1. Introduction
Annonaceae acetogenins (ACGs) are functional aliphatic molecules of 35 to 37 carbons, bearing a terminal γ-lactone, a variable number of attached hydroxyl groups, and one, two, or three tetrahydrofuran rings in their central region or other functional groups. These compounds are unique to the Annonaceae family and are widely studied in ethnobotany because they present biological activities such as antimicrobial, antiviral, and antitumoral [
1].
ACGs are found in the roots, stem-bark, leaves, pulp, peel, and seeds from Annonanaceae plants. Seeds from
A. muricata contain the highest concentration of ACGs. Seeds represent 5–10% of the whole fruit and are considered a waste; therefore, they can be used as a rich source of ACGs [
2]. However, until now, the yield of ACGs by conventional extraction methods is low. Using maceration was obtained 1% ACGs [
3]; while using the Soxhlet method was extracted 0.04–0.1% ACGs [
4], and with supercritical fluids, 1.68–2.09 mg ACGs/g [
5,
6], all from
A. muricata seeds. Nonetheless, ultrasound-assisted extraction (UAE) is a current technology used to extract ACGs from
A. muricata pulp and by-products, including seeds [
7]. The optimization of the UAE conditions at 25 °C to obtain an ACGs crude extract from
A. muricata seeds was developed by Aguilar-Hernández et al. [
7]. The authors found that the optimal UAE conditions to get a 1.3% yield of ACGs crude extract from whole seeds were 15 min of extraction time, 0.7 s pulse-cycle, and 100% sonication amplitude at 25 °C, using chloroform as solvent. However, the yield of ACGs extraction could increase if the thermosonication conditions are proven.
Combining ultrasound and heat to extract metabolites from natural sources is called thermosonication-assisted extraction (TSAE). It is a promising alternative for removing thermostable bioactive compounds from complex matrices at temperatures from 28 °C to 60 °C [
8]. The ultrasonic waves and heat increase the yield of compounds from plant tissues because heat causes the growth and rapid implosion of microbubbles, increasing the diffusivity of the solvent in the matrix [
9]. Physical changes more frequently reported with thermosonication are fragmentation, detexturation (disruption), erosion, and sonoporation with damage to cell membranes and the internal distortion of the organelles that causes the release of substances from the solid phase to the solvent [
9,
10]. Agcam et al. [
8] reported that TSAE maximized the yield of five different anthocyanins from black carrot pomace using the extraction conditions at 183.1 W/g, 50 °C, and 20 min. On the other hand, López-Ordaz et al. [
11] demonstrated that the optimal TSAE conditions to increase the oil yield (61.12%) from the seeds of
Ricinus communis L were 50% sonication amplitude, 35 min, and a solid:liquid ratio of 1:10 (w:v), compared with Soxhlet extraction since the yield was 57.3% after 8 h of extraction. Moreover, the extraction of peanut oil was investigated using TSAE. The optimum TSAE conditions were 4 min, 60 °C, a ratio solvent: solid of 9:l (
v/
w), and 100% sonication amplitude with a maximum extraction yield of 39.86% [
12].
On the other hand, it has been demonstrated that
A. muricata extracts exhibit antifungal activity. Rizwana et al. [
13] evaluated the in vitro and in vivo effect of extracts from the pulp and seeds of
A. muricata at different concentrations (0.5, 1, 1, 2, 2, 4, 6%
w/
v) as an alternative to synthetic fungicides against
Alternaria alternata causal agent of tomato fruit black spot. These authors reported that methanol extracts of seeds (6%
w/
v) were more potent in inhibiting
A. alternata than pulp extracts. The in vitro assay showed maximum inhibition of mycelial growth of
A. alternata (90%) and a marked reduction in lesion diameter (2.1 mm) in the in vivo assay with 84% disease inhibition in fruit treated with seed extracts. Likewise, León-Fernández et al. [
14] tested the effect of 15 fractionated extracts (chloroform: methanol) of soursop pulp against
Colletotrichum gloeosporioides and
Rhizopus stolonifer, finding that three rich ACGs fractions were the most effective against
C. gloeosporioides and
R. stolonifer with 59% and 38% inhibition, respectively. Until now, there is no information on the application of TSAE in the extraction of ACGs from
Annona seeds nor the antifungal activity of isolated ACGs.
The objective of this work was to extract ACGs from A. muricata seeds using the TSAE and compare the optimal TSAE conditions with ultrasound-assisted extraction (UAE) at 25 °C and the Soxhlet method measuring total ACGs and their antioxidant capacity. In addition, ACGs crude extract was purified (named isolated ACGs) and analyzed by HPLC-DAD. Moreover, the antifungal activity of ACGs crude extract or isolated ACGs was evaluated.
3. Materials and Methods
3.1. Chemicals and Reagents
Two acetogenin samples (Annonacin and pseudoannonacin) from A. muricata pulp were provided by Zepeda-Vallejo’s research group (Department of Organic Chemistry of the National School of Biological Sciences, National Polytechnic Institute, Mexico City, Mexico). The chemical structure of the above acetogenins was established based on semipreparative-HPLC, HPLC–MS, and NMR analysis. The purity of each sample was determined to be above 95%. Silica gel, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid; 2,2-diphenyl-1-picrylhydrazyl; methanol, acetonitrile, 3,5-hydroxybenzoic acid, 2,4,6-tripyridyl-s-triazine, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, ferric chloride hexahydrated and water-HPLC grade were purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). Petroleum ether, potassium hydroxide, dichloromethane, ethyl acetate, hexane, chloroform, ethanol, and acetone, all analytical grade, were purchased from Jalmek Scientific S.A., Guadalajara, Jalisco, Mexico.
3.2. Collection of Raw Material and Its Preparation
The ripened A. muricata fruits were harvested in an orchard in Compostela, Nayarit, Mexico (21°05′00.4″ N 105°08′50.8″ W). The seeds were obtained after manual depulping. They were dried in an oven (Memmert LL-50, Schwabach, Germany) for 24 h at 50 °C to 34.9 ± 1.14% of moisture. The tegument was eliminated from dried seeds, and the endosperms were pulverized in a high-speed multifunctional mill (CGoldenWall HC-2000, San Francisco, CA, USA) to 80–300 µm of particle size.
Endosperm Defatting
Endosperm defatting was performed using Soxhlet equipment (Novatech VH-6, Jalisco, Mexico) with 10 g of the sample placed in the extraction cartridge and 250 mL of petroleum ether. Defatting was carried out for 24 h, and the defatted endosperm was dried for 4 h at 50 °C [
35].
3.3. Thermosonication-Assisted Extraction (TSAE) of Crude Acetogenins from the Defatted Endosperm of Annona muricata Seeds
A Box-Behnken design was used. The individual and interaction effects of sonication amplitude (XSA 80, 90, and 100%), pulse-cycle (XPC 0.5, 0.7, and 1 s), and sonication temperature (XTE 40, 50, and 60 °C) were evaluated. Temperatures were selected as a strategy to avoid solvent evaporation considering that the boiling temperature of methanol is 64.7 °C and they were stable (
Table S1, Supplementary Materials); however, when there was a loss of dissolvent (only at 60 °C), methanol was added (2–3 mL) to complete total volume (35 mL). The range of the analyzed experimental conditions were based on experiments published by Aguilar-Hernández et al. [
7], López-Ordaz et al. [
11], and Ketenoglu [
12]. TSAE was developed using a UP400S ultrasonic system (ultrasound power of 400 W, 24 kHz frequency) (Hielscher Ultrasonic, Teltow, Germany) with an ultrasonic probe (H7 Tip 7, Hielscher, Teltow, Germany) of maximum amplitude (175 µm) and an ultrasound intensity of 300 W/cm
2. The defatted endosperm (2 g) was mixed with 35 mL of methanol previously heated at temperature according to design. The ultrasonic probe was immersed 2 cm in the methanolic solution, and extraction was by 50 min. The temperature was regulated with a cold-water recirculating bath (Thermo Scientific 2870, Waltham, MA, USA). The samples were centrifuged (Hermle Z32HK, Wehingen, Germany) at 11,624×
g for 15 min at 4 °C. The residue was resuspended with methanol, and the extraction was repeated. The supernatants were joined and used for the ACGs analysis. The design was performed for three replicates.
