Microwave treatment of rice bran and its effect on phytochemical content and antioxidant activity

An alternative approach for rice bran stabilization is microwave treatment. However, the effects of the microwave treatment on the contents of bioactive compounds and antioxidant activities of the rice bran have rarely been reported in detail. In this study, microwave pretreatment (130–880 W for 0.5–5.0 min) of rice bran was proposed where the antioxidant activity, total flavonoids, and total phenolic contents were determined using UV–Vis spectrometry. Tocols, γ-oryzanols, squalene, phytosterols and phenolic compounds were quantified using high-performance liquid chromatography. The results showed an increase in the antioxidant activity (0.5 folds), total phenolic contents (1.3 folds), total flavonoid contents (0.9 folds), total tocols (2.6 folds), total γ-oryzanols (1.6 folds), and total phytosterols (1.4 folds). Phytochemicals were enhanced, especially trans-p-coumaric acid (10.3 folds) and kaempferol (8.6 folds). The microwave treatment at 440 W for 2.5 min provided the best contents of the bioactive compounds and antioxidant activity. This work revealed the microwave treatment as a potential tool for stabilizing rice bran and increasing the usability of its phytochemicals, which applies to several industries concerning the use of rice bran as an ingredient.

Phenolic compounds content. Eleven phenolic compounds in the RB sample were analyzed with HPLC (Table 2), and the chromatographic elution order was gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, catechin, vanillic acid, chlorogenic acid, caffeic acid, kaempferol, epigallocatechin, trans-p-coumaric acid, and sinapic acid ( Supplementary Fig. 1). It was interesting to note that the content of trans-p-coumaric acid had increased by 10.3 folds from 1.82 μg/g in the control to 20.53 μg/g after roasting at 440 W for 1.5 min. Kaempferol, a curative agent against cancer cell growth, was increased markedly by up to 9.6 folds (6.53 μg/g) after MWT at 440 W for 2.0 min. The MWT at 880 W showed a negative effect which reduced the catechin content (18.67-22.86 μg/g) from the control (23.30 μg/g). Furthermore, a reduction in gallic acid content was obtained after MWT at 130 W (3 and 5 min) and 880 W (1.5 and 2 min). The chlorogenic acid, caffeic acid, epigallocatechin, and sinapic acid in RBs showed positive changes when applied with the microwave roasting process. Protocatechuic acid and vanillic acid were the only two phenolic compounds that decreased after MWT. Similar observations were reported in a study of blue poppy seed 16 . The combination of high power and long operation time exhibited a significant decrease in the phenolic content, which might be due to the partial burning of the RB and thermal damage from hydrothermal treatments on nutraceutical contents of RB as reported by Prateep Table 1. Total phenolic, flavonoid, and antioxidant activity of rice bran treated in microwave oven. Rice bran was burnt partially (treatment in 260 W for 5 min, 440 W for 3 and 5 min as well as 620 W and 880 W for 2.5, 3, 5 min). Values are means ± standard deviations (n = 3). Tocols, γ-oryzanols, phytosterols, squalene, cholecalciferol and phylloquinone content. RB is an abundant source of tocols (α-, β-, γ-, δ-tocopherol (T), α-, β-, γ-, δ-tocotrienol (T3)), γ-oryzanols, phytosterols and squalene. The changes in tocols content after MWT is shown in Table 3 and chromatographic results are shown in Supplementary Fig. 2 (left side). The vitamin E in the raw RB were γ-T3 (84.86 µg/g), followed by α-T (12.43 µg/g), α-T3 (8.84 µg/g), γ-T (4.29 µg/g), β-T (1.85 µg/g), δ-T3 (1.74 µg/g), and δ-T (0.37 µg/g), respectively. The MWT had positive effects on the tocols content of the RB, especially at 440 W. The changes of the tocols were dependent on the exposure time and microwave power, in which the MWT at 440 W for 2.5 min obtained the highest contents of total tocols (367.09 µg/g, equivalent to 2.6-fold increase from the control of 101.95 µg/g). The results for other functional compounds are shown in Table 4, and the chromatographic result is shown in Supplementary Fig. 2 (right side). These include γ-oryzanols, a fundamental substance with several healthbeneficial effects, such as anti-oxidant activity, anticarcinogenic, and antidiabetic 17,18 . The main γ-oryzanols in the raw RB was 24-methylene cycloartanyl ferulate (24-MCFer) (716.55 μg/g), followed by cycloartenyl ferulate (CycloFer) (442.77 μg/g), campesteryl ferulate (CampFer) (270.05 μg/g), and β-sitosteryl ferulate (β-SitFer) (119.94 μg/g) with the total γ-oryzanol content of control at 1549.31 μg/g. The current study showed an enhancement of γ-oryzanols after MWT. The optimum exposure power was 260 W, which the CycloFer, 24-MCFer, CampFer, and β-SitFer increased 1.3, 2.4, 0.6, and 1.4 folds than those of the control, respectively. The MWT of KDML 105 RB in this study showed a 1.6-fold increase of total γ-oryzanols while the parboiled and steamed of Sona masuri RB showed 0.7 and 0.4-fold increase 13 . Generally, the MWT contributed to the positive changes in total phytosterol contents.
The highest total phytosterol content was found in the RB treated at 440 W for 1 min (3059.56 µg/g), which increased 1.4 folds from the control (1252.01 µg/g). In most cases, microwave-treated RB showed higher levels of β-sitosterol (β-SIT) than the raw RB (424.76 μg/g). The highest content of β-SIT was found in the RB treated The content of squalene (99.55 µg/g), cholecalciferol (3.01 µg/g), and phylloquinone (2.45 µg/g) in the control showed improvements after the MWT. Roasting at 440 W for 1.5 min obtained the highest content of squalene (303.89 µg/g, increased by 2.0 folds, and roasting at 880 W provided the highest content of cholecalciferol (14.15 µg/g, increased by 3.7 folds) and phylloquinone (11.91 µg/g, increased by 3.9 folds). The impact of exposure time on phylloquinone determination exhibited the same trend as the effect of time on squalene content. Pokkanta et al., (2019) reported that RBs were an abundant source of phytosterols (stigmasterol, campesterol and β-sitosterol) and squalene 19 .
Based on our results, the changes of phytochemicals when exposed to MWT with increasing power and exposure time share the same trend. The phytochemical content in RBs after MWT proportionally increased with increasing MW power and exposure time until it reaches its highest value. Sequentially, a decrease in phytochemical content was observed for MWT at high power and long exposure time. This could be because the phytochemicals in plant cell walls, such as phenolic compounds, dissolve due to the breakage of the bonds that connect them. Solubility of the phytochemical increased as a result of its dissolution in cell tissue, increasing the released phytochemical 20 . The heat generated from the MWO can inactivate enzymes such as lipase and oxidase, causing deterioration of the phytochemicals. The antioxidant activity was increased partly due to the formation of the Maillard reaction products, an antioxidant in foods 21 . On the other hand, after each phytochemical increased to its highest content, it began to degrade. The MWT at high power and long operation times can lead to the elevated temperature of the sample. The generated heat acts particularly on polar bonds of the compounds, contributing to chemical reactions such as oxidation, dehydration, structural changes, and esterification that can react or transform secondary plant metabolites into other structures 22 . Furthermore, excessive microwave exposure can degrade phytochemicals of natural products due to the electromagnetic force of microwave, thermalaccelerated oxidative deterioration, especially in heat-sensitive substances (e.g., polyphenols) 23 .
In general, MWT could improve the nutrients of food samples, however, the appropriate MW power and exposure time are required for different crop material to retain high amounts of phytochemicals. The results found different optimum conditions for the content of γ-oryzanols (260 W for 2 min), tocols (440 W for 2.5 min), phytosterols (440 W for 1 min), squalene (440 W for 1.5 min), cholecalciferol, and phylloquinone (880 W for 1 min). However, the MWT at 440 W for 2.5 min was concluded as the best overall condition, which provided the highest content of the studied bioactive compounds and antioxidant activity. Table 3. Tocols content (α-, β-, γ-and δ-tocopherol (T) and α-, β-, γ-and δ-tocotrienol (T3)) of rice bran treated in microwave oven (µg/g). ND non detectable. Values are means ± standard deviations (n = 3).

