Consumption of Cucurbitaceae Seeds Oils is Better in Risk of Cardiovasular Disease Prevention on Rats Than The Consumption of Re ned Palm Oil

Howélé OUATTARA (  ouattarahowele@gmail.com ) Peleforo Gon Coulibaly University: Universite Peleforo Gon Coulibaly https://orcid.org/0000-0003-1400-8241 Albert Atsamo University of Yaounde I: Universite de Yaounde I Bazoumanan Ouattara Peleforo Gon Coulibaly University: Universite Peleforo Gon Coulibaly Vazoumana Koné Peleforo Gon Coulibaly University: Universite Peleforo Gon Coulibaly Séraphin Kati-Coulibaly University of Felix Houphouet-Boigny: Universite Felix Houphouet-Boigny


Introduction
Five species of Cucurbitaceae, improperly called «pistaches» in French or «pistachio» in English, are found in Côte d'Ivoire. These species are Lagenaria siceraria (Molina), Citrullus lanatus (Thunb), Cucubita moschata (Duchesne), Cucumeropsis mannii (Naudin) and Curcumis melo (Subsp) [1]. Among them, the most important, because of its economic role, are Citrullus lanatus (Thunb), Lagenaria siceraria (Molina) and Cucumeropsis mannii (Naudin). The names of these cucurbitaceae, in Senoufo the local language in the North of Côte d'Ivoire where they are most cultiviated, are «Taho or Pkèlègué » for Lagenaria siceraria, « Woilai » for Citrullus lanatus and « Fonguelé » for Cucumeropsis mannii. According to Zoro Bi et al. [2], the cost of cucurbitaceae seeds was 1500 FCFA per kilogramme of seeds in the year 2006. The price is at present 2000F CFA per kilogramme of seeds. The seeds are consumed after being prepared in sauce in several regions of Africa especially during celebrations. They are sources of energy, proteins, vitamins, trace element and particularly they are high sources of lipids [3,4,5,6,7,8] which value can be increased of. Unfortunately, cucurbitaceae seeds oils (CSO) are neglected in favour of palm oil, soya been oil, colza oil, sun ower oil, palm seed oil, cottonseed oil, groundnut oil, coconut oil and olive oil which degree of consumption in the world are 33 %, 27 %, 16 %, 10 %, 4 %, 3 %, 3 %, 2 % and 2 % respectively [9]. In Côte d'Ivoire, vegetables oils used in human diet are produced from shea seed, cotton seed, groundnut and palm tree. According to the quantity of palm oil production in a year, Côte d'Ivoire held the 10 th place in the world and the second place after Nigeria in Africa [10]. Palm oil is the most oil produced in the world [11,12]. It is also the rst oil produced and used in Côte d'Ivoire. Nowadays, the consumption of palm oil is often questioned for various reasons : on the one hand, its consumption has effects on cardiovascular health [13,14,15] because of its high saturated fatty acid contents which are palmitic fatty acid and stearic fatty acid. On the other hand, it threatens food security due to the possible accrual of pollutants during re ning; and nally, there is the debate on the sustainability of its cultivation in the countries of origin [16]. The food and nutrition sectors are increasingly adopting a more holistic view, which encompasses not only health, but also environmental protection and traceability. And yet, all ecologists are in agreement to say that industry of palm oil production destroyed forests and enhanced carbon dioxide [17]. Considering the castigation of palm oil, it appeared important to look for other sources of oils available which do not enhance environment pollution and which contain few saturated fatty acids in particular few palmitic acids. CSO can be an alternative solution, in Côte d'Ivoire, if the comparison of the effects of these oils give satisfactory results concerning the decrease of the Oils extraction Oils were extracted in the mixture of each seed species using an electric grinder. The powder of each seed species was put in an expanded polysterene bag and squeezed with a mechanical press for four hours. Oil which owed out was caught in a can with a help of a funnel. At the end, the different oils obtained were ltered three times using thin sieves. These oils, shown on gure 1, were utilized for differents studies.

