Phenolic profiles and antioxidant activity in four tissue fractions of whole brown rice

Abstract

In view of the fact that different types of processed rice contain different tissue fractions, the present study quantified free and bound phenolic profiles and antioxidant activity in the pericarp, aleurone layer, embryo and endosperm fractions of japonica and indica whole brown rice. Significant differences were found in the total phenolic content, oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) of the different fractions. The ratios of free and bound phenolics to total phenolics varied between the fractions. Thirteen individual phenolic compounds (gallic, protocatechuic, hydroxybenzoic, chlorogenic, vanillic, caffeic, syringic, isoferulic, coumaric and ferulic acids, as well as catechin, epicatechin and quercetin) were detected in both free and bound forms. The contributions of the pericarp, aleurone layer and embryo fractions to whole brown rice were 13.0%, 28.5% and 8.8% for total phenolics, 14.1%, 29.7% and 9.1% for total flavonoids, 18.2%, 38.0%, and 11.1% for total ORAC values and 14.6%, 38.0%, and 16.9% for CAA values, respectively. These findings indicate that the phenolics in brown rice can be concentrated by processing different fractions or by milling to different levels because of the uneven distribution of chemical constituents.

---

1. Introduction

Rice is one of the most important grain crops worldwide. Rice production is the highest of all foodstuffs (total output about 600 million tons) and provides the staple food for more than half of the global population.¹ Rice has two major subspecies: Oryza sativa L. japonica, mainly consumed in Southeast Asia, Northern China, Japan and the United States, and Oryza sativa L. indica, mainly consumed in India, Southern China, and Southeast Asia. Whole brown rice is a rice grain from which only the husk has been removed. The hull, or outer covering, corresponds to 18–20% of the total weight of the rice. It is removed from the brown rice by dehulling.¹ Rice, like other cereals, does not have a homogeneous structure in the hulled kernel. However, the rice kernel is differentiated from its outer surface to its inner central part into four tissue fractions: the pericarp (2–3% of whole brown rice by total weight), the aleurone layer (4–6%), the embryo (2–3%) and the endosperm (about 90%). The endosperm fraction, also called white or polished rice, is a major part of the human daily diet in many countries.²

Because of the poor sensory quality of brown rice, the embryo and bran layers, including the pericarp and aleurone layer, are removed for human consumption. White rice, which commands a higher price in the market, is obtained after removing 10–15% of the total weight of brown rice. However, white rice lacks nutrients, as they are lost from brown rice during the process of milling.³ Epidemiological studies have associated increased whole grain consumption with a reduced risk of many chronic diseases. Phenolics are important antioxidants, shown to be responsible for many health benefits, such as anti-allergenic, anti-atherogenic, anti-inflammatory, anti-microbial, antioxidant, anti-thrombotic, cardioprotective and vasodilatory effects.^4–8 The concentrations of total phenolics in rice bran from five rice varieties grown in Southern China were 13.1 times higher than in the endosperm fraction.⁹ The higher concentration and activity of phenolics in the rice bran fraction may account for the potentially beneficial medical effects of brown rice. Furthermore, analysis of successive milling fractions has shown that nutrients are not uniformly distributed in brown rice.^10–14 Rice with different degrees of milling contains different tissue fractions. At present, many types of rice are available in the food markets of Asian countries: semi-brown rice (brown rice with the pericarp/testa removed), embryo rice (brown rice with the pericarp/testa and a small percentage of the aleurone layer removed), lightly milled rice (brown rice with the pericarp/testa, embryo and most of the aleurone layer removed) and polished rice (brown rice with the pericarp/testa, embryo and most of the aleurone layer removed) as well as brown rice.

The contents of brown rice constituents have been analyzed in fractions obtained at different stages of multistep milling. Researchers have previously reported the effect of milling on the nutritional constituents of brown rice, but have focused on minerals, starch and proteins.^2,11–17 The protein and mineral contents decreased, whereas the starch content increased, from the outer bran layers to the endosperm. Data on phytochemical profiles have been limited to a few studies.^18,19 Phytic acid, vitamin E and γ-oryzanol compounds have also been analyzed; Monks et al. (2013) reported that the phytic acid content decreased in brown rice as the degree of milling, as defined by the machining accuracy of the milling equipment, increased.²⁰ Shobana et al. (2011) evaluated changes in the contents of phytochemicals, dietary fiber, and γ-oryzanol in different fractions according to the degree of milling in two Indian rice varieties.²¹ There have been fewer studies on other compounds, including carotenoids (lutein, zeaxanthin, β-cryptoxanthin, and β-carotene), and phenolics. Phenolics include phenolic acids (p-coumaric, caffeic, ferulic, vanillic, and syringic acids) and flavonoids (flavonols, flavones, catechins, and anthocyanins). Some studies have focused on the total phenolic contents from the outer to the inner layers.²² In recent years, others have compared the total phenolic contents in rice bran, the rice bran layer (rice bran except embryo) and the rice embryo.²³ Overall, these studies suggest that the beneficial phytochemicals in whole grain rice are distributed in the free, soluble-conjugated and bound forms in the inner and outer layers of whole brown rice. However, the rice samples were from different species, thus complicating the direct comparison of data on these four fractions. Finally, most of the previous studies obtained the changes of nutrients at different milling times using the rice machining accuracy as an index, which does not correspond to the four different fractions of brown rice. Because of this, information on the beneficial phytochemicals of the different fractions of indica and japonica rice is of great importance to researchers. Therefore, studying the phytochemicals of these different fractions has important scientific significance and economic value for determining the degree to which brown rice and embryo rice should be processed.

The overall objective of the present study is to provide information, which quantifies the content of these antioxidant phytochemicals in the different fractions of whole brown rice, to satisfy the needs of food producers and rice consumers. The specific objectives of this study were: (1) to reveal the distribution and difference of phenolic contents and antioxidant activity in four tissue rice fractions—pericarp, aleurone layer, embryo and endosperm; (2) to demonstrate the differences of the ratios of free and bound phenolics/antioxidant activity to the totals; and (3) to determine the percentages of the contributions of the different fractions to the total phenolic content and antioxidant activity of whole brown rice.

2. Materials and methods

2.1. Chemicals and reagents

2′,7′-Dichlorofluorescin diacetate (DCFH-DA), 6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid (Trolox), 2,2′-azobis-(2-amidinopropane) dihydrochloride (ABAP), 3′,6′-dihydroxyspiro[isobenzofuran-1(³H), 9′-(⁹H)-xanthen]-3-one disodium salt and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gallic, protocatechuic, chlorogenic, hydroxybenzoic, vanillic, caffeic, syringic, coumaric, ferulic, syringic and isoferulic acids, as well as catechin and epicatechin, were purchased from Aladdin Reagents (Shanghai, China). High-performance liquid chromatography-grade acetic acid and acetonitrile were obtained from Fisher (Suwanee, GA, USA). HepG2 human liver cancer cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Williams' Medium E and Hank's Balanced Salt Solution (HBSS) were purchased from Gibco Life Technologies (Grand Island, NY, USA). Fetal bovine serum was obtained from Atlanta Biologicals (Lawrenceville, GA, USA). All other chemicals used were of analytical grade or above.

