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Abstract

In this study, the n-hexane fraction of soft coral Nephthea sp. gathered from the Red Sea was evaluated for its antidermatophyte activity. The antidermatophyte activity was performed versus different fungi, including Microsporum canis, Trichophyton gypseum, and Microsporum mentagrophytes, using a broth microdilution method. The n-hexane fraction showed minimum inhibitory concentrations (MICs) against the tested dermatophytes of 104.2 ± 20.8, 125 ± 0.0, and 83.33 ± 20.83 μg/mL respectively. The chemical constitution of the lipoidal matter (n-hexane fraction) was characterized by gas chromatography coupled with a mass spectrometer (GC-MS). The unsaponifiable fraction (USAP) of Nephthea sp. showed relative percentages of hydrocarbons and vitamins of 69.61% and 3.26%, respectively. Moreover, the percentages of saturated and unsaturated fatty acids were 53.67% and 42.05%, respectively. In addition, a molecular networking study (MN) of the GC-MS analysis performed using the Global Natural Products Social Molecular Networking (GNPS) platform was described. The molecular docking study illustrated that the highest binding energy score for spathulenol toward the CYP51 enzyme was -8.3674 kcal/mol, which predicted the mode of action of the antifungal activity, and then the results were confirmed by the inhibitory effect of Nephthea sp. against CYP51 with an IC50 value of 12.23 μg/mL. Our results highlighted the antifungal potential of Nephthea sp. metabolites.
Potential Inhibitors of CYP51 Enzyme in Dermatophytes by Red Sea
Soft Coral Nephthea sp.: In Silico and Molecular Networking Studies
Nevine H. Hassan, Seham S. El-Hawary, Mahmoud Emam, Mohamed A. Rabeh,
Usama Ramadan Abdelmohsen,*
,#
and Nabil M. Selim*
,#
Cite This: https://doi.org/10.1021/acsomega.2c00063
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ABSTRACT: In this study, the n-hexane fraction of soft coral
Nephthea sp. gathered from the Red Sea was evaluated for its
antidermatophyte activity. The antidermatophyte activity was
performed versus dierent fungi, including Microsporum canis,
Trichophyton gypseum, and Microsporum mentagrophytes, using a
broth microdilution method. The n-hexane fraction showed
minimum inhibitory concentrations (MICs) against the tested
dermatophytes of 104.2 ±20.8, 125 ±0.0, and 83.33 ±20.83 μg/
mL respectively. The chemical constitution of the lipoidal matter
(n-hexane fraction) was characterized by gas chromatography
coupled with a mass spectrometer (GC-MS). The unsaponiable
fraction (USAP) of Nephthea sp. showed relative percentages of
hydrocarbons and vitamins of 69.61% and 3.26%, respectively.
Moreover, the percentages of saturated and unsaturated fatty acids
were 53.67% and 42.05%, respectively. In addition, a molecular networking study (MN) of the GC-MS analysis performed using the
Global Natural Products Social Molecular Networking (GNPS) platform was described. The molecular docking study illustrated that
the highest binding energy score for spathulenol toward the CYP51 enzyme was 8.3674 kcal/mol, which predicted the mode of
action of the antifungal activity, and then the results were conrmed by the inhibitory eect of Nephthea sp. against CYP51 with an
IC50 value of 12.23 μg/mL. Our results highlighted the antifungal potential of Nephthea sp. metabolites.
INTRODUCTION
Dermatophytes are keratin-loving fungi that commonly cause
cutaneous infections in animals and humans such as ringworm
and tinea.
13
Usually, dermatophytes do not violate the living
tissues but colonize the external layer of the skin.
4
In addition,
dierent symptoms typically appear within 2 weeks after direct
contact between the human part and fungi.
5
The most
identied colonies belong to the three main genera
Trichophyton,Microsporum, and Epidermophyton.
6
The possible track of dermatophyte login to the host body is
injured skin, scars, and burns.
6
The fungal pathogens induce
both immediate hypersensitivities as well as cell-mediated or
delayed-type hypersensitivity.
7
Microsporum canis (M. canis) causes tinea capitis and has a
higher incidence in the winter season,
8
while Trichophyton
mentagrophytes (T. mentagrophytes) causes tinea pedis and and
its incidence is increased in the hot season.
810
Also, the
geophilic dermatophyte of Microsporum gypseum (M. gypseum)
appears during the rainy season and usually occurs from
August to November when people come into direct contact
with the soil.
10,11
Despite the development of dermatophytosis treatment
science and technology, it is still treated with commercially
available topical and oral antifungal agents from Whitelds
ointment to azoleswith many side eects.
6
Nature is considered an untapped source of biologically
active metabolites. Recently, marine habitats have provided the
drug market with unique skeletons with various diverse
pharmacological activities.
12,13
In addition, the treatment of
cutaneous infections using natural plants and/or marine
sources has displayed antidermatophyte potency.
6,1417
Marine Nephthea sp. is a known genus of the family
Nephtheidae (20 genera) that comprises 12 species, and the
members of this family are known as carnation corals, tree
corals, or colt corals. They are distributed in the Indo-Pacic
region.
18
Received: January 5, 2022
Accepted: March 30, 2022
Article
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Members of the genus Nephthea exhibit diverse bioactivities
such as cytotoxic, antiviral, antihypertriglyceridemia, and
antiphlogistic activities due to the presence of various chemical
entities such as sesquiterpenes, diterpenes, and steroids.
19,20
To the best of our knowledge, the antidermatophyte
activities of the investigated Nephthea sp. fraction have not
been previously evaluated. Therefore, the target of this study is
the investigation of the antidermatophyte activity of the
lipoidal matter of Nephthea sp. gathered from the Red Sea
region. Additionally, the inhibitory action on the fungal CYP51
enzyme was determined to conrm the fungicidal activity.
Moreover, an in silico study of the major identied components
against the CYP51 enzyme was constructed. Finally, the
chemical constituents of the investigated fraction and
molecular networking based on the GC-MS data were reported
(Scheme 1).
MATERIALS AND METHODS
Soft Coral Collection, Identication, and Extraction.
The investigated soft coral was gathered from the shores of the
Red Sea (Hurghada, Egypt) by snorkeling in January 2020.
The specimen was authenticated by Dr. El-Sayd Abed El-Aziz
(Department of Invertebrates Laboratory, National Institute of
Oceanography and Fisheries, Red Sea Branch, Egypt).
Nephthea sp. (500 g) was stored at 4 °C, cut into small slices,
and then extracted several times with a mixture of methanol
and methylene chloride (1/1) and concentrated under vacuum
to yield 6 g. The obtained extract was fractionated using n-
hexane, concentrated to dryness to yield 2 g, and kept at 4 °C
for further investigation.
Antifungal Activity. Microsporum canis,Trichophyton
mentagrophytes,andMicrosporum gypseum were collected
from dierent habitats of dogs, humans, and soil, respectively.
They were authenticated by Dr. Mona M. H. Soliman
(Microbiology and Immunology Department, National
Research Centre, Giza, Egypt).
