Dietary administration of rice in improving the antioxidant status in Long-Evans Rat

INTRODUCTION

Free radicals have been implicated in the progression of numerous conditions including cancer, diabetes, cardiovascular disease, ageing and neurological disorders. Human body has three levels of defense against free radical attack. Preventative antioxidants are to inhibit the formation of free radicals e.g. metal binding proteins like Ceruloplasmin, Metallothionine, Albumin, Transferrin, Ferritin and Myoglobin. Scavenging antioxidants are to remove any reactive species once formed. e.g. Superoxide Dismutase, Glutathione Peroxidase, Catalase and small molecules such as Ascorbate, Tocopherol, Bilirubin, Uric Acid, Carotenoids and Flavonoids. Finally repair enzymes has to correct the damaged biomolecules e.g. DNA repair enzymes. Rice has the potential to promote human health, due to its content of phenolic compounds that are able to inhibit the formation or reduction of the concentrations of reactive cell-damaging free radicals, thereby reducing the risk of coronary heart disease and cancer (Victor et al, 2009, Wahle et al, 2010) and preventing oxidative damage of lipid and low-density lipoproteins (Vauzour et al, 2010). Alak et al (2012) reported that BR5 rice contained the highest Total Phenolic Content (TPC), Ferric Reducing Antioxidant Power (FRAP) and Total Antioxidant Capacity (TAC), BRRI dhan28 and BRRI dhan29 had intermediate level and BR16 had the lowest among all the tested HYV rice varieties. Bangladesh Rice Research Institute (BRRI) has developed about 72 high yielding variety (HYV) but have not yet tested its’ antioxidant effects although rice is the staple food in Bangladesh and in some Asian countries. In the present study, we have selected BR5, BR16, BRRI dhan28 and BRRI dhan29 among the HYV rice developed by BRRI to test the effect of antioxidant properties in mammalian host like rat. Our study resembled the potential impact of antioxidant properties in the studied rice varieties on the improvement of mammalian immunity.

METHODS AND MATERIALS

Twenty-three adult male Long-Evans rats from the stock colony of our Grain Quality and Nutrition (GQN) laboratory, BRRI, Gazipur (Collected mating rats from Bangladesh University of Health Sciences, Mirpur, Dhaka, Breeding at GQN laboratory under controlled condition), three months old (~12 weeks), weighing 150 ± 2g, were used in this study. Animals were housed individually in cages in a room maintained at 22–24°C with a controlled 12 hrs light–dark cycle, and had free access to tap water and cooked rice feed. Generally, rat is not accustom with eating cooked rice so, we used to accustom rat providing cooked rice before starting experiment. We systematically withdrawal commercial rat diet gradually and replace cooked rice instead. We weighted rat before and after experiment but did not get any significant weight loss in this regards (data not shown). In case of rice, we get carbohydrate, fat, protein and vitamin content. So, rice is an ideal source of energy for maintaining healthy life. We used cooked rice instead of powder or grinding rice because if we provide powdery rice then it would digest very quickly and it will not reflect our pattern of consuming rice. Since we are consuming cooked rice, the experiment was subjected to reflect our pattern of consuming. Four rice BR5, BRRI dhan28, BRRI dhan29, BR16 samples were cleaned and milled on a Satake test mill (Satake Corporation, Japan) for separating into bran and brown rice fraction. Brown rice was then polished 10% as milled rice. These milled rice were subjected to feed individual rat group for four weeks’ time at twice meal per day as per requirement (0.89g cooked rice per meal for each rat equivalent to 5.94 gKg^-1 body weight of rat).  Following two week acclimatization with BRRI dhan29 at twice a day, the rats were allocated randomly to three groups of five animals each, ensuring the groups were balanced for body weight. After one week of interval for washout the effect of BRRI dhan29, commercial rat food were served. Then after Group1, Group2 and Group3 were fed BR5, BRRI dhan28 and BR16 respectively. Feeding of the experimental diets to rats lasted four weeks at twice meal a day.  Unhemolyzed serum or heparinized or EDTA plasma was needed for clinical analysis. Thus, we anesthetized the rat(s) by using Diethyl ether and collected blood from jugular vein. Then blood samples were centrifuge at 6000 rpm for 15 minutes to get serum. Serum was stored at 4°C in a refrigerator until analysis. Serum Albumin, Uric acid and Iron were measured at 623 nm, 550 nm, 546 nm wavelength respectively and individual methodology describes the manual procedure to use the BioMed-Albumin kit (ALB100240), BioMed-Uric acid L.S kit (UA119100 and BioMed-IRON kit respectively. In quantitation of Serum TIBC and Transferrin, Excess ferric iron (FeCl₃) was added to rat serum specimen to saturate the transferrin. Remaining ferric iron was absorbed on MgC0_3. Bound iron in the supernatant is termed as TIBC, and assayed by Iron detection procedure of BioMed-IRON kit by Ferrozine method. Duncan's multiple range test (DMRT) was applied on Iron, TIBC, Transferrin, Uric acid and Albumin parameter for statistical analysis using SPSS, version 20.0.