2.2. Grain samples and sample preparation

Grains of the indica cultivar Zaomiao and the japonica cultivar Wujingyun 27, which are consumed primarily in Southern and Northern China, respectively, were obtained from the Experimental Farm of the Rice Research Institute of Guangdong Academy of Agricultural Sciences in 2013. The rice plants were sown in late March 2013 and harvested in mid-July. The rice grains were air-dried until their moisture content was reduced to approximately 13% and stored at room temperature for three months. These rice samples were milled to separate the husks from the brown rice. The husk was not included in the analysis. The embryos were separated manually from the brown rice sample (w₀, ∼5 g) and weighed (w₁). The degermed brown rice samples were successively polished to collect different tissue fractions (pericarp, aleurone layer and endosperm) of the brown rice samples using a Satake mill (Satake Corp., Tokyo, Japan). The weights of the pericarp and aleurone layers were sequentially marked as w₂ and w₃. The brown rice samples, divided into four tissue fractions, were ground separately to a powder that could pass through a 60-screen mesh, then stored at −20 °C until further analysis. The percentages of pericarp, aleurone layer, embryo and endosperm were calculated using the following equations:²⁴

Pericarp (%) = w₂/w₀ × 100

Aleurone layer (%) = w₃/w₀ × 100

Endosperm (%) = (w₀ − w₁ − w₂ − w₃)/w₀ × 100

Embryo (%) = w₁/w₀ × 100

2.3. Extraction of phenolic compounds

2.3.1. Extraction of free phenolic compounds. The method was adapted from a study by Zhang, Zhang, Zhang and Liu (2010)^25–27 with a few modifications. First, 0.5 g pericarp, 0.5 g aleurone layer, 0.5 g embryo and 2 g endosperm were weighed precisely and transferred into a 100 mL centrifuge tube. 50 mL 80% acetone (v/v) pre-cooled at 5 °C for 20 min was then added. The mixture was homogenized for 5 min using a homogenizer at 10 000 rpm and centrifuged at 2500 g for an additional 10 min. The supernatant was then obtained. 50 mL 80% acetone was added to the precipitate and the extraction procedure mentioned above was repeated. The supernatants obtained from the two centrifugations were pooled and rotary-evaporated at 45 °C. The residue was dissolved in 10 mL methanol to obtain the free phenolic extract solution. The solution was then divided and stored at −20 °C. The weighing and extraction procedures were performed in triplicate.

2.3.2. Extraction of bound phenolic compounds. The method was adapted from Adom, Sorrells and Liu (2003)²⁸ with a few modifications. 40 mL 2 M NaOH solution was added to the precipitate that had undergone the free phenolic extraction process described in Section 2.3.1. The solution obtained was then protected with nitrogen and shaken at room temperature for 1 h. After adjustment to pH 1 using 6 mol L⁻¹ HCl solution, the solution was extracted and degreased with 100 mL n-hexane and then extracted five times with ethyl acetate. All the ethyl acetate extract phases were pooled and rotary-evaporated to dryness at 45 °C. The residue was dissolved in 10 mL methanol to obtain the bound phenolic extract solution, which was then stored at −20 °C. All the procedures were performed in triplicate.

2.3.3. Determination of total phenolic content. The method was adapted from Dewanto, Wu, Adom and Liu (2002)²⁹ with a few modifications. 0.125 mL of the free/bound phenolic extract solution was pipetted and added to 0.5 mL distilled water and 0.5 mL Folin's phenol reagent. After mixing well, the solution was allowed to react for 6 min at 25 °C and then 1.25 mL 7% (m/v) Na₂CO₃ solution and 1.25 mL distilled water were added. After mixing well, this solution was maintained at 25 °C in the dark for 90 min. The absorbance was then determined at a wavelength of 760 nm. The blank control sample was prepared by substituting the sample extract solutions with 0.125 mL methanol. The standard curve was plotted with gallic acid as the standard. The total phenolic content was determined as mg gallic acid equivalents per 100 g dry weight (mg GAE/100 g DW). The abovementioned determinations were performed in triplicate.

2.4. Determination of total flavonoid content

The determination method for total flavonoid content was adapted from Dewanto et al. (2002)²⁹ with a few modifications. 0.3 mL free/bound phenolic extract solution was pipetted and then 1.5 mL distilled water and 0.09 mL of 5% (m/v) NaNO₂ solution were added. After thorough mixing, the solution was allowed to react for 6 min at 25 °C and then 0.18 mL of 10% (m/v) AlCl₃·6H₂O was added. After reacting at 25 °C for a further 5 min, 0.6 mL of 1 mol L⁻¹ NaOH solution was added and the volume was increased to 3 mL with distilled water. The absorbance of the solution was determined at a wavelength of 510 nm. The blank control sample was prepared by substituting the extract solution with 0.3 mL methanol. The standard curve was plotted using catechin as the standard. The total flavonoid content was determined as mg catechin equivalents per 100 g dry weight (mg CE/100 g DW). The abovementioned determinations were performed in triplicate.

2.5. Determination of phenolic composition

The phenolic composition was determined using an Agilent 1200 high performance liquid chromatograph (Waldbronn, Germany) equipped with a VWD ultraviolet detector. The chromatographic conditions were: Zorbax SB-C18 column (4.6 mm × 250 mm, 5 μm) (Agilent, Palo Alto, CA, USA); mobile phase A: acetonitrile, B: 0.4% glacial acetic acid; flow rate: 1.0 mL min⁻¹; column temperature: 30 °C; detection wavelength: 280 nm; gradient elution procedure: 0–40 min, A 5–25%; 40–45 min, A 25–35%; 45–50 min, A 35–50%. The total run time was 50 min and the sample volume was 20 μL. The recovery test, performed using the standards, showed over 96–99% recovery, meeting the requirement for quantitative analysis. The determination, performed in triplicate, was performed by comparing the retention times of the samples with those of the standards.

2.6. Measurement of oxygen radical scavenging capacity (ORAC)

The method was adapted from Zhang et al. (2010)²⁵ with a few modifications. The free/bound phenolic extract solution from the brown rice was dried by nitrogen flow and then diluted with 75 mmol L⁻¹ phosphate buffer so that the total phenolic content was controlled within a certain range. The dilution of the standards and dissolution of samples all used 75 mmol L⁻¹ potassium phosphate buffer (pH 7.4). 20 μL buffer solution (blank), 20 μL Trolox standard solution, 20 μL phenolic extract solution and 200 μL 0.96 μmol L⁻¹ fluorescein working solution were added to the wells of a 96-well plate. The plate was then incubated at 37 °C for 10 min. Furthermore, 20 μL freshly prepared 119 mmol L⁻¹ ABAP solution was added quickly to each well using a multichannel pipettor. The multi-functional microplate reader was immediately launched to detect the fluorescence intensity of each well continuously to monitor the fluorescence decay (37 °C, excitation wavelength 458 nm, emission wavelength 538 nm). The detection was performed for 35 cycles (4.5 min each cycle). The total oxygen radical absorbance capacity (ORAC) index value was determined as Trolox equivalent per gram dry weight (μmol TE per g DW). All procedures were performed in triplicate.

2.7. Measurement of cellular antioxidant activity

The cellular antioxidant activity (CAA) test was conducted using the method of Wolfe and Liu (2007).³⁰ Briefly, human hepatoma cells (HepG2 cells) were inoculated onto the 96-well plate at a cell density of 6 × 10⁴ with 100 μL culture solution (DMEM containing 10% fetal calf serum) in each well. Each inoculated well was rinsed with PBS. Then, 10 μL free/bound phenol extract solution (containing 25 μmol L⁻¹ DCFH-DA) was added to each well and incubated at 37 °C under a 5% CO₂ atmosphere for 1 h. The plate was then taken out and 100 μL HBSS culture medium (containing 600 μmol L⁻¹ ABAP) were added to each well, except for the blank well, wherein 100 μL HBSS culture medium without ABAP were added. The plate was then placed in a fluorescence microplate for scanning, and the fluorescence values of all wells were detected continuously (at 37 °C, excitation wavelength 485 nm, emission wavelength 538 nm) for 12 cycles (each cycle 5 min). The calculation formula used for CAA was:

CAA (unit) = 1 − (∫SA/∫CA)

where ∫SA and ∫CA are the integral areas under the sample time-fluorescence value and control time-fluorescence value curves, respectively. The median effective concentrations (EC50) of the sample polyphenol extracts were calculated according to the median effect principle of log(fa/fu) vs. log(dose), where fa represents the effects of actions of the samples (CAA unit) and fu represents 1 − CAA unit. The calculation of EC50 was based on three parallel tests and then converted into CAA values as μmol quercetin equivalents/100 g dry weight (μmol QE/100 g DW). All the abovementioned procedures were performed in triplicate.

