Minor Constituents in Rice Bran Oil and Sesame Oil Play a Significant Role in Modulating Lipid Homeostasis and Inflammatory Markers in Rats

Abstract

The effects of feeding rats with groundnut oil (GNO), rice bran oil (RBO), and sesame oil (SESO) on serum lipids, liver lipids, and inflammatory markers were evaluated in rats. Male Wistar rats were fed with AIN-93 diet supplemented with 10 wt% of GNO, RBO, and SESO in the form of native (N) and minor constituent-removed (MCR) oils. Rats given RBO and SESO showed significant reduction in serum and liver lipids, 8-hydroxy-2-deoxyguanosine, cytokines in liver, and eicosanoids in leukocytes as compared with the rats given GNO and MCR oils. The rats fed with native oils of RBO and SESO showed an upregulation of sterol regulatory element-binding protein (SREBP)-2 and peroxisome proliferator-activated receptor gamma (PPARγ) and downregulation of nuclear factor-kappa B (NF-κB) p65. These effects of native oil were significantly compromised when rats were given MCR oils. In conclusion, the minor constituents significantly support the hypolipidemic and anti-inflammatory properties of RBO and SESO.

Introduction

The levels of total cholesterol, low-density lipoprotein (LDL) cholesterol, and triacylglycerol (TAG) in the plasma are often used as risk factor indicators for cardiovascular diseases. Dietary oils containing saturated fatty acids are known to elevate total and LDL cholesterol and thereby increase the risk for cardiovascular diseases, whereas polyunsaturated fatty acid (PUFA)-containing oils lower the plasma lipid levels.^1
–3 In addition to plasma lipid levels, the uncontrolled production of inflammatory mediators and oxidative stress are also recognized as contributors for cardiovascular diseases, which can also be significantly modulated by dietary lipids.^4,5

Dietary oils are composed of saponifiable as well as unsaponifiable fractions (UF). While TAG form the bulk of saponifiable fraction, various minor constituents contribute to UF of the oils. Very often the effects of dietary oils on serum lipid levels are attributed to the overall fatty acid composition as well as to the positional distribution of these fatty acids in TAG of oils.⁴ The contributions of minor constituents of oils and fats toward health benefits are seldom recognized.⁶ The minor constituents, such as γ-oryzanol and lignans such as sesamin and sesamolin are uniquely present in rice bran oil (RBO) and sesame oil (SESO), respectively. These oils are widely used in India for cooking purposes.

γ-Oryzanol is a group of ferulic acid esters of phytosterols and triterpene alcohols found in UF of RBO. It is known to reduce cholesterol biosynthesis and exhibit hypolipidemic activity.⁷ The UF of SESO contain a unique minor constituent known as lignans, such as sesamin and sesamolin, which exhibit antiatherogenic effects.^8,9 These bioactive compounds are known to inhibit the nuclear factor-kappa B (NF-κB) activation and translocation into the nucleus, which downregulate the proinflammatory genes. These then reduce the secretion of reactive oxygen species, cytokines, eicosanoids, hydrolytic enzymes, and matrix metalloprotienases, which are involved in the promotion of inflammation.¹⁰ The activation of NF-κB is regulated by peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ exerts anti-inflammatory properties by interfering with NF-κB signaling in transcriptional pathways involved in inflammatory responses.^10

–13 Hence NF-κB and PPARγ play a major role in controlling inflammation and maintaining cardiovascular health.¹⁴

Recently, we have demonstrated that minor constituents in RBO and SESO contribute to the antioxidant potential of oils. The dietary RBO and SESO significantly activated antioxidant defense systems in rats. However, there was a considerable decrease in the antioxidant activity of RBO and SESO, which were stripped of their minor constituents by removing the UF of the oils.¹⁵ Dietary γ-oryzanol also played a significant role in the anti-inflammatory activity exhibited by RBO.¹⁰ It was concluded from these studies that the unique minor constituents contributed to the antioxidant and anti-inflammatory activities exhibited by RBO and SESO in rats. However, the effect of minor constituent-removed (MCR) oils on serum and hepatic lipids, oxidative stress markers, pro- and anti-inflammatory cytokines, and eicosanoids having a bearing on cardiovascular health was not adequately addressed in these studies. We evaluated these parameters by feeding rats with diets containing groundnut oil (GNO), RBO, and SESO with UF or after removing minor constituents by stripping UF. These three oils are used for cooking purpose. They contain similar amounts of monounsaturated and PUFA, but differ in the unique bioactive compounds present in their UF. While RBO contained γ-oryzanol (1386 ± 13.42 mg/100 g oil), SESO contained sesamin (91.8 ± 8.6 mg/100 g oil) and sesamolin (147.6 ± 12 mg/100 g oil), but GNO did not show the presence of any unique bioactive compounds in the UF.¹⁵

Materials and Methods

Materials

Commercially available, branded GNO, RBO, and SESO used for domestic cooking were purchased from a local super market in Mysuru, India and are designated as native oils (N). 8-hydroxy-2-deoxyguanosine (8-OHdG), cholesterol, tripalmitin, calcium ionophore, eicosanoid standards, and fatty acid standards were obtained from Sigma Chemical Company (St. Louis, MO, USA). Vitamins, minerals, cellulose, choline bitartrate were obtained from Himedia (Mumbai, India). All the general chemicals and solvents used for the analysis were purchased from Ranbaxy Fine Chemicals Ltd. (Delhi, India).

Preparation of MCR oils

The minor constituents from GNO, RBO, and SESO were removed following the method of Khan and Shahidi.¹⁶ Silicic acid (100 g) was washed three times with a total of 3 L double-distilled water and filtered on Whatman filter paper using a Buchner funnel. The washed silicic acid was dried at 110°C for 20 h. The dried silicic acid (22.5 g) and activated charcoal (5.5 g) were suspended in 100 and 70 mL of hexane, respectively. A chromatographic column (3.0 cm i.d. × 35 cm height) was packed sequentially with 22.5 g of silicic acid, followed by 5.5 g of activated charcoal, and then topped with 22.5 g of silicic acid. Thirty grams of oil (GNO, RBO, SESO) was dissolved in 30 mL of hexane and passed through the column. The oil was eluted from the column with 270 mL of hexane. The eluted oil, which was devoid of minor constituents and UF, were collected in round bottom flasks. Hexane was removed using a rotary evaporator operating at 37°C under vacuum. Traces of remaining solvent were removed by flushing with nitrogen. The oils, which were stripped of minor constituents by the removal of UF, were aliquoted into brown glass bottles, flushed with nitrogen, and stored at −20°C until use. By this procedure more than 95% of UF and minor constituents were removed from the oils.^15,17