Isolation of Fungi and Characterization. The skin
scraping and hair samples, collected from infected humans and
cats, were taken from the lesions using a blunt scalpel blade
and cleaned with 70% alcohol. They appeared clinically as
ringworm lesions. Hairs were pulled out from the lesion using
sterile forceps and kept in a sterile envelope for further
mycological examination.
21
Soil samples were collected in
sterile plastic bags. The hair bait technique was used to isolate
dermatophytes from soil according to Em and Cu.
22
The
isolation and identication of dermatophytes were imple-
mented according to Scott and Miller,
23
while the isolates were
identied microscopically according to Monika and Chinna.
24
Broth Microdilution Method (MIC). A microdilution
assay was used to evaluate the antifungal susceptibility testing,
according to Clinical and Laboratory Standards Institute
(CLSI) guidelines in the M38-A mold document.
25
The
dermatophyte strains were subcultured on potato dextrose agar
(PDA) (Merck Co., Darmstadt, Germany) and incubated at 30
°C for 57 days. Conidia were kept for 15 min in sterile saline,
prior to being counted by a hemocytometer. The suspension
was adjusted to 1 ×104CFU mL1in RPMI 1640 medium
(Roswell Park Memorial Institute Medium) (with L-glutamine,
without, according to Scott and Miller, sodium bicarbonate;
Gibco-BRL, Grand Island, New York) buered with MOPS (3-
(N-morpholino)propanesulfonic acid; Serva, Feinbochemica
GmbH, Germany). As well, a series of 2-fold serial dilutions
were carried out for the Nephthea sp. n-hexane fraction from
1000 to 1.9 μg/mL. The inoculum and dierent concen-
trations of the fraction, as well as positive and negative
controls, were incorporated in 96-well microtiter plates and
incubated at 32 °C for 5 days. The minimum inhibitory
concentration (MIC) was determined and compared with the
positive control. Both positive and negative control wells were
included in all of the tested plates. The experiments were
performed in triplicate for each fungus sample.
26
Sample Preparation for GC-MS analysis. Preparation
of Unsaponiable Matter (USM) and Fatty Acid Methyl Ester
Scheme 1. Illustrated Steps for the Extraction of the Marine Sample, Antidermatophytes, Chemical Investigation, Molecular
Networking (MNW), In Silico Evaluations, and CYP51 Inhibition
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(FAME). An n-hexane fraction (1.42 g) was saponied under
reux using 10% alcoholic potassium hydroxide.
27
The
unsaponiable matter was extracted with diethyl ether and
evaporated to dryness after removal of excess alkali and kept
for GC-MS analysis. The USM obtained (0.44 g) represented
30.99% of the total lipoidal matter of the n-hexane fraction.
The free fatty acids were liberated after acidication, extracted
with diethyl ether, dried in vacuo, and weighed. The fatty acids
obtained (0.88 g) represented 61.97% of the total lipoidal
matter of the n-hexane extract. The methylation of the
obtained fatty acids was carried out with anhydrous methanol
and diazomethane,
27
and the samples were then kept for GC/
MS analysis.
Gas ChromatographyMass Spectrometry Conditions.
The GC-MS analysis was performed using a Thermo Scientic
Trace GC Ultra/ISQ Single Quadrupole MS with a TG-5MS
fused silica capillary column (30 m, 0.251 mm, 0.1 mm lm
thickness). An electron ionization (EI) system with a power of
70 eV was used. The mobile phase was helium gas at a
constant ow rate of 1 m min1. The injector and MS transfer
line temperatures were set at 280 °C. The oven temperature
was programmed at an initial temperature of 50 °C (2 min),
50150 °C at a rate of 7 °C min1, 150270 °C at a rate of 5
°C min1(2 min), and 270310 °C as the nal temperature at
a rate of 3.5 °C min1(10 min). Quantication of all identied
components was carried out using the relative peak area. The
identication of the constituents was carried out by a
comparison of their relative retention times and mass spectra
with those of the NIST and Wiley library data of the GC-MS
system.
GC-MS Molecular Networking. Molecular networking
(MN) is a simple computational process that may visualize and
interpret mass data analysis. In addition, it can suggest the
identical structures for all mass spectra within the data set and
correlate the annotation between the unknown molecules and
related molecules through the identical mass fragments.
2831
A
molecular network (MN) for the GC-MS analysis data of the
studied SAP and UNSAP Nephthea sp. lipoidal matters was
constructed as follows. Thermo raw data les were transformed
into the open format (mzML.) using MS conversion that was
supported by GNPS.
32
The spectra in the network were then
searched against GNPS GC-MS spectral libraries. The created
MN was investigated and predicted using Cytoscape (ver.
3.9.0) which is open-source software for the analysis and
exploration of MNs.
33
Molecular Docking Study. All of the molecular modeling
studies were carried out using Molecular Operating Environ-
ment (MOE, 2019.0102) software. All minimizations were
performed with MOE until an RMSD gradient of 0.1 kcal
mol1Å1with the MMFF94x force eld and the partial
charges were automatically calculated. The X-ray crystallo-
graphic structure of human lanosterol 14α-demethylase
(CYP51) complexed with ketoconazole (KKK) (PDB ID:
3LD6) was downloaded from the Protein Data Bank (https://
www.rcsb.org/structure/3LD6). Water molecules and ligands
were removed for each cocrystallized enzyme not involved in
the binding. The protein was equipped for a docking study
using the Protonate 3D protocol in MOE with default options.
Docking binding sites were determined through a cocrystal-
lized ligand (KKK). Moreover, the Triangle Matcher place-
ment method and London dG scoring function were used for
docking.
Inhibitory Activity of Lanosterol 14α-Demethylase
(CYP51). Plate Reader Assay. The n-hexane fraction of
Nephthea was screened for its inhibitory activity against
lanosterol 14α-demethylase (CYP51) in comparison with the
drug uconazole as a reference at the Conrmatory Diagnostic
Unit VACSERA, Cairo, Egypt. 7-Ethoxyresorun (7-ER) is a
uorescent substrate and competitive suppressor of cyto-
chrome P450 (CYP) isoform CYP1A1 (IC50 = 0.1 μM). Upon
enzymatic cleavage by CYP1A1 resorun was released and its
uorescence was used to quantify CYP1A1 activity. Resorun
displays excitation/emission maxima of λmax 572/580 nm,
respectively. Resorun and 7-ethoxyresorun obtained from
Sigma-Aldrich (St. Louis, MO, USA) are light-sensitive;
therefore, this procedure should be carried out under yellow
light to protect the integrity of the stock solutions. Incubations
were prepared in a black 96-well plate, consisting of a substrate
(7ER) and CaCYP51 bactosomes in 1pH 7.4 00 mM
potassium phosphate buer containing 5 mM magnesium
chloride. Reactions were initiated by adding 40 μLofa5x
NADPH generating system (this can be omitted from wells
containing blanks and standards). The formation of resorun
was measured uorometrically every 30 s through the use of
detection wavelengths (excitation/emission at 572/604 nm)
chosen to minimize interference from NADPH and 7ER. The
substrate 7-ethoxyresorun and its metabolite resorun are
both available from Cypex.
