Development of Wheat Bran Oil Concentrates Rich in Bioactives with

Oct 19, 2017 - Wheat bran, an abundant byproduct of the milling industry, comprises fat-soluble bioactives and fibers. In the present study, two conce...
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Cite This: J. Agric. Food Chem. 2017, 65, 9838-9848

Development of Wheat Bran Oil Concentrates Rich in Bioactives with Antioxidant and Hypolipidemic Properties Sharanappa T. Talawar,† Nanishankar V. Harohally,‡ Chetana Ramakrishna,‡ and G. Suresh Kumar*,† †

Department of Biochemistry and ‡Spice, Flavour Science, CSIR−Central Food Technological Research Institute, Mysuru 570020, India ABSTRACT: Wheat bran, an abundant byproduct of the milling industry, comprises fat-soluble bioactives and fibers. In the present study, two concentrates were prepared from wheat bran oil (WBO) using silicic acid coupled with acetone (WBA) and hexane (WBH). WBA extract had enhanced color and viscosity and was enriched with fat-soluble bioactives (sterols, oryzanollike compounds, tocopherols, and carotenoids) as evidenced from NMR and other techniques. In in vitro studies, WBA exhibited significant free radical scavenging activity, limited DNA and LDL oxidation, and inhibiting HMG-CoA reductase and lipase activity better than WBH and WBO. Further, an in vivo study with WBA 2 or 3.5% containing high fat diet ameliorated malonaldehyde (MDA) level, lipid profile, and antioxidant enzyme (SOD, catalase, GPx, and GR) activities in liver. A possible reason for this effect is downregulation of HMG-CoA reductase expression with WBA. Thus, WBA has significant potential as an ingredient in health food formulations. KEYWORDS: wheat bran oil concentrate, fat-soluble bioactives, hyperlipidemia, lipid profile

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INTRODUCTION

ments fed a high fat diet have been carried out to demonstrate the beneficial effect of wheat bran oil bioactives.

Wheat is a major staple food around the globe and second in India. Wheat bran (WB) and germ are byproducts of wheat milling industries. Wheat bran is a rich source of dietary fiber and known to have health beneficial effects against obesity and diabetes. A substantial number of WB-based food products are already available commercially.1 It is well-documented that the removal of lipids and lipid-soluble components from WB increases colon tumorigenesis, whereas fortification of defatted bran diet with bran oil significantly inhibits it, suggesting that the bioactive compounds present in WB oil possess inhibitory properties against colon carcinogenesis.2 The biological activities of molecules are mainly attributed to 3D structure and intactness of functional groups.3 In the context of wheat bran oil, several harsh methods are employed for the oil treatment as well as for the preparation of oil concentrate and other crystallization processes that could alter the structure and in turn the activity of bioactive molecules. Supercritical fluid extraction is one advanced technique, but has limitations at larger scale, including high cost. Enrichment of such various bioactives in a convenient medium is very important, but attempts at such have been scarce. The fat-soluble bioactives, sterols, carotenoids, tocols, and so forth, as well as their isoforms, are very sensitive to various extraction conditions that may affect their bioavailability. The presence of dietary fat has been shown to increase bioavailability of such bioactives.4,5 Any food product developed from this oil concentrate will have enhanced bioactives with superior market value as an ingredient or nutraceutical concentrate. The present study was attempted to develop a process for fat-soluble bioactives from wheat bran oil and subsequent identification of the bioactives. Furthermore, evaluation of antioxidant activity, hypolipidemic properties by in vitro specific enzyme inhibition, and in vivo animal model experi© 2017 American Chemical Society

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MATERIALS AND METHODS

Fresh wheat bran was obtained from the International School of Milling Technology (ISMT), a mill located at our institute, Department of Flour Milling, Baking & Confectionery Technology (FMBCT), CSIR-CFTRI. Standard oryzanol, sitosterol, α-tocopherols, β-carotene, lutein, lipase, bovine serum albumin, cholesterol, calf thymus DNA, Tris-HCl, NaCl, CaCl2, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). BODIPY 493/503 stains were from Thermo Scientific, Invitrogen (Carlsbad, CA, USA). HPLCgrade acetonitrile, methanol, hexane, and ethyl acetate were purchased from RANKEM Ltd. (New Delhi, India). Kits for lipid profile studies were obtained from Agappe Diagnostics Ltd. (Kerala state, India). Silicic acid was procured from Loba Chemie Pvt Ltd. (Mumbai, India). Alumina was obtained from Himedia Laboratories Pvt Ltd. (Maharashtra, India), and other chemicals used were of analytical grade. Preparation of Wheat Bran Oil Concentrate and Evaluation of Their Physicochemical Properties. Wheat bran (Triticum estivum) was powdered and passed through a sieve (No. 12/1.68 μm). The Soxhlet extraction method was used for lab scale studies, and a solvent extractor (Arm field, product code: FT29, England) was used for pilot scale extraction of oil. As given in Figure 1, extraction was carried out using 14 kg of bran using 57 L of hexane at room temperature for ∼10 h, and desolventalization was carried out at 45 °C. The extracted oil was clarified by adding cold ethanol (3 times the weight of oil), left overnight at 4 °C, and then centrifuged. The oil obtained was subjected to rotary vacuum evaporation to remove ethanol and then passed through a glass column (prototype made at CSIR-CFTRI, Mysore 50 cm length × 6 cm i.d.) containing activated Received: Revised: Accepted: Published: 9838

July 25, 2017 October 18, 2017 October 19, 2017 October 19, 2017 DOI: 10.1021/acs.jafc.7b03440 J. Agric. Food Chem. 2017, 65, 9838−9848