2.8. Statistical analysis

The data analysis and plotting were performed using SPSS 13.0 (SPSS Inc. Chicago, IL, USA). The data were provided in the form of mean ± SD. One-way analysis of variance was used to compare the mean values of the phenolic content and the antioxidant capacity of the different fractions of brown rice using the LSD method at a significance level of p < 0.05.

3. Results

3.1. Weight of pericarp, aleurone layer, embryo and endosperm

The percentages of pericarp, aleurone layer, embryo and endosperm were determined to be 2.0% ± 0.1%, 4.7% ± 0.4%, 2.5% ± 0.4%, and 90.8% ± 0.8% of japonica rice and 2.0% ± 0.2%, 4.7% ± 0.5%, 2.7% ± 0.8%, and 90.6% ± 1.2% of indica rice, respectively. The average percentages of the pericarp, aleurone layer, embryo and endosperm fractions of brown rice were 2.0%, 4.7%, 2.6% and 90.7%, respectively. These measurements were only for research purposes and were not involved in the milling process.

3.2. Total phenolic content

Table 1 provides the free, bound and total phenolic contents of the four tissue fractions of the two types of brown rice.

Table 1 Total phenolic and flavonoid content of four fractions in two types (subspecies) of whole brown rice^a

Rice type	Tissue fraction	Free	Bound	Total
a # values with no letters in common in each column are significantly different (p < 0.05), ## values in parentheses indicate percentage contribution to the total phenolics, * values of each fraction in japonica and indica rice are significantly different (p < 0.05).b mg GAE/100 g DW.c mg CE/100 g DW.
Phenolics^b
Japonica	Pericarp	484.4 ± 5.2a^# (55.7)^##	385.1 ± 3.1a (44.3)	869.5 ± 4.3a
	Aleurone layer	473.3 ± 9.0a (59.7)	319.5 ± 8.6b (40.3)	792.8 ± 6.3b
	Embryo	351.6 ± 5.8b (70.9)	144.5 ± 3.4c (29.1)	496.1 ± 2.7c
	Endosperm	38.1 ± 1.3c (65.3)	20.3 ± 0.6d (34.7)	58.4 ± 1.9d
Indica	Pericarp	327.4 ± 2.2a* (62.3)	198.0 ± 3.2a* (37.7)	525.5 ± 1.2a*
	Aleurone layer	323.3 ± 4.2a* (64.2)	179.9 ± 6b* (35.8)	503.2 ± 8.6b*
	Embryo	171.2 ± 3.1b* (65)	92.2 ± 2c* (35)	263.4 ± 4.3c*
	Endosperm	37.2 ± 0.6c (67.2)	18.1 ± 0.5d (32.8)	55.3 ± 0.3d
Flavonoids^c
Japonica	Pericarp	457.6 ± 9.8a (69.8)	198.0 ± 3.2a (30.2)	655.6 ± 8.2a
	Aleurone layer	383.5 ± 15.1b (66.3)	194.8 ± 6.2a (33.7)	578.4 ± 15.6b
	Embryo	299.7 ± 12.2c (76.5)	92.2 ± 2.0b (23.5)	391.9 ± 13.6c
	Endosperm	33.8 ± 2.1d (63)	19.8 ± 2.8c (37)	53.6 ± 1.9d
Indica	Pericarp	462.7 ± 4.4a* (58.8)	324.8 ± 4.5a* (41.2)	787.5 ± 5.4a*
	Aleurone layer	413.7 ± 1.4b* (57.8)	301.6 ± 15.8b* (42.2)	715.3 ± 15.7b*
	Embryo	239.7 ± 0.5c (68.8)	108.5 ± 3.2c* (31.2)	348.1 ± 3.0c*
	Endosperm	35.0 ± 2.3d (67.3)	17.0 ± 2.0d (32.7)	52.0 ± 1.1d

---

There were significant differences in the free, bound and total phenolic contents between the four tissue fractions of japonica and indica brown rice (p < 0.05). For japonica rice, the total phenolic content was highest in the pericarp (p < 0.05), followed by the aleurone layer and the embryo. The total phenolic content was lowest in the endosperm (p < 0.05). The order of the four tissue fractions of indica brown rice was very similar to the ranking of japonica brown rice.

Comparing indica and japonica rice, the distribution of free, bound and total phenolic contents among the four fractions was very similar. However, the values of free, bound and total phenolic contents of japonica compared with indica were 48.0%, 94.5% and 65.5% higher (p < 0.05), in the pericarp, 46.4%, 77.6% and 57.6% higher (p < 0.05), in the aleurone layer, 105.4%, 56.7% and 88.3% higher (p < 0.05), in the embryo and 2.4%, 12.2% and 5.6% higher (p < 0.05) in the endosperm, respectively. This may have been due to the genetic differences between the subspecies or types.

3.3. Total flavonoid content

Table 1 shows the free, bound and total flavonoid contents in the four fractions of the two types of brown rice.

There were significant differences in the free, bound and total flavonoid contents between the four fractions of japonica and indica brown rice (p < 0.05). The trend of the flavonoids of the two types of brown rice was the same as that of the phenolics.

Comparing indica and japonica rice, there was a similar distribution of free, bound and total phenolic contents among the four fractions. The values of bound and total flavonoid content of indica compared with japonica were 64.0% and 20.1% higher (p < 0.05), in the pericarp and 54.8% and 23.7% higher (p < 0.05) in the aleurone layer, respectively. The genetic differences between these subspecies or types may have led to these differences. The values of free, bound and total flavonoid contents were more or less similar in both the embryo and endosperm fractions for the two types.

3.4. Phenolic composition

Table 2 shows the individual phenolic composition and contents of four successive fractions in the two types of brown rice. There were significant differences in free and bound phenolic contents among the four fractions in the two types of brown rice (p < 0.05). The composition and forms of the phenolic compounds present were similar for the two types of brown rice. Ferulic and coumaric acid were predominant in brown rice, both existing mainly in the bound form. Of the phenolics, ferulic acid had the highest content and was richest in the pericarp (mean value = 2204.7 μg g⁻¹ DW). Coumaric acid was also richest in the pericarp (mean value = 944.7 μg g⁻¹ DW). These results suggest that phenolic acids are concentrated mainly in the pericarp fraction of whole brown rice. The much smaller amounts of epicatechin detected in the embryo existed in the free form. The epicatechin content in the embryo (mean value = 535.9 μg g⁻¹ DW) was significantly higher than in the other fractions. Gallic, protocatechuic, hydroxybenzoic, chlorogenic, vanillic, caffeic, syringic and isoferulic acids, as well as catechin and quercetin were detected at low or trace levels in all extracts of the identified phenolic acids.

Table 2 Phenolic composition of four fractions in two types of whole brown rice

Rice type	Tissue fraction	Free (μg g^−1)	Bound (μg g^−1)	Total (μg g^−1)
a Values with no letters in common in each column are significantly different (p < 0.05).b Values in parentheses indicate percentage contribution to the total phenolic acids.
Japonica rice
Gallic acid	Pericarp	tr	nd	tr
	Aleurone layer	3.2 ± 0.3a^a (72.9)^b	1.2 ± 0.1 (27.1)	4.4 ± 0.3a
	Embryo	1.3 ± 0.1b (100)	nd	1.3 ± 0.1b
	Endosperm	tr	nd	tr
Protocatechuic acid	Pericarp	9.0 ± 0.5a (56.1)	7.1 ± 0.8a (43.9)	16.1 ± 1.4a
	Aleurone layer	10.5 ± 0.8b (83.9)	2.0 ± 0.2b (16.1)	12.5 ± 0.5b
	Embryo	4.2 ± 0.7c (100)	nd	4.2 ± 0.7c
	Endosperm	tr	nd	tr
p-Hydroxybenzoic acid	Pericarp	11.2 ± 0.9b (39.4)	17.2 ± 0.8b (60.6)	28.4 ± 0.9b
	Aleurone layer	12.8 ± 1.1a (38.6)	20.3 ± 0.3a (61.4)	33.0 ± 0.8a
	Embryo	12.2 ± 0.8a (63.7)	7.0 ± 0.1c (36.3)	19.2 ± 0.8c
	Endosperm	2.0 ± 0.1c (100)	nd	2.0 ± 0.1d
Chlorogenic acid	Pericarp	nd	14.8 ± 0.6b (100)	14.8 ± 0.6c
	Aleurone layer	9.3 ± 0.1 (36.2)	16.4 ± 1.1b (63.8)	25.7 ± 1a
	Embryo	nd	23.0 ± 1.3a (100)	23.0 ± 1.3b
	Endosperm	2.4 ± 0.1 (20.7)	9.1 ± 0.5c (79.3)	11.5 ± 0.5d
Vanillic acid	Pericarp	1.9 ± 0.6a (19)	8.2 ± 0.6a (81)	10.1 ± 1.1b
	Aleurone layer	4.0 ± 1.7a (31.3)	8.8 ± 1.6a (68.7)	12.8 ± 0.2a
	Embryo	2.6 ± 1.1a (100)	tr	2.6 ± 1.1c
	Endosperm	tr	nd	tr
Caffeic acid	Pericarp	tr	tr	tr
	Aleurone layer	tr	nd	tr
	Embryo	9.7 ± 1 (100)	nd	9.7 ± 1
	Endosperm	nd	nd	nd
Syringic acid	Pericarp	11.9 ± 0.7b (54.4)	10 ± 0a (45.6)	22 ± 0.7b
	Aleurone layer	13.1 ± 1.6b (56.5)	10.1 ± 0.3a (43.5)	23.2 ± 1.9b
	Embryo	19.4 ± 0.6a (67.3)	9.4 ± 1a (32.7)	28.8 ± 0.9a
	Endosperm	1.5 ± 0.1c (100)	nd	1.5 ± 0.1c
Coumaric acid	Pericarp	20.4 ± 2.9b (2.1)	929.6 ± 14.8a (97.9)	950 ± 13.9a
	Aleurone layer	15.6 ± 2.4c (1.8)	832.6 ± 65.8b (98.2)	848.2 ± 63.5b
	Embryo	35.7 ± 2.9a (8.7)	373 ± 10.2c (91.3)	408.7 ± 7.8c
	Endosperm	1.5 ± 0.2d (7.5)	18.9 ± 0.4d (92.5)	20.5 ± 0.2d
Ferulic acid	Pericarp	45.5 ± 1.4b (2)	2192.7 ± 38.3a (98)	2238.2 ± 38.5b
	Aleurone layer	40.6 ± 3.8b (1.5)	2692.2 ± 166b (98.5)	2732.8 ± 162.5a
	Embryo	140.4 ± 6.7a (14.3)	843 ± 12.3c (85.7)	983.4 ± 9.3c
	Endosperm	4.7 ± 0.1c (3.7)	123.9 ± 4.2d (96.3)	128.6 ± 4.2d
Isoferulic acid	Pericarp	1.2 ± 0.3b (1.4)	86.2 ± 6.6b (98.6)	87.4 ± 6.8b
	Aleurone layer	tr	124.5 ± 13.3a (100)	124.5 ± 13.3a
	Embryo	4.8 ± 2a (9.3)	46.4 ± 7.4c (90.7)	51.1 ± 8.9c
	Endosperm	nd	6.3 ± 0.3d (100)	6.3 ± 0.3d
Catechin	Pericarp	4.1 ± 0.6b (100)	nd	4.1 ± 0.6b
	Aleurone layer	6.2 ± 1.3a (100)	nd	6.2 ± 1.3a
	Embryo	tr	nd	tr
	Endosperm	nd	nd	nd
Epicatechin	Pericarp	5.7 ± 3.1b (5)	109.2 ± 4a (95)	114.9 ± 7b
	Aleurone layer	tr	106.9 ± 11.4a (100)	106.9 ± 11.4b
	Embryo	612.5 ± 34.2c (87.6)	86.8 ± 3b (12.4)	699.4 ± 36.1a
	Endosperm	tr	tr	tr
Quercetin	Pericarp	8.8 ± 0.5c (28.1)	22.6 ± 0.6a (71.9)	31.4 ± 1.1a
	Aleurone layer	11.6 ± 0.2b (37.1)	19.6 ± 1.1b (62.9)	31.2 ± 1a
	Embryo	15.4 ± 1.8a (62.8)	9.1 ± 0.3c (37.2)	24.5 ± 2b
	Endosperm	2.6 ± 0.4d (100)	tr	2.6 ± 0.4c
Indica rice
Gallic acid	Pericarp	1.2 ± 0.1a (100)	nd	1.2 ± 0.1a
	Aleurone layer	1.3 ± 0.1a (100)	nd	1.3 ± 0.1a
	Embryo	tr	nd	tr
	Endosperm	tr	nd	tr
Protocatechuic acid	Pericarp	1.6 ± 0.2b (24.3)	5 ± 0.7 (75.7)	6.7 ± 0.7a
	Aleurone layer	1.8 ± 0.2b (100)	nd	1.8 ± 0.2b
	Embryo	7.3 ± 0.8a (100)	nd	7.3 ± 0.8a
	Endosperm	tr	nd	tr
p-Hydroxybenzoic acid	Pericarp	5.1 ± 0.1b (38.3)	8.3 ± 0.3a (61.7)	13.4 ± 0.3a
	Aleurone layer	6.3 ± 0.1a (51.6)	5.9 ± 0.4b (48.4)	12.1 ± 0.5a
	Embryo	6.2 ± 0.1a (100)	nd	6.2 ± 0.1b
	Endosperm	1.6 ± 0.1c (100)	nd	1.6 ± 0.1c
Chlorogenic acid	Pericarp	nd	23.6 ± 2.2b (100)	23.6 ± 2.2b
	Aleurone layer	nd	17.9 ± 2.5c (100)	17.9 ± 2.5c
	Embryo	9.1 ± 0.1 (23.5)	29.8 ± 3.0a (76.5)	38.9 ± 3.1a
	Endosperm	2.9 ± 0.1 (26.7)	7.8 ± 0.4d (73.3)	10.7 ± 0.4d
Vanillic acid	Pericarp	tr	15.9 ± 2.0a (100)	15.9 ± 2.0a
	Aleurone layer	tr	3.3 ± 2.2b (100)	3.3 ± 2.2b
	Embryo	tr	tr	tr
	Endosperm	nd	nd	nd
Caffeic acid	Pericarp	1.7 ± 0.6b (100)	nd	1.7 ± 0.6b
	Aleurone layer	2.3 ± 0.1ab (100)	nd	2.3 ± 0.1b
	Embryo	3 ± 0.2a (100)	nd	3 ± 0.2a
	Endosperm	tr	nd	tr
Syringic acid	Pericarp	8 ± 0.6a (34.8)	15 ± 1.1a (65.2)	22.9 ± 0.8a
	Aleurone layer	8 ± 0.6a (43.8)	10.3 ± 1.7b (56.2)	18.3 ± 2.2b
	Embryo	5.9 ± 0.2b (43.6)	7.6 ± 0.7c (56.4)	13.5 ± 0.9c
	Endosperm	1.2 ± 0.1c (100)	nd	1.2 ± 0.1d
Coumaric acid	Pericarp	10.2 ± 2.0a (1.1)	929.2 ± 97.6a (98.9)	939.3 ± 98.0a
	Aleurone layer	6.3 ± 0.5b (1.5)	408.1 ± 117.5b (98.5)	414.4 ± 117.4b
	Embryo	4.2 ± 0.6c (2.9)	140.7 ± 39.2c (97.1)	144.9 ± 39.2c
	Endosperm	tr	11.5 ± 0.8 (100)	11.5 ± 0.8d
Ferulic acid	Pericarp	27.7 ± 1.2b (1.2)	2216.7 ± 229a (98.8)	2244.4 ± 229.4a
	Aleurone layer	21.3 ± 1.2c (1.6)	1304 ± 370.3b (98.4)	1325.3 ± 371.5b
	Embryo	36.8 ± 1a (10.5)	313.3 ± 84.4c (89.5)	350.1 ± 85.3c
	Endosperm	3.5 ± 0.3d (3.4)	101.3 ± 3.8d (96.6)	104.8 ± 4d
Isoferulic acid	Pericarp	tr	119.2 ± 14.6a (100)	119.2 ± 14.6a
	Aleurone layer	tr	81.7 ± 25.8b (100)	81.7 ± 25.8b
	Embryo	5.3 ± 0.7 (21.2)	19.9 ± 6.9c (78.8)	25.2 ± 6.7c
	Endosperm	nd	6.9 ± 0.9d (100)	6.9 ± 0.9d
Catechin	Pericarp	5.1 ± 1b (100)	nd	5.1 ± 1b
	Aleurone layer	7.4 ± 1.1a (100)	nd	7.4 ± 1.1a
	Embryo	tr	nd	tr
	Endosperm	nd	nd	nd
Epicatechin	Pericarp	tr	150.3 ± 16.6a (100)	150.3 ± 16.6b
	Aleurone layer	tr	74.9 ± 24b (100)	74.9 ± 24.0c
	Embryo	459.2 ± 17.2 (93.5)	32.2 ± 10.2c (6.5)	491.3 ± 25.5a
	Endosperm	tr	tr	tr
Quercetin	Pericarp	9.5 ± 0.9b (26)	26.9 ± 2a (74)	36.4 ± 2.6a
	Aleurone layer	10.3 ± 1.3b (39.9)	15.6 ± 2.8b (60.1)	25.9 ± 3.7b
	Embryo	16.3 ± 1a (100)	nd	16.3 ± 1c
	Endosperm	2.6 ± 0.2c (100)	nd	2.6 ± 0.2d