Physicochemical parameters determination
The acid and peroxide values were determined by the method of Devine and Williams [18]. The iodine value was obtained by the method of Strong and Koch [19]. Speci c gravity was determined by the ratio of the mass of equal volume of cucurbitaceae oil and water both kept at a temperature of 20°C using a pycnometer according to the formula : (m 2 -m 0 )/(m 1 -m 0 ) with m 0 : mass of empty pycnometer, m 2 : mass of pycnometer full of oil and m 1 : mass of pycnometer full of water.
Characterization of CSO using High Performance Liquid Chromatography method After each CSO was extracted, total fat acid and sterol composition were determined by molecular characterization. The characterization of total fatty acid was made in two steps. Firstly, compounds were separated using a High Performance Liquid Chromatography (Thermo sher, France) and secondly the molecules separated were analyzed using also High Performance Liquid Chromatography (Thermo sher, France) as peace of apparatus. Sterol compounds were determinated in three stages. Compounds which cannot be saponi ed were, in the rst time, extracted. In the second time, they were isolated using thin coat silica gel chromatography method. At last, they were measured out using gaseous phase chromatography (chromatograph, GC8000).

Formulation of diets used in the experimentation
Four diets differing by the kind of oil, re ned palm oil (RPO) or Citrullus lanatus seeds oil (HCL) or Lagenaria siceraria seeds oil (HLS)or Cucumeropsis mannii seeds oil (HCM), were prepared. Each diet was composed by the mixture of 45.9 % of maize starch, 10 % of sugar cane, 5 % of agar agar used as dietary bre, 18 % of lipids which were RPO or HCL or HLS or HCM, 13 % of casein used as proteins sources, 3.5 % of mineral mixture and 1 % of vitamins mixture. All the diets were made as recommended by the American Institute of Nutrition according to Reeves et al. [20]. The study was absout young rats. In table 1, it is shown the differents percentages of ingredients used. Preliminary work before the beginning of the experimentation Twenty four young albino Wistar rats with a mean weight of 51,5±6,6 g were used. At the beginning of the experimentation, animals were teamed in six homogeneous young groups of rats containing three males and three females. They were put individually in cages and maintained under standard laboratory conditions (temperature 25±2 °C) with dark and light cycle (12h/12h). In the cages, rats were acclimatized to this condition and fed with the diet which will be utilized as experimentation diet during the duration of the experimentation and the ve days before the beginning of the real experimentation.

Experimentation
After that, each group of rats constituted was fed ad libitum with one of the different diets formulated which are « diet RHDI » for RPO oil, « diet RHCL » in which CSO oil was used, « diet RHLS » that included HLS oil and « diet RHCM » for HCM oil for a 45 days diet. The food served was made in paste by adding to it a quantity of water in order to minimize the waste.
Every day, during the experimentation, when it was time to feed the rats, each kind of food which are, « diet RHDI », « diet RHCL », « diet RHLS » and « diet RHCM », was made in paste by adding to it a quantity of water clearly determined in order to minimize the waste. After that, a quantity of each mashed food made, was weighted and was given to each animal according to the group. Little mashed of the different food was, every day, weighted after being dried during 4 hours in an oven (MMM Medcenter Gmbh (D-82152, Munich, Germany) at 105°C and the weight obtained was written in a notebook. Then, with this sample the dry matter in each food given to animals can be calculated. The following day, before distributing the diets, the rests of food given the day before were separately collected and were weighted after being dried during 4 hours in an oven at 105°C. The different weights obtained were also written in a notebook. This methodology permitted us to determine the total dry matter consumed every day by each animal which is the difference between the dry matter of food given the day before and the rest collected and dry the following day. Then, the total dry matter of food consumed by each group during the time of the experiment (45 days) is obtained by the summation of the dry matter consumed per day by each rat of the group during the 45 days. The mean Dry Matter Ingested every day (DMI/d) by each animal is obtained by the difference between Total Dry Matter of food consumed divided by 45.

Calculation of the Mean Weight Gain
The mean weight gain of each rat of each group during the time of the experimentation was calculated. The weight gain of each rat is obtained by the difference between the nal body weight of a rat and the initial body weight of the same rat.

