Determination of lipophilic and hydrophilic bioactive compounds in raw and parboiled rice bran

Abstract

Rice bran is one of the most important rice by-products and represents about 10% of rice production. This rice fraction contains large amounts of bioactive compounds; because of that, the aim of this study was to investigate the impact of parboiling on the concentration of lipophilic bioactive compounds (fatty acids, tocols, γ-oryzanol and, free + esterified and bound sterols and triterpenic alcohols) and free and cell wall-bound phenolic compounds. Parboiling treatment caused changes in fatty acid composition and increased the total tocols and γ-oryzanol content. GC-qTOF-MS analysis of phytosterol and triterpenic alcohols allowed the determination of 14 compounds that were quantified in free and bound form; free phytosterol and triterpenic alcohol compounds represented 10 and 43% of the total fraction in raw and parboiled rice bran, respectively. The analysis of free and bound phenolic compounds was carried out by HPLC-DAD-TOF-MS. The TOF analyzer permitted the determination of 24 free and 27 bound phenolic compounds and, to our knowledge, some of them have been quantified for the first time in rice.

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1. Introduction

Rice is one of the most important cereals produced in the world and about 50% of the world production is located in Asia.¹ As other cereals, it is considered a good source of energy and nutrients and, as is known, its quality depends on the variety, method of cultivation, processing and cooking conditions.²

Rice contains several bioactive compounds that are related to health benefits, among them antioxidant, anti-inflammatory and other functional properties.¹ The principal bioactive compounds that are linked with these properties are tocols, sterol derivatives (particularly γ-oryzanol) and phenolic compounds.^1–3 However, it is known that these compounds are mostly located in the external layer of rice grain and they are present in rice by-products such as husk, bran and germ. Because of that, in the past years, rice bran has received particular attention as a functional ingredient for nutraceutical and functional foods.^4,5

Recent data showed that rice bran extract (RBE) protects from mitochondrial dysfunction; in fact RBE increased ATP production and respiratory rates as well as PGC1α protein levels in PC12APPsw cells, improving the mitochondrial function assessed in a cell culture Alzheimer's disease model. Therefore, RBE has been proposed by Hagl and co-workers⁶ as a promising nutraceutical for the prevention of Alzheimer's disease.

Candiracci et al.⁷ investigated the properties of rice bran enzymatic extract to ameliorate the inflammatory state existing in visceral adipose tissue of obese Zucker rats; they demonstrated that a rice bran enzymatic extract-supplemented diet decreased the overproduction of tumor necrosis factors; because of that, this extract ameliorates the obesity-associated proinflammatory response. Ham et al.⁸ noticed that the administration of rice bran unsaponifiable matter reduced the body weight gain, food efficiency ratio and size of the epididymal fat tissue, serum triglyceride, total cholesterol and low-density lipoprotein-cholesterol level, as well as the atherogenic index and cardiac risk factor in mice.

Similar results were reported by Wang et al.;⁹ they reported that γ-oryzanol and ferulic acid ester with phytosterols from rice bran exhibited similar effects in alleviating high-fat and high-fructose diet-induced obesity, hyperlipidemia, hyperglycemia, and insulin resistance index. Moreover, γ-oryzanol treatment was more effective to decrease the liver index, hepatic triglycerides content and serum levels of C-reactive protein.

The cited health benefits are related to sterols, tocols, γ-oryzanol and phenolic compounds present in rice bran.¹⁰ Because of that, the aim of this work was to determine the major lipids and phenolic compounds in raw and parboiled rice bran using different analytical methodologies.

2. Materials and methods

2.1. Chemicals

All the solvents and reagents were purchased from Merck (Darmstadt, Germany). The standard compounds were supplied by Sigma-Aldrich (Saint Louis, MO, USA). GLC-463 mix was from Nu-Check (Elysian, MN, USA).

2.2. Samples

Hulled rice (Ribe) was used for parboiling test. The parboiling process was carried out using a LABPAR (Colombini & Co. srl, Abbiategrasso, Milano, Italy) parboiling pilot plant. 3 kg of paddy rice was soaked for 6 hours at 50 °C in water to reach about 40% of moisture (using a humidity data logger). Subsequently, the rice was dried at 50 °C under vacuum until the moisture was about 12%.

Rice grains were dehulled by a G.390/R dehuller (Colombini & Co. srl, Abbiategrasso, Milano, Italy).

Rice brans were milled (sieve size 0.50 mm) using a laboratory mill (Retsch Ultra Centrifugal Mill ZM 200, Haan, Germany) and stored at −43 °C until analysis.

2.3. Extraction of oil by Folch method

The rice bran oil was extracted using the Folch procedure described by Boselli et al.¹¹ Approximately 2 g of sample was homogenised with 100 mL of a chloroform/methanol solution (1/1, v/v) in a glass bottle with a screw-cap. The bottle was kept at 60 °C for 20 min before adding an additional 100 mL of chloroform. After 3 min of homogenisation, the content of the mixture was filtered through a filter paper. The filtrate was mixed thoroughly with 70 mL of 1 M KCl solution and left overnight at 4 °C in order to phase separation. The organic phase was collected and dried with a rotary evaporator at 40 °C, dissolved in 5 mL n-hexane/isopropanol solution (4/1, v/v) and stored at −18 °C until it was analysed.

2.4. Fat extraction by acid hydrolysis and purification of total unsaponifiable fraction

Total sterols (TS) fraction was obtained by acid hydrolysis performed using AAAC method 30.10 (ref. 12) with some modifications. As internal standard (IS), 1 mg of dihydrocholesterol (Sigma-Aldrich Co. St Louis, MO, USA) was added to the sample before extraction. About 3 g of rice bran was weighed in a glass tube and 30 mL of 30% HCl was added. The tube was shaken vigorously and placed into an 80 °C water bath for 30 min. After heating, the tube was cooled and 25 mL diethyl ether and 25 mL petroleum ether were added to the digested sample. The organic phase was transferred to a glass flask and the lower phase was washed twice with 25 mL of a diethyl ether : petroleum ether (1 : 1, v/v) mixture. The organic fractions were pooled and then evaporated at 35 °C under vacuum using a rotary evaporator. The residue obtained was saponified at room temperature using 40 mL methanolic 0.5 M KOH for 18 h in the dark under continuous agitation.¹³

The organic fraction was washed with deionised water and extracted three times with diethyl ether. The solvent was removed under vacuum and the unsaponifiable fraction was stored on n-hexane/2-propanol (4 : 1, v/v) at −18 °C until GC analysis.

2.5. Free + esterified phytosterol extraction

To determine the free + esterified phytosterols (FES), approximately 0.5 mL of dihydrocholesterol (c = 2.0 mg mL⁻¹) was added to 250 mg of fat obtained by Folch extraction and saponification was conducted at room temperature. After saponification, the organic fraction was washed with 10 mL diethyl ether/water (1/1 v/v), and further the unsaponifiable matter was extracted in sequence; twice with 10 mL diethyl ether, and washed twice with 10 mL 0.5 N aqueous KOH and again twice with 10 mL of distilled water. The diethyl ether solvent was removed under vacuum and the unsaponificable matter was used for the free + esterified sterol determination.

2.6. Determination of sterols and triterpenic alcohols in rice bran unsaponifiable fractions

The unsaponificable matter from TS and FES extracts were silylated according to Sweeley et al.,¹⁴ and they were analysed using a GC-QTOF-MS (Agilent 7200B GC/Q-TOF, Agilent Technologies, Santa Clara, CA, USA) with the chromatographic conditions reported by Kim et al.¹⁵

2.7. Determination of bound sterols

Bound sterol fraction was calculated by:

Total sterol fraction − (free sterol fraction + esterified sterol fraction).