Conclusions
The study revealed that the MWT increased antioxidant activities and amounts of released bioactive compounds from the RB. The MWT was able to increase the capability of the phytochemical compounds to be released from their bound structures. The MWT required very little time, therefore, enabling the preservation of nutraceutical values and properties of the RB. The long exposure time and high power in the microwave process might cause degradation of the nutrients. The findings suggested that the MWT could be a powerful tool for the stabilization, enhancement of usability, and retainment of RB phytochemicals.

Methods
The study complies with local and national guidelines.  Spectrometry analysis of phenolics, flavonoids, and antioxidant activity in RB. RBs (0.5 g)

Microwave stabilization.
were extracted with 5.00 mL of 80% methanol under sonication for 1 h. The extraction solvent was chosen because it is proven to be the most effective extraction solvent for phenolics and antioxidant activity in rice 24 . Sonication was used to maximize extraction efficacy of the targeted compounds 25 . The resulting solution was centrifuged at 3500 rpm for 10 min, and the supernatant was filtered through a 0.45 μm nylon filter. The resulting extract was subjected to determination of phenolics 26 , flavonoids 27 , and antioxidant activity 28 with a UNICO 2150-UV Spectrophotometer.
HPLC analysis of individual phenolic, tocols, γ-oryzanols, phytosterols, squalene, cholecalciferol and phylloquinone. Two HPLC systems were employed. The first system was applied for the analysis of the eleven phenolic compounds 29 . Phenolic compounds were extracted with the same method used in the spectrometric analysis. The system utilized a Kinetex C18 column (150 × 4.6 mm; 2.6 µm, Phenomenex) and a gradient elution system consisting of water/acetic acid (99:1, v/v) as mobile phase A and water/acetonitrile/ acetic acid (67:32:1, v/v/v) as mobile phase B. The phenolics were detected at 275 nm. The other HPLC system was for analysis of the other functional phytochemicals (total of seventeen compounds) 19 . RBs (0.30 g) were extracted with methanol (3.00 mL) for 5 min, and the extracted RBs were then re-extracted with dichloromethane (3.00 mL) and hexane (3.00 mL), respectively. Supernatants of these three solvents were combined, evaporated, and re-dissolved with dichloromethane before analysis. The HPLC system employed a Kinetex PFP column (4.6 × 250 mm, 5 µm, Phenomenex) and a mobile phase of methanol and water. A fluorescent detector was set at 294 nm (excitation) and 326 nm (emission) for detection of tocols, and a variable wavelength detector was set to detect cholecalciferol at 265 nm (0-8 min), phytosterols and squalene at 210 nm (8-18 min), and phylloquinone and γ-oryzanols at 328 nm (18-30 min).

Statistical analysis.
Quantitative data were expressed as the mean ± standard deviation (n = 3). Statistical analysis in this study was analyzed using one-way ANOVA with RStudio version 1.2.5042. Differences are statistically significant at P < 0.05.