Effect of rice parboiling on the functional properties of an enzymatic extract from rice bran

Accepted Manuscript Effect of rice parboiling on the functional properties of an enzymatic extract from rice bran Consuelo Santa María, Elisa Revilla, Bruno Rodríguez-Morgado, Angélica Castaño, Pilar Carbonero, Belén Gordillo, Rosa Cert, Juan Parrado PII:

S0733-5210(16)30256-9

DOI:

10.1016/j.jcs.2016.09.010

Reference:

YJCRS 2218

To appear in:

Journal of Cereal Science

Received Date: 23 May 2016 Revised Date:

13 September 2016

Accepted Date: 14 September 2016

Please cite this article as: Santa María, C., Revilla, E., Rodríguez-Morgado, B., Castaño, A., Carbonero, P., Gordillo, B., Cert, R., Parrado, J., Effect of rice parboiling on the functional properties of an enzymatic extract from rice bran, Journal of Cereal Science (2016), doi: 10.1016/j.jcs.2016.09.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Effect of rice parboiling on the functional properties of an enzymatic extract from rice

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bran

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Consuelo Santa María , Elisa Revilla* , Bruno Rodríguez-Morgado , Angélica Castaño , Pilar

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Carbonero, Belén Gordillo , Rosa Cert , Juan Parrado

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Sevilla. 41012 Sevilla, Spain

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Sevilla, Spain.

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Departamento de Bioquímica y Biología Molecular.Facultad de Farmacia, Universidad de

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Departamentode Nutrición.Facultad de Farmacia, Universidad de Sevilla. 41012 Sevilla, Spain

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Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide - Edificio 46.41013

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*Corresponding author:

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Dr. Elisa Revilla Torres

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Keywords: Rice bran, parboiling, enzyme extract, functional properties

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Abbreviations:

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AAPH: 2-2’-azobis-(2-amidinopropane)-hydrochloride

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CH: cumene hydroperoxide

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DMACA: p-dimethylaminocinnamaldehyde

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DNPH: dinitrophenylhydrazine

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MDA: malondialdehyde

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ORAC: Oxygen Radical Absorbance Capacity Assay

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PBS: phosphate buffer

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RB: untreated rice bran

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RBp: parboiled rice bran

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RBEE: rice bran enzymatic extract

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RBEEp: parboiled rice bran enzymatic extract

Departamento de Bioquímica y Biología Molecular. Facultad de Farmacia, Universidad de Sevilla. 41012 Sevilla, Spain

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[email protected]. Phone: 34-954 55 67 50. Fax: 34-954 55 65 98

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ABSTRACT We have studied the influence of the parboiling process on enzymatic extract obtained

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from rice bran. Two rice bran enzymatic extracts have been obtained; one from non-parboiled

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(RBEE), the other from parboiled rice (RBEEp), and their chemical composition and antioxidant

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capacities have been compared. These extracts differ in their main chemical composition;

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RBEEp has less carbohydrates and more fat content than RBEE. No differences in protein

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content were found.

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With regard to bioactive compounds, both extracts are rich in phytosterols, tocopherols,

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tocotrienols and γ-oryzanol, being γ-oryzanol similar in both extracts. However RBEEp is richer

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in total tocols and phytosterols and RBEE has the highest content in hydrophilic phenols.

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Functional properties such as total antioxidant activity revealed that RBEE has higher capacity

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to scavenger peroxyl radicals than RBEEp. Accordingly, specific antioxidant studies showed

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that RBEE has greater protective capacity against lipid and protein oxidation than RBEEp.

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Therefore, we can conclude that RBEE has better bioactive properties than RBEEp.

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In conclusion, these findings suggest that the parboiling pretreatment of rice modifies

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the bioactive composition of derived products such as rice bran enzymatic extract,that have

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been shown to exert bioactive properties with application in the nutraceutical and cosmetic

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fields.

