Hypocholesterolemic Effect of Dietary Fish Oil in Albino Wistar Rats

 

Thilakavathi Arunagiri¹, Arjun Pandian², Samiraj Ramesh³*

¹Department of Zoology, PRIST University, Thanjavur - 614904, Tamil Nadu, India

²Department of Biotechnology, PRIST University, Thanjavur - 614904, Tamil Nadu, India

³Department of Microbiology, PRIST University, Thanjavur - 614904, Tamil Nadu, India

 

ABSTRACT:

The present study was undertaken to analyze the influence of omega 3 Fish oil and Cod liver oil on serum cholesterol levels and triglycerides in albino wistar rats. Serum total cholesterol was determined by an enzymatic method using a diagnostic kit. HDL and LDL cholesterol were measured by an enzymatic method using automated analyzer. Triglycerides were analyzed using enzymatic method. VLDL cholesterol was calculated from triglycerides values.  The atherogenic index was calculated as: serum total cholesterol/HDL cholesterol.  Serum total cholesterol level in rats fed with diet containing 15% omega 3 Fish oil (15% OF) supplemented with cholesterol (0.5% CH) decreased significantly (P < 0.05) compared to those fed with the diet having only cholesterol (0.5% CH) and 10% Fluvastatin (10% FS) supplemented with cholesterol. Rats fed with diet containing 15% omega 3 fish oil (15% OF) supplemented with cholesterol had significantly lower serum total cholesterol than 15% Cod liver oil (15% CO) blended with cholesterol. Omega 3 fish oil is a strong hypocholesterolemic agent as that of fluvastatin (a cholesterol reducing drug). Therefore, regular ingestion of omega 3 Fish oil or supplementation with diets is important in the maintenance of human health.

 

 

 

INTRODUCTION:

Fish oil is basically a by-product from the fish meal industry and it is sold today as sources of vitamins A, D and LCn-3 PUFA^1,2.  In addition, fish oil concentrates contain different proportions of EPA and DHA are available for use in diverse clinical conditions.  A major challenge to the food industry is to prepare EPA and DHA concentrates with good stability (microencapsulation and addition of antioxidants) which can be used in infant formulae processed foods, margarines, bakery products and milk powders etc. Extensive research done during the past two decades has documented that fish oils can reduce coronary heart disease (CHD) through a variety of mechanisms. There are number of factors that influence a person’s cholesterol levels including diet, gender, age, weight, genetics, diseases and lifestyle.

 

There are two dietary factors associated with increases in blood cholesterol levels i.e. eating food that is high in saturated fat and eating food containing high level of cholesterol^3,4. Hence the present investigation was undertaken to analyze the effect of omega 3 fish body oil and cod liver oil on serum cholesterol levels and triglycerides in albino wistar rats.

 

MATERIAL AND METHODS:

The rats were assigned to eleven groups (four rats in each group weighing each around 150 ± 10g) (Table 1).  These were fed with diets identical in all respects, except the treatment source. Cholesterol at 0.5% level was added to one of the groups (group 2). Omega 3 fish oil, cod liver oil and fluvastatin (cholesterol reducing drug) at different levels (each containing 5%, 10% and 15%) were incorporated with 0.5% of cholesterol in the diet (groups 3-11). The other ingredients in the diet were fat-free casein to provide 10% proteins, 4% USP salt mixer, 5% cellulose, 1% vitamin mixture⁵ and 1% methionine.  The diet was made up to 100% with corn starch⁶.  The control group was fed with a commercial rat feed (Amrut laboratory rat feed) containing 22.08% crude protein, 4.38% crude lipid and 7.77% ash (group 1).  The diets and water were provided ad libitum for 45 days. At the end of the experimental period and after a 24h fast, the rats were sacrificed by cervical decapitation and blood sample was collected by retro orbital bleeding. This protocol was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) at PRIST University.  

 

Serum total cholesterol was determined by an enzymatic method using a diagnostic kit (ERBA Diagnostics Mannheim GmbH, Germany). HDL and LDL cholesterol were measured by an enzymatic method using automated analyzer (Dia Sys Diagnostic Systems GmbH, Germany).  Triglycerides were analyzed using a kit based enzymatic method (Accurex Biomedical Pvt. Ltd., Mumbai). VLDL cholesterol was calculated from triglycerides values. The atherogenic index was calculated as: serum total cholesterol/HDL cholesterol.  Statistical analyses were carried out with SPSS (2003) Windows version 12.0.  Data were subjected to one-way analysis of variance (ANOVA) and differences between means were assessed by Duncan’s multiple range test (DMRT). All statistical tests were considered significant at 5% level (P < 0.05).