Article

Journal of Agricultural and Food Chemistry

mL/min. β-Carotene used as the reference standard for identification and quantification. Tocopherol and Tocotrienol Composition. Total tocopherols and tocotrienols (total tocols) content and composition were determined by HPLC (model LC-10 AVP Shimadzu Corp.,Tokyo, Japan) coupled with a silica column (250 mm × 4.6 mm, 5 μm i.d.) and fluorescence detector. Conditions used were λ Ex 290 nm, λ Em 330 nm, mobile phase of hexane/isopropanol (99.5:0.5 v/v), flow rate of 1 mL/ min, and α-tocopherol as the standard for identification and quantification. Oryzanol-like Compound Analysis. Total γ-oryzanol-like compounds were determined using an HPLC Shimadzu LC 10A system coupled with Shimadzu C18 column (150 mm × 4.6 mm, 5 μm i.d.) and Shimadzu SPD-M10 AVP UV detector. Conditions used were a mobile phase of acetonitrile/methanol/ isopropyl alcohol (10:9:1 v/v/v), flow rate of 1 mL/min, wavelength of 325 nm, and γ-oryzanol as a standard for steryl ferulate quantification.8 Nuclear Magnetic Resonance (NMR) Spectroscopy. Samples for NMR spectroscopy were prepared by dissolving 20 mg of oil in CDCl3 for the measurement of 1H and 13C spectra, respectively. All NMR experiments were performed on a Bruker 400 MHz spectrometer at 25 °C using a BBO probe. Proton chemical shifts are given relative to residual CDCl3 signal (7.26 ppm), whereas carbon chemical shifts are given relative to the residual CDCl3 (77.16 ppm). In Vitro Antioxidant and Inhibition of HMG-CoA Reductase Assay. Free radical scavenging activity of the fractions and oil were determined by using 2,2-diphenyl-1picrylhydrazyl (DPPH) radicals in toluene in accordance with procedure of Ramadan9 and was expressed in terms of concentration of oil required to scavenge 50% DPPH free radicals. The inhibition of 3-hydroxy-3-methyl glutaryl-CoA (HMG-CoA) reductase activity was carried out according to the method of Hulcher et al.10 Lipase Inhibition Assay by Titration Method. The inhibitors used for the lipase assay were prepared using DMSO to achieve a concentration of 10 μg/mL. The substrate emulsion was prepared by ultrasonication of olive oil in a solution containing lecithin (1 mM), bovine serum albumin (15 mg/mL), cholesterol (0.1 mM), 2 mM Tris-HCl (pH 8), NaCl (100 mM), and CaCl2 (1 mM). This composition of the test emulsion was chosen to closely mimic the in vivo conditions of the intestine. The reaction was started by the addition of porcine pancreatic lipase and terminated by adding ethanol. The released fatty acid was estimated by titrating against 0.02 N NaOH.11 In Vitro DNA Protection Assay. DNA protection assays were carried out according to the method of Rodriguez et al.12 The reaction mixture of calf thymus DNA (10 μg/mL) and Fenton’s reagent (50 mM ascorbic acid, 80 mM FeCl3, and 30 mM H2O2) followed by the addition of extracts and final volume of the mixture was brought up to 20 μL with distilled water. The mixture was then incubated for 30 min at 37 °C, and the DNA was analyzed on 1% agarose gel with TAE buffer followed by ethidium bromide staining. In Vitro LDL-Oxidation Prevention Assay. The LDL was isolated from a single donor by discontinuous ultracentrifugation of freshly isolated plasma.13 The obtained LDL protein concentration was determined by Lowry’s method. Oxidized LDL was prepared by exposing freshly isolated LDL to CuSO4 (10 mM/L) at 37 °C for 12 h.

Figure 1. Extractions of wheat bran oil concentrate from wheat bran into different fractions (WBO, WBH, and WBA). silicic acid (100−200 mesh) and alumina (in the ratio of oil-silicic acidalumina of 1:1:2.5; w:w:w). Activation was carried out by keeping both silicic acid and alumina in a hot air oven at 108 ± 2 °C for 8 h. Hexane (three bed volume of the column) was used as a first eluent solvent followed by acetone (three bed volume of the column), and the flow rate was maintained at 2 mL/min. Both oil fractions obtained were individually subjected to vacuum evaporation followed by air drying to remove residual solvent completely and stored in brown bottles at 4 °C. Using standard methods of AOCS,6 the free fatty acid value (FFA) (Ca 5a-40), peroxide value (PV) (Cd 8−53), and unsaponifiable matter (Ca 6a-40) were determined. Color was measured by CIELAB parameters using a Hunter colorimeter (Hunterlab D25 A-9, USA) equipped with the setting 10°/D 65 and expressed according to the Commission Internationale de L’Eclairaige (CIE) system and reported as L* (lightness), a* (redness), and b* (yellowness); ΔE represents the total color difference between the samples. A Brookefield (Viscometer model DV-II+Pro) rotational type viscometer was used to measure the viscosity of oil samples. Before use, the viscometer (accuracy, ±1% full-scale range; repeatability, 0.2% full-scale range) was calibrated with silicone oil as a standard. The viscosity of the oils was measured in triplicate at ten different shear rates. SC4-21 spindle was operated at different speeds between 3 and 100 rpm (Brookfield Engineering Lab, Inc., Stoughton, MA). Viscosity measurements were carried out at ambient temperature (27 ± 1 °C) with a rotational speed of 50 rpm. The viscosity value from the viscometer was based on the built-in calculation as part of the physical rotational torque sensor. Fatty Acid Analysis. Methyl esters of fatty acids were prepared using boron trifluoride/methanol according to the AOCS method,7 Ce 1-62, and were analyzed using a gas liquid chromatograph (GC-15A, Shimadzu Corp. Koyto, Japan) coupled with a data processor (model CR-4A, Shimadzu Corp., Japan) and flame ionization detector. Column: capillary column (50 m length × 0.25 mm i.d.) from Varian-CP7419 with the nitrogen flow conditions of a 1 mL/min temperature program with initial column temperature 120 °C/5 min increased to 260 °C at 5 °C/min and held for 5 min, injection temperature 220 °C, and detector temperature 230 °C. The fatty acids were identified by using reference standards of fatty acid methyl esters and expressed in terms of relative peak area percentage.

