Enantiomeric Composition, Antioxidant Capacity and Anticholinesterase Activity of Essential Oil from Leaves of Chirimoya (Annona cherimola Mill.)

Annona cherimola Mill. is a native species of Ecuador cultivated worldwide for the flavor and properties of its fruit. In this study, hydrodistillation was used to isolate essential oil (EO) of fresh Annona cherimola leaves collected in Ecuadorian Sierra. The EO chemical composition was determined using a non-polar and a polar chromatographic column and enantiomeric distribution with an enantioselective column. The qualitative analysis was carried out by gas chromatography coupled to a mass spectrometer and quantitative analysis using gas chromatography equipped with a flame ionization detector. The antibacterial potency was assessed against seven Gram-negative bacteria and one Gram-positive bacterium. ABTS and DPPH assays were used to evaluate the radical scavenging properties of the EO. Spectrophotometric method was used to measure acetylcholinesterase inhibitory activity. GC-MS analysis allowed us to identify more than 99% of the EO chemical composition. Out of the fifty-three compounds identified, the main were germacrene D (28.77 ± 3.80%), sabinene (3, 9.05 ± 1.69%), β-pinene (4, 7.93 ± 0.685), (E)-caryophyllene (10.52 ± 1.64%) and bicyclogermacrene (11.12 ± 1.39%). Enantioselective analysis showed the existence of four pairs of enantiomers, the (−)-β-Pinene (1S, 5S) was found pure (100%). Chirimoya essential oil exhibited a strong antioxidant activity and a very strong anticholinesterase potential with an IC50 value of 41.51 ± 1.02 µg/mL. Additionally, EO presented a moderate activity against Campylobacter jejuni and Klebsiella pneumoniae with a MIC value of 500 μg/mL.


Introduction
Worldwide, the Annonaceae family comprises more than 128 genera and approximately 2106 species and they are mainly distributed in tropical and subtropical regions [1]. For Ecuador, 25 genera, 106 species and 20 endemic species are reported [2]. In a review of the antimalarial properties of the Annonaceae family, 11 species from Annona and Xilopia genus were recognized for their antiparasitic potential. Annonaceae species used in traditional medicine, over the tropical regions, are well documented for having potential for the treatment of parasitic diseases such as Malaria, Chagas and Leishmaniasis as well as other illnesses [3]. Indeed, Annona muricata was one of the most cited species with a variety of medicinal properties including the treatment for the symptoms of malarial infection, fever, liver ailments and headaches [4].
Annona cherimola, A. crassiflora, A. muricata, A. squamosa and A. reticulata are the commercial species, highly valued by their exotic edible fruit. Furthermore, different parts of the tree from these species have been used in folk medicine to treat several conditions including gastrointestinal diseases, diabetes and hypertension [5]. Many secondary metabolites have been reported such as phenols and other bioactive compounds but, the main chemical marker of the genus is a diverse group of polyketides called acetogenins, compounds closely associated to their antiproliferative effect on cancer cell lines [5,6].
In a related study, nine species from Annona genus, including A. cherimola, where reviewed in relation to their phytochemical composition and biological activity and found that mainly polar extracts obtained from these plants, induce a reduction in blood sugar levels in chemically induced type-2 diabetic rats which demonstrate the antidiabetic potential of species from this genus. Likewise, seven out of nine of the studied species reported a good antioxidant capacity profile in different in vitro assays. A. cherimola was only tested trough the oxygen radical absorbance capacity (ORAC) assay [7].
Annona cherimola Mill. is a native shrub, widely distributed in the Andean, Coastal, Amazon and Insular regions of Ecuador, between 0-3000 m a.s.l [2]. Currently, this species is cultivated in the subtropical and tropical regions worldwide, especially for its fruit, which is considered exotic. The A. cherimola species is commonly known as "chirimoya" or "chirimoyo" (Spanish language) and "custard-apple" (English language) [8]. The vernacular name "chirimoya" is derived from the Quechua (indigenous language) word "chirimuya", "chiri" that means cold and "muya" seeds. The fruit of this species is considered one the most appreciated inside the genus. The plant has been used in traditional medicine for the treatment of boils and others skin diseases [9].
Anthropological evidence suggests that the species A. cherimola was cultivated since the times of the Incan Empire and its fruit was considered as an active ingredient in the their diet [10]. Some important compounds have been isolated from this species such as alkaloids as cherimoline, annocherine A, annocherine B, cherianoine and rumocosine H [11], and some amides as cherinonaine, cheritamide, (N-trans-feruloyltyramine, N-trans-caffeoyltyramine, N-cis-caeoyltyramine, dihydro feruloyltyramine, N-transferuloylmethoxytyramine, N-cis-feruloylmethoxytyramine, and N-p-coumaroyltyramine [12]. Recent studies have shown that custard apple leaves contain flavonoids and other phenolic compounds with biological properties [13,14] and that alcoholic extracts from the leaves have proapoptotic and antidepressant activities [15].
Several studies on the species of the Annona genus have reported the occurrence of compounds with potential application for pharmaceuticals, food, agrochemicals products and cosmetics [3,5,6,16]. Extracts from these species are used for a wide range of beneficial purposes, however, the research have been focused mainly on the non-volatile fraction of the fruits, meanwhile, the enantiomeric distribution and biological properties of the EOof Annona cherimola have not been reported previously. This fact stimulated our interest in studying the chemical composition, enantiomeric distribution and antimicrobial, antioxidant and anticholinesterase activities of the essential oil of custard apple leaves.

