Introduction

Hypercholesterolemia is a risk factor in the development of atherosclerosis ₁ . Therapeutic agents which control the levels of serum cholesterol have proven to be effective in the treatment of coronary heart diseases (CHD) ₂,₃ . While agents exist that can modulate circulating levels of cholesterol carrying lipoproteins by inhibiting cholesterol synthesis, these agents have little or no effect on the intestinal absorption of cholesterol. Dietary cholesterol can increase the level of serum cholesterol to levels which can place an individual at increased risk for the development or exacerbation of atherosclerosis ₁,₄ .

The liver plays a central role in the storage, synthesis, and metabolic transformations of lipids by packaging triglycerides and cholesterol, which are insoluble in the plasma, into particles called lipoproteins which can be carried in the bloodstream. Atherosclerosis weakens the arterial wall and narrows the path of blood within the vessels. Atherosclerotic lesions frequently appear in the coronary arteries, producing CHD. As the plaque increases in size, the coronary arteries may become completely blocked, when that occurs, the heart muscles are deprived of oxygen from the blood and the victim suffers a “heart attack”, or a myocardial infarction ₁,₅ . The risk of CHD increases dramatically as the plasma concentration of LDL cholesterol increases ₆ . Consequently, development of methods for lowering LDL cholesterol levels has become a major focus of medical research. The approach of reducing dietary cholesterol suffers from two limitations. The first is that cholesterol is present in all animal fats and many people are unwilling to scarify their preferred diet. The second is that the liver and other tissues synthesize cholesterol de novo if the dietary supply is inadequate.

In West Africa, egusi melon (Citrullus lanatus) seeds (fig. 1b) from egusi melon plant (fig. 1a) are a common component of daily meals. Little nutritional detail on egusi melon oil is readily available to an international readership. Research studies have shown that these seeds contained about 50% oil ₇ , 42-57% oil ₈ , 44-53% oil ₉ for seeds cultivated in different bioclimatic regions of Cameroon. These studies showed that egusi melon seeds contained good amounts of oil that can be exploited. On this regard, the present study is aimed at establishing the fatty acid profile of egusi melon oil and the effect of the oil on serum lipids and on the activities of some selected serum enzymes used to aid diagnostic tools in cardiovascular disease.

Figure 1

Figure 1a: (egusi melon) plant

Figure 2

Figure 1b: Citrullus lanatus (egusi melon) seeds

Materials And Methods

Chemicals: All chemicals used were of analytical grade and were products of BDH Chemicals Ltd, Poole England unless otherwise stated.

Collection and preparation of seeds sample: Egusi melon seeds used for this study were obtained from a local market in Iwaro-Oka Akoko, Ondo State, Nigeria and were identified as Citrullus lanatus (egusi melon) by a taxonomist in the Department of Crop Science, Faculty of Agriculture, University of Benin, Nigeria. The seeds were screened to remove bad ones, shelled manually and further screened. The seeds were then dried to constant weight in an oven at 70 °C, ground using mechanical grinder, put in air-tight containers and stored in desiccators for further analysis, some of the seeds was subsequently deposited at the herbarium of the faculty.

Oil extraction: Oil from the seeds of egusi melon was extracted by continuous extraction in Soxhlet apparatus (Cehmglass) for 8 hours using petroleum ether (60-80° C boiling range) as solvent according to the method described ₁₀ . At the end of the extraction the extraction solvent was evaporated in a rotary evaporator (Cehmglass). The extracted oil was used for feed formulation and the remaining stored in light proof, airtight and moisture proof container at -4°C for further analysis.

Fatty acid composition analysis: The fatty acid profile of egusi melon oil was determined by gas liquid chromatography (Hewlett Packard, model 5750).

