Biocatalytic modifications of ethynodiol diacetate by fungi, anti-proliferative activity, and acetylcholineterase inhibitory of its transformed products

Article abstract of DOI:10.1016/j.steroids.2021.108832

The fungal transformations of ethynodiol diacetate (1) were investigated for the first-time using Botrytis cinerea, Trichothecium roseum, and R3-2 SP 17. The metabolites obtained are as following: 17α-Ethynyl-17β-acetoxyestr-4-en-3-one-15β-ol (2), 19-nor-

Full text of DOI:10.1016/j.steroids.2021.108832

Steroids 171 (2021) 108832
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Biocatalytic modifications of ethynodiol diacetate by fungi,
anti-proliferative activity, and acetylcholineterase inhibitory of its
transformed products
a
,
b
b,
c
c,d
Sharifah Nurfazilah Wan Yusop , Syahrul Imran , Mohd Ilham Adenan
Kamran Ashraf , Sadia Sultan
,
a,
b
a,b,*
a
Faculty of Pharmacy, Universiti Teknologi MARA Puncak Alam Campus, Bandar Puncak Alam, 42300 Kuala Selangor, Selangor, Malaysia
b
Atta-ur-Rahman Institute for Natural Product Discovery (AuRins), Universiti Teknologi MARA Puncak Alam Campus, Bandar Puncak Alam, 42300 Kuala Selangor,
Selangor, Malaysia
c
Faculty of Applied Sciences, Universiti Teknologi MARA Shah Alam, 40450 Shah Alam, Selangor, Malaysia
d
Universiti Teknologi MARA, Pahang Branch, Bandar Tun Abdul Razak, 26400 Jengka, Pahang, Malaysia
A B S T R A C T
The fungal transformations of ethynodiol diacetate (1) were investigated for the first-time using Botrytis cinerea, Trichothecium roseum, and R3-2 SP 17. The me-
tabolites obtained are as following: 17
α-Ethynyl-17β-acetoxyestr-4-en-3-one-15β-ol (2), 19-nor-17a-ethynyltestosterone (3), and 17α-ethynyl-3β-hydroxy-17β-ace-
toxyestr-4-ene (4). The new metabolite, 2 (IC50 = 104.8 µM), which has ketone group at C-3, and the β-hydroxyl group at C-15, resulted in an almost equipotent
strength with the parent compound (IC50 = 103.3 µM) against proliferation of SH-SY5Y cells. The previously reported biotransformed product, 3, showed almost
equal strength to 1 against acetylcholinesterase. Molecular modelling studies were carried out to understand the observed experimental activities, and also to obtain
more information on the binding mode and the interactions between the biotransformed products, and enzyme.
1
. Introduction
process. Ethynodiol diacetate (1) is a semi-synthetic steroidal drug. It is
a potent progestin that inhibits the ovulation process; therefore, it is
used as an oral contraceptive. To date, only one study has published
information on the microbial transformation of ethynodiol diacetate.
Zafar et al., (2012) reported that the microbial transformation of (1)
could be achieved by Cunninghamella elegans. They also indicated that
the biotransformation of ethynodiol diacetate resulted in three new
Climates, geographical location, substrate type, and microhabitat are
among the factors that affect the distribution of fungi globally [1]. These
results in a broad range of their physiologies and metabolic assortments.
Bioprospection of other less established settings such as the Antarctic
continent offers a unique source of microorganisms and their metabo-
lites, presenting a vast capacity of biotechnological applications [2]. For
instance, extracellular enzymes such as chitinase activity were also
indicated in Antarctic yeasts isolates [3]. Another good example of
biotechnological application of unique microorganisms is the discovery
of biotransformation ability of Mortierella minutissima. Mortierella min-
utissima is a novel psychrotolerant fungus that is used in the biotrans-
formation of D-limonene [4]. Furthermore, Chrysosporium pannorum is a
hydroxylated compounds: 17
α
-ethynylestr-4-en-3β,17β-diacetoxy-6
α
-
ol, 17
α
-ethynylestr-4-en-3β,17β-diacetoxy-6β-ol, and 17
α
-ethynylestr-4-
en-3β,17β-diacetoxy-10β-ol [6]. In addition to the main role of repro-
ductive regulation, progesterone along with progestin (synthetic pro-
gesterone) have shown promising outcomes in animal studies of myelin
repair, neurodegeneration, and also recovery of brain damage [7].
Cholinergic function was also improved by treatment with progesterone
and 17β-estradiol [8]. It has been demonstrated that amyloid-β levels
were significantly reduced in the mice when they are subjected to
treatment of cyclic progesterone alone [9].
novel psychrotolerant fungus that is used in bioconversion of
α-pinene
[
5]. This is important as microbial transformation supports sustainable
uses of resources under defined culture conditions, freeing them from
pathological restrictions and seasonal fluctuations. These microbial-
catalysed reactions aids in generating diverse organic molecules with
complex structures such as steroids, and also boosting up drug discovery
In a continuation of our work on microbial transformation [10–18],
we report here for the first time the microbial transformation of ethy-
nodiol diacetate by Botrytis cinerea, Trichothecium roseum, and R3-2 SP
*
Corresponding author at: Faculty of Pharmacy, Universiti Teknologi MARA Puncak Alam Campus, Bandar Puncak Alam, 42300 Kuala Selangor, Selangor,
Malaysia.
E-mail address: drsadia@uitm.edu.my (S. Sultan).
https://doi.org/10.1016/j.steroids.2021.108832
Received 10 September 2020; Received in revised form 22 March 2021; Accepted 28 March 2021
Available online 6 April 2021
0
039-128X/© 2021 Published by Elsevier Inc.

S. Nurfazilah Wan Yusop et al.
Steroids 171 (2021) 108832
1
7. Ethynodiol diacetate (1) along with its biotransformed metabolites
2.5. Structure elucidation of biotransformed products
were subjected to anti-proliferative activity against SH-SY5Y cells and
anti-acetylcholinesterase activity.
The structure of metabolites was identified by their comprehensive
spectral data. Nuclear magnetic resonance (NMR) spectra were
completed based on solubility of the samples in deuterated solvents
2
. Experimental
(
3 6 3 3 4
chloroform-d (CDCl ), acetone‑d (CD COCD ), and methanol‑d
2
.1. Chemicals
(CD
3
OD)) CDCl
3
on Bruker Ultra Shield Plus 600 MHz and 500 MHz
(
Bruker, USA) using a 5 mm probe. All solvents were purchased from
1
13
Ethynodiol diacetate (ED) was obtained from Sigma-Aldrich. All
Merck. H NMR, C NMR, DEPT, HMQC, HMBC, COSY, and NOESY
spectra were recorded using a 5 mm probe. HRMS analysis was per-
formed on Agilent LC/TOF-MS 6210 mass spectrometer system. IR ab-
chemicals used were of HPLC grade and analytical grade from Merck.
ꢀ 1
sorbances (cm
) were measured on Bruker Tensor II FT-IR
2
.2. Microorganisms
spectrophotometer. UV (in nm) absorbances were recorded on Jasco J-
8
15 spectrophotometer.