---

3.5. Antioxidant capacity

3.5.1. ORAC. Table 3 shows the free and bound ORAC values in the four fractions of the two types of brown rice.

Table 3 ORAC antioxidant capacities (Trolox equivalent/g dry weight) of four fractions in two types of whole brown rice

Rice type	Tissue fraction	Free	Bound	Total
a Values with no letters in common in each column are significantly different (p < 0.05).b Values in parentheses indicate percentage contribution to the total ORAC value.c Values of each fraction in japonica and indica rice are significantly different (p < 0.05).
Japonica rice	Pericarp	297.1 ± 32a^a (49.4)^b	303.9 ± 19.5a (50.6)	601.0 ± 13.1a
	Aleurone layer	293.1 ± 25.9a (54.1)	248.5 ± 10.8a (45.9)	541.6 ± 15.8b
	Embryo	110.3 ± 1.2b (72.5)	41.9 ± 4.7b (27.5)	152.2 ± 5.7c
	Endosperm	18.7 ± 0.4c (81.1)	4.3 ± 0.5c (18.9)	23.0 ± 0.8d
Indica rice	Pericarp	251.3 ± 27.1a (50.8)	243 ± 14.2a^c (49.2)	494.3 ± 15.6a^c
	Aleurone layer	238.1 ± 27.8a (54.7)	197.4 ± 19.8a^c (45.3)	435.5 ± 8.4b^c
	Embryo	99.5 ± 25.6b (78.1)	28 ± 7.2b^c (21.9)	127.5 ± 32.6c
	Endosperm	15 ± 0.8c^c (73.7)	5.3 ± 0.3c^c (26.3)	20.3 ± 0.4d^c

---

There were significant differences in the free, bound and total ORAC values among the four fractions of japonica and indica brown rice (p < 0.05). In both types of brown rice, similar to the phenolics, the total ORAC values were highest in the pericarp (p < 0.05) and lowest in the endosperm (p < 0.05). The sequence of the total ORAC values was pericarp > aleurone layer > embryo > endosperm.

Comparing indica and japonica rice, the distribution of free, bound and total ORAC values among the four fractions was similar. However, the free, bound and total ORAC values in the pericarp of japonica were 18.2%, 25.1% and 21.6% higher (p < 0.05) than those of indica, respectively, and the same values in the aleurone layer were 23.1%, 25.9% and 24.4% higher (p < 0.05), respectively, than those of indica. These differences may have been caused by the genetic differences between the subspecies or types. The free, bound and total ORAC values in both the embryo and endosperm fractions of japonica were slightly higher or very similar to those of indica.

3.5.2. CAA. Table 4 shows the free and bound CAA values in the four fractions of the two types of brown rice.

Table 4 CAA antioxidant capacity (μmol QE/100 g DW) of four fractions in two types of whole brown rice

Rice type	Tissue fraction	Free	Bound	Total
a Values with no letters in common in each column are significantly different (p < 0.05).b Values in parentheses indicate percentage contribution to the total CAA value.c Values of each fraction in japonica and indica rice are significantly different (p < 0.05).
Japonica rice	Pericarp	147.8 ± 9.3b^a (28.6)^b	368.6 ± 19a (71.4)	516.4 ± 28a
	Aleurone layer	93.4 ± 8.5c (19.8)	379.4 ± 23.9a (80.2)	472.8 ± 39.7b
	Embryo	279.7 ± 12.2a (83.1)	56.7 ± 1.4b (16.9)	336.5 ± 11.7c
	Endosperm	10.4 ± 0.4d (44.3)	13.1 ± 1.1c (55.7)	23.5 ± 0.8d
Indica rice	Pericarp	117.1 ± 14.3b^c (29.4)	281.2 ± 8.5b^c (70.6)	398.3 ± 13.3b^c
	Aleurone layer	120.8 ± 8b^c (22.4)	419.1 ± 17.7a (77.6)	539.9 ± 27.4a^c
	Embryo	359.5 ± 10.6a^c (70.5)	150.1 ± 6.9c^c (29.5)	509.5 ± 13.7a^c
	Endosperm	8 ± 0.3c^c (43.1)	10.6 ± 0.5d^c (56.9)	18.6 ± 0.8c^c

---

There were significant differences in the free, bound and total CAA values among the four fractions of japonica and indica brown rice (p < 0.05). For japonica rice, the total CAA values were highest in the pericarp (p < 0.05) and lowest in the endosperm (p < 0.05). The sequence of the total CAA values was pericarp > aleurone layer > embryo > endosperm, whereas the sequence of the total CAA values of indica rice was aleurone layer > embryo > pericarp > endosperm. The discrepancy between the ORAC and CAA activity of these compounds could be at least partly attributed to their difference in chemical structure, which affects their ability to scavenge free radicals and the levels of cellular absorption and metabolism.