2.8. Total fatty acid analysis (FAMEs)

The fatty acid composition of rice bran samples was determined from fat Folch extract as FAMEs by capillary gas chromatography analysis after alkaline treatment. The chromatographic conditions were the same as reported by Verardo et al.¹⁶

2.9. Tocols determination in rice bran

Tocols determination was carried out using Folch extract. Briefly, 0.1 g of extract was diluted in 1 mL of n-hexane. Tocols was determined according to Gómez-Caravaca et al.¹⁷

α-Tocopherol standard solutions were used to obtain a calibration curve and it was used for quantification.

2.10. γ-Oryzanol determination in rice bran

γ-Oryzanol was determined spectrophotometrically according to Ha et al.¹⁸ Briefly, bran was extracted with n-hexane and γ-oryzanol was determined using the absorbance at 315 nm using the specific extinction coefficient 358.9.

2.11. Extraction of free/esterified and bound phenolic compounds in rice bran samples

To isolate the free/esterified polar fraction, the protocol of Verardo et al.¹⁹ was used. Briefly, 2 grams of rice hull was extracted twice in an ultrasonic bath (20 min) with 20 mL of a solution of ethanol/water (4 : 1 v/v). The residues from ethanolic extraction were digested with 100 mL of 2 M NaOH at room temperature for 20 h by shaking under nitrogen gas. The mixture was then brought to pH 2–3 by adding 10 M hydrochloric acid in a cooling ice bath and extracted with 500 mL of hexane to remove the lipids. The final solution was extracted three times with 100 mL of diethyl ether/ethyl acetate (1/1, v/v). The organic fractions were pooled and evaporated to dryness. The phenolic compounds were reconstituted in 2 mL of methanol/water (1 : 1 v/v).

The final extracts were filtered through 0.22 μm RC syringe filters and stored at −18 °C until the analyses.

2.12. Determination of phenolic compounds by HPLC-ESI-TOF-MS

HPLC analysis was performed by an Agilent 1200 series rapid resolution LC system (Agilent Technologies, CA, USA) consisting of a vacuum degasser, autosampler, and a binary pump equipped with a Kinetex C18 column (4.6 × 100 mm, 2.6 μm) from Phenomenex (Torrance, CA, USA). The mobile phase and gradient program were used as previously described by Gómez-Caravaca et al.²⁰ All HPLC components were controlled by Hystar 3.1 software (Bruker Daltonik, Bremen, Germany). The HPLC system was coupled to a microTOF (Bruker Daltonics, Bremen, Germany), an orthogonal-accelerated TOF mass spectrometer (oaTOF-MS), equipped with an ESI interface. Parameters for analysis were set using negative ion mode with spectra acquired over a mass range from m/z 50 to 1100. The other optimum values of the ESI-TOF-MS parameters were drying gas temperature, 210 °C; drying gas flow, 8 L min⁻¹; and nebulising gas pressure, 2 bar.

The accurate mass data of the molecular ions were processed through the newest software Data Analysis 4.0 (Bruker Daltonics, Bremen, Germany), which provided a list of possible elemental formula by using the Smart Formula Editor. The Editor uses a CHNO algorithm, which provides standard functionalities such as minimum/maximum elemental range, electron configuration, and ring-plus double bonds equivalents, as well as a sophisticated comparison of the theoretical with the measured isotope pattern (sigma value) for increased confidence in the suggested molecular formula. The widely accepted accuracy threshold for confirmation of elemental compositions has been established at 5 μg mL⁻¹ (Ferrer et al., 2005).²¹

During the development of the HPLC method, external instrument calibration was performed using a Cole Palmer syringe pump (Vernon Hills, Illinois, USA) directly connected to the interface, passing a solution of sodium acetate cluster containing 5 mM sodium hydroxide in the sheath liquid of 0.2% acetic acid in water/isopropanol 1 : 1 (v/v). Using this method, an exact calibration curve based on numerous cluster masses each differing by 68 Da (NaCHO₂) was obtained. Due to the compensation of temperature drift in the microTOF, this external calibration provided accurate mass values for a complete run without the need for a dual sprayer setup for internal mass calibration.

2.13. Statistical analyses

One-way analysis of variance, ANOVA (Tukey's honest significant difference multiple comparison) and Pearson's correlations were evaluated using Statistica 8.0 software (2007, StatSoft, Tulsa, OK, USA). p values lower than 0.05 were considered statistically significant. All extractions and analytical determinations were carried out in triplicate, and the analytical data were used for statistical comparisons.

3. Results and discussion

The major bioactive compounds present in rice bran are lipids and hydrophilic compounds; however, the technological treatments influence their location in the rice caryopsis.²²

3.1. Determination of oil content and FAMEs in rice bran samples

Table 1 shows the total oil content and FAMEs composition of rice brans. As expected, parboiling process caused the increase of fat content in parboiled rice bran (PRB) compared to raw rice bran (RRB); in fact, the total fat content increased twice in PRB. These data confirmed the previous results obtained by Padua and Juliano²³ which demonstrated that PRB had a higher fat and protein content than RRB due to the greater resistance of the starchy endosperm of parboiled rice to milling.

Table 1 Oil content (g per 100 g of bran) and fatty acids composition (%) in rice bran samples. Different letters in the same line indicate significantly different values (p < 0.05)^a

	RRB	PRB
a RRB: raw rice bran samples; PRB: parboiled rice bran samples.
Oil content	13.3 ± 1.1 b	25.7 ± 1.4 a
C16:0	14.2 ± 0.3 b	18.1 ± 0.6 a
C18:0	1.6 ± 0.1 b	2.2 ± 0.1 a
C18:1 cis 9	39.1 ± 0.4 a	37.2 ± 0.3 b
C18:1 cis 11	1.0 ± 0.2 a	1.1 ± 0.3 a
C18:2	39.4 ± 0.1 a	37.5 ± 0.3 b
C18:3	1.4 ± 0.1 a	0.9 ± 0.2 b
C20:0	0.4 ± 0.0 a	0.3 ± 0.0 a
C20:1	0.7 ± 0.1 a	0.8 ± 0.1 a
C22:0	0.7 ± 0.0 a	0.6 ± 0.0 a
C22:2	0.5 ± 0.0 a	0.4 ± 0.0 a
C24:0	1.0 ± 0.1 a	0.9 ± 0.1 a
SFA	17.9 ± 0.8 b	22.1 ± 1.0 a
MUFA	40.8 ± 0.3 a	39.1 ± 0.4 b
PUFA	41.3 ± 0.5 a	38.8 ± 0.4 b
UFA/SFA	4.59 ± 0.3 a	3.52 ± 0.2 b

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Five saturated fatty acids (C16:0, C18:0, C20:0, C22:0 and C24:0), three monounsaturated (C18:1 c9, C18:1 c11 and C20:1) and three polyunsaturated fatty acids were identified in both samples. Oleic and linoleic acids were the principal fatty acids reported in both samples. Palmitic acid was the third fatty acid. These data agreed with the results reported by Przybylski et al.²⁴ in brow rice and by Chia et al.²⁵ in rice bran oils.

RRB sample showed a higher level of unsaturation; in fact, the sum of MUFA and PUFA content in RRB represented more than 82% of total FAMEs; instead, this content was lower in PRB sample (78%). Contrary, SFA content was significantly higher (p < 0.05) in PRB than in RRB sample. These data confirmed the results showed by Rizk and co-workers²⁶ which demonstrated that the parboiling process decreases the ratio between total unsaturated (UFA) and total saturated fatty acids (SFA) in rice bran oil; in fact, this ratio in PRB was about 23% lower than in RRB. This aspect could be related to a higher lipid stability of PRB sample compared to RRB.

Finally, it can be affirmed that parboiling process, in addition to prejudicing the production of free fatty acids,²⁶ it increases the saturation ratio of rice bran oil. These two factors may favour the increase of shelf life of rice bran.

3.2. Determination of tocols and γ-oryzanol content in rice bran samples

The content of tocols (expressed as tocopherols and tocotrienols) in rice bran samples is listed in Table 2.

Table 2 Content of tocols in rice bran samples (μg g⁻¹ bran d.w.). Different letters in the same line indicate significantly different values (p < 0.05)^a

Tocols	RRB	PRB
a RRB: raw rice bran samples; PRB: parboiled rice bran samples.
α-Tocopherol	5.8 ± 0.1 b	7.9 ± 0.4 a
α-Tocotrienol	12.7 ± 0.3 b	16.4 ± 0.6 a
γ-Tocopherol	15.8 ± 0.2 b	27.3 ± 0.3 a
γ-Tocotrienol	94.4 ± 1.1 b	169.2 ± 0.6 a
δ-Tocopherol	1.9 ± 0.1 a	2.0 ± 0.3 a
δ-Tocotrienol	3.5 ± 0.1 a	3.6 ± 0.2 a
Sum	134.1 ± 1.4 b	226.4 ± 1.5 a

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The total amounts of vitamin E (the sum of tocopherols and tocotrienols) in rice bran samples were 134.1 and 226.4 μg g⁻¹ rice bran in RRB and PRB, respectively. According to literature, parboiling process affects significantly the content of vitamin E.^27,28

As reported by several authors^27–30 the principal tocol of rice bran was the γ-tocotrienol. Its content in PRB was 1.8 times higher than RRB. A similar increase was reported by Pradeep et al.²⁷ in Jyothi rice bran.

The second and third tocols in rice bran were γ-tocopherol and α-tocotrienol, respectively; the relative amounts were in the same order of magnitude of those reported by Goufo and Trindade.³⁰ As reported for γ-tocotrienol, the parboiling process increased 1.7 and 1.3 times the content of γ-tocopherol and α-tocotrienol, respectively.

The tocols namely α-tocopherol, δ-tocotrienol and δ-tocopherol were also determined; α-tocopherol was 1.4 times higher in PRB than RRB. Instead, no statistical differences were noticed for δ-tocopherol and δ-tocotrienol in RRB and PRB samples.

Tocopherols represented the 16–17% of total tocols; therefore, tocotrienols ranged between 83 and 84% of total tocols. These values agreed with the results reported by Goufo and Trindade.³⁰ The high amounts of tocotrienols in rice bran promote this by-product as a functional ingredient because, as demonstrated by Friedman,⁵ 100 mg per day of tocotrienol-rich fraction of rice bran reduced significantly LDL cholesterol, apolipoprotein B, and triglycerides in hypercholesterolemic human subjects.

Pearson's correlation analysis showed positive correlations between α-tocopherol and α-tocotrienol (r = 0.9905 p < 0.05) and, γ-tocopherol and γ-tocotrienol (r = 0.9998 p < 0.001).

The amount of γ-oryzanol in the rice bran samples was also determined. As reported by Friedman,⁵ γ-oryzanol reduces the aortic cholesterol, modulate the immune system, presents anti-oxidative activity in vitro and in vivo and preserves tocols during food processes.

As expected PRB sample showed the highest amount of γ-oryzanol (387.2 ± 1.9 mg per 100 g of bran). In RRB sample γ-oryzanol was 253.9 ± 1.6 mg per 100 g of bran. Similar amounts were noticed by other authors.^27–30

3.3. Determination of free/esterified, bound and total sterols and triterpenic alcohols in rice bran samples

Table 3 shows the sterols and triterpenic alcohols identified in rice brans. The identification was carried out studying the mass spectra, and comparing the mass data with literature and NIST library.

Table 3 Sterols and triterpenic alcohols identified in rice bran samples by GC-ESI-QTOF-MS^a

	Sterol or triterpenic alcohol	RRT	Molecular formula	M^+	Principal fragments (m/z)
a RRT: relative retention time.
1	Campesterol	0.87	C28H48O	472	457, 382, 367, 343, 315, 255, 253, 213, 129
2	Campestanol	0.89	C28H50O	474	459, 384, 369, 358, 345, 305, 257, 255, 215
3	Stigmasterol	0.91	C29H48O	484	469, 394, 379, 355, 343, 255, 253, 213, 129
4	Stigmastanol	0.93	C29H52O	486	471, 396, 381, 345, 305, 257, 255, 215
5	Clerosterol	0.97	C29H48O	484	469, 394, 379, 343, 255, 253, 213, 129
6	23-Dehydrositosterol	0.98	C29H48O	484	469, 394, 379, 355, 343, 255, 253, 213, 129
7	Sitosterol	1.00	C29H50O	486	471, 396, 381, 343, 357, 255, 253, 213, 129
8	Sitostanol	1.02	C29H52O	488	473, 398, 383, 345, 305, 215
9	Δ^5-Avenasterol	1.03	C29H48O	484	469, 394, 379, 355, 343, 255, 253, 213, 129
10	Gramisterol	1.08	C29H48O	484	469, 400, 394, 379, 359, 357, 317, 269, 267, 227
11	Cycloartenol	1.12	C30H50O	498	483, 408, 393, 365, 339, 286, 271
12	Δ^7-Avenasterol	1.16	C29H48O	484	469, 394, 386, 379, 345, 343, 303, 255, 253, 213
13	24-Methylen-cycloartanol	1.26	C31H52O	586	422, 407, 379, 353, 300
14	Citrostadienol	1.38	C30H50O	498	483, 400, 408, 393, 357, 269, 267, 227

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All the sterols and triterpenic alcohols identified in this work had previously been identified in rice and rice bran.^24,31

Six Δ⁵-sterols namely campesterol, stigmasterol, clerosterol, 23-dehydrositosterol, sitosterol and Δ⁵-avenasterol were identified. According to Pelillo et al.,³² these compounds presented three typical ions at m/z 213 (derived from the loss of the side chain and the D ring), m/z 129 and its complement [M − 129]⁺. Other typical fragments that have been reported in mass spectra were the ions at m/z 343 (corresponding to the loss of side chain [M − SC − 2]⁺) and the ions at m/z 255 ([M − SC − 90]⁺) and 253 ([M − SC − 90 − 2]⁺). Finally the typical fragments corresponding to [M − CH₃]⁺, [M − TMSOH]⁺ and [M − CH₃ − TMSOH]⁺ were also reported.

Three stanols (campestanol, stigmastanol and sitostanol) were identified in rice brans. They showed two common principal fragments at m/z 215 and 305 corresponding to [M − SC − D − 90]⁺ and [M − SC − D]⁺, where D correspond to D ring of cholestane structure. Other common fragments were [M − SC − 2]⁺, [M − CH₃]⁺, [M − TMSOH]⁺ and [M − CH₃ − TMSOH]⁺; as expected, the ions corresponding to [M − SC − 90]⁺ and [M − SC − 90 − 2]⁺ were noticed only in campestanol and stigmastanol compound but not in sitostanol.³²

Δ⁷-Avenasterol was identified due to its characteristic fragmentation pattern; mass spectra showed two principal ions at m/z 386 and 343 corresponding to [M − 98]⁺ and [M − SC − 2]⁺. Other typical ions were at m/z 253, 255 and 213 corresponding to [M − SC − 90 − 2]⁺, [M − SC − 90]⁺ and [M − SC − D − 90]⁺, respectively. Moreover, the ions corresponding to [M − CH₃]⁺ and [M − CH₃ − TMSOH]⁺ were also detected.

Two 4-methyl-Δ⁷-sterols, called gramisterol and citrostadienol were also identified in rice brans. Their presence was confirmed by two typical fragments of this sterol class at 357 and 400 m/z. The first was the most abundant fragment representing the fragmentation [M − SC − 2]⁺; instead, the second fragment was the result of a bond break in the β-position of the 24–28 double bond.

Finally, two triterpenic alcohols (cycloartenol and 24-methylen-cycloartanol) were identified.