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ANALYSIS OF MINOR COMPONENTS IN WBA, WBH, AND WBO Carotenoids Analysis. β-Carotene and lutein were determined through RP-HPLC analysis by injecting known amounts of oil in acetone using LC-6A, a Shimadzu instrument equipped with a PDA detector and fitted with Bondapak, C18 column (25 cm × 4.6 mm, 5 μm i.d.), detector set at 450 nm; mobile phase of acetonitrile/methanol/dichloromethane (6:2:2 v/v) containing 0.1% ammonium acetate and a flow rate of 1 9839

DOI: 10.1021/acs.jafc.7b03440 J. Agric. Food Chem. 2017, 65, 9838−9848

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Journal of Agricultural and Food Chemistry Animal Studies. The animal studies were initiated after obtaining IAEC (296/2014) clearance from CSIR-CFTRI, Mysore. Experiments were conducted in weaning male Wistar rats (40−50 g) to evaluate the hypolipidemic property of the WBO concentrate (WBA) at 2 and 3.5%. Animals with equal body weights were divided into six groups: ND, normal/control diet fed group; HFC, high fat fed group; WBA 2 and 3.5%, groups treated with wheat bran acetone fractions of 2 and 3.5% levels. A total of six rats were taken in each group and housed in polycarbonate cages (2 rats per cage) and maintained in a 12 h light/dark cycle. The animals were given free access to diet and water for 2 weeks for acclimatization. Animal diet was prepared according to AIN −76. Normal diet was fed to the normal group (SFC), and other groups were fed the high fat diets (30%) with or without wheat bran oil concentrate (WBA) as a supplement for 45 days. During the experimental period, dietary intake and body weights were measured. After sacrificing the animals, blood was collected, and the separated serum was subjected to lipid profile analysis including TG, cholesterol, LDL, and HDL using kits. Evaluation of Antioxidant Enzyme Activities and TBA Reactant (MDA) from Liver Tissue. Approximately 100 mg of liver was used to extract protein for analyzing SOD, GPx, CAT, and GR enzyme activities and expressed in terms of units/mg of protein. Superoxide Dismutase (SOD). The enzyme activity of SOD was carried out by xanthine-xanthine oxidase ferricytochrome C (X/XOD/Cyt C3+) method.14 The reaction mixture consisted of solution A, 5 μmol xanthine in sodium hydroxide (0.001 N), and Cyt C (2 μmol) in potassium phosphate buffer (50 mM) containing 0.1 mM EDTA in a 50 μL sample. The reaction was initiated by adding solution B (50 μL), prepared xanthine oxidase in EDTA phosphate buffer of pH 7.8 (0.1 mM), and mixed well, and enzyme kinetics were measured at 580 nm. Glutathione Peroxidase (GPx). The enzyme activity was carried out according to the method by Flohe et al.15 Briefly, the reaction mixture contained 0.4 mL of GSH (0.1 mM), 0.2 mL of TBS solution (Tris 50 mM, NaCl 150 mM pH 7.4), and 0.2 mL of sample. After 5 min incubation at 25 °C, 0.2 mL of H2O2 (1.3 mM) was added to the mixture, which was incubated at 37 °C for 10 min. The reaction was stopped by adding 1 mL of 1% TCA and centrifuged. Absorbance was taken at 412 nm. Catalase. The activity was followed according to the method of Aebi16 and determined by monitoring the rate of H2O2 consumption in a mixture containing 50 mM potassium phosphate buffer (pH 7.0), 10 mM H2O2, and sample for 3 min at 240 nm. Glutathione Reductase (GR). Reduced glutathione was monitored by the rate of formation of 5-dithio-2-nitrobenzoic acid at 412 nm.17 In brief, liver and tissue homogenates (10%, w/v in PBS, pH 7.4) were deproteinized using tricarboxylic acid (10%, v/v). The deproteinized sample (100 μL) was reacted with 50 μL of 10 mM DTNB in 4.75 mL of sodium phosphate buffer (0.1 M, pH 8.0). Formation of 5-dithio-2-nitrobenzoic acid was read spectrophotometrically (Shimadzu Corporation, UV-1800, Japan) using standard reduced glutathione at 412 nm. Lipid Peroxidation by TBARS Method (TBA Reactant/ MDA). Lipid metabolites were measured as MDA (TBA reactant) as an indicator of lipid peroxidation using TBARs method.18 The supernatant of tissue homogenized with KCl and centrifuged was used as the test sample. Solutions were