Animal experiments

Male Wistar rats (OUBT-Wistar, IND-cft [2c]) (Rattus norvegicus) weighing 45–50 g were grouped (six rats in each group) by random distribution and housed in individual cages, under a 12-h light–12h dark cycle, in an approved animal house facility of CSIR-CFTRI at Mysuru, India. Animals were provided a fresh diet (AIN-93 diet) daily and leftover food was weighed and discarded. The growth of rats was monitored by weighing at regular intervals. The rats had free access to food and water throughout the study. AIN-93 diet¹⁸ was supplemented with N or MCR oils of GNO, RBO, or SESO. The composition of the AIN-93 diet was (g/kg diet): corn starch, 500; casein, 200; sucrose, 100; fat, 100; cellulose, 50; mineral mix, 35; vitamin mix, 10; L-cysteine, 3; choline bitartrate, 2. After 60 days of feeding the diets containing N or MCR oils, rats were fasted overnight and sacrificed under diethyl ether anesthesia. Blood was drawn by cardiac puncture and serum was separated by centrifugation at 1100 g at 4°C for 20 min. The liver was removed, rinsed with ice-cold saline, blotted, weighed, and stored at −80°C until the analysis was completed.

Isolation of circulating white blood cells

Whole blood from rats was collected after cardiac puncture into a vacutainer tube containing 3.6 mg of K₂ EDTA. Whole blood (500 μL) was mixed with 4 mL of RBC lysis buffer and gently rocked for 5 min. The sample was centrifuged at 300 g for 7 min to obtain a white blood cells (WBC) (leukocyte) pellet. After careful removal of the supernatant, the WBC pellet was washed with phosphate-buffered saline (PBS) and used for the analysis of eicosanoid production as described by Ramaprasad et al. ¹⁹

Analytical Methods

Total lipid extraction from serum and liver

Total lipid was extracted from serum by the method of Bligh and Dyer.²⁰ Liver lipid was extracted by the method of Folch et al. ²¹

TAG and cholesterol estimation in serum and liver

TAG from serum and liver were estimated by the method of Fletcher.²² The total cholesterol in the serum and liver lipid extracts was quantified as described by Searcy and Bergquist.²³ Serum LDL+VLDL was separated from high-density lipoprotein (HDL) by adding heparin (5000 U/mL) and MnCl₂ (2 M).²⁴ The solution was vortexed thoroughly, left overnight at 4°C, and centrifuged at 4000 g for 15 min. The precipitate was suspended in saline and cholesterol from LDL+VLDL was extracted in acetone:alcohol (1:1 v/v). The cholesterol was estimated as described earlier. Fatty acid composition of the dietary oils and tissue lipids were determined by gas chromatography using the method described by Morrison and Smith.²⁵

Western blotting

The liver proteins (30 μg) were separated on 8% SDS-PAGE at 60 V for 6 h. The proteins were transferred on to PVDF membranes and incubated for 8 h with polyclonal antibodies (anti-rat raised in rabbit) for sterol regulatory element-binding protein (SREBP)-2, PPARγ, and NF-κB p65 in Tris buffer saline at 4°C. The membranes were washed and hybridized with secondary antibody conjugated with horseradish peroxidase (anti-rabbit IgG-HRP; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h and washed with Tris buffered saline. Finally, the membranes were developed with the Enhanced Chemiluminescent Detection Kits (Pierce Biotechnology Co., Ltd., Rockford, IL, USA) for 2 min. The protein bands were scanned by densitometry (Syngene Gel Doc, Gi Box; ChemixT4, Cambridge, UK) and the results were expressed as mean relative densitometric units.²⁶

Oxidative stress markers in liver

The antioxidant values of the liver lipid were determined by photochemiluminescence method following the protocol given by the instrument manufacturer (Photochem^®; Analytik, Jena, Germany). Liver lipid (1 mg) was dissolved in 200 μL hexane and 30 μL aliquots were taken for measuring the antioxidant activity.¹⁵ Values are expressed in terms of trolox equivalents. Protein carbonyl content in the liver homogenate was determined as described by Levine et al. ²⁷ The 8-OHdG in liver was separated by HPLC using a C18 column Bondapak (300 × 3.9 mm, 10 μm i.d), mobile phase consisting of water:acetonitrile (83:17 v/v), adjusted to pH 3.0 at a flow rate of 0.5 mL/min and peaks were monitored at 254 nm. The 8-OHdG levels were calculated from standard curve generated using 8-OHdG (1–20 nmol) as described by Abu-Quare and Abou-Donia.²⁸ Malondialdehyde (MDA) formed in liver was measured at 532 nm using the method of Buege and Aust,²⁹ and 1 g liver was homogenized in 20 mM phosphate buffer (pH 7.4). It was centrifuged at 600 g for 10 min and thiobarbituric acid reagent was added to the supernatant. Tubes were kept in a boiling water bath for 30 min. The MDA was measured at 532 nm and quantitated using extinction coefficient of 1.56 × 10⁻⁵ cm⁻¹. The protein content in liver was estimated by the method of Lowry et al. ³⁰ using bovine serum albumin as the reference standard. All spectrophotometric measurements were carried out in Shimadzu ultraviolet 1601A spectrophotometer (Shimadzu, Kyoto, Japan).

Estimation of eicosanoids in leukocytes

The leukocytes in PBS (1.0 × 10⁶ cells/mL) were stimulated with 0.5 μM calcium ionophore for 15 min. The reaction was stopped by adding ethanol and 3% formic acid. The cells were sonicated and centrifuged. The cell supernatant was passed through Sep-Pak C18 column (Waters; Millipore Corp., Milford, MA, USA) and loaded on RP-C18 column (Supelco, Discovery, 25 cm × 4.6 mm, 5 μm i.d; Bellefonte, PA, USA) fitted to a SPD 20A HPLC (Shimadzu Corp., Tokyo, Japan). The prostaglandins and thromboxanes were eluted with methanol:water:trifluoroacetic acid:triethylamine (80:20:0.1:0.05 v/v) at a flow rate of 0.7 mL/min and monitored at 200 nm using UV detector as described by Dutrieu and Delmotte.³¹ The leukotrienes were eluted with acetonitrile:methanol:acetic acid:water (65:10:1:24 v/v) adjusted to pH 5.6 with ammonia and monitored at 280 nm as described by Abe et al. ³² The eicosanoids were quantified by comparing with respective standards.

