1. Introduction
Gac (
Momordica cochinchinensis Spreng.) is a tropical vine popularly grown in Southeast Asia, China and India. Gac fruit has been reported as one of the richest natural sources of carotenoids [
1,
2]. In the processing of Gac fruit, only the aril (seed membrane) is used to produce commercial products like Gac oil and Gac powder while other parts of the fruit such as seeds, pulp and peel are discarded as wastes [
3]. Gac seed has traditionally been used in Chinese medicine due to its beneficial bioactivities for human health [
4]. The pulp (yellow fruit meat) and the peel (spiny red skin) of Gac fruit have also been found to contain a significant amount of bioactive compounds and have significant antioxidant ability [
5,
6].
The peel of Gac fruit has been reported to have a comparable carotenoid content as the well-known carotenoid-rich sources like tomatoes and carrots [
7,
8,
9]. Thus, if carotenoids from Gac peel can be efficiently recovered, this may be a good source of these compounds instead of being regarded as a by-product of Gac processing. In order to recover carotenoids and other bioactive compounds from the peel, drying and extraction techniques have been recently developed. For example, hot-air drying combined with ascorbic acid pre-treatment was found as the most suitable method for preserving content of carotenoids in the dehydrated Gac peel in comparison to other drying methods such as heat pump drying, vacuum drying and freeze drying [
7]. For the recovery of carotenoids and antioxidant capacity from dried Gac peel, the use of ethyl acetate as the solvent with the liquid–solid ratio of 80:1 (mL/g) at temperature of 40.7 °C and the extraction time 150 min were reported as optimal conditions of the extraction process [
10].
Although conventional extraction is the most popular method for recovering bioactive compounds, it requires a large solvent amount, high energy consumption and long extraction time to achieve an efficient extraction yield. Therefore, many advanced extraction techniques such as microwave-assisted extraction, ultrasound-assisted extraction, supercritical fluid extraction and pressurized liquid extraction have been developed in order to overcome the disadvantages of the conventional extraction method [
11,
12]. Among the recently developed extraction techniques, ultrasound-assisted extraction (UAE) is considered as one of the most practical extraction methods for recovering bioactive compounds from plant sources because of its high efficiency and the popularity of the ultrasonic equipment [
13]. UAE can increase the rate of mass transfer of the extraction based on the cavitation generated within the material. The cell wall of the material is destructed when the cavitation bubbles are produced and collapsed by ultrasound and thereby the release of the soluble compounds from material into the liquid phase is promoted [
14].
The literature has shown that the application of UAE in for extracting carotenoids can enhance the recovered carotenoid yield, reduce amount of solvent and shorten extraction time in comparison to the classical solvent extraction. For instance, Nowacka and Wedzik (2016) reported an increase up to 50% in carotenoid extractability from the ultrasound-treated carrots compared to the untreated carrots [
8]. The use of UAE have also led to higher extraction yield of carotenoids including lycopene and β-carotene from plant sources with shorter time, lower temperature and smaller solvent volume compared to the conventional extraction process [
15,
16,
17]. In our previous studies, the ultrasound-assisted extraction of Gac peel using ethyl acetate as the solvent for 80 min resulted in a comparable carotenoid yield and a higher antioxidant capacity compared to those obtained from the conventional extraction of Gac peel for 150 min which used the same amount of the solvent [
10,
18].
Although the advantages of UAE have been proven and this technique has been widely applied for recovering carotenoids from plant sources, its application in carotenoid extraction from Gac fruit and particularly Gac peel is limited. Beside the investigation into the influences of extraction conditions to the extraction yield, the application of modeling techniques to exactly determine optimal conditions to achieve the maximum carotenoid yield from the material is necessary. Response surface methodology (RSM) is one of the most efficient techniques used for investigating both the impacts of single parameters and their interactive effects on the dependent responses [
19]. In comparison to the classical single variable optimizing method, RSM shows a number of advantages such as the lower number of experiments and the clear expression of the interactive impacts of the parameters on the responses via 2D contour as well as 3D surface profilers [
19,
20,
21].
In this study, the response surface methodology (RSM) using the Box–Behnken design was applied to investigate the effects of extraction time, extraction temperature and ultrasonic power on the recovery of carotenoids and antioxidant capacity from Gac peel. The optimal values of these extraction parameters were also determined to maximize the recovery of total carotenoid content and antioxidant capacity from Gac peel.
2. Materials and Methods
2.1. Chemicals
Analytical graded ethyl acetate and HPLC graded acetonitrile, dichloromethane and methanol were purchased from Merck Millipore (Bayswater, VIC, Australia). ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), potassium persulfate, trolox ((S)-(-)-6-hydroxy-2,5,7,8 tetramethylchroman-2-carboxylic acid, 98%) and β-carotene, lycopene and lutein standards were purchased from Sigma-Aldrich Pty Ltd. (Castle Hill, NSW, Australia).
2.2. Materials
Fully ripe Gac fruits (full-red surface) were harvested from Wootton, NSW, Australia and then transported to the laboratories of the University of Newcastle, Australia (Central Coast campus). Gac peel was separated from other parts of the fruit and then freeze dried to the moisture content below 4%. The dried peel was then ground, well mixed and sieved to obtain powder with particle size ranging from 250–500 µm. The peel powder was stored in a freezer at −18 °C until being used for the experiments.
