Microwave‐assisted extraction and ultrasound‐assisted extraction for recovering carotenoids from Gac peel and their effects on antioxidant capacity of the extracts

Abstract The peel of Gac fruit (Momordica cochinchinensis Spreng.) contains high levels of bioactive compounds, especially carotenoids which possess significant antioxidant capacities. However, the peel of Gac is regarded as a waste from the production of carotenoid‐rich oil from Gac fruit. In this study, carotenoids of Gac peel were extracted by microwave‐assisted extraction (MAE) and ultrasound‐assisted extraction (UAE) using ethyl acetate as extraction solvent. The effect of extraction time and different levels of microwave and ultrasonic powers on the yield of total carotenoid and antioxidant capacity of the extracts were investigated. The results showed that an extraction at 120 W for 25 min and an extraction at 200 W for 80 min were the most effective for MAE and UAE of the Gac peel samples, respectively. The maximum carotenoid and antioxidant capacity yields of UAE were significantly higher than those of the MAE. The antioxidant capacity of extract obtained by the UAE was also significantly higher that of the conventional extraction using the same ratio of solvent to material. The results showed that both MAE and UAE could be used to reduce the extraction time significantly in comparison with conventional extraction of Gac peel while still obtained good extraction efficiencies. Thus, MAE and UAE are recommended for the improvement of carotenoid and antioxidant capacity extraction from Gac peel.

. β-carotene is well-known as a precursor to vitamin A, while lycopene and lutein have been reported as beneficial bioactive compounds for human health due to their antioxidant, anticancer and macular-protective activities (Bernstein et al., 2016;Bhuvaneswari & Nagini, 2005;Vuong, Dueker, & Murphy, 2002). Thus if the carotenoids in Gac peel are recovered effectively, the peel may be a potential source of natural carotenoids for food, cosmetic and medicinal uses.
Our studies on the conventional extraction of bioactive compounds from Gac peel showed that carotenoids and antioxidant capacity from the peel can be efficiently extracted using organic solvents (Chuyen, Roach, Golding, Parks, & Nguyen, 2017c;Chuyen, Tran, et al., 2017). However, conventional methods require large volumes of solvents, high energy use and long extraction times for an efficient extraction of bioactive compounds. Recently, many advanced techniques for the extraction of bioactive compounds have been investigated to improve the extraction efficiency and overcome the disadvantages of conventional extractions. Among the newly developed techniques, microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE) have been regarded as two of the most practical methods for the industrial scale due to the availability of equipment, the convenient operation and the high extraction efficiency (Wani, Bishnoi, & Kumar, 2016).
The MAE is based on the assistance of electromagnetic radiation with frequencies from 0.3 to 300 GHz, which induce heat inside the material via dipolar rotation and ionic conduction of the molecules (Camel, 2001). The activation of these molecules and the heat generated in this process may weaken or break the cell walls thereby the bioactive compounds can be released more easily from material matrix to the extraction solvents (Kaufmann & Christen, 2002). In another extraction technique, UAE improves the mass transfer of the extraction process by generating cavitation within the material. When the cavitation bubbles are produced and collapsed, the cell walls of the material will be destructed and the release of the solutes is promoted (Toma, Vinatoru, Paniwnyk, & Mason, 2001).
Previous studies have shown that the applications of MAE and UAE in carotenoid extraction can enhance efficiency, reduce solvent amount and save the extraction time compared with the conventional extraction methods. For example, the extraction time for carotenoids from carrots and algae was significantly reduced using continuous and intermittent microwave radiations (Hiranvarachat, Devahastin, Chiewchan, & Vijaya Raghavan, 2013;Pasquet et al., 2011). The UAE of lycopene from tomato waste has shown to occur with shorter extraction times, lower temperatures and smaller solvent volumes with higher extraction yields compared to the conventional extractions (Kumcuoglu, Yilmaz, & Tavman, 2014). These studies suggest that extraction of carotenoids from Gac peel may be improved with the assistance of microwave and ultrasound.
In this study, different power levels of microwave radiation and ultrasound and extraction times were investigated for enhancing the extractability of carotenoids from Gac peel. The effects of these parameters on antioxidant capacity of the extracts from the peel were also determined.

| Material
Gac fruits at fully ripe stage were harvested at Wootton, NSW and transported to the laboratories at Central Coast campus of the University of Newcastle, Australia. The peel of the fruits was separated by a knife and dried to a moisture content of 4 ± 0.2%. The dried peel was then ground, mixed into a uniform lot and sieved by different size meshes. The ground peel with particle size of 250-500 μm was selected and stored in vacuum sealed bags in a freezer at −18°C in the dark until the extraction.
The diagram of sample preparation and experimental design is presented in Figure 1.