34
Statistical Analysis. All results are stated as mean ±SE (n
= 3), and the statistical analysis of the inhibition activity (IC50)
against sterol 14α-demethylase (CYP51) was analyzed by a t
test utilizing SPSS statistics 18.0 (Chicago, USA). The
statistical signicance was considered to be p< 0.05. GraphPad
Prism 8.0 (GraphPad Prism Software Inc., San Diego, CA,
USA) was used to visualize the results.
RESULTS AND DISCUSSION
The potential activity of the n-hexane fraction of Nephthea sp.
was evaluated against the healthiest and puried dermatophy-
tosis isolates M. canis,T. mentagrophytes, and M. gypseum.
Antifungal Results. Figure 1 shows that the n-hexane
fraction of Nephthea sp. has antifungal activity against isolated
dermatophytes. The highest activity against M. gypseum was
observed with MIC = 83.33 ±20.83 μg/mL, followed by M.
canis with MIC = 104.2 ±20.8 μg/mL and T. mentagrophytes
with MIC = 125 ±0.0 μg/mL.
Chemical Prole of USAP Fraction. The unsaponiable
fraction (USAP) was subjected to a GC-MS analysis, and the
relative percentages of the total hydrocarbons and total
Figure 1. Antifungal activity (MIC) of Nephthea sp. against
dermatophyte isolates. All results are given as means ±SE.
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oxygenated compounds were 37.34% and 33.68%, respectively.
The sesquiterpene widdrene was the major identied
component of nonoxygenated hydrocarbons with a content
of 20.24% (Table 1 and Figure 2), followed by methyl 3,5-
tetradecadiynoate (13.16%). Spathulenol with a content of
7.14% is a tricyclic sesquiterpene alcohol that has a basic
skeleton similar to that of the azulenes. Additionally, an acyclic
diterpene alcohol, phytol, was identied with a content of
1.39% and can be used as a precursor for the manufacturing of
the synthetic forms of vitamin E and vitamin K1. A polyene
chromophore structure (retinal), which is considered as the
active skeleton of vitamin A, was detected with a content of
1.87%.
Molecular networking based on GC-MS data was used to
visualize the identical compounds having similar fragments
(Figure 3). Eighteen clusters were connected to visualize 152
nodes together through 289 edges. The MN of the USAP
fraction of Nephthea sp. revealed that widdrene (thujopsene),
methyl 3,5-tetradecadiynoate, and spathulenol were the most
predominant skeletons in the tested sample. The compounds
were recognized on the basis of GNPS libraries and the highest
matching factor (SI 700).
Chemical Prole of the Fatty Acid Methyl Ester
(FAME) Fraction. Table 2 and Figure 4 show that the relative
percentages of saturated and unsaturated fatty acids are 55.98%
and 39.80%, respectively. In addition, Cyclohexylpropanoic
acid and undecenoic acid were the major identied fatty acids
in Nephthea sp. with relative percentages of 43.99% and
29.87%, respectively. Stearic acid and arachidonic acid,
polyunsaturated omega 6 fatty acids, were detected with
relative percentages of 8.15% and 6.37%, respectively.
Table 1. Chemical Compositions of USM of Nephthea sp. Identied by GC-MS Analysis
no. Rt(min) rRt
a
Kovats index molecular formula identied compound molecular weight base peak (m/z) rel area (%) library
1 11.31 0.53 1085 C7H16O neoheptanol 116 43 0.05 GNPS
2 13.85 0.65 1162 C9H20 2,2,3,4-tetramethylpentane 128 57 0.18 GNPS
3 16.28 0.77 1229 C9H18 2,4,4-trimethyl-1-hexene 126 71 0.13 GNPS
4 17.85 0.84 1270 C13H20Oα-ionone 192 121 0.62 GNPS
5 18.18 0.86 1278 C15H24 1,4-cadinadiene 204 161 0.19 GNPS
6 18.54 0.88 1287 C16H20O4deoxysericealactone 276 43 0.31 GNPS
7 19.22 0.91 1305 C15H24 α-cubebene 204 161 0.84 Wiley9
8 19.95 0.94 1332 C15H24 aristolene 204 105 1.23 Mainlib
9 20.32 0.96 1346 C15H24 α-muurolene 204 105 2.14 Wiley9
10 21.04 1 1371 C15H24 widdrene (thujopsene) 204 119 20.24 Wiley9
11 21.82 1.03 1398 C15H24 α-selinene 204 93 3.11 Wiley9
12 22.36 1.06 1419 C15H24 α-gurjunene 204 81 3.38 Wiley9
13 22.87 1.08 1439 C15H24 cadinene 204 161 3.19 Wiley9
14 23.86 1.13 1476 C15H22O2methyl 3,5-tetradecadiynoate 234 91 13.16 Mainlib
15 24.03 1.14 1482 C15H22 α-vatirenene 202 159 1.93 Mainlib
16 24.58 1.16 1502 C15H24 O lanceol, cis 220 93 0.58 Mainlib
17 25.02 1.19 1516 C15H24 O spathulenol 220 43 7.14 Mainlib
18 25.53 1.21 1532 C20H28O retinal 284 91 1.87 Wiley9
19 30.96 1.47 1700 C13H20O3verticellol 290 121 1.48 Wiley9
20 31.75 1.50 1728 C15H24 α-elemene 204 81 1.28 Mainlib
21 32.40 1.54 1750 C19H38O nonadecanone 282 58 1.37 Wiley9
22 33.90 1.61 1800 C17H36O heptadecanol 256 55 2.74 Mainlib
23 34.43 1.63 1819 C20H40O phytol 296 71 1.39 Mainlib
24 35.10 1.66 1843 C15H26O elemol 222 59 3.95 GNPS
25 35.19 1.67 1846 C15H24 β-elemene 204 81 0.04 GNPS
26 35.38 1.68 1853 C10H16 terpinolene 136 93 0.05 GNPS
27 35.48 1.68 1856 C30H50O lanosterol 426 95 0.04 GNPS
28 37.48 1.78 1954 C36H74O dotriacontyl isobutyl ether 523 57 0.04 GNPS
29 37.75 1.79 1974 C20H26O4lobohedleolide 330 53 0.03 GNPS
30 40.61 1.93 2174 C30H62 squalane 422 57 0.08 GNPS
rel % of total identied compounds 72.87
rel % of identied hydrocarbons 69.61
rel % of identied vitamins 3.26
a
rRt: retention time relative to that of widdrene (Rt= 21.04 min)
Figure 2. Total ion chromatogram (TIC) for GC-MS of the
unsaponiable matter of Nephthea sp.
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Figure 5 illustrates the molecular network constructed from
the GC-MS data and visualizes the identical fragments inside
the same cluster. Fifty-ve nodes were connected through 148
edges and visualized in 10 clusters. The designed MN reveals
that cyclohexyl propanoic acid, undecenoic acid, and
arachidonic acid skeletons are the most prevalent in the tested
sample (Figure 5).
Molecular Docking Analysis. Fungal CYP51 has been
demonstrated to be the biochemical target for commercial
fungicides; through an examination of the binding interactions
of KKK (ketoconazole) to the active site of the CYP51
enzyme, it shows strong hydrogen-bond interactions with
Phe77, Phe234, Gly307, Ile379, Cys449 and Met487 (Figure
6).
Figure 3. Molecular network of the GC-MS investigation of the
Nephthea sp. UNSAP nodes are labeled with the compound name
from the GNPS GC-MS spectral libraries.
Table 2. Chemical compositions of FAME of Nephthea sp. identied by GC-MS Analysis
no. Rt(min) rRt
a
Kovats
index molecular
formula identied compound molecular
weight base peak
(m/z)rel area
(%) library
1 4.24 0.32 769 C10H7NO4xanthurenic acid 205 187 0.01 GNPS
2 4.77 0.36 807 C13H26O2lauric acid 214 87 0.04 GNPS
3 8.58 0.65 992 C14H28O2myristic acid 228 74 0.70 Wiley9
4 10.76 0.82 1069 C11H20O2cyclopropane pentanoic acid, 2-
undecyl 310 43 0.05 Wiley9
5 12.55 0.96 1123 C16H30O2palmitoleic acid 254 237 1.92 Wiley9
6 13.04 1 1138 C9H16O2cyclohexylpropanoic acid 156 74 43.99 Wiley9
7 14.47 1.10 1179 C16H32O2palmitic acid 256 74 0.15 Mainlib
8 15.20 1.16 1199 C17H34O2margaric acid 270 73 0.29 Wiley9
9 16.52 1.26 1236 C18H30O2α-eleostearic acid 278 67 2.19 Mainlib
10 16.83 1.29 1244 C11H20O2undecenoic acid 198 55 29.87 Wiley9
11 17.41 1.33 1259 C18H36O2stearic acid 284 74 8.15 Wiley9
12 18.21 1.39 1279 C18H34O2oleic acid 282 73 0.06 GNPS
13 18.72 1.43 1291 C18H32O2linoleic acid 280 67 0.13 Mainlib
14 20.17 1.54 1340 C20H32O2arachidonic acid 304 79 6.37 Wiley9
15 20.31 1.55 1354 C22H32O2docosahexanoic acid 328 79 1.05 Mainlib
16 21.50 1.64 1387 C20H40O2arachidic acid 312 74 0.30 Wiley9
17 27.51 2.10 1591 C22H36O2adrenic acid 332 79 0.46 Mainlib
rel % of total identied compounds 95.73
rel % of saturated fatty acids 53.67
rel % of unsaturated fatty acids 42.05
others 0.01
a
rRt: retention time relative to cyclohexylpropanoic acid (Rt= 13.04 min).
Figure 4. Total ion chromatogram (TIC) for GC-MS of the fatty acid
methyl esters of Nephthea sp.
Figure 5. Molecular network of the GC-MS investigation of the
Nephthea sp. FAME. Nodes are labeled with the compound name
from the GNPS GC-MS spectral libraries.
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The docking setup was rst validated by self-docking of the
cocrystallized ligand (KKK) in the proximity of the binding
site of the enzyme; the docking score (S) was 12.6869 kcal/
mol, and the root mean square deviation (RMSD) was 2.3919
Å(Figure 7.).
Fungal CYP51 was docked with the major detected
components of GC-MS, revealing that spathulenol showed
the highest binding energy score (8.3674 kcal/mol) among
the tested compounds, indicating a higher tting ability,
followed by cyclohexylpropanoic acid (8.0406 kcal/mol) and
then undecanoic acid and widdrene. However, the cyclo-
hexylpropanoic acid derivative showed a higher number of
interactions with the amino acids in the active site of the tested
enzyme. The results are summarized in Table 3 and Figures
811).
Inhibition of Fungal CYP51 Enzyme. One-third of the
agrochemical fungicides used are azole drugs that target
inhibition of the CYP51 enzyme which belongs to the
cytochrome P450 monooxygenase (CYP) superfamily.
CYP51 enzyme is considered a critical step in the synthesis
of ergosterol, that is fungal-specic sterol.
35
The therapeutic
azole antifungal compounds emerged in orally administrated
forms during the 1980s, rst with ketoconazole and then later
with uconazole and itraconazole.
36
These drugs are used
extensively due to the widespread incidence of fungal
infections associated with AIDS but also are associated with
cancer chemotherapy and organ transplantation and are used
in the intensive care unit. Thus, more detailed information on
the activity of CYP51 inhibitors is important toxicologically so
that further applications may emerge.
37
The most popular antifungal agent that inhibitsthe biosyn-
thesis of lanosterol 14αdemethylase (CYP51) and ergosterol
in the fungal cell membrane is the drug uconazole, which was
used in this study as a reference drug (ST) with IC50 = 2.27 ±
0.05 μg/mL. In addition, the n-hexane fraction of Nephthea sp.
showed inhibitory activity against CYP51 with IC50 = 12.23 ±
0.29 μg/mL (Figure 12).
Figure 6. 2D interactions of KKK within the CYP51 active site.
Figure 7. 3D symbolism of the superimposition of the cocrystallized
(red) and docking poses (green) of KKK in the energetic site of the
CYP51 enzyme.
Table 3. Docking Results of Major Detected Components of
GC-MS of Nephthea sp. on the Binding Sites of Fungal
CYP51
S(kcal/mol) amino
acids interacting
group type of interaction length
(Å)
Cyclohexylpropanoic Acid
8.0406 His236 CH2
(cyclohexyl) H-bond
(nonclassical) 4.17
Met378 O (CO) H-bond acceptor 3.99
Ile379 OH H-bond donor 3.14
Met381 CH2
(cyclohexyl) H-bond
(nonclassical) 4.03
Met487 CH3H-bond
(nonclassical) 3.96
His489 O (CO) H-bond acceptor 3.07
Spathulenol
8.3674 Met378 OH H-bond acceptor 3.87
Ile379 CH3H-bond
(nonclassical) 3.89
Met487 CH2H-bond
(nonclassical) 3.60
Met487 CH H-bond
(nonclassical) 4.38
His489 OH H-bond acceptor 2.89
Undecanoic Acid
7.7217 Tyr145 O (CO) H-bond acceptor 3.08
Pro376 CH H-bond
(nonclassical) 3.43
Ile379 CH2H-bond
(nonclassical) 3.71
Ile379 CH2H-bond
(nonclassical) 3.98
Met487 CH2H-bond
(nonclassical) 4.39
Widdrene
7.9324 Ile379 CH H-bond
(nonclassical) 4.09
Met487 CH2H-bond
(nonclassical) 3.99
Met487 CH2H-bond
(nonclassical) 4.17
Figure 8. 2D and 3D interactions of cyclohexylpropanoic acid within
the CYP51 active site.
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DISCUSSION
Most dermatophyte infections are not life threatening and
respond well to currently available topical treatment with over
the counter (OTC) fungal agents. However, some dermato-
phyte infections require complex treatment regimens and are
more resistant to traditional antifungal therapies.
38
In addition,
the emergence of the resistance of microbes, including fungi
and yeasts, toward the available antimicrobial agents requires a
search for other antimicrobials.
Moreover, this is the rst report of the antifungal activity of
the n-hexane fraction of soft coral Nephthea sp. using a broth
microdilution method and a Candida albicans CYP51 plate
reader assay. Undoubtedly, there is a relationship between the
antifungal activity of the investigated fraction and its main
components.
In the present study, the major chemical constituents of the
unsaponiable fraction are sesquiterpene, widdrene (thujop-
sene), and the tricyclic sesquiterpene alcohol spathulenol,
which have been previously reported to have high antimicro-
bial activities.
39,40
In addition, several fatty acids (methylated
and hydroxylated fatty acids) and their derivatives were
identied from the FAME fraction that previously exhibited
antifungal activity targeting the cell membrane. They cause
leakage of intracellular components and cell death through an
increase in membrane uidity.
41
In previous studies, widderene (thujopsene) was a volatile
component of the heartwood extract of the cedar Callitropsis
nootkatensis with activity against Phytophthora ramorum
42
and
showed potent antifungal activity at low concentrations (0.1%,
1% ,and 10%), against 16 fungal strains, particularly against
Gonytrichum macrocladum (GMB), Eurotium herbariorum
(EHA), and Penicillium decumbens (PDT) using a disk
diusion method.
43
Likewise, it showed antibacterial activity
against several strains of Cryptococcus neoformans.
44
Addition-
ally, Ashe juniper showed signicant antifungal activity against
four species of wood-rot fungi, due to its high content of
thujopsene (over 30%).
45
There are few data concerning the
eect of thujopsene on dermatophytes. This may be the rst
study concerning the eect of this compound on dermato-
phytes. The previous ndings suggested that the content of
widderene (thujopsene) might explain its vital role in
antidermatophytosis.
In addition, essential oils showed antifungal activity against
dermatophytes and Candida spp.;
4649
for instance, the
essential oil of Croton argyrophylloides showed antifungal
activity against M. canis due to its contents of spathulenol
and bicyclogermacrene through a synergistic eect.
50
Fur-
thermore, the extracts and fractions of Jatropha neopauciora
(Pax) were also shown to have antifungal activity, particularly
against Trichophyton mentagrophytes, due to its major contents
of β-sitosterol, spathulenol, coniferyl alcohol, and lupeol.
51
These ndings exhibited the roles of terpene, sterol, and
phenylpropanoid activities.
Methyl 3,5-tetradecadiynoate is a methylated fatty acid
detected in the USAP of Nephthea sp. as a major component. It
was previously reported that 12-methyltetradecanoic (12-Me
14:0) acid inhibits the formation of appressorium in the rice
pathogen Magnaporthe oryzae.
52
However, its mechanism of
action is still unknown and needs further investigation.
Garg in 1993 showed that saturated fatty acids having short
chains ranging from C7 to C11 are more toxic to skin fungi in
comparison to the corresponding long chains of >12. As well as
odd-numbered carbon, chain fatty acids are slightly more toxic
than their corresponding even-numbered one carbon-less fatty
acid. Polyunsaturated fatty acids were found to be more toxic
than their corresponding saturated fatty acids.
53
This
conclusion is in agreement with our present study, which
stated that cyclohexylpropanoic acid C3:0 and undecenoic acid
(11:1) may be responsible for the antifungal activity against
selected dermatophytes.
In addition, Garnier et al. in 2020 showed that propanoic
(propionic) and acetic acids were the most abundant
Figure 9. 2D and 3D interactions of spathulenol within the CYP51
active site.
Figure 10. 2D and 3D interactions of undecenoic acid within the
CYP51 active site.
Figure 11. 2D and 3D interactions of widdrene within the CYP51
active site.
Figure 12. Inhibition activity (IC50)ofNephthea sp. against sterol
14α-demethylase (CYP51). The dierent letters represent statistically
signicant dierences (p< 0.05).
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fermentation products for Propionibacterium jensenii and were
shown to exhibit promising antifungal activities in dairy
products.
54
However, propionic acid was only quantied at
high levels in the P. jensenii fermentate 322 (59.94 ±21.28
mg/g). The antifungal activity of propanoic acid (propionic
acid) has been previously reported in the literature.
55,56
Moreover, unsaturated fatty acids such as undecenoic acid
(11:1), which contains a xed bent CCbondwere
identied; when undecenoic acid is inserted into the
membrane, it increases motional freedom inside the membrane
and increases oxidative stress, encouraging its fungicidal
activity.
57
Also, undecenoic acid is used for the production
of the bioplastic nylon-11, which is used in the treatment of
fungal infections of the skin.
58
Undecenoic acid (C11:1) is a
short-chain unsaturated fatty acid and is more toxic to
dermatophytes than long-chain fatty acids (>C 12:0); it
completely inhibited the growth of species such as as T.
mentagrophytes,T. mentagrophytes var. interdigitale, T. rubrum,
M. canis and M. gypseum at <0.5 mM, suggesting the highest
activity of this fatty acid in the range of C7C13 series.
53
Likewise, it has been previously used in treating tinea pedis
produced by T. mentagrophytes and T. rubrum.
59
In addition, it
has been utilized in curing dermatomycosis caused by T.
rubrum,Epidermophyton inguinale and M. audouini (Carolina et
al., 2011). Moreover, onychomycosis is caused by T. rubrum.
60
Additionally, McDonough et al. in 2002 found that the
medium-chain fatty acids (MCFAs) undecanoic acid (11:0),
10-undecenoic acid (11:1 Delta 10), and lauric acid (12:0) can
aect the growth of Saccharomyces cerevisiae in a dose-
dependent manner.
61
This study is a companion to that of
Ells and co-workers, who in 2009 demonstrated that the
polyunsaturated fatty acid arachidonic acid (20:4) may
increase the antifungal sensitivity of biolms formed by two
closely related Candida species, thus resulting in a decrease in
the dose of the antimycotic agent needed to inhibit biolm
formation.
62
The previous data regarding 3-cyclohexylpropanoic acid,
dierent fatty acids, and its methylated compounds explain
their prospective eect against dermatophytes. In addition, our
ndings are consistent with the previously reported literature,
which stated that undecenoic acid was the best of the
fungicides tested for the routine treatment of dermatophy-
tosis.
41
Furthermore, the conrmation studies for several natural
structures of volatile compounds (e.g., eugenol) were reported
to inhibit the ergosterol synthesis, targeting the sterol 14α-
demethylase (CYP51) enzyme.
63
Also, the terpenoidal
structure of tormentic acid isolated from Callistemon citrinus
decreased the ergosterol content.
64
These results indicate that
the presence of volatile compounds and terpene skeletons has
an inhibitory eect on CYP51, in addition to fatty acids and
hydrocarbons.
In conclusion, the molecular network based on a GC-MS
analysis of n-hexane fraction of Nephthea sp. revealed that the
most predominant skeletons in FAME are cyclohexylpropanoic
acid, undecenoic acid, and arachidonic acid. However, the
most predominant skeletons in USAP are widdrene, methyl
3,5-tetradecadiynoate, and spathulenol in the tested sample.
These results are in agreement with the results of the docking
study, which exposed that spathulenol has the highest binding
energy score, followed by cyclohexylpropanoic acid, then
undecanoic acid, and nally widdrene (thujopsene). However,
cyclohexylpropanoic acidshowedahighernumberof
interactions with the amino acids in the active site of the
tested enzyme. From all of the previous reports, our present
study suggested that the antidermatophyte potential of the soft
coral Nephthea sp. is due to the presence of widdrene
(thujopsene), spathulenol, undecenoic acid, and cyclohexyl-
propanoic acid structures that might act synergistically as
antifungal components.
CONCLUSION
In this study, we investigated the unsaponiable and
saponiable materials of Nephthea sp. by GC-MS that could
be helpful in the authentication of marine soft coral. This is the
rst documentation of molecular networks toward the lipoidal
matter by GC-MS analysis in addition to the promising
antidermatophyte activity of Nephthea species against M.
gypseum,M. canis, and T. mentagrophytes through inhibition of
fungal CYP51. The Global Natural Products Social Molecular
Networking rostrum and a molecular docking study predicted
spathulenol ecacy against the CYP51 enzyme that might be
responsible for antifungal activity, which was then conrmed in
vitro by the inhibitory eect of Nephthea sp. against CYP51.
Further clinical studies will support these ndings and explore
the detailed mechanism of action.
AUTHOR INFORMATION
Corresponding Authors
Nabil M. Selim Pharmacognosy Department, Faculty of
Pharmacy, Cairo University, Giza 11562, Egypt;
Email: dr_nabilselim79@yahoo.com
Usama Ramadan Abdelmohsen Pharmacognosy
Department, Faculty of Pharmacy, Minia University, 61519
Minia, Egypt; Pharmacognosy Department, Faculty of
Pharmacy, Deraya University, 61111 New Minia, Egypt;
orcid.org/0000-0002-1014-6922;
Email: usama.ramadan@mu.edu.eg
Authors
Nevine H. Hassan Pharmacognosy Department, Faculty of
Pharmacy, Modern University for Technology and
Information, Cairo 11571, Egypt
Seham S. El-Hawary Pharmacognosy Department, Faculty
of Pharmacy, Cairo University, Giza 11562, Egypt
Mahmoud Emam Phytochemistry and Plant Systematics
Department, National Research Centre, Cairo 12622, Egypt
Mohamed A. Rabeh Pharmacognosy Department, Faculty of
Pharmacy, Modern University for Technology and
Information, Cairo 11571, Egypt; Pharmacognosy
Department, Faculty of Pharmacy, Cairo University, Giza
11562, Egypt
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsomega.2c00063
Author Contributions
#
U.R.A. and N.M.S. contributed equally.
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
The authors are beholden to Dr. Mona M. H. Soliman,
Microbiology and immunology Department, National Re-
search Centre, Dokki, Egypt, for providing the dermatophyte
isolates and carrying out the assay. In addition, the authors
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https://doi.org/10.1021/acsomega.2c00063
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H
wish to thank Dr. Essam Rashwan, Head of the conrmatory
diagnostic unit VACSERA-EGYPT, for performing lanosterol
14-α-demethylase (CYP51) inhibitory assays.
REFERENCES
(1) Patel, A.; Lloyd, D.; Lamport, A. Survey of dermatophytes on
clinically normal cats in the southeast of England. J. Small Anim. Pract.
2005,46 (9), 436439.
(2) Cafarchia, C.; Romito, D.; Capelli, G.; Guillot, J.; Otranto, D.
Isolation of Microsporum canis from the hair coat of pet dogs and cats
belonging to owners diagnosed with M. canis tinea corporis. Vet.
Dermatol. 2006,17 (5), 327331.
(3) Gnat, S.; Nowakiewicz, A.; Zięba,P.Taxonomyof
dermatophytesthe classification systems may change but the
identification problems remain the same. Adv. Microbiol. 2019,58
(1), 4958.
(4) Braunwald, E.; Fauci, A. S.; Kasper, D. L.; Hauser, S. L.; Longo,
D. L.; Jameson, J. L. Harrisons Principles of Internal Medicine, 15th ed.;
McGraw-Hill: 2001.
(5) Degreef, H. Clinical forms of dermatophytosis (ringworm
infection). Mycopathologia 2008,166 (56), 257.
(6) Lakshmipathy, D. T.; Kannabiran, K. Review on dermatomy-
cosis: pathogenesis and treatment. Nat. Sci. 2010,02 (07), 726.
(7) Sharma, V.; Kumawat, T. K.; Sharma, A.; Seth, R.; Chandra, S.
Distribution and prevalence of dermatophytes in semi-arid region of
India. Adv. Microbiol. 2015,05 (02), 93.
(8) Lee, W. J.; Park, J. H.; Kim, J. Y.; Jang, Y. H.; Lee, S.-J.; Bang, Y.
J.; Jun, J. B. Low but continuous occurrence of Microsporum gypseum
infection in the study on 198 cases in South Korea from 1979 to 2016.
Ann. Dermatol. 2018,30 (4), 427.
(9) Frías-De-León, M. G.; Martínez-Herrera, E.; Atoche-Diéguez, C.
E.; González-Cespón, J. L.; Uribe, B.; Arenas, R.; Rodríguez-Cerdeira,
C. Molecular identification of isolates of the Trichophyton
mentagrophytes complex. Int. J. Med. Sci. 2020,17 (1), 45.
(10) Woodfolk, J. A. Allergy and dermatophytes. Clin. Microbiol. Rev.
2005,18,3043.
(11) Souza, B. d. S.; Sartori, D. S.; Andrade, C. d.; Weisheimer, E.;
Kiszewski, A. E. Dermatophytosis caused by Microsporum gypseum in
infants: report of four cases and review of the literature. An. Bras.
Dermatol. 2016,91 (6), 823825.
(12) Chen, J.; Zhang, P.; Ye, X.; Wei, B.; Emam, M.; Zhang, H.;
Wang, H. The Structural Diversity of Marine Microbial Secondary
Metabolites Based on Co-Culture Strategy: 20092019. Mar. Drugs
2020,18 (9), 449.
(13) Mulzer, J.; Bohlmann, R. Role of natural products in drug
discovery; SSBM: 2013p Vol. 32.
(14) Bajpai, V. K.; Yoon, J. I.; Kang, S. C. Antifungal potential of
essential oil and various organic extracts of Nandina domestica
Thunb. against skin infectious fungal pathogens. Appl. Microbiol.
Biotechnol. 2009,83 (6), 11271133.
(15) Deepika, T.; Kannabiran, K. A report on antidermatophytic
activity of actinomycetes isolated from Ennore coast of Chennai,
Tamil Nadu, India. Int. J. Integr. Biol. 2009,6(3), 132136.
(16) Soares, L. A.; Sardi, J. d. C. O.; Gullo, F. P.; Pitangui, N. d. S.;
Scorzoni, L.; Leite, F. S.; Giannini, M. J. S. M.; Almeida, A. M. F. Anti
dermatophytic therapy: prospects for the discovery of new drugs from
natural products. Braz. J. Microbiol. 2013,44, 10351041.
(17) Lopes, G.; Pinto, E.; Salgueiro, L. Natural products: an
alternative to conventional therapy for dermatophytosis? Mycopatho-
logia 2017,182 (12), 143167.
(18) Amir, F.; Koay, Y. C.; Yam, W. S. Chemical constituents and
biological properties of the marine soft coral Nephthea: A review
(Part 1). Trop. J. Pharm. Res. 2012,11 (3), 485498.
(19) Abdelhafez, O. H.; Fahim, J. R.; Desoukey, S. Y.; Kamel, M. S.;
Abdelmohsen, U. R. Recent updates on corals from Nephtheidae.
Chem. Biodivers. 2019,16 (6), No. e1800692.
(20) Abdelhafez, O. H.; Ali, T. F. S.; Fahim, J. R.; Desoukey, S. Y.;
Ahmed, S.; Behery, F. A.; Kamel, M. S.; Gulder, T. A.; Abdelmohsen,
U. R. Anti-inflammatory potential of green synthesized silver
nanoparticles of the soft coral Nephthea sp. supported by
metabolomics analysis and docking studies. Int. J. Nanomed. 2020,
15, 5345.
(21) Quinn, P.; Markey, B. K.; Carter, M.; Donnelly, W.; Leonard, F.
Veterinary microbiology and microbial disease. BSL: 2002.
(22) EM, E.; CU, E. Prevalence of keratinophiic fungi and other
dermatophytes from soils of Nnewi in Anambra state, Nigeria. Nov.
Res. Microbiol. J. 2019,3(3), 379386.
(23) Scott, D. W.; Miller, W. H. Equine Dermatology, 2nd ed.;
Elsevier Health Sciences: 2010.
(24) Monika, G. S.; Chinna, D. Clinico-Mycological correlation of
superficial fungal infect ions. J. Dermatol. Venereol. 2016,23 (1), 613.
(25) Wayne, P. Reference method for broth dilution antifungal
susceptibility testing of lamentous fungi; Clinical and Laboratory
Standards Institute: 2008; CLSI Document M38-A2.
(26) Maurya, V. K.; Kachhwaha, D.; Bora, A.; Khatri, P. K.; Rathore,
L. Determination of antifungal minimum inhibitory concentration and
its clinical correlation among treatment failure cases of dermatophy-
tosis. J. Family Med. Prim. Care. 2019,8(8), 2577.
(27) Ahmed, W.; Kamal, A.; Ibrahim, R. Phytochemical Screening
and Chemical Investigation of Lipoidal Matter of Arenga engleri
Leaves. J. Adv. Pharm. Res. 2019,3(2), 8389.
(28) Wang, M.; Carver, J. J.; Phelan, V. V.; Sanchez, L. M.; Garg, N.;
Peng, Y.; Nguyen, D. D.; Watrous, J.; Kapono, C. A.; Luzzatto-Knaan,
T. Sharing and community curation of mass spectrometry data with
Global Natural Products Social Molecular Networking. Nat.
Biotechnol. 2016,34 (8), 828837.
(29) El-Mekkawy, S.; Shahat, A. A.; Alqahtani, A. S.; Alsaid, M. S.;
Abdelfattah, M. A.; Ullah, R.; Emam, M.; Yasri, A.; Sobeh, M. A
Polyphenols-Rich Extract from Moricandia sinaica Boiss. Exhibits
Analgesic, Anti-Inflammatory and Antipyretic Activities In Vivo.
Molecules 2020,25 (21), 5049.
(30) Emam, M.; Abdel-Haleem, D. R.; Salem, M. M.; Abdel-Hafez,
L. J. M.; Latif, R. R. A.; Farag, S. M.; Sobeh, M.; El Raey, M. A.
Phytochemical Profiling of Lavandula coronopifolia Poir. Aerial Parts
Extract and Its Larvicidal, Antibacterial, and Antibiofilm Activity
Against Pseudomonas aeruginosa. Molecules 2021,26 (6), 1710.
(31) Raheem, D. J.; Tawfike, A. F.; Abdelmohsen, U. R.; Edrada-
Ebel, R.; Fitzsimmons-Thoss, V. Application of metabolomics and
molecular networking in investigating the chemical profile and
antitrypanosomal activity of British bluebells (Hyacinthoides non-
scripta). Sci. Rep. 2019,9(1), 113.
(32) Nothias, L.-F.; Petras, D.; Schmid, R.; Dührkop, K.; Rainer, J.;
Sarvepalli, A.; Protsyuk, I.; Ernst, M.; Tsugawa, H.; Fleischauer, M.
Feature-based molecular networking in the GNPS analysis environ-
ment. Nat. Methods 2020,17 (9), 905908.
(33) Shannon, P.; Markiel, A.; Ozier, O.; Baliga, N. S.; Wang, J. T.;
Ramage, D.; Amin, N.; Schwikowski, B.; Ideker, T. Cytoscape: a
software environment for integrated models of biomolecular
interaction networks. Genome Res. 2003,13 (11), 24982504.
(34) Morcoss, M. M.; El Shimaa, M.; Ibrahem, R. A.; Abdel-
Rahman, H. M.; Abdel-Aziz, M.; Abou El-Ella, D. A. Design, synthesis,
mechanistic studies and in silico ADME predictions of benzimidazole
derivatives as novel antifungal agents. Bioorg. Chem. 2020,101,
103956.
(35) Zhang, J.; Li, L.; Lv, Q.; Yan, L.; Wang, Y.; Jiang, Y. The fungal
CYP51s: their functions, structures, related drug resistance, and
inhibitors. Front. Microbiol. 2019,10, 691.
(36) Zuza-Alves, D. L.; Silva-Rocha, W. P.; Chaves, G. M. An update
on Candida tropicalis based on basic and clinical approaches. Front.
Microbiol. 2017,8, 1927.
(37) Arınç, E. The role of polymorphic cytochrome P450 enzymes
in drug design, development and drug interactions with a special
emphasis on phenotyping. J. Mol. Catal., B Enzym. 2010,64 (34),
120122.
(38) Parrish, N.; Fisher, S. L.; Gartling, A.; Craig, D.; Boire, N.;
Khuvis, J.; Riedel, S.; Zhang, S. Activity of Various Essential Oils
ACS Omega http://pubs.acs.org/journal/acsodf Article
https://doi.org/10.1021/acsomega.2c00063
ACS Omega XXXX, XXX, XXXXXX
I
Against Clinical Dermatophytes of Microsporum and Trichophyton.
Front. Cell. Infect. Microbiol. 2020,10, 567.
(39) Ghaffari, T.; Kafil, H. S.; Asnaashari, S.; Farajnia, S.; Delazar,
A.; Baek, S. C.; Hamishehkar, H.; Kim, K. H. Chemical composition
and antimicrobial activity of essential oils from the aerial parts of
Pinus eldarica grown in Northwestern Iran. Molecules 2019,24 (17),
3203.
(40) Ghavam, M.; Manca, M. L.; Manconi, M.; Bacchetta, G.
Chemical composition and antimicrobial activity of essential oils
obtained from leaves and flowers of Salvia hydrangea DC. ex Benth.
Sci. Rep. 2020,10 (1), 110.
(41) Pohl, C. H.; Kock, J. L.; Thibane, V. S. Antifungal free fatty
acids: A Review. Science against microbial pathogens: communicating
current research and technological advances 2011,3,6171.
