Chemical and Biological Evaluation of Omega-3 Fatty Acids Rich Foods

CHEMICAL AND BIOLOGICAL EVALUATION OF OMEGA-3 FATTY ACIDS RICH FOODS

KARIMA SAID MOHAMED HAMMAD B.Sc. Agric. Sci. (FoodScience), Fac. Agric., Cairo Univ., 2006 M.Sc. Agric. Sci. (FoodScience), Fac. Agric., Cairo Univ., 2011

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Agricultural Sciences (Food Science)

Department of Food Science Faculty of Agriculture Cairo University EGYPT

2016

ABSTRACT Hypercholesterolemia is the most common pathologic process underlying atherosclerosis and cardiovascular disease. Alpha–linolenic acid (ALA, n- 3) is believed to protect the cardiovascular system. This study was carried out to investigate the protective and antiatherogenic effects of linseed oil and walnut as omega-3 rich foods in rats upon feeding hypercholesterolemic diet (HCD). Furthermore, study the therapeutic effect of linseed oil and walnut, rich diets on lipid profile, oxidative status and histopathologic changes of hypercholesterolemic (HC) rats. Sixty male Sprague- Dawley rats weighed 121.3±10.6 g were divided into six groups. Rats were fed for 10 weeks on rodent diets contained 2% cholesterol , 3% corn oil and 20% fat whose source was from either palm stearin (saturated fat), corn oil, linseed oil or walnut at different levels of ALA that ranged from zero to 9.71%. Weight gain, liver weight, serum lipid profile and liver function parameters were determined. HCD based on saturated fat resulted in significant alterations in serum lipids, increase in body weight gain and relative liver weight accompanied by a negative effect on liver function parameters. Dietary ALA could counteract the detrimental effects brought about by the HCD. Histopathological examination confirmed the protective effect of ALA against the histopathological alterations induced by the HCD in the liver, heart, aorta and prostate of rats. Rats fed HCD based on linseed oil showed less macroscopic and microscopic liver damage and less degenerative changes of the epithelial cells of the prostate. These results suggest that feeding rats on a high level of ALA could ameliorate the inflammation and damage in the investigated tissues induced by HCD. Results indicated that feeding HC rats (total cholesterol, TC≥240mg/dl) on a cholesterol free high fat diet (HFD) rich in walnut or linseed oil for 6 weeks significantly (P<0.05) decreased TC, low-density lipoprotein cholesterol , triglycerides, and atherogenic indices to the normal levels.Hepatic and brain levels of superoxide dismutase ofHC rats that were fed on a HFD rich in walnut or linseed oil were significantly higher than those fed on the diet rich in palm stearin. Hepatic and brain levels of malondialdehyde and cholesterol, elevated significantly (p<0.05) upon feeding HC rats on HCD compared to those of rats that were fed on a HFD rich in linseed oil by 2 fold. The overall beneficial effects provided by high level of ALA were better than those given by low levels of ALA. Linseed oil exerted a hypolipidemic effect and could be considered as a promising functional food in cardiovascular disease. Key words: alpha-linolenic acid, linseed oil, walnut, hypercholesterolemia, lipid profile, liver function parameters

INTRODUCTION Hyperlipidemia is a condition of excess fatty substances called lipids, largely cholesterol and triglycerides, in the blood.(NCEP, 1994). The American Heart Association reports that the lipids contained in the bloodstream include cholesterol, triglycerides, cholesterol compounds (esters) and phospholipids. Terms applied to various forms of hyperlipedemia include hypercholesterolemia, hypertriglyceridemia and hyperlipoproteinemia(Niroshaet al., 2014).

Hypercholesterolemia is one of the most important risk factors in the development and progression of atherosclerosis that lead to cardiovascular diseases (CVD) (Rerkasem et al., 2008 andPriya etal.,2010). Hypercholesterolemia is characterized by high blood serum cholesterol and low-density lipoprotein levels besides low level of high-density lipoprotein. Excessive saturated fat and cholesterol consumption for a long time are believed to be directly related to hypercholesterolemia (Asahina et al., 2005). Atherosclerosis is a progressive, pathologic process and the primary contributing factor to CVD (Xu et al., 2014). High fat diets can be used to generate a rodent model for the analysis of the pathophysiology of dyslipidemia (Estadellaet al., 2004). Feeding animals on cholesterol-rich diets has been used to increase serum or tissue cholesterol level or content in order to study the etiology of hypercholesterolemia-related metabolic disorders (Kabiri et al., 2011). Hypercholesterolemic animals are useful models for studies on cholesterol homeostasis, and drug trials to better understand the relationship between disorders in cholesterol metabolism, atherogenesis as well as possible treatments for the reduction of circulatory cholesterol levels (Jung and Wang, 2009). National Heart Associations have recommended increased consumption of fatty fish or ω-3 polyunsaturated fatty acids (PUFA) supplements to prevent CVD. Non- fish sources of ω-3 PUFA vary in their capacity to regulate blood levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (C20–22, ω-3 PUFA) and CVD risk factors (Jump, 2012).Increased intakes of dietary alpha linolenic acid (ALA) elicit anti-inflammatory effects (Zhao et al., 2007).

