1. Introduction
In recent years, there was an increased interest in natural sources that could provide active components to prevent the impact of free radicals on cells. For this reason, the number of studies on natural antioxidants increased considerably. Mangosteen (
Garcinia mangostana L.) is a tree with a height of 6–25 m that belongs to the Clusiaceae family, and it is thought to originate in Southeast Asia.
Mangostana garcinia Gaertn was approved as a synonymous name, and its vernacular names include Mangosteen (English); Manggis, Semetah, and Semontah (Malay); Dao nian zi (Chinese); and Sulambali (Tamil) [
1]. The mangosteen fruit is reddish/dark purple with a juicy, soft, edible pulp and delectable taste. The pericarp of
G. mangostana was used as a cure for chronic intestinal catarrh and dysentery, as a lotion [
2], as a treatment of respiratory disorders [
3], to heal skin infections and relieve diarrhea [
4], and as an astringent [
5]. Several biological activities were reported for the pericarp extract of
G. mangostana, such as antioxidant [
6,
7], antimicrobial [
8], antidiabetic [
9], antiproliferative [
10], and antitumor activities [
11]. The biological activities of herbs/crops are related to their phytochemical constituents. The phytochemical analysis of mangosteen pericarp showed that it is rich in α-mangostin, phenolics (for example, ferulic acid, p-coumaric acid, veratric acid, t-cinnamic acid, vanillic acid, cinnamic acid, caffeic acid, mandelic acid, gentisic acid, and sinapic acid), and flavonoids (for example, epicatechin and quercetin) [
7,
12,
13].
Previous studies reported that most of the biological activities of
G. mangostana are significantly correlated with the concentration of α-mangostin [
14,
15]. Alpha-mangostin isolated from the extract of dried
G. mangostana rind showed antioxidant, anticancer, and cytotoxicity activities [
15]. The extraction process of α-mangostin from the mangosteen pericarp is critical [
14], and the polarity and concentration of the extraction solvents were reported to be important factors. To extract different types of secondary metabolites from plant sources, various types of solvents, such as methanol, ethanol, and acetone, are commonly used [
16]. The use of these organic solvents in an extraction process depends on the plant variety and the compounds targeted [
16]. However, to extract on a large scale, an essential step is the optimization of the variables that are critical in the extraction process to obtain the maximal yield of the targeted compound. More useful information and optimal experimental conditions can be achieved using a good design and a suitable experimental model. Response surface methodology (RSM) was developed to optimize various extraction processes, including the extraction variables such as solvent polarity, extraction time, and temperature [
17,
18]. The various parameters and their interactions could be evaluated efficiently using this data analytical technology, thus reducing the experimental group number [
19]. Previously, only two extraction methods (the supersonic wave and the supercritical CO
2 method) of α-mangostin were optimized using RSM [
20,
21]. However, these techniques require specific equipment to extract α-mangostin. In recent years, a trans-ferulic microwave extraction method was developed for the extraction of bioactive compounds from herbs [
22,
23,
24]. Finding a simple method with a higher extraction yield such as the microwave extraction method could be useful for extracting α-mangostin from the mangosteen pericarp on a large scale. Therefore, we are interested in the preparation of α-mangostin extracts from mangosteen pericarp using green extraction concepts. A green extraction concept is based on the design of extraction procedures that can reduce energy consumption, allow for the use of alternative safe solvents and renewable natural products, and ensure a safe and high-quality extract. To the best of our knowledge, there is no information regarding the optimization of the microwave-assisted extraction of α-mangostin from mangosteen pericarp using RSM.
This study was designed in order to enhance the extraction yield and quality of α-mangostin from the mangosteen pericarp using a green extraction method and RSM. Therefore, individual parameters such as microwave power, extraction time, and solvent polarity were optimized to extract the α-mangostin from G. mangostana using central composite design (CCD) and RSM. In addition, individual secondary metabolite (flavonoids and phenolic acid) profiling and the antioxidant and antimicrobial activity of the optimized extracts were evaluated.
3. Materials and Methods
3.1. The Sampling of the Mangosteen Fruit
Mangosteen fruits were harvested from a mangosteen farm located in Johor, Malaysia. All harvested fruits were washed with pure water. The pericarps of the fruits were separated and dried in a 45 °C oven for five days. The dried pericarps were powdered using a grinder (0.355 mm) and were sieved (80 mesh). The samples were kept at −20 °C for future analysis.
