), is variously named winter melon, white gourd, ash pumpkin, tallow gourd, white pumpkin, ash gourd, wax gourd, gourd melon and Chinese watermelon or Chinese preserving melon in English [1
]. The Cucurbitaceae
family is mostly distributed around the tropical regions and the winter melon, which has been cultivated for at least 2,000 years, originated from south-east Asia [2
]. This fruit is large and seedy with white colored and spongy flesh. Depending on the shape, type and maturity of the fruit, the seeds, which are smooth and white to yellowish-colored, fill the centre of the fruit [2
]. Index of Nutritional Quality (INQ) data shows that Benicasa hispida
is valued as a high quality vegetable [2
]. Several investigations on the biologically active components of Benincasa
species have proven its antioxidant activity on different tissues like liver and brain [4
]. To the best of our knowledge, studies on chemical composition of seed oil from Benincasa hispida
and its antioxidant activity have not been reported yet.
Extraction is a major step for the isolation, identification and use of valuable compounds from different plants [5
]. The Soxhlet method which was developed by von Soxhlet in 1879 has been performed as a standard method for extraction of valuable compounds from different plant sources. This technique is not always acceptable for an extraction due to the fact that this process is very slow and degradation of targeted compounds is common [6
]. Numerous studies have been carried out to develop novel extraction processes which are applicable to various compounds [6
]. In recent years, ultrasound-assisted extraction has received considerable attention for the recovery of different compounds from different sources [8
]. This technique is attractive because of its simplicity and low equipment cost compared with other extraction techniques such as supercritical fluid or microwave-assisted extraction [11
]. Moreover, other advantages include drastically reduced processing time, consumption of less energy and reduced thermal degradation effects [12
]. The higher efficiency of ultrasound-assisted extraction is attributed to disruption of cell walls, particle size reduction and enhanced mass transfer of the cell content via cavitation bubble collapses [13
]. Various applications of ultrasound in extraction of bioactive compounds from plant materials were reviewed by Vinatoru [15
]. So far, there is no report on ultrasound-assisted extraction of crude oil from Benincasa hispida
seed. Furthermore, the application of ultrasound in food technology is reviewed by Chemat et al
Optimization of the experimental conditions is a critical step in developing a successful ultrasound-assisted extraction process due to the effect of various process variables such as ultrasound power, process temperature and sonication time on the extraction efficiency [11
]. Response surface methodology (RSM) is an effective statistical method for optimizing experimental conditions and investigation of critical processes, while at the same time reducing the number of experimental trials [17
]. Therefore, the objectives of this study were to investigate the effect of process variables including power level, temperature and sonication time on crude extract yield (CEY). RSM was employed to optimize extraction conditions in order to obtain the maximum crude yield. Furthermore, the antioxidant activity, total phenolic content and fatty acid composition of the extract obtained under optimized conditions were determined and then compared with those obtained by the Soxhlet method.
Whole winter melons (Benincasa hispida L.) were purchased from a local market in Serdang, Selangor, Malaysia. Fruits were chosen at commercial maturity according to their similarity of color, size and absence of surface defects. The fruits were cut, and then seeds were separated manually and washed under tap water. Seeds were dried at 40 °C in a ventilated oven (1350FX, Cornelius, OR, USA) for 24 h and then stored at an ambient temperature in the dark. The seeds were ground in a grinder mill (MX-335, Panasonic, Shah Alam, Malaysia) for 10 s to produce a powder with an approximate size of 1.5–2.5 mm.
3.2. Chemicals and Reagents
Carbon dioxide (CO2, SFE grade) contained in a dip tube cylinder was purchased from MOX-Linde Gases Sdn. Bhd. (Petaling Jaya, Malaysia). Analytical grade ethanol and n-hexane were obtained from Scharlau (Port Adelaide, Australia). Sodium methoxide, potassium persulphate, catechin, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS˙+), 1,1-diphenyl-2-picrylhydrazyl (DPPH˙), gallic acid and Folin-Ciocalteu reagent (FCR) were purchased from Fisher (Pittsburgh, PA, USA). Fatty acid methyl ester (FAME) standards were obtained from Sigma-Aldrich (St. Louis, MO, USA). All chemicals were either of chromatography or analytical grade.
