*2.5. 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% (SC50). Trolox was used as a positive control.

**Table 5.** Antioxidant activity of essential oils of *Annona cherimola*.


Through the DPPH method, the essential oils of *A. cherimola* showed strong antioxidant activity with a SC50 value of 470 ± 30 μg/mL. Employing the ABTS technique the SC50 could not be calculated at the concentration ranges tested (Figure 1).

#### *2.6. 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 IC50 value. The IC50 value obtained for chirimoya essential oil was 41.51 ± 1.02 μg/mL. The positive control (donepezil) exhibited an IC50 value of 13.80 ± 1.01 nM.

**Figure 1.** Scavenging capacity vs. concentration of *Annona cherimola* essential oil obtained by DPPH and ABTS assays.

**Figure 2.** Half-maximum inhibitory concentration of *Annona cherimola* essential oil against acethylcholinesterase.

#### **3. 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].

The major components (>5%) identified in the *A. cherimola* EO were germacrene D (28.77%), bicyclogermacrene (11.12%), (E)-caryophyllene (10.52%), sabinene (9.05%) and βpinene (7.93%). The results are different to those reported by Elhawary et al. fβ-caryophyllene with 9.50%, germacrene-D with 17.71% an β-elemene with 25.02% [21], and those reported by Rios et al. reported bicyclogermacrene (18.20%), trans-caryophyllene (11.50%), α-amorphene (7.57%), α-copaene (5.63%) and germacrene D (3.75%) [20]. In addition, Pino observed that the major compounds were α-thujene (18.7 ppm), α-pinene (23 ppm), terpinen-4-ol (19.8 ppm) and germacrene D (17.6 ppm) [16]. Despite the differences in their concentrations, the main component that is common in all the studies is germacrene D. It is well known the influence of different cultivation and climatic factors over the chemical composition of the essential oils.

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 (−)-α-pinene (1S,5S) (63.99%), (−)-sabinene (1S,5S) (95.91%), (−)-limonene (4R) (27.50%) and (−)-germacrene D (8S) (95.91%).

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 SC50 of 470 μg/mL for the DPPH assay while the SC50 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 SC50 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 IC50 value of 41.51 μg/mL, this inhibitory activity could be considered very strong compared to the related EO of *Piper carpunya* (IC50 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].

#### **4. Materials and Methods**

#### *4.1. Materials*

Dichloromethane (DMC), methanol (MeOH), sodium sulfate anhydrous, DPPH (2,2 diphenyl-1-picrylhydryl), ABTS (2,2 -azinobis-3-ethylbenzothiazoline-6-sulfonic acid), acetylcholinesterase (AChE), acetylthiocholine (ATC), phosphate buffered saline, Ellman's reagent (donepezil, 5,5 -dithiobis(2-nitrobenzoic acid)), tris hydrochloride (Tris-HCl), magnesium chloride hexahydrate and 2,3,5-Triphenyl tetrazolium chloride (TTZ) were purchased from Sigma-Aldrich (San Luis, MO, USA). The microbiological media such as Mueller Himton broth, Mueller Hinton II broth and fluid thioglycollate medium were purchased from DIPCO (Quito, Ecuador). Horse serum and Oxoid CampyGen were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The standard aliphatic hydrocarbons were purchased from ChemService (West Chester, PA, USA). Helium was purchased from INDURA (Quito, Ecuador). All chemicals were of analytical grade and used without further purifications.
