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Background Mangroves plants and their endophytes represent a natural source of novel and bioactive compounds. In our ongoing research on mangrove endophytes from the Panamanian Pacific Coast, we have identified several bioactive endophytic fungi. From these organisms, an isolate belonging to the genus Zasmidium(Mycosphaerellaceae) showed 91.3% of inhibition against α-glucosidase enzyme in vitro. Results Zasmidium sp. strain EM5-10 was isolated from mature leaves of Laguncularia racemosa, and its crude extract showed good inhibition against α-glucosidase enzyme (91.3% of inhibition). Bioassay-guided fractionation of the crude extract led to obtaining two active fractions: L (tripalmitin) and M (Fungal Tryglicerides Mixture). Tripalmitin (3.75 µM) showed better inhibitory activity than acarbose (positive control, IC50 217.71 µM). Kinetic analysis established that tripalmitin acted as a mixed inhibitor. Molecular docking and molecular dynamics simulations predicted that tripalmitin binds at the same site as acarbose and also to an allosteric site in the human intestinal α-glucosidase (PDB: 3TOP). Conclusions Zasmidium sp. strain EM5-10 represents a new source of bioactive substances that could possess beneficial properties for human health.
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Lopézetal. BMC Chemistry (2019) 13:22
https://doi.org/10.1186/s13065-019-0540-8
RESEARCH ARTICLE
α-Glucosidase inhibitors fromamangrove
associated fungus, Zasmidium sp. strain EM5-10
Dioxelis Lopéz1,2, Lilia Cherigo3, Luis C. Mejia1, Marco A. Loza‑Mejía4 and Sergio Martínez‑Luis1*
Abstract
Background: Mangroves plants and their endophytes represent a natural source of novel and bioactive compounds.
In our ongoing research on mangrove endophytes from the Panamanian Pacific Coast, we have identified several bio‑
active endophytic fungi. From these organisms, an isolate belonging to the genus Zasmidium (Mycosphaerellaceae)
showed 91.3% of inhibition against α‑glucosidase enzyme in vitro.
Results: Zasmidium sp. strain EM5‑10 was isolated from mature leaves of Laguncularia racemosa, and its crude extract
showed good inhibition against α‑glucosidase enzyme (91.3% of inhibition). Bioassay‑guided fractionation of the
crude extract led to obtaining two active fractions: L (tripalmitin) and M (Fungal Tryglicerides Mixture). Tripalmitin
(3.75 µM) showed better inhibitory activity than acarbose (positive control, IC50 217.71 µM). Kinetic analysis estab‑
lished that tripalmitin acted as a mixed inhibitor. Molecular docking and molecular dynamics simulations predicted
that tripalmitin binds at the same site as acarbose and also to an allosteric site in the human intestinal α‑glucosidase
(PDB: 3TOP).
Conclusions: Zasmidium sp. strain EM5‑10 represents a new source of bioactive substances that could possess ben‑
eficial properties for human health.
Keywords: α‑Glucosidase, Laguncularia racemosa, Zasmidium, Triglycerides, Mangroves, Diabetes mellitus
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Background
Mangrove plants and their endophytes represent a natu-
ral source of novel and bioactive compounds that have
been attracting the attention of scientists in the past dec-
ade. Mangroves are a group of 73 species of shrubs or
trees found in tropical and subtropical areas. Mangroves
are highly adapted to tolerate extreme conditions such
as high levels of salinity, high temperature, and moisture
[1]. In several parts of the world, mangroves have been
used for traditional medicine. Chemical studies of man-
grove species have led to the identification of more of 200
bioactive compounds [24]. Endophytic fungi of man-
groves have been shown to be a good source of novel,
bioactive, and exceptional compounds with unique and
unusual structures. So far, more than 322 fungal metabo-
lites, isolated from mangrove associated fungi, have been
obtained and have showed promising biological activities
[5, 6].
Hyperglycemia is a rapid increase in blood glucose
levels due to starch hydrolysis by α-amylase and glucose
releasing into the small intestine by α-glucosidase action.