3.3. Ultrasound-Assisted Extraction
In present study, a 500 W ultrasound equipment (Sonics and Materials Inc., Model VC505, Danbury, CT, USA) with a titanium ultrasonic probe (13 mm diameter) was used for ultrasound-assisted extraction of crude oil from Benincasa hispida
seeds. The nominal frequency was 20 kHz. Ground seed (about 5 g) was placed in a 100 mL beaker containing ethanol as a food grade solvent which is recommended by the US Food and Drug Administration for extraction purposes. The solid/solvent ratio was 1:10 (g/mL). The beaker placed in a temperature controlled water bath (Memmert WNE14. Memmert GmbH Co. KG, Schwabach, Germany). Extraction was carried out at temperatures ranging from 45 to 55 °C. Temperature was verified with a digital thermometer (Ellab CTD-85, Ellab, Hilleroed, Denmark) and a thermocouple (1.2 mm needle diameter constantan type T) and no significant increase in temperature (below 2 °C) was detected due to circulation of water in water bath during extraction. The applied power levels were adjusted to 25, 50 and 75% of the maximal equipment power (500 W), corresponding to 125, 250 and 375 W through the variation of amplitude of piezocrystals. The corresponding ultrasound intensities were 94, 189 and 284 W/cm2
. The immersed samples in extraction solvent were subjected to ultrasonic waves for 20 to 40 min. The input range of the selected variables was determined by preliminary experiments. After extraction, the extracts were filtered through the Whatman No. 1. filter paper. Then, ethanol was removed from the extracts by evaporation under vacuum at 40 °C using a rotary evaporator (Eyela, A-1000S, Koishikawa Bunkyo-ku, Japan). Subsequently, the residual solvent was removed by drying in an oven at 40 °C for 1 hr and flushing with 99.9% nitrogen. The scheme of experimental set-up was presented in Figure 6
3.4. Soxhlet Method
Ground Benincasa hispida seed (about 5 g) was put into extraction thimble and covered with wool. Then the thimble was transferred into a Soxhlet apparatus. Extraction was performed with ethanol (99.5%, 150 mL) for 6 h. The temperature of extraction corresponded with the boiling point of the solvent in use. After extraction, solvent was removed under vacuum at 40 °C using a rotary evaporator (Eyela). Subsequently, the residual solvent was removed by drying in an oven at 40 °C for 1 h and flushing with 99.9% nitrogen.
3.5. Crude Extract Yield Measurement
The extracts were weighed gravimetrically using a Mettler Toledo analytical balance (±0.0001 g) (Mettler Toledo GmbH, Greinfensee, Switzerland) and then the CEY was calculated according to the following equation:
is the crude extract mass (g) and ms
is the extracted sample mass (g). The measurement was performed in triplicate and the mean values of CEY were expressed as mg-extract/g-dried matter.
3.6. Determination of Radical Scavenging Activity
The extracts of Benincasa hispida seeds were subjected to antioxidant activity analysis using DPPH˙ and ABTS˙+ free radical scavenging assays. All determinations were done in triplicate and expressed as means ± Standard Deviation.
3.6.1. Determination of DPPH˙ Radical Scavenging Activity
This assay was carried out as described by Zengin et al.
