Free phenolics were extracted under the optimum conditions of 80% acetone, 58.3˚C temperature, 1:10.5 solid to solvent ratio, for 90 min based on parameters established in our previous studies [5]. Extractions were performed twice in a shaking incubator (G24 Environmental Incubator Shaker, New Brunswick Scientific Co., NJ, USA).

BMR samples were autoclave treated (3021-S, Gravity, AMSCO, Mentor, OH, USA) under the standard temperature (121˚C) and pressure (15 psi) for 20 min, which is typically used for sterilization and enzyme inhibition. The samples were cooled to room temperature and used for extraction. Extractions were performed twice for APSE (80% aqueous acetone, 1:10 solid to solvent, 40˚C, 60 min) in a shaking incubator (G24 Environmental Incubator Shaker, New Brunswick Scientific Co., Edison, NJ, USA).

The antioxidant phenolic compounds were extracted using a microwave assisted extraction system (Model: MARS 6, CEM Corporation, Matthews, NC). The optimum extraction conditions of 5.4 min extraction time, 42.1˚C and 79.1% of solvent concentration as designed in previous research were used [5]. After careful extraction in Xpress vessels, the samples were cooled down and transferred to centrifuge tubes for further extraction process of free and bound phenolic compounds.

After extraction, samples were centrifuged (Model: accuSpin^TM 1R, Fisher Scientific, Pittsburg, PA, USA) at 3500 rpm for 15 mins. The supernatants were filtered using Whatman filter No. 1 and the extracts were pooled and dried at 35˚C under nitrogen using an evaporator (34 Position N-EVAP Nitrogen Evaporator, Organomation, Berlin, MA, USA). The dried extracts were reconstituted with 80% methanol and kept at 4˚C until used for analysis. All the extractions were conducted in duplicates. The residues from free phenolics were used to extract bound phenolics.

Alkaline hydrolysis using sodium hydroxide (1N) was carried out for the extraction of bound phenolics from BMR. The crude free phenolics residues obtained after the free phenolic extraction was dried overnight and hydrolyzed using NaOH (10 mL/g) for 16 h at ambient temperature. The sample was then acidified with 1 N HCl to a pH of 2 and then extracted thrice with ethyl acetate (3 × 10 mL). The extracts were then pooled together and dried at 35˚C under nitrogen gas. The extracts were reconstituted with 80% methanol and kept under 4˚C until used for analysis.

Method for determination of TPC was adopted from Devanto et al., [15] with slight modifications. Gallic acid (0 - 300 µg/mL) was prepared for a standard curve. Extract of 0.125 mL from the reconstituted solution was added to 0.5 mL of distilled water and followed by the addition of 0.125 mL of Folin-Ciocalteu reagent (FCR). After 6 mins of reaction time, 1.25 mL of Na₂CO₃ (7% aqueous) was added. The total volume was adjusted to 3 ml by adding distilled water. Samples were incubated at room temperature in the dark for 90 mins and the absorbance was measured at 760 nm using a spectrophotometer (Model: UV-1800, Shimadzu Corporation, Kyoto, Japan). All values were expressed as mg of gallic acid equivalents (GAE) per gram of dry weight of the BMR. All experiments were done in quadruplicate and the mean values were used for the analysis.

The total antioxidant activity of the BMR extracts was measured using DPPH assay with slight modifications [16] [17] [18] and was expressed as µg of trolox equivalents (TE) per gram of dry weight of the sample. Trolox stock solution of concentrations (0 to 500 µM) was prepared for producing a standard curve. All the reagents were prepared fresh every time before the analysis. About 100 µL of reconstituted extracts/standard were added to 3900 µL of DPPH reagent and were incubated for 1 hour in dark environment at room temperature. The absorbance was recorded at 515 nm using the above mentioned spectrophotometer. All the analyses were conducted in quadruplicate and the mean data were considered for statistical analysis.

The method outlined by Duh et al., [19] was used to measure the formation of secondary lipid peroxidation products in corn oil during heating at a frying temperature of 185˚C. Malondialdehyde (MDA) of 10⁻⁵ concentration was used to prepare the calibration curve. Equal volumes of trichloroacetic acid (15% w/v) and 2-thiobarbituric acid (0.375% v/v) along with 0.25 N of hydrochloric acid were added to prepare the TBARS reagent. To 4 ml of TBARS reagent 0.2 ml of oil was added and the mixture was heated for 15 mins in a boiling water bath. After 15 mins of heating the samples were cooled, and the absorbance was measured at 535 nm by a spectrophotometer. All the measurements were performed in triplicates and the mean values were expressed as µ mol MDA equivalents per gram of oil. The analysis was also conducted in triplicates and mean values of the data were used for statistical analysis.