(42) Manter, D. K.; Kelsey, R. G.; Karchesy, J. J. Antimicrobial
activity of extractable conifer heartwood compounds toward
Phytophthora ramorum. J. Chem. Ecol. 2007,33 (11), 21332147.
(43) Polizzi, V.; Fazzini, L.; Adams, A.; Picco, A. M.; De Saeger, S.;
Van Peteghem, C.; De Kimpe, N. Autoregulatory properties of
(+)-thujopsene and influence of environmental conditions on its
production by Penicillium decumbens. Microb. Ecol. 2011,62 (4),
838852.
(44) Costa, T. R.; Fernandes, O. F.; Santos, S. C.; Oliveira, C. l. M.;
Lião, L. M.; Ferri, P. H.; Paula, J. R.; Ferreira, H. D.; Sales, B. H.;
Maria do Rosário, R. S. Antifungal activity of volatile constituents of
Eugenia dysenterica leaf oil. J. Ethnopharmacol. 2000,72 (12), 111
117.
(45) Tumen, I.; Eller, F. J.; Clausen, C. A.; Teel, J. A. Antifungal
activity of heartwood extracts from three Juniperus species.
BioResources 2012,8(1), 1220.
(46) Tepe, B.; Daferera, D.; Sokmen, A.; Sokmen, M.; Polissiou, M.
Antimicrobial and antioxidant activities of the essential oil and various
extracts of Salvia tomentosa Miller (Lamiaceae). Food Chem. 2005,90
(3), 333340.
(47) Magwa, M. L.; Gundidza, M.; Gweru, N.; Humphrey, G.
Chemical composition and biological activities of essential oil from
the leaves of Sesuvium portulacastrum. J. Ethnopharmacol. 2006,103
(1), 8589.
(48) Matasyoh, J. C.; Kiplimo, J. J.; Karubiu, N. M.; Hailstorks, T. P.
Chemical composition and antimicrobial activity of essential oil of
Tarchonanthus camphoratus. Food Chem. 2007,101 (3), 11831187.
(49) Fontenelle, R.; Morais, S.; Brito, E.; Kerntopf, M.; Brilhante, R.;
Cordeiro, R.; Tomé, A.; Queiroz, M.; Nascimento, N.; Sidrim, J.
Chemical composition, toxicological aspects and antifungal activity of
essential oil from Lippia sidoides Cham. J. Antimicrob. Chemother.
2007,59 (5), 934940.
(50) Fontenelle, R.; Morais, S.; Brito, E.; Brilhante, R.; Cordeiro, R.;
Nascimento, N.; Kerntopf, M.; Sidrim, J.; Rocha, M. Antifungal
activity of essential oils of Croton species from the Brazilian Caatinga
biome. J. Appl. Microbiol. 2008,104 (5), 13831390.
(51) Hernández-Hernández, A.; Alarcón-Aguilar, F.; Jiménez-
Estrada, M.; Hernández-Portilla, L.; Flores-Ortiz, C.; Rodríguez-
Monroy, M.; Canales-Martínez, M. Biological properties and chemical
composition of Jatropha neopauciflora Pax. AJTCAM. 2016,14 (1),
3242.
(52) Jeon, Y.-T.; Jun, E.-M.; Oh, K.-B.; Thu, P. Q.; Kim, S.-U.
Identification of 12-methyltetradecanoic acid from endophytic
Senotrophomonas maltophilia as inhibitor of appressorium formation
of Magnaporthe oryzae. J. Korean Soc. Appl. Biol. Chem. 2010,53 (5),
578583.
(53) Peres, N.; Cursino-Santos, J.; Rossi, A.; Martinez-Rossi, N. In
vitro susceptibility to antimycotic drug undecanoic acid, a medium-
chain fatty acid, is nutrient-dependent in the dermatophyte
Trichophyton rubrum. World J. Microbiol. Biotechnol. 2011,27 (7),
17191723.
(54) Garnier, L.; Penland, M.; Thierry, A.; Maillard, M.-B.; Jardin, J.;
Coton, M.; Salas, M. L.; Coton, E.; Valence, F.; Mounier, J. Antifungal
activity of fermented dairy ingredients: identification of antifungal
compounds. Int. J. Food Microbiol. 2020,322, 108574.
(55) Bian, X.; Muhammad, Z.; Evivie, S. E.; Luo, G.-W.; Xu, M.;
Huo, G.-C. Screening of antifungal potentials of Lactobacillus
helveticus KLDS 1.8701 against spoilage microorganism and their
effects on physicochemical properties and shelf life of fermented
soybean milk during preservation. Food Control 2016,66, 183189.
(56) Lind, H.; Sjögren, J.; Gohil, S.; Kenne, L.; Schnürer, J.; Broberg,
A. Antifungal compounds from cultures of dairy propionibacteria type
strains. FEMS microbiology letters 2007,271 (2), 310315.
(57) Thibane, V. S.; Kock, J. L.; Ells, R.; Van Wyk, P. W.; Pohl, C. H.
Effect of marine polyunsaturated fatty acids on biofilm formation of
Candida albicans and Candida dubliniensis. Mar. Drugs 2010,8(10),
25972604.
(58) Shi, D.; Zhao, Y.; Yan, H.; Fu, H.; Shen, Y.; Lu, G.; Mei, H.;
Qiu, Y.; Li, D.; Liu, W. Antifungal effects of undecylenic acid on the
biofilm formation of Candida albicans. Int. J. Clin. Pharmacol. Ther.
2016,54 (5), 343.
(59) Nikolov, A.; Ganchev, D. Effect of zinc undecylenates on plant
pathogenic fungi. Bulg. J. Agric. Sci. 2010,16, 220226.
(60) Rehder, P.; Nguyen, T. T. Clinical Research: A new concept in
the topical treatment of onychomycosis with cyanoacrylate,
undecylenic acid, and hydroquinone. Foot Ankle Spec. 2008,1(2),
9396.
(61) McDonough, V.; Stukey, J.; Cavanagh, T. Mutations in erg4
affect the sensitivity of Saccharomyces cerevisiae to medium-chain
fatty acids. Biochim. Biophys. Acta. Mol. Cell Biol. Lipids 2002,1581
(3), 109118.
(62) Ells, R.; Kock, J. L.; Van Wyk, P. W.; Botes, P. J.; Pohl, C. H.
Arachidonic acid increases antifungal susceptibility of Candida
albicans and Candida dubliniensis. J. Antimicrob. Chemothe. 2008,
63 (1), 124128.
(63) Lone, S. A.; Khan, S.; Ahmad, A. Inhibition of ergosterol
synthesis in Candida albicans by novel eugenol tosylate congeners
targeting sterol 14α-demethylase (CYP51) enzyme. Arch. Microbiol.
2020,202 (4), 711726.
(64) Bvumbi, C.; Chi, G. F.; Stevens, M. Y.; Mombeshora, M.;
Mukanganyama, S. The Effects of Tormentic Acid and Extracts from
Callistemon citrinus on Candida albicans and Candida tropicalis
Growth and Inhibition of Ergosterol Biosynthesis in Candida albicans.
Sci. World J. 2021,2021,113.
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