RESULT

We fed our rats with BRRI dhan29 as a control for 14 days to accustom with food habit (only cooked rice) and controlled conditioned environment in a rat room of GQN laboratory, BRRI, Gazipur. Then we had use eight healthy rats to established normal range of different clinical parameters like Albumin, Uric acid, TIBC and Transferrin in rat serum for our laboratory. Clinical data explains the normal range of Albumin, Uric acid, TIBC and Transferrin in Lang Evan rat by 2.59±0.18 g dL-¹, 2.32±0.29 mg dL-¹, 257.59±16.36 µg dL-¹ and 214.66±13.63 µg dL-¹ (Fig 1, n=8, All data are not shown graphically) respectively. After one week of interval for washout the effect of BRRI dhan29, commercial rat food were served. Then after cooked BR5 rice was fed for 28 days twice meal per day in group1 rats (n=5).

Table 1. Clinical parameters (Iron, TIBC, Transferrin, Uric acid and Albumin) of measuring Antioxidant status in rat serum (n=5 rats for each group).

Group /Variety	Iron (µg dL-1)	TIBC (µg dL-1)	Transferrin (mg dL-1)	Uric Acid (mg dL-1)	Albumin (g dL-1)
Group 1 (BR 5)	53.75a	311.23a	259.36a	3.23a	3.36a
Group 2( BRRI dhan28)	88.79b	266.38b	221.99b	2.86b	3.06b
Group 3 (BR16)	103.74c	161.26c	134.39c	2.40c	3.01c
Any two means having common letter (s) are not statistically different at a P< 0.05, as measured by the Duncan Multiple Range Test (DMRT).

We found the lowest level of iron content 53.75 µg/dL but very elevated level of TIBC 311.23 µg/dL, Transferrin 259.36 µg/dL, Uric acid 3.23 mg dL^-1and Albumin 3.36 g dL^-1in rat blood serum of group1 (Table 1). On the other hand in group3 where BR16 cooked rice were fed, we found the highest level of iron content 103.74 µg dL-¹but the lowest amount of TIBC 161.26 µg dL-¹, Transferrin 134.39 µg dL-¹, Uric acid 2.40 mg dL^-1 and Albumin 3.01 g dL^-1 in rat blood serum (Table 1). In Group 2 rats revealed intermediate range of values for iron 88.79 µg dL-¹, TIBC 266.38 µg dL-¹, Transferrin 221.99 mg dL^-1, Uric acid 2.86 mg dL^-1and Albumin 3.06 g dL^-1 in rat blood serum (Table 1).