Results
A total of 15,150 g of fresh custard apple leaves, with a moisture of 53 ± 5% (w/w), were used as raw material for the extraction. The isolation of the essential oil was carried out by hydrodistillation using a Clevenger-type apparatus. The amount of EO obtained was 38.5 mL, which represents a yield of 0.25 ± 0.02 (v/w).

Physical Properties of Essential Oil
The essential oil isolated from leaves of A. cherimola was presented as a viscous liquid with a characteristic texture. The physical properties of EO are shown in Table 1. In addition, this table shows the subjective color and its RGB and CMYK values.

Enantioselective Analysis
The enantioselective analysis from Annona cherimola EO was achieved for the first time. The Table 3 shows the enantiomeric distribution, linear retention indices and enantiomeric excess (e.e.) of each enantiomer. Using a chiral column could be quantified four pairs of enantiomers, whose peaks were well separated at the base. The β-pinene (−) was found practically pure, while (−)-α-pinene and (−)-sabinene and (−)-germacrene D exhibited a high enantiomeric excess, whereas (−)-limonene and (+)-limonene were found in a racemic mixture.

Antibacterial Activity
Microdilution broth method was used to assess the antibacterial activity of essential oil of A. cherimola leaves. Tetracycline was used as a positive control and the maximum evaluated concentration was 1000 µg/mL. The minimum inhibitory concentration (MIC) values and the microorganisms used (seven Gram-negative bacteria and one Gram-positive bacterium) are shown in Table 4. The A. cherimola essential oil reported MIC values of 500 µg/mL against Campylobacter jejuni (ATCC 33560). EO dissolved in aqueous media caused the formation of an emulsion that difficulted the visual observation of bacterial growth particularly with C. jejuni. The reduction of 2,3,5-Triphenyl tetrazolium chloride (TTZ) yield a red color product only in the wells were bacteria developed a well growth. A blank of EO with the same range of concentrations and media was included to discard interferences due to contamination which was also confirmed by reading at 405 nm (data not shown) For the other bacteria, the essential oil did not show activity at the maximum dose tested.

Antioxidant Capacity
The results obtained for DPPH and ABTS radical scavenging of the EO are shown in Table 5. The results are expressed as the concentration of the EO that scavenge or decrease the concentration of the radical at 50% (SC 50 ). Trolox was used as a positive control. Through the DPPH method, the essential oils of A. cherimola showed strong antioxidant activity with a SC 50 value of 470 ± 30 µg/mL. Employing the ABTS technique the SC 50 could not be calculated at the concentration ranges tested (Figure 1).

Anticholinesterase Activity
Three different concentrations of the essential oil from A. cherimola leaves were used to determine its anticholinesterase potential. The data obtained by measuring the rate of reaction of AChE against EO are shown in Figure 2. The results plotted as Log (concentration essential oil) vs. normalized response rate of reaction allowed us to calculate the IC 50 value. The IC 50 value obtained for chirimoya essential oil was 41.51 ± 1.02 µg/mL. The positive control (donepezil) exhibited an IC 50 value of 13.80 ± 1.01 nM.