Removal of organs
At the end of the experimentation, animals were anaesthetized with chloroform and after that were sacri ced. Heart, liver, kidney, spleen, stomach and lung were removed and weighted. The mean weight of each kind of viscera according to the group was calculated. The different means weight obtained were after that compared.

Hematological parameters
Also, at the end of the experimentation, blood sample was collected at the vena cava level of all animal and put in individually tube containing Ethylene Diamine Tetra Acetic (EDTA). These blood samples were used for full blood count. Then, hematological parameters including hemoglobin content, total count of red blood cells (RBC) and white blood cells (WBC), differential count of leukocytes such as granulocyte (%), lymphocyte (%), monocyte (%), hematocrit (Hct), mean cell volume (MCV), mean corpuscular hemoglobin concentration (MCHC) and platelet count were measured using an automatic hematological analyzer (Symex-kx-21N).

Statistical analysis
The experimental results were expressed mean and standard error (mean ± standard error). Data were assessed by the method of analysis of ANOVA and Newman-Keuls test thanks to STATISTICA 6.0 Software. The level p ≤ 0.05 was considered as the cut-off value for signi cance. Curves were drawn using Microsoft Execl 2010.

Results And Discussion
Physico-chemal parameters of the differents oils The acid index of the three cucurbitaceae oils are signi cantly higher (p ≤ 0.01) than that of RPO. But, there is signi cant difference (p ≤ 0.05) among the acid index of the three cucurbitaceae oils. In fact, HLS has an acid index signi cantly lower (p ≤ 0.05) than that of HCL and that of HCM and HCL acid index is signi cantly lower (p ≤ 0.05) than that of HCM.
Iodine value of HCL is signi cantly higher (p ≤ 0.01) than that of HCM which in turn is signi cantly higher (p ≤ 0.01) than that of HLS. Iodine value of RPO value is signi cantly higher (p ≤ 0.01) than that of HLS but it is signi cantly lower (p ≤ 0.05) than that of HCL and it is slightly lower (p > 0.05) than that of HCM.
Peroxide index of HCL and HCM are signi cantly lower (p ≤ 0.01) than that of RPO and HLS. That of HCL is also very lower (p ≤ 0.01) than that of HCM. On the other hand, that of RPO is higher (p ≤ 0.05) than that of HLS.
Speci c gravity of HCM is almost the same with that of RPO. The Speci c gravity of HCM and RPO are higher (p ≤ 0.05) than that of HCL and lower than (p ≤ 0.05) that of HLS.
All these results are shown on table 2 The results of the characterization are shown that Citrullus lanatus seeds oil (HCL) and Lagenaria siceraria seeds oil (HLS) contain high quantity of lauric acid. The percentage of lauric acid found in these oils is up above 15 % of the total component. The quantity of lauric acid found in Cucumeropsis mannii seeds oils (HCM) is the half of that found in Citrullus lanatus seeds oil and Lagenaria siceraria seeds oil when lauric acid found in re ned palm oil do not reach 1 % of it total component.
Among the experiment oils, re ned palm oil is the most saturated followed respectively by Cucumeropsis mannii seeds oil, Lagenaria siceraria seeds oil and Citrullus lanatus seeds oil. Re ned palm oil is also the most rich in palmitic acid and stearic acid among the four oils. Citrullus lanatus seeds oil contain more polyinsaturated fatty acids than the others oils and re ned oil is the most lower in polyinsaturated fatty acids. All these results are shown in table 3.  Growth performance of rats which are consumed the different diets differing by the kind of oil used are shown on gure 2. On this gure, it is shown a rapid growth of rats whatever the kind of oil consumed. However, this increase is higher in case of Lagenaria siceraria seeds oil consumed than the other oils which have practically the same in uence on rats growth.

Evaluation of parameters in relation with diets consumption
Dry matter ingested by rats fed with diet RHDI is signi cantly low (p < 0.05) than that of rats fed with diet RHCL and high signi cantly low (p < 0.01) than that of rats fed with diet RHLS. On the other hand, dry matter calculated in case of diet RHDI consumed is high than that of rats fed with diet RHCM. Dry matter ingested by rats fed with diet RHCL is virtually the same with that of rats fed with diet RHLS.     7).