3.2. Extraction Parameters and Preliminary Study of Extraction Parameters
Extraction was conducted using a microwave extractor (Multivalve 3000, Graz, Austria). Specifically, 2 g of powdered mangosteen pericarp was extracted with 20 mL of green solvent (water, ethanol, ethyl acetate, or dichloromethane). The microwave power during extraction was adjusted using a microwave power control panel. An extraction time of 2–12 min, a microwave power of 100–500 W, and solvent percentages of 20–100% (v/v) were chosen as the variables for the extraction process. A one-factor-at-a-time method was used to investigate the influence of each factor on the targeted yield in extracts. The effect of different concentrations of ethyl acetate (diluted with ethanol) on the extraction yield was also evaluated. The extracts were filtered through Whatman No. 1 paper, transferred to Falcon tubes, and kept at −20 °C for future analysis. Experiments were carried out in triplicate to ensure reproducibility.
3.3. RSM Analysis
RSM is an experimental statistical technique applied to the multiple regression analysis using quantitative data obtained from properly designed experiments. Various parameters that influenced the extraction efficiency were optimized to efficiently extract active compounds, including phenolic acids and flavonoids, from pigmented rice bran. In this study, the relationships among time (X
1), microwave power (X
2), and solvent percentage (X
3) were investigated using CCD to obtain the optimal extraction conditions. The quadratic polynomial step-by-step regression method and data were analyzed using the Design Expert (Version 7, Stat-Ease, Inc., Minneapolis, MN, USA) software. The model shown below was used to predict the response variables.
where Y is the predicted dependent variable; b
0 is a constant that fixes the response at the central point of the experiment; b
1, b
2, and b
3 are the regression coefficients for the linear effect terms; b
1b
2, b
1b
3, and b
2b
3 are the interaction effect terms; and b
12, b
22, and b
32 are the quadratic effect terms. The regression coefficients of the individual linear, quadratic, and interaction terms were determined according to an analysis of variance (ANOVA). To visualize the relationship between the response and experimental levels of each factor and to deduce the optimal conditions, the regression coefficients were used to generate three-dimensional (3D) surface plots and contour plots from the fitted polynomial equation. The factor levels were coded as −1.682, −1, 0, +1, and +1.682. The variables were coded as described by the following equation:
where
Xi is the (dimensionless) coded value of the variable
Xi,
X0 is the value of
X at the central point, and Δ
X is the step change.
3.4. HPLC Analysis of α-Mangostin
Alpha-mangostin in the extracts was identified using an Agilent HPLC 1200 system (Agilent Technologies, Santa Clara, CA, USA). The separation was conducted at 25 °C on a Lichrocart column (5 μm, 4 mm × 250 mm). The mobile phase for the method developed consisted of acetonitrile (solvent A) and 0.2% aqueous formic acid in water (solvent B). The method employed a step-wise linear gradient. In addition, the injection volume and flow rates were 20 μL and 1 mL/min, respectively. The UV wavelength was set at 240 nm. The calibration curve of α-mangostin was performed at different concentrations (15, 30, 60, 120, and 240 μg/mL). The amount of α-mangostin was calculated based on a linear equation: Y = 30871.46X + 1941.82, R2 = 0.9983. Each calibration point was conducted in triplicate.
3.5. HPLC Analysis of Phenolics and Flavonoids
Qualitative and quantitative analysis of the samples was performed using an Agilent HPLC 1200 system (Agilent Technologies, Santa Clara, CA, USA). A C18 column with ZORBAX (5 μm, 2.1 mm × 12.5 mm) was equipped. The mobile phase for the method developed consisted of 0.03 M ortho-phosphoric acid (solvent A) and HPLC-grade methanol (solvent B). The method employed a step-wise linear gradient. The column was maintained at 35 °C. In addition, the injection volume and flow rates were 10 μL and 1 mL/min, respectively. A standard solution of each compound was prepared at different concentrations, and a calibration curve was prepared. Linear equations of each compound were as follows: gallic acid (Y = 872.62X + 119.20), trans-ferulic acid (Y = 594.39X + 85.46), cinnamic acid (Y = 294.50X + 60.29), caffeic acid (Y = 317.69X + 57.03), quercetin (Y = 314 X + 86.29), catechin (Y = 438.11X + 106), and rutin (Y = 297.36X + 84.25).