Discussion
The essential oil from Annona cherimola exhibited a low yield of 2.5 ± 0.2 mL/Kg [17]. The extraction yield of essential oils is very variable between plant species and depends on different aspects related to the plant such as the part, the age and the time after plant collection and other aspects related to the isolation process such as the pretreatment of the material (drying, grinding, etc.) and the extraction time [18].
The aroma of the Annona species is well recognized and has been studied in some species, however, little has been reported about the essential oil composition of Annona cherimola. In the present study, the main chemical components identified were aliphatic monoterpenes (25.68%) and aliphatic sesquiterpenes (69.40%), which was similar to the information reported by Rabelo et al. [19]. Furthermore, Rios et al. in 2003 [20] reported monoterpenes (6.09%) and sesquiterpenes (76.56%) as the main type of compounds in the A. cherimola EO. On the other hand, the same type of volatile compounds were meaningful in fruits of Annona cherimola (monoterpene 40.3% and sesquiterpene 24.3%) [16].
Due to the relevance of aromatic compounds of the Annona species Ferreira et al. in 2009 compared the essential oil and the volatile compounds of the leaves and fruits of Annona cherimola. The chemical composition for the EO was different to the volatile compounds in fruits, the main compounds in the leaves essential oil were identified in lower quantities, germacrene-D (0.11% to 0.22%), sabinene (not identified), β-pinene (0.79% to 3.60%), (E)-caryophyllene (0.23% to 0.32%) and bicyclogermacrene (not identified) while the main compounds analyzed by headspace solid phase microextraction were methyl butanoate, butyl butanoate, 3-methylbutyl butanoate, 3-methylbutyl 3-methylbutanoate and 5-hydroxymethyl-2-furfural [22]. This is the first report of enantioselective GC-MS analysis of A. cherimola EO, this analysis showed the ocurrence of five pairs of enantiomers and one enantiomerically pure chiral monoterpenoid, β-pinene. The enantiomeric ratio of an essential oil is an important information which could be related with the biological activity, metabolism and organoleptic quality of the enantiomeric pairs [23]. The enantiomeric excess (e.e %) were Regarding their biological activity, the essential oil of Annona cherimola showed moderate antibacterial activity against Campylobacter jejuni (ATCC 33560) and Klebsiella pneumonia (ATCC 9997), both with MIC at 500 µg/mL and no activity for the other bacteria tested (MIC was higher than 1000 µg/mL). Compared to data reported in the literature, Rios et al. in 2003 reported a significant activity against two Gram-positive bacteria Staphylococcus aureus (MIC 250 µg/mL) and Enterococcus faecalis (MIC 500 µg/mL), however, the MIC values for Gram-negative bacteria were higher than 5000 µg/mL [20]. Elhawary et al. in 2013 reported the MIC of EO A. cherimola for Bacillus subtilis (130 µg/mL), Staphylococcus aureus (285 µg/mL), Escherichia coli (110 µg/mL), Pseudomonas aeruginosa (140 µg/mL), and Candida albicans (152 µg/mL) [21]. When the antibacterial activity of pure compounds was analyzed [20] the MIC of trans-caryophyllene, β-pinene, linalool, and other compounds was higher than the value for the essential oil, therefore suggesting that the antibacterial potency could be exerted by a synergistic effect among the constituents above mentioned. The essential oil of Annona species showed a wide range of biological activity, for A. vepretorum Costa et al. in 2012 reported a moderate activity (MIC 500 µg/mL) against Staphylococcus aureus and Staphylococcus epidermis and a significant activity against Candida tropicalis (MIC 100 µg/mL) [24]. Another study, in 2013, Costa et al. observed the antibacterial activity of essential oil of A. salzmannii and A. pickelii against Staphylococcus aureus, Staphylococcus epidermis and Candida tropicalis with MIC of 500 µg/mL [25].