The ATCC fungi (Tricothecium roseum, and Botrytis cinerea) were
subcultured on (PDA) plates and stored at 2 8˚ C in an incubator. The
Antarctic fungus, R3-2 SP 17 was subcultured, and stored at 4˚C in the
chiller. Petri dishes were incubated for 5 days for all fungi. Microbial
cultures were transferred into a broth medium flask containing freshly
prepared sterilised fermentation medium (40 mL in 100 mL flask) from
the agar plates.
2
2
.6. Spectroscopic data of biotransformed products
.6.1. 17
Yellow-brown residue, yield (percentage yield): 7.00 mg (2.33%);
HRMS m/z:
M + Na] at m/z 379.1872, for C22
α-Ethynyl-17β-acetoxyestr-4-en-3-one-15β-ol: (2)
+
[
H
28
O
4
Na (calcd. 379.1885). UV
1
(
MeOH) λmax nm (241). IR (MeOH, cmꢀ ):
υ
= 3418, 2924, 1744, and
2
.3. Medium preparation and fungal cultivation for biotransformation
1
1
680. H NMR (600 MHz, CDCl
3
, δ, ppm): 5.897 (1H, s, H-4), 4.318 (1H,
experiments
s, H-15), 2.188 (1H, m, H-16b), 2.010 (1H, m, H-16a), 1.059 (1H, t, J =
0.62 Hz, H-14), 2.736 (1H, m, H-2a), 2.169 (1H, m, H-2b), 2.349 (1H,
m, H-1a), 1.652 (1H, m, H-1b), 1.961 (1H, m, H-7a), 1.148 (1H, m, H-
1
All biotransformation experiments for all fungi were set as followed.
The fermentation medium was prepared according to a specific recipe;
the specific recipe contains the measured ingredients (Glucose (10.0 g),
7
2
b), 1.823 (1H, m, H-11a), 1.448 (1H, m, H-11b), 2.494 (1H, m, H-12a),
.284 (1H, m, H-12b), 2.116 (1H, m, H-10), 1.487 (1H, m, H-9), 2.458
2 4
KH PO (5.0 g), peptone (5.0 g), yeast extract (5.0 g), NaCl (5.0 g),
(
1H, m, H-8), 2.326 (1H, m, H-6a), 2.308 (1H, m, H-6b), 1.172 (3H, s,
glycerol (10.0 mL)) in 1.0 L of purified water. The medium was then
distributed evenly among conical flasks (100 mL in 250 mL flask).
Sterilisation was completed using autoclave. 2.0 L of culture medium for
each fungus was prepared. Spores were transferred aseptically into the
13
CH
3
-18), 2.646 (1H, s, H-21), 2.078 (3H, s, CH
, δ, ppm): Table 1.
3
-25). C NMR (151
MHz, CDCl
3
2
.6.2. 19-nor-17a-ethynyltestosterone: (3)
2
50 mL conical flasks containing 100 mL of sterile medium and were
White amorphous powder, yield (percentage yield): 20.00 mg
◦
incubated for 48 h at 28 C. The cultures were shaken at 80 rpm on an
orbital shaker. Aliquots (300 µL) from the seed flask were transferred
aseptically to twenty flasks (for each fungus), and grown for a further 72
h. After 3 days of culture, the substrate solution (300 mg/ 8 mL) was
transferred equally into all the flasks (15 mg/ 0.40 mL/ flask) under
sterilized conditions. The flasks were maintained under the same con-
ditions for an additional 12 days for ethynodiol diacetate.
+
(
6.67%); HRMS m/z: [M + Na] at m/z 361.2373, for C20
26 2
H O Na
calcd 321.1830). UV (MeOH) λmax nm (240). 1H NMR (600 MHz,
(
CDCl
3
, δ, ppm): 5.858 (1H, s, H-4), 1.758 (1H, m, H-15a), 1.305 (1H, m,
H-15b), 2.316 (1H, m, H-16a), 2.003 (1H, m, H-16b), 1.565 (1H, m, H-
1
1
4), 2.354 (1H, m, H-2a), 2.287 (1H, m, H-2b), 2.265 (1H, m, H-1a),
.552 (1H, m, H-1b), 1.848 (1H, m, H-7a), 1.083 (1H, m, H-7b), 1.931
(
1H, m, H-11a), 1.263 (1H, m, H-11b), 1.883 (1H, m, H-12a), 1.624 (1H,
2
.4. HPLC analysis and isolation of the biotransformed products
Table 1
1
3
C NMR data for compounds 1–4.
The fermentation media were extracted three times with ethyl ace-
No.
1
2
3
4
tate. The organic extract was evaporated under reduced pressure on a
rotary evaporator. One mg of the extract was dissolved in 1 mL of
methanol (MeOH). The sample was filtered through a nylon membrane
1
23.38
34.99
70.28
26.02
37.32
26.58
36.50
22.65
35.14
64.54
2
3
199.87
124.84
167.15
36.51
30.79
35.1
199.61
124.61
166.51
35.47
30.66
41.04
49.12
42.56
26.21
32.43
46.86
49.16
22.88
38.82
79.71
12.60
87.26
74.17
–
filter 0.45
DAD). The detector was also set to display the absorbance at the
following wavelengths: 220, 254, 280, 320 and 360 nm. All analyses
were carried out in a reverse phase mode, using a Synergy 4 m Hydro-
RP 80 Å column (150 × 4.6 mm, 4 m particle size, Phenomenex®, USA)
μm. The extracts were analysed using a diode array detector
4
119.98
144.74
31.36
25.72
41.21
49.40
41.65
25.21
32.91
47.71
47.67
23.39
37.27
84.49
13.42
83.33
78.48
169.50
21.40
170.85
21.40
121.85
145.27
32.98
31.26
41.13
49.42
42.02
26.20
30.35
47.75
47.70
23.43
37.28
84.53
13.43
83.35
74.80
–
(
5
6
7
μ
8
μ
9
48.77
37.51
23.19
35.12
47.27
53.06
66.73
41.38
84.55
15.95
83.28
75.47
169.55
21.40
–
with a guard column filled with the same material. The column tem-
perature was maintained at 3 6˚ C. The mobile phase comprises of purified
water (solvent A) and acetonitrile (solvent B). The assessment was car-
ried out at a flow rate of 1 mL/min with the following elution gradient: 0
min 10% B, 10 min 46% B, 15 min 70% B, and 20 min 100% B. The
qualitative analysis of ED metabolites was carried out by Agilent HPLC-
10
11
1
1
1
1
2
3
4
5
16
1
1
2
2
7
8
0
1
1
200 using a 10
μL injection. Purification of sample mixtures from
GILSON-PLC 2020 was achieved using Recycling Preparative HPLC (RP-
HPLC JAI LC-9103) fitted with JAIGEL ODS-AP, 20 X 250 mm column.