DISCUSSION

Oxidative stress occurs when the body is unable to eliminate the free radicals which are implicated in the pathogenesis of various neurological as well as physiological disorders. Maintenance of oxidative balance in the brain is tightly regulated by antioxidants (Becker, 1993). Free radicals are generated largely during the production of ATP in mitochondria.  During this process, radicals leaking from the mitochondria form reactive oxygen species such as the superoxide anion and hydroxyl radicals.  These species lead to the production of hydrogen peroxide from which further hydroxyl radicals are generated in a reaction that appear to depend on the presence of iron ions.  These radicals have both beneficial and harmful actions in biological tissues.  They are known to have a crucial role in stimulation of phagocytosis, induction of drug detoxification pathways and stimulation of signal transduction pathways (Droge 2002, Salganik 2001).  However, these same radicals can be potentially       dangerous products of cellular metabolism in that they can directly influence cell growth and development, cell survival and likely increase the pathogenesis of atherosclerosis, cancer, aging and several other conditions, including inflammatory disease.  Uric acid is the antioxidant present in highest concentration in human blood (Bindu et al, 2014). It functions as a paradox as it acts as an antioxidant in plasma or pro-oxidant within the cell (Enomoto, 2005, Sautin, 2008). It measurements are used in the diagnosis and treatment of numerous renal and metabolic disorders including renal failure, gout, leukemia and psoriasis. Uric acid is a potent antioxidant contributing to around half the antioxidant capacity of blood plasma. It is a scavenging antioxidant that acts by inactivating free radicals such as ^-HO and HOCI. Total Iron Binding Capacity (TIBC) measures the blood’s capacity to bind iron with transferrin and is therefore an indirect measurement of transferrin. Iron is capable of stimulating the production of harmful free radicals. Plasma levels of transferrin are regulated by the availability of iron and increase when plasma levels of iron are low.  Transferrin can be described as a preventative antioxidant and acts by binding iron in a redox inactive form. This process is extremely important as free iron is capable of stimulating the production of harmful free radicals. Albumin represents a very abundant and important circulating antioxidant. Albumin is the most abundant protein in serum representing 55-65% of the total protein. Its’ main biological functions are to maintain the water balance in serum and plasma and to transport and store a wide variety of ligands e.g. fatty acids, calcium, bilirubin and hormones such as thyroxine. Recent evidence suggests albumin may exert antioxidant properties by functioning as a serum peroxidase in the presence of reduced glutathione and it has an important role in ligand binding and free radical-trapping activities (Marjolaine, 2008). In Bangladesh, BRRI dhan28 and BRRI dhan29 are two mega high yielding rice varieties which covers most of the rice production area during boro season. On the other hand, BR5 is a popular aromatic rice with strong fragrance and BR16 is a low glycemic rice (Howlader, 2009). In 2012, Alak et al. in a comparative study on antioxidant properties of ten high yielding rice varieties of Bangladesh, reported that TPC was the highest in BR5 (25.30±0.52 mg GAE 100g^-1) and the lowest was in BR16 (10.78±0.70 mg GAE 100g^-1) among all ten tested varieties . BRRI dhan28 and BRRI dhan29 had an intermediate score of 18.42±0.45 mg GAE 100g^-1and 17.67±0.08 mg GAE 100g^-1 respectively. Both antioxidant parameters like FRAP and TAC of these rice varieties were positively correlated with TPC (Alak, 2012). We have selected these four varieties for this experiment based on their performance in previous study by Alak et. Al, 2012. Since there were three deferent groups having three treatments, so Duncan's multiple range test (DMRT) was applied on Iron, TIBC, Transferrin, Uric acid and Albumin parameter for statistical analysis using SPSS, version 20.0. Our data revealed that high antioxidant enriched rice BR5 showed the lowest iron content 53.75 µg/dL among other rice varieties BR16 and BRRI dhan28. Since plasma levels of transferrin are regulated by the availability of iron and increase when plasma levels of iron are low, we found the content of transferrin in group 1 (rats) was a mean of 259.36 µg/dL. Mean values of TIBC, Uric acid and Albumin were elevated in group 1 compare to group2 and group3. Group3 had the highest free iron content of 103.74 µg dL-¹ and lowest level of TIBC, Transferrin, Uric acid and Albumin among three groups (Table 1). Comparative analysis of antioxidant status for dietary administration of high, intermediate and low antioxidant enrich  HYV rice varieties of BR5, BRRI dhan28 and BR16 in rat model significantly correlate between the content of antioxidant properties of rice and antioxidant status in rat blood serum. Thus, we concluded that dietary administration of antioxidant enriched rice in improving the antioxidant status in rat blood serum has a significant impact indeed.

