Chemical Composition, Enantiomeric Distribution and Biological Activity of Essential Oil from Morella pubescens (Humb. & Bonpl. ex Willd.) Wilbur

The species Morella pubescens, commonly known as wax laurel, is a tree belonging to the Myricaceae family that can be found from Costa Rica to Bolivia. In this study, the chemical composition, enantiomeric distribution, and biological activity of essential oil isolated from the leaves of this species was determined. Hydrodistillation was used to isolate the essential oil (EO). Gas chromatography coupled with mass spectrometry was used to determine the qualitative composition, gas chromatography 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. The broth microdilution method was employed to assess the antibacterial capacity of the essential oil against seven opportunistic microorganisms, including three Gram-positive cocci bacteria, a Gram-positive bacilli bacterium and three Gram-negative bacilli bacteria. 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical cation and 2,2-diphenyl-1-picrylhydryl free radical were used as reagents to determine the antioxidant activity of essential oil. The spectrophotometric method was used to analyze the acetylcholinesterase inhibitory effect of the essential oil. The extraction method afforded a low yield of around 0.076 ± 0.008% (v/w). Fifty-eight chemical compounds, which represent 97.9% of the total composition, were identified in the essential oil. Sesquiterpene hydrocarbons were the most representative group with 24 compounds (67.8%). The principal constituents were (E)-caryophyllene (27.5 ± 1.3%), limonene (11.8 ± 0.6%), δ-selinene (9.1 ± 0.2%), β-selinene (8.0 ± 0.2%), selina-3,7(11)-diene (5.3 ± 0.2%) and germacrene B (5.0 ± 0.5%). Three pairs of enantiomers were identified in the essential oil of Morella pubescens. Essential oil presented strong activity against the bacterium Enterococcus faecium (ATCC 27270) with an MIC of 250 μg/mL. The antioxidant activity of essential oil was very strong in the ABTS method with an SC50 of 46.4 ± 1.0 µg/mL and was strong in the DPPH method with an SC50 of 237.1 ± 1.8 µg/mL. Additionally, the essential oil reported strong anticholinesterase activity with an IC50 of 133.5 ± 1.06 µg/mL.


Introduction
Medicinal plants are the first line of defense in the remediation of diseases, and are widely used globally, especially in developing countries where it is the only available therapeutic remedy [1]. The World Health Organization (WHO) defines traditional medicine as the sum total of the knowledge, skill, and practices based on the theories, beliefs, and experiences that are indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement, or treatment of physical and mental illness. It includes diverse health practices that incorporate plant, animal, and/or mineral-based medicines to maintain well-being, as well as to treat, diagnose and prevent disease. The study of phytochemicals in medicinal plants is of continuous of the essential oil isolated from the leaves of Morella pubescens, as well as its antibacterial, antioxidant and anticholinesterase activities. In this way, we can contribute information on the aromatic and medicinal flora of Ecuador.

Essential Oil Obtained
A total of 15,000 g (three distillations of 5000 g) of fresh (with a moisture of 72 ± 4% w/w) M. pubescens leaves were hydrodistilled in a Clevenger-type apparatus to isolate its essential oil (EO). The amount of EO obtained was 11.4 mL, which represents a yield of 0.076 ± 0.008% (v/w), or 0.76 ± 0.08 mL/Kg.

Physical Properties of Essential Oil
The EO from M. pubescens leaves presented as an unctuous liquid with a strong odor characteristic of this species. Table 1 shows the mean values and standard deviations (SD) of the physical properties of essential oil. In general, the essential oil of M. pubescens was a yellow liquid less dense than water.

Enantiomeric Analysis
Using a column with the enantioselective stationary phase, it was possible to separate three pairs of enantiomers from the EO of M. pubescens leaves. 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 and (+)-limonene were found to be practically pure with 94.8% and 91.3% of enantiomeric excess, respectively.

Antimicrobial Activity
The microdilution broth method was used to determine the antibacterial activities of EO from leaves from M. pubescens. Table 4 shows the tested microorganisms and minimum inhibitory concentration (MIC) values of both the EO and positive control. The values of the negative control are also shown. Ampicillin was used as a positive control for Enterococcus faecalis, Enterococcus faecium, and Staphylococcus aureus, and ciprofloxacin was used as a positive control for Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, and Salmonella enterica. The M. pubescens EO reported MIC values of 250 µg/mL against Enterococcus faecium, 2000 µg/mL against Staphylococcus aureus, and 4000 µg/mL against Listeria monocytogenes.

Antioxidant Activity
The antioxidant activity of essential oil from M. pubescens was determined using the methods DPPH and ABTS. Table 5 shows the scavenging capacity (SC 50 ) in µg/mL of both the essential oil and the positive control. The maximum evaluated concentration was 1000 µg/mL. M. pubescens EO presented a SC 50 of 46.37 µg/mL with method ABTS, a value close to that of the positive control.

