The human neuroblastoma SK-N-SH cells line was obtained from the ATCC (Rockville, MD, USA) and maintained in minimum essential medium (Life Technology, Inc.) containing 2 mM L-glutamine in a humidified incubator at 37˚C and 5% CO₂. The medium was supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 0.1 mM minimal essential medium nonessential amino acids (Life Technology, Inc.), 100 units/ml penicillin, and 100 mg/ml streptomycin (Biofluids, Rockville, MD). Cells were plated at 5 × 10⁵ cells/ml were cultured in 96 well plates (for Reactive Oxygen Species (ROS) formation and for cell viability (MTT assay). Cell were plated at 1 × 10⁶ cells/ml on 6 well plates for DNA fragmentation then maintained in serum free optimal minimal modified Eagles medium (MEM) supplemented with Fetal bovine serum (Invitrogen, Carlsbad, CA). Cultures were maintained for 24 h before treatments.

Pre-germinated brown rice (Oryza sativa L.) was supplied by Innofood (Thailand) Co., Ltd. in Pratumthani and was germinated by soaking in the following solutions at 25˚C - 26˚C in the dark for 72 h: was air dried, frozen in liquid nitrogen pre-germinated brown rice extracts were prepared as previously described [10] Briefly, the pregerminated brown rice (PGBR), brown rice (BR), polish rice or white rice (WR) were ground with a mortar and pestle, and then added with distilled water mix with Vortex for 10 min and then kept in water bath at 70˚C for 30 min. The samples were centrifuged at 15,000 rpm at 4˚C for 30 min, and the supernatants were collected, passed through filters with 0.45 μm pores, and used as extracts.

To determine the influence of brown rice extract on cell viability which induced by 150 μM H₂O₂. Cells were plated 5 × 10⁵ cells/ml in 96-well plates (Falcon, Germany) in medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. After additions by treated with increasing concentrations of brown rice extract (0 - 4000 μg/ml) at 37˚C for 3 h and further added with 150 μM H₂O₂ in culture incubation at 37˚C for 24 h as indicated. Viabilities of the cells were assay by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] method, MTT solution was added at a final concentration of 0.5 mg/ml at 37˚C for 3 h incubation. Then solubilize solution (DMSO) was then added, the plates were shaken for 10 min and absorbance was read at 570 nm in a microplate reader.

Detection of intracellular reactive oxygen species (ROS). The production of intracellular reactive oxygen species was estimated by using a fluorescent probe, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) [21]. DCFH-DA is transported across the cell membrane and hydrolyzed by intracellular esterases to form nonfluorescent 2′,7′-dichloro fluorescein (DCFH), which is then rapidly converted to highly fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of reactive oxygen species. The DCF fluorescence intensity is believed to be parallel to the amount of reactive oxygen species formed intracellular. To study the free radical scavenging effect of Brown rice extracts (rice extract at final concentration 2000 μg/ml) was added 3 h before added with 150 μM H₂O₂ to the culture and incubated at 37˚C for 24 h. To determine the amounts of ROS induced by H₂O₂, DCFH-DA (50 μM final concentration in DMSO) was added to the cells culture and then incubated at 37˚C for 2 h. The production of reactive oxygen species was measured immediately by microplate reader using excitation and emission at 485 nm and a 530 nm.

Cultured cells were prepared at 1 × 10⁶ cells/well in 6 well plates. Rice extracts were added into the cultures at 2000 μg/ml and cultured at 37˚C for 3 h and further added with 150 μM H₂O₂ in culture incubation at 37˚C for 24 h then cells were collected. Genomic DNA was extracted using the Apoptotic DNA Ladder Kit, (Roche Applied Science, Mannheim, Germany) with slightly modification. In brief, cells were washed twice with ice cold 1 × PBS, then 200 uL binding/lysis buffer was added and mixed immediately. After holding for 10 min at 22˚C, 100 μL of 100% isopropanol was added and the solution was vortexed for 10 sec. The lysate was run through the column then washed twice with washing buffer. DNA was eluted with 200 μL of pre-warmed (70˚C) elution buffer and concentrated with a speedvac. Extracted DNA was subjected to gel electrophoresis, and the image was captured with GelDoc^TM EQ (Bio-Rad Laboratories, Ltd. Hercules, CA).

The results were expressed as mean ± SEM of triplicate assays. The statistical comparison between control and treated experimental groups were carried out using Student’s t-test. P-value less than 0.05 were considered to be significantly different.

