Study of Essential Oil Isolated from Achiote (Bixa orellana) Leaves: Chemical Composition, Enantiomeric Distribution and Antimicrobial, Antioxidant and Anticholinesterase Activities

Bixa orellana is a native and cultivated species of Ecuador commonly known as achiote (annatto), this species is very versatile with a wide variety of uses and applications of its leaves, fruits and seeds. In this study, the chemical composition, enantiomeric distribution and biological activity of essential oil isolated from the leaves of Bixa orellana were determined. Hydrodistillation was used to isolate the essential oil. Gas chromatography coupled with mass spectrometry was used to determine the qualitative composition, a gas chromatograph equipped with a flame ionization detector was used to determine quantitative composition and gas chromatography on an enantioselective column was used to determine enantiomeric distribution. Antibacterial activity was determined using the broth microdilution method, for which we used three Gram-positive cocci bacteria, a Gram-positive bacilli bacterium and three Gram-negative bacilli bacteria. 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic (ABTS) acid radical cation and 2,2-diphenyl-1-picrylhydryl (DPPH) free radical were used as reagents for determining the antioxidant activity of the essential oil. The spectrophotometric method was used to analyze acetylcholinesterase inhibitory effect of the essential oil. The yield of leaves in essential oil was 0.13 ± 0.01% (v/w). A total of 56 chemical compounds, which represent 99.25% of the total composition, were identified in the essential oil. Sesquiterpene hydrocarbons were the most representative group in number of compounds and relative abundance with 31 compounds and 69.06%, respectively. The principal constituents were found to germacrene D (17.87 ± 1.20%), bicyclogermacrene (14.27 ± 0.97%), caryophyllene < (E)– > (8.56 ± 1.24%) and pinene  (6.34 ± 0.13%). Six pairs of enantiomers were identified in the essential oil of Bixa orellana. The essential oil presented strong activity against the Enterococcus faecium (ATCC 27270) with an MIC of 250 μg/mL and weak activity against Enterococcus faecalis (ATCC 19433) and Staphylococcus aureus (ATCC 25923) with an MIC of 1000 μg/mL. The antioxidant activity of the essential oil was strong according to ABTS methods with a SC50 of 61.49 ± 0.04 µg/mL and was moderate in DPPH with a SC50 of 224.24 ± 6,4 µg/mL. Additionally, the essential oil reported moderate anticholinesterase activity with an IC50 of 39.45 ± 1.06 µg/mL.


Introduction
Medicinal plants have been used for thousands of years throughout the world as a form of traditional medicine, and are an important source of active ingredients for the modern pharmaceutical industry [1]. Bixaceae are a family of various medicinal plants found in some tropical and subtropical countries, including Ecuador, Brazil, Colombia, Peru, Mexico and other Latin American countries. This family includes about 15 genera and more than 200 species. Some of the genera included in the Bixaceae family are Cochlospermum, Ochroma, Heliocarpus, Ellipanthus, Dicella, Bixopsis, Gyrocarpus and Bixa [2,3]. pigments, such as bixin and norbixin, which have antioxidant and anti-inflammatory properties and are widely used in the food industry as natural colorants [18]. The presence of essential oil in the leaves and seeds from B. orellana has been reported previously [12,19,20]. Bixa orellana seeds contain essential oils rich in volatile organic compounds, which are mainly terpene and sesquiterpene in nature [12,21]. Likewise, volatile compounds have been detected in the aqueous and organic extracts of B. orellana seeds [22]. Despite the applications in traditional medicine and common and industrial uses of the species B. orellana, to the best of our knowledge, not enough information has been found in the literature on the biological activity of the essential oil of Bixa orellana leaves. That is why the aim of this study is to determine the chemical composition, enantiomeric distribution and antimicrobial, antioxidant and anticholinesterase activities of the essential oil isolated from leaves of this species, in order to determine its potential in pharmacological applications.

Essential Oil Isolated
Approximately 25 kg (24,930 g divided into three distillations of 6130 g, 9290 g and 9510 g) of fresh (with a moisture of 64 ± 1% w/w) B. orellana leaves, were hydrodistilled in a Clevenger type apparatus in order to isolate the essential oil (EO). The amount of EO obtained was approximately 32 mL, which represents a yield of 0.13 ± 0.01% (v/w) or 1.3 ± 0.1 mL/kg.

Physical Properties of Essential Oil
The EO from B. orellana presented as an unctuous liquid. Table 1 shows the mean values and standard deviations (SD) of the physical properties of essential oil. In general, the essential oil of B. orellana was a yellow liquid less dense than water.

