resuspended in ice-cold 1X binding buffer. One microliter of Annexin V-FITC solution and 5 μL of propidium iodide were added to 100 μL of cells suspension. The tube was incubated on ice for 15 min in the dark followed by addition of 400 μL ice-cold 1X binding buffer and mixing gently. The samples were analyzed using flow cytometer with Summit software (Version 4.3; CyAN ADP, Beckman Coulter: Brea, CA, USA).
3.10. DPPH Radical Scavenging Assay
Stock solutions of GBR, BR and GABA were prepared as 5 mg/mL and dissolved in DMSO. Fifty microlitres of individual solutions were pipetted into a 96-well microtitre plate and mixed with 195 μL of 0.1 mM DPPH methanolic solution. The plate was swirled gently for 1 min and incubated in the dark at room temperature up to 60 min. Absorbance was read at 540 nm using Microplate reader (Opsys MR, Thermo Labsystem, Franklin, MA, USA). Trolox was used as standard and analyses were carried out in triplicate. Percent inhibition of DPPH radical was calculated using the following formula:
DPPH scavenging activities of GBR, BR and GABA were expressed as mg Trolox equivalent per gram of extract (mg TE/g extract).
3.11. ABTS Radical Cation Scavenging Assay
ABTS radical cation was generated through oxidation of 7 mM ABTS with 2.45 mM potassium persulfate and incubated overnight in the dark at room temperature. ABTS radical cation solution was then diluted with ethanol to obtain an absorbance of 0.70 ± 0.02 at 734 nm spectrophotometerically (Pharmaspec UV-1700, Shimadzu, Kyoto, Japan). GBR, BR and GABA (50 μL each) were individually mixed with 950 μL of diluted ABTS solution and incubated for 10 min at room temperature. The absorbance was measured again at 734 nm. All the determinations were carried out in triplicate and the readings were averaged. Trolox was used as standard and percentage of ABTS radical cation decolorization inhibition was calculated using the following formula:
ABTS radical cation scavenging activity of GBR, BR and GABA was expressed in mg Trolox equivalent per gram extracts (mg TE/g extract).
3.12. Ferrous Ion Chelating Ability Assay
GBR, BR and GABA (1 mL each) was mixed with FeCl2 (0.1 mL; 2 mM) and ferrozine (50 μL; 5 mM) solutions. The absorbance at 562 nm was determined spectrophotometerically (Pharmaspec UV-1700, Shimadzu, Kyoto, Japan) 10 min after mixing the contents. The ferrous ion chelating ability was calculated as follows:
The ferrous ion chelating ability of GBR, BR and GABA was expressed as mg EDTA equivalent per g extract (mg EDTA eq/g extract).
3.13. Statistical Analysis
Statistical analysis was conducted by one-way ANOVA, Tukey’s multiple comparisons and Student’s t-test using Statistical Package for Social Science (SPSS) version 20. p < 0.05 was considered as statistically significant difference.