Inhibition of both enzymes should result in postpran-
dial hyperglycemia decline, which could be an important
strategy for the control of diabetes mellitus. Unfortu-
nately, available therapeutic α-glucosidase inhibitors have
a strong α-amylase inhibitory activity which can lead to
digestive tract disorders such as abdominal distension,
flatulence, meteorism, and diarrhea [7]. us, new inhibi-
tors are required, especially ones with low α-amylase
inhibition activity, because they could represent an
effective therapy for postprandial hyperglycemia with
minimal side effects. e first inhibitor of α-glucosidase
introduced in the market was acarbose, a metabolite dis-
covered in microorganisms from the Actinoplanes genus.
In recent years, several fungal compounds, including
Open Access
BMC Chemistry
*Correspondence: smartinez@indicasat.org.pa
1 Centro de Biodiversidad y Descubrimiento de Drogas, Instituto de
Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT
AIP), Edificio 208, Ciudad del Saber, Apartado, 0843‑01103 Panama City,
Panama
Full list of author information is available at the end of the article
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Lopézetal. BMC Chemistry (2019) 13:22
those from mangrove endophytes, have been reported as
inhibitors of α-glucosidase [813], which is evidence that
these kinds of microorganisms are prolific producers of
α-glucosidase inhibitors.
Recently, our lab group decided to establish a research
line aimed at the systematic biological evaluation of
extracts from mangrove organisms to find them potential
biomedical applications [14]. In our ongoing research on
mangrove plant species and their endophytes from the
Panamanian Pacific Coast, several bioactive endophytic
fungi have been identified. From these fungi, an isolate
belonging to the genus Zasmidium (Mycosphaerellaceae)
showed good activity against α-glucosidase enzyme
invitro. Its organic extract inhibited 91.3% of the enzyme
function. Bioassay-guided fractionation allowed us to
obtain two active fractions, one of which was composed
by tripalmitin and the other for a triglyceride mixture.
Here, we report some results obtained in this study.
Results
Fungal isolation andcharacterization
An endophytic fungus, isolate EM5-10, was obtained from
mature leaves of Laguncularia racemosa (Combretaceae),
collected from Mangroves and wetlands located in an
area of the Bay of Panama known as Juan Diaz, Panama.
is isolate was identified as Zasmidium sp., based on
99% DNA sequence identity of the ITS region of this iso-
late with that from the holotype of Stenella musae (culture
CBS 122477, Accession Number EU514291.1), now under
the genus Zasmidium [15]. e isolate is here identified as
Zasmidium sp. strain EM5-10 with ITS sequence labeled
as Genbank Accession Number KX898455. Further sys-
tematic work is required for accurate phylogenetic rela-
tionships of this isolate with congeneric species and for
assessing the generality of the bioactive activity described
in this work. In our view, this is the first report of the isola-
tion of a species belonging to Zasmidium genus as endo-
phytic fungi of L. racemosa leaves, and this finding allows
us to determine that this species can tolerate a relatively
high percentage of salt in its culture conditions.
Chemical study
In the initial screening, the crude extract showed good
inhibition against α-glucosidase enzyme (91.3% of inhibi-
tion). Following the protocols of our laboratory, we per-
formed a primary fractionation by Solid-Phase Extraction
to obtain 16 fractions. All 16 fractions were submitted for
bioactivity testing. Only two fractions, L and M, exhibited
97% and 96% of α-glucosidase inhibition, respectively, at
concentrations of 6.25 µg/mL. rough spectroscopic
analysis, we detected that both fractions had compounds
of triglycerides type. Additionally, Fraction L contained
one major component with approximately 97% of purity
(compound 1), and Fraction M consisted of a mixture
of triglycerides (with at least two main components).
Comparison of the obtained NMR data with those of the
literature allow the identification of the compound as
tripalmitin(Fig.1) [16, 17]. Additionally, chemical shifts
of the isolated compound were compared with those of
authentic sample of tripalmitin obtained from Sigma-
Aldrich, and the NMR spectra of both samples showed
complete concordance (Fig.2). In order to corroborate
the presence of triglycerides, we proceeded to perform
a methanolysis reaction to release the fatty acid methyl
esters (FAME). e FAME formed after methanolysis
were extracted and analyzed by NMR and TLC. Analysis
of the results of these tests revealed that the methyl ester
of palmitic acid was the main component of the reaction
mixture (Fig.2).