Sample (10–70 mg) was injected into the system in a single injection,
and the RP-HPLC was set to an isocratic condition of ACN in water (Flow
rate: 4 mL/min) with pre-set UV of 220 nm/250 nm depending on the
chromatographic profiles of sample mixtures.
22
23
–
–
2
2
4
5
–
169.61
21.45
–
–
2

S. Nurfazilah Wan Yusop et al.
Steroids 171 (2021) 108832
m, H-12b), 2.112 (1H, td, J = 4.62, 10.32 Hz, H-10), 0.914 (1H, m, H-9),
2.8. Acetylcholinesterase inhibition assay
1
.352 (1H, m, H-8), 2.422 (1H, dt, J = 5.88, 10.02 Hz, H-6a), 2.273 (1H,
m, H-6b), 0.936 (3H, s, CH
MHz, CDCl
, δ, ppm): Table 1.
3
-18), 2.603 (1H, s, H-21). 13C NMR (151
2.8.1. In-vitro anti-acetylcholinesterase activities of biotransformed
products and their precursors
3
Slight modification to Ellman’s developed spectrophotometric
method was used for measurement of AChE inhibiting activity of bio-
transformed compounds. Physostigmine was used as a reference for
AChE inhibitor and measurement of anticholinesterase activity was
2
.6.3. 17
White residue, yield (percentage yield): 7.96 mg (2.65%); HRMS m/
z: [M + H] + at m/z 365.4188, for C22 Na (calcd. 365.2093). UV
MeOH) λmax nm (240). H NMR (600 MHz, CDCl , δ, ppm): 4.159 (1H,
m, H-3), 5.581 (1H, d, J = 4.5 Hz, H-4), 1.363 (1H, m, H-15a), 1.776
1H, m, H-15b), 2.011 (1H, m, H-16a), 2.754 (1H, m, H-16b), 2.036 (1H,
m, H-2a), 2.262 (1H, m, H-2b), 1.406 (1H, m, H-1a), 1.845 (1H, m, H-
α-ethynyl-3β-hydroxy-17β-acetoxyestr-4-ene: (4)
30 3
H O
1
′
(
3
done using 5, 5 -Dithio-bis (2-nitrobenzoic) acid (DTNB). The reference,
physostigmine, and all the test samples were dissolved in a buffer prior
to assay at a stock concentration of 3.125 mM and serial dilution was
(
done accordingly. In brief, 190
μ
L DTNB, 20
μ
L of sample solution, and
1
1
1
b), 1.148 (1H, m, H-7a), 1.961 (1H, m, H-7b), 1.227 (1H, m, H-11a),
.942 (1H, m, H-11b), 1.578 (1H, m, H-12a), 1.803 (1H, m, H-12b),
.729 (1H, m, H-10), 0.815 (1H, m, H-9), 1.286 (1H, m, H-8), 1.703 (1H,
20 L of AChE enzyme solution were added using multichannel auto-
μ
matic pipette) into a 96-well microplate and incubated for 10 min at
◦
37 C. Next, addition of 20
μ
L acetylcholine iodide (substrate) initiated
m, H-6a), 1.889 (1H, m, H-6b), 0.927 (3H, s, CH
3
-18), 2.610 (1H, s, H-
the reaction. The reaction of thiocholines and DTNB catalysed by en-
zymes gave rise to the formation of the yellow 5-thio-2-nitrobenzoate,
which signifies the degree of hydrolysis of acetylcholine iodide. The
degree of hydrolysis of acetylcholine iodide was measured using a
microplate reader SPECTROstar Nano (BMG LABTECH), where the
absorbance was read kinetically for 15 min at 412 nm. Reaction rates for
the samples to the blank were compared in order to measure enzyme
activity of the isolated biotransformed products. GraphPad Prism was
used to determine IC50 values.All the samples were tested in triplicates,
and the results were expressed as mean ± SEM.
2
1), 2.064 (3H, s, CH
3
-25). 13C NMR (151 MHz, CDCl
3
, δ, ppm): Table 1.
2
.7. Anti-proliferative study of biotransformed products against human
neuroblastoma cells (SH-SY5Y cell line)
Anti-proliferative activity of biotransformed metabolites were eval-
uated against SH-SY5Y cells (human neuroblastoma cells) by using the
MTT (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide)
assay. SH-SY5Y cells were maintained in Nacalai Tesque Minimal
essential medium (MEM) containing 4-(2-hydroxyethyl)-1-piper-
azineethanesulfonic acid (HEPES) supplemented with F-12 K nutrient
mixture, 10% (v/v) foetal bovine serum, and 1% pen-
Percentage inhibition (%) =
Absorbanceofcontrol ꢀ Absorbanceoftestsam ple
× 100%
Absorbanceofcontrol
◦
icillin–streptomycin in T175 flasks. The cells were kept at 37 C in hu-
midified 5% CO incubator and they were passaged every three days.
2
2
.8.2. In-silico studies of acetylcholinesterase inhibition
Cells were introduced into 96-well plates at a concentration of 25 000
cells/well and incubated for 24 h. After 24 h, medium was removed, and
cells were treated for 24 h with various concentrations of the bio-
transformed compounds (3.906, 7.813, 15.625, 31.250, 62.5, 125, 250
and 500 µM). Twenty-four hours later, 20 µL of MTT solution (5 mg
dissolved in 1 mL PBS) was added into each well including controls and
The crystal structure of recombinant human AChE (PDB ID: 4EY7)
was obtained from the Research Collaboratory for Structural Bioinfor-
matics (RCSB) Protein Data Bank. The structure contained a known co-
crystallized inhibitor Donepezil. The 3D structure of recombinant
human AChE (rhAChE) was prepared using BIOVIA Discovery Studio
Visualizer Version 17.2.0.16349. The preparation of the structure in-
cludes the process of deletion of ligand, removal of all water molecules,
addition of the missing hydrogen atoms, and minimisation of energy.
Validation of the docking protocol was performed in order to ensure its
reliability for later analysis of the studied compounds. Donepezil was
extracted from the co-crystal ligand from the PDB file and later re-
docked to the co-crystal recombinant human AChE protein. The
computation was performed for the root mean squared deviation
◦
incubated for another 4 h at 37 C. Next, 100 µL dimethyl sulfoxide
(
DMSO) was added to each well to dissolve resultant formazan crystals.
Reduction of MTT to formazan crystals by mitochondrial dehydrogenase
which is present in viable cells was visualized by the development of
blue formazan product. The absorbance was read at 570 nm with a
microplate reader SPECTROstar Nano (BMG LABTECH). Cisplatin was
used as positive control for human neuroblastoma cells, whereas DMSO
was added in the negative control instead of compounds (Table 2).