] with some modifications. A total of 0.1 mg/mL of the extracts and synthetic antioxidant (catechin) in the ethanol were added into an ethanolic solution of DPPH˙
(3 mL, 6 × 10−5
M). The mixture was vortexed for 20 s at room temperature. Absorbance measurements at 515 nm commenced immediately in a 1 cm quartz cell after 1 min up to 60 min with 10 min intervals using a UV-260 visible recording spectrophotometer (Thermo 4001/4 UV–Vis Spectrophotometer, Thermo Fisher Scientific, West Palm Beach, FL, USA). The blank test was conducted with 0.1 mL ethanol instead of extracts and the absorbance was recorded as Ablank
. The inhibition percent of DPPH˙
which was scavenged (%DPPHsc
) was calculated according to the following equation:
% DPPHsc = 100 × (Ablank − Asample)/Ablank
are the absorbance values of the blank and of the tested samples, respectively, checked after 60 min.
3.6.2. Determination of ABTS˙+ Radical Scavenging Activity
The 2,2´-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS˙+
) assay was carried out according to the method of Cai et al.
]. The ABTS˙+
radical solution was prepared by mixing 7 mM ABTS and 2.45 mM potassium persulphate, and incubating the mixture in the dark at room temperature for 16 h. The ABTS˙+
solution was then diluted with 80% (v/v) ethanol to obtain an absorbance of 0.70 at 734 nm. ABTS˙+
solution (3.9 mL) was added to sample (0.1 mg/mL) and mixed vigorously. The absorbance of the mixtures at room temperature was recorded immediately using UV-260 visible recording spectrophotometer (Thermo 4001/4 UV–Vis Spectrophotometer) at 734 nm for 10 min at 2 min intervals. The blank test was conducted with ethanol instead of extracts and the absorbance was recorded as Ablank
. The inhibition percent of ABTS˙+
which was scavenged (%ABTSsc
) was calculated using the following equation:
%ABTSsc = (Ablank – Asample) × 100/Ablank
are the absorbance values of the blank and of the tested samples, respectively.
3.7. Determination of Total Phenolic Content
The total phenolic content (TPC) of the Benincasa hispida
seed extracts was determined using Folin-Ciocalteu reagent (FCR) according to the procedure reported by Singleton et al.
] with some modifications. This method is based on measuring color change caused by reduction of the Folin-Ciocalteu reagent by phenolates in the presence of sodium carbonate. Extract (about 10 mg) was dissolved in deionised water (1 mL). This solution was mixed with Folin-Ciocalteu reagent (diluted 10 fold with distilled water, 1 mL). The solution was kept at room temperature for 5 min and then 60 mg/mL of aqueous carbonate sodium (Na2
) solution (7.5 mL) was added. The color change was determined by scanning the wavelength at 765 nm (Thermo 4001/4 UV–Vis Spectrophotometer) since maximum absorbance was obtained. TPC of the extract was determined as mg gallic acid equivalent using the standard curve prepared at different concentrations of gallic acid (25–500 ppm) and reported as mg GAE/g extract. The determination was carried out in triplicate and expressed as means ± Standard Deviation.
3.8. Preparation of Fatty Acid Methyl Esters
Samples were brought to a temperature of 50–60 °C and homogenized thoroughly before taking a test sample in order to obtain the fatty acid methyl esters (FAMEs). An aliquot of the test sample (100 µL) was mixed with n-hexane (1 mL) in a 2 mL vial. An aliquot of sodium methoxide (1 µL, 1% w/v) was added to the vial which was mixed vigorously using a vortex mixer. The mixture first became clear and then turbid as sodium glyceroxide was precipitated. After a few minutes, the clear upper layer of methyl ester was pipetted off and injected into the gas chromatograph (GC) for further analysis.
3.9. Gas Chromatography Analysis
Fatty acids composition analysis was performed in a Hewlett-Packard 6890 gas chromatography (Wilmington, DE, USA), equipped with a flame ionization detector (FID) and a BPX70 (30 m × 0.25 mm × 0.25 µm, Victoria, Australia) GC column. Oven temperature was programmed isothermally to 115 °C during 2 min, then was raised at 4 °C/min to 163 °C and then at 1 °C/min to 170 °C. Finally, temperature increased to 200 °C at 10 °C/min and held at this temperature for 2 min. Helium was used as a carrier gas which flowed at a rate of 1 mL/min. The injection volume was 1 µL. Standard methyl esters of fatty acids were used as authentic samples. The fatty acids determination was accomplished by comparing with standards and was valued by the area percentage of each fatty acid. The fatty acid determination was performed in triplicate for each sample and expressed as means ± Standard Deviation.