Flax is an annual plant belonging to the genus Linum and the family Linaceae. Different varieties of Linum bred for fiber use are called flax, whereas the oilseed varieties are called linseed (Linumusitatissimum L.), oilseed flax or flax (Popaet al., 2012).Linseeds (Linumusitatissimum) contain 32–45% of their mass, as oil. Linseed oil is the only oil of plant origin known to have the highest concentration of ALA. ALA serves as a precursor for long chain ω-3 polyunsaturated fatty acids such as C20–22, ω-3 PUFA. The ω-3 fatty acid found in linseed differs from those in fish. Linseed oil has been tested in clinical trials that have described its potential beneficial effect against disorders, such as dyslipidemia and cardiovascular disease (Makniet al., 2008 andLemoset al., 2012).

Walnut, JuglansregiaL. (Juglandacea) is classified as a strategic species for human nutrition and is included in the Food and Agriculture Organization of United Nations (FAO) list of priority plants (Gandev, 2007).The high protein and oil contents of the kernels of walnut make this fruit essential for human nutrition (Aryapak and Ziarati, 2014) Among nut oils, walnut oil contains the highest amounts of PUFAs (Amaralet al., 2003). The fatty acid composition of walnut oil is unique compared to other nuts oil because walnut oil contains predominantly linoleic acid (49 to 63%), and a considerable amount of ALA (8 to 15.5%) (Moigradeanet al., 2013). However, FDA (2004) stated that, there is no significant scientific evidence that consumption of walnuts may reduce the risk of coronary heart disease”. Other researchers (Berryman et al., 2013 and Kris-Etherton, 2014) reported that frequent consumption of walnuts and/or walnut oil may improve cardiovascular risk via mechanisms that extend beyond their established cholesterol- lowering action. Beneficial effects of ALA on plasma lipid and lipoproteins are controversial (Vijaimohanet al., 2006, Rizoset al., 2012 andVlachopouloset al., 2013).The discrepancies between these results may be due to differences in study protocols, diets, amounts of n-3 PUFA supplement and durations (Adkins and Kelley, 2010)

Accumulation of lipids within artery walls caused atherosclerosis. It is considered an important risk factor for cardiovascular diseases (Paoletti et al., 2004). Therefore, many studies have been conducted to verify the effectiveness of certain foods in the prevention of these cardiovascular diseases ( de Miranda et al., 2014). Hypercholesterolemic diet is a crucial factor in the development of hypercholesterolemia (Deepa and Varalakshmi, 2005). Plant based dietary therapies and natural food components are being proposed for the prevention of dyslipidemia. Hypercholesterolemia causes morphological changes in various organs within the body, including the liver and blood vessels (Green et al., 2011). In hypercholesterolemia, cholesterol alters the vascular structure. It builds within the lining of the vascular wall, leading to lesions, plaques, and ischemia reperfusion injury and causes endothelial cell dysfunction (Stapleton et al., 2010).

AIM OF INVESTIGATION

This study was designed to examine the effect of dietary walnut kernel and linseed oil on lipid profile, liver function and antioxidant status of rats upon feeding hypercholesterolemic diets.In addition, this study investigated therapeutic effect of different levels of dietary ALA (omega-3) on the hypercholesterolemic rats and the histological features of liver, heart, aorta prostate andbrain structure in the rat.

a. Biological experiment 1. Experimental animals Animals were housed on a 12 h light-dark cycle at 25 ± 2ºC and relative humidity of 30 - 60%. Rats were individually housed in rodent stainless steel mesh cages in the Animal House of Research Institute of Ophthalmology, Egypt. Rats were fed water and diet ad- libitum. Rats were fed on a basal diet (AOAC, 2012) for 7 days prior to experimentation. Rats were humanely treated in accordance with the WHO guidelines for animal care, and the study design was approved by the Ethics of Animal was used in Research Committee, Cairo University with approval number (CUFA/F/FS/2015/44). a, USA). 2. Experimental design a. Part one After the adaptation period, rats were randomly divided into six groups. Each group consisted of six rats except (G6) that contained 30 rats

Group 1 (G1): Rats were fed on a standard basal diet (control). Group 2 (G2): Rats were fed on a hypercholesterolemic diet (HCD) containing 20 % linseed oil and 3% corn oil+ 2% cholesterol.Group 3 (G3):Rats were fed on HCD containing 10% linseed oil and 13 % corn oil + 2% cholesterol. Group 4 (G4):Rats were fed on a HCD containing 10% linseed oil ,15.82% walnut and 3% corn oil + 2% cholesterol.Group 5 (G5):Rats were fed on a HCD containing 31.65 % walnut and 3% corn oil + 2% cholesterol.