mixed with SDS (8%) and acetic acid (20%). The mixture was kept in a boiling water bath along with thiobarbutric acid (0.8%) for 1 h, and butanol was added after cooling. Butanol fractions were separated by centrifugation, and absorbance was measured at 532 nm for formation of the TBA reactant (MDA) using 1,1,3,3-tetraethoxy propane (TEP) as standard. Histopathology Evaluation and BODIPY 493/503 Staining. The sections of liver and kidney (5 μm) were mounted on super-frosted slides. Gill’s hematoxylin and eosine (H&E) staining was carried out using kit reagents and bodipy stain by incubating with 0.1 mg/mL of BODIPY@493/503 in DMSO for 30 min in the dark. Images were captured at 60× magnification using bright field and oil immersion objectives for confocal microscopy to evaluate histopathological changes. Protein and Gene Expression. Western Blot Analysis. An aliquot of 30 μg of the protein sample was diluted in 2× sample buffer (50 mM Tris (pH 6.8), 2% SDS, 10% glycerol, 0.1% bromophenol blue, and 5% β-mercaptoethanol) and heated for 5 min at 95 °C before SDS-PAGE gel analysis (10%). Subsequently, it was transferred to a PVDF membrane (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated overnight with 5% blocking reagent (TBST-Tris-buffered solution containing 0.1% Tween-20) at 4 °C. The membrane was rinsed with TBST thrice. It was then incubated with blocking solution for 2 h containing 1:200 dilution of primary antibody HMG-CoA reductase (rabbit monoclonal HMGCR, Abcam, Cambridge, UK). After four washes, the membrane was incubated again for 2 h in horseradish peroxidase conjugated with mouse antirabbit IgG secondary antibody (1:1000, Santa Cruz Biotechnology, Dallas, Texas, USA) and developed using enhanced chemiluminescence kit (ECL Western blot analysis kit, Thermo Fischer, Waltham, USA). The signals were digitalized using image analysis software (Biorad, ChemiDoc imaging system, USA). For comparing the differences between control (SFC), high fat (HFC), and WBA-treated (2 and 3.5%) groups, the relative band intensities of each protein normalized with β-actin were analyzed. RNA Extraction and cDNA Synthesis. RNA was extracted from snap-frozen liver tissue stored at −80 °C using a previous method.19 The cDNA synthesis was carried out according to the manufacturer’s instructions (Takara Bio Inc., Shiga, Japan #RR037A). Gene Expression Analysis. Real-time PCR was carried out using a previously described method of Ohtomo et al.20 using one step RT-PCR and SYBR Premix Ex TaqII (TliRNaseH Plus, RR820) (Takara Bio Inc., Shiga, Japan) kits. The experiment was performed as per the manufacturer’s instructions. CFX96 Touch Real-Time PCR Detection System was used (Bio-Rad laboratories, Hercules, CA) to evaluate the mRNA expression of HMG-CoA Reductase21 and β-actin.22 Primer sequences for HMG-CoA reductase forward: 5′AGCCGAAGCAGCACATG-3′, reverse: 3′-CTTGTGGAATGCCTTGTG-5′, and β-actin forward: 5′- CAAAAGCCACCCCCACTCCTAAGA-3′, reverse: 3′- GCCCTGGCTGCCTCAACACCTC-5′ were used, and expression of HMG-CoA reductase gene was normalized to β-Actin mRNA (annealing temperature of 57 °C). Comparative cycle threshold (Ct) values were calculated relative to the control group. 22S Ct (βActin) value was subtracted from the Ct values of the gene of interest to give a ΔCt value. ΔΔCt values were obtained by subtracting the average control ΔCt value for each treatment group, and the target gene expression relative to control was 9840

DOI: 10.1021/acs.jafc.7b03440 J. Agric. Food Chem. 2017, 65, 9838−9848

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Journal of Agricultural and Food Chemistry

Figure 2. (upper panel) Wheat bran oil and its fractions: (a) wheat bran oil (WBO), (b) wheat bran hexane (WBH), (c) wheat bran acetone (WBA). (lower panel) HPLC chromatogram of oryzanol-like molecules and tocopherol in the different fractions of wheat bran oil concentrate for (A, E) standard oryzanol and carotenoids, (B, F) WBO, (C, G) WBH, and (D, H) WBA. (I) Sterols and esters of sterol identified using 13CNMR.

derived using the eq 2−ΔΔCt. The derived normalized values were the mean of triplicate runs. Statistical Analysis. The statistical analysis of the data was conducted with statistical software SAS version 9.2 using Analysis of Variance (ANOVA) by Duncan’s multiple range test (DMRT). All data are expressed as mean ± SEM, and statistical significance was considered at p < 0.05.

had a lower value when compared to WBO oil, indicating lower content of bioactives. The color intensity or total color difference (ΔE*) was higher in WBA fractions, indicating the presence of denser amounts of both total carotenoids and tocols. Physical properties such as viscosity of WBA fractions showed highest value of 1312 mPa and lowest in WBH of 913 mPa when compared to WBO (1071 mPa). The quality of the oil in both fractions (Table 1) was then analyzed, wherein the FFA level was observed to be more in the WBH (9% as oleic acid) fraction and less in the WBA (3%) and WBO (4%) fractions. The increment in the unsaponifiable matter was observed in WBA (8.2%) and was decreased in the WBH (2.8%) fraction. The peroxide value was found to be higher in the WBA fraction; however, it was not significant when compared to those of WBH and WBO. The results suggested that WBA has more unsaturated fatty acids. Fatty acid compositions revealed that there was not much change between the fractions and oil; however, a slight increase in unsaturated and reduced saturated fatty acids was observed in the WBA fractions (Table 1). Analysis of Various Fat-Soluble Bioactives of WBO and Its Fractions WBH and WBA. The present study showed that the fat-soluble bioactives content varied by different folds in WBH and WBA fractions in which total steryl ferulate

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RESULTS Preparation of Oil and Its Fractions from Wheat Bran. The oil obtained from wheat bran after clarification overnight and centrifugation yielded WBO. Two fractions were obtained from WBO after passing through the column (Figure 2A−C). The fractions obtained were WBH and WBA, which yielded 45.9 and 41.2% of oil, respectively (Table 1). Analysis of Physicochemical Properties of WBO and Its Fractions WBH and WBA. Physicochemical properties such as color measurement L* (lightness), a* value (redness), and ΔE* (difference in their color) are presented in Table 1. L*, which describes the lightness of a product, showed the lowest value of 71 for WBA and highest for WBH at 84, indicating WBH is a lighter-colored oil. Similarly, the a* value was higher in WBA, indicating a darker color and hence the presence of increased fat-soluble bioactives; the WBH fractions 9841