On the other hand, active fraction M was a triglycerides
mixture (FTGm), and this mixture presented an inher-
ent difficulty for the separation of its constituents, due
to this, we were unable to separate the compounds with
the equipment available to us. Hence, we proceeded to
identify some of its major components with the acquired
spectra. e mass spectrum of fraction M exhibited
two peaks sticking out over the rest of the components,
with pseudomolecular ions at m/z 889.8211 and at m/z
887.8057. e molecular formula of these ions were
C57H109O6 and C57H107O6, which together with NMR
data analysis allows us to infer that both compounds are
triglycerides containing oleic acid and stearic acid in their
structure. For molecular Docking study, a hypothetical
fungal triglyceride (FTG) structure was proposed which
contained one chain of oleic acid located in C-2 and two
chains of stearic acid in C-1 and C-3 (Fig.3).
α‑Glucosidase inhibition evaluation andkinetic studies
Tripalmitin inhibited α-glucosidase enzyme in a con-
centration-dependent manner with an IC50 value of
3.02µg/mL (3.75µM, Fig.4a). On the other hand, FTG
from fraction M inhibited α-glucosidase enzyme in a
concentration-dependent manner with an IC50 value
of 0.92 µg/mL (Fig. 4b). Both fractions showed better
inhibitory activity than acarbose (positive control, IC50
217.71 µM/140.55 µg/mL) (Fig. 4d). Kinetic analysis
Fig. 1 Compound 1 (tripalmitin)
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was carried out to understand the interaction of tripal-
mitin with α-glucosidase. Lineweaver–Burk plots were
constructed using different concentrations of substrate
and tripalmitin. Lineweaver–Burk plots in Fig.5a dem-
onstrated tripalmitin acts as a mixed inhibitor against
α-glucosidase enzyme. Also, methyl palmitate inhib-
ited α-glucosidase enzyme in a concentration-depend-
ent manner (Fig.4c) with an IC50 value of 0.13µg/mL
(0.46 µM). Lineweaver–Burks plots in Fig. 5b showed
that methyl palmitate acts as mixed inhibitors against
α-glucosidase. e kinetic parameters of α-glucosidase
inhibition by tripalmin and methyl palmitate are in
Table1.
Docking study
Experimental results showed that methyl palmitate and
tripalmitin acted as mixed inhibitors. Two different
docking studies were carried out: one in the active site
and the second one in a close allosteric site [18] which
included Acarbose as a “cofactor” to block the entrance
Fig. 2 13C NMR Spectra (100 MHz). (a) Tripalmitin, (b) tripalmitin standard, (c) methyl palmitate (obtained from the methanolysis reaction)
Fig. 3 Fungal triglyceride mixture
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to the active site. Results from the docking study are
shown in Figs.6, 7 and Table2. Rerank scores are cal-
culated in Molegro Virtual Docker as an estimate of
ligand binding, where lower values are associated with
higher affinity.Molecular dynamics (MD) simulations
and molecular mechanics with Poisson–Boltzmann and
surface area solvation (MM-PBSA) calculations were also
performed to have a better understanding of the inter-
actions found in the molecular modeling analysis. e
results obtained are shown in Fig.8.
Artemia salina bioassay
Lethality assay using Artemia salina is a fast and cheap
bioassay for assessing natural organic extracts biological
activity which correlates well with cytotoxic activ-
ity. e LC50 value of Artemia salina obtained for the
crude extract and active fractions (M and L) was higher
than 1000, indicating an absence of cytotoxicity in these
samples.