Some studies have shown that the enantiomers of a compound have different biological activities. Lis-Balcnin et al. reported that 18 out of 25 different bacteria were more affected by the (−)-α-pinene in comparison with the (+) enantiomer, 19 out of 20 different Listeria monocytogenes strains were affected more by (+)-α-pinene isomer and two of three filamentous fungi were affected more by the (+) enantiomer [26]. The MIC and minimal microbicidal concentration (MMC) showed that the positive enantiomers of pinene exerted a microbicidal effect against all the fungi and bacteria tested with MIC values ranging from 117 to 4150 µg/mL. However, with concentrations up to 20 mg/mL of the negative enantiomers, no antimicrobial activity was observed [27]. The MIC values against three Gram-positive (B. cereus, E. faecalis and S. aureus) and four Gram-negative (E. coli, K. pneumoniae, M. catarrhalis and P. aeruginosa) bacteria were in the ranges of 3 to 27 mg/mL for (+)-limonene and 2 to 27 mg/mL for (−)-limonene. The greatest difference was obtained against Staphylococcus aureus ATCC 12600 where the (+)-limonene showed a MIC of 14 mg/mL and the (−)-limonene a MIC of 4 mg/mL [28]. Omran et al. found that (−)-limonene had better antifungal activity than (+)-limonene against Aspergillus niger, Aspergillus sp., Candida albicans and Penicillium sp. [29]. It was not possible to find previous studies about the antifungal or antibacterial activity of the (+) and (−) enantiomers of the main compound germacrene D, however, Stranden et al. determined that the two enantiomers of this compound mediate the same kind of information to the receptor neurons of the moth Helicoverpa armigera, but (−)-germacrene D had approximately 10 times stronger effect than (+)-germacrene D [30]. The difference in biological activity of the enantiomers is maintained even when they are mixed with other compounds [28]. The enantiomers of a compound have different biological activities, then, the enantiomeric distribution of the compounds could influence the biological activity for an essential oil.
Regarding their antioxidant effect, the Annona cherimola essential oil showed an SC 50 of 470 µg/mL for the DPPH assay while the SC 50 was >1000 µg/mL in the ABTS assay. Costa et al. reported as strong the antioxidant activity of EO A. salzamannii and A. pickelii measured by a TLC-based DPPH assay, however, the individual components β-pinene and α-pinene did not show antioxidant activity [25]. Araújo, et al. [31] and Costa, et al. [24] reported a weak antioxidant activity for the EO of A. vepretorum. Another study, Gyesi, et al. in 2019 [32] reported an SC 50 of 244.8 µg/mL from the DPPH assay for the EO of A. muricata. The differences between the antioxidant activity of EO and pure compounds could correspond to synergistic effects among the components in the essential oil.
The acetylcholinesterase inhibitory activity of A. cherimola EO has not been previously reported. Chirimoya EO showed an AChE IC 50 value of 41.51 µg/mL, this inhibitory activity could be considered very strong compared to the related EO of Piper carpunya (IC 50 of 36.42 µg/mL) [33]. The inhibition of AChE due to EO is of relevant interest in the treatment of Alzheimer disease since different studies report in vitro and clinical AChE inhibitory activity. Benny and Tomas summarize the neuroprotective effects of EO and its relevance on Alzheimer disease stating that EO could rebuild the antioxidant status of brain which confer neuroprotective effect as in the case of EO of Coriandrum sativum L., Syzygium aromaticum (L.), Juniperus communis, Rosmarinis officinalis (L.), and other species. The same activity has been observed for pure compounds such as thymol, linalool, αterpinene, α-terpineol, carvacrol, (E)-β-caryophyllene, α-pinene, and eugenol [34].

