In ternationa l Scholars Journa ls African Journal of Food Science Research ISSN 2375-0723 Vol. 4 (5), pp. 054-058, December, 2016. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper A study of atherogenic property of oils extracted from Cucumeropsis mannii Mercy Bih Achu 1* , Elie Fokou 1 , Clergé Tchiégang 2 , Martin Fotso 3 and Felicité Mbiapo Tchouanguep 4 1 Department of Biochemistry, Faculty of Science, P. O. Box 812, University of Yaoundé I, Cameroon. 2 Department of Food Science and Nutrition, ENSAI/IUT, Ngaoundéré, Cameroon. 3 Centre for Research in Food and Nutrition, IMPM, Yaoundé, Cameroon. 4 Department of Biochemistry, Faculty of Science, University of Dschang, Cameroon. Accepted 03 February, 2016 The atherogenicity of Cucumeropsis mannii and Cucumis sativus oils was compared to that of corn oil and palm oil. Female wistar albino rats of 6 weeks old weighing 58 – 65 g were randomly assigned to one of four diet groups: C. mannii and C. sativus oils (test diets) and corn and palm oil (control diets). There was weight gain in all the groups amounting to 128.65% (palm oil), 132.75% (C. mannii), 140.8% (C. sativus) and 153.45 (corn oil) with no significant difference. The weights of the livers ranged from 4.22 (palm oil) to 5.17 g (corn oil), ratio of weights of liver to that of rat, from 0.029 - 0.034 and percentage weight gain from 128.65 (palm oil) to 153.45% (corn oil). There was no significant difference in the values. For the atherogenic parameters measured, the triglyceride level ranged from 73 (palm oil) to 79.4 (C. sativus oil) with no significant difference. Total cholesterol levels ranged from 49.6 (corn oil) to 64.2 mg/dl ( C. sativus oil) with significantly lower values in the corn oil group but similar values in the rest of the groups. HDL ranged from 18.94 (C. mannii oil) to 32.8 (palm oil) which was significantly high, LDL from 6.2 (palm oil) to 25.06 mg/dl ( C. mannii oil) and atherogenic ratio (AR) from 0.2 (palm oil) to 1.61 (C. mannii) which was significantly high. The levels of these atherogenic parameters are far below the borderline level for oils to cause atherosclerosis, indicating that they could be potential good edible oils for reducing cardiovascular illnesses. Keywords: Atherogenicity, oils, Cucumeropsis mannii, Cucumis sativus. INTRODUCTION In Africa, obesity has become a major problem in line with HIV. Though obesity is traditionally seen as a sign of wealth, it is becoming a very serious health issue due to its complications (BBC News, 2004). In Cameroon, according to information from an Obesity Clinic in the Yaounde Central Hospital, 35% of the adult population is either overweight or obese and this often leads to dia- betes and cardiovascular diseases. In addition to exer- cises and drugs (which usually have undesirable side effects), one of the ways of overcoming this health pro- blem can be through the use of diets and foodstuffs, especially those that are locally available. Cucurbitaceae (egusi) seeds are one of such foodstuffs. These seeds have been shown to be rich in proteins and oils and the *Corresponding author. E-mail: lohmercy@yahoo.fr. oils contain mostly linoleic (an essential fatty acid which can only be got from the diet) followed by oleic acid (Silou et al., 1999; Murkovic et al., 1996; Younis et al., 2000; Achu et al., 2005; Achu, 2006). Essential fatty acids are important for normal foetal and infant growth and deve- lopment, brain development and visual acuity (FAO, 1994). The other essential fatty acids; linolenic acid, can be got from linoleic acid, and arachidonic acid from lino- lenic acid. Arachidonic acid is a component of the liver, plasma phospholipids and cholesterol esters. It is an important precursor of prostaglandins, thrombosanes and prostacyclins. Prostaglandins stimulate contraction of smooth muscles. Prostacyclins have an antagonistic effect to that of thrombosanes by inhibiting platelet aggre- gation, relaxing coronary arteries and lowering blood pressure (Ottaway and Apps, 1984). Linoleic acid (poly- unsaturated) moderately reduces serum cholesterol and LDL