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1. Introduction There is an emerging interest in the use of agroalimentary byproducts as potential

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source of functional ingredients for nutraceutical, medicinal or cosmetic purposes. Rice is the

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most important staple crop, feeding more than half of the world’s population and generating tons

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of waste (Sharif et al., 2014). Thus, rice bran, the greatest abundant byproduct obtained in the

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milling process is generated in large quantities and is underutilized despite having singular

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products with a biological significance (Hagl et al., 2013; Okai and Higashi-Okai, 2006), such as

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naturally-occurring antioxidant compounds (Goufo and Trindade, 2014). These antioxidants

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principally include γ-oryzanol, tocopherols, tocotrienols and polyphenols. Rice bran also

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contains high amounts of fiber and vitamin B complex. Moreover, proteins from rice bran have a

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high nutritional value, so balanced amino acid profile and hypoallergenic nature of bran protein

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are suggestive for its application in infant foods (Sharif et al., 2014). Nevertheless, several

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factors, including the presence of antinutrients and the lipid deterioration that starts soon after

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bran removal (Sharif et al., 2014), limit the nutritional use of rice bran. It is therefore necessary

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to find a good method for preventing the rapid deterioration of the bran, thus ensuring a quality

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material for further processing. Our group has developed an enzymatic method that inactivates

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the lipase, preventing rancidity and thus turning it into waste products that are rich in soluble

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compounds with applications in different fields (Parrado et al., 2006).

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The new products obtained from rice bran are water-soluble rice bran enzymatic extracts

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(RBEE) which preserve functional properties and improve both the proteins solubility and the rice

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branantioxidant components (Parrado et al., 2006). The enzymatic treatment also increases the

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concentration of minor functional components making the RBEE rich in bioactive compounds

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(Parrado et al., 2003). We have previously described that RBEE has antioxidant properties

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(Santa María et al., 2010) and hypocholesterolemic activity (Revilla et al., 2009), as well as

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antiproliferative and inmunoactivatory abilities (Revilla et al., 2013). Moreover, it has shown

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beneficial activities against hyperinsulinemia and hypertension, restoring endothelial function and

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vascular contractility in obese Zucker rats (Justo et al., 2013a, 2013b).

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Traditionally, rice is consumed as polished white rice with the husk, bran, and germ

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fractions removed. However, there is another variation of white rice called parboiled (partially

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boiled) rice, where the hulled rice is hydrated and steamed in order to retain the nutrition of the

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bran within the rice grain. Parboiled rice is widely used in cooking as the process alters the

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nature of the starch, resulting in transparent grains that will be less sticky and more separate

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when cooked. The aim of the current study was to evaluate how the parboiling process affects

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the chemical composition and antioxidant capacity of a rice bran extract obtained according to

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our enzymatic protocol.

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2. Materials and Methods

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2.1. Preparation of rice bran For this work it has been selected two types of rice bran (Oryza sativa, var. indica):

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untreated rice bran (RB) and parboiled rice bran (RBp), provided by Herba Ricemills, S.L.U

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(Sevilla, Spain). RB is obtained during the polishing/milling of raw rice grains once they have

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been stripped of the husk. It looks similar to flour, with a slightly granular texture, a light/beige

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brown color and it has a smell reminiscent of rice. Parboiled rice bran differs from untreated rice

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bran in one respect only; the grains have undergone a steaming process. This process consists

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of four consecutive steps: gross grain is soaked in water at 50-70 ºC for 3-4 hours and drained

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of the free water; steam and hot water are introduced into the cooking vessel and the rice is

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kept under pressure to gelatinize the starch in the rice grain and finally the grain is dried with hot

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air. Once dry, the grain undergoes the same process of husking and polishing/milling as the

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untreated white rice.

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2.2. Preparation of enzymatic extracts

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RBEE and RBEEp were prepared according to an enzymatic process previously

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described by our group (Parrado et al., 2006). Briefly, both types of rice brans were modified by

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enzymatic hydrolysis, using subtilisin (EC 3.4.21.62), a protease from Bacillus licheniformis as

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hydrolytic agent (Biocom, Spain) and a bioreactor at controlled temperature of 60 ºC and pH 8.

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The processing of this product follows different steps, including centrifugation, filtration, and

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concentration. The final product is brown syrup that is completely water-soluble.

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2.3. Chemical characterization Rice bran and ezymatic extracts were chemically characterised using the AOAC

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standard protocols (1990). Total protein content was determined by method 954.01. Total fat

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content was determined by method 920.39. Fatty acids were determined by method 969.33 and

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total ash content was determined by method 942.05.

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Carbohydrates were determined by HPLC as previously described (Zhang et al., 2003).