| Microwave-assisted extraction (MAE)
A quantity of 0.5 gram of the dried Gac peel was extracted with 40 ml of ethyl acetate in a conical flask that was covered by glass fiber. The extraction was then carried out with a microwave oven (Sharp Carousel, Abeno-ku, Osaka, Japan) that was placed in a fume hood for the ventilation of the evaporated solvent. An intermittent microwave radiation F I G U R E 1 Preparation of Gac peel sample and experimental design with 30 s of heating ("on") and 30 s of non-heating ("off") alternatively was applied to avoid overheating of the extraction mixture. The extraction processes were terminated when the temperature reached 60°C.
Three power levels (120, 240, and 360 W) were investigated for the extraction of carotenoids and antioxidant capacity of the extract.
The temperature of the extract was measured every minute using a digital thermometer (ThermoFisher Scientific, North Ryde, NSW, Australia). Following each minute of the extraction, the liquid phase was separated and filtered with a 0.45 μm cellulose syringe filter (Phenomenex Australia Pty. Ltd., NSW, Australia) to determine the total carotenoid content and antioxidant capacity.

| Measurement of absorbed microwave and ultrasonic powers
The microwave and ultrasonic powers absorbed by a mass unit of the extraction solution were determined using the following equation (Hiranvarachat & Devahastin, 2014;Ordóñez-Santos, Pinzón-Zarate, & González-Salcedo, 2015): where P is the power absorbed by a mass unit of the extraction solution (W/g), C p is the specific heat capacity (J/g. o C), ΔT is the temperature increase by the irradiation process ( o C) and t is the irradiation time (s).

| Determination of total carotenoid content
The absorbance at 450 nm of the extracts from Gac peel or standard solutions was determined using a Cary 50 Bio UV-Visible spectrophotometer (Varian Australia Pty. Ltd., Mulgrave, VIC, Australia). The total carotenoid content of the extracts was expressed as mg β-carotene equivalent/100 g dry weight (DW) based on the standard curve of βcarotene in ethyl acetate.

| Determination of antioxidant activity
To evaluate the antioxidant capacity of a bioactive compound or an extract, different antioxidant assays are usually required as an individual compound or group of compounds may exhibit different antioxidative powers on different assays (Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Hawkins Byrne, 2006). However, the results of our previous studies on Gac peel have shown that carotenoid extracts from Gac peel do not possess DHPH radical scavenging activity and also do not have significant activity on an iron reducing power assay. Only ABTS radical scavenging activity of the carotenoid extracts from Gac peel was found to be significant and that was highly correlated with the total carotenoid content in the extracts (Chuyen, Roach, Golding, Parks, & Nguyen, 2017a;Chuyen et al., 2017b). Thus, the ABTS radical scavenging activity was selected to represent the total antioxidant capacity of carotenoid extracts from Gac peel in this study.
The ABTS antioxidant assay of Gac peel extracts was carried out based on the methods described by Thaipong et al., 2006

| Statistical analysis
All experiments were repeated in triplicate and the results were expressed as the mean values ± standard deviations. The overall statistical significance for each experiment was determined using the analysis of variance test (ANOVA) and the LSD post-hoc test was used for comparisons amongst the mean values if the ANOVA was significant. Differences were considered to be significant at p < .05.

| Effect of microwave power on the temperature of the extract
Preliminary experiments showed that the continuous microwave radiation brought the extraction solvent to boil very quickly at all applied power levels (data not shown). Therefore, an intermittent microwave radiation with 30 s "on" and 30 s "off" alternatively was applied to prolong the extraction time. The temperature of 60°C was selected as upper P = C p ΔT t limit for the extraction process because the results of published studies which have shown that carotenoids are severely degraded above this temperature (Fratianni, Cinquanta, & Panfili, 2010;Pasquet et al., 2011).
The variation in the used microwave power led to a significant difference in the temperature increase in the extract (Figure 2). The temperature of the extraction at 360W rapidly increased from room temperature (20°C) to 63°C in 4 min while the MAE at 240W also reached 61°C after 6 min. When the microwave power was reduced to 120W, the temperature of the extract was retained below 60°C for 30 min (Figure 2). The determination of the absorbed power showed that the microwave energy absorbed by the extraction mixtures at 120W, 240W, and 360W was 0.32, 0.69 and 0.83 W/g, respectively. The high levels of microwave power absorbed at 240W and 360W were responsible for the rapid increase in the temperature of the extraction mixtures even the intermittent microwave radiation procedure has been applied. In contrast, the extraction at 120W could be maintained for a much longer time because its absorbed power was significant lower than that of the other extractions.

| Carotenoid extraction yield
The total carotenoid extraction yield of the MAE at 360W increased rapidly from 156 to 236 mg/100 g DW during 4 min of extraction time ( Figure 3), which is comparable to a 6 min extraction and a 10 min extraction with microwave power at 240 W and 120 W, respectively.
The extraction at 240W also caused a relatively high extraction yield of carotenoid (150 mg/100 g DW for 1 min) that rose steadily to 235 mg/100 g DW by the end of the process. For the extraction at 120W, the total carotenoid yield also slowly increased along with the slow increase in the temperature (Figure 3). The highest total carotenoid yield of this extraction (262 mg/100 g DW) was achieved at 25 min before being reduced slightly for the extended extraction time.