Cytokine assay

After sacrificing the rats, the liver was removed and washed with ice-cold PBS. The liver (200 mg) was homogenized in extraction buffer (NaCl: 150 mM; Tris: 50 mM, pH 7.2; Triton X-100: 1% containing protease inhibitor) and centrifuged at 1500 g at 4°C. One hundred microliters of supernatants were added to a 96-well microtiter plate precoated with monoclonal antibody. The cytokines were analyzed by ELISA (Invitrogen Corporation, Camarillo, CA, USA) by following the manufacturer's instructions for using the kits. The plates were read at 450 nm in ELISA reader Infinite M200 PRO (Tecan Austria GmbH, Grödig, Austria).³³

Statistical analysis

Data were analyzed using SPSS statistical software package version 17.0. Results are expressed as mean ± SD for each experimental group. Statistical analysis was performed using one-way ANOVA followed by a post hoc Tukey's test. P-values of less than .05 were considered as statistically significant.

Results

Fatty acid and UF composition of N or MCR oils

No significant difference in the fatty acid composition of N and MCR oils was observed (provided in Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/jmf). The monounsaturated fatty acids (MUFA) and PUFA composition of GNO, RBO, and SESO were similar, but differed in the levels and composition of UF. The amount of UF present in N oils of GNO, RBO, and SESO were 0.9%, 4.2%, and 2.1%, respectively. The UF in the MCR oils were reduced to 0.1% for GNO and 0.2% for RBO and SESO. This also significantly reduced the minor constituents present in MCR oils. While sesamin and sesamolin could not be detected in MCR oils of SESO, about 66 mg of oryzanol/100 g oil was present in MCR oil of RBO. These values are in agreement with our earlier reports.^10,15 The total tocopherol concentrations in the native oils were in the range of 50–110 mg/100 g oil.

Serum and liver lipid profile

The serum lipid levels were altered depending on the type of oils fed to rats. The serum TAG and total cholesterol levels were significantly lower in the serum of rats given native oils of RBO and SESO when compared with rats fed with native oil of GNO. The TAG levels in the serum of rats given native oils of RBO and SESO were lower by 31% and 21%, respectively, when compared with rats fed with native oil of GNO (Table 1). The TAG levels in the serum of rats given MCR oils of RBO and SESO were less by 23% and 12%, respectively, when compared with rats fed with MCR oil of GNO. The total cholesterol levels in the serum of rats given native oils of RBO and SESO were less by 34% and 25%, respectively, when compared with those fed native oil of GNO. The total cholesterol levels in the serum of rats given MCR oils of RBO and SESO were lowered by 25% and 16%, respectively, when compared with those fed MCR oil of GNO. The total cholesterol level in serum of rats given native oils of RBO and SESO were lowered by 15% and 14%, respectively, when compared with MCR oils of RBO and SESO (Table 1). The ability of MCR oils of RBO and SESO to lower serum TAG and cholesterol was considerably reduced when compared with respective native oil. However, the native or MCR oil of GNO had similar effect on serum TAG and cholesterol levels.

Table 1.

Serum and Liver Lipid Profile of Rats Given Native and Minor Constituent-Removed Oils

	GNO		RBO		SESO
	N	MCR	N	MCR	N	MCR
Serum (mg/dL)
TAG	120.5 ± 4.9^d	123.2 ± 4.1^d	83.1 ± 3.6^a	94.6 ± 3.5^b	95.3 ± 3.1^b	108.1 ± 3.9^c
Total cholesterol	110.5 ± 4.7^d	112.1 ± 3.6^d	73.2 ± 2.9^a	84.5 ± 1.8^b	82.6 ± 2.2^b	94.2 ± 3.1^c
HDL cholesterol	39.7 ± 2.0^b	38.6 ± 3.2^b	33.0 ± 2.5^ab	31.0 ± 3.0^a	34.9 ± 2.6^ab	33.4 ± 2.9^ab
LDL+VLDL cholesterol	70.8 ± 3.2^d	73.5 ± 4.7^d	40.2 ± 1.3^a	53.5 ± 2.8^b	47.7 ± 2.9^b	60.8 ± 3.1^c
Liver (mg/g)
TAG	18.8 ± 0.6^d	19.4 ± 0.7^d	13.3 ± 0.6^a	16.4 ± 0.6^c	14.6 ± 0.5^b	17.1 ± 1.1^c
Total cholesterol	9.8 ± 0.3^cd	10.3 ± 0.5^d	6.9 ± 0.4^a	8.7 ± 0.5^c	7.8 ± 0.4^b	9.1 ± 0.3^c

Values are mean ± SD of six rats. Mean in a row with different superscript differs significantly at P < .05.

GNO, groundnut oil; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MCR, minor constituents removed; N, native; RBO, rice bran oil; SESO, sesame oil; TAG, triacylglycerol.

HDL levels were lowered by 17–22% in rats fed with RBO and by 13–16% in SESO-fed rats as compared with those given GNO. Nevertheless, there was no difference in the levels of HDL altered by native oil or MCR oil of RBO- or SESO-fed rats. The LDL+VLDL cholesterol levels in the serum of rats given native oils of RBO and SESO were less by 43% and 33%, respectively, when compared with those fed with native oil of GNO (Table 1). Similar changes were observed in TAG and cholesterol levels in liver (Table 1). The results indicated that rats given RBO and SESO containing minor constituents had higher efficacy in reducing the serum and liver lipids as compared with rats given the same oils after removing endogenous minor constituents from them.

Levels of 18:2 in liver lipids of rats fed with native oils of RBO and SESO were lower by 36% and 19%, respectively, when compared with those fed with native oil of GNO. Levels of 20:4 in rats fed with native oils of RBO and SESO were lowered by 47% and 24%, respectively, when compared with rats fed with native oil of GNO. There were no significant changes in the fatty acid composition of liver lipids in rats fed with native and MCR oils. Thus, feeding the oils from which UF was removed did not show any significant impact on hepatic fatty acid composition in rats as compared with those given respective native oils (results provided in Supplementary Table S2).