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Lipids containing γ-oryzanol were extracted using ethylacetate: hexane (1:1). The γ-oryzanol

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components were separated and quantified by analytical reversed-phase HPLC (Miller et al.,

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2003). Concentrations of tocopherols and tocotrienols isomers in RBEE were determined

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according to IUPAC standard method2432 (IUPAC, 1987). Tocopherols and tocotrienols were

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purified using a silica column. The oil sample was diluted with hexane and an aliquot of the

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diluted sample was subjected to HPLC analysis using a fluorescence detector (Agilent

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Technologies, USA) with a silica column packing with LiChrosorb SI 60.

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The phytosterols concentrations were determined by gas chromatography using 5-

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cholestane as an internal standard. A gas chromatograph (Varian 3800; Varian Inc., Walnut

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Creek, CA, USA) was equipped with an SAC-5 fused-silica capillary column (30 m 60.32 mm,

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Supelco, Bellefonte, PA, USA) and a flame ionization detector. The column was held at 280 ºC

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for 1 min and programmed to rise to 300 ºC at a rate of 2 ºC/min. It was then held at 300 ºC for

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20 min. The carrier gas was helium and the total gas flow rate was 20 ml/min. Injector and

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detector temperatures were 310 ºC and 320 ºC, respectively. Comparing the retention times

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with standard times enabled the sterols to be identified.

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2.5. Analyses of phenolics by spectrophotometry

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The spectrophotometric determination of total flavonols, flavanols and phenolic content

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of

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spectrophotometer (Palo Alto, CA, USA), using 10-mm path length glass cells and distilled

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water as reference. For this purpose, 5 mg of samples (RBEE and RBEEp, respectively) were

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dissolved in 2 ml of ultrapure water and filtered through Millipore-AP 20 filters (Bedford, MA)

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prior to the spectrophotometric analysis. Total flavanols content was determined following a

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modification of the method described by Vivas et al. (1994). Ten microlitres of supernatant was

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mixed with 190 µL of methanol and 1 mL of p-dimethylaminocinnamaldehyde (DMACA)

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reagent. The absorbance was recorded at 640 nm after 10 min of reaction. A calibration curve

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of (+)-catechin was used for quantification. Results were expressed as mg of flavanols

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(expressed as catechin equivalents) per g of dry weight.

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Total flavonols content was determined using a modification of the method originally

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described by Glories for wine phenols (Glories, 1979). The method consisted of placing 0.25 mL

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of sample or standard in a test tube and adding 0.25 mL of 0.1% HCl in 95% ethanol and 4.55

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mL of 2% HCl. The solution was mixed and allowed to sit for approximately 15 min before

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reading the absorbance at 360 nm. Standard used was quercetin in 95% ethanol and the results

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were expressed as mg of flavonols (expressed as quercetin equivalents) per g of dry weight. Total phenolics content was determined using a modification of the Folin-Ciocalteau

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method (Singleton and Rossi, 1965). Briefly, 0.25 mL of sample, 1.25 mL of Folin-Ciocalteu

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reagent, and 3.75 mL of a solution of sodium carbonate at 20% were mixed, and distilled water

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was added to make up a total volume of 25 mL. The solution was homogenized and left to stand

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for 120 min for the reaction to take place and stabilize. Absorbance was measured at 765 nm.

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Gallic acid was used as a calibration standard, and results were expressed as mg of polyphenol

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(expressed as gallic acid equivalents) perg of dry weight.

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2.6. Oxygen Radical Absorbance Capacity Assay (ORAC)

Antioxidant capacity was assayed using the method described by Ou et al. (2001).The

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assay uses the protein fluorescein as a free-radical-sensitive fluorescent indicator of the

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antioxidant capacity of the samples. 50 µL of the working solution containing 78 nM of

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fluorescein, that was prepared daily in phosphate buffer (PBS) (75 mM, pH 7.0), was

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preincubated at 37 °C for 30 min, in both the absen ce and presence of 50 µL of the appropriate

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dose of the antioxidants Trolox (referenced as standard solution), PBS or extracts. Oxidation

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was triggered by adding 25 µL of the water soluble free-radical initiator 2-2’-azobis-(2-

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amidinopropane)-hydrochloride (AAPH) to a final concentrationof 4 mM. The fluorescence was

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measured immediately and measurements were taken every 5 min. The measurements were

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taken in triplicate. The final ORAC values were expressed as nmoles Trolox equivalents/mg

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(nmol TE/mg).

