Antioxidative effects of stabilized and unstabilized defatted rice bran methanolic extracts on the stability of rice bran oil under accelerated conditions

Many studies have focused on rice bran as a source of natural antioxidants e.g. rice bran (Chotimarkorn et al., 2008) and defatted rice bran (Devi and Arumughan, 2007), which is the predominant byproduct of rice bran oil and a good source of insoluble dietary fiber, protein, phytic acid, inositol and vitamin B (Hargrove, 1994). The antioxidant potential of defatted rice bran was also determined (Shin et al., 1992; Devi et al., 2007), but it still remains a relatively unexplored source material, which demands further investigation especially with regards to its phytochemical composition related to possible health benefits as antioxidants. According to literature reports, several authors have demonstrated the antioxidative effects of rice bran extracts such as free radical scavenging, chelating ability and reducing power in vitro systems (Iqbal et al., 2005; Nam et al., 2006). However, stabilized and unstabilized defatted rice bran extracts have not been investigated as a source of natural antioxidant substitutes for synthetic antioxidants in bulk oil during storage. Moreover, the changes in the antioxidative components of crude defatted rice bran extracts have not been explored during autoxidation. The objectives of this research were to investigate the phenolic compounds of stabilized and unstabilized defatted rice bran extracts and to evaluate their antioxidant activity (AOA) using different in vitro methods. The different extracts were dissolved in a small amount of methanol and applied to rice bran oil at levels of 0.25 and 0.5% to examine their antioxidative activity; the development of the peroxide, anisidine, conjugated deine (CD), and TBRAS values during the oxidation of rice bran oil were also evaluated at 70°C for 168 hour.


INTRODUCTION
Many studies have focused on rice bran as a source of natural antioxidants e.g.rice bran (Chotimarkorn et al., 2008) and defatted rice bran (Devi and Arumughan, 2007), which is the predominant byproduct of rice bran oil and a good source of insoluble dietary fiber, protein, phytic acid, inositol and vitamin B (Hargrove, 1994).The antioxidant potential of defatted rice bran was also determined (Shin et al., 1992;Devi et al., 2007), but it still remains a relatively unexplored source material, which demands further investigation especially with regards to its phytochemical composition related to possible health benefits as antioxidants.According to literature reports, several authors have demonstrated the antioxidative effects of rice bran extracts such as free radical scavenging, chelating ability and reducing power in vitro systems (Iqbal et al., 2005;Nam et al., 2006).However, stabilized and unstabilized defatted rice bran extracts have not been investigated as a source of natural antioxidant substitutes for synthetic antioxidants in bulk oil during storage.Moreover, the changes in the antioxidative components of crude defatted rice bran extracts have not been explored during autoxidation.The objectives of this research were to investigate the phenolic compounds of stabilized and unstabilized defatted rice bran extracts and to evaluate their antioxidant activity (AOA) using different in vitro methods.The different extracts were dissolved in a small amount of methanol and applied to rice bran oil at levels of 0.25 and 0.5% to examine their antioxidative activity; the development of the peroxide, anisidine, conjugated deine (CD), and TBRAS values during the oxidation of rice bran oil were also evaluated at 70°C for 168 hour.

Antioxidative effects of stabilized and unstabilized defatted rice bran methanolic extracts on the stability of rice bran oil under accelerated conditions.
The present research assessed the antioxidant activity against the synthetic 1, 1-diphenyl-2-picrylhydrazyl radical, and b-carotene-linoleic acid assay of the methanolic extracts of defatted rice bran from stabilized and unstabilized rice bran.The effects of the extracts (0.1 and 0.25% w/w) on the oxidative stability of refined-bleached rice bran oil were determined and compared with those of BHA (synthetic antioxidant).The study was carried out over a 168 hr period at 70°C and the progression of oxidation was measured by peroxide value, p-anisidine value, and thiobarbituric acid-reactive substances (TBARS).The relative % of residual a-tocopherol and γ-oryzanol of the rice bran oil containing methanolic extracts of stabilized and unstabilized defatted rice bran during storage at 70°C were studied.

KEY-WORDS: Antioxidant activity -Rice bran oil -Stability -Stabilized defatted rice bran.
ABDALBASIT ADAM MARIOD, HADIzA ALTINE ADAMU, MAzNAH ISMAIL AND NORSHARINA ISMAIL 1,1-diphenyl-2-picrylhydrazyl (DPPH) according to Gordon et al.(2001) and using b-carotene-linoleic acid assay according to the method of Amarowicz et al. (2003).All determinations were performed in triplicate.The collected different methanolic rice bran extracts were applied to 100 g commercial edible rice bran oil obtained from a local market (free of any antioxidant) at levels of 0.1% and 0.25% to examine their antioxidative activity.BHA at a level of 0.02 % was used as a standard.A control sample was prepared using the same amount of methanol used to dissolve the antioxidant and the extracts (Moure et al., 2001).The commercial rice bran oil with added antioxidants was heated at 70°C for 168 hours.Samples (5 g) were removed periodically every 24,48,72,96,120,144 and 168 hours for analysis.Peroxide value (PV) and p-anisidine value (AV), and thiobarbituric acid-reactive substances (TBARS) were determined by the (AOCS, 1993) methods.
TBARS test.Each sample (200 mg) was weighed into a 25-mL volumetric flask.It was then made up to the mark with 1-butanol (ACS grade) and mixed thoroughly.A 5-mL portion of this solution was transferred into a dry test tube, and a 5-mL quantity of fresh TBA reagent (200 mg TBA in 100 mL 1-butanol) was added to it.The tube was placed in a 95°C water bath for 120 min.The tube was cooled, and the absorbance of the solution was read at 532 nm.The TBARS value was calculated as follows: TBARS value = (A*0.415)/m(Chirinos et al., 2009).
where A = absorbance at 532 nm and m = mass of the sample