 

RESULTS AND DISCUSSION:

About 85% of our body cholesterol is endogenous i.e. produced by our body and the rest 15%, or so comes from an external source i.e. diet. However, the level of cholesterol already present in our body can be increased because of high consumption of cholesterol diet and saturated fat in the diet. The increase in dietary cholesterol has been associated with atherosclerosis, the build up of plaques that can narrow or block vessels⁷. In the present study, the serum total cholesterol level was found to be low in rats fed with diet containing 15% omega 3 fish oil (15% OF) (40.75 mg) than in 15% cod liver oil (15% CO) (52.74 mg) blended with cholesterol (Table 2). This shows that omega 3 fish oil is a strong hypocholesterolemic agent like that of fluvastatin.

 

In rats fed with 15 % OF, 15% CO and 15% FS diets blended with cholesterol (CH), serum total cholesterol (40.75 mg, 52.74 mg and 40.55 mg respectively) and LDL cholesterol (21.03 mg, 24.38 mg and 19.23 mg respectively) were significantly lower than in rats fed with cholesterol alone supplemented diet (serum total cholesterol - 80.75 mg; LDL cholesterol - 45.25 mg) (Table 2).  This may be attributable to the relatively high polyunsaturated fatty acid content of the oils, since it is known that consumption of these fatty acids may increase nitric oxide synthesis⁸. Studies indicated that fish oil lowered serum cholesterol about twice as well and more rapidly than corn oil. The P/S ratio (polyunsaturated/saturated fatty acid) became the dietary key to lower total serum cholesterol.  The omega-3 fatty acids of fish oils suppress fatty acid biosynthesis and enhance fatty acid oxidation, there by reducing serum triglycerides and VLDL production leading to lowered total plasma cholesterol and sometimes LDL cholesterol⁹.  This is confirmed by the chief sources of omega-3 fatty acids in fish oils in the present study.

 

Table 1. Dietary combinations used in the experiments

Rats	Dietary Treatments
Group 1 (CF)	Control diet
Group 2 (0.5% CH)	0.5% Cholesterol1 diet
Group 3 (0.5% CH + 5% OF)	0.5% Cholesterol + 5% Omega 3 fish oil2 diet
Group 4 (0.5% CH + 10% OF)	0.5% Cholesterol + 10% Omega 3 fish oil diet
Group 5 (0.5% CH + 15% OF)	0.5% Cholesterol + 15% Omega 3 fish oil diet
Group 6 (0.5% CH + 5% CO)	0.5% Cholesterol (0.5%) + 5% Cod liver oil3 diet
Group 7 (0.5% CH + 10% CO)	0.5% Cholesterol + 10% Cod liver oil diet
Group 8 (0.5% CH + 15% CO)	0.5% Cholesterol + 15% Cod liver oil diet
Group 9 (0.5% CH + 5% FS)	0.5% Cholesterol + 5% Fluvastatin4 diet
Group10 (0.5% CH + 10% FS)	0.5% Cholesterol + 10% Fluvastatin diet
Group 11 (0.5% CH + 15% FS)	0.5% Cholesterol + 15% Fluvastatin diet

 

¹Cholesterol obtained from Merck (India) Ltd., Mumbai, India 

²Omega 3 fish oil obtained from Coastal Aquatic Proteins, Mangalore, India 

³Cod liver oil obtained from Universal Medicare Pvt. Ltd., Mumbai, India 

⁴Fluvastatin obtained from Ranbaxy, Ahmedabad, India

 

In the present study, the triglyceride level was found to be significantly lower in rats fed with diets containing 15% OF with cholesterol (51.23 mg) than in cholesterol only supplemented diet (89.25 mg) (Table 2). The predominant effect of long chain n-3 fatty acids on lipid and lipoprotein metabolism in cell culture systems, animals or humans is to reduce triglyceride concentrations^10,11, by decreasing the activity of acyl-CoA: 1, 2-diacylglycerol acyl transferase, the last enzyme in triglyceride synthesis^11,12. The average reduction of triglycerides is dose related^13,14, the minimal effective dose of n-3 fatty acids being about 1 g daily and a plateau being reached at between 5 and 10 g¹³.  Studies on the impact of fatty acids on serum lipids and lipoproteins have shown that saturated fatty acids (SFA) increase LDL, MUFA (oleic acid), PUFA (linoleic acid (LA) and α-linolenic acid (ALNA)¹⁵. Fish and fish oils have a remarkable effect in lowering serum VLDL and triglycerides as well as postprandial lipemia both in normal subjects and hyperlipidemic patients¹⁶.  In support of the above, VLDL cholesterol and triglycerides in the present study showed low levels in the rats fed with diets having 15% OF (11.24 mg) and 15% CO (13.51 mg) blended with cholesterol (Table 2).  The decrease in VLDL is due to inhibition of hepatic synthesis of triglycerides and increased fractional catabolic rate of triglyceride rich VLDL¹⁷.