levels. Oleic acid (monounsaturated) appears to be Achu et al. 055 neutral in regard to LDL but modestly raises HDL (FAO, 1994). There is little data on the atherogenicity of oils from Cucurbit seeds cultivated in Cameroon. The main objective of the study was to investigate the atherogenic property of oils extracted from Cucumeropsis mannii and Cucumis sativus seeds from Cameroon which can be exploited at the alimentary levels. This was in order to determine the levels of serum triglycerides, total choles- terol (TC), high density (HDL) and low density (LDL) lipo- protein cholesterol, atherogenic ratio (AR) and proteins of rats fed with oils extracted from the seeds of C. mannii (egusi melon) and C. sativus (“Ibo” egusi) seeds com- pared to rats fed with palm and corn oils diets. MATERIALS AND METHODS Sample collection and treatment C. mannii seeds were collected from Ebolowa (in the South Province) and C. sativus seeds from Bafia (in the Centre Province), which are amongst the regions of great cultivation of these seeds in Cameroon. The seeds were bought already sun-dried by the farmers, transported in polyethylene bags to the laboratory, wiped with filter paper and dried in an Oven at 70°C to constant weight. They were ground in an electric grinder, put in airtight bottles and stored in the desiccator for analyses. Extraction of the oils Oils were extracted from the ground seeds by continuous extraction in a Soxhlet apparatus for 8 h using hexane as solvent (AOAC, 1980). The hexane was evaporated on a rotary evaporator and the oil obtained was dried in an oven at 60°C for 24 h to remove all traces of solvent. The Experimental design The experimental design was 4 types of oils tested x 4 groups of rats x 5 rats per group. That is: 4 oils x 4 groups = 4 x 4 groups. Formulation of the diets given to rats The diet was formulated by weighing the various components according to the American Institute of Nutrition-76 (AIN-76) compo- sition, modified for casein level as follows: casein 10%, starch 36%, -cellulose 5%, vitamin mixture 1%, choline chloride 0.1%, DL- methionine 0,3%, sucrose 27.6%, salt mixture 5%, oil 5% (Oil = corn oil, palm oil, oil extracted from C. mannii and C. sativus seeds). This diet was mixed with 10% of water to moisten the food for easy consumption by the rats. The food was given to the rats in the form of a paste of 20 g of food per rat. Treatment of the rats 20 wistar albino rats of 6 weeks old weighing 58 – 65 g were used. One rat was put in a metabolic cage for an adaptation period of 5 days. During this time, they received the same food (that is, the food that was consumed by rats of the corn oil group which is the corn oil diet) and water ad libitum. They were then weighed and randomly distributed into 4 groups of 5 rats each, receiving 4 types of food differing only by the type of oil (corn oil and palm oil for the control groups and C. mannii and C. sativus oils for the experi- mental or test groups). The feeding took 3 months. This was to allow enough time to follow up the effect of the oils tested on the metabolism of the rats. The rats were weighed every 3 days. 12 h before sacrifice, these rats were left to fast. They only took water. On the morning of the sacrifice, they were weighed and sacrificed by decapitation. The blood of each rat was collected into dry 10 ml test tubes and centrifuged at 3000 rpm/min for 5 min. The separated serum was collected into 2 ml ependoff tubes. The serum was divided into 2 parts. The first part was preserved in aliquots at 4°C for HDL cholesterol analysis and the second part at -28°C for the rest of the analysis. The liver of each rat was equally collected and weighed in order to see the effect of the oils tested on the size of the liver. Analysis of serum samples Triglycerides, total and HDL cholesterol were assayed enzyma- tically according to the method on the Randox kits used, while LDL levels were calculated using the formula of Friedewald et al. (1972). The AR ratio was also calculated and the serum protein levels were assayed according to the method of Gornall et al. (1949). Statistical analysis The Kruskal-Wallis test (for the distribution of the sample as shown by the Kolmogorov test was non normal) was used to find differences between the parameters measured in the rats that received diets prepared with the different oils. The Student- Newman- Keuls (S-N-K) test was used to locate these differences. The tests were done at 5% level of significance, using SPSS 10.1. RESULTS Table 1 shows the amounts of food ingested, the evolu- tion of the body weights of the rats and the ratio, weight of liver/weight of rat. The amounts of food ingested per rat per day ranges from 9.68 (corn oil) to 11.06g ( C. mannii oil). At the start of the experiment, the weights of the rats ranged from 58.28 (C. sativus oil) to 64.91 g (C. mannii). At the end of the experiment, they ranged from 139 (C. sativus) to 151.07g (C. mannii). There is an increase in weight in all the groups with a gain of 80 (palm oil) to 90.52 g (corn oil) amounting to 128.65% (palm oil), 132.75% (C. mannii), 140.8% (C. sativus) and 153. 45% (corn oil), showing no significant difference. The weights of the livers range from 4.22 (palm oil) to 5.17g (corn oil), ratio of weights of liver to that of rat, from 0.029 (palm and C. mannii oils) to 0.034 (corn oil). There is no significant difference between the food ingested and the weights of these rats that received diets prepared with the different types of oils. Table 2 shows the serum levels of triglycerides (TG), total cholesterol (TC), HDL and LDL cholesterol, athero- genic ratio (AR) and serum proteins. No significant differ- rences were observed in the total TG levels among the four groups. However, total serum cholesterol was significantly higher in the rats on C. sativus compared to the rats on corn oil diet. There was no significant differ- rence in serum HDL cholesterol among palm oil and C. 056 Afr. J. Food Sci. Res. Table 1. Evolution of body weights of rats and the ratio, weight of liver/weight of rat. Food Initial weight Final weight weight gain Weight of Liver/rat Groups ingested % weight gain of rats (g) of rats (g) (g) liver (g) weight (g/rat/day) Corn oil 9.68 ± 2.58 58.89± 3.82 149.40± 19.03 90.52± 16.67 153.45± 24.68 5.17± 1.26 0.034± 0.005 Palm oil 9.77 ± 1.93 63.06 ± 3.36 143.73 ± 6.11 80.66 ± 8.36 128.65 ± 19.09 4.22 ± 0.37 0.029± 0.002 C. sativus oil 10± 1.67 58.28 ± 5.23 139.85 ± 6.37 81.57± 2.83 140.80± 12.58 4.42 ± 0.26 0.032 ± 0.001 C. mannii oil 11.06 ± 1.72 64.91 ± 3.17 151.07± 12.86 86.16 ± 11.49 132.75 ± 16.76 4.32 ± 0.28 0.029± 0.002 No significant differences were observed between groups in the amount of food ingested and in the total weight gain. Table 2. Serum levels of triglycerides, total cholesterol, HDL and LDL cholesterol, atherogenic ratio and serum proteins. 0 Triglycerides Total HDL LDL Atherogenic 0 Total Groups (mg/dl) Cholesterol Cholesterol Cholesterol Ratio, Proteins (g/l) (mg/dl) (mg/dl) (mg/dl) (LDL/HDL) Corn oil 76.2± 12.66 49.6± 4.93 b 25.92± 2.52 ab 8.44± 1.14 b 0.33± 0.05 b 79.04 ± 13.4 Palm oil 73 ± 10.2 53.6± 7.99 ab 32.8± 6.53 a 6.2 ± 4.57 b 0.20± 0.13 b 81.57± 8.01 C. sativus oil 79.4 ± 10.06 64.2± 8.64 a 27.72± 4.18 ab 20.6± 8.65 a 0.77± 0.38 a 78.17± 11.83 C. mannii oil 75 ± 6.96 59 ± 6.82 ab 18.94± 7.14 b 25.06± 7.25 a 1.61 ± 1.05 a 84.71±15.37 Kruskal-Wallis test: 0 = there is no significant difference (p>0.05) between values in the same column. Student-Newman-Keuls test: Values in the same column with different letter superscripts are significantly different (p<0.05). sativus oil. However, HDL cholesterol was significantly higher in the rats on palm oil compared to those on C. mannii oil (p<0.05). LDL cholesterol was significantly higher in the rats on C. mannii and C. sativus oils com- pared to the control diets, corn oil and palm oil. Similarly the atherogenic ratio (AR) was significantly higher in the rats on C. mannii and C. sativus oils compared to corn and palm oil diets. Total serum protein was not signify- cantly different among groups. DISCUSSIONS The triglyceride (TG) levels were 75 in C. mannii and 79.4 in C. sativus oil rats compared to 73 