DOI: 10.1021/acs.jafc.7b03440 J. Agric. Food Chem. 2017, 65, 9838−9848

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Journal of Agricultural and Food Chemistry Table 1. Physicochemical Properties of Wheat Bran Oil and Its Fractionsa sample fractions yield (%) L* (D 65) a* (D 65) b* (D 65) ΔE* (D 65) torque (%) viscosity (mPa)

WBO

WBH

100 45.9 ± Color Measurements 082.0 ± 2.97b 084.0 ± 010.0 ± 0.91a 008.0 ± 114.0 ± 4.62 115.0 ± 117.1 ± 6.14a 116.0 ± Physical Characteristics 10.21 ± 0.77a 008.7 ± 1071 ± 14.1b 914.0 ± Quality Parameters 65.0 ± 68.21 ± 2.41b

WBA

fat-soluble bioactivesa

41.1 ± 1.45a

1.91b

± ± ± ±

3.05c 0.09a 3.54 2.97a

071.0 031.0 121.0 128.0

0.64a 17.6a

12.5 ± 0.98b 1312 ± 21.3c

peroxide value 1.90a (meqO2 kg−1) FFA (% as oleic 4.57 ± 0.57a 9.00 ± 0.72b acid) unsaponifiable 3.71 ± 0.45a 2.80 ± 0.50a matter (%) IC50 (μg/mL) 1.65 ± 0.17a 1.99 ± 0.21a HMG-CoA Reductase Assay (nmol mg−1 h−1) 0.21 ± 0.01a 8.79 ± 0.71c Fatty Acid Composition (%) palmitic (16:0) 17.23 ± 1.05ab 19.6 ± 1.38b b stearic (18:0) 18.75 ± 1.68 13.0 ± 2.37ab a linoleic (18:1) 60.71 ± 3.41 61.2 ± 3.21a linolenic (18:2) 03.33 ± 0.70a 06.0 ± 0.91b AIN-76 Diet Composition (g)

steryl ferulates (mg %)

1.71a 1.09b 5.85 5.52b

campesteryl ferulate campestanyl ferulate β-sitosteryl ferulate + β-sitostanyl ferulate total tocols (mg/ 100 g wrt α-T) α-T (%) α-T3 (%) γ-T (%) γ-T3 (%) carotenoids (mg/ 100 g) β-carotene (mg/ 100 g) lutein (mg/100 g)

69.0 ± 1.68b 3.40 ± 0.59a 8.20 ± 1.13b 1.20 ± 0.20a 2.29 ± 0.22b 16.9 15.8 63.0 04.2

± ± ± ±

1.02ab 1.33a 3.02b 0.81a

composition

SFC

HFD (30%)

WBA (2%)

WBA (3.5%)

starch casein vitamin mix mineral mix choline chloride DL-methionine lard ground nut oil WBA

653 200 10 35 2 0.020

453 200 10 35 2 0.020 200 100

453 200 10 35 2 0.020 200 80.0 20.0

453 200 10 35 2 0.020 200 65.0 35.0

100

Table 2. Composition of Bioactive Components of Wheat Bran Oil and Its Fractions WBO

WBH

WBA

317.0 ± 13.62b

31.1 ± 3.14a

1417.6 ± 32.1c

Composition (%) 026.2 ± 3.01b 02.2 ± 0.50a

147.03 ± 07.91c

202.3 ± 8.24b

19.1 ± 2.01a

884.00 ± 20.12c

088.6 ± 07.77b

09.9 ± 1.25a

386.8 ± 16.87c

185.0 ± 13.24b

31.0 ± 1.41a

405.0 ± 15.14c

043.4 025.1 009.7 096.8 02.76

± ± ± ± ±

04.25b 02.52b 01.30b 04.32b 00.21b

08.7 00.7 01.8 19.4 00.9

± ± ± ± ±

0.71a 0.15a 0.31a 0.91a 0.10a

108.8 065.4 208.5 021.7 08.43

± ± ± ± ±

07.78c 05.22c 07.66c 01.65a 00.75c

01.86 ± 00.14a

00.9 ± 0.10a

06.41 ± 00.35c

00.90 ± 00.10b

0.03 ± 0.005a

02.04 ± 00.07c

a

Abbreviations: T, tocopherol; T3, tocotrienol, total tocols, total tocopherols and tocotrienols.

(WBA) fraction had complex patterns mainly due to the presence of a substantial amount of fatty acids and their interference with other constituents. The 13C NMR was more resolved, and in combination with HSQC experimental data, clearly established the presence of various steryl ferulates and sterols. For the WBA fraction, 13C NMR analyses revealed the presence of two sterols, namely, β-sitosterol and sitostanol, characterized by chemical shifts of C3 carbon at 72.1 and 71.8 ppm, respectively. In addition, 13C NMR also detected βsitosterol ferulate and campesterol ferulate having a chemical shift for C3 carbon at 73.8 and 72.9 ppm, respectively (Figure 2 (I)). The peaks due to ferulates of β-sitosterol and campesterol were clearly observed. Further, unsaponifiable matter was prepared for all the fractions and subjected to NMR analysis to understand the occurrence of sterols and sterol ferulates. The WBA fraction via 13C NMR peaks and HSQC cross peaks clearly confirmed the occurrence of β-sitosterol and sitostanol. The sitostanyl and campestanyl ferulates, which were analyzed by HPLC, were not detected in NMR possibly because of lower concentration compared to those of the other ferulates. The WBO and WBH had a similar occurrence of sterol ferulates and sterol as indicated by 13C NMR; however, the concentration was much lower compared to that of WBA. The 1H and 13C NMR of WBO, WBH, and WBA fractions did not indicate the peaks due to tocopherols and carotenoids, probably due to the low level of occurrence. Biological Activities of Wheat Bran Oil and Its Fractions in Vitro. The antioxidant activity was evaluated via DPPH method for all of the fractions, wherein the WBA fraction exhibited higher activity (IC50 1.2 μg/mL) compared with that of the WBH (1.99 μg/mL) fraction followed by that of the WBO fraction (1.65 μg/mL). The WBA fraction, even with lower concentrations, had better free radical scavenging effect, whereas the same free radical scavenging required more concentrated WBO and WBH fractions. HMG-CoA reductase (whose extraction was performed using liver microsome) in

a

Different superscripts in rows are significantly different (p < 0.05). Abbreviations: WBO, wheat bran oil; WBH, wheat bran hexane fraction; WBA, wheat bran acetone fraction.