Leucocytic bloodline parameters
White Blood Cells (WBC) White blood cells count on blood sample of rats fed with diets RHDI, RHLS, RHCL and RHCM are not signi cantly different (p > 0.05) to each other (table 7).

Neutrophil
The number of neutrophil in blood sample of rats fed with diets RHDI, RHLS, RHCL and RHCM are not signi cantly different (p > 0.05) to each other (table 7).

Eosinophil
The number of neutrophil in blood sample of rats fed with diets RHDI, RHLS and RHCL are not signi cantly different (p > 0.05) to each other. On the other hand, there are signi cantly high (p ≤ 0.01) compared to that of rats fed with diet RHCM (table 7).

Monocytes
The number of monocytes in blood sample of rats fed with diets RHDI, RHLS and RHCM are not signi cantly different (p > 0.05) to each other. On the other hand, there are signi cantly high (p ≤ 0.01) compared to that of rats fed with diet (table 7).

Lymphocytes
The number of neutrophil in blood sample of rats fed with diets RHDI, RHLS, RHCL and RHCM are not signi cantly different (p > 0.05) to each other (table 7).

Platelet
The detailed account of platelet in blood sample of rats fed with the differents diets are not signi cantly different (p > 0.05) to each other (table 7).

Discussion
According to the acid index determined, it's appeared that RPO contain low free fatty acid than LSO which in turn contain low free fatty acid than HCL. CMO is the oil which contain the most quantity of free fatty acid among the differents oils used in our study. Compare the quantity of soda used to neutralize 1 g of free fatty contain in the differents oils to the standard norm of Codex alimentarius (Maximum of 6.6 mg of NaOH/g) and the International Olive Council (Maximum 4 mg of NaOH/g of oil), RPO and LSO are in the norm suggesting that these two oils can be consumed without any risk of damage on health but not the others. But, acid index value obtained are lower than the minimum safe limit (15%) mean for consumption. This suggests that these oils have low deteriorating rate and can therefore be stored for relatevely long period [21].
According to the results obtained, the ranking of the different oils per low peroxide index value is HCL, CMO, LSO and RPO. That mean that the degree of the oxidation of unsaturated fatty acids contain in RPO is higher than that contain in LSO which is also higher than that contain in LSO and which in turn is lower than that contain in RPO [22,23]. Then, RPO is the oil in which unsaturated fatty acids are the most oxidized. That proves that, cucurbitaceae oils are good for consumption.
Norm of Codex alimentarius and the International Olive Council standard about peroxide index are xed to a maximum level of 20 meq O 2 /Kg of oil [24]. All our study sample have a peroxide index value which are in accordance with the commercial norm. This testify that the methodology we used to make extraction of the cucurbitaceae oils do not provoke major oxidation of these oils. Also, thiat mean that the time of storage do enhance the raise of rancidity [25].  [29] are found a quantity of 71 % of unsaturated fatty acid in HCM from Soudan. The different of composition can depend of the type of ground and the pluviometry. In fact, it is know that the factor of the variation in nutrient content in crops depend to the cultivation environment [30].
The quantity of dry matter ingested by rats which are consumed diet RHCL and diet RHLS were pratically the same but were superior than that of rats which consumed diet RHDI and diet RHCM. Diet RHCL and diet RHLS may have the same appetence due probably to their good arome and avour which may be better than that of diets RHDI and RHCM. In fact, according to several researchers [31,32], food consumption depend to several factor such as physiological state of the organism and also the characteristics of the food which are their arome, their avour and their chemical composition. It is observed a low dry matter ingested in our study than the result of the study made by Meite et al. [33] when they utilized defatted cake of Citrillus lanatus to make bread which served to feed rats. That is due to the fact that bread make contain low quantity of lipids which are food constituent the most energetic.
Mean body weight are in the same way with the dry matter ingested. In any case, mean alimentary e cacy coe cient calculated were not signi cantly different (p > 0,05) because the differents food have the same constituent and group of animals constituted were in the same physiological state. The difference beetwen food was the kind of oil. Even if the different oil used do not have the same arome and avour, these characteristics do not impact on the differents mean alimentary e cacy coe cient.
