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Effect of Solvents and Extraction Methods on Recovery of Bioactive Compounds from Defatted Gac (Momordica Cochinchinensis Spreng.) Seeds

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Effect of Solvents and Extraction Methods on Recovery of Bioactive Compounds from Defatted Gac (Momordica Cochinchinensis Spreng.) Seeds separations Article Effect of Solvents and Extraction Methods on Recovery of Bioactive Compounds from Defatted Gac (Momordica cochinchinensis Spreng.) Seeds Anh V. Le 1,2,* ID , Sophie E. Parks 1,3 ID , Minh H. Nguyen 1,4 and Paul D. Roach 1 1 School of Environmental and Life Sciences, University of Newcastle, Ourimbah, NSW 2258, Australia; [email protected] (S.E.P.); [email protected] (M.H.N.); [email protected] (P.D.R.) 2 Faculty of Bio-Food Technology and Environment, University of Technology (HUTECH), Ho Chi Minh City 700000, Vietnam 3 Central Coast Primary Industries Centre, NSW Department of Primary Industries, Ourimbah, NSW 2258, Australia 4 School of Science and Health, Western Sydney University, Penrith, NSW 2751, Australia * Correspondence: [email protected] Received: 3 July 2018; Accepted: 23 July 2018; Published: 30 July 2018 Abstract: Gac (Momordica cochinchinensis Spreng.) seeds contain bioactive compounds with medicinal properties. This study aimed to determine a suitable solvent and extraction technique for recovery of important compounds, namely, trypsin inhibitors, saponins, and phenolics. The antioxidant capacity and total solids of derived extracts were also measured. Water with conventional extraction method gave the highest value of trypsin inhibitor activity (118.45 ± 4.90 mg trypsin g−1) while water-saturated n-butanol and methanol extracts were characterized by their highest content of saponins (40.75 ± 0.31 and 38.80 ± 2.82 mg AE g−1, respectively). Aqueous extract with microwave assistance achieved the highest phenolics (3.18 ± 0.04 mg GAE g−1). As a measure of antioxidant capacity, the 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay gave highest value to the aqueous microwave extract (23.56 ± 0.82 µmol TE g−1) while the ferric reducing antioxidant power (FRAP) assay gave highest values to water-saturated n-butanol and 70% ethanol extracts (5.25 ± 0.04 and 4.71 ± 0.39 µmol TE g−1, respectively). The total solids value was highest using water with microwave assistance (141.5 g kg−1) while ultrasound treatment did not improve any extractions. Therefore, trypsin inhibitors are suitably recovered using water while water-saturated n-butanol or methanol is for saponins, both using a conventional method. Microwave extraction is suitable for phenolics recovery. These conditions are recommended for an efficient recovery of bioactive compounds from defatted Gac seeds. Keywords: Gac seeds; MAE; UAE; Momordica cochinchinensis; trypsin inhibitors; saponins; phenolics 1. Introduction Gac (Momordica cochinchinensis Spreng.) is a plant species of the family Cucurbitaceae, whose fruit is also known as red melon, baby jackfruit, spiny bitter gourd, sweet gourd and cochinchin gourd. It is native to the Southeast Asian region and is commonly grown as a crop in countries like Vietnam, Thailand, Laos, Myanmar, and Cambodia [1,2]. The most important part of the mature fruit is the red flesh surrounding the seeds, called the aril, which is used as a colourant in rice or as a material for further processing into functional food ingredients [3]. The seeds are not eaten; they are removed from the aril and are mostly considered waste [3]. However, in traditional medicine, Gac seeds are purported to have a wide array of therapeutic effects Separations 2018, 5, 39; doi:10.3390/separations5030039 www.mdpi.com/journal/separations Separations 2018, 5, 39 2 of 13 on a wide variety of conditions, such as fluxes, liver and spleen disorders, haemorrhoids, wounds, bruises, swelling, and pus [2,4]. Several constituents have been identified, which could be involved in the medicinal effects of Gac seeds. These include trypsin inhibitors, such as MCoTI-I, MCoTI-II, and MCoTI-III [5–7]; saponins, such as Momordica Saponin I (Gypsoside), Momordica Saponin II (Quillaic acid) [8,9]; and phenolic compounds such as gallic acid and p-hydroxybenzoic acids [10]. However, studies on how to efficiently extract these various components from Gac seeds are scarce and they are vital for facilitating future applications for these bioactives. For any given plant bioactive, extractive yield depends on the extraction solvent, the chemical nature of the targeted component, and the characteristics of the extraction procedure. When other factors are kept constant, the extraction solvent plays a key role in obtaining the target constituents in terms of desired quality and quantity [11,12]. The choice of the solvent is mainly done based on the chemical properties, that is, polarity or hydrophobicity, of the target compounds. Organic solvents with medium or high polarity have been most commonly used in laboratories dealing with natural products and there is evidence that some extracts from these solvents have better activity compared with aqueous extracts [11]. However, organic solvents present safety and environmental issues. Due to the hydrophylic nature of the trypsin inhibitors and