Expression of SREBP-2, PPARγ, and NF-κB p65

Cholesterol homeostasis is regulated by changes in the expression of enzymes involved in cholesterol biosynthesis. SREBP-2 plays a vital role in this. PPARγ is a nuclear anti-inflammatory protein, which downregulates the proinflammatory markers through inhibition of NF-κB p65.^34,35 The expression of SREBP-2 and PPARγ was upregulated and that of NF-κB p65 was downregulated in the liver of rats given native oils of RBO and SESO, when compared with those fed with native oil of GNO (Fig. 1). The rats fed with native oils of RBO and SESO showed upregulation in expression of SREBP-2 by 93% and 54%, respectively, as compared with rats fed with native oils of GNO (Fig. 2a). The rats fed with native oils of RBO and SESO showed upregulation of PPARγ by 136% and 83%, respectively, as compared with rats fed with MCR oils of RBO and SESO (Fig. 2b). The rats fed with native oils of RBO and SESO showed downregulation of NF-κB p65 by 74% and 56%, respectively, as compared with rats fed with native oils of GNO (Fig. 2c). However, rats fed with MCR oils of RBO and SESO downregulated NF-κB p65 expression by 35% and 25%, respectively as compared with rats fed with MCR oils of GNO (Fig. 2c) The ability of RBO to upregulate SREBP-2 and PPARγ or to downregulate the NF-κB p65 expression was compromised to a great extent when minor constituents were removed from RBO and SESO. This reflected on cholesterol levels observed in rats fed with GNO, RBO, and SESO, in which UF of oils were retained or removed.

FIG. 1.

Effect of feeding N and MCR oils on expression of (a) SREBP-2, (b) PPARγ, and (c) NF-κB p65 in liver homogenate. β-Actin was used as housekeeping protein for quantification. The pattern is a representative of three independent experiments with similar results. GNO, groundnut oil; MCR, minor constituents removed; N, native oil; NF-κB, nuclear factor-kappa B; PPARγ, peroxisome proliferator-activated receptor gamma; RBO, rice bran oil; SESO, sesame oil; SREBP, sterol regulatory element-binding protein.

FIG. 2.

Effect of feeding N and MCR oils on expression of (a) SREBP-2, (b) PPARγ, and (c) NF-κB p65 in liver homogenate. Expression of proteins in GNO-fed animals was taken as reference with fold expression of 1. Data are expressed as mean ± SD of three independent experiments. Mean not sharing the same superscript are not statistically significant at P < .05.

Oxidative stress markers

The total antioxidant activity of liver lipids of rats given native oils of GNO, RBO, and SESO were 7.3, 21.6, and 17.2 μg trolox equivalent, respectively (Table 2). The total antioxidant activity of hepatic lipids in rats fed MCR oils of GNO, RBO, and SESO were 1.3, 2.7, and 2.4 μg trolox equivalents, respectively. Thus, total antioxidant activity of liver lipid was decreased by 82%, 87%, and 86% when MCR oils of GNO, RBO, and SESO were given to rats. Hence, minor components present in GNO, RBO, and SESO had a greater influence on total antioxidant activity in liver. The formation of protein carbonyls, 8-OHdG, and lipid peroxidation was significantly lower in liver homogenate of rats fed with native oils of RBO and SESO when compared with those fed with native oil of GNO. The protein carbonyl levels in liver of rats given native oils of RBO and SESO were less by 45% and 33%, respectively, when compared with rats given native oils of GNO. The 8-OHdG levels in liver of rats given native oils of RBO and SESO were less by 60% and 46%, respectively, when compared with rats given native oils of GNO. The lipid peroxidation levels in liver of rats given native oils of RBO and SESO were less by 50% and 34%, respectively, when compared with rats given native oils of GNO (Table 2). Thus, dietary consumption of native oils of RBO and SESO significantly reduced oxidative stress markers and enhanced the antioxidant activity. This effect was, however, compromised to some extent when UF was removed from these oils and fed to rats.

Table 2.

Effect of Native and Minor Constituent-Removed Oils on Antioxidant Activity and Oxidative Stress Markers in Rat Liver

	GNO		RBO		SESO
Liver	N	MCR	N	MCR	N	MCR
Total antioxidant activity (μg of trolox equivalent/mg lipid)	7.3 ± 0.6^c	1.3 ± 0.2^a	21.6 ± 1.3^e	2.7 ± 0.3^b	17.2 ± 0.9^d	2.4 ± 0.4^b
Protein carbonyls (nmol/mg protein)	28.2 ± 1.3^d	33.6 ± 2.1^e	15.6 ± 1.4^a	22.7 ± 1.7^c	18.8 ± 1.2^b	26.5 ± 1.8^d
8-OHdG (nmol/mg protein)	4.8 ± 0.3^d	5.4 ± 0.4^d	1.9 ± 0.2^a	3.3 ± 0.4^c	2.6 ± 0.3^b	4.2 ± 0.4^d
Lipid peroxidation (nmol of MDA/mg protein)	12.3 ± 0.6^d	14.4 ± 1.0^e	6.2 ± 0.3^a	8.9 ± 0.4^c	8.1 ± 0.6^b	11.2 ± 0.8^d

Values are mean ± SD of six rats. Mean in a row with different superscript differs significantly at P < .05.

8-OHdG, 8-hydroxy-2-deoxyguanosine; MDA, malondialdehyde.

Eicosanoids

The PGE₂ levels in leukocytes of rats given native oils of RBO and SESO were less by 35% and 19%, respectively, when compared with rats fed with native oil of GNO. The PGE₂ levels in leukocytes of rats given native oils of RBO and SESO were less by 24% and 20%, respectively, when compared with rats given MCR oils of RBO and SESO (Fig 3a). The 6-keto PGF_1α levels in leukocytes of rats given native oils of RBO and SESO were less by 16% and 8%, respectively, when compared with rats fed with native oil of GNO. Significant differences were not observed in the levels of 6-keto PGF_1α in the leukocytes of rats given MCR and native oils of GNO, RBO, and SESO (Fig. 3b). The TXB₂ levels in leukocytes of rats given native oils of RBO and SESO were less by 34% and 14%, respectively, when compared with rats fed diet containing native oil of GNO (Fig. 3c). The TXB₂ levels in leukocytes of rats given MCR oils of RBO and SESO were decreased by 17% and 11%, respectively, when compared with rats given MCR oils of GNO (Fig. 3c). The secretion of LTB₄, LTC_4, and LTD₄ also followed similar trends (Fig. 3d–f).

FIG. 3.

Effect of feeding N and MCR oils on the generation of (a) PGE_2, (b) 6-keto PGF_1α, (c) TXB_2, (d) LTB_4, (e) LTC_4, and (f) LTD₄ by rat leukocytes. Values are mean ± SD of six rats. *P < .05 and **P < .05. NS is not significant. Data analyzed by one-way ANOVA followed by post hoc Tukey's test (*P < .05). Significances were determined by making comparisons between control (GNO) versus RBO, SESO, and N versus MCR oils of RBO, SESO.

Cytokines

The proinflammatory cytokine levels in rats were influenced by dietary lipids. The levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were significantly lower in liver homogenate of rats given native oils of RBO and SESO as compared with rats fed with native oil of GNO (Table 3). Rats given native oils of RBO reduced the levels of IL-1β, IL-6, and TNF-α by 50%, 74%, and 72%, respectively, as compared with that observed in rats given native GNO. Similarly, rats given native SESO showed reduced levels of IL-1β, IL-6, and TNF-α by 33%, 58%, and 57%, respectively, as compared with rats given native GNO.

Table 3.

Effect of Native and Minor Constituent-Removed Oils on Cytokines in Rat Liver

	GNO		RBO		SESO
Cytokines (ng/mg protein)	N	MCR	N	MCR	N	MCR
Proinflammatory
IL-1β	60.9 ± 3.2^d	65.5 ± 2.2^d	30.4 ± 2.0^a	48.8 ± 2.6^c	40.7 ± 3.1^b	62.9 ± 3.2^d
IL-6	40.8 ± 3.5^d	43.7 ± 3.0^d	10.5 ± 0.6^a	15.1 ± 1.0^b	17.2 ± 1.2^b	31.6 ± 2.3^c
TNF-α	43.9 ± 2.9^d	49.5 ± 2.6^d	12.4 ± 0.4^a	21.3 ± 2.1^b	18.7 ± 0.9^b	35.1 ± 2.5^c
Anti-inflammatory
IL-4	6.5 ± 0.6^a	5.3 ± 0.4^a	14.6 ± 1.0^c	9.1 ± 0.9^b	9.8 ± 0.6^b	6.2 ± 0.4^a
IL-10	5.9 ± 0.4^a	5.0 ± 0.3^a	11.9 ± 0.8^c	7.4 ± 0.4^b	8.1 ± 0.5^b	5.5 ± 0.3^a

Values are mean ± SD of six rats. Mean in a row with different superscript differs significantly at P < .05.

IL, interleukin; TNF, tumor necrosis factor.

Even though dietary MCR oils of RBO and SESO also reduced the levels of IL-1β, IL-6, and TNF-α, it was to a lesser degree as compared with that observed in rats given native oils of RBO and SESO. Rats given MCR oils of RBO showed reduced levels of IL-1β, IL-6, and TNF-α by 25%, 63%, and 56%, respectively, as compared with rats given MCR GNO. Similarly, rats given MCR SESO showed reduced levels of IL-1β, IL-6, and TNF-α by 4%, 28%, and 29%, respectively, as compared with rats given MCR GNO (Table 3). However, in contrast to inflammatory cytokines, the levels of anti-inflammatory cytokines, such as IL-4 and IL-10, were increased significantly in the liver of rats given native oils of RBO and SESO as compared with rats fed with native oil of GNO. The IL-4 and IL-10 levels in the liver of rats given native oils of RBO were increased by 146% and 102%, respectively, when compared with rats given native oils of GNO. Similarly, IL-4 and IL-10 levels in the liver of rats given native oils of SESO were increased by 51% and 37%, respectively, when compared with rats given native oils of GNO (Table 3). The IL-4 and IL-10 levels in the liver of rats given MCR oils of RBO were increased by 12% and 48%, respectively, when compared with rats given MCR oils of GNO. The IL-4 and IL-10 levels in the liver of rats given MCR oils of SESO were increased by 17% and 10%, respectively, when compared with rats given MCR oils of GNO (Table 3). Thus, the ability of dietary RBO and SESO to modulate pro- and anti-inflammatory cytokines was significantly decreased when minor constituents were removed from the oil. However, the levels of pro- and anti-inflammatory cytokines observed in rats given native or MCR oils of GNO did not differ significantly.

Discussion

The primary objective of this investigation was to evaluate the importance of minor constituents present in UF of selected oils in modulating serum lipids, liver lipids, and oxidative stress markers in liver and eicosanoids production in leukocytes of rats. Earlier we had demonstrated that removal of minor constituents from RBO and SESO significantly decreased the antioxidant activity associated with the oil. Removal of minor constituents from these oils also reduced its impact on the antioxidant defense systems in rats.¹⁵ However, the effect of minor constituent-removed oils on lipid parameters as well as on eicosanoids, which have a bearing on cardiovascular health, was not monitored in the earlier study. To evaluate this, we have selected three oils; GNO, RBO, and SESO. These oils had similar amounts of MUFA and PUFA contents, but they differed in the composition of minor constituents present. RBO and SESO contain unique minor constituents such as γ-oryzanol and lignans, respectively, which are reported to have several health benefits.^34

–38 GNO did not contain any unique minor bioactive compounds which are not found in other vegetable oils. RBO and SESO are promoted as heart-healthy oils in South East Asia and in Japan which is now being recognized in Western countries also.

Rats fed with RBO and SESO had significantly lower amounts of serum TAG, total cholesterol, and LDL cholesterol as compared with rats given GNO. However, the ability of RBO and SESO to lower serum lipids was reduced considerably when MCR oils of RBO and SESO were fed to rats. However, no changes were observed in the levels of serum TAG, total cholesterol, and LDL cholesterol when rats were fed MCR oils of GNO in place of native GNO. Similarly, rats fed with native oils of RBO and SESO showed reduced levels of TAG and cholesterol in liver, but the extent of decrease was reduced when MCR RBO and SESO were fed to rats. These studies indicated that RBO and SESO retain their ability to lower serum and liver lipids, even after removing minor constituents, but with lesser efficacy compared with native oils. Based on the values obtained for serum lipids in rats following the dietary studies, it was observed that minor constituents in UF contributed to lowering of serum cholesterol and TAG by RBO and SESO to an extent of ∼15%. UF of RBO and SESO also contributed to an extent of ∼30% in lowering LDL cholesterol. Similarly, minor constituents of RBO and SESO contributed to lowering of liver cholesterol and TAG to an extent of 17–23%. Thus, the minor constituents contribute significantly to the hypolipidemic activity of RBO and SESO. However, no differences in serum lipid levels were observed when native GNO or minor constituent-removed GNO was fed to rats.