Comparing indica and japonica rice, a similar distribution of free, bound and total CAA values among the four fractions was observed. The values of free, bound and total CAA values in the pericarp of japonica were 26.2%, 31.1% and 29.7% higher (p < 0.05) than those of indica, respectively, whereas the values of free, bound and total CAA in the aleurone layer of indica were 29.3%, 10.5% and 14.2% higher (p < 0.05) than those of japonica, respectively, and in the embryo, 28.5%, 164.7% and 51.4% higher (p < 0.05) than those of japonica, respectively. The genetic differences between these subspecies or types may account for these differences. The free, bound and total CAA values in the endosperm fraction were almost the same in the two types of rice.

4. Discussion

4.1. Phenolic contents of four tissue fractions of whole brown rice

Brown rice is botanically defined as the fruit of the rice plant; however, its seed is entirely covered with a thin pericarp. The testa that covers the seed is also very thin; it contains the aleurone layer, the embryo and the endosperm.³¹ The distribution of phenolics has not been examined in the four tissue fractions of brown rice. At the tissue level, higher concentrations of phenolic compounds are found in the outer layers of plants, e.g. in the epidermis, rather than in the inner layers.³² A previous study indicated that the concentrations of phenolic acids decreased from the aleurone layer to the endosperm in brown rice.³³ Another study also found that the bran and embryo exhibited higher free and bound phenolic contents than the endosperm.²⁴ The results of the present study provide a more comprehensive understanding of the distribution of antioxidants in four tissue fractions of brown rice: the pericarp, aleurone layer, embryo and endosperm. Analysis of the free and bound phenolic contents in these tissue fractions suggested that they were highest in the pericarp and lowest in the endosperm. The phenolic content tended to decrease progressively from the outside to the center of brown rice, in a similar way to other nutrients.^16,34 These results have demonstrated that the concentration of phenolics varies in the different fractions of brown rice, suggesting the potential for the greater use of these fractions as different sources of concentrated antioxidants from natural whole brown rice.

Phenolic acids and flavonoids are important polyphenols in plants. Our results have shown that the phytochemicals (phenolics and flavonoids) in these four tissue fractions (pericarp, aleurone layer, embryo and endosperm) exist mainly in the free and bound forms. The bound forms of the phenolics in the pericarp, aleurone layer, embryo and the endosperm provided, on an average, 41.8%, 38.5%, 31.2% and 33.8% and the bound forms of the flavonoids provided 36.2%, 38.4%, 27.1% and 34.9%, respectively (sum of the bound values of two types of rice/sum of the total values of two types of rice). Liu (2007) has shown that bound phenolics cannot be decomposed by human digestive enzymes; after the phenolics reach the colon, the ester bond and the macromolecules on the cell wall will be destroyed during fermentation by microbial flora, thus releasing the phenolics and providing a constant source for humans.³⁵ The data in the present study has shown that the pericarp and aleurone layer fractions contributed more bound phenolics than the embryo and endosperm fractions. This indicated that the pericarp and aleurone layer may deliver a higher level of phenolics to the colon and therefore have positive health effects because of these higher proportions of bound phenolics. One study has demonstrated that the bound phenolic content of whole grain brown rice contributed more than 60% to the total.²⁸ Another reported that the bound fraction provided 12.2% of phenolics and 29.3% of flavonoids in rice bran and 26.7% of phenolics and 40.7% of flavonoids in polished rice.¹⁰ The different levels of bound phenolics are primarily not only attributable to the various phenolic acids in the different tissue fractions, but also to the rice types. Phenolic acids in plants are not uniformly distributed at either the tissue or cellular levels. At the same tissue level, higher concentrations of phenolic acids are found in the outer layers of plants than in the inner layers.^24,32 The data in the present study have comprehensively documented the distribution of free and bound phytochemicals (phenolic and flavonoids) in the four tissue fractions of whole brown rice.

In whole brown rice, the pericarp, aleurone layer, and embryo fractions contribute 13.0%, 28.5% and 8.8% of the total phenolics and 14.1%, 29.7% and 9.1% of the total flavonoids, respectively. The endosperm fraction contributes the remaining 49.7% of the total phenolics and 47.1% of the total flavonoids (Fig. 1a and b). Thus, whole brown rice is more abundant in phenolic resources than the endosperm fraction, which agrees with Zhou, Robards, Helliwell and Blanchard (2004) who reported that phenolics were distributed mainly in the rice bran.³⁶ This indicates that brown rice is a good dietary source of antioxidants when compared with the polished rice/endosperm generally consumed in the human diet. Importantly, our results have shown that in the rice bran fraction, the aleurone layer contributed most of the phenolics and flavonoids, followed by the embryo and endosperm fractions. Therefore, the present analysis of the contributions of the four tissue fractions of whole brown rice has provided data that can lead to improving its applications; for example, whole brown rice may be further processed into bran, aleurone layer and embryo rice and used in food products.

Fig. 1 Percentage contributions of tissue fractions to total values (weight percentage × total values/sum of percentage contributions of embryo, aleurone layer and endosperm) of (a) phenolics, (b) flavonoids, (c) ORAC, and (d) CAA in whole brown rice samples based on the naturally occurring proportions of pericarp, aleurone layer, embryo and endosperm fractions.

These food products may have different requirements regarding sensory properties, quality and different health benefits for different groups of consumers. In the past, because the distribution of phenolics in the different fractions of brown rice had not been clear, the consumption of whole brown rice had been overemphasized to consumers; thus, its further promotion to consumers should be re-evaluated. In fact, embryo rice is popular in cooking because of its taste, and because it retains some nutrients from the embryo and part of the aleurone layer. The results of the present study provide the necessary information for evaluating the health benefits of consuming embryo rice. These data also provide help for judicious control of the degree of milling during the processing of whole brown rice with regard to its sensory qualities and phenolic content. In general, brown rice milled to a higher degree has a better appearance; however, the phytochemicals, beneficial for human health, are discarded.² This study presents data of the percentages of phenolics, flavonoids and antioxidant activity in different rice forms found in the market, including semi-brown rice, embryo rice, lightly milled rice and polished rice, compared to whole brown rice. Therefore, the present study has provided knowledge that may encourage the consumption of semi-brown rice, embryo rice, and lightly milled rice rather than brown rice or polished rice (Fig. 2).

Fig. 2 Percentage contributions of each type of sample to the total values of weight, phenolics, flavonoids, ORAC and CAA in whole brown rice. *These samples are (a) brown rice, (b) semi-brown rice (detached pericarp/testa from brown rice), (c) embryo rice (detached pericarp/testa and a small percentage of aleurone layer from brown rice), (d) lightly milled rice (detached pericarp/testa, embryo and most of aleurone layer from brown rice), and (e) polished rice (detached pericarp/testa, embryo and most of aleurone layer from brown rice), commercially available, based on the milling process. **Percentage contributions of whole brown rice, semi-brown rice (sum of percentage contributions of embryo, aleurone layer and endosperm), embryo rice (sum of percentage contributions of embryo, 2/3 aleurone layer and endosperm), lightly milled rice (sum of percentage contributions of 1/2 aleurone layer and endosperm) and polished rice (percentage contribution of endosperm) to total values of weight, phenolics, flavonoids, ORAC and CAA in whole brown rice were used to estimate their nutritional values.