| Antioxidant capacity of the extracts
The results of the different MAE extraction conditions on antioxidant capacity is presented in Figure 4 and shows that the antioxidant capacity of the extracts from Gac peel was similar in trend to that of the total carotenoid yield. The antioxidant capacity of the extract using 360 W of microwave power sharply increased to 659 μmol/L TE/100 g DW the end of the process, which was statistically comparable to the antioxidant values of 6 min extraction at 240 W (664 μmol/L TE/100 g DW) and 15 min extraction at 120 W (679 μmol/L TE/100 g DW).
Although the antioxidant capacity of the extract at 120 W was lower than that of the other extractions when compared at correlative points of time, its maximum antioxidant yield (716 μmol/L TE/100 g DW at 25 min) was significantly higher than the maximum values of the others.

| Carotenoid extraction yield
The results of the total carotenoid extraction yield of the UAE carried

| Antioxidant capacity of the extracts
The results in Figure 6 show that a very high antioxidant capacity of the extracts (568-583 μmol/L TE/100 g DW) was achieved after only

| DISCUSSION
In the microwave-assisted extraction, the temperature of the extraction mixture increases by absorbing microwave energy.
The increase in the temperature results in the lower viscosity of the solvent that promotes the diffusion rate of the desired compounds from the materials into the extraction medium (Eskilsson & Björklund, 2000). The heating using microwave energy also causes the rupture of the material cell walls which allows solvent to penetrate into the solid matrix to dissolve and release the compounds inside the cells into the liquid phase (Zhou & Liu, 2006). However, one of the obstacles of the MAE is the rapid increase in temperature of the extraction mixture that may terminate the extraction process quickly due to the boil of the solvent. When the extraction is terminated early, the desire compounds are not sufficiently diffused from the material into the solvent and consequently the extraction yield is reduced (Hiranvarachat & Devahastin, 2014;Nguyen et al., 2016). In this study, the use of intermittent microwave operation using 30 s "on" then 30 s "off" to cool down the extraction mixture could extend the extraction time significantly compared to the continuous microwave heating. In comparison with a conventional extraction using the same batch of Gac peel sample and ratio of solvent to material (Chuyen et al., 2017c), the MAE at 120W obtained a lower carotenoid yield and a comparable antioxidant capacity yield (Table 1). However, the total extraction time of the MAE was sixfold shorter than that of the other (25 min compared with 150 min). Previous studies on extraction of carotenoids also showed that MAE resulted in higher carotenoid yields and shorter extraction times compared to conventional extraction methods. For example, MAE extraction of astaxanthin from Haematococcus pluvialis at 141 W for 5 min resulted in a higher extraction yield compared to the conventional stirring extraction for 12 hr (Zhao, Chen, Zhao, & Hu, 2009). Similar extraction efficiencies of carotenoids from a microalga (Cylindrotheca closterium) were also obtained with 5 min of MAE at 50W and 60 min of a conventional soaking extraction in acetone (Pasquet et al., 2011).
For the ultrasound-assisted extraction, the higher extraction yield of the UAE carried out at 200W compared to that carried out at 150W may be due to the greater cell wall disruption of Gac peel material when the higher ultrasonic power was applied (Chemat et al., 2017). However, the mechanism of the mass transfer based on the cell breakage is not adequate for explaining the reduction in the extraction yield resulted by the UAE carried out at 250W compared to that at 200W. The previous studies have found that lutein and β-carotene, the major carotenoids in Gac peel (Chuyen et al., 2017a), were significantly degraded by ultrasound treatments and the degradation was greater with the increase in ultrasonic power (Sun, Ma, Ye, Kakuda, & Meng, 2010;Sun, Xu, & Godber, 2006 (Li, Fabiano-Tixier, Tomao, Cravotto, & Chemat, 2013).
In comparison to the MAE extraction, the results in this study showed that UAE resulted in significantly higher extraction yields of both total carotenoid and antioxidant capacity (Table 1). This improved yield could be related to the greater amount of bioactive compounds being diffused into the solvent over a longer period. The lower thermal degradation of bioactive compounds caused by the UAE, which was carried out at 20°C, could have also contributed to its higher extraction efficiency compared with the MAE.
Although the ultrasound-assisted extraction showed an improvement in the extraction efficiency compared to the microwave-assisted extraction and the conventional extraction for carotenoids and antioxidant capacity from Gac peel, its energy consumption was much higher than that of the others (

| CONCLUSION
MAE and UAE at different power levels were investigated for the extraction of carotenoids and antioxidant capacity from Gac peel.
The applied microwave and ultrasonic powers significantly in- Gac peel, the interactive effects of the parameters should be studied and the determination of optimal conditions for MAE and UAE is recommended.

ACKNOWLEDGMENT
The first author (Hoang Van Chuyen) acknowledges the Australian Awards Scholarship for financial support.

CONFLICT OF INTEREST
The authors declare no conflicts of interest. T A B L E 1 A comparison of carotenoid and antioxidant capacity extractions from Gac peel using different extraction methods