SREBP-2 is a transcriptional factor which regulates cholesterol levels. The rats fed with native oils of RBO and SESO showed an upregulation of SREBP-2 as compared with rats given GNO. Earlier we had demonstrated that ingestion of RBO or blended oils containing RBO, which provide 0.104–0.13 wt% γ-oryzanol, upregulated SREBP-2, CYP7A1, and LDL receptor expressions in rats.³⁹ This significantly reduced serum cholesterol levels. In the present study, based on food composition, it was estimated that rats ingested 0.14 wt% γ-oryzanol from RBO. These groups of rats showed an upregulation of SREBP-2 by 42% as compared with rats given GNO, which lacked γ-oryzanol. Sesame lignans are also reported to exhibit hypocholesterolemic effect.³⁴ In the present study, rats were given SESO containing sesamin (926 mg/kg oil), sesamolin (1453 mg/kg oil), and sesamol (8 mg/kg oil), which could have contributed to the upregulation of the SREBP-2 and cholesterol-lowering effects of SESO. Studies have shown that dietary SESO containing sesamin (2 g/kg oil) and sesamolin (0.6 g/kg oil) alters the expression of sterol transporters involved in cholesterol absorption in rats.³⁹ These studies indicated that in addition to PUFA, the minor constituents in RBO and SESO show beneficial effect on cardiovascular health by lowering serum cholesterol levels.

The minor constituents in RBO and SESO significantly decreased the susceptibility of hepatic lipids to peroxidation. The oxidative stress markers such as protein carbonyls and 8-OHdG were increased in rats given MCR of RBO and SESO as compared with those given respective native oils. Furthermore, the total antioxidant activity in the hepatic tissue was decreased drastically by 90% in rats fed with MCR of GNO, RBO, and SESO, which concurs with the increase in oxidative stress markers. These studies indicated that oxidative stress in rats is increased when RBO and SESO with the MCR are fed to rats. These observations are in agreement with that of Khan and Shahidi¹⁶ for the antioxidant properties of minor constituents in vegetable oils.

Prostaglandins, such as TXB₂ and 6-keto PGF_1α, have direct impacts on thrombosis. In the present study, it was observed that leukocytes from rats given RBO and SESO produced significantly lower amounts of TXB₂ as compared with that produced by rats given GNO. Nevertheless, the ability of dietary RBO and SESO to lower TXB₂ was reduced when rats were fed MCR RBO. However, the levels of TXB₂ remained the same when rats were given MCR GNO or native oils of GNO. No significant changes were observed in 6-keto PGF_1α levels when GNO, RBO, or SESO was given with UF or after removal of minor constituents. PGE₂, LTB₄, LTC₄ and LTD₄ levels remained unaltered when rats were given GNO as native oil or with MCR. However, the ability of RBO and SESO to lower the eicosanoids was significantly reduced when they were fed with MCR RBO or SESO. These studies indicated complementary nature of minor constituents in RBO or SESO in controlling proinflammatory eicosanoids.

PPARγ is a ligand-dependent transcription factor of nuclear receptor family. PPARγ plays an important role in the immune response by inhibiting inflammatory mediators and by directly triggering the immune cells to differentiate toward anti-inflammatory phenotype. The native oils of RBO and SESO upregulated the expression of PPARγ and downregulated that of NF-κB p65. These changes modulated the secretion of proinflammatory mediators such as eicosanoids. Studies have shown that oryzanol and phytosteryl ferulates from rice bran exhibit anti-inflammatory effects by downregulating the transcription factor (NF-κB), which lowers the expression of enzymes such as COX and LOX, which generate proinflammatory mediators.^7,12,36 Studies have also shown that sesamin downregulates the expression of NF-κB.⁴⁰

The anti-inflammatory effects of PPARγ include decreases in proinflammatory cytokines, ROS, and metalloprotienases. Notably, PPARγ inhibits the activation of NF-κB p65, whereas it enhances the transcription of anti-inflammatory and antioxidant-related proteins.¹⁴ In the present study, we observed that the rats fed with native oils of RBO and SESO showed upregulation of the anti-inflammatory cytokines, such as IL-4 and IL-10, and downregulation of proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α when compared with rats given the GNO-containing diet. Rats fed with MCR oils of RBO and SESO showed lesser degree of increase in anti-inflammatory ILs when compared with rats fed with native oils of RBO and SESO. The minor constituents contributed to lowering of proinflammatory cytokines IL-1β, IL-6, and TNF-α by RBO to an extent of 40%, 56%, and 28%, respectively. The contributions of minor constituents in lowering IL-1β, IL-6, and TNF-α by SESO were 36%, 46%, and 12%, respectively. The contribution of minor constituents in enhancing anti-inflammatory cytokines IL-4 and IL-10 by RBO was to an extent of 38% and 39%, respectively, whereas that in SESO the minor constituents contributed for enhancement of IL-4 and IL-10 to an extent of 37% and 53%, respectively. Thus, minor constituents of RBO and SESO play an important role in controlling levels of pro- and anti-inflammatory cytokines. A critical balance between proinflammatory and anti-inflammatory cytokines is essential in managing risk factors for cardiovascular diseases.⁴¹

The health benefits of minor constituents of oils and fats have been documented in many studies.^6,10,17 But most of these studies are carried out either using oils after enriching them with a specific minor constituent or using minor constituents that are isolated from natural sources. Rukmini and Raghuram⁴² have reported that bioactive compounds from RBO lower cholesterol levels in rats and human subjects. γ-oryzanol also differentially modulated the expression of antioxidant and oxidative stress-related genes in rats, thereby giving relief from oxidative stress.⁴³ The phytosterols of rice hull extracts inhibited LPS-induced nitric oxide production and mRNA expression of inducible nitric oxide synthase as well as the COX-2 enzyme in RAW264.7 macrophages. It also downregulated m-RNA and protein expression for vascular endothelial growth factors COX-2 and LOX. The cytokines, IL-1β and TNF-α released by macrophages, were also reduced by these phytosterols. Furthermore, the extracts attenuated the activation of NF-κB p65 as well as the phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinases, and c-Jun N-terminal kinase in LPS-stimulated macrophage cell lines.⁴⁴ Lignans from sesame enhance antioxidant activity of vitamin E and showed inhibitory effects on lipid peroxidation initiated in cumene hydroperoxide systems.⁴⁵ Sesamin ameliorated oxidized LDL-induced ROS generation. It also attenuated the oxidized LDL-induced activation of NF-κB. Sesamin also showed inhibitory effect on IL-8 and ET-1 release, adhesion molecule expression, and the adherence of THP-1 cells mediated through the blockade of NF-κB activation.⁴⁶ All these studies have shown that minor constituents, such as γ-oryzanol and sesamin, exhibit anti-inflammatory activity as well as suppression of oxidative stress. However, these studies did not address the independent effects of fatty acids per se and that of minor constituents in RBO and SESO on serum and tissue lipid levels and on pro- and anti-inflammatory mediators. In the present study, we were able to delineate the effect of fatty acids and minor constituents of RBO and SESO on serum lipids, liver lipids, as well as on inflammatory markers.