4.2. Phenolic components of four tissue fractions of brown rice

The distribution of phenolic acids in brown rice was not uniform, being more concentrated in the bran layer and less in the endosperm.^33,37 Recent studies have concentrated on phenolics in brown rice or the rice bran layers of whole brown rice. A previous study found that seven different free and bound phenolic acids (gallic, protocatechuic, caffeic, clove, chlorogenic, coumaric and ferulic acids) existed mainly in the rice bran fractions, with most present in the bound form.¹⁰ The present results were consistent with this conclusion that the phenolic acids were mainly present in the rice bran fraction. Zhou et al. (2004) found that brown rice contained more phenolic acids (ferulic, coumaric, gallic, vanillic, caffeic and syringic acids) than polished rice.³⁶ The present study has shown that the coumaric acid contents in the pericarp, aleurone layer and embryo were 59, 39 and 17 times higher, respectively, than in the endosperm, whereas the ferulic acid contents were 19, 17 and 8 times higher, respectively. The main active phenolic acids in rice bran were coumaric acid and ferulic acid, which mainly connect to sugar residues or side chains of xylan polysaccharides in the cell wall through ester linkages.³⁴ These phenolic acids are also commonly present in the bound form and as components of complex structures. Overall, the significant differences in the distribution of these phenolic acids between the pericarp, aleurone layer, embryo and endosperm fractions have now been defined.

In the present study, flavonoids such as catechin, epicatechin and quercetin were detected in different parts of whole brown rice, an aspect previously unreported. The class of flavonoids containing (−)-epicatechin, (+)-catechin and quercetin are widespread in fruits and whole grains. The present study detected them in both the free and bound form, another aspect previously unreported. In the pericarp, aleurone layer and embryo fractions, the content of (−)-epicatechin was relatively higher, with average values of 132.6, 90.9 and 595.4 μg g⁻¹, respectively, and the quercetin content had average values of 33.9, 28.6 and 20.4 μg g⁻¹, respectively. These compounds were not detected in the endosperm fraction. The content of (+)-catechin was found to be low or not detected in the pericarp, aleurone layer, embryo and endosperm fractions. It should be noted that in the present study, these individual flavonoid compounds have been analyzed by a direct comparison with corresponding standards based on retention times. Therefore, this tentative identification method needs further confirmation using other methods, such as HPLC-MS, to give a positive identification.

4.3. Antioxidant activity of four tissue fractions of whole brown rice

Four different fractions of brown rice have been surveyed using both the ORAC and CAA assays. The ORAC assay is a traditional chemical method based on the hydrogen atom transfer reaction mechanism. The CAA assay is an improvement over the traditional chemistry-based antioxidant activity assay. It provides a better prediction of antioxidant behavior in biological systems, as it takes into account some aspects of cell uptake, metabolism and distribution of bioactive compounds.^26,27,38 The results of the present study have shown that the four different fractions of whole brown rice exhibited a wide range of antioxidant potentials. Although the range of ORAC and CAA mean values did not vary greatly, the total antioxidant activity showed statistically significant differences between the fractions (p < 0.05).

Phenolic compounds contribute to antioxidant activity; however, reports examining the contributions of free and bound antioxidant activity in the pericarp, aleurone layer, embryo and endosperm fractions are limited. In the present study, the results of the total ORAC values agreed with those of Ti et al. (2014), who determined these values in the rice bran and milled rice fractions of five indica rice varieties. The ORAC values ranged from 182.2 to 221.2 μmol TE per g DW in rice bran and from 16.1 to 24.5 μmol TE per g DW in milled rice, with the antioxidant activity mainly distributed in the free form. It is difficult to compare the CAA values, as there are no other reports of these values for brown rice. However, the present study has shown that the CAA values in the pericarp, aleurone layer and endosperm exist mainly in the bound form, whereas in the embryo, they exist mainly in the free form. This result disagreed with previous articles that claimed that free phenolics formed the majority of antioxidants in brown rice. This difference may be due to differences in the rice varieties and growing environments. The composition of free and bound individual phenolics was also different, with significant differences in CAA values between the individual phenolics. Finally, differences in the solubility, molecular size and polarity of the wide variety of compounds present in grains, fruits and vegetables confer unique bioactivity and distribution at the cellular, organ and tissue levels.³⁰ Thus, different phenolic compounds showed significant differences in CAA antioxidant activity.

As mentioned for whole brown rice (Fig. 1c and d), the distributions of the total ORAC and CAA values in the pericarp, aleurone layer, embryo and endosperm fractions were 18.2%, 38.0%, 11.1% and 32.8% for ORAC, and 14.6%, 38.0%, 16.9% and 30.5% for CAA, respectively. The endosperm fraction, the whitest portion of the grain, is generally favored because of its better taste and appearance. The present data has indicated that although the endosperm fraction was proportionately larger than the other fractions of brown rice, its antioxidant activity was not the highest. A previous study has reported that the rice bran/embryo fraction had a higher antioxidant activity than the endosperm fraction,¹⁰ which was basically consistent with the results of the present research. However, the antioxidant activity contribution of each fraction to brown rice after actual processing was previously unclear because of the complex structure of rice bran. The present research has found that, although the aleurone layer only forms a small proportion of whole brown rice, this layer was the largest contributor of antioxidant activity; moreover the endosperm was ranked second despite forming a larger proportion of whole brown rice. The pericarp and embryo fractions contributed nearly 30% of the antioxidant activity, but represent only 4.6% of the weight of whole brown rice. The present study has provided information on the contribution of four successive fractions to the free, bound and total antioxidant activities of brown rice using two assays. This information is necessary for processing whole brown rice and its products for the food and pharmaceutical markets. The present study is part of ongoing efforts to promote added value to the production and use of brown rice for preventing human chronic diseases related to oxidative stress.

5. Conclusions

To summarize, this study has shown that the contents of free and bound phenolics and antioxidant activity in four tissue fractions were significantly different. The highest phenolic content and antioxidant activity were in the pericarp fraction, whereas those in the endosperm fraction were lower. The phenolic contents and antioxidant activity in the pericarp, aleurone layer, embryo and endosperm in indica and japonica rice were present both in the free and bound forms. The aleurone layer fraction contributed greater or similar total phenolic content, composition and antioxidant activity compared with the endosperm fraction, although it forms a smaller proportion of whole brown rice. Thirteen phenolic compounds (gallic, protocatechuic, hydroxybenzoic, chlorogenic, vanillic, caffeic, syringic, isoferulic, coumaric and ferulic acids, catechin, epicatechin, and quercetin) were detected in the four tissue fractions; ferulic acid content was found in the highest amount, followed by coumaric acid. Measuring the antioxidant activity of grains using cell culture is an important step in screening for potential bioactivity and is biologically more representative than data obtained from chemistry-based antioxidant activity assays. The present study used ORAC and CAA assays to confirm the significant antioxidant effects of different fractions of brown rice. Therefore, this study will have significant value for deciding the type of rice processing, including different tissue fractions, and will provide guidance on the consumption of whole brown rice.

Acknowledgements

This study was supported by the National Key Technology Research and Development Program for the 12th Five-year Plan (No. 2012BAD33B10 and 2012BAD33B08), the National “948” project (2014-Z52), the Special Fund for Public Welfare Industry (Agriculture) (Research projects 201303071 and 201403063-2) and the Innovation Capacity Construction Special Project of the Main Scientific Research Organization of Guangdong Province (2011).