Plant Material
Annona cherimola leaves were collected under permission granted for Ministerio del Ambiente de Ecuador (Ecuadorian Environmental Ministry) by means of the authorization No. 001-IC-FLO-DBAP-VS-DRLZCH-MA. The leaves of chirimoya were collected between the months of October and January in the surroundings of the locality of Cango Bajo, canton Calvas, province of Loja, Ecuador, at 1950 m a.s.l. at a latitude of 4 • 20 34 S and a longitude of 79 • 34 24 W. Collection, store and transfer of chirimoya leaves were performed according to what is described by Valarezo et al. [35]. Botanist Nixon Cumbicus made the identification of the plant material. A voucher specimen was deposited at the Herbarium of Universidad Técnica Particular de Loja (HUTPL).

Postharvest Treatments
Between 2 and 3 h after being collected, the plant material was subjected to the postharvest treatments, which consist of the separation of degraded leaves and foreign material.

Moisture Determination
Method Loss on drying (Moisture) in plants, AOAC 930.04-1930, was used to determine the moisture of plant material, for this an analytical balance (Mettler AC 100, Mettler Toledo, Columbus, OH, USA) was used. Moisture was calculated according to Equation (1).
where wi is the initial weight of sample and wo is weight of sample after drying.

Essential Oil Extraction
The isolation of the essential oil from leaves of A. cherimola was carried out by hydrodistillation using a Clevenger-type apparatus according to the procedure described by Valarezo et al. [36]. After being collected, the essential oil was dried using anhydrous sodium sulphate. Finally, the EO was stored at 4 • C in amber sealed vials until being used in the subsequent analysis.

Determination of the Physical Properties of the Essential Oil
Density of the essential oil was determined using the ISO 279:1998 standard (equivalent to the AFNOR NF T 75-111 standard). Density measurement was performed using an analytical balance (Mettler AC 100, Mettler Toledo, Columbus, OH, USA) and a pycnometer of 1 mL. Refractive index was determined using the standard ISO 280:1998 [37] (similary to AFNOR NF T 75-112), for which a refractometer (model ABBE, BOECO, Hamburg, Germany) was used. An automatic polarimeter (Mrc-P810, MRC, Holon, Israel) was used to measure the optical rotation of the EO according to the standard ISO 592:1998. All measurements were taken at 20 • C.

Quantitative and Qualitative Analysis
The dentification of the chemical constituents of the essential oil was carried out using an Agilent gas chromatograph (GC) (6890N series, Agilent Technologies, Santa Clara, CA, USA). For the quantitative analysis gas chromatograph was equipped with a flame ionization detector (FID) and for qualitative analysis gas chromatograph was coupled to a mass spectrometer (quadrupole) detector (MS) (model Agilent 5973 inert series, Agilent Technologies, Santa Clara, CA, USA). The GC-FID and GC-MS analyses were performed according to the procedure described by Valarezo et al. [35]. The injection of the samples was carried out by an automatic injector (Agilent 7683 automatic liquid sampler, Agilent Technologies, Santa Clara, CA, USA) in split mode. Chromatographic runs were performed using a nonpolar and a polar column. The nonpolar was an Agilent J&W DB-5ms Ultra Inert GC column with stationary phase 5%-phenyl-methylpolyxilosane and the polar was an Agilent J&W HP-INNOWax GC column with stationary phase polyethylene glycol. Both columns with a length of 30 m, an outer diameter of 0.25 mm and a stationary phase thickness of 0.25 µm. Identification of the EO compounds was based on a comparison of relative retention indices (RIs) and mass spectra data with those of the published literature [38,39] according as described by Valarezo et al. [36].

Enantioselective Analysis
The enantiomeric distribution was performed using an enantioselective column with stationary phase 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin. The chromatographic run was performed with a temperature ramp of 2 • C/min from 50 • C (maintained for 2 min) to 220 • C (maintained for 2 min) in an Agilent gas chromatograph (model 6890N series, Agilent Technologies, Santa Clara, CA, USA) coupled to a mass spectrometer (quadrupole) detector (model Agilent 5973 inert series, Agilent Technologies, Santa Clara, CA, USA). The injection of enantiomerically pure standards was used to determine the order of elution of the enantiomers.

