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

Article abstract of DOI:10.3390/ph10040084

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.

Full text of DOI:10.3390/ph10040084

pharmaceuticals
Article
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
James Calva 1,*, Nicole Bec 2,3,4, Gianluca Gilardoni 1, Christian Larroque 2,3,4, Luis Cartuche 1,
Carlo Bicchi 5 and José Vinicio Montesinos 1
1
Department of Chemistry and Exact Sciences, Private Technical University of Loja (UTPL),
Calle Marcelino Champagnat s/n, 1101608 Loja, Ecuador; gianluca.gilardoni@gmail.com (G.G.);
lecartuche@utpl.edu.ec (L.C.); jvmontesinos@utpl.edu.ec (J.V.M.)
IRCM, Cancer Research Institute of Montpellier, INSERM, U1194, 34298 Montpellier, France;
2
nicole.bec@inserm.fr (N.B.); christian.larroque@inserm.fr (C.L.)
Montpellier Regional Cancer Institute, 34298 Montpellier, France
Institut de Recherche en Cancérologie de Montpellier, INSERM, U1194, University of Montpellier,
3
4
34298 Montpellier, France
5
Department of Medicine Science and Technology, University of Turin, Via Pietro Giuria 9, 10125 Turin, Italy;
carlo.bicchi@unito.it
*
Correspondence: jwcalva@utpl.edu.ec; Tel.: +593-7370-1444 (ext. 3049)
Received: 19 September 2017; Accepted: 25 October 2017; Published: 31 October 2017
Abstract: 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 (+)/( )- -pinene was 86.9:13.1, while the one of (+)/(
oil showed a weak inhibitory activity, expressed as Minimal Inhibitory Concentration (MIC), against
−
β
−
)-sabinene was 80.9:19.1. The essential
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.
Keywords: Niphogeton dissecta; essential oil; enantiomeric distribution; acorenone B; AChE; BChE
1. Introduction
Medicinal plants have been used for a long time as sources of new pharmaceuticals due to the
presence of bioactive compounds [
displaying a wide range of biological activities, including possible leads for the treatment of
neurodegenerative diseases [ ]. Natural products have contributed greatly as sources for drug
discovery for Alzheimer’s disease [ ]. Earlier studies have shown that the maintenance of correct
levels of acetylcholinesterases is directly related to different diseases such as Alzheimer’s disease (AD),
1]. Plants are rich in structurally diverse secondary metabolites
2,3
4
bipolar disorder, depression, and schizophrenia [
5]. There are two distinct basic types of cholinesterases:
Pharmaceuticals 2017, 10, 84; doi:10.3390/ph10040084
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acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The main physiological function
of AChE is the splitting of acetylcholine (ACh), a mediator of cholinergic synapses, during the
transduction of nerve impulses [6].
AD is a prevalent disease that affects more than 26 million people globally, and is the
most common neurodegenerative disease worldwide [7]. AChE inhibitors (AChEIs) have medical
applications and are particularly important for the symptomatic treatment of Alzheimer’s disease [
8
].
The butyrylcholinesterase enzyme (BChE) is synthesized in the liver and its main function is to
hydrolyze hydrophobic and hydrophilic carboxylic or phosphoric acid ester containing compounds.
Its toxicological and pharmacological importance becomes clear when an individual is exposured to
poisonous compounds targeting the acetylcholine binding sites. When there is a hepatic alteration,
its concentration decreases in direct relation with the altered hepatocytes [9,10].
The anti-AChE activity of the essential oils of Eryngium campestre and Eryngium amethystine,
two species of the Apiaceae family, is described in the work conducted by Cianfaglione et al. [11],
showing that the inhibitory activity is very low (10.5% at 19.5 mg/mL) compared to the commercial
inhibitor galanthamine. The activity was expressed as galanthamine equivalent inhibitory activity and
the dose tested is similar to an inhibition exerted by 3
Likewise, the essential oil of Daucus aristidis (Apiaceae) tested against AChE and BChE enzymes, by the
Ellman method, at a concentration of 100 g/mL appeared to have a moderate level of inhibitory effect
µg/mL of the positive control galanthamine.
µ
(between 13% to 50%) against both enzymes as compared to that of galanthamine. The moderate AChE
inhibitory activity of Daucus aristidis essential oil could be explained by the highest concentration of its
individual components, α- and β-pinene (over 70%) [12].
Species belonging to the Apiaceae family such as cumin, coriander, carrot, celery, and parsley
are considered foods or spices; however, some of them contain highly toxic substances [13].
Niphogeton dissecta is a native herb of the Ecuadorian Andes found at 2500–4500 m above sea level [14],
and is widely distributed in the provinces of Loja, Azuay, Cañar, Carchi, Chimborazo, Cotopaxi,
Morona Santiago, Napo, Pichincha, and Zamora-Chinchipe. Traditionally, it is believed to have
medicinal properties and it is used for the treatment of diarrhea, vomiting, inflammation of the belly,
colds, and rheumatism [15].
The aim of the present study is to assess the potential antimicrobial, antifungal, and anticholinesterase
(AChE and BChE) activity of the essential oil of Niphogeton dissecta, as well as to determine its chemical
and physical properties. In addition, the phytochemical study of the essential oil led to the isolation
and characterization of the known sesquiterpene acorenone B.
2. 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
enantiomeric analysis, and biological activity are discussed below.
±
0.03% (w/w). The physical properties, chemical composition,
2.1. Physical Properties
Three physical properties were determined: refractive index (n = 1.499
±
0.002), relative density
(d = 0.906
±
0.012 g.m/L), and optical rotation ([∝]2D0 = −7.50
±
0.98 in CHCl3, c = 10.0). In this
context, some authors state that the physical properties are determined by the genetic characteristics,
geographical location, and phenological stages of the plant [16,17].
2.2. Chemical Composition
The chemical composition of the essential oil was defined based on calculated linear retention
indices (LRIc) and mass spectra compared with literature [18–23]. 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

Pharmaceuticals 2017, 10, 84
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(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.
Table 1. Chemical composition of Niphogeton dissecta essential oil of province of Loja, Ecuador.
c
LRI lit b
Component
LRI a
%
δ
Literature for LRI
α-Pinene
Sabinene
β-Pinene
β-Myrcene
p-Cymene
Limonene
(Z)-β-Ocimene
(E)-β-Ocimene
γ-Terpinolene
Unidentified
α-Pinene oxide
Unidentified
(E,E) 2,6-Dimethyl-3,5,7-octatriene-2-ol
Citronellol
Unidentified
Geranial
Methyl geranate
Unidentified
α-Copaene
β-Funebrene
(E)-Caryophyllene
β-Cedrene
cis-Thujopsene
(E)-β-Farnesene
allo-aromadendrene
α-Himachalene
cis-Cadina-1(6),4-diene
γ-Muurolene
Germacrene-D
ar-Curcumene
α-Zingiberene
(E,E)-α-Farnesene
Unidentified
δ-Cadinene
β-Sesquiphellandrene
(E)-Nerolidol
Unidentified
Spathulenol
Unidentified
Unidentified
Geranyl isovalerate
Cedrol
Unidentified
α-Cadinol
Unidentified
Acorenone
Acorenone B
(3E)-Butylidene phthalide
Unidentified
Sandaracopimaradiene
Biformene
930
969
974
987
932
969
974
988
1020
1024
1032
1044
1056
–
1099
–
1207
1223
–
3.94
1.41
0.30
2.14
trace
0.13
0.10
29.64
0.29
trace
trace
trace
trace
0.16
0.15
0.07
0.51
0.06
trace
0.17
0.24
1.23
0.97
1.13
trace
trace
1.70
0.36
0.38
0.15
0.34
trace
0.23
0.36
0.89
0.08
0.42
0.13
0.40
0.15
0.57
0.33
0.07
0.08
1.20
0.11
41.01
5.54
0.11
trace
trace
1.79
0.28
0.09
0.37
–
0.03
0.04
1.63
0.30
–
[18]
[18]
[18]
[18]
[18]
[18]
[18]
[18]
[19]
–
[18]
–
[20]
[18]
–
1022
1027
1034
1045
1055
1072
1093
1155
1205
1224
1256
1264
1318
1334
1371
1411
1414
1417
1420
1450
1454
1457
1466
1469
1475
1477
1491
1501
1506
1513
1519
1557
1565
1569
1581
1585
1594
1600
1609
1645
1655
1666
1683
1718
1777
1943
1983
–
–
–
0.07
0.01
0.01
0.04
0.01
–
0.02
0.02
0.13
0.10
0.13
–
1264
1322
–
[18]
[18]
–
1374
1413
1417
1419
1429
1454
1458
1449
1461
1478
1484
1479
1493
1505
–
[18]
[18]
[18]
[18]
[18]
[18]
[18]
[18]
[18]
[18]
[18]
[18]
[18]
[18]
–
–
0.17
0.04
0.05
0.01
0.02
–
0.02
0.03
0.08
0.01
0.07
0.01
0.03
0.01
0.05
0.03
0.01
0.01
0.05
0.01
3.35
0.93
0.04
–
1522
1521
1561
–
1577
–
[18]
[18]
[18]
–
[18]
–
–
–
1606
1600
–
1652
–
1655
1675
1717
–
1942
1990
[18]
[18]
–
[18]
–
[21]
[22]
[18]
–
[23]
[18]
–

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Table 1. Cont.
c
LRI lit b
Component
LRI a
%
δ
Literature for LRI
Unidentified
(E,E)-Geranyl linalool
Unidentified
2008
2016
2047
2052
-
-
-
-
-
-
–
2026
–
–
-
-
-
-
-
trace
trace
trace
0.64
37.97
0.25
9.71
42.31
6.22
–
–
–
–
[18]
–
Unidentified
0.19
–
Monoterpene hydrocarbons
Oxygenated monoterpene
Sesquiterpene hydrocarbons
Oxygenated sesquiterpene
Others
Total amount of compounds
-
96.46%
a
Calculated linear retention indices (LRI) on DB-5MS capillary column; b Linear retention indices according to
literature; c Relative percentage values are means of four determinations with a Relative Standard Deviation (RSD%)
below 5% for the most abundant components. Traces % < 0.05.
A typical chromatogram of the essential oil from N. dissecta is shown in Figure 1.
Figure 1. Typical gas-chromatogram of essential oil of Niphogeton dissecta.
2.3. Isolation and Characterization of Acorenone B
Acorenone B was isolated by column chromatography on silica gel G60 (1 kg). The mixture (5.0 g)
was eluted in isocratic condition with hexane-ethyl acetate 90:10. The compound (1.48 g) was obtained
as a pale yellow oil, with optical rotation [∝]2D0 = −17.6 (CHCl3; c = 11.4). Results similar to ours were
reported by Zalkow [24].
The molecular structure of acorenone B (Figure 2) was confirmed by 1H NMR, 13C NMR, and MS
analysis, and compared with data present in the literature [24–27].

Pharmaceuticals 2017, 10, 84
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O
7
6
1
2
8
5
3
4
10
9
Figure 2. Chemical structure of acorenone B.
δ (ppm): 0.74 (3H, d, J = 6.8 Hz, CH3 of isopropyl), 0.83 (3H, d, J = 6.8 Hz,
1H NMR (400 MHz, CDCl3),
CH3 of isopropyl), 0.92 (3H, d, J = 6.4 Hz, 4-CH3), 1.73 (3H, m, 8-CH3), 2.04 (1H, m, H-10), 2.20 (1H, m,
H-6) 2.27 (1H, m, H-10), 2.67 (1H, m, H-6), 6.62 (1H, m, H-9).
13C NMR (100 MHz, CDCl3),
δ (ppm): 200.6 (C-7), 144.4 (C-9), 135.4 (C-8), 56.9 (C-1), 49.4 (C-6),
48.4 (C-5), 46.1 (C-4), 29.8 (C-3), 29.2 (C-11), 26.0 (C-10), 25.3 (C-2), 24.2 (C-13), 21.4 (C-14), 17.1 (C-12),
15.6 (C-15).
EI-MS m/z (%): 220 (M+, 58), 177 (76), 149 (45), 135 (100), 121 (60), 109 (100), 93 (46), 82 (84), 69 (28),
55 (22), 41 (29).
In our study, the fractionation of the essential oil of Niphogeton dissecta afforded a pure sequiterpene:
acorenone B. Several authors reported the occurrence of acorenone B in other volatile fractions,
such as the ones of Bothriochloa intermedia (47%) [24], Bothriochloa pertusa (9.8%) [28], Chaerophyllum
hirsutum (9.47% to 18.49%) [26], Euphorbia macrorrhiza (16.72% and 25.80%) [29], Levisticum persicum
(8.3% to 12.6%) [30], and as a major compound in Daucus littoralis subsp. Hyrcanicus (19.7% to 57.5% in
all parts) [31], and Bothriochloa bladhii (18.2%) [32].
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.
2.4. 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).
Table 2. Enantiomeric excess of some essential oil constituents from Niphogeton dissecta.
LRI b
Components
RT a (min)
Enantiomeric Distribution (%)
e.e. (%)
(+)-β-pinene
(−)-β-pinene
(+)-Sabinene
(−)-Sabinene
a
11.08
11.52
957
965
86.9
13.1
73.8
12.47
13.15
983
997
80.9
19.1
61.8
Retention Time (RT); b Calculated on MEGA-DEX-DET chiral stationary phase.
It has been documented that sometimes different enantiomers may present dissimilar biological
activities [33]. In our essential oil, the enantiomeric excess of (+)- -pinene was 73.8% with respect to
(–)- -pinene. Some studies have shown that the positive enantiomer has antimicrobial activity against
Candida albicans, Cryptococcus neoformans, Rhyzopus oryzae, and methicillin-resistant Staphylococcus aureus
β
β

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(MRSA) [34]. In contrast, the negative enantiomer exhibits antiviral properties against infectious
bronchitis virus (IBV) [35].
Abundance (PA)
β-pinene
140,000.00
(+)
120,000.00
100,000.00
80,000.00
60,000.00
40,000.00
20,000.00
0.00
(-)
Time(min)
11.65
10.95
11.05
11.15
11.25
11.35
11.45
11.55
(a)
Abundance (PA)
700,000.00
Sabinene
600,000.00
500,000.00
400,000.00
300,000.00
200,000.00
100,000.00
(+)
(-)
Time (min)
13.4
0.00
12.2
12.4
12.6
12.8
13
13.2
(b)
Figure 3. Enantiomeric separation in the essential oil Niphogeton dissecta: (a) β-pinene; (b) sabinene.
2.5. 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.

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Table 3. Strains used for biological tests.
E.O, (Niphogeton dissecta) mg/mL
Microorganism
Acorenone B (mg/mL)
Candida albicans
Enterococcus faecalis
Escherichia coli
Micrococcus luteus
Staphylococcus aureus
NA
10
NA
NA
5
NA
NA
NA
NA
NA
NA = non-active.
2.6. 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 IC50 concentrations of
40.8 and 10.9
reference compound donepezil (6.7 nM) versus AChE [38], are close to those previously published for
galanthamine (2.2 g/mL and 11.7 g/mL) or other plant extracts [ 39 40]. Interestingly, in spite of
µg/mL, respectively (Figure 4). These inhibitory potentials, even far from that of the
µ
µ
4, ,
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].
Table 4. Cholinesterase inhibitory activity.
Compound
AChE (IC50) µg/mL
BChE (IC50) µg/mL
Niphogeton dissecta essential oil
NA
40.8
11.5
10.9
Acorenone B
NA = non-active.
Figure 4. Determination of the IC50 values for the Acorenone B vs. (
a
) acetylcholinesterase (AChE) and
(b) butyrylcholinesterase (BChE). IC50: half maximal inhibitory concentration.
3. Materials and Methods
3.1. Plant Material
The aerial parts of Niphogeton dissecta in flowering state were collected in September and October
2016 in the sector Loma la Torre, Loja, at an altitude of 3210 m a.s.l., with coordinates 696030 N,
9593252 E. The specimen was identified by Dr. Fani Tinitana of the UTPL herbarium, and deposited

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with voucher number 5835. The plant was collected under permission of the Ministry of Environment
of Ecuador (MAE-DNB-CM-2016-0048).
3.2. 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.
3.3. Physical Analysis
The relativity density (d20) was determined using a 1-cm3 pycnometer. The refractive index (n20)
was measured by an Abbe’s refractometer, manufactured by Boeco, Germany. The specific optical
rotation [∝]2D0 was determined in a Hanon P 810 automatic polarimeter. All these properties were
expressed as mean values and standard deviations of four measurements.
3.4. 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%).
3.5. 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.
3.6. 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 m) from Mega (Legnano, MI, Italy) was used. The essential oil was diluted
µ

Pharmaceuticals 2017, 10, 84
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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.).
3.7. 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
(1.48 g). The identification was performed by spectroscopic techniques such as EI-MS, H, and
13C NMR. 1H and 13C NMR spectra were acquired using a VARIAN NMR spectrometer (400 MHz for
1H and 100 MHz for 13C), tetramethylsilane was used as an internal standard, and chemical shifts are
given in δ (ppm).
3.8. 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.
3.9. 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 IC50 values were calculated using the GNUPLOT package on line (www.ic50.
tk, www.gnuplot.info). Donepezil was used as reference ChE inhibitor with an IC50 = 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.
4. Conclusions
The physical properties, chemical composition, biological activity, and enantiomeric distribution
of the essential oil distilled from Niphogeton dissecta were determined for the first time. Forty-one

Pharmaceuticals 2017, 10, 84
10 of 12
compounds, representing 96.46% of the total oil, were identified. The major compounds were
acorenone B (41.01%), (E)- -ocimene (29.64%), (3E)-butylidene phthalide (5.54%), and -pinene (3.94%).
β
α
The whole volatile fraction and its pure major constituent were assayed for two biological activities:
inhibition of two bacteria strains and enzymatic inhibition against AChE and BChE. Concerning the
first activity, the oil showed an MIC value of 10 mg/mL against Enterococcus faecalis and 5 mg/mL
against Staphylococcus aureus. No activity was detected for pure acorenone B. Concerning the enzymatic
inhibition, the Essential Oil (E.O.) showed an IC50 value of 11.5 µg/mL in the inhibition of BChE and
no inhibition of AChE. On the other hand, acorenone B was active against both enzymes, with an
IC50 of 40.8 µg/mL for AChE and 10.9 µg/mL for BChE. The chiral analysis of the E.O. indicated the
following enantiomeric distribution: (+)-
and (−)-sabinene (19.1%).
β
-pinene (86.9%), ( )-β-pinene (13.1%), (+)-sabinene (80.9%),
−
Acknowledgments: We are very grateful to the Master Program in Applied Chemistry of the Universidad Técnica
Particular de Loja, Ecuador, for financial support.
Author Contributions: All of the authors listed in this work provided academic contributions to the development
of this manuscript: James Calva, Gianluca Gilardoni, and José Vinicio Montesinos collected the plant material and
performed the hydrodistillation; James Calva performed the chemical analyses and wrote the article; Gianluca
Gilardoni supervised the experiments and interpreted analytical data; Nicole Bec, Christian Larroque, and Luis
Cartuche conducted the biological activity tests; Carlo Bicchi furnished enantiomerically pure standards and gave
technical support to chiral analysis. All the authors have revised and approved the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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