3.3.1. Total Acetogenins Content (TAC) and Yield
The TAC was determined according to the established procedure by Aguilar-Hernández et al. [
7]. The colorimetric reaction was carried out with 50 µL of the methanolic extract and 2 mL of Kedde’s reagent (3,5-hydroxybenzoic acid dissolved in methanol and 5.7% potassium hydroxide solution). The absorbance was measured at 505 nm in a spectrophotometer (Jenway 6705, Dunmow, UK). The analysis was performed in triplicate. The TAC was calculated with a standard annonacin curve, and the results were expressed as milligram annonacin equivalents per gram of dry weight (mg/g DW). The yield percentage was calculated with Equation (3).
3.4. Response Surface Analysis to Obtain the Optimal Tsae Conditions of Acetogenin Crude Extract from the Defatted Endosperm of Annona muricata Seeds
The optimal TSEA conditions for the acetogenin extraction and yield from the defatted endosperm of
A. muricata seeds were obtained by applying the RSM. The predicted values were calculated from the second-order polynomial Equation (4).
where Y is the predicted response (TAC or Yield), Xi is the coded or uncoded value for the factors (X
TE, X
PC, and X
AS), β0 is a constant, βi is the main effect of the coefficient for each variable, and βij are the interaction effect coefficients.
Model adequacy was evaluated by analysis of variance (ANOVA) to determine the significant effect of independent variables and correlation coefficients (square R) at a significance level of 5%. Moreover, data were adjusted (adjusted R) with the second-order polynomial equation by multiple regression using Statistic software (v. 10 Statsoft, Tulsa, OK, USA).
3.5. Model Reliability and Comparison of the Optimal TSAE Conditions with Ultrasound and Soxhlet Method to Obtain Acetogenin Crude Extract
The optimal TSAE conditions on TAC extraction were experimentally verified with three replicates to validate the model reliability. The antioxidant capacity (AOX) was also evaluated. Moreover, the results were compared with those obtained by UAE at 25 °C and the Soxhlet method.
UAE at 25 °C was performed using the same UP400S ultrasonic equipment (Hielscher Ultrasonics, Teltow, Germany) and ultrasonic probe. The procedure was started with 2 g of defatted endosperm sonicated with 35 mL of methanol at 100% amplitude, pulse-cycle of 0.5 s, and extraction time of 50 min at 25 ± 2 °C using a cold-water recirculating bath (Thermo Scientific 2870, Waltham, MA, USA). The extracts were centrifuged (11,624× g, 5 min, 4 °C). The residue was resuspended with methanol, and the extraction was repeated. The supernatants were joined and analyzed.
The defatted endosperm (10 g) was weighed and placed in extraction cartridges, and 150 mL of methanol was added to the flask and placed in the Soxhlet equipment at 70 ± 2 °C for 10 h [
26]. Then, the extract was concentrated in a rotary evaporator (Yamato RE300, Tokyo, Japan) for 20 mL and analyzed.