(31.37−1417.83 mg/100 g), tocopherols (30.70 and 404.53 mg/100 g), and carotenoids (0.96−8.43 mg/100 g) were observed. Table 2 shows the content of individual components of each bioactives. The composition of steryl ferulate such as campesteryl ferulate, campestanyl ferulate, and β-sitosteryl ferulate and sitostanyl ferulate varied in the fractions (WBH and WBA) as presented in Figure 2 (C, D). The tocopherol compositions were varied in the fractions; however, changes were clearly noticed in the WBA fractions relative to those of WBH (Figure 2 G, H). Changes in the individual components of carotenoids were also observed in the fractions. The results showed increased concentration of individual bioactives of WBA along with unsaponifiable matter, indicating an enrichment of bioactives in the unaltered state in their biological activities. NMR Studies of Different Fractions Obtained from the Wheat Bran Oil. NMR analysis of different fractions of wheat bran oil including unsaponifiable matter was based on the combination of 1H, 13C, DEPT135, HSQC, and HMBC experiments. The 1H NMR of wheat bran oil (WBO), wheat bran oil hexane (WBH) fraction, and wheat bran oil acetone 9842

DOI: 10.1021/acs.jafc.7b03440 J. Agric. Food Chem. 2017, 65, 9838−9848

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Journal of Agricultural and Food Chemistry

intake in the treated groups. The average body weight of the HFC group was increased (180 g) when compared to control (160 g) and was reduced with WBA in a dose-dependent manner (WBA 2%, 169 g; WBA 3.5%, 163 g) and is statistically significant when compared to the HFC group. The study indicates that body weight gain in the high fat fed animals was due to consumption of high calorie diet and was reduced significantly in concentration fed groups at WBA 2 and 3.5% levels. However, dietary intake was higher in the SFC groups, which could be due to less calorie value (starch-based diet) in the HFC groups. The consumption of diet in treated groups was increased when compared to that of the HFC group. After 45 days, the animals were sacrificed under over-anesthesia. Blood was collected and subjected to serum separation followed by analysis of the lipid profile including triglycerides (TG), total cholesterol (TC), and LDL levels, which were increased in HFC when compared to those of the SFC groups. However, after treatment with concentrates, levels of TG were significantly reduced in a dose-dependent manner (WBA 2%, 84.2; 3.5%, 75.5 mg/dL). Cholesterol and LDL levels were also reduced significantly during the 45 day experimental period with WBA treatment (Table 3). Effect of Antioxidant Enzyme Activities in Control, High Fat Fed Diet, and Treated Groups. Antioxidant enzyme activities were analyzed in the livers of different groups including superoxide dismutase (SOD) and glutathione peroxidase (GPx), which were increased by 1.02- and 1.3fold, respectively, in HFC groups when compared to those of SFC, whereas catalase and glutathione reductase were decreased by 1.7- and 1.6-fold, respectively. Treatment with WBA at 2 and 3.5% levels ameliorated activities significantly when compared with those of HFC however between the treated groups, although there is slight difference in their values. However, there was a much more significant difference between HFC and treated groups. The level of MDA (TBA reactant) was increased in the HFC groups (0.168 nm/mg of protein) with respect to that of the normal diet fed group (0.122 nm/mg of protein), which decreased dose dependently after treatment with WBA (0.152 and 0.145 nm/mg of protein) (Table 3). The liver sections of the control group (SFC) and treated groups (WBA 2 and 3.5%) showed normal hepatic architecture and hepatocytes with prominent nuclei and central vein and portal areas. However, in the HFC group, lipid droplets or vacuolelike structures were noted both in H&E (black arrows) and BODIPY (white arrows) stains. Similarly, in kidney histology, none of the experimental groups had changes in their renal tubules or glomeruli of the kidneys as shown in Figure 5. Protein and Gene Expression. The HMG-CoA reductase enzyme activity and its expression, which is necessary for cholesterol synthesis, mainly take place in the liver and adipose tissue and depot lipids. Expression of HMG-CoA reductase protein was performed in the liver and analyzed with its specific antibody (Figure 6A). The results clearly indicate that HMGCoA reductase increased with the high fat diet fed group with respect to control, whereas in the treated groups at 2 and 3.5% levels, it decreased. Furthermore, its gene expression was also analyzed in liver and adipose tissue. Treatment with WBA at different levels (2 and 3.5%) showed downregulation of HMGCoA in the liver (1.58 and 2.04, Figure 6B) and adipose (1.22 and 1.46, Figure 6C) tissues when compared with those of the HFC group. Overall, the study clearly suggests that fat and its soluble portion of the wheat bran have beneficial health effects apart from that of the bran fiber.

vitro activities were analyzed in the presence of WBO and its fractions WBH and WBA. Activity was significantly reduced (11-fold) with the treatment of WBA, whereas the observed enhancement by 3.8-fold was statistically significant (Table 1). Lipase inhibitory activity of WBO, WBH, and WBA fractions were carried out along with known standards sitosterol and orlistat (Figure 3). The inhibition of pancreatic lipase was

Figure 3. Pancreatic lipase inhibition assay by titration method. Values represent the mean ± standard deviation (n = 3).

