Chemical composition and bioactive compounds of Cucurbitaceae seeds: Potential sources for new trends of plant oils

2. Materials and methods 
All reagents used in the assessment procedure were of analytical grade or HPLC 
grade and were supplied by Merck (Darmstadt, Germany). 
2.1. Raw material 
Seeds of pumpkin (Cucurbita pepo) of variety “Essahli”, of watermelon (Citrullus 
lanatus) of variety “Ananas”, and of melon (Cucumis melo) of variety “Ananas” were used. 
Approximately 4 kg of each Cucurbitaceae variety were brought from a local market in 
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Chebika region (latitude 35°37’38’’; longitude 10°2.15’38’’; elevation 86 m), located in the 
south east of Tunisia. The seeds were first selected, and only the ones that were completely 
and totally formed were used, washed to remove impurities, and eventually air-dried in a 
forced circulation oven at 40 ± 0.5 °C for 48 h and stored in glass jars.
2.2. Chemical composition of Cucurbitaceae seeds 
2.2.1. Moisture and protein content 
Moisture was determined according to (AOAC, 1990) whereas the protein content was 
calculated on the basis of the nitrogen content as follows: N (%) × 6.25 using Kjeldahl’s 
method. 
2.2.2. Ash and mineral content 
Ash was determined after mineralisation of the sample seeds at 600° C for 8h. The 
mineral content was determined by atomic absorption spectrophotometer (Varian 220 FS, 
Belgium). Potassium and sodium were both assayed by flame emission spectrophotometer (IL 
151), whereas the phosphorus content was determined using the molybdenum-blue method 
(Hwang, 1989). 
2.2.3. Fat, fibre, and total sugar contents 
 
The fat content was measured by using a Soxhlet extraction apparatus with petroleum 
ether as a solvent for 8h. Crude fibre was determined by the acid detergent fibre method (Van 
Soest et al., 1991). Total sugars (TS) were calculated as follows: TS= 100 – (% moisture + 
%protein+ %ash+ %fibre +%fat). 
2.3. Oil extraction 
Pumpkin, watermelon, and melon seed oils were obtained by cold pressing using a 
Komet DD 85 G vegetable oil screw press (IBG MonfortsOekotec GmbH & Co. KG, 
Mönchengladbach, Germany). This technique obviously provides a cold extraction of the 
solid sample oil contained within a plant hopper. The oil extraction was carried out when the 
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Cucurbitaceae seeds (2 kg for each studied variety) were ground and squeezed under the 
pressure that was exerted by the conical screw rotation. The oil was forced into the perforated 
tube. The meal was then evacuated at the end of the shaft by a calibrated orifice that often 
interchangeably acts as a barrier to the flow of the meal (residual fat, nutritional value, etc.). 
The remaining oil flows to the centrifuge for 15 min in order to separate the oil from plant 
material debris. This is automatically followed by filtration. The seed oils are then stored in a 
freezer at (- 20° C) for further analysis. 
2.4. Chemical analysis 
 
Official methods (AOCS, 1997) were used for the determination of the acid value 
(method Cd 3d-63), peroxide value (method Cd 8-53), iodine value (method Cd 1-25), 
saponification value (method Cd 3-25), unsaponifiable matter (method Ca 6a-40), specific 
gravity (using a 10 ml pycnometer at 25° C), and the refractive index (using Abbé 
refractometer at 40° C) of the Cucurbitaceae seed oils. 
2.5. Fatty acid composition 
Fatty acids were determined by the analytical methods described by the European 
Parliament and the European Council in EEC regulation 2568/91 (European Economic 
Community, 1991). The fatty acids were converted to fatty acid methyl esters (FAMEs) 
before being analysed. This is done through shaking off a solution of 0.2 g of oil and 3 mL of 
hexane with 0.4 mL of 2 N methanolic potassium hydroxide. The FAMEs were then analysed 
in a Hewlett-Packard model 4890D Gas Chromatograph furnished with an HP-INNOWax 
fused silica capillary columns (Cross-Linked PEG), 30 m × 0.25 mm × 0.25 m and a flame 
ionization detector (FID). Inlet and detector temperatures were held at 230
◦ 
C and 250
◦ 
C, 
respectively. The initial oven temperature was held at 120
◦ 
C for 1 min and then it was raised 
to 240
◦ 
C at a rate of 4.0
◦ 
C/min for 4 min. The FAMEs injected volume was 1 µL and 
nitrogen (N
2
) was used as the carrier gas at 1 mL/min with a split inlet flow system at a 1:100 
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split ratio. Eventually, the fatty acid contents were measured using a 4890A Hewlett-Packard 
integrator. The FAMEs’ peaks were identified by comparison with the retention times of a 
standard mixture. The peak areas were computed and the percentages of the FAMEs were 
obtained as area percentages by direct normalization. 
2.6. Triacylglycerol analysis 
Triacylglycerol (TAG) profile was obtained by high-performance liquid 
chromatography (HPLC) (Agilent 1100, Santa Clara, CA, USA) equipped with an auto-
injector and refractive index detector. The TAGs were separated using an RP-18 column (250 
4 mm) with a particle size of 5 µm and eluted from the column with a mixture of acetonitrile-
acetone (25: 75) at a flow rate of 1 mL min
-1
. Twenty microlitres of the mixture (0.05 g oil 
diluted in 1 mL choloroform–acetone (50:50, v/v) was injected into the HPLC column and the 
total run time was 1 h. TAG peaks, observed in the high-performance liquid chromatograph, 
were identified by comparison with the retention times of standard TAG peaks and the 
retention times observed in the chromatographs of other vegetable oils such as olive, 
sunflower, soybean and corn oils. The TAG peaks were subsequently examined under similar 
analytical conditions, as previously described. 
2.7. Determination of the free radical scavenging activity 
2.7.1. Preparation of extracts
10 g of seed oil samples were weighed, dissolved in 20 ml of hexane and homogenized 
with 20 ml of methanol (60 %) at room temperature for 30 min in a shaker. The solvent mix 
was transferred to a separating funnel. The lower fraction was washed with 20 ml of methanol 
(60 %), twice homogenized for another 10 min, separated and then added to the 
corresponding separating funnel. The methanolic fraction was concentrated to dryness under 
reduced pressure in a rotary evaporator at 40°C to yield dried methanolic extract. The dried 
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methanolic extract was used to prepare solutions at different concentrations in order to 
determine the antioxidant activity (Nakbi et al., 2010). 
2.7.2. Free radical scavenging activity of the Cucurbitaceae seed oils 
The DPPH assay of seed oil extracts was determined according to the method 
described by Lopes-Lutz et al. (2008) and modified by Sarwar Alam et al. (2007). To 1 ml of 
the extract solution (in methanol), 0.5 ml of 0.15 mM DPPH solution (in methanol) was 
added. The contents were strongly mixed and kept at ambient temperature for 30 min. The 
absorbance was measured at 517 nm. The scavenging activity was examined based on the 
percentage of DPPH radical scavenged (P) defined according to the following formula: