Acorenone B: AChE and BChE Inhibitor as a Major Compound of the Essential Oil Distilled from the Ecuadorian Species Niphogeton dissecta (Benth.) J.F. Macbr

This study investigated the chemical composition, physical proprieties, biological activity, and enantiomeric analysis of the essential oil from the aerial parts of Niphogeton dissecta (culantrillo del cerro) from Ecuador, obtained by steam distillation. The qualitative and quantitative analysis of the essential oil was realized by gas chromatographic and spectroscopic techniques (GC-MS and GC-FID). Acorenone B was identified by GC-MS and NMR experiments. The enantiomeric distribution of some constituents has been assessed by enantio-GC through the use of a chiral cyclodextrin-based capillary column. We identified 41 components that accounted for 96.46% of the total analyzed, the major components were acorenone B (41.01%) and (E)-β-ocimene (29.64%). The enantiomeric ratio of (+)/(−)-β-pinene was 86.9:13.1, while the one of (+)/(−)-sabinene was 80.9:19.1. The essential oil showed a weak inhibitory activity, expressed as Minimal Inhibitory Concentration (MIC), against Enterococcus faecalis (MIC 10 mg/mL) and Staphylococcus aureus (MIC 5 mg/mL). Furthermore, it inhibited butyrylcholinesterase with an IC50 value of 11.5 μg/mL. Pure acorenone B showed inhibitory activity against both acetylcholinesterase and butyrylcholinesterase, with IC50 values of 40.8 μg/mL and 10.9 μg/mL, respectively.


Introduction
Medicinal plants have been used for a long time as sources of new pharmaceuticals due to the presence of bioactive compounds [1]. Plants are rich in structurally diverse secondary metabolites displaying a wide range of biological activities, including possible leads for the treatment of neurodegenerative diseases [2,3]. Natural products have contributed greatly as sources for drug discovery for Alzheimer's disease [4]. Earlier studies have shown that the maintenance of correct levels of acetylcholinesterases is directly related to different diseases such as Alzheimer's disease (AD), bipolar disorder, depression, and schizophrenia [5]. There are two distinct basic types of cholinesterases:

Results and Discussion
The essential oil of the aerial parts of Niphogeton dissecta was obtained by steam distillation for 4 h, yielding an average of 0.33 ± 0.03% (w/w). The physical properties, chemical composition, enantiomeric analysis, and biological activity are discussed below.

Chemical Composition
The chemical composition of the essential oil was defined based on calculated linear retention indices (LRIc) and mass spectra compared with literature [18][19][20][21][22][23]. Table 1 presents the components of the essential oil determined by GC-MS and quantified by GC-FID. Forty-one compounds were separated, which represented 96.46% of the total essential oil. The major compounds were acorenone B (41.01%), (E)-β-ocimene (29.64%), (3E)-butylidene phthalide (5.54%), and α-pinene (3.94%). Oxygenated sesquiterpenes (42.31%) and monoterpene hydrocarbons (37.97%) were the most representative groups. This is the first report on the characterization of the essential oil distilled from N. dissecta. A typical chromatogram of the essential oil from N. dissecta is shown in Figure 1. A typical chromatogram of the essential oil from N. dissecta is shown in Figure 1.
The molecular structure of acorenone B ( Figure 2) was confirmed by 1 H NMR, 13 C NMR, and MS analysis, and compared with data present in the literature [24][25][26][27].
The occurrence of acorenone B in N. dissecta oil appears somewhat unusual because of the diversity of the sesquiterpenoid skeleton involved. According to Zalkow et al. [24], the proposed structure of acorenone B is considered to be derived from trans-cis-farnesol via the β-bisabolyl cation.
The occurrence of acorenone B in N. dissecta oil appears somewhat unusual because of the diversity of the sesquiterpenoid skeleton involved. According to Zalkow et al. [24], the proposed structure of acorenone B is considered to be derived from trans-cis-farnesol via the β-bisabolyl cation.