3.10. Experimental Design and Statistical Analysis
Response surface methodology (RSM) was applied to optimize the process variables including power level (25–75%), temperature (45–55 °C) and sonication time (20–40 min) to achieve the highest amount of crude oil from Benincasa hispida
seeds. A central composite design (CCD) with axial points was used for designing the experimental data. This generated 20 treatments with six replications at the centre points to estimate the repeatability of the method (Table 1
). The effect of unexplained variability induced by extraneous factors on the observed responses was minimized by randomizing the order of experiments. Blocks are assumed to have no impact on the nature and shape of the response surface. The following second-order polynomial model was fitted to the data:
Yi = β0 + β1X1 + β2X2 + β3X3 + β11X12 + β22X22 + β33X32 + β12X1X2 + β13X1X3 + β23X2X3
is predicted response, β0
is offset term, β1
are the regression coefficients for linear effect terms, β11
are quadratic effects and β12
are interaction effects. In this model, X1
represent power level, temperature and sonication time, respectively. The significant terms (p
< 0.05) in the model were found by analysis of variance (ANOVA) based on p
-value. The terms statistically found non-significant (p
> 0.05) were dropped from the initial model and the experimental data was refitted only to significant (p
< 0.05) variables in order to obtain the final reduced model [34
The three-dimensional response surface plot was generated for the graphical interpretation of the interaction effect of independent variables on the response. Numerical optimization was carried out to predict the exact optimum level of independent variables leading to the desirable response goal. The model adequacy was determined using model analysis, lack of fit test, coefficient of determination (R2
) and adjusted-R2
]. Furthermore, experimental data were compared with predicted values (method validation) in order to verify the adequacy of final reduced model. In addition, the quality of fit between the experimental and predicted data was determined according to value of the mean relative deviation modulus (E). The criteria can be calculated as follows:
are the experimental and predicted values, respectively, n is the number of experimental data. A model is considered acceptable if E value is less than 10% [36
]. The experimental design matrix, data analysis, regression coefficients, generation of 3D graph and numerical optimization procedure were created using Design Expert Version 8.0.7 software (Stat-Ease Inc., Minneapolis, MA, USA, trial version).
In this study, response surface methodology with a central composite design (CCD) was applied to investigate the ultrasound-assisted extraction of crude oil from Benincasa hispida seed. The experimental results showed that all three process variables, including power level, temperature and sonication time, contributed to the extraction of crude oil. It was found that ultrasound power is the most significant variable among the process variables studied. An empirical quadratic polynomial correlation has been proposed to estimate the optimum operating condition of the process. The highest crude extract yield (108.62 mg-extract/g-dried matter) was obtained when the extraction process was carried out at 65% power level, 52 °C temperature and sonication time of 36 min. The results of the comparative study revealed that the CEY of ultrasound-assisted extraction was lower than that obtained by the Soxhlet method, whereas the extract obtained using the ultrasound-assisted technique had higher quality in terms of antioxidant activity and total phenolic content. Furthermore, it was revealed that the both extracts were rich in unsaturated fatty acids, with the majority corresponding to linoleic acid and oleic acid. Therefore, it may be said that plant sources like winter melon (Benincasa hispida) may provide new natural products to the food industry with safer and better antioxidants that provide good protection against oxidative damage which occurs both in the body and in the processed food. In addition, it could be suggested that ultrasound-assisted extraction is an effective and indeed feasible method for the extraction of crude oil rich in valuable compounds from the Benincasa hispida seed.