If the hypolipidemic effect of dietary oils were solely attributable to their fatty acid composition, then rats given GNO, RBO, and SESO should have similar levels of serum and liver lipids as all the three oils have comparable levels of linoleic acid, which is considered to have cholesterol-lowering properties. But our results indicated that the hypolipidemic effects of RBO and SESO are significantly greater than that of GNO. Therefore, the minor constituents present in these oils made a significant difference in their hypolipidemic effects. It is not only the fatty acid composition per se, but also the distribution of PUFA in TAG molecular species of oils that contributes to its hypolipidemic effects. The distribution of fatty acids in molecular species of TAG in GNO, RBO, and SESO are shown to be different.³⁹ We have earlier demonstrated that interesterified fats and blended oils with similar fatty acid composition exhibit differences in their hypolipidemic activity. Interesterified oils were more effective in lowering serum lipids compared with blended oils with similar fatty acid composition.³⁹

It is also interesting to note that GNO, RBO, and SESO feeding differentially affected the 18:2 and 20:4 levels in hepatic tissues. Both RBO and SESO feeding resulted in reduced levels of 18:2 and 20:4 in liver lipids, whereas GNO-fed rats showed higher levels of these essential fatty acids. Conversely, the fatty acid composition of liver lipids remained the same in rats given oils which were stripped of minor constituents as compared with those given native oils. This is similar to our earlier observation that no differences were noticed in the overall fatty acid composition of serum lipids when rats were fed with native oils or oils that were stripped of minor constituents.¹⁵

Many studies have indicated that the minor constituents have a vital role in providing stability to oils. Based on such studies it was suggested that the refining methods for oils should be optimized to retain minor constituents so that the enriched oils can be used in designing functional foods with improved stability.¹⁶ In our study, we have used vegetable oils, which are sold in the market for domestic use. Commercially available RBO and SESO contained sufficient amounts of endogenous minor constituents to support cardiovascular health. The commercially available vegetable oils are subjected to regulatory guidelines under the Food Safety and Standards Act in which the maximum amounts of UF that could be present are defined. Accordingly, the UF should not be more than 1% for GNO, not more than 4.5% for physically refined RBO, and not more than 2.5% for SESO.⁴⁷ This poses a constraint on the amount of minor constituents that could be made available in individual oils. Consumer preference for clear oils may prompt the manufacturers to refine the oils to such an extent that the bioactive components in the UF may be removed during refining of the oils. Therefore, attention should be given while refining of oils to retain endogenous minor constituents so that the health benefits of vegetable oils can be fully exploited.

Footnotes

Acknowledgments

The authors thank Prof. Ram Rajasekharan, Director, CSIR-CFTRI, for encouragement and support to this work. Y.P.C.R. and D.S. acknowledge Indian Council of Medical Research, New Delhi for granting a Senior Research Fellowship.

Author Disclosure Statement

No competing financial interests exist.

References

Kuller

: Nutrition, lipids, and cardiovascular disease. Nutr Rev, 2006; 64:S15–S26.

Layden

, Anqueira

, Brodsky

, et al.: Short chain fatty acids and their receptors: New metabolic targets. Transl Res, 2013; 161:131–140.

Schwager

, Richard

, Riegger

, et al.: ω-3 PUFAs and resveratrol differently modulate acute and chronic inflammatory processes. BioMed Res Int, 2015; 2015:535189.

Jung

, Torrejon

, Chang

, et al.: Fatty acids regulate endothelial lipase and inflammatory markers in macrophages and in mouse aorta. Arterioscler Thromb Vasc Biol, 2012; 32:2929–2937.

Harijith

, Ebenezer

, Natarajan

: Reactive oxygen species at the crossroads of inflammasome and inflammation. Front Physiol, 2014; 5:352.

Beardsell

, Francis

, Ridley

: Health promoting constituents in plant derived edible oils. J Food Lipids, 2002; 9:1–34.

Chauhan

, Chauhan

: Rice bran oil: Its therapeutic potential. Int J Agric Sci Res, 2015; 5:205–222.

Moazzami

, Haese

, Kamal-Eldin

: Lignan contents in sesame seeds and products. Eur J Lipid Sci Technol, 2007; 109:1022–1027.

Bhaskaran

, Santanam

, Penumetcha

, et al.: Inhibition of atherosclerosis in low-density lipoprotein receptor-negative mice by sesame oil. J Med Food, 2006; 9:487–490.

10.

Rao

, Sugasini

, Lokesh

: Dietary gamma oryzanol plays a significant role in the anti-inflammatory activity of rice bran oil by decreasing pro-inflammatory mediators secreted by peritoneal macrophages of rats. Biochem Biophys Res Commun, 2016; 479:747–752.

11.

Minatel

, Francisqueti

, Corrêa

, et al.: Antioxidant activity of γ-oryzanol: A complex network of interactions. Int J Mol Sci, 2016; 17:1107–1122.

12.

Baker

, Hayden

, Ghosh

: NF-κB, inflammation and metabolic disease. Cell Metab, 2011; 13:11–22.

13.

Jeng

, Hou

, Wang

, et al.: Sesamin inhibits lipopolysaccharide induced cytokine production by suppression of p38 mitogen-activated protein kinase and nuclear factor-κB. Immunol Lett, 2005; 97:101–106.

14.

Delerive

, Fruchart

, Staels

: Peroxisome proliferator-activated receptors in inflammation control. J Endocrinol, 2001; 169:453–459.

15.