in palm oil and 76.2mg/dl in corn oil rats, but with no significant differ- rence. However, from the results of analysis of the quality of C. mannii and C. sativus oils, C. sativus oil shows lower levels of free fatty acids (acid index: C. sativus oil = 1.76 and C. mannii oil = 4.56), higher degree of unsatu- ration (iodine index: C. sativus oil = 114.4 and C. mannii oil = 106.78; unsaturated fatty acids: C. sativus oil = 78.4% and C. mannii oil = 54.7%; saturated fatty acids or SFA: C. sativus oil = 21.7% and C. mannii oil = 42%), lower level of peroxides (peroxide index: C. sativus oil = 3.31 and C. mannii oil = 13.68), lower R1 value (R1 is the ratio of the sum of saturated to that of unsaturated fatty acids. The R1 of C. sativus oil = 0.28 and C. mannii oil = 0.77) and lower linolenic acid level (linolenic acid: C. sativus oil = 0.1% and C. mannii oil = 0.3%) (Achu, 2006). These suggest that the quality of C. sativus oil is better than that of C. mannii oil and that C. sativus oil contains more unsaturated fatty acids than C. mannii oil. C. sativus and C. mannii oils have high levels of polyun- saturated fatty acids, PUFA (39.3% in C. mannii oil and 62.2% in C. sativus oil) as in corn oil (58.5%) while palm oil has a low level (8.5%) (Table 3). These results are similar to those of Noubissi in Djamen (1998), who after 2 months of experiment, found a non significant decrease in the TG level of rats fed with palm oil diet (rich in SFA) compared to those fed with cotton oil diet (poor in SFA). However, these results show that these egusi oils can be good edible oils (do not cause hypertriglyceridaemia) because their TG levels which are 75 in C. mannii oil and 79.4 mg/dl in C. sativus oil rats, are far below 150 mg/dl, the borderline level of TG, above which the person has hypertriglyceridaemia (Randox Assayed Multisera, 2004b). The total cholesterol (TC) level of C. sativus oil (64.2) is similar to those of C. mannii oil (59) and palm oil (53.6) but significantly higher than that of corn oil rats (49.6mg/dl). C. sativus and corn oils all have high levels of PUFA which are 62.2 and 58.5% respectively, due to their high linoleic acid levels. But C. sativus oil has higher levels of SFA (21.7%) than corn oil (16.5%) (Table 3). Hence, the high TC level in C. sativus oil compared to corn oil rats may be due to its higher SFA levels. However, the TC levels of all these four groups of rats were low when compared to levels that cause hypercho- lesterolaemia. Secondly, other studies have shown that the hypercholesterolaemic effect of SFA is mostly due to C14:0 and C12:0 fatty acids rather than C16:0 (Ng, 1994). C. sativus and C. mannii oils contain little or no Achu et al. 057 Table 3. Fatty acid composition of oils used. Corn oil Palm oil C. sativus oil C. mannii oil (Achu, Fatty Acid Symbol (Ngogang et al., (FAO, 1981) (Achu, 2006) 2006) 1996) Caprylic acid C8:0 - - - - Capric acid C10:0 - - - - Lauric acid C12:0 - - - 1.5 Myristic acid C14:0 1 3.5 - 4.7 Palmitic acid C16:0 12 40.6 10.7 24.4 Palmitoleic acid C16:1 0.5 - - 0.8 Stearic acid C18:0 2 4 10.6 11.2 Oleic acid C18:1 24 43 16.2 14.6 Linoleic acid C18:2 56.5 8.5 61.8 38.7 Linolenic acid C18:3 2 - 0.1 0.3 Arachidonic acid C20:0 1 - 0.4 0.2 Gadoleic acid C20:1 0.5 - - - Arachidonic acid C20:2 - - 0.3 0.3 Behenic acid C22:0 - - - - Lignoceric acid C24:0 0.5 - - - Total Saturated fatty acids 16.5 48.1 21.7 42 Total MUFA 25 43.4 16.2 15.4 Total PUFA 58.5 8.5 62.2 39.3 Total unsaturated fatty acids 83.5 51.9 78.4 54.7 PUFA/SFA (R2) 3.55 0.17 2.87 0.93 = Not found, MUFA = monounsaturated fatty acids, PUFA = poly unsaturated fatty acids. C14:0 and C12:0 fatty acids. Their SFA are mostly C16:0 as in corn and palm oils (Table 3) which do not lead to much rise in cholesterol levels. Also, the non-hypercho- lesterolamic action of these oils is possibly due to their presence of tocotrienols, the unsaturated analogue of tocopherols. Tocotrienols inhibit cholesterol synthesis in vivo, exerting a hypocholesterolamic action in humans and animals. This has been shown in palm oil by Rukmini (1994). However, these