determined by release rate of oleic acid per hour. Orlistat had the highest inhibiting activity (0.32 mg g−1 h−1), whereas sterols and WBA fractions exhibited similar inhibitory activity (0.34 mg g−1 h−1) and were significant when compared to those of the WBO and WBH fractions (Table 1). Effect of Wheat Bran Oil Concentrates on in Vitro Studies of DNA and LDL Oxidation. The Fenton’s reaction responsible for the creation of hydroxyl radicals from hydrogen peroxide and iron are strong enough to achieve scavenging of DNA as presented in Figure 4A. Incubation of DNA with or without oxidizing agent, WBO, WBH, and WBA fractions at concentrations of 0.06, 0.012, and 0.025 μg/mL scavenged free radicals in a dose-dependent manner. Among the fractions, WBA had better scavenging effect and prevented degradation of DNA against free radicals. The WBA fraction showed complete protection against oxidizing agent on damaged calf thymus DNA at the 0.025 μg/μL concentration. It is well-known that antioxidants donate electrons to hydroxyl radicals or free radicals to make it stable; thus, WBA is a potent antioxidant that attenuates oxidative DNA damage. The isolated LDL was incubated with or without oxidizing agent along with WBO, WBH, and WBA for ∼12 h (Figure 4B). The relative mobility of the oxidized LDL was observed with that of native LDL. Incubated LDL fraction with 10 μM copper sulfate decreased the electrophoretic mobility of LDL (lane 1) compared to that of native LDL (lane 2). WBA fractions significantly prevented oxidation (200 μg/mL). From these studies, it can be concluded that WBA fractions significantly prevent the oxidation of LDL from free radicals. Animal Experiments. Hypolipidemic Effect of Concentrated WBA in High Fat Fed Animals. Hypolipidemic and hypocholesterolemic properties of the concentrates (WBA) were analyzed in the high fat fed rat model. Animals were maintained for ∼45 days with different diets of normal and high fat with and without WBA. The doses were fixed based on previous reports as well as the trial experiments. All of the presented results are obtained by comparing the treated group with the high fat fed groups. Body weights and dietary intake were recorded and are presented in Table 3. Dietary intake was observed more (9.3 g) in control animals (SFC) and least (7.6 g) in the HFC group, and there were no changes in the dietary 9843

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Figure 4. Effect of wheat bran oil concentrate on free radical-induced damage. Electrophoretic image of oryzanol-like molecules in the different fractions of wheat bran oil concentrate (A) and DNA (B); LDL protection assay with sterols and ferulic acid used as positive controls. L1, 2, 3 and so forth represent different lanes with different concns of concentrates.

Table 3. Effect of Wheat Bran Oil Concentrate on Lipid Profile and Antioxidant Enzyme Activitiesa group dietary intake (g/day) body weight (g) TG (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) TC (mg/dL) SOD (UA/dL) GPX (UA/dL) catalase (UA/dL) GR (UA/dL) MDA (nm/mg protein)

SFC 9.3 160.8 70.4 55.1 10.4 60.8 3546.6 80.3 4311.9 129.57 0.122

± ± ± ± ± ± ± ± ± ± ±

HFD a

0.5 6.7a 4.2a 6.5 1.0a 2.3a 10a 2.04a 164a 02.1a 0.04a

7.65 180.8 97.1 42.2 25.7 89.1 3928.0 106.8 4097.8 78.71 0.168

± ± ± ± ± ± ± ± ± ± ±

WBA 2% b

0.4 8.3b 5.3b 5.0 1.3b 4.6b 12b 8.04b 124b 8.4b 0.02b

7.23 173.4 84.2 50.9 20.1 75.4 3622.2 091.1 4391.4 118.5 00.1

± ± ± ± ± ± ± ± ± ± ±

WBA 3.5% b

0.26 5.8a 2.1a 6.2 2.0b 2.0a 11a 6.08a 158a 6.8a 0.01a

7.75 168.9 78.5 53.6 16.9 68.0 3600.3 84.0 4282.5 103.85 0.135

± ± ± ± ± ± ± ± ± ± ±

0.75b 6.0a 4.8a 6.9 1.3a 2.9a 10a 2.86a 118a 4.25b 0.02a

a

Abbreviations: SFC, starch-fed control; HFD, high fat control; WBA 2%, high fat along with wheat bran oil concentrate fed group; WBA 3.5%, high fat along with wheat bran oil concentrate fed group. Values are the average of six animals. Different superscripts in rows are significantly different (p < 0.05).

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Figure 5. Effect of WBA on lipid accumulation in liver. The liver sections of theHFC group showed the accumulation of lipid as indicated by black arrows (H&E stains, A) and white arrows (BODIPY stain, B; relative intensity, b). Treated-group WBA (2 and 3.5%) accumulate less lipid. However, there were not many changes observed in the kidney sections of all the groups (H&E stains, C).

Figure 6. Effect of WBA on HMG CoA reductase in high fat fed animals. Protein expression of HMG-CoA in liver (A). HMG-CoA reductase gene expression in liver (B) and adipose (C) tissues.

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DISCUSSION The fortification of defatted bran diet with wheat bran oil is shown to inhibit colon cancer.2 These results inspired us to work with the fat-soluble bioactives of wheat bran oil in particular. The present study focused on the preparation of wheat bran oil concentrates enriched with bioactives. Two fractions were obtained using hexane (WBH) and acetone (WBA). Fraction WBA had a higher amount of bioactives including steryl ferulate, carotenoids, and tocopherols; however, there was not much change in the fatty acid compositions between the oil and other fractions. The present study also focused on antioxidant activity (DPPH assay) and DNA and

LDL protection from free radical-induced damage using the different fractions. The WBA fraction had a better effects with its lowest concentrations. Furthermore, in vitro HMG-CoA reductase and intestinal lipase activities were also carried out, and the results revealed good inhibition of their activities with WBA fractions when compared to those of the WBH and WBO fractions. Both enzymes play a major role in cholesterol and lipid metabolism in terms of synthesis and absorption motives. On the basis of the in vitro investigations described above, animal experiments were conducted for a short duration of 40 days, wherein the WBA fraction at two different dose levels, 2 and 3.5%, were tested. Results clearly indicated that there is 9845