Cell proliferation inhibition (%) = 100-{(As-Ab)/ (Ac-Ab)} x100
Where,
(
RMSD) of the atomic position between the original orientation of the
co-crystal ligand and the re-docked ligand. The value is deemed
acceptable if the RMSD value is less than or equal to 2.0. The ligands
As = Absorbance value of sample compound
(
two most potent biotransformed compounds) were prepared using
ChemDraw Ultra Version 12.0.2.1076 and ChemDraw 3D Pro Version
2.0.2.1076. Grid box was prepared to maintain coverage of the active
Ab = Absorbance value of blank
Ac = Absorbance value of control
1
site of a recombinant human with a dimension of 50X50X50 Å (X, Y, and
Z axes of 10.634, ꢀ 56.163, ꢀ 23.873, respectively. Molecular docking
was consequently performed using AutoDock 4.2 using refined protein
and ligands. Finally, AutoDock 4.2 was run continually to get best 150
different docked conformational poses against a target molecule. The
parameters such as inhibition constant, binding energy and intermo-
lecular energy for the two most potent biotransformed products were
studied. The three-dimensional structure of protein–ligand interaction
was created and visualised using BIOVIA Discovery Studio Visualizer
Version 17.2.0.16349.
Table 2
In-vitro anti-proliferative activities of ED, and its metab-
olites against human neuroblastoma cell line, SH-SY5Y.
Compound
IC50 (µM)
1
2
3
4
103.30 ± 0.03776
104.80 ± 0.07355
169.30 ± 0.07735
206.80 ± 0.03967
0.61 ± 0.09010
3
. Results and discussion
b
Cisplatin
3
.1. Structure elucidation
Results are expressed as IC50 values (µM). IC50 ± S.E.M are
also given. All value is the mean of three replications.
33 3
Microbial transformation of ethynodiol diacetate (C24H O ) (1)
b
Control used in the assays.
3

S. Nurfazilah Wan Yusop et al.
Steroids 171 (2021) 108832
using Botrytis cinerea, and Trichothecium roseum are reported here for the
first time. Fermentation of 1 with B. cinerea yielded one new metabolite
group at C-15 was deduced to be β on the basis of NOESY correlations
between H-15 (δ 4.318), and H-14 (δ 1.059). Therefore, a β-OH was
placed at C-15. Thus, the structure of the new compound was charac-
2
(Fig. 1). Biotransformation of 1 by T. roseum yielded one known
metabolite 3 (Fig. 1). A known compound 4 was obtained from
biotransformation of 1 with the Antarctic fungus, R3-2 SP 17 (Fig. 1).
The isolated new metabolite 2 is obtained as yellow–brown residue
from RP-HPLC (60% ACN in water with pre-set UV of 250 nm).The
terized as 17
α-Ethynyl-17β-acetoxyestr-4-en-3-one-15β-ol (Fig. 3).
Fermentation of 1 with T. roseum yielded a known product (as the
main transformation product). The metabolite 3 was characterized as
norethisterone based on detailed spectrometric studies. Compound 3
was previously reported by Zafar et al. (2012) where it was obtained via
biotransformation technique of using plant cell suspension cultures of
Ocimum basilicum (20 mg product yield and required 35 days for the
whole process) & Azadirachta indica. (Yield not disclosed, and also
required 30 days for the whole process). Freudenthalet. al., 1977 re-
ported that incubation of 1 with rat and human liver cells has also
resulted in the formation of metabolite 3. To date, no formation of 3 via
microbial transformation has been reported. Our findings have short-
ened the process to about half of the time reported with comparable
yield, used less starting material, and no photoperiod required when
compared with the studies done by Zafar et al. (2012). Metabolite 3,
isolated as a white amorphous powder from the isocratic condition of
50% ACN in water with pre-set UV of 250 nm on RP-HPLC. The HRMS
28 4
molecular formula of 2 was established as C22H O as supported by the
presence of adduct ions surfacing at m/z 379.1872 (calcd. 379.1885)
and and m/z 735.3874 (calcd. 735.3873) corresponding to sodium ad-
+
+
ducts [M + Na] and [2 M + Na] , respectively in the (+) HRMS
analysis. HPLC-DAD analysis showed the UV spectrum having absorp-
tion at 241 nm. The IR spectrum of 2 displayed absorptions at 3418,
2
924, 1744, and 1680 cm-1 indicative for hydroxyl and conjugated
1
carbonyl functionalities, respectively. The H NMR spectrum of metab-
olite 2 displayed two methyl groups where both methyls appeared as
3 3
singlets at δ 1.172 and δ 2.078, assigned to CH -18 and CH -23,
respectively. The loss of one of the methyl groups when compared to the
substrate, 1 suggested removal of acetate at C-3 leading to ketone for-
mation. The loss of one methylene proton signals was in line to an
additional downfield signal (δ 4.318), suggesting the hydroxylation of
the methylene group. The 13C NMR and DEPT spectra of the bio-
transformed product gave a total of 22 carbon signals. The HMQC cor-
relations presented the proton signal at δ 5.897, which corresponded to
an olefinic methine carbon signal at δ 124.84 (C-4). The olefinic methine
proton and carbon signals have become deshielded and this could be due
to an adjacent carbonyl group. Hence, a carbonyl group at C-3 is
established by the occurrence of downfield quaternary carbon value at δ
+
analysis provided a [M + Na] at m/z 321.6121 which is consistent with
20 26 2
C H O Na formula (calcd 321.1830). The UV spectrum displayed an
absorption at 240 nm. The IR spectrum of 3 exhibited absorption bands
–
–
at 3400 (OH), 3300 (C ≡ CH), 1650 (C
O), and 1445 (C-H bending)
2
1
1
cm- . The H NMR spectrum of 3 was remarkably different from the
substrate, in many aspects, especially the loss of C-3 methine, C-23
methyl, and C-25 methyl protons. The C-4 methine proton resonated at a
downfield shift δ 5.858, instead of δ 5.350. The disappearance of the C-
23 and C-25 methyl signals furthers suggested the hydrolysis of the C-3
and C-17 ester groups. A quaternary carbon signal at δ 199.61, and a
1
99.73. The loss of one methylene carbon signal and the presence of a
downfield signal at δ 66.73 was attributed to the C-11 methine, which
gave additional evidence of the presence of an OH group at this carbon.
Methyl carbons resonated at δ 15.95, and δ 21.40 and they were
attributed to C-18 and C-19 methyl carbons respectively. The position of
carbonyl carbon was assigned at C-3 (δ 199.87) based on HMBC corre-
lations of H-1a (δ 1.652) and H-1b (δ 2.349) with C-3 (δ 199.87). HMBC
correlations of C-4 proton (δ 5.897) to C-2 (δ 37.32) and C-6 (δ 36.51)
also supported the assignment of ketonic carbonyl carbon at C-3. The
position of the hydroxyl group was assigned at C-15 (δ 66.73) was also
based on the key HMBC correlations of H-16a (δ 2.010) with C-15 (δ
1
3
downfield methine signal at δ 124.61 exhibited in the C NMR spectrum
were assigned to C-3 and C-4, respectively, thus providing evidence for
the presence of carbonyl signal at C-3. An upfield carbon signal was
found resonating at δ 79.71, and this was attributed to quaternary C-17
in the 13C NMR spectrum of metabolite 3, which further supported hy-
droxylation has occurred. In the HMBC spectrum of 3, the C-1 methylene
3
protons (δ 1.552, δ 2.265) showed J heteronuclear couplings with C-3 (δ
199.91). The C-6 methylene protons (δ 2.273, δ 2.422) showed J het-
eronuclear couplings with C-5 (δ 166.51). Changes to C-17 was sup-
ported by the J heteronuclear couplings of C-14 proton (δ 1.565), and
C-15 proton (δ 1.758) with C-17 (δ 79.71). C-17 (δ 79.71) also have J
heteronuclear couplings with C-18 methyl protons (δ 0.936) and C-16
2
3
6
6.73), and H-15 (δ 4.318) with C-13 (δ 47.27) (Fig. 2). In the COSY
spectrum, H-15 (δ 4.318) showed correlations with H-14 (δ 1.059), H-
6a (δ 2.010) and H-16b (δ 2.188). The stereochemistry of the hydroxyl
2
1
Fig. 1. Biotransformation of ethynodiol diacetate (1) with Botrytis cinerea, Tricothecium roseum, and R3-2 SP 17.
4

S. Nurfazilah Wan Yusop et al.
Steroids 171 (2021) 108832
Fig. 2. Key HMBC correlations in metabolites 2, 3, and 4.
Fig. 3. Key COSY and NOESY correlations in metabolites 2, 3, and 4.
proton (δ 2.003) (Fig. 2). In the COSY spectrum, C-4 methine proton
which resonated at δ 5.858, showed cross-peaks with C-2 proton (δ
towards the cells [20]. Norethisterone and medroxyprogesterone ace-
tate are significantly metabolised in human cervical tissue although at
lesser degree than progesterone [21]. The steroid norethisterone is also
one of the examples of novel autophagy inducers apart from oxaprozin,
methyltestosterone, and zidovudine [22].
2
.287), C-6 proton (δ 2.422), and C-10 methine proton (δ 2.112). The
experiment also showed C-18 methyl protons has homonuclear cou-
plings with H-10 (δ 2.109), and C-15 methylene protons (δ 1.305, δ
1
.758) (Fig. 3). In 1951, norethisterone or 19-nor-17a-ethynyltestoster-
Fermentation of 1 with R3-2 SP 17 yielded 7.96 mg of metabolite 4 as
the main transformation product. Metabolite 4 was identified as 2 based
on the spectrometric studies and evaluation of the reported data by Zafar
et al. (2012) as a biotransformation product of 1 by plant cell suspension
cultures of Ocimum basilicum. The research team also reported that they
only managed to obtain 3.5 mg of product yield 4 from the use of 600 mg
one was first synthesized in the lab of Carl Djerassi, marking the
chemical birthday of the pill [19]. Norethisterone is a progestin that is
used as oral contraceptive pills. Minami, Kosugi, Suganuma, Yamanaka,
&
Kusuki (2013) reported that norethisterone induces apoptosis in
human endometriotic stromal cells, and have anti-proliferative activity
5

S. Nurfazilah Wan Yusop et al.
Steroids 171 (2021) 108832
of the substrate and required a total of 35 days for the whole process.
Again, the findings here have shortened the process to about half of the
time reported, used less starting material and yielded more of the
product when compared with the studies done by Zafar et al. (2012).
Our technique has no photoperiod required. Another research was done
in 1977 by Freudenthalet. al. has described the formation of metabolite
study of sex steroid hormone neurobiology involves the use of human
neuroblastoma SH-SY5Y cells as part of experimental cell models.
In the search of new active compounds with prospective anti-tumour
activity, ethynodiol diacetate (1) and its metabolites were evaluated for
their anti-proliferative activity against human neuroblastoma cell line,
SH-SY5Y.
4
by incubation of 1 with rat and human liver cells (Freudenthalet. al.,
The neuronal cells, SH-SY5Y, were exposed to the parent compound,
1, and its purified biotransformed products. Removal of acetate and
formation of OH-group at C-3 in 4 has caused a major loss of activity
against SH-SY5Y, giving rise to the least active biotransformed metab-
olite of ethynodiol diacetate (IC50 = 206.8 µM). The new metabolite, 2
(IC50 = 104.8 µM), which has ketone group at C-3, and the β-hydroxyl
group at C-15, resulted in an almost equipotent strength with the parent
compound (IC50 = 103.3 µM). On the other hand, the removal of C-3 and
C-17 acetate, and the formation of the β-hydroxyl group at C-17 led to a
significant loss of anti-proliferative activity against SH-SY5Y in 3 (IC50
= 169.30 µM). It is interesting to note the trend in activities of 2 and 3, as
both have C-3 acetate removal, but with additional hydroxylation at
different positions. An extra removal of C-17 acetate and formation of
OH-group at C-17 in 3 is shown to be unfavourable to anti-proliferative
activity.
1
977, as cited in Zafar et al., 2012). To date, no formation of 4 via mi-
crobial transformation has been reported. Metabolite 4 was isolated as
white residue from the isocratic condition of 80% ACN in water with
pre-set UV of 220 nm on RP-HPLC. The HRMS analysis of the metabolite
+
displayed a [M + Na]
at m/z 365.4188 which is consistent with
22 30 3
C H O Na formula (calcd. 365.2093). This showed the loss of 42 a.m.
u, when compared to 1, and this was indicative of the modification of the
acetate region in the transformed product. This was further supported by
the existence of adduct ions appearing at m/z 707.1213 (calcd.
+
7
07.4288) that also corresponds to sodium adduct [2 M + Na] . The UV
spectrum showed an absorption at 240 nm. The IR spectrum exhibited
–
–
–
–
ꢀ 1
C) cm .
absorption bands at 3415 (OH), 1648 (C
O) and 1598 (C
1
The absence of δ 5.233 signal of starting material in the H NMR spec-
trum that is a proton geminal signal to ester group at C-3 suggests
1
modification might have occurred at this region. However, the H NMR
spectrum of compound 4 displayed a broad singlet at δ 4.159 and also
another broad singlet at δ 5.581 which are attributed to C-3 and C-4,
respectively. The presence of a singlet for methyl group at δ 2.064 (H-
3.3. Acetylcholinesterase inhibition assay
3.3.1. In-vitro anti-acetylcholinesterase activities of biotransformed
products and their precursor
2
5), indicates that the ester at C-17 is still intact. The missing methyl
group at C-3 indicates hydrolysis of the C-3 ester into an –OH. Another
methyl group that still remains is represented by a singlet at δ 0.927 that
is due to the C-18 methyl group. The rest of the spectrum was almost
similar to the 1. The 13C NMR spectra exhibited the presence of two
methyls, eight methylene, seven methine and five quaternary carbons.
Two quaternary carbon signals at δ 84.53 and δ 83.35 were assigned to
C-17 and C-20, respectively. This further confirms the C-17 ester group
is still intact and the loss of an ester group is due to hydrolysis of C-3
ester into an –OH. The position and hydrolysis of C-3 ester into an –OH
was concluded from distinct assignments of carbons and protons that
were made through the analysis of HMQC, HMBC and COSY outcomes.
The cross-peaks in the HMBC spectrum further validated the hydrolysis
of the ester group at C-3 into an –OH. In the HMBC spectrum, Me-25
protons (δ 2.064) showed heteronuclear correlations with C-17 (δ
Depletion of acetylcholine level is associated with a malfunction of
the cholinergic neurotransmission leading to loss of memory or intel-
lectual abilities in Alzheimer’s disease (AD) patients. Elevation of
acetylcholine level at synaptic junctions can be achieved via inhibition
of acetylcholinesterase. The elevated level of acetylcholinesterase am-
plifies the cholinergic transmission which could lead to improvement of
cognitive function in AD patients. Octohydroaminoacridine succinate is
one of the new acetylcholinesterase inhibition therapies that is currently
in phase III of Alzheimer’s disease drug development is [24]. To date,
only drugs of this class are approved by the Food and Drug Adminis-
tration (FDA). Donepezil, galantamine, and rivastigmine are the three
acetylcholinesterase inhibitors approved by the FDA [25].
To determine the inhibitory potential of the ethynodiol diacetate
analogues, the inhibitory activities of 1 and its biotransformed products
(2, 3, and 4) were evaluated against acetylcholine (AChE) using the
modified Ellman’s method [26]. Physostigmine was used as a reference
inhibitor for comparison purposes. Table 3 summarises IC values of
8
4.53), C-20 (δ 83.35), and C-24 (δ 169.61). Along with the assignments
of protons and carbons in the HMQC spectrum, this confirms that the C-
3
1
7 ester is still present. C-17 also exhibited J correlations with H-18 (δ
5
0
2
0
.927) and H-21 (δ 2.610) protons, while also having a J correlation
biotransformed products of ED for the inhibition of AChE.
with C-16 methylene protons (δ 2.011, δ 2.754). H-21 was also seen
The impact of the modification pattern of the starting material on the
inhibition activity of acetylcholinesterase of isolated biotransformed
derivatives tested in this assay was examined. None of the transformed
metabolites of 1 has improved AChE inhibitory activity than the starting
material, 1, under the same experimental conditions. Removal of both
acetates and formation of ketone at C-3 and introduction of the hydroxyl
group at C-17 in the isolated biotransformed product of 3 (IC50 = 27.22
µM) proved to be an ineffective strategy to increase enzyme inhibition. A
minor change to starting material, which involves the removal of
having a 2J correlation with C-20 (δ 83.35). The HMBC spectrum also
3
displayed J correlations of H-3 (δ 4.159) with C-1 (δ 22.65), while C-1
3
protons (δ 1.406, δ 1.845) also have J correlations with C-3 (δ 64.54). C-
3
4
3
6
proton (δ 5.581) was observed to have J correlations with C-2 (δ
2
5.14) and C-10 (δ 42.02), while also having a J correlation with C-3 (δ
4.51). These too supported no C-3 ester group correlation detected as
the C-3 ester group has hydrolysed into –OH and the ester at C-17 is still
intact (Fig. 2). The COSY spectrum revealed the presence of cross-peaks
between H-3 (δ 4.159) to H-4 (δ 5.581) and these were indicative of the
closeness of these protons. C-25 methyl protons have correlations with
H-15 methylene protons (δ 1.363, δ 1.776) and one of the H-16 meth-
ylene protons (δ 2.754). These too verified that C-17 ester group remains
and the hydrolysis of C-3 into –OH as no correlation of ester detected at
C-3 region (Fig. 3).
Table 3
In-vitro anti-acetylcholinesterase activities of ED, and its
metabolites.
Compound
IC50 (µM)
1
24.44 ± 0.04704
40.19 ± 0.05158
27.22 ± 0.04102
42.65 ± 0.06331
0.067 ± 0.04448
2
3
.2. Anti-proliferative activity of biotransformed products against human
3
neuroblastoma cell line, SH-SY5Y
4
b
Physostigmine
Sex steroid hormones such as estrogens, androgens, and pro-
gesterones (progestins included) have activities on cell differentiation,
cell proliferation, and homeostasis [23]. According to the authors, the
Results are expressed as IC50 values (µM). IC50 ± S.E.M are
also given. All value is the mean of three replications.
b
Positive control used in the assays.
6

S. Nurfazilah Wan Yusop et al.
Steroids 171 (2021) 108832
acetate, and the introduction of a β-hydroxyl group at C-3 resulted in the
formation of 4. Further compound 4 exhibited the weakest AChE inhi-
bition (IC50 = 42.65 µM) among the isolated biotransformed products as
compared to 3, and its starting material, ED. Ketone formation at C-3
and hydroxylation at C-15 in 2 was also detrimental to acetylcholines-
terase inhibition strength (IC50 = 40.19 µM).
would eventually lead to an increase in cholinergic transmission [28].
The simulations suggest that ligand interactions of 1 with residues VAL
294, PHE 295, TRP 86, HIS 447, and GLY 48 of rhAChE. These residues
are parts of binding pocket 78 (P78), which is also known as β-anionic
site of AChE are significant for its inhibitory activity [29]. Although 3
has hydrogen bond interactions with residues that are also part of P78,
the smaller number of hydrogen bond interactions formed in 3 when
compared to 1 may have caused the slight loss of activity. In this study, it
was also observed only in 3 that the carbonyl group was located nearby
to one of the catalytic triad residues, HIS 447 of acetylcholinesterase.
HIS 447 along with SER 203 are believed to be directly involved in the
forming and breaking of bond for the natural mechanism[30]. The
imidazole group of HIS 447 acts as a catalytic base, accepting one proton
transferred from SER 203 where the SER 203 functions as a nucleophilic
attacking site [31]. The result of 3 suggests that a nucleophilic can occur
between SER 203 and carbonyl group in the inhibitor with the presence
of HIS 447 nearby. However, this nucleophilic attack is not observable
through molecular docking, and no interaction is observed. In previous
study, it was reported that the catalytic serine residue of acetylcholin-
esterase promotes a nucleophilic attack in the carbonyl group of
physostigmine [31]. PHE 295 hydrogen bond interaction was observed
in 1. In addition, PHE 295 is one of the main residues in the acyl binding
pocket, and it is responsible for interacting with acetyl group [32]. PHE
295 along with PHE 297 function as gatekeepers that limit the dimen-
sion of substrates that can enter the active site [33]. The loss of this
interaction in 2, 3, and 4 implies the decline in inbition of acetylcho-
linesterase. Thus, all these residues must be considered for the devel-
opment of rational structure-based acetylcholinesterase inhibitors with
B. In-silico (molecular modelling) studies of acetylcholinesterase
Molecular modelling studies of the enzyme-ligand binding in-
teractions of the stating material, and biotransformed products, within
the active site of rhAChE produces a structural insight into the inhibition
mechanism of biotransformed products.