For the various free BMR extracts (p < 0.05) using various concentrations for this study showed significant differences. The TBARS profile for all the free phenolic extracts used in the study and the formation of secondary lipid peroxidation products expressed as MDA equivalents (µg/mL of corn oil) are shown in Figures 1-4 for 25, 50, 100 and 150 ppm, respectively. Accordingly, with the increase in the heating time, the MDA values increased significantly from –0.652 ± 0.0 µg /mL to 2.748 ± 0.0 µg /mL of corn oil. This indicates the formation of the secondary oxidation products in the oil due to the heat treatment for an extended period of 5 hours.

The added APSE extracts exhibited significant antioxidant activity at all concentrations (25, 50, 100, 150 ppm) over the 5 hour of heating period. With increase in the concentrations of the BMR extract from 25 to 50 ppm the inhibition of lipid oxidation increased by 12%. The addition of 100 ppm the MDA value was 1.948 ± 0.57 µg /mL of corn oil compared to 1.694 ± 0.30 µg /mL of corn oil at 150 ppm.

At lower concentrations, CSE extracts could not exhibit any antioxidant activity. The MDA values after 5 hours of heating at 185˚C for 25 ppm and 50 ppm CSE extracts were found to be 2.389 ± 0.77 µg/mL of corn oil, 2.72 ± 0.146 µg /mL of corn oil compared to 2.748 ± 0.282 µg /mL of corn oil (Control). With increase in concentration from 100 ppm to 150 ppm of CSE extracts, the MDA equivalents increased from 1.784 ± 0.181 µg /mL of corn oil to 2.180 ± 0.093 µg /mL of corn oil. APSE extracts at 50 ppm concentration performed equally as good as the CSE extracts at 100 ppm.

MAE extracts effectively reduced the lipid oxidation in heated corn oil at all the concentrations. The MDA equivalents from the TBARS assay at 25 ppm, 50 ppm, 100 ppm and 150 ppm are 2.101 ± 0.43 µg/mL of corn oil, 2.217 ± 0.0 µg/mL of corn oil, 1.642 ± 0.131 µg/mL of corn oil, and 1.896 ± 0.770 µg/mL of corn oil, respectively. There was no significant difference in the antioxidant activity between 25 and 50 ppm concentrations of MAE extracts. The highest inhibition of oxidation was observed at 100 ppm. There were no significant differences between the MDA value of BHT and MAE added samples at 100 ppm and 150 ppm. The order of inhibition of oxidation is as follows: BHT (100 ppm) > APSE (50 ppm) > MAE (100 ppm) > CSE (100 ppm).

It is evident that autoclaving as a pre-treatment (APSE) or microwave assisted extraction treatment (MAE) has significantly increased the total phenolic content and the antioxidant power of barley malt rootlet extracts. These results are in agreement to the fact that thermal treatments can enhance not only the yield of phenolic extracts but also the antioxidant activity, by breaking the lignocellulose of the cell components and creating disordered structures with or without the removal of inherent components [20] [21] [22] [23].

The bound phenolic extracts followed a different pattern of antioxidant activity compared to the free phenolic extracts. The antioxidant activity of bound phenolics was comparatively low compared to the free phenolics. All values were significantly different between free and bound phenolics with all concentrations tested in the present study. The MDA values for all the bound phenolic extracts are presented in Figures 5-8, for 25, 50, 100 and 150 ppm concentrations of BMR extracts, respectively.

The MDA values for autoclave bound phenolic extracts at 25 ppm, 50 ppm, 100 ppm, 150 ppm concentrations are 2.285 ± 0.40 µg /mL of corn oil, 2.105 ± 0.04 µg /mL of corn oil, 1.941 ± 0.18 µg/mL of corn oil, and 2.419 ± 0.66 µg /mL of corn oil, respectively. With increase in the concentration from 25 ppm to 50 ppm there was an increase in inhibition of oxidation by 7%. However, there was a relative decline in antioxidant power after 100 ppm. The order of antioxidant activity of bound phenolic extracts with respect to the concentration is as follows 100 ppm > 50 ppm > 25 ppm > 150 ppm.

The bound phenolics of conventional solvent extracts (CSE) have shown poor antioxidant power compared to the autoclave treated extracts (APSE). The formation of MDA at 25 ppm of CSE was 2.913 ± 0.771 µg/mL of corn oil compared to the MDA value of the control (2.748 ± 0.0 µg /mL of corn oil).

For bound MAE extracts, at lower concentration the values were significant high for MDA (3.264 ± 0.207 µg/mL of corn oil), however with the increase in concentration of MAE extracts, there was an increase in the inhibition of the oxidation. The highest level of inhibition of oxidation was observed at 100 ppm concentration of MAE, and it is similar to free phenolics from MAE. It is interesting to note that, among the bound phenolics, the MAE extracts at 100 ppm

exhibited similar antioxidant activity to BHT. The order of inhibition by the various bound extracts is as follows: MAE (100 ppm) > BHT (50 ppm) > autoclave (100 ppm) > CSE (100 ppm). Overall it was found that the bound phenolic extracts did not exhibit as good antioxidant activity as the free phenolics. In the present study, the antioxidant activity declined in around 100 ppm concentrations for all the BMR extracts.