Chemical Composition of Essential Oil
The qualitative identification of the B. orellana compounds was carried out by gas chromatography coupled to mass spectrometry (GC-MS) and the quantification of their relative abundances was performed by means of gas chromatography equipped with the flame ionization detector (GC-FID). The information on the compounds and their relative abundances (%) are shown in Table 2. A total of 56 chemical compounds were identified in the EO of leaves from B. orellana, which represent 99.25% of the total composition. The compounds were classified into four groups: monoterpene hydrocarbons (MH), sesquiterpene hydrocarbons (SH), oxygenated sesquiterpene (OS) and diterpene hydrocarbons (DH). In terms of number of compounds and relative abundance, SH was the most representative group with 31 compounds and 69.06%, respectively. Only one compound (bifloratriene) of the DH group was identified and no oxygenated monoterpene compounds or non-terpene compounds were found. The principal constituents (> 5%) were found to be SH germacrene D (CN: 25

Enantiomeric Analysis
Using a column with enantioselective stationary phase it was possible to separate six pairs of enantiomers from EO of B. orellana leaves, although it was not possible to establish the order of elution between the enantiomers (+) and (-) in four of them (α-copaene, αtrans-bergamotene, bicyclogermacrene and spathulenol). Table 3 shows the retention time (RT), enantiomers, retention indices (RI), enantiomeric distribution (ED) and enantiomeric excess (e.e.), for each pair of compounds. The (-)-α-pinene was found practically pure with e.e. of 99.63%.

Antimicrobial Activity
Microdilution broth method was used to determine the antibacterial activities of EO of leaves from B. orellana. Table 4 shows the tested microorganisms and minimum inhibitory concentration (MIC) values of EO and positive control, in addition, the values of the negative control. Ampicillin was used as a positive control for Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus, and ciprofloxacin for Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica. Dimethyl sulfoxide at 5% was used as a negative control. Bixa orellana EO reported MIC values of 250 µg/mL against Gram-positive cocci Enterococcus faecium and MIC values of 1000 µg/mL against Enterococcus faecalis and Staphylococcus aureus.

Antioxidant Activity
The antioxidant activity of essential oil from B. orellana was determined using the DPPH and ABTS methods. The DPPH method is based on the scavenging capacity of the essential oil against the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) and in the ABTS method scavenging capacity was determined against the radical ion 2,2 -azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS •+ ). Table 5 shows the scavenging capacity (SC 50 ) in µg/mL of the essential oil and the positive control (trolox), as well as the standard deviation (SD). The maximum evaluated concentration was 1000 µg/mL.

Anticholinesterase Activity
The anticholinesterase (anti-AChE) activity was determined using the spectrophotometric method. Figure

Discussion
This article presents the study of the essential oil from B. orellana leaves obtained by hydrodistillation. The extraction yield was 1.3 ± 0.1 mL/Kg (moisture of 64%), which could be considered low, since according to the categorization proposed by the "Science and Technology for Development" (CYTED), for plant species, the yield values in EO less than 5 mL/Kg are considered low, values between 5 mL/Kg and 10 mL/Kg intermediate and values greater than 10 mL/Kg high [23]. Caixeta et al. in 2020 determined that the yield in the EO of dry leaves (moisture of 10.6%) from B. orellana was 0.21% (2.1 mL/kg) [24]. The potential application of essential oils should be evaluated from a quantitative and qualitative point of view. From a bioeconomy perspective, the biodiversity has limitations of production, and different efforts have been applied to improve extraction yields and make it sustainable. Some strategies include the use of co-products or by-products of the industrialization of primary cultivars; this could be the case of industrialization of annatto as a food additive where the isolation of other natural active principles from the leaves could be implemented for obtaining essential oils. The EO of B. orellana was characterized by its physical properties; the density and refractive index are useful measurements to declare the purity, quality and chemical composition [25], and it is known that these properties vary with the composition and represent the average weighting. According to previous reports of the chemical composition of the EO of B. orellana leaves, Lawrence et al. in 1973 reported ishwarane (54%) a sesquiterpene hydrocarbon