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2.7. Preparation of tissue homogenates

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Wistar rats (3 months old) were used in all of the experiments. The rats were provided

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by the animal care facility of the University of Seville and all experiments were approved by the

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local ethical committees and complied with international animal welfare guidelines. They were

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housed in groups of three or four in a temperature- and light-controlled room, with a pathogen-

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rapidly removed. The liver was homogenised in 6 volumes of homogenisation buffer: 20

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mMTris/HCl, 1 mMNaCl, and pH 7.5. A cocktail of protease inhibitors (0.2 mM

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phenylmethylsulphonyl fluoride, 1 mM EDTA, chymostatin, leupeptin and pepstatin: 1µg/mL of

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each) was added to the incubation mixture to prevent proteolysis. The homogenates were

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centrifuged for 30 min at 1000 g. The process was performed at 0-4°C.

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2.8. Dot blot analysis of protein carbonyl groups

The protein carbonyl determination was performed as described previously (Conrad et

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al., 2000). Briefly, 200 µL liver homogenates (17.5 mg/mL) were incubated with increasing

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doses of the enzymatic extracts (2.5 mg/mL, 5 mg/mL, 10 mg/mL) or 500 µL of quercetin

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(0.01mg/mL) as the antioxidant control, respectively. The samples were incubated for 30 min at

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37°C. Oxidation was initiated by adding 25 µL cumen e hydroperoxide (CH) to a final

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concentration of 0.5 mM. After an additional incubation for 30 minutes at 37°C, reaction was

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stopped with ice-cold TCA and samples were centrifuged. Protein pellets were analyzed by dot

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blot. Samples were post-derivatized with DNPH (dinitrophenylhydrazine) on membrane and

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after derivatization membranes were blocked with 5% low-fat milk in Tris buffered saline (50 mM

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Tris-ClH,

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dinitrophenylhydrazone polyclonal antibody (1:1000, Sigma D-9656) overnight in cold and

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understirring, to identify the oxidized proteins. After washing, we used anti-rabbit HRP (DAKO

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P0399, 1:3000) as a secondary antibody that would be recognized by chemiluminescence

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(Thermo Scientific 34080). Images were acquired with a system FUJIFELM LAS 3000 mini and

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analyzed using the MULTI GAUGE V3.0 program. Results are expressed as percentage of

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protein oxidation respect to control (sample without antioxidant).

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2.9. Determination of malondialdehyde (MDA)

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An aliquot (0.5 mL) of the supernatant obtained after the treatment with oxidant was

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used for MDA determination (Esterbauer and Cheeseman, 1990). Samples were treated with

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TBA reagent (20 mM TBA in 50% v/v glacial acetic acid), and then heated at 100 ºC for 1 h. The

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absorbance of the upper layer was measured at 532 nm with a spectrophotometer, and the

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MDA level was expressed as percentage of lipid peroxidation respect to control (sample without

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antioxidant).

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2.10. Statistical analysis All data are presented as the mean ± S.E.M. of five independent experiments. Data

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were statistically analyzed using a two tailed t-test. The significance was set at 95% of

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confidence. Significant differences are referenced as p < 0.05 in the text.

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3. Results and discussion

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3.1. Chemical composition First, the chemical composition of two rice brans and their respective enzymatic extracts

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was analyzed. The content of carbohydrates, fat, protein and ash of the rice brans and

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enzymatic extracts are shown in Table 1. The main components of rice brans are

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carbohydrates, having RBp less carbohydrates and higher fat content than RB. This could be

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due to the parboiling process thatleads to the solubilization and diffusion of soluble components

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allowing some nutrients to transfer from the hull into the grain. Finally, proteins represent the

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smallest component in both brans, with no differences between them having been found.The

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enzymatic process leads to a drastic change in the chemical composition of extracts. The

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protein content in both extracts is clearly enriched when compared with the corresponding rice

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bran, becoming the main component. This is due to the use of proteases which extract,

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solubilize and hydrolyze the initial insoluble proteins in brans, reducing the size of original rice

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bran proteins into soluble peptides (Parrado et al., 2006), without any difference between both

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extracts. It has been described that proteins from RB have a high nutritional value with balanced

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amino acid profile and hypoallergenic nature that make it suggestive for its application in infant

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foods (Sharif et al., 2014).