For quantitative purposes, free/esterified and total sterols and triterpenic alcohols were extracted using two different protocols. To obtain the total sterols and triterpenic alcohols, the acid hydrolysis of rice bran and subsequent saponification of lipid extracts was carried out. As reported by Iafelice et al.,³³ a direct saponification (in alkaline conditions) of cereals did not permit the hydrolysis of acetal bond between sterol and carbohydrate moiety, because of that acid hydrolysis in combination with saponification are necessary.

Table 4 shows the content of free/esterified, bound and total sterols and triterpenic alcohols in rice brans. Free/esterified sterols and triterpenic alcohols content has been compared with previous research; contrary, literature lacks of information about the bound and total sterols and triterpenic alcohols content.

Table 4 Sterols and triterpenic alcohols content in rice bran samples (mg g⁻¹ of bran d.w.). Different letters in the same line indicate significantly different values (p < 0.05)^a

	Sterols and triterpenic alcohols	RRB			PRB
		Free/esterified	Bound	Total	Free/esterified	Bound	Total
a RRB: raw rice bran samples; PRB: parboiled rice bran samples; n.d. = not detected.
1	Campesterol	0.04 ± 0.00 d	1.21 ± 0.02 b	1.25 ± 0.02 b	1.01 ± 0.07 c	1.39 ± 0.14 b	2.40 ± 0.21 a
2	Campestanol	0.04 ± 0.00 d	0.99 ± 0.02 b	1.03 ± 0.02 b	0.81 ± 0.03 c	0.86 ± 0.07 c	1.67 ± 0.01 a
3	Stigmasterol	0.31 ± 0.01 d	0.94 ± 0.13 c	1.25 ± 0.12 b	0.82 ± 0.08 c	1.17 ± 0.03 b	1.99 ± 0.26 a
4	Stigmastanol	0.06 ± 0.00 d	1.63 ± 0.11 c	1.70 ± 0.11 b, c	2.08 ± 0.20 b	2.25 ± 0.30 b	4.33 ± 0.50 a
5	Clerosterol	0.16 ± 0.00 e	0.28 ± 0.06 d	0.44 ± 0.02 c	0.47 ± 0.09 c	1.03 ± 0.21 b	1.50 ± 0.13 a
6	23-Dehydrositosterol	0.04 ± 0.00 e	0.36 ± 0.03 c	0.40 ± 0.01 c	0.25 ± 0.03 d	1.00 ± 0.11 b	1.25 ± 0.05 a
7	Sitosterol	0.97 ± 0.01 e	5.75 ± 0.16 c	6.72 ± 0.15 b	4.89 ± 0.28 d	5.89 ± 0.35 c	10.78 ± 0.17 a
8	Sitostanol	0.19 ± 0.01 e	1.41 ± 0.18 a	1.60 ± 0.17 a	0.54 ± 0.06 c	0.35 ± 0.05 d	0.89 ± 0.09 b
9	Δ^5-Avenasterol	0.14 ± 0.00 d	2.46 ± 0.25 a	2.60 ± 0.25 a	0.55 ± 0.06 c	0.10 ± 0.00 e	0.65 ± 0.01 b
10	Gramisterol	0.10 ± 0.01 d	1.11 ± 0.06 a	1.21 ± 0.07 a	0.39 ± 0.02 c	0.75 ± 0.06 b	1.13 ± 0.09 a
11	Cycloartenol	0.05 ± 0.00 d	1.42 ± 0.02 a	1.47 ± 0.02 a	1.11 ± 0.04 b	0.27 ± 0.01 c	1.38 ± 0.09 a
12	Δ^7-Avenasterol	0.03 ± 0.00 c	0.45 ± 0.02 a	0.48 ± 0.02 a	0.08 ± 0.00 b	n.d.	0.08 ± 0.00 b
13	24-Methylen-cycloartanol	0.02 ± 0.00 c	1.66 ± 0.32 a	1.68 ± 0.32 a	1.40 ± 0.05 a	0.35 ± 0.06 b	1.75 ± 0.01 a
14	Citrostadienol	0.08 ± 0.00 d	0.13 ± 0.00 c	0.20 ± 0.00 b	0.67 ± 0.04 a	0.05 ± 0.00 e	0.72 ± 0.04 a
Sum		2.24 ± 0.02 e	19.80 ± 0.86 c	22.04 ± 0.88 b	15.07 ± 0.45 d	15.46 ± 0.31 d	30.55 ± 0.48 a

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The two types of brans showed the same qualitative profile in terms of sterols and triterpenic alcohols; however, statistical differences (p < 0.05) were noticed in terms of total compounds amounts in bran samples. In fact, PRB showed total content that is 38.5% higher than RRB.

Sitosterol was the most abundant compound in each sample and represented the 30.5 and 35.3% of total triterpenic fraction in RRB and PRB, respectively.

Δ⁵-Avenasterol was the second sterol in RRB that contained 2.6 mg g⁻¹ but, its content decreased to 0.65 mg g⁻¹ of bran in PRB sample. Stigmastanol, stigmasterol, campesterol and campestanol represented other principal sterols in rice bran; they increased their content about 1.6–2.5 times from RRB to PRB sample.

Sitostanol and Δ⁷-avenasterol were the only sterols that decreased from RRB to PRB sample.

Cycloartenol and 24-methylen-cycloartanol were the major triterpene alcohols present in the rice bran and their content in RRB was 1.5 and 1.7 mg g⁻¹ of bran d.w. and no statistical differences were showed in PRB sample.

Free + esterified sterol (FES) compounds were only the 10% of total sterols in RRB sample; instead, parboiling process influenced the sterol composition, in fact FES content in PRB was 49.3% of total sterols. Probably, thermal treatment caused the breaking of sterol bonds in the cellular structures.

Statistical differences were noticed in terms of total FES amounts; in fact, the FES content was 2.2 and 15.1 mg g⁻¹ of bran d.w. in RRB and PRB, respectively (Table 4). This content was in the same order of magnitude of the results reported by Jiang and Wang³¹ in rice bran.

Sitosterol was the most abundant FES and accounted the 43.3 and 32.4% of FES in RRB and PRB, respectively.

The other FES compounds in RRB, in decreasing order of abundance, were stigmasterol, clerosterol, sitostanol, Δ⁵-avenasterol and gramisterol. Minor FES compounds that were determined were Δ⁷-avenasterol, campesterol, campestanol, stigmastanol, 23-dehydrositosterol, cycloartenol, 24-methylen-cycloartanol and citrostadienol.

Surprisingly, parboiling process changed the order of most abundant FES and each of them statistically increased from 2.6 to 70 times in PRB.

Bound sterols represented the 89.8 and 50.6% of total sterol fraction in RRB and PRB, respectively. These data confirmed the influence of thermal treatment such as parboiling on the breakage of chemical bonds between sterols and cell structures.

3.4. Determination of free phenolic compounds in rice bran

The analysis by HPLC-ESI-TOF-MS of the free phenolic extract revealed that three phenolic classes were noticed in rice bran; briefly, two hydroxybenzoic, fourteen hydroxycinnamic and six flavone derivatives were determined and they are summarized in Table 5.