results show that these oils are good edible oils (do not cause hypercholesterolaemia) because their TC levels (59 in C. mannii oil and 64.2 mg/dl in C. sativus ) are far below 200 mg/dl, the desirable blood cholesterol level, above which the person has hypercholesterolaemia (Randox, 2004a). The HDL level of C. sativus (27.72) is similar to those of C. mannii (18.94), corn oil (25.92) and palm oil (32.8 mg/dl) rats. The HDL level of C. mannii oil is similar to those of C. sativus and corn oil rats but significantly lower (p<0.05) than that of palm oil rats (Table 2) . This lower HDL value in C. mannii compared to palm oil rats may be due to the lower level of MUFA in C. mannii (15.4%) than palm oil (43.4%) and higher PUFA in C. mannii (39.3%) than palm oil (8.5%) (Table 3) . This is in line with the results of a study carried out by O’Callaghan et al. in During et al. (2000) who found that patients fed PUFA dairy products showed lower plasma HDL levels than patients fed MUFA dairy products. This might be the case with these oils where C. mannii had higher PUFA, lower MUFA and showed lower HDL levels than palm oil with lower PUFA, higher MUFA and high HDL levels. The LDL level of C. mannii oil rats (25.06) is similar to that of C. sativus (20.6) and these are significantly higher than those of corn (8.44) and palm (6.2) oil rats, with similar LDL levels. Apart from increasing HDL levels, high MUFA also reduce LDL levels. This high LDL level and the eventual higher atherogenic ratio in C. mannii and C. sativus oils may be due to their lower MUFA and higher PUFA levels as opposed to corn and palm oil rats. This is seen in the study carried out by During et al. (2000) where rats fed with experimental cheeses containing mostly MUFA resulted in a significant increase of HDL- cholesterol (11%) and a significant reduction of LDL- cholesterol (31%). Other findings have shown that, in addition to unsaturated TG, dietary phytosterols in vege- table oils can also reduce plasma LDL levels (Jones et al. in During et al., 2000). Hence, high HDL and low LDL levels are due to high MUFA (low PUFA) and dietary phytosterols (as in palm oil) and low HDL and high LDL levels are due to low MUFA, high PUFA and possibly low phytosterols (as in C. mannii and C. sativus oils). However, the HDL levels of C. mannii (18.94) and C. sativus oil rats (27.72mg/dl) are below the desirable HDL level (>40 mg/dl) and their LDL levels 25.06 and 20.6 mg/dl respectively are far below the borderline (<130 058 Afr. J. Food Sci. Res. mg/dl) (American Academy of Family Physicians, 2005). This means that these oils could be very good in reducing LDL and to a lesser extent, raise HDL levels. However, these oils need to be tested on humans in order to better confirm these findings that were carried out on wistar albino rats. The atherogenic ratio, AR (LDL/HDL) in C. mannii oil rats (1.61) is similar to that of C. sativus (0.77) and these are significantly higher (p<0.05) than those of corn (0.33) and palm (0.2) oil rats with similar AR levels. These high AR levels in C. mannii and C. sativus oil rats are due to their high LDL levels, caused by their low MUFA levels. Although the atherogenic ratio is higher in the test groups (than the palm oil and corn oil groups), these ratios are good, for they are below 3.55 in men and 3.22 in women, the borderline levels for lipids to cause atherosclerosis. Above these values, the risk of appearance of cardiovascular illness is high (Laboratoires Fournier, 1981). The serum protein levels are 84.71 g/l in C. mannii and 78.17 in C. sativus oil rats compared to 79.04 in corn and 81.57 g/l in palm oil rats but with no significant difference in all these values. Conclusion This study which was aimed at investigating the athero- genic property of oils extracted from C. mannii and C. sativus seeds from Cameroon showed that these oils are good in reducing LDL cholesterol levels thereby reducing the atherogenic ratio, for the AR levels were far below the threshold values expected to cause athero-sclerosis. 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