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The enzymatic extracts are also enriched in fat content, the lipidic content being higher

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in the RBEEp. The fat components present in the extracts are mainly soluble due to protein

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interactions, a similar process to lipid extraction and solubilization (emulsion) by proteases was

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previously described (Grodji et al., 2006). Rice bran contains mainly unsaturated fatty acids and

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considerable amounts of essential fatty acids (Sharif et al., 2014). The fatty acid composition of

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enzymatic extracts has also been determined and data show (Table 2) that both extracts have

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similar fatty acid content, being linoleic and oleic acids the most abundant fatty acids found.

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The phytosterols content is shown in Table 3. γ-oryzanol is the most characteristic

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phytochemical in rice. This molecule is antitumorogenic, hypocholesterolemic and anti-

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inflammatory (Cicero, 2001). There is an increasing interest in the use of this molecule for

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drugs, nutraceuticals and functional foods, as well as cosmetics. Our study shows that both

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enzymatic extracts have a similar γ-oryzanol content. Both extracts were γ-oryzanol-enriched

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with respect to its respective rice bran. So, the amount of γ-oryzanol in RB and RBp was 7.20

ACCEPTED MANUSCRIPT and 7.72 mg/g respectively; therefore the increase of γ-oryzanol in extracts was 24 % and 30%

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respectively. The RBEEp was richer in phytosterols than RBEE, probably because hydrosoluble

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molecules diffuse into the grain during the parboiling process. Several properties have been

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attributed to phytosterols such as antiinflammatory, antitumor-like, bactericidal and fungicidal.

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Nevertheless, the best-characterized property is its hypocholesterolemic activity (de Jong et al.,

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2003). Phytosterols are highly effective in competing with cholesterol for incorporating into

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mixed micelles, which is the supposed mechanism for the cholesterol absorption-inhibiting

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action of plant sterol (de Jong et al., 2003). Overall, in both extracts the most abundant is β-

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sitosterol, followed by campesterol.

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Rice is also a good source of other lipid molecules such as tocopherols and tocotrienols

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that are known collectively as vitamin E or tocols. Vitamin E occurs in nature in at least eight

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different isoforms, four tocopherols and four tocotrienols. The concentration of tocols was also

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analyzed (Table 4). Seven homologues (α-, β-, γ- and δ-tocopherols and α-, γ- and δ-

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tocotrienols) were identified. Of these, γ-tocotrienols werethe most abundant, followed by α-

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tocopherols and α-tocotrienols. The highest total tocopherol (174 µg/g) and tocotrienol (260

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µg/g) concentrations were observed in RBEEp, probably also due to the lipid enrichment found

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in the parboiled product. In general, the tocotrienols content was greater than that of

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tocopherols. Interestingly, the composition of tocols identified in our samples was similar to that

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described for bran rice oil (Yoon et al., 2014).

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Phenolic acids and flavonoids are major classes of phenolic compounds that are widely

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found in the plant kingdom (Yao et al., 2012). Total water-soluble flavonols, flavanols and

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phenolics content in both enzymatic extracts are represented in Table 5. Total phenolic content

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was higher in RBEE than in RBEEp. The flavanols content was higher in RBEE, as also

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occurred for total phenolics content.

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3.2. Antioxidant capacity

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The antioxidant activity of both enzymatic extracts has been analyzed by the ORAC

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methods. The ORAC assay measured the scavenging capacity against peroxyl radicals. RBEE

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reached an ORAC value (696.1 ± 63.4 nmol TE/mg), significantly higher (p<0.001) than that for

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RBEEp (487.8 ± 10.2 nmol TE/mg).

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capacity for protecting proteins and lipids against oxidative damage caused by the peroxyl

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radical, using a liver homogenate as anex vivo model. The MDA and carbonyl groups content

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was measured as an index of lipid and protein damage respectively. Figure 1A shows the effect

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of CH on liver homogenate lipids in the absence or presence of antioxidants. The CH produced

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a significant increase in MDA content without any protective substance. The protection against

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lipid oxidation was higher in RBEE, showing a significantly lower MDA content at minimal doses

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tested (2.5 mg/mL), reaching 50% lipid protection at doses of 10 mg/mL. This was similar to the

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observed lipid protection exerted by quercetin (0.01 mg/mL). In contrast, the RBEEp only

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showed a significant protection at the maximal concentration assayed (10 mg/mL).