Discussion
Microorganisms tend to use different strategies to com-
bat adverse conditions within the ecosystem in which
they develop, one of them being the production of
metabolites [19]. In many cases, the specific function of
each compound produced remains unknown, although
it is possible to infer it based on the type of compound
structure and function described in other areas. ese
Fig. 4 α‑Glucosidase inhibition a tripalmitin, b fungal triglycerides mixture, c methyl palmitate, d acarbose
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correlations can be very useful to find potential applica-
tions of many of the compounds obtained from micro-
organisms. Triglycerides are the main form of energy
storage in living organisms. In animals, most cells store
small amounts of triglycerides, which appear as scat-
tered droplets in the cytoplasm. is kind of metabolites
are a reservoir of energy and carry out thermal insula-
tion and protection functions [20]. e presence of high
amounts of triglycerides in Zasmidium sp. suggests that
these compounds could be performing functions similar
to those mentioned above in this fungus because its host
(the mangrove) lives in an ecosystem with great adverse
conditions as mangroves such as extreme solar radiation
during daytime.
In our understanding, this is the first report about the
α-glucosidase inhibitory effect of triglyceride-type com-
pounds, in order to be sure of our results we also evalu-
ated the activity of tripalmitin and triolein standards
purchased from Sigma-Aldrich. Both standards exhibited
inhibitory activity of the enzyme α-glucosidase (IC50:
3.20 and 1.52M, respectively). We also evaluated the
activity of methyl palmitate and glycerol (both obtained
from methanolysis reaction) to get more information
about the biological activity of tripalmitin, and we found
that methyl palmitate inhibited α-glucosidase enzyme,
while glycerol was inactive. Finally, the activity of oleic
and stearic acids, constituent units of the triglycerides
present in Fraction M, were also tested and both showed
inhibitory activity with IC50 values of 0.23 and 0.81µM.
All these activity results corroborate the inhibitory effect
of the α-glucosidase enzyme by triglyceride-type com-
pounds, in addition to pointing out that the fatty acid
units constituting the triglyceride are responsible for this
activity.
Lineweaver–Burks plots showed that tripalmitin
and methyl palmitate act as mixed inhibitors against
α-glucosidase, suggesting that tripalmitin and methyl pal-
mitate could bind to the free enzyme or the enzyme–sub-
strate complex. When we analyze the obtained Km values,
we find that it decreases when the inhibitor binds to the
enzyme–substrate complex, which in this case denotes
an increase in the apparent affinity of the enzyme for the
substrate.
Taking into account this evidences, molecular dock-
ing was used to predict binding conformations between
inhibitors (tripalmitin and methyl palmitate) and the
α-glucosidase enzyme, which could generate information
for understanding the mechanism of action that could be
used as the basis for the design of new inhibitors.
Rerank scores agree with previous reports that state
that potency of inhibition is roughly correlated with the
number of unsaturations [2123] as fungal triglycerides
Fig. 5 Lineweaver–Burk plots of α‑glucosidase inhibitors a tripalmitin, b methyl palmitate
Table 1 Kinetic parameters of α-glucosidase inhibition
bytripalmitin andmethyl palmitate
Km Michaelis–Menten constant, Vmax maximum reaction velocity, Ki inhibitory
constant, n/a not applicable
Samples Km (mmol/L) Vmax (mmol/(Lmin) Ki (mM)
α‑Glucosidase 0.365 ± 0.001 0.0041 ± 0.0001 n/a
Tripalmitin 0.511 ± 0.003 0.0027 ± 0.0001 332 ± 39
Methyl palmitate 0.483 ± 0.002 0.0024 ± 0.0002 34 ± 2
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(FTG) and methyl oleate had the lowest Rerank scores.
Due to their size, both triglycerides, FTG, and tripalmi-
tin can bind to both allosteric and active sites (Fig. 6a,
b). FTG has a lower Rerank score than tripalmitin which
coincides with experimental data. Analysis of the interac-
tion of tripalmitin with specific residues of α-glucosidase
reveals that hydrophobic interactions have an essential
contribution to ligand binding as expected. However,
hydrogen bond interactions of the carbonyl groups of the
triglyceride with polar residues located inside the active
site cavity (Gln1372 and Arg1377) seems also crucial for
ligand binding.
Results from docking studies (Table2) allow us to pro-
pose that while FAMEs prefer to bind to the active site,
it is possible that they could bind, with slightly lower
affinity, to the allosteric site even when the active site is
occupied, explaining the mixed-inhibition behavior. Fig-
ure7 shows the binding of methyl ester to the active site
(Fig.7a, b) and the allosteric site (Fig.7c, d). Analysis of
the interaction patterns reveals that, as excepted, most
interactions are of hydrophobic nature. e presence of
a hydrogen bonding group seems important for enzyme
binding in both active and allosteric sites. is aspect
would be necessary to take in consideration in the design
of new inhibitors based on FAME template.
Fig. 6 a Predicted pose of tripalmitin bound to the active and allosteric site of glucosidase. b Interaction diagram of the same binding pose.
Allosteric site is comprised between Tyr 1251 to Ser 1292 sites
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Even though the results of the docking study cor-
relate with the results found in our experiments, it
is a well-known fact that the efficiency of a docking
study may decrease as the number of rotatable bonds
increases [24]. Thus, we carried out MD simulations
and MM-PBSA calculations, which are considered
more rigorous than molecular docking [25]. Analy-
sis of variations of Root-Median Standard Deviation
(RMSD) along time (Fig. 8) suggests that the three
complexes predicted from molecular docking are
stable since RMSD fluctuations did not surpass 3 Å
along all simulation time. In other hand, ligand bind-
ing energy (LBE) calculated using MM-PBSA shows
that methyl palmitate has a slightly better affinity to
the active site than to the allosteric site which coin-
cides with docking scores. LBE calculated for tripalmi-
tin complex suggest that it could have better binding
than methyl palmitate, a fact that does not precisely
correlate with experimental evidence from inhibi-
tion kinetics. The high flexibility of tripalmitin could
be detrimental for binding to the active or allosteric
Fig. 7 Predicted poses of methyl palmitate within the a active and b allosteric sites of α‑glucosidase. 2D diagrams of enzyme‑inhibitor complexes
in c active and d allosteric site
Table 2 Results of molecular docking studies (Rerank
score, more negative values indicate better theoretical
anity)
a These compounds occupy both sites simultaneously
Ligand Active site Allosteric site
Methyl oleate 94.2873 84.4339
Methyl stearate 85.5979 79.7206
Methyl palmitate 80.5525 78.3820
FTG 133.662 a
Tripalmitin 94.8291 a
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site, and this could explain the lack of correlation with
experimental results.
Conclusions
Bioassay-guided fractionation of active extract from Zas-
midium sp. strain EM5-10 against α-glucosidase allowed
us to isolate one triglyceride and to detect a complex
triglycerides mixture from a second active fraction. e
structure of the active compound was established by
spectroscopic analyses and comparison of the obtained
data with those of the literature.
Tripalmitin (3.67 µM) and fungal triglycerides mix-
ture (0.95µg/mL) showed better inhibitory activity than
Acarbose (positive control, IC50 217.71 µM). To our
knowledge, this is the first report on α-glucosidase inhib-
itory activity of triglycerides (tripalmitin and triolein, in
our case). On the other hand, this type of compounds
exhibited a mixed type of inhibition against S. cerevisiae
α-glucosidase. Results from docking studies reveals that
hydrophobic interactions have an essential contribution
to ligand binding as expected. However, hydrogen bond
interactions of the carbonyl groups of the tripalmitin
with polar residues located inside the active site cav-
ity (Gln1372 and Arg1377) seems also crucial for ligand
binding.
erefore, this fungus represents a new source of
α-glucosidase inhibitors, which could possess beneficial
properties for human health.
Methods
General experimental procedures
NMR spectra were acquired on Jeol Eclipse 400 MHz.
One-dimensional spectra were referenced to δH 7.26,
δC 77.0 for CDCl3 (used solvent). ESI mass spectra were
recorded on a micrOTOF-Q II spectrometer, Bruker
Daltonics, Germany. e purification of the compounds
was carried out on SPE cartridge on C18 (6mL/1000mg,
Macherey–Nagel, Düren, Germany). Purification sol-
vents were HPLC grade and used without further purifi-
cation. Methanolysis chemicals were reagent grade.