Total Acetogenin Content and Antioxidant Capacity
TAC was determined, as was mentioned in
Section 3.3.1. The antioxidant capacity of the acetogenin extracts was evaluated by three in vitro methods. ABTS radical (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) assay was evaluated according to Re et al. [
36] with some modifications. The samples (35 µL) were mixed with 265 µL of the ABTS (7 mM) and shaken in the dark for 7 min at 30 °C. The absorbance was measured at 734 nm. DPPH (1,1-Diphenyl-2-picrylhydrazyl) assay was performed based on the Prior et al. [
37] method with some modifications. Briefly, the extract (40 µL) was reacted with 260 µL of DPPH solution (190 µM), and after 10 min in the dark, the absorbance was measured at 517 nm. Ferric reducing antioxidant power (FRAP) assay was carried out with the methodology described by Benzie and Strain [
38]. The extracts (36 µL) were mixed with 9 µL of distilled water and 264 µL of FRAP reagent (solution 10:1:1 (
v/
v/
v) of 0.3 M sodium acetate buffer, 10 mM 2,4,6-tripyridyl-s-triazine and 20 mM ferric chloride hexahydrated). After 30 min in the dark, the absorbance was measured at 595 nm. All absorbances were measured on a microplate reader (Bio-Tek Synergy HT, Winooski, VT, USA). Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) was used as a standard for the calibration curve. The results were expressed in micromoles of Trolox equivalents per gram of dry weight (μmol TE/g DW).
3.6. Solubility and Identification by HPLC-DAD of Isolated Acetogenins
3.6.1. Isolation of Acetogenins by Column Chromatography
First, a methanolic extract (500 mL) was obtained under optimal TSAE conditions. The methanolic extract was evaporated to dryness in a rotavapor (Yamato RE300, Tokyo, Japan) to get a crude extract. Approximately 2 g of ACGs crude extract was subjected to a chromatographic column (6.4 × 57.0 cm). Silica gel (60 mesh) was used as a stationary phase, and dichloromethane: ethanol mixture (9:1
v/
v) was used as an initial eluent with a gradual increase in polarity until finishing with 100% ethanol. The obtained fractions were subjected to thin-layer chromatography (TLC) and revealed with Kedde reagent to confirm the presence or absence of ACGs [
7]. The ACG-rich fractions were joined (F1). F1 was subjected to a flash chromatography column (6 cm × 25 cm, SiO
2, 230–400 mesh), using a mixture of dichloromethane: ethyl acetate as eluent. The ratio of eluents during the elution was 98:2 (
v/
v) until ACGs stopped eluting (91:9
v/
v). The fractions were submitted to TLC. Those rich in ACGs were joined (F2) and subjected to an additional assay by flash column chromatography for the final isolation process. The resultant fractions were submitted to TLC to select four ACG-positive fractions, and they were joined (F3). Finally, F3 was concentrated to dryness and used to evaluate its solubility to be analyzed by HPLC-DAD.
3.6.2. Solubility of Isolated Acetogenins
Ten solvents (water, petroleum ether, acetonitrile, hexane, dichloromethane, chloroform, ethyl acetate, ethanol, methanol, and acetone) were used for the solubility test. Briefly, 1 mg isolated acetogenins (F3) was dissolved in 1 mL of each solvent. Subsequently, the mixture was shaken for 1 min in a vortex until complete homogenization. The test was repeated until the saturation of ACGs in each solvent. The results were reported in dissolved milligrams per milliliter of solvent (mg/mL).
3.6.3. Analysis of Acetogenins by HPLC-DAD
Partial identification of acetogenins was carried out according to the conditions established by Yang et al. [
5,
6]. Isolated acetogenins (F3) were resuspended in methanol (0.22 mg/mL) and, before injection, were filtered through 0.22 µm membrane filters. Samples (10 µL) were injected into an HPLC system (Agilent Technologies 1260 Infinity, Waldbronn, Germany) equipped with a photodiode array detector (DAD) and an Agilent Zorbax Extend C18 reverse-phase column (250 mm × 4.6 mm, 5 µm) at 30 °C. The mobile phase consisted of methanol (eluent A) and water (eluent B), using a linear gradient: 0–40 min (85% A) and 40–60 min (85–95% A) at a flow rate was 1 mL/min. In addition, other separation tests were made by changing the column temperature to 10 °C, 15 °C, 20 °C, or 25 °C. ACGs were detected at 220 nm.