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Carbonyl groups are introduced into proteins by oxidative mechanisms and are an

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established marker of protein oxidation. After incubation of liver homogenates with CH in the

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absence of a protective substance there was also a remarkable increase in protein oxidation

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which produced a significant rise in the generation of protein carbonyls (Figure 1B). The

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inclusion of RBEE showed a significant protein protection, preventing the oxidation of the liver

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proteins and lowering the carbonyl groupseven under the values of control samples. However,

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the RBEEp showed no protection at any of the doses assayed.

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In the last two decades, research has shown that rice bran contains a unique complex

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of naturally occurring antioxidant compounds. The most characteristic antioxidant of rice bran is

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γ-oryzanol, followed by tocopherols, tocotrienols and polyphenols (Goufo andTrindade, 2014).

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The antioxidant capacity observed in both enzymatic extracts may be related to these

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compounds. So, γ-oryzanol up-regulates antioxidant genes and down-regulates the oxidative

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stress gene markers (Ismail et al., 2010). However, the concentration of γ-oryzanol is similar in

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both extracts and would not explain the difference in antioxidant capacity found between the

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two extracts assayed. Tocols have also antioxidant activity because of their ability to donate

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phenolic hydrogens (electrons) to lipid radicals. Several researchers have reported that

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tocotrienols have a greater antioxidant activity than tocopherols and that they exert a more

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efficient protection against some free radical related diseases (Qureshi et al., 2002). However,

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the difference in antioxidant activity is neither explained by tocols content as is higher in RBEEp

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than in RBEE.

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ACCEPTED MANUSCRIPT As described above, a third important group of antioxidants in enzymatic extracts are

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polyphenols. They exert multiple biological activities including vasodilatory, anticarcinogenic,

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antiinflammatory, antibacterial, immune-stimulating, antiallergic, etc (Rice-Evans et al., 1996).

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Polyphenols present in cereals have received less attention than those present in fruit and

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vegetables, although nutritional guidelines put grains and grain products at the base of the food

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guide pyramid. The explanation for the discrepancy in antioxidant activity may be found in the

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phenols content. They are water-soluble antioxidants that act as reducing agents, hydrogen

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donating antioxidants, and singlet oxygen quenchers. Thesecompounds pass from the hull to

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the grain during the parboiling processing. Therefore the bran obtained from parboiled rice has

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fewer soluble antioxidants than that obtained from the white rice bran and consequently RBEEp

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has lower phenolic content than RBEE. Accordingly, a plausible explanation for the lower

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antioxidante activity in RBEEp maybe a better synergic effect between lipophilic and hydrophilic

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antioxidants found in RBEE. The activity of antioxidants in foods and biological systems is

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dependent on a multitude of factors, including the localization of antioxidants in different phases

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(Frankel and Meyer, 2000).

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Conclusions

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This comparative study shows that RBEE, the enzymatic extract obtained from

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untreated rice contains fewer amounts of antioxidant compounds such as sterolsand tocols but

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higher general and specific antioxidant capacity thanRBEEp, the extract obtained from

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parboiled rice. This may be due to the higher content in hydrosoluble antioxidants in the former

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which keep a better synergistic effect between hydrophilic and lipophilic antioxidants. Altogether

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we can conclude that although parboiled rice has advantages in cooking, parboiling process is

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clearly inconvenient when bran is used as functional foods sources since alters in a negative

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way the bioactive properties of the enzymatic extract.

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Funding

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This work was supported by Ministerio de Ciencia e Innovación, CTM2011-29930-01, and Junta

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de Andalucía, Proyecto de Excelencia P11-RNM-7887.

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ACCEPTED MANUSCRIPT References

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endothelial function and vascular contractility in obese rats by reducing vascular inflammation

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and oxidative stress. J. Nutr. Biochem. 24,1453-1461.