Table 5 Free phenol compounds identified in rice bran samples by HPLC-ESI-TOF-MS^a

	Phenolic compound	Rt (min)	Molecular formula	m/z experimental	m/z calculated	Error (ppm)	mSigma
a Rt: retention time.
1	3-p-Cumaroylquinic acid	5.34	C16H18O8	337.1097	337.0999	−4.9	48.1
2	p-Hydroxybenzoic acid	5.76	C7H6O3	137.0237	137.0244	4.2	37.7
3	Dehydrotriferulic acid	6.57	C30H26O12	577.1359	577.1351	−1.3	6.7
4	3-Feruloylquinic acid	7.74	C17H20O9	367.0965	367.1035	4.9	20.1
5	Vanillic acid	8.16	C8H8O4	167.0330	167.0350	4.7	34.8
6	Synapoyl-sucrose I	8.50	C23H32O15	547.1654	547.1668	2.6	13.5
7	Cistanoside F	9.22	C21H28O13	487.1458	487.1457	−0.2	16.5
8	Diferulic acid	9.42	C20H18O8	385.0957	385.0929	−4.2	22.6
9	Dehydrotriferulic acid	9.89	C30H26O12	577.1361	577.1351	−1.6	46.2
10	Feruloyl-sucrose (Arillatose B)	10.22	C22H30O14	517.1587	517.1563	−4.8	2.5
11	Synapoyl-sucrose II	10.54	C23H32O15	547.1659	547.1668	−1.9	10.2
12	4-Feruloylquinic acid	10.75	C17H20O9	367.0995	367.1035	4.8	15.8
13	Diferulic acid	11.06	C20H18O8	385.0936	385.0929	−3.8	17.4
14	6-C-arabinosyl-8-C-glucosyl apigenin	11.61	C27H28O14	563.1406	563.1405	0.2	18.3
15	p-Coumaric acid	11.67	C9H8O3	163.0369	163.0401	3.1	7.8
16	6-C-glucosyl-8-C-arabinosyl apigenin	12.14	C27H28O14	563.1422	563.1405	2.8	9.0
17	Apigenin-6,8-di-C-glycoside	12.56	C27H30O15	593.1502	593.1512	5.0	15.7
18	Apigenin-6,8-di-C-glycoside	12.79	C27H30O15	593.1532	593.1512	3.3	5.1
19	Ferulic acid	13.0	C10H10O4	193.0485	193.0506	5.0	17.9
20	Sinapic acid	13.14	C11H12O5	223.0582	223.0612	4.9	40.1
21	C-dipentosyl apigenin	13.24	C25H26O13	533.1272	533.1301	4.4	34.9
22	Di-sinapoyl-sucrose	16.81	C34H42O19	753.2222	753.2248	3.3	11.3
23	Feruloyl-sinapoyl-sucrose	17.31	C33H40O18	723.2107	723.2142	4.8	7.0
24	Tricin	23.84	C17H14O7	329.0618	329.0637	4.9	10.1

---

Hydroxybenzoic acid derivatives were the compounds 2 and 5; due to their molecular formulas, m/z and UV spectra (maximum at ∼280 nm), they were identified as p-hydroxybenzoic and vanillic acid. Moreover, the co-elution with commercial standards further confirmed the identity. Their presence in rice has been noticed by several authors.^34–36

The hydroxycinnamic derivatives were glycosylated compounds of p-coumaric, ferulic, sinapic and caffeic acids. Compounds 1, 4 and 12 were identified, based on molecular ions and molecular formulas, as 3-p-coumaroylquinic acid, 3-feruloylquinic acid and 4-feruloylquinic acid, respectively; the presence of these hydroxycinnamoylquinic acid derivatives in rice was noticed by Bordiga et al.³⁷

Compounds 3 and 9 showed a molecular ion at m/z 577 and molecular formula C₃₀H₂₆O₁₂; they produced the UV absorption spectra with maximum at 220, 235, 280 and 315 nm. These compounds, according to Bunzel et al.³⁸ were identified as dehydrotriferulic acid isomers. They identified this compound in maize bran; however, to our knowledge, its presence in rice bran has not previously been noticed.

Compounds 6 and 11 reported the same molecular ion and molecular formula; according to Tian et al.,³⁴ these compounds were identified as sinapoyl-sucrose isomers.

The compound 7 showed a molecular ion at 487 m/z, molecular formula C₂₁H₂₈O₁₃ and UV spectra 240, 262 and 310 nm. Moreover, it reported a fragment at 341.1084 m/z with molecular formula C₁₅H₁₈O₉ corresponding to a caffeic hexoside fragment. Because of that, this fragmentation pattern and UV data were assigned to cistanoside F; to our knowledge, this compound has not previously been identified in rice.

Compounds 8 and 13 produced a molecular ion at m/z 385 and molecular formula C₂₀H₁₈O₈, because of that these compounds were identified as diferulic acid and their presence in rice has extensively been reported by several authors.^39,40

Compound 10 showed a molecular formula C₂₂H₃₀O₁₄ and a molecular ion at 517 m/z; according to Tian et al.³⁴ this MS data were assigned to feruloyl-sucrose.

Peaks 14 and 16 reported a molecular ion at 563 m/z and molecular formula C₂₇H₂₈O₁₄; these compounds were identified as apigenin-di-hexoside. Qiu and coworkers⁴¹ noticed the presence of 6-C-arabinosyl-8-C-glucosyl apigenin and 6-C-glucosyl-8-C-arabinosyl apigenin in rice; however, compound 14 showed a fragment at 473 m/z corresponding to [M − H − 90]⁻, because of that and according to Qiu et al.,⁴¹ this compound was identified as 6-C-arabinosyl-8-C-glucosyl apigenin. Consequently, compound 16 was assigned as 6-C-glucosyl-8-C-arabinosyl apigenin.

Mass data and co-elution with commercial standards permitted the identification of compounds 15, 19 and 20 as p-coumaric, ferulic and sinapic acids, respectively.

Compound 17 and 18 showed a molecular ion at 593 m/z and according to Qiu et al.⁴¹ these compounds were identified as apigenin-diglycoside isomers. According to the same authors, compound 21 was identified as C-dipentosyl apigenin.

Compound 22 showed a molecular ion at 753 m/z with molecular formula C₃₄H₄₂O₁₉ and a fragment at 547 m/z, and an UV spectrum that exhibited absorption at 268 and 326 nm. This fragmentation pattern was assigned to di-sinapoyl-sucrose.

Compound 23 reported a molecular ion at 723 m/z and molecular formula C₃₃H₄₀O₁₈, its UV spectrum showed absorption at 267 and 328 nm; this compound was tentatively identified as feruloyl-sinapoyl-sucrose.

To our knowledge, compounds 22 and 23 have been identified and quantified for the first time in rice bran by HPLC-MS; however their presence in rice was reported by Nakano et al.⁴² that identified this compounds by NMR.

Finally, compound 24 reported a molecular ion at 329 m/z and molecular formula C₁₇H₁₄O₇; according to Lam et al.⁴³ this compound was identified as tricin.

Table 6 reported the content of each phenolic compound in rice bran samples. The total phenolic content was 108.2 and 66.2 μg g⁻¹ of dry weigh rice bran for RRB and PRB, respectively. These contents are in the same order of magnitude of those reported by other authors.^35,44 As reported by Pradeep and co-workers²⁷ and Walter and co-workers,⁴⁵ parboiling process caused a decrease of free phenolic content.

Table 6 Content of free phenol compounds in rice bran samples (μg g⁻¹ d.w.) determined by HPLC-ESI-TOF-MS. Different letters in the same line indicate significantly different values (p < 0.05)^a