Fungal isolation
e endophytic fungus isolate reported in this study was
isolated from mature leaves of L. racemosa (Combreta-
ceae), collected in Juan Diaz, Panama, in 2012. It was
isolated in Bacto Malt Extract agar with 1% artificial
sea salt (MEA) and 0.02% antibiotic, using a standard
protocol. Briefly, leaves were surface-sterilized, first they
were immersed in 70% ethanol solution, followed by 1%
sodium hypochlorite solution, then in 70% ethanol solu-
tion, and finally leaves were washed with sterile distilled
water. Each immersion process was 2min. Leaves were
cut into several 1mm2 pieces and placed in individual
Petri dishes with Bacto Malt Extract agar with 1% arti-
ficial sea salt (MEA) and Chloramphenicol as antibiotic.
Petri dishes were checked daily to observe the growth of
fungi, which were isolated and transferred to new Petri
dishes for purification.
DNA sequence identication
Identification of the endophytic fungus isolate was
made by comparison of the nuclear ribosomal inter-
nal transcribed spacer region (ITS) nucleotide sequence
of the fungus with those in the National Institutes of
Health genetic sequence database (GenBank), using the
Basic Local Alignment Search Tool (BLAST) [26]. DNA
sequence identity equal or above 99% for the entire ITS
region (ITS 1, the 5.8S gene, and ITS2) with type culture
sequence and evaluation of taxonomic literature [15] was
considered as the criterion for assigning genus name to
the isolated fungus. Extraction, PCR and sequencing of
DNA were done as reported in Mejia etal. [27].
Small‑scale culture
For screening of the initial activity against α-glucosidase
enzyme, the fungus was grown in small-scale using 10
Petri dishes with MEA inoculated with small pieces
actively growing mycelium (0.5cm2). e cultures were
incubated for 21days at room temperature.
Large‑scale culture
For chemical studies, Zasmidium sp. strain EM5-10 was
grown in 500 Petri dishes, each containing MEA, dishes
were individually inoculated with a 1cm2 agar plug taken
from a stock culture with the fungus under study. e
cultures were incubated for 21days at room temperature.
Fig. 8 RMSD variations along MD simulation of a‑glucosides
complexes of tripalmitin and methyl palmitate in the active site and
methyl palmitate in allosteric site. Ligand binding energies calculated
by MM‑PBSA are also included
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Production ofextracts
After the incubation period, the Petri dishes were freeze-
dried for 48 h. Mycelia and MEA were extracted with
ethyl acetate. e solvent was evaporated, and extract
concentrated under reduced pressure to a semisolid paste
using a Buchi rotary Evaporator (R-215) to obtain 1.5g of
crude extract.
Isolation ofcompounds
e organic extract was fractionated by SPE cartridge on
C18 (6mL/1000mg). e SPE cartridge was first condi-
tioned with methanol (12mL) and then equilibrated by
water (12mL). e crude extract (1.5g) was dissolved
with 6mL of 10% MeOH and sample was loaded onto
the SPE column. e column was eluted with a gradi-
ent of water:MeOH (9:1 0:1). Altogether, 15 fractions
(12ml each) were collected as primary fractions (FA to
FP). Evaluation of all fractions showed that only fractions
L and M exhibited activity (97% and 96% of α-glucosidase
inhibition, respectively) at a concentration of 6.25µg/mL.
Methanolysis
Tripalmitin was subjected to methanolysis using Kato
etal. and Ichibara etal. protocols, with small modifica-
tions [28, 29]. Briefly, to 50mg of sample diluted in 1mL
of ethyl ether was added 9ml of 0.7M KOH in MeOH
and 1 mL of MeOH. e reaction mixture was kept
overnight at room temperature without agitation. Next
day, pH of the reaction mixture was adjusted to 3.0 with
0.5N H2SO4, distilled water was added to obtain solvent
phases. e reaction was monitored by thin layer chro-
matography using as a mobile phase hexane/acetone/
acetic acid (95:5:0.5 v/v). Spots were visualized using sul-
phuric acid solution and heating the TLC to 135°C [30].
FAMEs were recovered washing the mixture solution
three times with hexane. Organic extraction was concen-
trated under reduced pressure using a Buchi rotary Evap-
orator (R-215).