References

1. FAOSTAT (2009). FAO Statistics Division 2009. 18 September 2009. <http://faostat.fao.org/>.
2. L. Lamberts, E. de Bie, G. E. Vandeputte, W. S. Veraverbeke, V. Derycke, W. de Man and J. A. Delcour, Effect of milling on colour and nutritional properties of rice, Food Chem., 2007, 100, 1496–1503.
3. M. W. Zhang, Special rice and its processing technology, 2000.
4. L. Liu, J. J. Guo, R. F. Zhang, Z. C. Wei, Y. Y. Deng, J. X. Guo and M. W. Zhang, Effect of degree of milling on phenolic profiles and cellular antioxidant activity of whole brown rice, Food Chem., 2015, 100, 1496–1503.
5. O. Okarter and R. H. Liu, Health benefits of whole grain phytochemicals, Crit. Rev. Food Sci. Nutr., 2010, 50, 193–208.
6. O. Benavente-Garcia, J. Castillo, F. R. Marin, A. Ortuno and J. A. Del Rio, Uses and properties of citrus flavonoids, J. Agric. Food Chem., 1997, 45, 4505–4515.
7. C. Manach, A. Mazur and A. Scalbert, Polyphenols and prevention of cardiovascular diseases, Curr. Opin. Lipidol., 2005, 16, 77–84.
8. E. Middleton, C. Kandaswami and T. C. Theoharides, The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer, Pharmacol. Rev., 2000, 52, 673–751.
9. S. Samman, P. M. Lyons Wall and N. C. Cook, Flavonoids and coronary heart disease: Dietary perspectives, 1998, pp. 469–482.
10. H. H. Ti, Q. Li, R. F. Zhang, M. W. Zhang, Y. Y. Deng, Z. C. Wei, J. W. Chi and Y. Zhang, Free and bound phenolic profiles and antioxidant activity of milled fractions of different indica rice varieties cultivated in Southern China, Food Chem., 2014, 159, 166–174.
11. K. L. Liu and Z. X. Gu, Selenium accumulation in different brown rice cultivars and its distribution in fractions, J. Agric. Food Chem., 2009, 57, 695–700.
12. A. Abdul-Hamid, R. R. Raja Sulaiman, A. Osman and N. Saari, Preliminary study of the chemical composition of rice milling fractions stabilized by microwave heating, J. Food Compos. Anal., 2007, 20, 627–637.
13. J. K. Park, S. S. Kim and K. O. Kim, Effect of milling ratio on sensory properties of cooked rice and on physicochemical properties of milled and cooked rice, Cereal Chem., 2001, 78, 151–156.
14. A. P. Resurreccion, B. O. Juliano and Y. Tanaka, Nutrient content and distribution in milling fractions of rice grain, J. Agric. Food Chem., 1979, 30, 475–481.
15. N. Singh, H. Singh, K. Kaur and B. M. Singh, Relationship between the degree of milling, ash distribution pattern and conductivity in brown rice, Food Chem., 2000, 69, 147–151.
16. T. Itani, M. Tamaki, E. Arai and T. Horino, Distribution of amylose, nitrogen, and minerals in rice kernels with various characters, J. Agric. Food Chem., 2002, 50, 5326–5332.
17. C. Prom-u-thai, C. Sanchai, B. Rerkasem, S. Jamjod, S. Fukai, I. D. Godwin and L. Huang, Effect of grain morphology on degree of milling and iron loss in rice, Cereal Chem., 2007, 84, 384–388.
18. J. Liang, Z. Li, K. Tsuji, K. Nakano, M. J. R. Nout and R. J. Hamer, Milling characteristics and distribution of phytic acid and zinc in long-, medium- and short-grain rice, J. Cereal Sci., 2008, 48, 83–91.
19. S. Yu, Z. T. Nehus, T. M. Badger and N. Fang, Quantification of vitamin E and γ-oryzanol components in rice germ and bran, J. Agric. Food Chem., 2007, 55, 7309–7313.
20. J. L. F. Monks, N. L. Vanier, J. Casaril, R. M. Berto, M. de Oliveira, C. B. Gomes, M. P. de Carvalho, A. R. G. Dias and M. C. Elias, Effects of milling on proximate composition, folic acid, fatty acids and technological properties of rice, J. Food Compos. Anal., 2013, 30, 73–79.
21. S. Shobana, N. Malleshi, V. Sudha, D. Spiegelman, B. Hong, F. Hu, W. Willett, K. Krishnaswamy and V. Mohan, Nutritional and sensory profile of two Indian rice varieties with different degrees of polishing, Int. J. Food Sci. Nutr., 2011, 62, 800–810.
22. S. Jang and Z. M. Xu, Lipophilic and hydrophilic antioxidants and their antioxidant activities in purple rice bran, J. Agric. Food Chem., 2009, 57, 858–862.
23. A. Moongngarm, N. Daomukda and S. Khumpika, Chemical Compositions, Phytochemicals, and Antioxidant Capacity of Rice Bran Layer, and Rice Germ, APCBEE Proc., 2012, 2, 73–79.
24. Y. F. Shao, F. F. Xu, X. Sun, J. S. Bao and T. Beta, Identification and quantification of phenolic acids and anthocyanins as antioxidants in bran, embryo and endosperm of white, red and black rice kernels (Oryza sativa L.), J. Cereal Sci., 2014, 59, 211–218.
25. M. W. Zhang, R. F. Zhang, F. X. Zhang and R. H. Liu, Phenolic profiles and antioxidant activity of black rice bran of different commercially available varieties, J. Agric. Food Chem., 2010, 58, 7580–7587.
26. W. Song, C. M. Derito, M. K. Liu, X. J. He, M. Done and R. H. Liu, Cellular Antioxidant Activity of Common Vegetables, J. Agric. Food Chem., 2010, 58, 6621–6629.
27. K. L. Wolfe, X. M. Kang, X. J. He, M. Done, Q. Y. Zhang and R. H. Liu, Cellular Antioxidant Activity of Common Fruits, J. Agric. Food Chem., 2008, 56, 8418–8426.
28. K. K. Adom, M. E. Sorrells and R. H. Liu, Phytochemical profiles and antioxidant activity of wheat varieties, J. Agric. Food Chem., 2003, 51, 7825–7834.
29. V. Dewanto, X. Wu, K. K. Adom and R. H. Liu, Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity, J. Agric. Food Chem., 2002, 50, 3010–3014.
30. K. A. Wolfe and R. H. Liu, Cellular antioxidant activity [CAA] assay for assessing antioxidants, foods, and dietary supplements, J. Agric. Food Chem., 2007, 55, 8896–8907.
31. K. Hoshikawa, Science of the Rice Plant, 1993, pp. 377–380.
32. M. Naczk and F. Shahidi, Extraction and analysis of phenolics in food, J. Chromatogr. A, 2004, 1054, 95–111.
33. S. Butsat and S. Siriamornpun, Antioxidant capacities and phenolic compounds of the husk, bran and endosperm of Thai rice, Food Chem., 2010, 119, 606–613.
34. J. I. Wadsworth, Rice: Science and technology, 1994, pp. 139–176.
35. R. H. Liu, Whole grain phytochemicals and health, J. Cereal Sci., 2007, 46, 207–219.
36. Z. Zhou, K. Robards, S. Helliwell and C. Blanchard, The distribution of phenolic acids in rice, Food Chem., 2004, 87, 401–406.
37. M. Naczk and F. Shahidi, The effect of methanol-ammonia-water treatment on the content of phenolic acids of canola, Food Chem., 1989, 31, 159–164.
38. J. Sun, Y. F. Chu, X. Wu and R. H. Liu, Antioxidant and antiproliferative activities of common fruits, J. Agric. Food Chem., 2002, 50, 7449–7454.

---