Group 6 (G6): Rats were fed on a HCD containing 20 % palm stearin and 3% corn oil + 2% cholesterol. Diets were prepared biweekly and were stored in the deep freezer at -20 °C. The experiment was carried out until mean of the blood serum cholesterol level of a rat group reached ≥240 mg/dl (induction of hypercholesterolemia). b. Part two

Hypercholesterolemic rats (≥240 mg cholesterol /dl) from (G6) were divided into four groups as follows (Table. 7)

Group (6): Rats were continued feeding on a HCD containing 20 % palm stearin and 3% corn oil + 2% cholesterol. Group (7): Rats were fed on a high fat diet, cholesterol free, (HFD) containing 20 % palm stearin and 3% corn oil. Group (8): Rats were fed on a HFD containing 31.65 % walnut and 3% corn oil. Group (9): Rats were fed on a HFD containing 20% linseed oil and 3% corn oil.

The consumed diets were recorded daily, the body weight of rats was recorded weekly to monitor body weight changes.

SUMMARY This study was carried out to investigate the protective and antiatherogenic effect of linseed oil and walnut as omega-3 rich food in rats upon feeding hypercholesterolemic diet(HCD)for 10 weeks. Furthermore, study the therapeutic effect of linseed oil and walnut, rich diets (Cholesterol free high fat diets) on lipid profile, oxidative status and histopathologic changes of hypercholesterolemic rats for another 6 weeks.

The results could be summarized as follows:

1. Fatty acid composition of the investigated oils indicated that linseed and walnut oils were rich (~66%) in the PUFA. ALA (ω-3) was the predominant fatty acid in linseed oil. Walnut oil and corn oil were characterized by a higher content of linoleic acid (18:2, ω- 6). Palm stearin was rich in saturated fatty acids (palmitic and stearic acids) (~60%). Chemical characteristics of oils were within the acceptable limits according to standards. 2. The oil, protein, fiber and ash contents of the walnut kernels were 63.2, 18.05, 5.33 and 1.7% on dry weight basis, respectively. These data were used to accommodate the oil, protein and carbohydrate contents of the added walnuts in the formula of diets. 3. After the adaptation period, rats were randomly divided into six groups. Each group consisted of six rats except (G6) that contained 30 rats as follows: Group 1 (G1): Rats were fed on a standard basal diet (control). Group 2 (G2): Rats were fed on a HCD containing 20 % linseed oil and 3% corn oil+ 2% cholesterol. Group 3 (G3): Rats were fed on a HCD containing 10% linseed oil and 13 % corn oil + 2% cholesterol. Group 4 (G4): Rats were fed on a HCD containing 10% linseed oil , 15.825% walnut and 3% corn oil + 2% cholesterol. Group 5 (G5): Rats were fed on a HCD containing 31.65 % walnut and 3% corn oil + 2% cholesterol. Group 6 (G6): Rats were fed on a HCD containing 20 % palm stearin and 3% corn oil + 2% cholesterol. Duration of this experiment extended for 10 weeks. Hypercholesterolemic rats from (G6) were divided into four groups as follows: Group (6): Rats were continued feeding on a HCD containing 20 % palm stearin and 3% corn oil + 2% cholesterol. Group (7): Rats were fed on a high fat diet, cholesterol free, (HFD) containing 20 % palm stearin and 3% corn oil. Group (8): Rats were fed on a HFD containing 33.5 % walnut and 3% corn oil. Group (9): Rats were fed on a HFD containing 20% linseed oil and 3% corn oil.Duration of this experiment extended for 6 weeks. 4. Feeding adult male rats on HCD based on palm stearin, caused the highest increase in weight gain (134.3 ± 18.9 g) when compared with the other investigated groups. Feed intake of the rats fed on HCD based on oils rich in PUFA, ALA >6% (G2 and G4) for the same duration was significantly lower than that of rats fed on the basal diet or HCD containing lower ALA (G3 and G5). Lowest weight gain was 69.0 ± 14.1 g for rats fed on HCD containing high walnut kernel level (31.65 %, G5). 5. Blood serum TC measurement was performed at the start (zero time) of the experiment and after 6 and 10 weeks in order to verify the development of hypercholesterolemia in rats (TC≥240 mg/dl).Feeding rats on HCD (G2-G6) for 6 weeks elevated their total serum TC level significantly (P<0.05) regardless of the type of oil (or type of fat used). TC level of rats that were fed on HCD based on palm stearin (G6) was significantly (P<0.05) higher than that of rats that were fed on the other investigated HCDs. Continuous feeding rats on palm stearin diet, four weeks thereafter, increased their serum TC level to >240 mg/dl. Continuous feeding of rats on HCDs based on oils rich in PUFA (G2 to G5), containing ≥2.56% ALA, for another 4 weeks was associated with a significant (P<0.05) reduction in their serum TC to the normal level. 6. Results showed that serum LDL-C and TG levels of rats fed HCD based on saturated fat (G6), for ten weeks, exceeded 180 mg/dl and 200 mg/dl, respectively. Atherogenic indices (CRI and AC) of the palm stearin group (G6) were14.24 and 13.24, respectively, These values were significantly higher than those of the control group and those fed on HCD s based on oils rich in PUFA (G2 - G5). It could be noticed that the TG level of rats fed HCD based on walnut as a source of ALA (G5) was lower than that of rats fed on the other HCD s based on oils rich in PUFA (G2-G4). 7. Hepatosomatic index (HI) of rats fed on a basal diet for 10 weeks was 2.34%. Meanwhile, HI of rats that received HCD s (G2-G6) for 10 weeks increased approximately threefold over that of low fat fed rats (control group, G1). However, feeding rats HCD based on linseed oil containing 9.71 % ALA alleviated this increase. ALT, AST and ALP levels were higher in rats fed the HCD s compared to those of rats that were fed on the control diet. Feeding rats on HCD based on palm stearin (G6), elevated their ALT, AST and ALP levels higher than those of the rats that were fed the HCD s based on oils rich in PUFA (G2- G5). 8. Higher AST/ALT ratio (2.06) was obtained fromHCD based on palm stearin (G6) compared to the control group (1.61). On the other hand, the AST/ALT ratio of the rats fed on HCD containing 9.71% ALA from linseed oil (G2) was equal to that of the normal control. LDH level of rats fed HCD based on palm stearin was 4 fold that of rats fed the control diet. Feeding rats on HCD based on linseed oil kept their LDH activity slightly different from that of the control group. 9. High level of dietary ALA (G2, 9.71%) was associated with elevation of ALA, EPA and DHA fatty acid content in the liver but feeding on palm stearin group (group 6) caused high level of SFA content in the liver. 10. The livers of animals fed on HCD were enlarged and had a pale yellow granular appearance. Histological examination of liver, cardiac, aorta and prostate tissues in animals fed on HCD based on palm stearin (G6) revealed severe pathological alterations. Meanwhile, feeding rats on HCD based on linseed oil or walnut ameliorated these effects. 11. Continuous feeding hypercholesterolemic (HC) rats on HCD for another 6 weeks exhibited significantly (P<0.05) an increase in blood serum total cholesterol, LDL-C, TG, AC, CRI, ALT and Alkaline phosphatase levels by >2 fold. Meanwhile, HI, LDH, and AST levels increased significantly (P<0.05) by 25, 29, and 70%, respectively. However, HDL-C level and AST/ALT ratio were not significantly (p>0.05) affected 12. Feeding HC rats on a HFD (cholesterol free) for 6 weeks insignificantly (P>0.05) affect cholesterol, LDL-C, AC, CRI, HI and alkaline phosphatase levels, however, it caused significant (P<0.05) increase in serum TG, LDH, levels and AST/ALT ratio. 13. Feeding HC rats on a HFD containing ALA at 2.56 % of the diet from walnut or 9.71% from linseed oil for 6 weeks significantly (P<0.05) decreased cholesterol, LDL-C, TG, AC and CRI to the normal levels. 14. Although feeding HC rats on a HFD (cholesterol free) with or without ALA did not significantly (p>0.05) affect the HI, it reduced significantly (p˂0.05) AST, and ALT activities. 15. SOD levels of HC rats that were fed on HFD rich in walnut or linseed oil were significantly (p<0.05) higher by 5-10 fold than those fed on either HCD or HFD (based on saturated fat, cholesterol free). Compared to linseed oil, walnut was more effective in elevating significantly the activity of hepatic and brain SOD. 16. The hepatic MDA level was significantly (p<0.05) higher upon feeding HC rats on HCD or HFD based on palm stearin than that of rats fed on HFD rich in linseed oil by 2 fold. 17. Excluding cholesterol from the diet of HC rats significantly (p<0.05) lowered the MDA concentration in the brain tissue to 50- 70% upon feeding for 6 weeks compared to that of HC rats that were fed on HCD. The same trend could be noticed in assessing the cholesterol level in liver and brain.The inclusion of dietary ALA at a higher level (9.71%) significantly (p<0.05) decreased cholesterol level in liver and brain to about 50% of that occurred when HC rats were fed on HCD for 6 weeks.