Sugasini

, Poorna

CRY

, Lokesh

: Total antioxidant activity of selected vegetable oils and their influence on total antioxidant values in vivo: A photochemiluminescense based analysis. Food Chem, 2014; 164:551–555.

16.

Khan

, Shahidi

: Oxidative stability of stripped and non stripped borage and evening primrose oils and their emulsions in water: Am Oils Chem Soc, 2000; 77:963–969.

17.

Chen

, McClements

, Decker

: Minor components in food oils: A critical review of their roles on lipid oxidation chemistry in bulk oils and emulsions. Crit Rev Food Sci Nutr, 2011; 51:901–916.

18.

Reeves

, Nielsen

, Fahey

: AIN-93 purified diets for laboratory rodents: Final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr, 1993; 123:1939–1951.

19.

Ramaprasad

, Zarini

, Sheibani

, et al.: Increased synthesis of leukotrienes in the mouse model of diabetic retinopathy. Invest Ophthalmol Vis Sci, 2010; 51:1699–1708.

20.

Bligh

, Dyer

: A rapid method of total lipid extraction and purification. Can J Biochem Physiol, 1959; 37:911–917.

21.

Folch

, Lees

, Stanley

GHS

: A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem, 1957; 226:497–509.

22.

Fletcher

: A colorimetric method for estimating serum triglycerides. Clin Chim Acta, 1968; 22:303–307.

23.

Searcy

, Bergquist

: A new color reaction for the quantification of serum cholesterol. Clin Chim Acta, 1960; 5:192–199.

24.

Warnick

, Albers

: A comprehensive evaluation of the heparin-manganese precipitation procedure for estimating high-density lipoprotein cholesterol. J Lipid Res, 1978; 19:65–76.

25.

Morrison

, Smith

: Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron trifluoride methanol. J Lipid Res, 1964; 5:600–608.

26.

Rao

, Pavan Kumar

, Lokesh

: Molecular mechanisms for the modulation of selected inflammatory markers by dietary rice bran oil in rats fed partially hydrogenated vegetable fat. Lipids, 2016; 51:451–467.

27.

Levine

, William

, Stadtman

, et al.: Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol, 1994; 233:346–357.

28.

Abu-Quare

, Abou-Donia

: Increased 8-hydroxy-2-deoxyguanosine, a biomarker of oxidative DNA damage in rat urine following a single dermal dose of DEET (N,N-diethyl-m-toluamide), and permethrin, alone and in combination. Toxicol Lett, 2000; 117:151–160.

29.

Buege

, Aust

: Microsomal lipid peroxidation. Methods Enzymol, 1978; 52:302–310.

30.

Lowry

, Rosebrough

, Farr

, et al.: Protein measurement with folin phenol reagent. J Biol Chem, 1951; 193:265–275.

31.

Dutrieu

, Delmotte

: Separation of prostaglandins by HPLC. Anal Chem, 1983; 315:360.

32.

Abe

, Kawazue

, Shigematsu

: Influence of salts on high performance of liquid chromatography of leukotrienes. Anal Biochem, 1985; 144:417–422.

33.

Rao

YPC

, Lokesh

: Down-regulation of NF-κB expression by n-3 fatty acid rich linseed oil is modulated by PPARγ activation, eicosanoid cascade and secretion of cytokines by macrophages in rats fed partially hydrogenated vegetable fat. Eur J Nutr, 2017; 56:1135–1147.

34.

Liang

, Chen

, Jiao

, et al.: Cholesterol-lowering activity of sesamin is associated with down-regulation on genes of sterol transporters involved in cholesterol absorption. J Agric Food Chem, 2015; 63:2963–2969.

35.

Sakai

, Murata

, Tsubosaka

, et al.: γ-Oryzanol reduces adhesion molecule expression in vascular endothelial cells via suppression of nuclear factor-κB activation. J Agric Food Chem, 2012; 60:3367–3372.

36.

Friedman

: Rice bran, rice bran oils, and rice hulls: Composition, food and industrial uses, and bioactivities in humans, animals, and cells. J Agric Food Chem, 2013; 61:10626–10641.

37.

Shirakawa

, Koseki

, Ohinata

, et al.: Rice bran fractions improve blood pressure, lipid profile, and glucose metabolism in stroke-prone spontaneously hypertensive rats. J Agric Food Chem, 2006; 54:1914–1920.

38.

Narasimhulu

, Selvarajan

, Litvinov

, et al.: Anti-atherosclerotic and anti-inflammatory actions of sesame oil. J Med Food, 2015; 18:11–20.

39.

Reena

, Gowda

, Lokesh

: Enhanced hypocholesterolemic effects of interesterified oils are mediated by up regulating LDL receptor and cholesterol 7-α-hydroxylase gene expression in rats. J Nutr, 2011; 141:24–30.

40.

, Jayakumar

, Duann

, et al.: Chondroprotective role of sesamin by inhibiting MMPs expression via retaining NF-κB signaling in activated sw1353 cells. J Agric Food Chem, 2011; 59:4969–4978.

41.

Vishal

, Vinod

, Bender

: Cytokines and cardiovascular disease. J Leukocyte Biol, 2005; 78:805–818.

42.

Rukmini

, Raghuram

: Nutritional and biochemical aspects of the hypolipidemic action of rice bran oil: A review. J Am Coll Nutr, 1991; 10:593–601.

43.

Ismail

, Al-Naqeeb

, Mamat

, et al.: Gamma-oryzanol rich fraction regulates the expression of antioxidant and oxidative stress related genes in stressed rat liver. Nutr Metabol, 2010; 7:1–13.

44.

, Sung

, Choi

, et al.: Oryza sativa (Rice) hull extract inhibits lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages by suppressing extracellular signal-regulated kinase, c-Jun N-terminal kinase, and nuclear factor-κB activation. Pharmacogn Mag, 2016; 12:295–301.

45.

Ghafoorunissa , Hemalatha

, Rao

: Sesame lignans enhance antioxidant activity of vitamin E in lipid peroxidation systems. Mol Cell Biochem, 2004; 262:195–202.

46.

Lee

, Ou

, Wu

, et al.: Sesamin mitigates inflammation and oxidative stress in endothelial cells exposed to oxidized low-density lipoprotein. J Agric Food Chem, 2009; 57:11406–11417.

47.

Food Safety and Standards Authority of India (FSSAI): Food products standards 2.2. In: The Food Safety and Standards Act, 2006. Commercial Law Publishers Pvt. Ltd., The Gazette of India, New Delhi, 2012.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.02 MB