phenolics, and the amphiphilic properties of saponins in Gac seeds, aqueous solvents and the alcohols are the safest and the most environmentally-friendly solvents for the extraction of these compounds [13,14]. The conventional aqueous solvent extraction technique, in which the solid material is suspended in water with no assistance for breaking the cell structure of the solid material, is often associated with a long heating time, which risks the degradation of bioactive compounds. This has led to the proposed use of advanced techniques such as microwave-assisted extraction (MAE) and ultrasonic-assisted extraction (UAE) that are efficient in terms of extraction time and water consumption. In MAE, microwave heating is able to disrupt the plant cell structure via an increase in the internal pressure of the cell, thereby releasing the bioactive compounds [15]. Similarly, ultrasonic cavitation during UAE produces shockwaves that are also capable of disrupting the plant cell structure and releasing the plant bioactives [16]. These two advanced extraction methods were reported to be more efficient than the conventional method for recovering carotenoids in Gac peel [17]. They have been widely used for the recovery of bioactive compounds from plant materials and are considered the dominant trends in “green chemistry” extractions [18]. In the present study, it was hypothesised that MAE and UAE would be better than the conventional aqueous extraction technique for the recovery of the important bioactive compounds from Gac seeds. Therefore, the extraction of trypsin inhibitors, saponins, and phenolic compounds, as well as the antioxidant activity of extracts from Gac seeds, using MAE and UAE was compared to conventional extraction with water. Furthermore, the efficiency of extraction for the MAE and UAE techniques was also compared to other alcohol solvents, namely methanol, 50% methanol, ethanol, 70% ethanol, and water-saturated n-butanol. To date, no study has focused on assisted systems of aqueous extraction for the recovery of bioactive compounds from Gac seeds. The findings will be useful in the selection of best extraction methods specifically for the recovery of trypsin inhibitors, saponins, and phenolic compounds. 2. Materials and Methods 2.1. Materials 2.1.1. Solvents, Reagents, and Chemicals Solvents, including ethanol, methanol, and n-butanol, and chemicals, including vanillin, sulphuric acid, and potassium persulfate were purchased from Merck (Darmstadt, Germany). Folin-ciocalteu’s phenol reagent, anhydrous sodium carbonate, sodium nitrite, ferric chloride, gallic acid, 2,4,6-Tris (2-pyridyl)-s-triazine; (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid (Trolox), aecsin, 2,20-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), trypsin (type I) from Separations 2018, 5, 39 3 of 13 bovine pancreas, benzyl-DL-arginine-para-nitroanilide (BAPNA), tris, and dimethylsulfoxide (DMSO) were from Sigma-Aldrich Co. (Castle Hill, NSW, Australia). Sodium acetate trihydrate was purchased from Government Stores Department (Kingswood, NSW, Australia). Acetic acid was obtained from BDH Laboratory Supplies (Poole, UK). Sodium hydroxide was from Ajax FineChem (Taren Point, NSW, Australia) and hydrochloride acid was obtained from Lab-scan Ltd. (Bangkok, Thailand). 2.1.2. Gac Seeds Gac seeds were collected from 450 kg of fresh Gac fruit from accession VS7 as classified by Wimalasiri et al. [1]. These fruits were bought at Gac fruit fields in Dong Nai province, Ho Chi Minh city, Vietnam (Latitude: 10.757410; Longitude: 106.673439). After their separation from the fresh fruit, the seeds were vacuum dried at 40 ◦C for 24 h to reduce moisture and increase the crispness of the shell, which would facilitate shell removal. The dried seeds were de-coated to get the kernels, which were then packaged in vacuum-sealed aluminium bags and stored at −18 ◦C until used. Preparation of Defatted Gac Seed Powder Gac seed kernels were ground in an electric grinder (100 g ST-02A Mulry Disintegrator), to pass through a sieve of 1.4 mm. The powder was then freeze-dried (Dynavac FD3 Freeze Dryer (Sydney, NSW, Australia)) for 48 h at −45 ◦C under vacuum at a pressure loading of 10−2 mbar (1 Pa), to reduce the moisture content to 12.1 ± 0.2 g kg–1. The powder was then defatted using hexane (1:5 w/v, 30 min, ×3) on a magnetic stirrer at room temperature. The resulting slurry was suction filtered and the residue (defatted powder) was air-dried for 12 h and stored in a desiccator at room temperature until used. The moisture content of the meal was recorded prior to weighing for extraction, using a MOC63u moisture analyzer (Shimazdu, Kyoto, Japan). 2.2. Methods 2.2.1. Extraction Methods Initially, for the conventional extraction (CE) method, there were six extraction solvents: water (DIW), pure methanol (Methanol), 50% methanol (Methanol50%), pure ethanol (Ethanol), 70% ethanol (Ethanol70%), and water-saturated n-butanol (Butanol), examined for the extraction of bioactive compounds from Gac seeds. For the two assisting methods of UAE and MAE, only water was chosen to investigate because in the CE method it was much better than the other solvents in terms of the extraction yield of trypsin inhibitors and phenolic compounds.
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