α‑Glucosidase inhibitory assay
e α-glucosidase inhibitory assay was performed
according to the protocol established in our labora-
tory [31]. α-Glucosidase from baker’s yeast purchased
from Sigma Chemical Co. e inhibition was measured
spectrophotometrically at pH 7.0 and 37 °C employing
2 mM p-nitrophenyl α--glucopyranoside (PNP-G) as
a substrate and 32mU/mL of the enzyme, in 100mM
potassium phosphate buffer (enzyme stock). Acar-
bose was dissolved in phosphate buffer, and serial dilu-
tions (in order to obtain the IC50) were prepared and
employed as positive control. e absorbance (A) of
4-nitrophenol released by the hydrolysis of PNP-G was
measured at 400nm by Synergy HT Bio Tek microplate
spectrophotometer. A 20µL of Acarbose or test com-
pounds solution was incubated for 7min with 150µL of
enzyme stock at 37°C. After incubating, 150µL of the
substrate was added and further incubated for 20 min
at 37°C. All assays are performed in 96-well microplates
(Greiner bio-one 655101) in duplicate. e activity of
samples was calculated as a percentage in comparison to
a control (DMSO or MeOH instead of sample solution)
according to the following equation:
e concentration required to inhibit activity of the
enzyme by 50% (IC50) was calculated by regression analy-
sis [32].
Standard reagents
Tripalmitin (glyceryl tripalmitate, purity 99%, T5888),
triolein (glyceryl trioleate, purity 99%, T7140), glycerol
(purity 99%, G6279), methyl stearate (purity 96%,
W504807) and methyl oleate (purity 99%, 311111) were
purchased from Sigma Chemical Co. (St. Louis, MO).
Docking study
Fungal triglyceride and methyl esters of fatty acids were
constructed in Spartan’10 [33], and its geometry was
optimized using MMFF force field. Protein–ligand dock-
ing studies were carried out in C-terminal domain of
human intestinal α-glucosidase (Accession Code: 3TOP)
[34] which was retrieved from the Protein Data Bank.
[35]. Molecular docking calculations were performed
using Molegro Virtual Docker v. 6.0.1. [36]. Before dock-
ing, all of the solvent and co-crystallized ligand molecules
were removed. As experimental data showed that fungal
triglyceride exhibits mixed inhibition of α-glucosidase,
some considerations were taken. A search for poten-
tial cavities was carried out finding five potential bind-
ing sites, one corresponding to the active site. Among
the other four cavities, the one that was closer to active
site was selected as the potential binding site of a non-
competitive inhibitor; this approach has been previously
reported [18]. As these cavities were close enough, they
were merged in one bigger cavity, to analyze if the stud-
ied compounds preferred the active or allosteric site. A
sphere of 18Å radius was centered in the merged cavity
for searching. Assignments of the charges and protona-
tion states were based on standard templates as part of
the program. Flexible ligand models and MolDock Opti-
mizer algorithm were used. Orientations of the ligands
into the cavity were searched and ranked based on their
scores. e RMSD threshold for multiple cluster poses
was set to < 1.00 Å. e docking algorithm was set to
10,000 maximum iterations with a simplex evolution
%
Inhibition =
Acontrol Asample
A
control
×
100%
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 10 of 11
Lopézetal. BMC Chemistry (2019) 13:22
population size of 250 and a minimum of 100 runs for
each ligand. Poses with the lowest Rerank scores were
selected for further analysis. Acarbose was also docked
into the cavity to assess the efficacy of this procedure.
e RMSD of the pose of the lowest Rerank score was
calculated. RMSD was lower than 2 Å, indicating that
the methodology used in the molecular docking studies
is appropriate. An additional docking study was carried
out maintaining the Acarbose in the active site, to ana-
lyze if the studied compounds could bind to the allosteric
site when a substrate is present. e same methodology
described above was carried out, except that for blocking
access to active site, Acarbose was kept in it.