Biotransformed metabolites of 1 interacted with various residues via
formation of multiple hydrogen bonds, and non-bonding interactions in
the docked rhAChE-ligand complex. Hydrogen bonding between a pro-
tein, and its ligands is important in molecular recognition as they
contribute to directionality, and specificity of interactions [27]. The
AChE residues found involved in hydrogen bonds in 1 were VAL 294,
PHE 295, TRP 86, HIS 447, and GLY 48 (Fig. 4 (1)A.). GLY 126, TYR
1
33, TYR 337, and TYR 124 were the AChE residues found associated
with hydrogen bonds in 2 (Fig. 4 (2)B.). Biotransformed metabolite 3
has hydrogen bond interactions with the AChE residues TYR 133, TYR
1
24, and GLY 120 (Fig. 4 (3)C.). Lastly, metabolite 4 has hydrogen bond
interactions with the AChE residues TYR 133, TYR 337, ALA 127, GLY
1
26, GLY 121 and GLY 120 (Fig. 4 (4)C.).
As acetylcholinesterase inhibitors, attachment of 1–4 at acetylcho-
linesterase inhibitors at binding site restrains the interaction of acetyl-
choline molecule with binding site residues leading to impairment of
hydrolysis reaction. As acetylcholine concentration intensifies, this
Fig. 4. Key hydrogen bonding interactions between AChE, and compounds 1, 2, 3, and 4.
7

S. Nurfazilah Wan Yusop et al.
Steroids 171 (2021) 108832
increased potency and high specificity.
s Disease Mice, Endocrinology 151 (2010) 2713–2722, https://doi.org/10.1210/
en.2009-1487.
[
10] S. Sultan, M.I. Choudhary, S.N. Khan, U. Fatima, M. Atif, R.A. Ali, A.U. Rahman, M.
Q. Fatmi, Fungal transformation of cedryl acetate and alpha-glucosidase inhibition
assay, quantum mechanical calculations and molecular docking studies of its
metabolites, Eur. J. Med. Chem. 62 (2013) 764–770, https://doi.org/10.1016/j.
ejmech.2013.01.036.
4
. Conclusions
Microbial transformation of compound 1 yielded metabolites 2, 3,
and 4 respectively with Botrytis cinerea, Trichothecium roseum, and R3-2
SP 17. Compound 2 was found to be new, while 3 and 4 were found to be
known products. The use of psychrotolerant fungus as biocatalytic agent
of 1 was reported here for the first time, resulting a significant, improved
yield of 3, and 4 than previous reported technique. Among all the tested
biotransformed compounds, the new biotransformed product, 2 is
almost as potent as parent compound, 1 for anti-proliferative activity
against SH-SY5Y tumour cell line. Compound 3 has comparable
acetylcholinesterase inhibition as 1. This is further supported by the
binding mechanism of 1, and 3 into the structure of rhAChE, which were
examined through molecular docking studies. The activities reported
here deserve to be put into consideration as they are good illustrations in
supporting the application of microbial transformation as a viable
method of future development of anti-proliferative drug candidates, and
acetylcholinesterase inhibitors.
[11] S.A.A. Shah, S. Sultan, H.S. Adnan, A whole-cell biocatalysis application of
steroidal drugs, Orient. J. Chem. 29 (2013) 389–403. doi:10.13005/ojc/290201.
[
12] S. Erum, S. Sultan, S.A.A. Shah, M. Ashraf, M.I. Choudhary, Microbial Oxidation of
Finasteride With Macrophomina Phaseolina(Kucc 730), Int. J. Pharm. Pharm. Sci.
9 (2017) 17. doi:10.22159/ijpps.2017v9i11.13576.
[13] M.I. Choudhary, S. Sultan, S. Jalil, S. Anjum, A. Rahman, H.-K. Fun, Atta-ur-
Rahman, Microbial Transformation of Mesterolone, C&B 2 (3) (2005) 392–400,
https://doi.org/10.1002/cbdv.200590019.
[
14] M. Atif, S.A.A. Shah, S. Sultan, M.I. Choudhary, Solid phase microbial fermentation
of anabolic steroid, dihydrotestosterone with ascomycete fungus fusarium
oxysporum, Int. J. Pharm. Pharm. Sci. 7 (2015) 104–107.
[
15] S. Sultan, M. Zaimi Bin Mohd Noor, E.H. Anouar, S.A.A. Shah, F. Salim, R. Rahim,
Z.B.K. Al Trabolsy, J.F.F. Weber, Structure and absolute configuration of 20β-
hydroxyprednisolone, a biotransformed product of predinisolone by the marine
endophytic fungus penicilium lapidosum, Molecules. 19 (2014) 13775–13787. doi:
1
0.3390/molecules190913775.
[
[
16] M.I. Choudhary, S. Sultan, M. Atif, S.A.A. Shah, S. Erum, Atta-ur-Rahman,
Microbial Metabolism of an anti-HIV and anti-malarial natural product
andrographolide, Int. J. Pharm. Pharm. Sci. 6 (2014) 20–23.
17] M.I. Choudhary, M. Atif, S.A. Ali Shah, S. Sultan, S. Erum, S.N. Khan, Atta-Ur-
Rahman, Biotransformation of Dehydroabietic acid with microbial cell cultures and
α
-Glucosidase inhibitory activity of resulting metabolites, Int. J. Pharm. Pharm.
Sci. 6 (2014) 375–378.
Declaration of Competing Interest
[
18] S.A.A. Shah, H.L. Tan, S. Sultan, M.A.B.M. Faridz, M.A.B.M. Shah, S. Nurfazilah, M.
Hussain, Microbial-catalyzed biotransformation of multifunctional triterpenoids
derived from phytonutrients, Int. J. Mol. Sci. 15 (2014) 12027–12060. doi:
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
1
0.3390/ijms150712027.
[19] C. Djerassi, Chemical birth of the pill (2006) 290–298, https://doi.org/10.1016/j.
ajog.2005.06.009.
[
20] Toshiyuki Minami, Keiji Kosugi, Izumi Suganuma, Kaoruko Yamanaka,
Izumi Kusuki, Tatsuya Oyama, Jo Kitawaki, Antiproliferative and apoptotic effects
of norethisterone on endometriotic stromal cells in vitro, European Journal of
Obstetrics & Gynecology and Reproductive Biology 166 (1) (2013) 76–80, https://
doi.org/10.1016/j.ejogrb.2012.08.023.
21] Salndave B. Skosana, John G. Woodland, Meghan Cartwright, Kim Enfield,
Maleshigo Komane, Renate Louw-du Toit, Zephne van der Spuy, Chanel Avenant,
Donita Africander, Karl-Heinz Storbeck, Janet P. Hapgood, Differential metabolism
of clinically-relevant progestogens in cell lines and tissue: Implications for
biological mechanisms, The Journal of Steroid Biochemistry and Molecular Biology
189 (2019) 145–153, https://doi.org/10.1016/j.jsbmb.2019.02.010.
22] Takeshi Kaizuka, Hideaki Morishita, Yutaro Hama, Satoshi Tsukamoto,
Takahide Matsui, Yuichiro Toyota, Akihiko Kodama, Tomoaki Ishihara,
Tohru Mizushima, Noboru Mizushima, An Autophagic Flux Probe that Releases an
Internal Control, Mol. Cell 64 (4) (2016) 835–849, https://doi.org/10.1016/j.
molcel.2016.09.037.