Discussion
This article presents the study of the essential oil from B. orellana leaves obtained by hydrodistillation. The extraction yield was 1.3 ± 0.1 mL/kg (moisture of 64%), which could be considered low, since according to the categorization proposed by the "Science and Technology for Development" (CYTED), for plant species, the yield values in EO less than 5 mL/kg are considered low, values between 5 mL/kg and 10 mL/kg intermediate and values greater than 10 mL/kg high [23]. Caixeta et al. in 2020 determined that the yield in the EO of dry leaves (moisture of 10.6%) from B. orellana was 0.21% (2.1 mL/kg) [24]. The potential application of essential oils should be evaluated from a quantitative and qualitative point of view. From a bioeconomy perspective, the biodiversity has limitations of production, and different efforts have been applied to improve extraction yields and make it sustainable. Some strategies include the use of co-products or by-products of the industrialization of primary cultivars; this could be the case of industrialization of annatto as a food additive where the isolation of other natural active principles from the leaves could be implemented for obtaining essential oils. The EO of B. orellana was characterized by its physical properties; the density and refractive index are useful measurements to declare the purity, quality and chemical composition [25], and it is known that these properties vary with the composition and represent the average weighting.
Ishwarane, a bioactive sesquiterpene hydrocarbon, has not been identified in our study, even as traces; however, it was reported by Lawrence [29], and in the essential oil of B. orellana seeds. Pino and Correa in 2003 presented the composition of EO of B. orellana seeds, from the 35 components identified the main were (Z,E)-farnesyl acetate (11.6%), occidentalol acetate (9.7%), spathulenol (9.6%) and ishwarane (9.1%) [21]. In the same way, Monzote et al. in 2013 reported ishwarane (18.6%) and geranylgeraniol (9.1%) as major compounds of B. orellana seeds [12]. The chemical composition of the EO of B. orellana of the present study was majorly grouped as sesquiterpene hydrocarbons (69.06%), similar to the report of Giorgi et al. in 2013 [11]. The chemical composition of EO of B. orellana leaves presents differences with the compared references. This is a fact that could be accounted for by the variations in cultivar practices, since compounds present in an EO depend on intrinsic factors (plant part, plant age, phenological state, etc.) and extrinsic factors (soil type, shade, amount of rainfall, etc.) of the plants. This species is cultivated in Central and South America and other tropical countries around the world where there are different climatic conditions and types of soil [17].
Regarding to the antimicrobial activity Van Vuuren and Holl in 2017 [31] recommended a scale for MIC to recognize the potency of natural extracts. They proposed for essential oils that values of MIC > 1001 µg/mL are considered inactive, MIC between 500 and 1000 µg/mL have moderate activity, MIC between 101 and 500 µg/mL have strong activity and MIC less than 100 µg/mL have very strong activity. When applying this scale to the EO of B. orellana leaves, it showed strong activity (MIC 250 µg/mL) against Enterococcus faecium (ATCC 27270), moderate activity (MIC 1000 µg/mL) against Enterococcus faecalis (ATCC 19433) and Staphylococcus aureus (ATCC 25923), while it was inactive (MIC > 1001 µg/mL) against Gram-positive bacilli and Gram-negative bacilli. The positive control shows MIC values of around 1 µg/mL, so it should be considered that the positive control is a pure synthetic substance while the EO is a mixture of natural compounds. Based on these results, EO of B. orellana leaves would have antimicrobial potential against three of the seven bacteria tested. Coelho dos Santos et al. in 2022 presented a review of the antimicrobial activity reported for extracts of different parts of B. orellana; however, there are no data for essential oils of the leaves [32].
The antioxidant activity was reported as the SC 50 , the results showed moderate scavenging capacity for the DPPH assay (SC 50 224.24 ± 6.4 µg/mL) and strong capacity for the ABTS assay (SC 50 61.49 ± 0.04 µg/mL) the differences could be related to the reaction mechanism of each antioxidant method, because in DPPH assay terpene compounds are less capable of donating a hydrogen atom [33]. The antioxidant activity has been related to the possible applications of the EO, and generally this activity responds to synergistic or antagonistic effects among the chemical components present in the mixture. As a comparison of the antioxidant activity, we cited Shilpi et al. (2006); they evaluated the methanol extract of B. orellana L. leaves and reported an IC 50 22.36 µg/mL in the DPPH assay and antibacterial activity against selected causative agents of diarrhea and dysentery, including Shigella dysenteriae [17].
Cholinesterase is a term referring to one of the two enzymes, named, acetylcholinesterase and pseudocholinesterase. Both compounds catalyze the hydrolysis of excess neurotransmitter acetylcholine, and the excessive activation caused by acetylcholine would produce damage to the neuron or muscle. A cholinesterase inhibitor is known as an anticholinesterase compound [34]. Anticholinesterase compounds are also used for treating myasthenia gravis, glaucoma and Alzheimer's disease. Synthetic anticholinesterase compounds are potent neurotoxins of moderate effectiveness, of high cost and short half-life that can also cause gastrointestinal disturbances, so currently compounds isolated from natural products (plants) are increasingly being explored for their anticholinesterase properties and their better secondary effects [35]. Therefore, different studies report the anticholinesterase activity of essential oils considering the potential uses in the treatment of Alzheimer's disease [36]. This is the first report of anticholinesterase activity for the EO of B. orellana; the IC 50 was 39.45 ± 1.06 µg/mL, this value could be considered as moderate potency when it is compared with the scale proposed by Santos et al. (2018) [35], which values high potency IC 50 < 20 µg/mL; moderate potency 20 < IC 50 < 200 µg/mL; and low potency 200 < IC 50 < 1000.