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Justo, M.L., Rodriguez-Rodriguez, R., Claro, C.M., Alvarez de Sotomayor, M., Parrado, J.,

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Herrera, M.D., 2013b. Water-soluble rice bran enzymatic extract attenuates dyslipidemia,

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hypertension and insulin resistance in obese Zucker rats. Eur. J. Nutr. 52, 789-797.

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Miller, A., Frenzel, T., Schmarr, H.G., Engel, K.H., 2003. Coupled liquid chromatography-gas

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chromatography for the rapid analysis of γ-oryzanol in rice lipids. J. Chromatogr.A 985, 403-410.

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Rice-Evans, CA., Miller, NJ., Paganga, G., 1996. Structure-antioxidant activity relationships of

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antioxidant

activity

and

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Parrado,

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des

procyanidines

du

raisin

et

du

vin

par

la

méthode

au

p-

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pyrazolone-labeled carbohydrates by liquid chromatography and micellar electrokinetic

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chromatography. J. Chromatogr. B. 793, 159-165.

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ACCEPTED MANUSCRIPT Figure legends:

431

Figure 1. A. Determination of malondialdehide levels. Lipid peroxidation was performed on

432

liver homogenate by induction with cumene hydroperoxide. Inhibitory capacity was evaluated by

433

malondialdehyde production in presence or absence of antioxidants. Samples with quercetin

434

were used as antioxidant control. All data are presented as mean ± S.E.M. of five independent

435

experiments. **p<0.001 vs RBEE; p<0.05 vs RBEEp B. Determination of carbonyl groups.

436

Carbonyl groups after incubation of a liver homogenate with cumene hydroperoxide alone or in

437

the presence of antioxidants were determined. Aliquots of the reaction mixture were subjected

438

to DOT-Blot to determine carbonyl groups. All data are presented as mean ± S.E.M. of five

439

independent experiments. **p<0.001, *p<0.05 vs RBEE

#

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ACCEPTED MANUSCRIPT Table 1 Chemical characterization

%

RB

RBEE

RBp

RBEEp ,##

Carbohydrates

51.06 ± 1.89

28.90 ± 2.10*

45.92 ± 0.90◊

22.21 ± 1.10*

Fat

22.50 ± 1.90

33.09 ± 2.70*

28.36 ± 3.00◊

39.57 ± 2.80*

Protein

16.98 ± 2.00

33.96 ± 4.00*

16.23 ± 2.30

32.46 ± 2.90*

9.46 ± 0.25

4.05 ± 0.15*

9.49 ± 0.20

5.76 ± 0.18*

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Ash

,#

,##

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All data are presented as mean ± S.E.M. of five independent experiments. Statistical # ## significance: ◊p<0.05 vs RB; *p<0.001 vs its respective RB; p<0.05, p<0.001 vs RBEE.

ACCEPTED MANUSCRIPT

RBEEp

C14:0 Mirístic acid

0.2 ± 0.03

0.2 ± 0.30

C16:0 Palmitic acid

14.6 ± 2.01

16.8 ± 2.16

C16:1 Palmitoleic acid

0.2 ± 0.05

0.2 ± 0.03

C17:0 Margaric acid

0.0 ± 0.00

0.0 ± 0.00

C17:1 Margaroleic acid

0.1 ± 0.16

0.0 ± 0.00

C18:0 Esteraric acid

1.6 ± 0.18

1.7 ± 0.15

C18:1 Oleic acid

37.3 ± 3.00

37.9 ± 3.00

C18:2 Linoleic acid

43.0 ± 3.90

40.1 ± 4.00

C20:0 Araquic acid

0.5 ± 0.12

0.6 ± 0.11

C18:3 Linolenic acidd

1.3 ± 0.15

C20:1 Eicosenoic acid

0.5 ± 0.09

C22:0 Behenic acid

0.2 ± 0.06

C24:0 Lignoceric acid

0.4 ± 0.05

SC

RBEE

M AN U

%

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Table 2 Fatty acid composition

1.2 ± 0.13

0.5 ± 0.08

0.2 ± 0.06

0.3 ± 0.05

TE D

All data are presented as mean ± S.E.M. of five independent

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experiments. No significant differences were found.