	Phenolic compound	RRB	PRB
a RRB: raw rice bran samples; PRB: parboiled rice bran samples.
1	3-p-Cumaroylquinic acid	0.10 ± 0.00	<LOQ
2	p-Hydroxybenzoic acid	4.34 ± 0.01 a	4.07 ± 0.03 b
3	Dehydrotriferulic acid	0.30 ± 0.00	<LOQ
4	3-Feruloylquinic acid	0.37 ± 0.02 a	0.21 ± 0.00 b
5	Vanillic acid	6.82 ± 0.12 a	3.93 ± 0.05 b
6	Synapoyl-diglucose I	0.70 ± 0.01	<LOQ
7	Cistanoside F	0.81 ± 0.04	<LOQ
8	Diferulic acid I	0.25 ± 0.01	<LOQ
9	Dehydrotriferulic acid	0.15 ± 0.01	<LOQ
10	Feruloyl-sucrose	14.62 ± 0.10 a	1.48 ± 0.05 b
11	Synapoyl-diglucose II	3.50 ± 0.03 a	0.40 ± 0.00 b
12	4-Feruloylquinic acid	0.99 ± 0.01 a	0.98 ± 0.01 a
13	Diferulic acid II	0.48 ± 0.02 b	0.99 ± 0.00 a
14	6-C-arabinosyl-8-C-glucosyl apigenin I	9.73 ± 0.12 a	10.24 ± 0.13 a
15	p-Coumaric acid	2.06 ± 0.06 b	11.54 ± 0.72 a
16	6-C-arabinosyl-8-C-glucosyl apigenin II	16.52 ± 0.09 a	6.45 ± 0.18 b
17	Apigenin-6,8-di-C-glycoside I	12.16 ± 0.07 a	4.92 ± 0.08 b
18	Apigenin-6,8-di-C-glycoside II	7.66 ± 0.06 a	4.35 ± 0.07 b
19	Ferulic acid	5.63 ± 0.11 a	1.83 ± 0.06 b
20	Sinapic acid	0.08 ± 0.01 a	0.04 ± 0.00 b
21	C-dipentosyl apigenin	6.45 ± 0.08 a	4.89 ± 0.04 b
22	Di-sinapoyl-sucrose	2.05 ± 0.18 a	0.50 ± 0.03 b
23	Feruloyl-sinapoyl-sucrose	7.46 ± 0.42 a	1.96 ± 0.15 b
24	Tricin	4.99 ± 0.40 b	7.41 ± 0.04 a

---

6-C-arabinosyl-8-C-glucosyl apigenin isomer II was the first free phenolic compound in RRB, followed by feruloyl-sucrose and apigenin-6,8-di-C-glycoside isomer I. PRB reported 6-C-arabinosyl-8-C-glucosyl apigenin isomer I and p-coumaric acid as principal free phenolic compounds, followed by tricin and 6-C-arabinosyl-8-C-glucosyl apigenin isomer II. Generally, most of the phenolic compounds decreased with parboiling process except diferulic acid isomer II, p-coumaric acid and tricin that increased their content after parboiling treatment. This increase should be attributed to the hydrolysis process during parboiling of bound phenolic compounds and tannins that are present in rice brans.

3.5. Determination of bound phenolic compounds in rice bran

Table 7 reports the identification of bound phenolic compounds in rice bran samples.

Table 7 Bound phenol compounds identified in rice bran samples by HPLC-ESI-TOF-MS

	Phenolic compound	tR (min)	Molecular formula	Detected ion	m/z experimental	m/z calculated	Error (ppm)	mSigma
1	Vanillic aldehyde	4.70	C8H8O3	[M − H]^−	151.0339	151.0401	4.1	22.4
2	p-Cumaroyl-hexose I	5.44	C15H18O8	[M − H]^−	325.0849	325.0829	4.7	13.8
3	Vanillic acid	8.16	C8H8O4	[M − H]^−	167.0299	167.0350	4.8	24.7
4	Benzoic aldehyde	8.46	C7H6O2	[M − H]^−	121.0283	121.0295	4.7	17.2
5	Syringic acid	9.15	C9H10O5	[M − H]^−	197.0376	197.0495	4.4	45.2
6	p-Cumaroyl-hexose II	10.51	C15H18O8	[M − H]^−	325.0860	325.0829	4.6	24.8
7	p-Coumaric acid	11.67	C9H8O3	[M − H]^−	163.0371	163.0401	3.7	0.6
8	trans Ferulic acid	13.01	C10H10O4	[M − H]^−	193.0495	193.0506	3.8	4.9
9	cis Ferulic acid	13.10	C10H10O4	[M − H]^−	193.0492	193.0506	4.4	4.5
10	Sinapic acid	13.14	C11H12O5	[M − H]^−	223.0588	223.0612	4.1	4.2
11	Disinapic acid	13.46	C22H22O10	[M − H − CO2]^−	401.1216	401.1242	2.6	6.0
12	Diferulic acid	14.28	C20H18O8	[M − H]^−	385.0885	385.0929	4.4	7.6
13	Diferulic acid	15.37	C20H18O8	[M − H]^−	385.0874	385.0929	4.8	33.6
14	Diferulic acid	16.37	C20H18O8	[M − H]^−	385.0884	385.0929	4.5	9.0
15	Dehydrotriferulic acid	16.97	C30H26O12	[M − H]^−	577.1364	577.1351	−2.2	30.9
16	Disinapic acid	17.46	C22H22O10	[M − H − CO2]^−	401.1158	401.1242	5.0	7.6
17	Disinapic acid	18.36	C22H22O10	[M − H − CO2]^−	401.1194	401.1242	3.1	5.4
18	Dehydrotriferulic acid	18.91	C30H26O12	[M − H]^−	577.1382	577.1351	−5.0	42.0
19	Diferulic acid	19.20	C20H18O8	[M − H]^−	385.0884	385.0929	4.7	9.6
20	Dehydrotriferulic acid	19.62	C30H26O12	[M − H]^−	577.1364	577.1351	−2.2	30.9
21	Diferulic acid	19.95	C20H18O8	[M − H]^−	385.0880	385.0929	4.8	6.3
22	Diferulic acid	20.72	C20H18O8	[M − H]^−	385.0870	385.0929	5.0	12.6
23	Diferulic acid	21.02	C20H18O8	[M − H]^−	385.0886	385.0929	4.3	11.1
24	Dehydrotriferulic acid	21.82	C30H26O12	[M − H]^−	577.1366	577.1351	−2.5	9.1
25	Dehydrotriferulic acid	22.22	C30H26O12	[M − H]^−	577.1346	577.1351	0.9	9.1
26	Dehydrotriferulic acid	23.69	C30H26O12	[M − H]^−	577.1343	577.1351	1.4	10.6
27	Caffeoyl-hexose	25.36	C15H18O9	[M − H]^−	341.0901	341.0878	4.1	24.0

---

Twenty-seven phenolic compounds were identified and quantified in rice brans; as expected, all the identified compounds belonged to phenolic acids.

Compounds 1, 3, 4 and 5 were identified as hydroxybenzoic acid derivatives; briefly, due to their mass and UV data they were assigned to vanillic aldehyde, vanillic acid, benzoic aldehyde and syringic acid, respectively. The presence of compounds 1 and 4 in rice has recently been noticed by Wang and co-workers.⁴⁶

Compounds 2 and 6 showed a molecular formula C₁₅H₁₈O₈ and a molecular ion at 325 m/z and a fragment at 163 m/z, because of that, these compounds were identified as p-coumaroyl-hexose isomers.

According to mass and UV data and based on the co-elution with commercial standards, compound 7, 8, 9 and 10 were identified as p-coumaric, trans ferulic, cis ferulic and sinapic acid, respectively.

Compounds 11, 16 and 17 showed molecular formula C₂₂H₂₂O₁₀ and molecular ion at 401 m/z; according to Grúz et al.⁴⁷ the molecular ion was assigned to [M − H − COO]⁻ representing the most abundant ion due to in source fragmentation of disinapic acid.

Compounds 12, 13, 14, 19, 21, 22 and 23 showed a molecular ion at 385 m/z and molecular formula C₂₀H₁₈O₈; because of that they were identified as diferulic acid.

Compounds 15, 18, 20, 24, 25 and 26 reported a molecular ion at m/z 577 and molecular formula C₃₀H₂₆O₁₂; as previously reported for free phenolic compounds; these compounds were identified as dehydrotriferulic acid.

Finally, compound 27 was identified as caffeoyl-hexose.

Total bound phenolics content was 603.4 and 974.3 μg g⁻¹ of d.w. rice bran for RRB and PRB, respectively. These amounts are in the same order of magnitude of those reported by other authors.^30,35 The increase of bound phenolic compounds in rice was also reported by Min and co-workers²⁸ that suggested that heat treatment causes the instability of cell-wall structure and binding properties, resulting in an increase in the extractability and/or release of bound phenolics. According to Shao et al.,³⁵ the bound phenolic content in rice bran was higher than free/esterified phenolic content.

