*2.2. Chemical Constituents of the Essential Oil*

The compounds ocurring in *A. cherimola* EO were identified and quantified by GC-MS and GC-FID using nonpolar and polar columns. The Table 2 shows the quantitative and qualitative data of chemical constituents of custard apple obtained using nonpolar column DB-5ms. In the essential oil from chirimoya leaves fifty-three compounds were

identified, which represent 99.80% of the total composition. According to their chemical nature, all the compounds were grouped in aliphatic monoterpene hydrocarbons (ALM), oxygenated monoterpenes (OXM), aliphatic sesquiterpene hydrocarbons (ALS) and oxygenated sesquiterpene (OXS). The ALS were the most representative compounds with twenty-seven compounds, which represents 69.40%, followed by ALM with 25.68%. Compounds belonging to the aromatic monoterpene hydrocarbons, aromatic sesquiterpene hydrocarbons and oxygenated sesquiterpene groups were not identified. The ALS germacrene D (compound 32, CF: C15H24, MM: 204.19 Da) was the main constituent with 28.77 ± 3.80%. Other main compounds (>5%) were sabinene (3, 9.05 ± 1.69%), β-pinene (4, 7.93 ± 0.68%), (E)-caryophyllene (24, 10.52 ± 1.645) and bicyclogermacrene (36, 11.12 ± 1.39%). Compounds 8 and 9 (limonene and β-phellandrene) co-eluted, both representing 0.66 ± 0.13%.

**Table 1.** Physical properties of the essential oil.


<sup>a</sup> Mean of nine determinations: three distillations × three collections, <sup>b</sup> Standard deviation.




**Table 2.** *Cont*.

RT: Retention Time; RI: Calculated Retention Indices; RI ref: References Retention Indices; SD: Standard Deviation; CF: Chemical Formula; MM: Monoisotopic Mass; tr: traces; -: not calculated. <sup>1</sup> Co-eluted compounds.

Coeluting compounds (limonene and β-phellandrene) were separated using an HP-INNOWax polar column. The retention index in this column for limonene was 1192 and for β-phellandrene was 1201. Limonene (mixture of (+)-limonene and (−)-limonene) presented a percentage of 0.55 ± 0.09% and β-phellandrene a value of 0.12 ± 0.01%.

#### *2.3. 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.

#### *2.4. 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.


**Table 3.** Enantiomeric distribution of chiral constituents occurring in the EO of *A. cherimola*.

**Table 4.** Antibacterial activity of essential oil from *Annona cherimola* leaves.


<sup>a</sup> Erythromycin for *Campylobacter jejuni* and tetracycline for other bacteria.