Plant Material
The leaves of B. orellana were collected in the surroundings of the El Dorado Parish, Francisco de Orellana Canton, Orellana Province. The collection was carried out in the place that is located at 0 • 29 48" south longitude and 76 • 54 41" west latitude and an altitude of 225 m.a.s.l. After being collected, the plant material was stored and transferred in airtight plastic containers.

Essential Oil Isolation
A Clevenger type apparatus was used for the isolation of essential oil, in which the extraction of the oil was carried out by hydrodistillation. An 80 L distiller was used, in which approximately 18 L of water was placed. The process was maintained for 3 h counted from the fall of the first drop of distillate. The condensed essential oil was separated from the water by decantation, then the essential oil was dried using anhydrous sodium sulfate and stored at 4 • C in amber sealed vials until it was used in analysis.

Determination of the Physical Properties of the Essential Oil
Density of the EO was determined using the ISO 279:1998 standard (equivalent to the AFNOR NF T 75-111 standard) 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 (similarly to AFNOR NF T 75-112) using a refractometer (model ABBE, BOECO, Hamburg, Germany). Optical rotation of the EO was determined according to the standard ISO 592:1998 using an automatic polarimeter (Mrc-P810, MRC, Holon, Israel). The subjective color was obtained online, for which a photograph of the EO taken with a white background was uploaded to the PINETOOL website (https://pinetools.com/, accessed on 20 July 2022). All measurements were taken at 20 • C.

Identification and Quantification of Essential Oil Compounds
The analysis of chemical composition was carried out in a gas chromatograph (GC) (model 6890N series, Agilent Technologies, Santa Clara, CA, USA). For qualitative analysis, the GC was coupled to a quadrupole mass spectrometer (MS) (model Agilent series 5973 inert, Agilent Technologies, Santa Clara, CA, USA) and for quantitative analysis the GC was equipped with a flame ionization detector (FID). In both cases, a nonpolar chromatographic column (Agilent J&W DB-5ms Ultra Inert GC column, Agilent Technologies, Santa Clara, CA, USA) with stationary phase 5%-phenyl-methylpolyxilosane, which was 30 m long, 0.25 mm of internal diameter and 0.25 µm of stationary phase thickness was used. The GC was equipped with a split/splitless autosampler (model 7683, Agilent Technologies, Santa Clara, CA, USA). The supply of hydrogen for the FID was carried out using a gas generator (model 9150, Packard, Conroe, TX, USA). The EO sample was prepared at 1% (v/v) by putting 10 µL of EO and 990 µL of dichloromethane in an amber vial. For the qualitative and quantitative analyses, a 1 µL of sample was injected in split mode with a partition ratio of 40:1, at a temperature of 220 • C and a pressure of 11 psi. In both cases, the chromatographic run began maintaining the initial temperature of 50 • C for 3 min, then the temperature was increased by 3 • C/min until reaching the final temperature of 230 • C, which was maintained for 3 min. For GC-MS, a constant flow of helium was maintained at a rate of 0.9 mL/min and a velocity of 23 cm/s, and for GC-FID the flow was 1.0 mL/min and the speed was 40 cm/s. Equation 1 [37] was used to determine the retention index (RI) of each compound. For the identification of the compounds, the IR and the mass spectra were compared with those existing in the bibliography [38,39].
where C is carbon number of aliphatic hydrocarbons (C 9 to C 25 ) that elute before the compound of interest, RTx is the retention time of the compound of interest, RTn is the retention time of aliphatic hydrocarbons that elute before the compound of interest and RTN is the retention time of hydrocarbons that elute after the compound of interest.