ACKNOWLEDGEMENT

Authors would like to give special thanks to Mr. Jamal Uddin Ahmed, Ms. Shahena Akter, Mr. Mohammad Ali, Md. Ashraful Islam and Mr. Alim of GQN Division for rats and rat room maintenance. We acknowledge Bangladesh Rice Research Institute, Gazipur, Department of Biochemistry and Molecular Biology, Dhaka University and Bangladesh University of Heath Science, for allocating GoB research fund, utilizing clinical analyzer and providing mating Long Evan rats for rat breeding purposes respectively.

ABBREVIATION

TIBC (Total Iron Building Capacity), Transferrin, Uric acid, TPC (Total Phenolic Content), FRAC (Ferric Reducing Antioxidant Power), TAC (Total Antioxidant Capacity), HYV(High yielding variety).

REFERENCE

1. Alak KD, Partha SG, Subrata B, Sukh M, Muhammad AS, Yearul K. Antioxidant properties of ten high yielding rice varieties of Bangladesh. Asian Pacific Journal of Tropical Biomedicine (2012)S99-S103

2. Becker BF. Towards the physiological function of uric acid. Free Radic Biol Med. 1993 Jun; 14(6):615-31.

3. Bindu M, Krishnan R, and Rajendiran VK. Low plasma antioxidant status in patients with epilepsy and the role of antiepileptic drugs on oxidative stress. Ann Indian Acad Neurol. 2014 Oct-Dec; 17(4): 398–404.

4. Droge W.  Free radicals in the physiological control of cell function. Physiol Rev 2002; 82:47-95.

5. Enomoto A, Endou H. Roles of organic anion transporters (OATs) and a urate transporter (URAT1) in the pathophysiology of human disease. Clin Exp Nephrol. 2005 Sep; 9(3):195-205.

6. J. A. Milner, New Insights into the Mechanism of Action of Antioxidants (Report). 2002. Nutrition Science Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892

7. Marjolaine Roche, Philippe Rondeau, Nihar Ranjan Singh, Evelyne Tarnus, Emmanue Bourdon. The antioxidant properties of serum albumin. FEBS Letters 582 (2008) 1783–1787.

8. Md Zakir Hossain Howlader, Shunil Kumar Biswas. Screening for Nutritionally Rich and Low Glycemic Index Bangladeshi Rice Varieties. National Food Policy Capacity Strengthening Programme. Research Grant Reports CF # 6/07.

9. Salganik RI. The benefits and hazards of antioxidants: controlling apoptosis and other protective mechanisms in cancer patients and the human population. J Am Coll Nutr 2001;20:464S-472S; discussion 473S-475S.

10. Sautin YY, Johnson RJ. Uric acid: the oxidant-antioxidant paradox. Nucleosides Nucleotides Nucleic Acids. 2008 Jun; 27(6):608-19.

11. Vauzour D, Rodriguez-Mateos A, Corona G, Oruna-Concha MJ, Spencer JPE. Polyphenols and human health: Prevention of disease and mechanisms of action. Nutrients 2010; 2: 1106-31.

12. Victor VM, Rocha M, Sola E, Banuls C, Garcia-Malpardita K, Hernandez-Mijares A. Oxidative stress, endothelial dysfunction and atherosclerosis. Curr Pharm Des 2009; 15: 2988-3002.

13. Wahle KW, Brown I, Rotondo D, Heys SD. Plant phenolics in the prevention and treatment of cancer. Adv Exp Biol 2010; 698:36-51.