2.3. Ultrasound-Assisted Extraction of GAC Peel
Of ethyl acetate 80 mL and 1 g of the dried Gac peel powder were added into a conical flask. After that, the flask was covered by glass fiber and placed in the Soniclean 1000 HD ultrasonic bath (Soniclean Pty Ltd., Thebarton, SA, Australia). The extraction was then carried out in a fume hood at different ultrasonic powers (150–250 W, 43.2 kHz of ultrasonic frequency) and different temperatures (30–50 °C) for different periods of time (60–100 min). After each extraction experiment, the liquid phase was collected, filtered through a 0.45 μm syringe filter and analyzed for the content of total carotenoid and antioxidant activity.
2.4. Experimental Design Using Response Surface Methodology (RSM)
Preliminary experiments were carried out to determine the likely ranges of the single parameters. The suitable ranges of extraction time (X1, min), extraction temperature (X2, °C) and ultrasonic power (X3, W) for the extraction of total carotenoid and antioxidant capacity from Gac peel were 60–100 min, 30–50 °C and 150–250 W, respectively.
A RSM using Box-Behnken design was applied to investigate the effects of the extraction parameters on the total carotenoid extraction yield and antioxidant capacity of the extracts and determine the optimal values of the variables. The coded levels for each variable were selected according to the results of the preliminary experiments and presented in
Table 1. The experimental design is shown in
Table 2, which consisted of 15 experimental runs (12 factorial points and three central points) and the combinations of the variables in each run.
The experimental results are expressed as mean values (n = 3).
A second-order polynomial was used to express the extraction yield of total carotenoid and antioxidant activity of the extracts as dependent responses of the independent variables as follows:
where
Yi is the dependent response,
Xi is the independent parameter and
βo,
βi,
βij and
βii are the regression coefficients of the intercept, linear, interaction and quadratic terms, respectively.
2.5. Measurement of Total Carotenoid Content
To determine total carotenoid content of Gac peel extract, the filtered extract was diluted by ethyl acetate using a volumetric flask to obtain an absorbance ranging from 0.2 to 1.0 at 450 nm.
A standard curve of β-carotene in ethyl acetate was established by measuring the absorbance of standard solutions at 450 nm using a spectrophotometer (Cary 50 Bio UV-Visible, Varian Australia Pty. Ltd., Mulgrave, VIC, Australia).
The total carotenoid content of the extract was calculated and expressed as mg β-carotene equivalent/100 g dry weight (DW) based on the standard curve of β-carotene in ethyl acetate.
2.6. HPLC Analysis of Carotenoid Composition
The composition of carotenoids of the extract from Gac peel obtained under optimal conditions was analyzed based on the method developed and validated by Kha et al. (2013) [
22]. A Kinetex C18 (150 mm × 4.6 mm i.d; 5 µm) column (Phenomenex, Lane Cove, NSW, Australia) was used for the HPLC analysis in combination with a 10A VP HPLC system (Shimadzu Corp., Kyoto, Japan). An isocratic elution was used with the mobile phase composition of acetonitrile (50%), dichloromethane (40%) and methanol (10%). The injection volume of 20 µL and the column temperature of 20 °C were used. The carotenoids were detected by an UV-vis detector at the wavelength of 450 nm. Chromatograms of individual carotenoids in the standard solution and Gac peel extract obtained under optimal extraction conditions are shown in
Figure 1. The retention times and the standard curves of the external standard carotenoids were used to calculate contents of individual carotenoids in the extracts.
2.7. Determination of Antioxidant Activity
The literature has shown that the estimation of total antioxidant capacity of a sample requires different antioxidant assays because an individual compound or an extract shows different antioxidant abilities on different assays [
23]. Consistently, our previous studies have showed that the carotenoid-rich extracts from Gac peel do not exhibit DPPH antioxidant activity and have insignificant FRAP activities (ferric reducing antioxidant power). The carotenoid-rich extracts from Gac peel only showed strong ABTS antioxidant activity, which was highly correlated with their carotenoid content [
6,
7]. Therefore, the ABTS was selected as the assay in this study to evaluate the antioxidant activity of carotenoid-rich extracts from Gac peel.
The method for determination of ABTS antioxidant assay described by Thaipong et al. (2006) [
23] was used for Gac peel extracts. The ABTS stock solution was obtained by the reaction of 7.4 mM ABTS solution with 2.6 mM potassium persulfate at a ratio of 1:1 (
v/
v) for 12–16 h at 20 °C in the dark. The ABTS working solution with an absorbance of 1.1 ± 0.02 at 734 nm was achieved by diluting the ABTS stock solution with methanol.
To determine ABTS antioxidant activity of an extract, 0.15 mL of extract and 2.85 mL of ABTS working solution were mixed in a test tube and left for reacting in the dark for 2 h. This reacted solution was then measured for absorbance at 734 nm using the above mentioned spectrophotometer. A standard curve of Trolox solutions was established to calculate antioxidant activity of the extracts from Gac peel. The ABTS was expressed as µmole Trolox equivalents (TE) per 100 g dry weight (g DW) of Gac peel.
2.8. Statistical Analysis
JMP 13.0 software (SAS, Cary, NC, USA) was used for establishing the experimental design of the optimization process. The experimental runs and the validated extractions at the predicted optimal conditions were carried out in triplicates.
The results are expressed as the mean values along with standard deviations. Multiples range test and LSD (least significant differences) were used for the comparisons of the mean values. A confidence interval of 95% (p < 0.05) selected for all the statistical tests.