Anticholinesterase Activity
The anticholinesterase activity was determined using a spectrophotometric method. Figure 1 shows the Log of the concentration of EO, and the normalized response rate of the reaction of acetylcholinesterase. The results were reported as a half-maximal inhibitory concentration (IC 50 ) value. The M. pubescens EO reported an IC 50 value of 133.5 ± 1.1 µg/mL. The positive control (donepezil) exhibited an IC 50 value of 12.4 ± 1.4 µg/mL. Salmonella enterica subs enterica serovar Thypimurium WDCM 00031, derived (ATCC 14028) >4000 0.39 + +: normal growth.

Antioxidant Activity
The antioxidant activity of essential oil from M. pubescens was determined using the methods DPPH and ABTS. Table 5 shows the scavenging capacity (SC50) in μg/mL of both the essential oil and the positive control. The maximum evaluated concentration was 1000 μg/mL. M. pubescens EO presented a SC50 of 46.37 μg/mL with method ABTS, a value close to that of the positive control.

Anticholinesterase Activity
The anticholinesterase activity was determined using a spectrophotometric method. Figure 1 shows the Log of the concentration of EO, and the normalized response rate of the reaction of acetylcholinesterase. The results were reported as a half-maximal inhibitory concentration (IC50) value. The M. pubescens EO reported an IC50 value of 133.5 ± 1.1 μg/mL. The positive control (donepezil) exhibited an IC50 value of 12.4 ± 1.4 μg/mL.

Discussion
The extraction yield of EO was 0.076 ± 0.008% (v/w), which could be considered a very low yield [16]. No reports about this value have been published in scientific articles for M. pubescens, but a preliminary work [17] reported a value of 0.24% for samples collected in Perú. The extraction yield is recognized to be dependent on the extraction process. Arango et al. [15] reported in 2009, regarding the hydrodistillation of M. pubescens, that the interaction between particle size and extraction time has an influence on the concentration of chemical components of the essential oil. Clearly, this is related to the extraction yield, but they did not report the value. For other species of this genus, Dolveni et al. published in 2016 an extraction yield between 0.3% to 0.5% for the essential oil of Morella parvifolia (Benth.) Parra-Os. [14]. The physical properties and the chemical composition could both be considered a characteristic of purity of the EO, as in this case for M. pubescens, the variations of refractive index are associated with changes in the chemical composition-as mentioned by Delgado Ospina, et al., this could allow its use as a quality parameter [18].
The enantiomeric analysis allows us to consider the potential applications of EO in pharmaceutical or food products [20] if bioactive chiral compounds are present in the complex mixture of an essential oil. For the EO of M. pubescenes, the occurrence of three pairs of enantiomers is reported in Section 2, and this is the first report of enantioselectivity GC-MS analysis.
The previous studies of M. pubescens did not perform analysis of bioactivity of the EO, and this study is the first to report on antibacterial, antioxidant, and anticholinesterase analyses. According to the criteria published by Van Vuuren and Holl in 2017 [21] about a scale for the MIC values of plant extracts and essential oils, the EO of M. pubescens (MIC 250 µg/mL) shows strong activity (MIC 101 to 500 µg/mL) against Enterococcus faecium (ATCC 27270), but it was inactive (MIC > 1001 µg/mL) against the other microorganisms tested ( Table 4) and Polhill, and reported antibacterial activity against S. aureus, Streptococcus agalactiae, E. coli, and Shigella flexneri, where the main components were hexadecanoic acid methyl ester (29.4%), (Z)-9-octadecenoic acid methyl ester (28.6%), and methyl tetradecanoate (10.7%) [23]. It is difficult to associate the antibacterial activity to the major components in an essential oil even though the individual components have shown strong antibacterial activity [24,25]. Rather, the additive or synergistic effects become an antagonistic one, causing a decrease or loss of activity. Lis-Balcnin et al. reported that 18 out of 25 different bacteria were more affected by the (−) enantiomer of α-pinene, while the (+) isomer affected more, about 19 out of 20 L. monocytogenes strains [26]. The different bioactivities could be referring to the different enantiomeric ratio of chiral compounds, and this was observed by Van Vuuren and Viljoen in 2007, who evaluated the antibacterial activity of both enantiomers (+) and (−) of limonene, and the combination with 1,8-cineole [27].
Regarding the antioxidant activity, the EO of M. pubescenes showed strong activity by the ABTS assay while the value was weak for the DPPH assay. In the literature, essential oils have shown strong, moderate, low, or no antioxidant activity. Anthony et al. in 2012 [28] reported that mono-and sesquiterpenes have less antioxidant activity after phenol compounds. The difference in composition explains the antagonist or synergistic activity, which was observed by Chandra et al. in 2017 [29]. Dahham et al. reported a strong antioxidant activity for β-caryophyllene with 1.3 ± 0.1 µM [25]. The antioxidant activity of limonene has been demonstrated to effectively attenuate oxidative stress in diabetic rats [30].
The anticholinesterase activity of the EO M. pubescens has not been published before.