Hydroxycinnamic acid derivatives represented about 97% of total bound phenolic compounds in both samples. Ferulic acid derivatives were the 61.3 and 63.0% of bound phenolic compounds in RRB and PRB, respectively (Table 8).

Table 8 Content of bound phenol compounds in rice bran samples (μg g⁻¹ d.w.) determined by HPLC-ESI-TOF-MS. Different letters in the same line indicate significantly different values (p < 0.05)^a

	Phenolic compound	RRB	PRB
a RRB: raw rice bran samples; PRB: parboiled rice bran samples.
1	Vanillic aldehyde	3.73 ± 0.06 b	4.57 ± 0.17 a
2	p-Cumaroyl-hexose I	0.13 ± 0.00 a	0.16 ± 0.01 a
3	Vanillic acid	5.73 ± 0.17 b	7.35 ± 0.34 a
4	Benzoic aldehyde	11.47 ± 0.48 b	15.27 ± 1.17 a
5	Syringic acid	0.07 ± 0.01 a	0.03 ± 0.00 b
6	p-Cumaroyl-hexose II	0.31 ± 0.04 a	0.18 ± 0.01 b
7	p-Coumaric acid	201.21 ± 4.15 b	301.28 ± 1.31 a
8	trans Ferulic acid	255.59 ± 0.92 b	382.42 ± 4.23 a
9	cis Ferulic acid	22.69 ± 0.96 b	26.09 ± 0.76 a
10	Sinapic acid	1.22 ± 0.08 b	6.96 ± 0.16 a
11	Disinapic acid	0.95 ± 0.05 b	3.70 ± 0.07 a
12	Diferulic acid	4.50 ± 0.22 b	11.41 ± 0.78 a
13	Diferulic acid	0.42 ± 0.05 b	1.32 ± 0.02 a
14	Diferulic acid	5.65 ± 0.03 b	16.55 ± 0.09 a
15	Dehydrotriferulic acid	0.55 ± 0.04 a	0.38 ± 0.02 b
16	Disinapic acid	3.09 ± 0.01 b	7.55 ± 0.62 a
17	Disinapic acid	0.58 ± 0.02 b	2.57 ± 0.12 a
18	Dehydrotriferulic acid	1.25 ± 0.02 b	2.33 ± 0.23 a
19	Diferulic acid	4.75 ± 0.13 b	11.48 ± 0.60 a
20	Dehydrotriferulic acid	0.70 ± 0.01 b	1.53 ± 0.11 a
21	Diferulic acid	16.26 ± 0.13 b	37.93 ± 0.05 a
22	Diferulic acid	1.39 ± 0.05 b	3.18 ± 0.16 a
23	Diferulic acid	35.39 ± 0.17 b	80.64 ± 2.34 a
24	Dehydrotriferulic acid	4.66 ± 0.24 b	12.90 ± 0.59 a
25	Dehydrotriferulic acid	13.82 ± 0.04 b	22.13 ± 0.02 a
26	Dehydrotriferulic acid	2.17 ± 0.01 b	3.34 ± 0.03 a
27	Caffeoyl-hexose	3.69 ± 0.15 b	8.43 ± 0.18 a

---

Trans ferulic and p-coumaric acid were the major bound phenolic compounds. All the bound phenolic compounds, except 2, 5, 6 and 15, increased their content after the parboiling treatment.

The results obtained in this work showed that parboiling treatment caused substantial changes in bioactive compounds distribution in rice grain. New information about the bound sterol and triterpenic alcohols content have been reported. Moreover, HPLC-ESI-TOF-MS permitted the identification of several phenolic compounds that, to our knowledge, have been identified and quantified for the first time in rice bran.

The obtained results suggested that raw rice bran is a good ingredient for free phenolic compounds enrichment; instead parboiled rice bran is a better source of tocols, γ-oryzanol, phytosterols, triterpenic alcohols and bound phenolic compounds.

Acknowledgements

The authors Ana María Gómez-Caravaca and Vito Verardo thank the Spanish Ministry of Economy and Competitiveness (MINECO) for “Juan de la Cierva” post-doctoral contracts, JCI-2012-12566 and JCI-2012-12574, respectively.