Acknowledgements
We would like to acknowledge Universiti Teknologi MARA for the
financial support under the reference number 600-IRMI 5/3 LESTARI
[
(
025/2019), Faculty of Pharmacy, UiTM and Atta-ur-Rahman Institute
For Natural Product Discovery (AuRIns), University Teknologi Mara
UiTM) for outstanding research facilities. Author would also like to
(
acknowledge Associate Prof Dr. Siti Aisah Binti Alias (University
MALAYA) for allow us to use Antarctic fungus R3-2 SP 17 for fermen-
tation of ED.
[
Appendix A. Supplementary data
[
[
[
23] C. Su, N. Rybalchenko, D.A. Schreihofer, M. Singh, B. Abbassi, R.L. Cunningham,
Cell Models for the Study of Sex Steroid Hormone Neurobiology, J Steroids Horm.
Sci. 2 (2011), https://doi.org/10.4172/2157-7536.S2-003.
24] J. Cummings, G. Lee, A. Ritter, K. Zhong, Alzheimer ’ s disease drug development
pipeline : 2018, Alzheimer’s Dement. Transl. Res. Clin. Interv. 4 (2018) 195–214.
doi:10.1016/j.trci.2018.03.009.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.steroids.2021.108832.
25] Andrea Haake, Kevin Nguyen, Lauren Friedman, Binu Chakkamparambil, George
T Grossberg, An update on the utility and safety of cholinesterase inhibitors for the
treatment of Alzheimer’s disease, Expert Opinion on Drug Safety 19 (2) (2020)
References
[
[
[
1] C. Manoharachary, I.K. Kunwar, A.B. Rajithasri, Advances in applied mycology and
fungal biotechnology *, KAVAKA. 43 (2014) 79–92.
1
47–157, https://doi.org/10.1080/14740338.2020.1721456.
[
[
26] G.L. Ellman, K.D. Courtney, V.J. Andres, R.M. Featherstone, A NEW AND RAPID
COLORIMETRIC OF ACETYLCHOLINESTERASE DETERMINATION, Biochem.
Pharmacol. 7 (1961) 88–95.
2] S. Castro-Sowinski (Ed.), Microbial Models: From Environmental to Industrial
Sustainability, Springer Singapore, Singapore, 2016.
3] M. Carrasco, J. Rozas, S. Barahona, J. Alcaíno, V. Cifuentes, M. Baeza, Diversity
and extracellular enzymatic activities of yeasts isolated from King George Island,
the sub-Antarctic region, BMC Microbiol 12 (1) (2012) 251, https://doi.org/
27] Asokkumar Kuppusamy, Madeswaran Arumugam, Sonia George, Combining in
silico and in vitro approaches to evaluate the acetylcholinesterase inhibitory
profile of some commercially available flavonoids in the management of
Alzheimer’s disease, Int. J. Biol. Macromol. 95 (2017) 199–203, https://doi.org/
1
0.1186/1471-2180-12-251.
[
[
[
4] M. Trytek, J. Fiedurek, A novel psychrotrophic fungus, Mortierella minutissima, for
D-limonene biotransformation, Biotechnol Lett 27 (3) (2005) 149–153, https://doi.
org/10.1007/s10529-004-7347-x.
1
0.1016/j.ijbiomac.2016.11.062.
[
[
28] M.E. García, J.L. Borioni, V. Cavallaro, M. Puiatti, A.B. Pierini, A.P. Murray, A.
B. Pe n˜ ´e n˜ ory, Solanocapsine derivatives as potential inhibitors of
5] M. Trytek, K. Jedrzejewski, J. Fieudurek, Bioconversion of
α – pinene by a novel
acetylcholinesterase : Synthesis, molecular docking and biological studies, Steroids
cold – adapted fungus Chrysosporium pannorum, J. Ind. Microbiol. Biotechnol.
1
04 (2015) 95–110, https://doi.org/10.1016/j.steroids.2015.09.001.
(
2015) 181–188. doi:10.1007/s10295-014-1550-0.
29] Lihu Zhang, Dongdong Li, Fuliang Cao, Wei Xiao, Linguo Zhao, Gang Ding, Zhen
zhong Wang, Identification of Human Acetylcholinesterase Inhibitors from the
Constituents of EGb761 by Modeling Docking and Molecular Dynamics
Simulations, CCHTS 21 (1) (2018) 41–49, https://doi.org/10.2174/
6] S. Zafar, S. Yousuf, H.A. Kayani, S. Saifullah, S. Khan, A.M. Al-Majid, M.
I. Choudhary, Biotransformation of oral contraceptive ethynodiol diacetate with
microbial and plant cell cultures, Chem. Cent. J. 6 (1) (2012), https://doi.org/
1
0.1186/1752-153X-6-109.
1
386207320666171123201910.
[
[
[
7] R. Sitruk-Ware, M. El-Etr, Progesterone and related progestins: potential new
[
30] N.N.S. Silva, J.R.A. Silva, C.N. Alves, E.H.A. Andrade, J.K.R. da Silva, J.G.. Maia,
Acetylcholinesterase Inhibitory Activity and Molecular Docking Study of 1-
Acetylcholinesterase Inhibitory Activity and Molecular Docking Study of 1-Nitro-2-
Phenylethane , the Main Constituent of Aniba canelilla Essential Oil, Chem Biol
Drug Des. 84 (2014) 192–198. doi:10.1111/cbdd.12304.
health benefits, Climacteric 16 (sup1) (2013) 69–78, https://doi.org/10.3109/
1
3697137.2013.802556.
8] R.B. Gibbs, Effects of gonadal hormone replacement on measures of basal forebrain
cholinergic function, Neuroscience 101 (4) (2000) 931–938, https://doi.org/
1
0.1016/S0306-4522(00)00433-4.
9] J.C. Carroll, E.R. Rosario, A. Villamagna, C.J. Pike, Continuous and Cyclic
Progesterone Differentially Interact with Estradiol in the Regulation of Alzheimer ’
8

S. Nurfazilah Wan Yusop et al.
Steroids 171 (2021) 108832
[
[
31] D.M. Quinn, Acetylcholinesterase: Enzyme Structure, Reaction Dynamics, and
Virtual Transition States, (1987).
32] Michal Harel, Daniel M. Quinn, Haridasan K. Nair, Israel Silman, Joel L. Sussman,
The X-ray Structure of a Transition State Analog Complex Reveals the Molecular
Origins of the Catalytic Power and Substrate Specificity of Acetylcholinesterase,
J. Am. Chem. Soc. 118 (10) (1996) 2340–2346, https://doi.org/10.1021/
ja952232h.
[33] S. Simeon, N. Anuwongcharoen, W. Shoombuatong, A.A. Malik, V.
Prachayasittikul, J.E.S. Wikberg, C. Nantasenamat, Probing the origins of human
acetylcholinesterase inhibition via QSAR modeling and molecular docking, PeerJ.
4
(2016) e2322. doi:10.7717/peerj.2322.
9