Plant Material
The leaves of M. pubescens were collected in the surroundings of the Guayllabamba parish, Quito canton, Pichincha province. The collection was carried out in a valley that is located at 0 • 04 43 south longitude and 78 • 20 59 west latitude, and at an altitude of 2171 m above sea level. After being collected, the plant material was stored and transferred in airtight plastic containers. The environmental conditions in the collection and transfer were a pressure of 79 KPa and a temperature of 18-20 • C.

Essential Oil Isolation
A Clevenger-type apparatus was used for the isolation of essential oil. The extraction of the oil was carried out by hydrodistillation according to the procedures previously described by Valarezo et al. [31], for which an 80 L distiller with approximately 18 L of water was used. The process was maintained for 3 h, counting from the fall of the first drop of distillate. The condensed essential oil was separated from the water by decantation, then it was dried using anhydrous sodium sulfate and stored at 4 • C in amber sealed vials until being used in analysis.

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, and 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, 30 m of length, 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, Detroit, MI, USA). The EO sample was prepared at 1% (v/v), putting 10 µL of EO and 990 µL of dichloromethane in an amber vial. For the qualitative and quantitative analyses, 1 µL of sample was injected in split mode with a partition ratio of 40:1, at a temperature of 220 • C, and at 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 3 • C/min until reaching a 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) [35] 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 in the bibliography [36,37].
where C is the carbon number of aliphatic hydrocarbons (C 9 to C 25 ) that elutes after before of the compound of interest, RTx is the retention time of the compound of interest, RTn is the retention time of aliphatic hydrocarbons that elutes before the compound of interest, and RTN is the retention time of hydrocarbons that elutes after the compound of interest.

Enantioselective Analysis
For enantiomeric analysis, a gas chromatography (Trace 1310, Thermo Fisher Scientific, Waltham, MA, USA) coupled to a mass spectrometer (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) with 30 m of length, 0.25 m of internal diameter, and 0.25 µm of thick stationary phase (2.3 -diethyl-6tert-butyldimethylsilyl-β-cyclodextrin). Sample preparation, amount injected, injection temperature, and partition radius were similar to 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 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. [38].

Antimicrobial Activity
The antibacterial activity of the essential oil was tested against seven opportunistic and nosocomial bacteria that are commonly found in hospitals, or which act as saprofitic organisms and can lead to a variety of infections in vital organs or systems such as the lungs, heart, urinary tract, gastrointestinal tract, skin, etc. Three Gram-positive cocci bacteria: 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: Escherichia coli O157:H7 (ATCC 43888), Pseudomonas aeruginosa (ATCC 10145), and Salmonella enterica subs enterica serovar Thypimurium WDCM 00031, derived from (ATCC 14028), were included in the assay. The broth microdilution method was used to determine this activity, and the procedures were performed as previously described by Valarezo et al. [39]. 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 M. pubescens. The DPPH method is based on the scavenging capacity of the essential oil against the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH • ), and the ABTS scavenging capacity was determined against the radical ion 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS •+ ). The antioxidant capacity of EO was determined according to the procedure described by Salinas et al. [33], 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 (DPPH • ) was produced from the reagent 2,2-diphenyl-1-picrylhydrazyl (DPPH), and the absorbance of the samples was measured at a wavelength of 515 nm. Instead, in the ABTS method, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS •+ ) was produced from the reagent 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and the measurement of the absorbance of the samples was carried out at a wavelength of 734 nm. SC 50 , which is the concentration value necessary for the EO to have half-radical scavenging capacity, 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 M. pubescens. The procedures were performed according to what was previously described by Valarezo et al. [32]. 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 for the identification of essential oil compounds, the enantioselective analysis, and identification of antimicrobial activity, which were performed nine times. The data were collected in a Microsoft Excel spreadsheet. 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 Morella pubescens was determined for the first time. Fifty-eight chemical compounds and three pairs of enantiomers were identified in the essential oil. The main compound was (E)-caryophyllene. Essential oil presented strong activity against Enterococcus faecium, and very strong antioxidant activity. With this research, new information is provided on the species of aromatic plants of Ecuador, thus contributing to the knowledge of Ecuadorian biodiversity. The biological activities displayed by the essential oil of leaves from Morella pubescens make this essential oil novel for the cosmetic, food, and pharmaceutical industries. For future studies, based on the good results of in vitro activity, it is proposed that in vivo studies be carried out-for example regarding anti-inflammatory activity in mice.