Subsequently, the identification of ACGs was analyzed according to the chromatographic results presented by Yang et al. [
5,
6]. The results are shown as area values and percentages of each ACG. In addition, two acetogenins were quantified with calibration curves.
3.7. Antifungal Activity of Crude Extract and Isolated Acetogenins
The antifungal activity of the crude extract and isolated acetogenins on
Candida albicans (ATCC),
C. glabrata (ATCC 15126),
C. tropicals (ATCC 1369), and
C. krusei (ATCC 14243) strains was carried out by the disk diffusion method according to Anaya-Esparza et al. [
39] with some modifications. The strains were grown aerobically in nutrient broth (8 g/L, pH 7.0 ± 0.1) for 24 h at 37 °C until the suspensions reached 1 × 10
6 CFU/mL compared to 0.5 of the McFarland standard. The disk diffusion assay was performed by passing each
Candida strain through nutrient agar (23 g/L, pH 6.8 ± 0.1). Subsequently, sterile filter paper discs of 7 mm diameter were impregnated (200 μL) with suspensions of the crude extract and isolated ACGs dissolved in dimethyl sulfoxide (DMSO) at concentrations of 12.5, 25, 50, 100, 200, 400, 800, 1000, 2000, and 4000 µg/mL and placed in inoculated Petri dishes using sterile forceps. The antibiotic ketoconazole (500 µg/mL) was used as a positive control, sterile distilled as the negative control, and DMSO as solvent. Subsequently, the Petri dishes were incubated at 37 °C for 48 h. The diameter of the inhibition zone formed around the disks was measured with a Vernier and reported in millimeters (mm). The procedure described above was performed in triplicate for each concentration and each
Candida strain in both extracts.
According to the highest inhibition (mm) presented by the ACGs crude extract and isolated ACGs in the different strains, two strains (C. albicans and C. tropicalis) were selected to calculate the medium inhibitory concentration (IC50). IC50 was used to carry out the sublethal damage and lethality assays in the same chosen strains. Moreover, the minimum inhibitory concentration (MIC) of crude extract and isolated ACGs in these strains was calculated.
Lethality and sublethal damage of crude extract and isolate ACGs on
C. albicans, and
C. tropicalis strains were evaluated by serial dilution assay using the pour-plate method established by Anaya-Esparza et al. [
32]. The nutrient broth (200 mL) was inoculated with 10 mL/L of cell suspension (10
6 CFU/mL) adjusted according to McFarland’s scale (0.5 of absorbance), spiked with the ACGs (100 µg/mL), and incubated for 15 min at 37 °C. Subsequently, 1 mL of the above solution was transferred to 9 mL of sterile saline (0.85%
w/
v) and homogenized. Serial dilutions (1 × 10
−1 to 1 × 10
−7 CFU/mL) were made in saline (9 mL), and 1 mL aliquots were taken and seeded on nutrient agar, and plates were poured. This procedure was repeated for each extract and with each strain, and the results were reported as log CFU/mL. Lethality was calculated as the difference between the logarithms of the colony counts in the control group without ACGs (N
0) and the colony counts in the samples treated with the extracts (N) (Log CFU/mL N
0-Log CFU/mL N).
The sublethal damage was calculated as the difference between the CFU of the control group and the CFU of strains treated with ACGs and expressed as a percentage (%).
3.8. Statistical Analysis
Data were expressed as means ± standard deviation (n = 6). The results from the Box–Behnken design were analyzed with the RSM. Subsequently, the other experiments were conducted in a one-factorial experimental design. The data were performed using ANOVA (p < 0.05) with Statistic software (v.10 Statsoft, Tulsa, OK, USA). Tukey test examined the mean differences between treatments (α = 0.05).