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The increase in HMG-CoA reductase gene or protein expression in the high fat fed group is not well understood. However, an increased expression of both gene and protein in the high fat-induced fatty liver in mice was observed.41 A further in-depth study is required in this area of research. There are widely used and commercialized extracts, such as rosemary, where the antioxidative behavior and synergistic actions of most of the compounds remain unknown.42 Nanoemulsions of WBO with water have been shown to have antioxidant and mushroom tyrosinase inhibitory activities.43 Aqueous enzymatic extraction from rice bran showed amelioration of various antioxidant enzymes in high fat fed animals.44 The present study revealed the presence of polar fractions in wheat bran oil that have good hypolipidemic effects. Rebolleda et al. have compared the supercritical fluid extraction and solvent extraction of wheat bran oil and found more alkyl resorcinols (phenolic lipids composed of long aliphatic chains) in the polar solvents.45 The present study also showed the amelioration of various antioxidant enzyme activities when compared to those of the high fat fed animal groups. There were no toxicological effects observed in the histologies of different groups because the samples tested were extracted from wheat. In the present study, we could develop a simple and effective method for concentrating fat-soluble bioactives of wheat bran oil (WBA). The WBA composition had a significant concentration of antioxidants, which reduces free radical-induced damage by quenching as well as improving the antioxidant defense system either directly or indirectly. Furthermore, downregulation of HMG-CoA reductase and inhibitory activities of HMG-CoA reductase in combination with lipase inhibitory activity were effective in bringing about the hypolipidemic effect of WBA. We attribute this to the bioactive components either individually or in combination having a synergistic effect on the beneficial health aspects.

reduction in body weights and lipid profile in HFD when compared to those of the SFC group; however, treatment with WBA reduced them significantly. This could be due to the combination of bioactives in the WBA fraction that may have a synergistic effect on reducing the lipid profile. The common plant sterols/stanols present in WBA are campesterol, sitosterol, sitostanol, and campestanol. Absorption of these phytochemicals is similar to that of cholesterol, through the sterol transporter is in the intestinal lumen. Only 50% absorption of sterols takes place with respect to cholesterol. The absorption efficiency of plant sterols is shown to be less than 2% and that of plant stanols is less than 0.2%.23 Consequently, the circulating serum levels of plant sterols and stanols are very low compared to that of serum cholesterol. Phytosterol reduces the serum cholesterol in animal studies and human trials.24−27 Oryzanol (group of steryl ferulates) is a nonsaponifiable fraction of rice bran oil that has been shown to reduce serum cholesterol and aortic fatty acid streak in hamsters.28 Several mechanisms are proposed to account for the action of phytosterols on lipid metabolism. These include competitive blocking of cholesterol absorption,29,30 increasing bile salt excretion,31 hindering cholesterol esterification,32 and displacement of cholesterol from bile salt micelles.33 Numerous studies have documented that phytosterols inhibit cholesterol absorption.29−33 The primary mechanism of action of phytosterols is to decrease cholesterol absorption in the intestine. In another mechanism, the oryzanol was cleaved into FA and sterols by intestinal lipase that showed inhibition of lipase and HMG-CoA, respectively, as well as elimination of the bile acids, thus lowering the lipid profiles. This suggests that important secondary mechanisms are taking place in the intestine, which was also proposed by our group.34 Tocopherols coadministered with tocotrienols (fat-soluble compounds) were shown to attenuate lipid and cholesterol metabolism by reducing Cpt-1a, Cyp7a1, and HMG-CoA reductase. 35 Fractions of tocotrienols obtained from rice bran DOD have been shown to have a hypolipidemic effect.36 Further in-depth research works are needed on lipid metabolism. The current in vitro study showed inhibition of HMG-CoA reductase, which was observed in the WBA fraction, and the in vivo animal experiments hold well with respect to lipid profile studies. In addition to this, HMG-CoA expression was reduced in the treated group when compared to that of the HFC group. The slides stained with H&E showed lipid accumulation in the HFC section as vacuole marks; thus, there is an acceleration of lipid metabolism over that of the control. However, in the treated group, no such observation was made, which indicates the inhibition of certain enzymes involved in lipid biosynthesis, thus showing reduced fat accumulation. There are several reports on feeding high fat diets that enhance the accumulation of lipids in the liver as well as in other organs.37−40 Several mechanisms have been proposed for lipid accumulation in the liver. There are reports that have shown in particular that the mice fed with high fat diets result in a reduction of hepatic lipid accumulation due to disruption of the perilipin-2 gene, and it was also observed that overexpression of the same gene enhanced lipid accumulation.38,39 In another study by Maho,40 feeding of a high fat diet increased the liver weight (lipid accumulation) when compared with a high sucrose fed group. A similar study on hepatic cells of the liver in high fat fed mice (C57BL/6J) showed adipogenic changes.37 Similar results were also observed in our study wherein high starch-containing diet (SFC) and high fat-containing (HFC) groups were maintained.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +91 08212514876; fax: +91 08212517233; e-mail: [email protected] or [email protected]. ORCID

Nanishankar V. Harohally: 0000-0003-2306-5897 G. Suresh Kumar: 0000-0002-4929-894X Funding

The work was carried out under funding of the Major Laboratory Project, CSIR-CFTRI, MLP-112, and GAP-424, DBT, New Delhi. S.T.T. also thanks the University Grant Commission (UGC), New Delhi, India, for financial support. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful to the Director, CSIR-CFTRI, Mysore, for providing the necessary infrastructure. The authors thank Dr. Gopala Krishna A.G. formerly Head LSTF dept, for his valuable suggestions toward chromatography studies.

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