Enantioselective Analysis
For enantiomeric analysis, gas chromatography (Trace 1310, Thermo Fisher Scientific, Waltham, MA, USA) coupled to mass spectrometry (quadrupole) (ISQ 7000, Thermo Fisher Scientific, Waltham, MA, USA) was used. Analyses were performed on an enantioselective GC column (MEGA-DEX DMT-Beta, Mega, Legnano, MI, Italy) of 30 m length, 0.25 m internal diameter and 0.25 µm thick stationary phase (2.3-diethyl-6-tert-butyldimethylsilylβ-cyclodextrin). Sample preparation, amount injected, injection temperature and partition radius were those described for GC-MS. The carrier gas used was helium with a flow of 1.0 mL/min and a speed of 40 cm/s. The chromatographic run began by maintaining the oven at 60 • C for 5 min, then the temperature was increased with a ramp of 2 • C/min up to 230 • C, finally this temperature was maintained for 5 min. The calculation of the enantiomeric excess and elution order was carried out according to the procedures previously described by Morocho et al. in 2023 [34].

Antimicrobial Activity
The antibacterial activity of the essential oil was tested against seven strains of bacteria: three Gram-positive cocci bacteria including Enterococcus faecalis (ATCC 19433), Enterococcus faecium (ATCC 27270) and Staphylococcus aureus (ATCC 25923); a Gram-positive bacilli bacterium Listeria monocytogenes ATCC 19115; and three Gram-negative bacilli bacteria including Escherichia coli O157:H7 (ATCC 43888), Pseudomonas aeruginosa (ATCC 10145) and Salmonella enterica subs enterica serovar Thypimurium WDCM 00031, derived (ATCC 14028). The broth microdilution method was used to determine this activity, the procedures were performed as previously described by Valarezo et al. in 2021 [40]. The maximum evaluated concentration was 4000 µg/mL. Ampicillin and ciprofloxacin were used as a positive control and DMSO was used as a negative control.

Evaluation of Antioxidant Capacity
The DPPH and ABTS methods were used to determine free radical scavenging activity of EO from B. orellana. The antioxidant capacity of EO was determined according to the procedure described by Salinas et al. [41], using a UV spectrophotometer (Genesys 10S UV-Vis Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). In the DPPH method, 2,2-diphenyl-1-picrylhydrazyl radical was produced from the reagent 2,2-diphenyl-1-picrylhydrazyl and the absorbance of the samples was measured at a wavelength of 515 nm. In the ABTS method, 2,2 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation was produced from reagent 2,2 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) and the measurement of the absorbance of the samples was carried out at a wavelength of 734 nm. The SC 50 , which is the concentration value necessary for the EO to have a scavenging capacity of half of the radicals, was used to express the antioxidant activity. Trolox and methanol were used as a positive and negative control, respectively.

Anticholinesterase Activity
The spectrophotometric method was used to determine the acetylcholinesterase inhibitory effect of the EO of leaves from B. orellana. The procedures were performed as previously described by Valarezo et al. [42]. Measurements were made in a microplate spectrophotometer (EPOCH 2, BioTek, Winooski, VT, USA) at a wavelength of 405 nm. The IC50 was used to express the anticholinesterase activity. IC50 is the concentration of EO required for 50% inhibition. Methanol and donepezil hydrochloride were used as a negative and positive control, respectively.

Statistical Analysis
All procedures were performed in triplicate, except the identification of essential oil compounds, enantioselective analysis and antimicrobial activity, which were performed nine times. The data were collected in a Microsoft Excel sheet. The statistical software Minitab 17 (Version 17.1.0., Minitab LLC., State College, PA, USA) was used to calculate the measures of central tendency and standard deviation.

Conclusions
The enantiomeric distribution, antimicrobial activity, antioxidant capacity and anticholinesterase activity of essential oil from leaves of Bixa orellana were determined for the first time. Fifty-six chemical compounds and six pairs of enantiomers were identified in the essential oil. The main compound was germacrene D. Essential oil exhibited strong antibacterial activity against one of the tested strains, making this oil suitable for formulations in the pharmaceutical industry of specific antimicrobial products for this type of bacterial strain. Furthermore, essential oil presented a strong antioxidant activity and moderate anticholinesterase activity, which makes it novel for the food industry where it could be used to formulate functional foods. With this research, new information is provided on the species of aromatic plants of Ecuador, thus contributing to the knowledge of Ecuadorian biodiversity. For future studies it is proposed to test the anti-inflammatory or anti-repellent activity.