Enantiomeric Analysis
The enantiomeric distribution and enantiomeric excess (e.e.) ( Table 2) of some chiral metabolites were determined on a cyclodextrine-based chiral stationary phase (MEGA-DEX-DET), comparing the retention time of separated enantiomers with enantiomerically pure standards. Two couples of chiral monoterpenoids were detected. The enantiomeric excesses of (+)-β-pinene and (+)-sabinene were quite considerable. These results further confirm that plants can also contain both enantiomers in the essential oil (Figure 3). It has been documented that sometimes different enantiomers may present dissimilar biological activities [33]. In our essential oil, the enantiomeric excess of (+)-β-pinene was 73.8% with respect to (-)-β-pinene. Some studies have shown that the positive enantiomer has antimicrobial activity against Candida albicans, Cryptococcus neoformans, Rhyzopus oryzae, and methicillin-resistant Staphylococcus aureus (MRSA) [34]. In contrast, the negative enantiomer exhibits antiviral properties against infectious bronchitis virus (IBV) [

Antimicrobial Activity
The essential oil showed a weak inhibitory activity against Enterococcus faecalis and Staphylococcus aureus, while acorenone B was non-active at the maximum dose tested (10 mg/mL) ( Table 3). According to Holetz et al. [36], an antibacterial activity is considered good when the Minimal Inhibitory Concentration (MIC) value is less than 100 µg/mL, demonstrating that both the essential oil and acorenone B do not show inhibitory activity against the evaluated strains.
The weak antimicrobial activity can be explained by the abundance of oxygenated sesquiterpenes in the investigated essential oil. In fact, hydrocarbon sesquiterpenes [29], carvacrol, thymol, eugenol, perylaldehyde, cinnamaldehyde, and cinnamic acid [37] are compounds generally more efficient as antimicrobial inhibitors.

Cholinesterase Inhibition Test
In the present work, we also evaluated the anti-AChE and anti-BChE activities ( Table 4). Acorenone B showed an inhibitory activity against AChE and BChE with IC 50 concentrations of 40.8 and 10.9 µg/mL, respectively (Figure 4). These inhibitory potentials, even far from that of the reference compound donepezil (6.7 nM) versus AChE [38], are close to those previously published for galanthamine (2.2 µg/mL and 11.7 µg/mL) or other plant extracts [4,39,40]. Interestingly, in spite of its moderate inhibitory potential, galanthamine is a typical drug used for the treatment of Alzheimer's disease [41], validating our efforts to identify new potential inhibitory compounds. Niphogeton dissecta essential oil exhibited selectivity for the inhibition of BChE, this property being particularly interesting in the treatment of Alzheimer's disease [40,42].

Plant Material
The

Extraction of Essential Oil
The essential oil was obtained from the aerial parts, by steam distillation in a Clevenger-type apparatus for 4 h. Four distillations were carried out with 1805, 751, 794, and 758 grams of fresh plant material, respectively. The oil was dried on anhydrous sodium sulfate and then stored at −14 • C. The yield was expressed as mean values and standard deviations of the four distillations and reported as percentages of w/w.

Physical Analysis
The relativity density (d 20 ) was determined using a 1-cm 3 pycnometer. The refractive index (n 20 ) was measured by an Abbe's refractometer, manufactured by Boeco, Germany. The specific optical rotation [∝] 20 D was determined in a Hanon P 810 automatic polarimeter. All these properties were expressed as mean values and standard deviations of four measurements.

Gas Chromatography Coupled to Mass-Spectrometry (GC-MS)
The chemical constituents of the Niphogeton dissecta essential oil were analyzed on an Agilent Technologies 6890 N gas chromatograph, coupled to a 5973N mass spectrometer (Santa Clara, CA, USA) and equipped with a DB-5MS capillary column (5%-phenyl-methylpolysiloxane, 30 m, 0.25 mm internal diameter., 0.25 µm film thickness; J & W Scientific, Folsom, CA, USA). For the separation of the volatile constituents, the following temperature program was used: 5 min at 60 • C, 3 • C/min up to 165 • C, 15 • C/min up to 250 • C, and held for 10 min. The injector and detector temperatures were kept at 220 • C. The carrier gas was helium, at a flow rate of 1 mL/min. The injector was operated in split mode, with a split ratio of 1:50. The acquisition mass range was set at 40-350 m/z. Ionization mode: electron-impact (70 eV). The essential oil was diluted 1:100 v/v in dichloromethane (Fisher Scientific, 99.9% purity) and 1 µL of the solution was injected.
For the identification of the essential oil components, linear retention indices were calculated according to Van Den Dool and Kratz. They were determined with a homologous series of linear alkanes (C9 from BDH, purity 99%, and C10-C25 from Fluka, purity 99%).

Gas Chromatography Coupled to Flame Ionization Detector (GC-FID)
Quantitative analysis of the essential oil was performed on an Agilent Technologies gas chromatograph (model 6890N) coupled to a flame ionization detector (FID) and using a 7683 series autoinjector (Agilent, Little Falls, DE, USA). The percentage composition of the oil was determined by correlating GC peak areas to the total chromatogram, without applying any correction factor, but normalizing values with nonane as an internal standard. The qualitative analysis is expressed as the mean values of four injections and standard deviations. The analytical parameters were the same as the GC-MS analysis.