Evaluation of Antibacterial Activity
The antibacterial activity of the essential oil from chirimoya leaves was assessed against Gram-negative and Gram-positive bacteria (Table 4) by the microdilution broth method according to the procedure described by Valarezo et al. [40]. The bacterial strains were incubated in Müeller-Hinton (MH) broth. Tetracycline was used as a positive control and DMSO was used as a negative control. Results are reported as minimum inhibitory concentration (MIC).
For Campylobacter jejuni (ATCC 33560) the broth microdilution method was carried out according to Valarezo et al. [40] with some specific requirements as described briefly. Fluid thioglycollate medium was used for reactivation of the strain, supplemented with 5% of Horse serum. An aliquot of a cryogenic reserve was resuspended in thioglycollate and incubated for 48 h at 37 • C in a microaerophilic atmosphere provided by an Oxoid CampyGen (2.5 L sachet). Sample solutions were made by dissolving 80 mg of EO in 1 mL of DMSO. Two-fold serial dilutions were employed to obtain decreasing concentrations of EO from 4000 to 31.25 µg/mL and cation-adjusted Muller Hinton II broth (pH 7.3) with 5% lyssed horse blood [41] as media for the antibacterial assay. The inoculum was prepared from thioglycollate culture and adjusted to 0.5 McFarland. Final concentration of bacteria was 5 × 10 5 CFU/mL. The microplate was incubated for 48 h at 37 • C in microaerophilic atmosphere (5% CO 2 , Oxoid CampyGen). Erythromycin was used as positive control with a MIC value of 15.65 µg/mL and DMSO as negative control. MIC was determined by visual examination of growth and through addition of a 1% solution of TTZ as bacterial viability indicator after incubation time to confirm the visual results. A blank with the same range of concentrations of EO was prepared simultaneously and measurements at 405 nm were made to discard reduction of TTZ by contamination. Optical density (OD) for TTZ reduction was measured in a microplate reader (EPOCH 2, BioTek, Winooski, VT, USA).

DPPH Radical Scavenging Capacity
The DPPH assay was performed using 2,2-diphenyl-1-picrylhydryl free radical (DPPH • ) based on the technique described by Brand Williams et al. [42] and Thaipong et al. [43] according to what was described by Valarezo et al. [33]. The concentrations of the EO from A. cherimola leaves used were 1000, 800, 600, 450, 300, 150 and 25 ppm. Trolox was used as a positive control and methanol as a blank control. The samples were evaluated in a UV spectrophotometer (Genesys 10S UV.Vis Spectrophotometer, Thermo Scientific, Waltham, MA, USA) at a wavelength of 515 nm. The percentage of scavenging capacity was calculated according to Equation (2). SC 50 is the EO concentration that provided 50% DPPH • scavenging effect.
where AEO is the absorbance of DPPH • mixed with EO and As is absorbance of DPPH mixed with methanol.

ABTS Radical Cation Scavenging Capacity
The ABTS assay was performed using 2,2 -azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS •+ ) according to the procedure report by Arnao et al. [44] and Thaipong et al. [43], as described by Valarezo et al. [33]. The concentrations of the essential oil from A. cherimola used were 1000, 500, 250, 100, 50 and 25 ppm. The samples were evaluated in a UV spectrophotometer (Genesys 10S UV.Vis Spectrophotometer, Thermo Scientific, Waltham, MA, USA) at a wavelength of 734 nm. Deionized water was used as a blank control and trolox was used as a positive control. The percentage of scavenging capacity was calculated according to equation.
where ASO is the absorbance of ABTS •+ with solvent mixture and Ai is the absorbance after reaction of ABTS •+ with the sample.

Anticholinesterase Activity
The AChE inhibitory effect was measured based on the methodology designed by Ellman, et al. [45], with slight modifications as suggested by Rhee, et al. [46], as previously described by Valarezo et al. [33]. The inhibition of AChE was detected after the addition of Acetylthiocholine as the enzyme substrate and several concentrations of EO dissolved in MeOH. The enzyme reaction was monitored in a microplate reader (EPOCH 2, BioTek, Winooski, VT, USA) at 405 nm for 60 min. Final concentrations of 1000, 100, and 10 µg/mL of the EO in MeOH were prepared to assess the enzyme inhibition. The assay was carried out by triplicate in 96-well microplates. Donepezil was used as positive control. The IC 50 value was calculated from the progression curve with the Graph Pad Prism software (v8.0.1.5., Graph Pad, San Diego, CA, USA)

Statistical Analysis
All procedures were repeated three times, except for the biological activity, which was repeated nine times. Microsoft Excel was used to collect the data and Minitab 17 (Version 17.1.0., Minitab LLC., State College, PA, USA) was used to calculate the measures of central tendency. The results are expressed as mean values.

Conclusions
Enantiomeric distribution, antibacterial activity against Campylobacter jejuni, antioxidant and anticholinesterase activities of the essential oil from Annona cherimola leaves were determined for the first time, in addition, the chemical composition and physical properties of this essential oil was studied. This research contributes to our knowledge about native species of Ecuador. The results obtained in this study motivate us to carry out new investigations in endemic and native species of this megadiverse country.