First, we attempted to establish the doses dependent of the brown rice extract, the cytotoxicity of brown rice extract on human neuronal SK-N-SH cells was observed. PGBR ex, BR ex and WR ex were applied in culture by increasing concentrations 0 - 4000 μg/ml to SK-N-SH cells for 24 h. We found that the data of cells survival showed that std. GABA up to 96.4 ± 0.052 (p > 0.48), PGBR ex 98.9 ± 0.032 (p > 0.30), BR ex 96.3 ± 0.039 (p > 0.39) and WR ex 95.3 ± 0.037 (p > 0.25), respectively. The data of viability cells had no significantly decrease cell survivals which had no toxicity affect on human neuronal SK-N-SH cells (Figure 1). However, treated with 150 μM H₂O₂ alone caused cells death > 56.9% ± 0.02% (p < 0.05) compared with control indicating at this concentration H₂O₂ had toxicity to SK-N-SH cells. (data not shown) Similar to Chan-Ho Oh and Suk-Heung Oh had tested the effect of the brown rice extracts on the viability of Hela cells, by treated cells with the brown rice ex extracts 2000 mg/ml in cultured, the viability of the cells was assayed. Their results had shown that brown rice extracts also had no effects on the retardation of HeLa cell proliferation.

Next we tested the protective effects of the brown rice extracts on the viability of SK-N-SH cells, cells were pretreated with the PGBR ex, BR ex and WR ex at 2000 μg/ml and then treated with 150 μM H₂O₂. These results showed that PGBR and BR ex can protect cells. The cells survival significantly increase up to 29.3% ± 0.01% and 13.4% ± 0.07% (p < 0.05), but not WR ex comparable with 150 μM H₂O₂ treated alone which caused cells death >56.9 ± 0.02% compared with control (Figure 2). PGBR ex contains approximately 13 times of GABA and the amount of

oryzanol in polished rice. Ferulic acid ester is one of the main components of oryzanol, a phytosterol derived from rice bran. Previous reports have hypothesized that oxidative stress is one of the mechanisms of Aβ-induced neurotoxicity. In vitro studies have shown that β-tocopherol, a representative antioxidant, inhibits Aβ-induced neuronal cell death and lipid peroxidation [22]. Ferulic acid also possesses free radical scavenging activity and reduces peroxidative damage [23].

DNA fragmentation is a marker of late stage of apoptosis. To verify the possible involvement of apoptosis in the 150 μM H₂O₂-induced death of neuronal SK-N-SH cells. We investigated its inhibitory effect of PGBR ex on the apoptosis induced by 150 μM H₂O₂ by observing DNA fragmentation levels in SK-N-SH cells. This figure demonstrates internucleosomal DNA degradation from gel electrophoresis. As a control, we used DNA isolated from untreated cells and no DNA ladder similar results was shown when treated with PGBR ex (Figure 3, lanes 2 & 3). Fragmented DNA was observed when cells were treated with 150 μM H₂O₂ resulted in the characteristic apoptotic DNA ladder. DNA fragments smeared the whole lane (Figure 3, lanes 4). These fragmentation patterns are entirely consistent with the molecular weight patterns expected to result from internucleosomal DNA cleavage [24]. But DNA fragments was decreased when co-treated with PGBR ex and 150 μM H₂O₂ (Figure 3, lanes 5). These DNA fragmentation patterns are entirely consistent with the molecular weight patterns expected to result from internucleosomal DNA cleavage. DNA fragmentations are indicative of early and late stage of apoptosis, respectively. From this results shown that the PGBR ex in our present study showed the anti-apoptotic effect on

SK-N-SH cells.

Intracellular ROS have been implicated with DNA fragmentation and apoptosis [25]. The intracellular radical scavenging activity of a given substance can be evaluated by DCFHDA assay. To determine whether PGBR ex inhibits the ROS generation induced by 150 μM H₂O₂ in neuronal SK-N-SH cells, and to determine whether the protection of SK-N-SH neuronal cells from apoptosis is accompanied by free radicals scavenging by the PGBR ex, changes in the concentrations of ROS in whole cell suspensions were analyzed over 2 h period after treatment with PGBR extract and/or 150 μM H₂O₂. SK-NSH cells showed relatively low levels of basal fluorescence but when treated with 150 μM/ml H₂O₂ for 2 h, a marked increase in fluorescence was observed. However, this increase in fluorescence by 150 μM H₂O₂ rapidly returned to the control level in PGBR ex pretreated (2000 μg/ml) to the cells (Figure 4).