Whatever the diet consumed, the weight of viscera was not signi cantly different to each other suggesting that they were not in a situation of pathology or if they were in pathologies situation, the different diets are caused the same pathologies and their damage provoke are equal.
There is certainly some difference beetween glyceamia measured on blood sample of the different group of rats but all these glyceamia may be normal because according to Durimel et al. [34] normal glycaemia on rat are between 0.7 g/l and 1.2 g/l and glyceamia measured in our study are between 0,60±0,39 g/l and 0,99±0,12 g/l. Then, these diets do not provoke mayor disruption on carbohydrate metabolism. Moreover, when we observed the standard deviation, these diets can be able to reduce glyceamia. The reduction of glycaemia could be a good thing for person who suffered from hypoglycaemia attact.
The fact that plasmatic calcium and phosphorus are not signi cantly different (p > 0.05) to each other explained similar bone metabolism whatever the diet consumed.
According to the plasma sodium level measured, we can believe that consumption of re ned palm oil is associated to low risk of cardiovascular disease than cucurbitaceae oils. In any case, seeing that oils are used in our experimentation beyond the norm, lipidic parameters are the main factor to estimate the cardiovascular risk. Values of lipid parameters (triglycerides, total cholesterol, LDL-cholesterol and HDL-cholesterol) measured in blood sample are di cult to explain. On the other hand, atherogenicity index calculation give the better way to evaluate risk of cardiovascular disease and obesity [35,36,37]. Then, according to atherogenicity index calculation, the consumption of diet RHLS may lead to low risk of cardiovascular disease than diet RHCL consumption and diet RHCL consumption in turn may lead to low risk of cardiovascular disease than diets RHDI and RHCM. At nisf, the rank of inducing cardiovascular disease highly is RHCM, RHDI, RHCL and RHLS. So, RPO come in the rd position, only befor HLS in the risk of inducing cardiovascular disease. We know that one of the arguments underpinning the questioning of palm oil healthfulness is its saturated fatty acids content, and speci cally the relationship of palmitic acid to cardiovascular health [38,39]. And in our work, the molecular characterization show that palmitic acid content in RPO is very higher than that found in all CSO. That is an argument which indicated that RPO may induce cardiovascular disease than all CSO used in our study.
Urea and creatinine measured on blood sample of the different group of rats do not show any signi cant difference (p > 0.05) when compared to each other suggesting that the functioning of kidneys is good whatever the diet consumed [40,41]. These results are corroborated by the differents kidneys weight which are not signi cantly differents to each other whatever the diet consumed.
TGO and TGP level on blood sample of rats fed with diets RHDI and RHCL are signi cantly higher (p ≤ 0.05) than that of rats fed with diets RHCM and RHLS.
That can explain a high risk of liver disease due to consumption of diets RHDI and RHCL than the consumption of diets RHCM and RHLS. HCL was recognized in previous study made by Madhavi et al. [42] for it protector effects on liver.
The fact that hematological parameters measured on rats fed with the different diets were not signi cantly different (p>0.05) from each other imply that medullar production of erythrocyte, leucocyte and platelet was similar on bone marrow cells of the different rats whatever the diet consumed.

Conclusion
We can notice that, in spite of no technological treatment done on «pistachio» oils, are better than RPO in reducing cardiovascular disease risk. Therefore, Pistachio oils are constituted a good alternative oils to replace palm oil in human diet if their extraction is made with cold-pressed as we do it. If cucurbitaceae seeds is used in industry of oil for human diet, the sector of cucurbit seeds could be an important activity for agriculture in Côte d'Ivoire. Another studies can be made in order to determinate their insaponi able (vitamins, antioxidant, carotin) content. Also, deodorization can permit to add some nutritioal value on these oils. author's while Kone Bazoumanan, a student, was a help in making the experimentation. All the authors contributed to data interpretation and drafting/revising the article for important intellectual content. All authors approved the nal version to be published.