Enantioselective GC Analysis
Enantioselective GC-MS analysis was performed with the same Agilent Technologies instrument previously described. The mass spectrometer operated in electron impact ionization mode at 70 eV, with a mass range of m/z 40-350 full scan mode. The ion source temperature was set at 220 • C. Helium was the carrier gas at a flow rate of 1.0 mL/min. The injector was operated in split mode (1:40) at 200 • C, with the transfer line at 230 • C. The oven thermal program was as follows: 60 • C for 2 min, then the temperature was raised to 220 • C with a gradient rate of 2 • C/min and held at 220 • C for 2 min. A chiral capillary column based on diethyl tertbutylsilyl-BETA-cyclodextrin (25 m × 0.25 mm × film thickness 0.25 µm) from Mega (Legnano, MI, Italy) was used. The essential oil was diluted 5:100 (v/v) in dichloromethane (Fisher Scientific, 99.9% purity) and 1 µL of the solution was injected. Enantiomerically pure standards, used to determine the elution order of enantiomers, were available in one of the authors' laboratory (C.B.).

Isolation and Identification of Acorenone B
The essential oil of N. dissecta (5 g) was subjected to column chromatography over a silica gel G60 column, applying an oil/silica weight ratio of 1:200, with a mixture of Hex:AcOEt 90:10 (isocratic elution), obtaining a total of five fractions and affording acorenone B as a pure compound (1.48 g). The identification was performed by spectroscopic techniques such as EI-MS, 1 H, and 13 C NMR. 1 H and 13 C NMR spectra were acquired using a VARIAN NMR spectrometer (400 MHz for 1 H and 100 MHz for 13 C), tetramethylsilane was used as an internal standard, and chemical shifts are given in δ (ppm).

Antimicrobial Activity
Five pathogenic bacteria (ATCC): Staphylococcus aureus ATCC 25923, E. faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Proteus vulgaris ATCC 8427, and Klebsiella pneumoniae ATCC 9997 were included in the investigation. For all bacteria, except for E. faecalis, a heart-heart infusion broth (BHI-DIFCO, DIFCO, Sparks, MD, USA) was used. All the strains were maintained at −80 • C until use, when they were withdrawn to prepare overnight cultures at 37 • C for 16 h. MIC values were determined by the micro-dilution broth method, using a final concentration of 5/105 CFU/mL. DMSO solutions of the sample were prepared at a concentration of 20 mg/mL. Assays were carried out in 96-well plates and a two-fold serial dilution was used to obtain decreasing concentrations from 1000 to 0.024 mg/mL. The incubation was performed at 37 • C for 24 h. Gentamicin was used a positive control with an MIC value of 0.40 mg/mL, except for E. faecalis where ampicillin (MIC 1.56 mg/mL) was used.

Cholinesterase Inhibition Test
The cholinesterase (ChE) activities were assayed following a colorimetric protocol adapted from Ellman et al. [43,44]. ChEs efficiently catalyze the hydrolysis of acetylthiocholine (ATCh), the sulfur analog of the natural substrate of these enzymes. Upon hydrolysis, this substrate analog produces acetate ion and thiocholine. Thiocholine, in the presence of the highly reactive dithiobisnitrobenzoate (DTNB) ion, generates a yellow color, which can be quantitatively monitored by spectrophotometric absorption at 412 nm. All reagents were obtained from the Sigma-Aldrich trading house. A typical 200 µL inhibition assay volume contained phosphate buffered saline solution (pH 7.4), DTNB (1.5 mM), test sample in DMSO (1% v/v final). Both acetylcholinesterase from Electrophorus electricus (Type V-S, lyophilized powder, 744 U/mg solid, 1 272 U/mg protein) and butyrylcholinesterase from equine serum (lyophilized powder, ≥900 units/mg protein) were dissolved in PBS pH 7.4 and used at 25 mU/mL for the assay. After 10 min of pre-incubation, the substrate acetylthiocholine iodide (1.5 mM) was added to start the reaction. During 1 h of incubation at 30 • C, 96-well microtiter multiplates were read on a PherastarFS (BMG Labtech) detection system. All measurements were made in triplicate. When possible, the IC 50 values were calculated using the GNUPLOT package on line (www.ic50. tk, www.gnuplot.info). Donepezil was used as reference ChE inhibitor with an IC 50 = 100 nM for AChE and 8500 nM for BChE. In this assay, we did not exclude the possibility of false-positive inhibition results previously described for high concentrations (>100 µg/mL) of amine or aldehyde compounds [45,46], but the lack of inhibition observed for the essential oil versus the AChE strongly minimized this possibility.