3.6. Evaluation of Antioxidant Activity
3.6.1. DPPH Assay
The optimized pericarp extracts of mangosteen were examined for their hydrogen-donating ability toward DPPH, which is a stable free radical. The sample extracts and ascorbic acid were adjusted to 100 μL with 3 mL of 0.1 mM DPPH in methanol and vortexed well. The solutions were incubated in the dark for 30 min. The scavenging activities of the extracts were determined from the absorbance at 517 nm against methanol as a blank solution [
41]. The following formula was used to calculate the scavenging activity:
3.6.2. Ferric Reducing Antioxidant Potential (FRAP) Assay
The FRAP assay was used to evaluate antioxidant activity. Briefly, 200 µL of the extracts were mixed with 2.0 mL of FRAP reagent (pH = 3.6). The mixture was incubated in a water bath at 25 °C for 30 min. The absorbance of the solution (blue color) was measured against acetate buffer (the blank) at 593 nm. A standard curve was prepared using concentrations of 100–1000 mM of FeSO
4 × 7 H
2O. The results are expressed in μM of Fe (II)/g DM [
41].
3.7. Antibacterial Test
Five reference bacterial strains and two laboratory strains from our laboratory stock culture confirmed to be multidrug-resistant bacteria were used for the antibacterial assay. The reference and laboratory strains are four Gram-positive bacteria (S. aureus (NCBI 50080), M. smegmatis (ATCC 700084), L. ivanovii, (ATCC 19119), and S. uberis (ATCC700407)), and three Gram-negative bacteria (E. cloacae (ATCC 13047), E. coli 180, and V. parahaemolyticus (ATCC 17802)) that were reported to be resistant to sulphamethoxazole, ampicillin, streptomycin, cefuroxime, cephalexin, tetracycline, and nalidixic. They were tested against the mangosteen optimized and non-optimized extracts.
The bacteria were cultivated in Mueller-Hinton broth at the appropriated temperature (34–37 °C) of the strains. Then, the turbidity of each culture of bacterium was adjusted to reach 1–5 × 10
8 colony-forming units (CFU)/mL. Briefly, 100 μL of a suspension containing 10
8 CFU/mL of bacteria cells was spread on Petri plates. The paper discs (6 mm in diameter) were separately impregnated with 20 μL of the extract (100 μg/mL) of mangosteen pericarp and placed on an agar plate which was previously inoculated with the selected test microorganisms. Ciprofloxacin was used as a positive reference for the bacteria. Discs without samples were used as a negative control. Plates were kept at 4 °C for 1 h. The inoculated plates were incubated at 37 °C for 24 h. The antimicrobial activity was assessed by measuring the diameter of the growth in millimeters (including disc diameter of 6 mm) for the test organisms compared to the controls [
49].
3.8. Data Analysis
The data were analyzed using the SAS (Statistical Analysis System) Version 9.2 software and Duncan’s multiple range test with significance set at the p < 0.05 level. The mean and standard deviation (n = 3) of each standard and sample were calculated.
4. Conclusions
This study investigated the optimization of the extraction process of α-mangostin from mangosteen pericarp using a microwave extraction method with ethyl acetate as a green solvent. A central composite design (CCD) was successfully employed to determine the optimal extraction conditions to obtain a high-quality extract with potential antioxidant and antimicrobial activities. The optimal microwave power, time, and ethyl acetate percentage of extraction to maximize the α-mangostin extract from mangosteen pericarp (120.68 mg/g DM) were 189.20 W, 3.16 min, and 72.40% (v/v), respectively. The OE of the mangosteen pericarp exhibited higher concentrations of TPC, TFC, and individual flavonoids and phenolic acids than the NOE. Trans-ferulic acid was found to be an abundant phenolic compound. In addition, the free-radical-scavenging power of the mangosteen pericarp extract obtained under optimal conditions was higher than that of the NOE. The OE exhibited the highest antibacterial activity, particularly against Gram-positive bacteria. This study is the first report of the optimization of the microwave-assisted green extraction process of α-mangostin from mangostin pericarp, and it provides a significant basis to further investigate the separation of this effective natural substance.