 

The HDL cholesterol was significantly higher in the groups fed with 15% OF and 15% CO diets blended with cholesterol (25.23 mg and 23.65 mg respectively) than in the diets containing 15% FS blended with cholesterol (21.56 mg).  Several previous studies have reported an increase in HDL-cholesterol with polyunsaturated fat consumption¹⁰. Another factor that may have contributed to the high HDL-cholesterol level in these groups is the relatively high arginine contents in the fish oils. Arginine is a precursor of nitric oxide. Furthermore, polyunsaturated fats may increase nitric oxide levels^18,19. Low values of the atherogenic index are associated with a low risk of coronary and atherosclerotic diseases^20-23. In the present study, the atherogenic index in rats fed with 15% OF diet blended with cholesterol (1.68) was lower than in rats fed with 15% FS (1.89), 10% FS (1.97), 10% OF (2.21) and 15% CO (2.27) diets supplemented with cholesterol (Table 2). 

 

CONCLUSION:

In the present study, the serum total cholesterol level was found to be low in rats fed with diet containing 15% omega 3 fish oil than in 15% cod liver oil blended with cholesterol.  This shows that omega 3 fish oil is a strong hypocholesterolemic agent as that of fluvastatin (a cholesterol reducing drug).  Therefore, regular ingestion of omega 3 fish oil or supplementation with diets is important in the maintenance of human health.

 

Table 2.  Serum cholesterol levels, triglycerides and atherogenic index in rats fed different diets 

Diets	Serum total cholesterol (mg/100ml)	Triglycerides (mg/100ml)	HDL cholesterol (mg/100ml)	LDL cholesterol (mg/100ml)	VLDL cholesterol (mg/100ml)	Atherogenic index
CF	57.46 ± 3.21ce	62.66 ± 1.63d	21.56 ± 1.42a	31.60 ± 1.74cde	11.77 ± 0.99	2.68
0.5% CH	80.75 ± 4.54f	89.25 ± 2.71f	15.13 ± 2.44a	45.25 ± 1.62f	15.55 ± 1.75	5.16
0.5% CH + 5% OF	66.42 ± 3.54de	80.35 ± 3.75ef	17.06 ± 1.05a	34.06 ± 1.44e	15.07 ± 1.22	3.82
0.5% CH + 10% OF	42.39 ± 1.21a	52.65 ± 2.55ac	19.45 ± 1.35a	22.05 ± 3.34abc	11.85 ± 0.95	2.21
0.5% CH + 15% OF	40.75 ± 5.36a	51.23 ± 6.24ac	25.23 ± 2.25b	21.03 ± 1.99abcd	11.24 ± 1.88	1.68
0.5% CH + 5% CO	70.71 ± 2.14d	81.76 ± 3.57ef	16.92 ± 1.73a	33.05 ± 2.75de	15.87 ± 1.75	3.95
0.5% CH + 10% CO	67.74 ± 1.64d	77.41 ± 4.97e	19.35 ± 2.31a	29.64 ± 2.55bcde	14.24 ± 1.34	3.47
0.5% CH + 15% CO	52.74 ± 4.63bc	60.53 ± 5.72bcd	23.65 ± 1.34a	24.38 ± 3.55abcde	13.51 ± 1.51	2.27
0.5% CH + 5% FS	44.28 ± 2.58ab	55.51 ± 3.33acd	19.21 ± 1.37a	33.68 ± 1.77e	10.53 ± 1.78	2.33
0.5% CH + 10% FS	39.26 ± 1.94a	48.35 ± 6.31a	20.52 ± 2.24a	31.65 ± 1.19cde	8.98 ± 1.82	1.97
0.5% CH + 15% FS	40.55± 2.65a	49.23 ± 2.42a	21.56 ± 1.97a	19.23 ± 1.67a	9.12 ± 1.96	1.89

Values are mean ± S.D. of four replicate samples

Atherogenic index was calculated with the mean value of each group

Means in the same column sharing different superscripts are significantly different (P < 0.05); Means within VLDL cholesterol are not significantly different (P > 0.05)

 

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Accepted on 20.04.2019         © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(9):4161-4164.