Identification of phenolic compounds
Phenolic compounds were identified and quantified by comparing their retention time and UV-Vis spectral data to known previously injected standards (Chirinos et al., 2009).The changes in gamma-oryzanol and a-tocopherol content in rice bran oil treated with rice bran methanolic extract during storage were measured according to the method of Rogers et al. (1993) The content of gamma-oryzanol and a-tocopherol were quantified using the HPLC system consisting of a Hewlett-Packard (Germany) Model G1311A HPLC connected to an ALS Auto-injector Series 1100 (Hewlett Packard, Germany) based on the peak area of the standard curve of standard gamma-oryzanol and a-tocopherol.

Statistical analyses
Statistical analyses were conducted using SPSS (Statistical Program for Social Sciences, SPSS Corporation, Chicago, IL) version 12.0 for Windows.The analysis of variance (ANOVA) and Pearson's correlation coefficients were performed to compare the data.All determinations were done at least in triplicate and all were averaged.The confidence limits used in this study were based on 95% (P < 0.05).hydroxyanisole (BHA), b-carotene, linoleic acid and Folin-Ciocalteau reagent as well as polyoxyethylene sorbitan monopalmitate (Tween 40) were obtained from Merck (Merck, Darmstadt, Germany).(+)-a-tocopherol (Sigma-Aldrich Co., St. Louis, MO, USA), Rice bran samples were obtained from a local milling factory in Tanjung Karang, Selangor, Malaysia.Samples were collected directly from the milling system and immediately placed in square plastic (polypropylene) containers that were halffilled and transported to the Molecular Biomedicine Laboratory of the Institute of Bioscince, IBS, UPM Serdang in a cool box.To stabilize the rice bran samples, they were heated for 2 min at 2450MHz, using a national microwave/convection oven (model NN-C2003S, Matsushita Electric Industrial Co., Japan).The heating was carried out one by one until all samples had been heated.The stabilized samples were left to cool at room temperature before sealing the lids with cellophane tape.Samples of stabilized rice bran (SRB) and unstabilized rice bran (USRB) were stored at 4°C in a cold room until further analyses.

Rice bran oil
The refined edible rice bran oil which was produced by Green Love Distribution Sdn Bhd, Kuala Lumpur, Malaysia was obtained from the local store Serdang, Malaysia; the oil was free of any synthetic antioxidant.

Extraction of phenolic compounds
Twenty grams of the dried defatted (stabilized SRB and unstabilized USRB) rice bran were extracted successively with 80% methanol (3x200ml) by sonication (Hwasin Technology, Seoul, Korea) to obtain methanolic extract at room temperature for 1 hour.The extracts were filtrated through Whatman no. 1 filter paper and the solvent was removed.The residual of defatted rice bran was further extracted twice with 80% methanol and the extracts were combined.Then the solvents were removed using a rotary evaporator (Buchi, Flawil, Switzerland) at 40°C and subjected to a nitrogen gas flow until dry (Devi and Arumughan, 2007).The yield of each extract was measured before storing at -80 o C for further analysis.The total phenolic (TPC) in the methanolic extracts of defatted rice bran was determined with the Folin-Ciocalteu reagent following the method of Kaur et al. (2008) using gallic acid as standard.The TPC of the sample was expressed as gallic acid in terms of mg gallic acid equivalents (GAE)/100 g dried samples.

The antioxidant activity
The antioxidant activity of phenolic extracts from defatted (stabilized SRB and unstabilized USRB) rice bran was measured using the stable radical ANTIOxIDATIVE EFFECTS OF STABILIzED AND UNSTABILIzED DEFATTED RICE BRAN METHANOLIC ExTRACTS ON THE STABILITy… DPPH radical were found to be 0.69 mg/ml, 0.79 mg/ ml, and 0.17 mg/ml for SRB, USRB and ascorbic acid (standard), respectively.The DPPH radicalscavenging activity was found to be in the order of ascorbic acid > SRB >USRB.The stabilized defatted rice bran extract showed the most potent activity.This radical-scavenging activity of extracts could be related to the nature of phenolic compounds or might be related to compounds other than phenolics.
Table 1 shows the individual phenolic acids found in the methanolic extract of SRB and USRB respectively, monitored at 280 nm.Gallic, chlorogenic, syringic, vanillin, and ferulic acids were the predominant phenolics showing the levels of 0.003, 0.008, 0.004, 0.006 and 0.005 mg/100 g DW, in USRB, respectively.While in SRB fewer levels of 0.002, 0.003 and 0.003 mg/100 g in SRB for syringic, vanillin, and ferulic acids were found.

Lipid oxidation compound assessment
Increasing primary and secondary lipid peroxidation products of rice bran oil treated with phenolic extract from SRB and USRB during 168 hr storage were measured by peroxide, p-anisidine and thiobarbituric acid reactive substance (TBARS) values.The changes in peroxide value (PV) of the treated oils as a function of time are shown in Table 2. Rice bran oil treated with SRB, and USRB exhibited lower PV (<50%) for up to 168 hr as compared with the control sample.Also, samples treated with synthetic antioxidants (BHA) showed lower peroxide values than the control tested in this work.Among all samples tested, SRB served best in lowering peroxide formation and it was superior to USRB samples and gave peroxide values of 3.2, 6.9, 10.4 and 22.5 (meq/kg oil) on hrs 0, 24, 96, and 168, respectively; corresponding values of the control sample were 3.5,

Phenolic compound analysis
Previous research has been interested in the identification of phenolic compounds from various natural sources (Moure, 2000;Mariod et al., 2009a;Mariod et al., 2009b).Rice bran was one of the most potential sources of plant phytochemicals.However, the content and stability of phenolic compounds in stabilized and unstabilized defatted rice bran extracts have not previously been investigated.The content of the total phenolic compounds of methanolic extracts from stabilized defatted rice bran (SRB) and unstabilized defatted rice bran (USRB) expressed as gallic acid equivalents are shown in Table 1.The results in this table reveal that the SRB exhibited a higher phenolic content of 519.6 mg/100g GAE followed by the USRB extract (480.1 mg/100g).These results were higher than the previous results of Devi and Arumughan (2007), who reported 5.3% of total phenolics in defatted rice bran, and Chatha et al. (2006) who reported that the TPC of different rice bran extracts ranged from 250-397 mg/100g with 80% methanol as the most efficient solvent system in extracting the phenolic antioxidants from rice bran as compared with other solvents.Gerard and Roberts (2004) reported that the total phenolic and flavonoid content of apple juice increased with increasing microwave heating.In contrast, Abdul-Hamid et al. (2007) reported that total phenolics present in stabilized rice bran showed decreasing concentrations in different milling fractions.

Antioxidant activity assessment
The antioxidant activity of SRB and USRB extracts was evaluated through the DPPH method and the results are presented in terms of IC 50 (Table 1).The IC 50 values of the scavenging activities on   The effects of the different extracts and synthetic antioxidants on the TBARS values of rice bran oil after accelerated oxidation at 70°C are shown in Table 2. TBARS formation of the control increased with an increase in storage time.However, the values for the samples treated with the defatted rice bran extracts and BHA were significantly (P < 0.05) lower than those of the control.Among the phenolic extracts used, SRB and USRB extracts at 250 mg showed better antioxidant activity than at 100 mg methanolic extract.The different extracts lowered the content of TBARS significantly (P < 0.05) after 168 hr of storage.TBARS measure the formation of secondary oxidation products, which may contribute to the off-flavor of oxidized oil (Wanasundara and Shahidi, 1994).These results indicate that the SRB and USRB extracts exhibited antioxidant activity not only in b-carotene linoleic acid (Fig. 1) but also during rice bran oil peroxidation (Table 2).

Endogenous antioxidant content
Tocopherols and oryzanols are the main antioxidants present in rice bran.The antioxidant activity of oryzanols is almost 10 times higher than that of tocopherols.The changes of gamma-oryzanol and a-tocopherol content in rice bran oil treated with rice bran methanolic extract during storage were measured according to method of Rogers et al., (1993), and the results are shown in Table 3.After 168 hrs of storage, the residual of a-tocopherol content was reduced by 17.9 and 20.1% of initial rice bran oil containing 100 and 250 mg USRB, respectively, 17.9 and 12.9% initial rice bran oil containing 100 and 250 mg SRB extracts, respectively.
Gamma-oryzanol contents in rice bran oil were decreased during the storage of defatted rice bran oil.The initial γ-oryzanol contents of rice bran oil containing 100 and 250 mg USRB extracts were 2.6, and 2.7 μg/100g oil, respectively.After 168 hrs of storage, the γ-oryzanol contents of rice bran oil added with 100 and 250 mg USRB extracts were 1.8 and 2.3 μg/100g oil, respectively.The initial γ-oryzanol contents of rice bran oil containing 100 and 250 mg SRB extracts were 2.7, and 2.7 μg/100g oil, respectively.After 168 hrs of storage, the γ-oryzanol contents of rice bran oil added with 100 and 250 mg SRB of rice bran extracts was 2.0 and 1.1 μg/100g oil, respectively.These results were in good agreement with Nystrom et al., (2007) who reported that, γ-oryzanol was more heat stable than a-tocopherol at high temperatures.Several studies found that the γ-oryzanol obtained from rice bran extracts had a strong stabilizing effect during storage and frying (Kochhar, 2000;Chotimarkorn and Silalai, 2008).

Table 3
Decrease in a-tocopherol, and γ-oryzanol contents