References

1. T. C. Lin, S. H. Huang and L. T. Ng, Effects of cooking conditions on the concentrations of extractable tocopherols, tocotrienols and γ-oryzanol in brown rice: Longer cooking time increases the levels of extractable bioactive components, Eur. J. Lipid Sci. Technol., 2015, 117, 349–354.
2. P. Roy, T. Orikasa, H. Okadome, N. Nakamura and T. Shiina, Processing Conditions, Rice Properties, Health and Environment, Int. J. Environ. Res. Public Health, 2011, 8, 1957–1976.
3. G. R. Kim, E. S. Jung, S. Lee, S. H. Lim, S. H. Ha and C. H. Lee, Combined mass spectrometry-based metabolite profiling of different pigmented rice (Oryza sativa L.) seeds and correlation with antioxidant activities, Molecules, 2014, 19, 15673–15686.
4. P. Loypimai, A. Moongngarm, P. Chottanom and T. Moontree, Ohmic heating-assisted extraction of anthocyanins from black rice bran to prepare a natural food colourant, Innovative Food Sci. Emerging Technol., 2015, 27, 102–110.
5. M. Friedman, Rice brans, rice bran oils, and rice hulls: composition, food and industrial uses, and bioactivities in humans, animals, and cells, J. Agric. Food Chem., 2013, 61, 10626–10641.
6. S. Hagl, R. Grewal, I. Ciobanu, A. Helal, M. T. Khayyal, W. E. Muller and G. P. Eckert, Rice bran extract compensates mitochondrial dysfunction in a cellular model of early Alzheimer's disease, J. Alzheimer's Dis., 2015, 43, 927–938.
7. M. Candiracci, M. D. M. L. Justo, A. Castaño, R. Rodriguez-Rodriguez and M. D. Herrera, Rice bran enzymatic extract–supplemented diets modulate adipose tissue inflammation markers in Zucker rats, Nutrition, 2014, 30, 466–472.
8. H. Ham, J. Sung and J. Lee, Effect of rice bran unsaponifiables on high-fat diet-induced obesity in mice, J. Food Biochem., 2015, 39, 673–681.
9. O. Wang, J. Liu, Q. Cheng, X. Guo, Y. Wang, L. Zhao, F. Zhou and B. Ji, Effects of ferulic acid and γ-oryzanol on high-fat and high-fructose diet-induced metabolic syndrome in rats, PLoS One, 2015, 10, e0118135,  DOI:10.1371/journal.pone.0118135.
10. M. K. Sharif, M. S. Butt, F. M. Anjum and S. H. Khan, Rice Bran: a novel functional ingredient, Crit. Rev. Food Sci. Nutr., 2014, 54, 807–816.
11. E. Boselli, V. Velazco, M. F. Caboni and G. Lercker, Pressurized liquid extraction of lipids for the determination of oxysterols in egg-containing food, J. Chromatogr. A, 2001, 917, 239–244.
12. AACC International, Approved Methods of the American Association of Cereal Chemistry, 10th edn, 2000, CD-ROM. Methods 30.10, 44.15A.
13. M. Sanders, P. B. Addis, S. W. Park and D. E. Smith, Quantification of cholesterol oxidation products in a variety of food, J. Food Prot., 1989, 52, 109–114.
14. C. C. Sweeley, R. Bentley, M. Makita and W. Wells, Gas–liquid chromatography of trimethylsilyl derivatives of sugars and related substances, J. Am. Chem. Soc., 2002, 85, 2497–2507.
15. J. K. Kim, S. H. Ha, S. Y. Park, S. M. Lee, H. J. Kim, S. H. Lim, S. C. Suh, D. H. Kim and H. Suk Cho, Determination of lipophilic compounds in genetically modified rice using gas chromatography–time-of-flight mass spectrometry, J. Food Compos. Anal., 2012, 25, 31–38.
16. V. Verardo, A. M. Gómez-Caravaca, A. Gori, G. Losi and M. F. Caboni, Bioactive lipids in the butter production chain from Parmigiano Reggiano cheese area, J. Sci. Food Agric., 2013, 93, 3625–3633.
17. A. M. Gómez-Caravaca, V. Verardo and M. F. Caboni, Chromatographic techniques for the determination of alkyl-phenols, tocopherols and other minor polar compounds in raw and roasted cold pressed cashew nut oils, J. Chromatogr. A, 2010, 1217, 7411–7417.
18. T. Y. Ha, S. N. Ko, S. M. Lee, H. R. Kim, S. H. Chung, S. R. Kim, H. H. Yoon and I. H. Kim, Changes in nutraceutical lipid components of rice at different degrees of milling, Eur. J. Lipid Sci. Technol., 2006, 108, 175–181.
19. V. Verardo, A. M. Gómez-Caravaca, E. Marconi and M. F. Caboni, Air classification of barley flours to produce phenolic enriched ingredients: comparative study among MEKC-UV, RP-HPLC-DAD-MS and spectrophotometric determinations, LWT--Food Sci. Technol., 2011, 44, 1555–1561.
20. A. M. Gómez-Caravaca, V. Verardo, A. Berardinelli, E. Marconi and M. F. Caboni, A chemometric approach to determine the phenolic compounds indifferent barley samples by two different stationary phases: A comparison between C18 and pentafluorophenyl core shell columns, J. Chromatogr. A, 2014, 1355, 134–142.
21. I. Ferrer, J. F. García-Reyes, M. Mezcua, E. M. Thurman and A. R. Fernández-Alba, Multi-residue pesticide analysis in fruits and vegetables by liquid chromatography–time-of-flight mass spectrometry, J. Chromatogr. A, 2005, 1082, 81–90.
22. V. R. Pestana, R. C. Zambiazi, C. R. B. Mendonça, M. H. Bruscatto and G. Ramis-Ramos, The influence of industrial processing on the physico-chemical characteristics and lipid and antioxidant contents of rice bran, Grasas Aceites, 2009, 60, 184–193.
23. A. B. Padua and B. O. Juliano, Effect of parboiling on thiamin, protein and fat of rice, J. Sci. Food Agric., 1974, 25, 697–701.
24. R. Przybylski, D. Klensporf-Pawlik, F. Anwar and M. Rudzinska, Lipid components of north american wild rice (Zizania palustris), J. Am. Oil Chem. Soc., 2009, 86, 553–559.
25. S. L. Chia, H. C. Boo, K. Muhamad, R. Sulaiman, F. Umanan and G. H. Chong, Effect of subcritical carbon dioxide extraction and bran stabilization methods on rice bran oil, J. Am. Oil Chem. Soc., 2015, 92, 393–402.
26. L. F. Rizk, A. E. Basyony and H. A. Doss, Effect of microwave and air drying of parboiled rice on stabilization of rice bran oil, Grasas Aceites, 1995, 46, 160–164.
27. P. M. Pradeep, A. Jayadeep, M. Guha and V. Singh, Hydrothermal and biotechnological treatments on nutraceutical content and antioxidant activity of rice bran, J. Cereal Sci., 2014, 60, 187–192.
28. B. Min, A. McClung and M. H. Chen, Effects of hydrothermal processes on antioxidants in brown, purple and red bran whole grain rice (Oryza sativa L.), Food Chem., 2014, 159, 106–115.
29. B. Min, A. McClung and M. H. Chen, Phytochemicals and antioxidant capacities in rice brans of different color, J. Food Sci., 2011, 76, C117–C126.
30. P. Goufo and H. Trindade, Rice antioxidants: phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols, tocotrienols, c-oryzanol, and phytic acid, Food Sci. Nutr., 2014, 2, 75–104.
31. Y. Jiang and T. Wang, Phytosterols in cereal by-products, J. Am. Oil Chem. Soc., 2005, 82, 439–444.
32. M. Pelillo, G. Iafelice, E. Marconi and M. F. Caboni, Identification of plant sterols in hexaploid and tetraploid wheats using gas chromatography with mass spectrometry, Rapid Commun. Mass Spectrom., 2003, 17, 2245–2252.
33. G. Iafelice, V. Verardo, E. Marconi and M. F. Caboni, Characterization of total, free and esterified phytosterols in tetraploid and hexaploid wheats, J. Agric. Food Chem., 2009, 57, 2267–2273.
34. S. Tian, K. Nakamura and H. Kayahara, Analysis of phenolic compounds in white rice, brown rice, and germinated brown rice, J. Agric. Food Chem., 2004, 52, 4808–4813.
35. Y. Shao, F. Xu, X. Sun, J. 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.
36. 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.
37. M. Bordiga, S. Gomez-Alonso, M. Locatelli, F. Travaglia, J. D. Coïsson, I. Hermosin-Gutierrez and M. Arlorio, Phenolics characterization and antioxidant activity of six different pigmented Oryza sativa L. cultivars grown in Piedmont (Italy), Food Res. Int., 2014, 65, 282–290.
38. M. Bunzel, J. Ralph, C. Funk and H. Steinhart, Structural elucidation of new ferulic acid containing phenolic dimers and trimers isolated from maize bran, Tetrahedron Lett., 2005, 46, 5845–5850.
39. H. Zhang, Y. Sha, J. Bao and T. Beta, Phenolic compounds and antioxidant properties of breeding lines between the white and black rice, Food Chem., 2015, 172, 630–639.
40. D. Doberstein and M. Bunzel, Separation and detection of cell wall-bound ferulic acid dehydrodimers and dehydrotrimers in cereals and other plant materials by reversed phase high-performance liquid chromatography with ultraviolet detection, J. Agric. Food Chem., 2010, 58, 8927–8935.
41. Y. Qiu, Q. Liu and T. Beta, Antioxidant activity of commercial wild rice and identification of flavonoid compounds in active fractions, J. Agric. Food Chem., 2009, 57, 7543–7551.
42. H. Nakano, H. Ono, N. Iwasawa, T. Takai, Y. Arai-Sanoh and M. Kondo, Isolation and Identification of Phenolic Compounds Accumulated in Brown Rice Grains Ripened under High Air Temperature, J. Agric. Food Chem., 2013, 61, 11921–11928.
43. P. Y. Lam, F. Y. Zhu, W. L. Chan, H. Liu and C. Lo, Cytochrome P450 93G1 is a flavone synthase II that channels flavanones to the biosynthesis of tricin o-linked conjugates in rice, Plant Physiol., 2014, 165, 1315–1327.
44. P. Wanyo, N. Meeso and S. Siriamornpun, Effects of different treatments on the antioxidant properties and phenolic compounds of rice bran and rice husk, Food Chem., 2014, 157, 457–463.
45. M. Walter, E. Marchesan, P. F. Sachet Massoni, L. Picolli da Silva, G. Meneghetti Sarzi Sartori and R. Bruck Ferreira, Antioxidant properties of rice grains with light brown, red and black pericarp colors and the effect of processing, Food Res. Int., 2013, 50, 698–703.
46. W. Wang, J. Guo, J. Zhang, J. Peng, T. Liu and Z. Xin, Isolation, identification and antioxidant activity of bound phenolic compounds present in rice bran, Food Chem., 2015, 171, 40–49.
47. J. Grúz, J. Pospíšil, H. Kozubíková, T. Pospíšil, K. Doležal, M. Bunzel and M. Strnad, Determination of free diferulic, disinapic and dicoumaric acids in plants and foods, Food Chem., 2015, 171, 280–286.

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