ACCEPTED MANUSCRIPT Table 3 Phytosterol composition

Sterols

RBEE

γ-Oryzanol (mg/g)

8.95 ± 0.85

RBEEp 10.51 ± 0.63 ##

15 ± 1.0

2,4 Metilencholesterol (µg/g)

6.9 ± 3.0

15 ± 2.4

Campesterol (µg/g)

871 ± 23

1627 ± 39

Campestarol(µg/g)

41 ± 3.0

64 ± 6.3

Stigmasterol (µg/g)

504 ± 28

δ-7-campesterol (µg/g)

18 ± 2.0

#

##

SC

#

##

947 ± 38 91 ± 10

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Clerosterol (µg/g)

29 ± 2.4

RI PT

Brassicasterol (µg/g)

##

#

58 ± 5.0

94 ± 9.8

1694 ± 32

3085 ± 34

58 ± 4.0

82 ± 7.1

127 ± 9.0

326 ± 23

δ-5-2,4- Stigmasterol (µg/g)

32 ± 2.3

49 ± 4.0

δ-7- Stigmasterol (µg/g)

17 ± 2.0

176 ± 17

δ-7-Avenasterol (µg/g)

18 ± 1.5

126 ± 11

Cholesterol (µg/g)

20 ± 3.0

41 ± 4.0

Others (µg/g)

73 ± 6.2

170 ± 15

Totals (µg/g)

3553 ± 66

6922 ± 118

β-Sitosterol (µg/g) Sitostanol (µg/g)

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δ-Avenasterol (µg/g)

##

#

##

#

##

##

#

##

##

All data are presented as mean ± S.E.M. of five independent experiments. Statistical # ## significance: p<0.05, p<0.001 vs RBEE.

ACCEPTED MANUSCRIPT Table 4 Tocols composition (µg/g)

RBEE

RBEEp

α-tocopherols

38.6 ± 4.0

96.7 ± 8.0

β-tocopherols

9.9 ± 0.5

8.3 ± 5.0

γ-tocopherols

20.4 ± 3.0

40.7 ± 6.0

δ-tocopherols

24.4 ± 4.0

28 ± 1.0

Total

93.4 ± 10

174 ± 20

α -tocotrienols

32 ± 2.0

82.9 ± 9.0

β-tocotrienols

N.D.

#

Tocotrienols

N.D.

132 ± 9.0

171 ± 11

δ-tocotrienols

5.9 ± 0.3

5.9 ± 1.0

Total

170 ± 15

SC

γ-tocotrienols

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Tocopherols

#

M AN U

260 ± 60

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All data are presented as mean ± S.E.M. of five independent experiments. Statistical # significance: p<0.05 vs RBEE.

ACCEPTED MANUSCRIPT

RBEE

RBEEp

Total flavonols

0.62 ± 0.01

0.70 ± 0.05*

Total flavanols

2.90 ± 0.05

2.50 ± 0.17**

Total phenolics

20.14 ± 1.12

RI PT

Table 5 Flavonoids and phenolic content

Compounds (mg/g DW)

16.65 ± 0.9**

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All data are presented as mean ± S.E.M. of five independent experiments. Statistical significance: *p<0.05 vs RBEE, **p<0.001 vs RBEE. DW: dry weight

AC C

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Figure 1. A. Determination of malondialdehide levels. Lipid peroxidation was performed on liver homogenate by induction with cumene hydroperoxide. Inhibitory capacity was evaluated by malondialdehyde production in presence or absence of antioxidants. Samples with quercetin were used as antioxidant control. All data are presented as mean ± S.E.M. of five independent #

experiments. **p<0.001 vs RBEE; p<0.05 vs RBEEp . B. Determination of carbonyl groups.

RI PT

Carbonyl groups after incubation of a liver homogenate with cumene hydroperoxide alone or in the presence of antioxidants were determined. Aliquots of the reaction mixture were subjected to DOT-Blot to determine carbonyl groups. All data are presented as mean ± S.E.M. of five

AC C

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M AN U

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independent experiments. **p<0.001, *p<0.05 vs RBEE

ACCEPTED MANUSCRIPT Highlights: Enzymatic hydrolysis of rice bran yields extracts rich in bioactive compounds Main bioactives in enzymatic extracts are γ-oryzanol, tocopherols and tocotrienols Parboiling of rice increases lipophilic antioxidant content in bran enzymatic extract Parboiling method decreases hydrophilic antioxidant content in bran enzymatic extract

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Parboiling method decreases total antioxidant capacity in bran enzymatic extract