Animals and diets: Albino Wistar rats (n=14) of both sexes and weighing 110-120g obtained from the animal laboratory of the Department of Biochemistry, University of Ilorin, Nigeria were used for the study. Animals were housed singly in stainless steel cages with raised wire floor in a room with a 12hour light/dark cycle at a temperature of about 30°C and fed rat chow (purchased from Guinea feeds, Nigeria) and water ad libitum for two weeks to acclimatize. The rats were then assigned randomly to two group of seven each designated: control and experimental respectively and placed on their respective diet for a period of 6 weeks. The composition of each diet is as shown in Table 1. Before the commencement of the feeding experiment, rats were fasted overnight but allowed access to water ad libitum. Rats had free access to diet and were weighed weekly.

Serum preparation: At weekly intervals, one rat from each group was sacrificed and 2ml blood collected into plain tubes, centrifuged at 10000g for 5min and the serum extracted and analyses were carried out immediately. The animal protocol was approved by the Animal Committee of the National Institute of Medical Laboratory Sciences, Nigeria.

Assays: Serum total and free cholesterol concentrations were determined by the method of Searcy and Bergquist ₁₁ , while the esterified cholesterol concentration was calculated as the difference between total and free cholesterol values. Triglyceride concentration was determined according to the method described by Tiez ₁₂ . Activity of lactate dehydrogenase (LDH) was determined using the method of Kubowitz and Otti ₁₃ . Serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using the methods described by Reitman and Fankel ₁₄ while gamma- glutamyl transpeptidase (γ-GT) activity was determined by Szaz method ₁₅ . Protein content in serum was measured by Lowry et al method ₁₆ .

Histological analysis: Histological preparations of the aorta of the rats at the end of the feeding period were made as described earliear ₁₇ . Frozen aorta samples were sliced (4µm) and stained in 60% Oil Red O stock solution (0.5g of Oil Red O in 100ml of isopropanol) for 5 minutes. Tissues were washed briefly in 60% polyethylene glycol and then rinsed in distilled water for microscopic observation and photography.

Statistical analysis: The data are presented as means ± SEM. The mean values of the control and test groups were compared using student's t-test performed using SPSS 10 software. P<0.05 was considered to be significant.

Figure 3

Table 1: Diet Composition (% by weight)

Results

All rats consumed about 70% of their daily ration and grew well during the study, gaining 15.4g mean weight weekly. No significant difference was found in the rate of weight gain for animals in the two groups. The oil extraction showed that about 47% by weight of egusi melon seed is composed of oil.

Fatty acid Composition: The fatty acid composition of egusi melon seed oil presented in Table 2 showed that the oil contained four main fatty acids: palmitic, stearic, oleic, and linoleic acids, linoleic acid being the most abundant. 71.9% of the fatty acid content of egusi melon seed oil are unsaturated out of which 14.5% are monounsaturated fatty acids and the remaining 57.4% are polyunsaturated fatty acids.

Figure 4

Table 2: Fatty acid composition of egusi melon oil

Serum Lipids: Changes in serum cholesterol concentration of the control and test rats over the entire feeding period are presented in Table 3. Cholesterol concentrations in the total, free and esterified fractions were significantly lower (p<0.05) in rats fed diet containing egusi melon seed oil compared with the control at the end of the feeding exercise. Similarly, serum triglyceride concentration was significantly lower (p<0.05) in the egusi melon oil-fed rats compared to the control (Table 4).

Figure 5

Table 3: Changes in serum cholesterol concentration (mg/dl).

Figure 6

Table 4: Serum triglyceride concentration (mg/dl).

Values are mean ± SEM of triplicate determinations. Values in the same row carrying different superscript are significant (p<0.05).

Serum enzymes' activities: As shown in Table 5, the egusi melon oil-fed rats presented significantly reduced (p<0.05) activities of the enzymes in serum compared with the control at the end of the feeding trial.

Figure 7

Table 5: Serum enzymes' activities (U/L)

Values are mean ± SEM of triplicate determinations. Values in the same row carrying different superscript are significant (p<0.05).

Note: LDH: Lactate dehydrogenase; ALT= alanine aminotransferase; AST= aspartate aminotransferase; γ-GT= gamma-glutamyl transpeptidase

Total protein concentration: the result shown in Table 6 shows that significant (p0.05) differences in the two groups studied only occurred in the last two weeks of the study. The experimental rats had significantly higher (p<0.05) serum protein concentration in these last two weeks.

Figure 8

Table 6: Total protein concentration (mg/dl)

Values are mean ± SEM of triplicate determinations. Values in the same row carrying different superscript are significant (p<0.05).

Histological analysis of the aorta of the control rat at the end of the feeding period showed massive degeneration of the arterial wall linings and movement of the smooth muscle cells into the intima of the arteries (see arrow on plate 1). There were also depositions of fatty streaks in the arterial lumen. No such observation was made for the egusi melon oil-fed rats (plate2).

Figure 9

Plate 1: Histological preparation of the aorta of control rat at the end of the feeding experiment

Figure 10

Plate 2: Histological preparation of the aorta of experimental rat at the end of the feeding experiment

Discussion

As earlier observed by Martin (1998) ₇ , Fokou et al (2004) ₈ and Achu et al (2005) ₉ , the result of our study showed that egusi melon seed contains considerable amount of oil. The fatty acid composition of egusi melon oil showed that the oil is very rich in polyunsaturated fatty acids. The most abundant fatty acid in egusi melon oil is linoleic acid, an essential fatty acid. Linoleic acid and linolenic acid as well as other (n-3) and (n-6) fatty acids have been reported to have protective effect against CHD ₁₈,₁₉,₂₀ . Studies have shown that linoleic acid lowers total and LDL cholesterol concentrations, which are established risk factors of CHD and also improves insulin sensitivity ₁₉,₂₀ . The linolenic acid content of egusi melon oil is rather too low by comparism to other polyunsaturated fatty acid rich oilseeds. Linolenic acid though an omega-3 fatty acid with positive health effects is easily susceptible to peroxidation, hence, undesirable in edible oils because of the off-flavours and potentially harmful oxidative products formed ₁₈ . The high linoleic acid and low linolenic acid content of egusi melon oil indicates that it is a good source of table oil, cooking oil and frying oil, making it good for the fight against CHD.

The results of this study show that the inclusion of egusi melon oil as a supplement to diet containing high cholesterol improves serum lipids. The reductions in total and esterified cholesterol observed in the egusi melon oil-fed rats observed in this study is of interest since the offending lipid in atherogenesis is the esterified cholesterol fraction. An increase in cholesteryl ester fraction above the plasma threshold level could possibly initiate atherogenesis. If the cholesteryl ester produced cannot be effectively catabolized due to its relatively high concentration, there would be consequent deposition of the excess in the peripheral and vascular tissues resulting in atherogenesis. The observed decrease in serum triglyceride concentration in the egusi melon-fed group also explains the positive benefits of the oil on serum lipids. Patients with hypertriglyceridemia have been demonstrated frequently to have lower plasma HDL cholesterol levels ₂₁ , which may contribute to increase risk for CHD ₂₂ . Lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (γ-GT) in addition to serum cholesterol and triglyceride are demonstrated to be constituent of some tissues (liver, kidneys, heart, adipose tissue, aorta and skeletal muscles etc). Damage to these tissues could result in leakage of these enzymes into the bloodstream. The observed low activities of LDH, ALT, AST, and γ-GT in the egusi melon-fed rats could be as a result of the possible role of linoleic acid in maintaining cellular integrity. This submission derived merit from the results of the histological preparations of the aorta at the end of the feeding exercise which showed an intact vasculature in the egusi melon-fed rats (plate 2) compared to that of the control group which showed massive degeneration of the arterial wall linings accompanied with movement of smooth muscle cells into the intima (see arrow on plate 1). In addition, the results of the serum protein concentration also justifies the fact that egusi melon may play some protective effect on cellular integrity by stimulating the secretion of certain proteinous molecules hence the higher protein concentration observed in the egusi melon fed rats. In conclusion the use of egusi melon oil as edible oil for cooking and frying and also as a food supplement especially in regions with high fat diets as staples is strongly recommended because of its high content of essential fatty acids and its positive health benefits on serum lipids. Further studies are warranted to confirm our results and to determine the exact mechanism(s) of action of the hypocholesterolemic effect of egusi melon oil.