Molecular dynamics simulations
e docking poses of tripalmitin (occupying both allos-
teric and active sites as shown in Fig.6) and methyl pal-
mitate (one pose with palmitate occupying the active site
and another one occupying the allosteric site as shown
in Fig.7) with α-glucosidase were further analysed using
molecular dynamics (MD) to analyse the stability and
conformational changes of the predicted complexes. e
MD simulations were performed using YASARA Dynam-
ics v.18.4.24 [37] using the AMBER 14 force field [38].
e initial structures were taken from the poses with the
lowest docking score of each complex and were placed in
a cell box that had an extension of 10 Å larger on each
side of the protein and was filled with water molecules.
Periodic boundary conditions (PBC) were applied. e
temperature was set at 298K, water density to 0.997g/
cm3 and pH to a value of 7.4. Sodium (Na+) and chlorine
(Cl) ions were included to provide conditions that simu-
late a physiological solution (NaCl 0.9%). Particle Mesh
Ewald algorithm was applied to evaluate long-range elec-
trostatic interactions, the cut-off for van der Waals inter-
actions was set to 8 Å. A multiple step of 2.5fs was set,
data were collected per 100ps to a final simulation time
of 40 ns. Results were analysed with a script included
as part of YASARA software and included Root Mean
Square Deviation (RMSD), Root Mean Square Fluc-
tuations (RMSF) and ligand binding energy calculations
using MM-PBSA.
Brine shrimp lethality assay
In vitro lethality assay of Artemia salina was used for
detecting toxicity from the crude fungal extract and
their fractions [39]. Brine shrimp eggs were placed in
seawater (3.8% w/v sea salt in distilled water) and incu-
bated at 28°C. Eggs were hatched within 48h providing
a large number of larvae (nauplii). Serial dilutions (1000,
500, 250 and 125ppm) were made in separated wells of
96-well microplate. Nauplii were placed in each well by
pipetting them until deposited 10–15 organism. Each
concentration was assessed by triplicate. e percentage
lethality was determined by comparing the mean surviv-
ing larvae of the test and control wells. Lethal concentra-
tions values were obtained from the best-fit line plotted
concentration versus percentage lethality. Potassium
dichromate was used as a positive control in the bioassay
while the negative controls were wells that contain only
the solvent used for the preparation of the test samples.
Authors’ contributions
Conceived and designed the experiments: SM‑L. Performed the experiments:
DL, SM‑L, LC, LCM, ML‑M. Analyzed the data: SM‑L, LC, ML‑M, DL. Wrote the
paper: ML‑M, LM, LC, DL, SM‑L. Reviewed the document: DL, SM‑L, LC, LCM,
ML‑M. All authors read and approved the final manuscript.
Author details
1 Centro de Biodiversidad y Descubrimiento de Drogas, Instituto de Investiga‑
ciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP), Edificio 208,
Ciudad del Saber, Apartado, 0843‑01103 Panama City, Panama. 2 Department
of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Gun‑
tur 522510, India. 3 Departamento de Química Orgánica, Escuela De Química,
Facultad de Ciencias Exactas y Tecnología, Universidad de Panamá, P.O.
Box 3366, Panama City, Panama. 4 Facultad de Ciencias Químicas, Universidad
La Salle, Benjamín Franklin 45, Cuauhtémoc, 06140 Mexico City, Mexico.
Acknowledgements
We want to thank the government of Panama (ANAM) for granting permission
to make the collections; to Alejandro De Sedas for the taxonomic identifica‑
tion of mangrove specimen. D.L. was supported by funds from the National
Secretariat of Science, Technology, and Innovation (SENACYT) doctoral Grant
270‑2011‑154. L.C and S.M‑L. were supported by funds from the National
Research System (SNI, SENACYT) [L. C. (SNI‑112‑2018) and S.M‑L. (SNI‑133‑
2018)]. LCM was supported by SENACYT Grant ITE11‑19. M.A.L.‑M. wishes
to thank Dirección de Posgrado e Investigación of Universidad La Salle for
additional computational resources and Juan Francisco Sánchez‑Tejeda for
assistantship during molecular dynamics studies. Finally, we thank Alberto E.
Morales (University of California, Irvine) for the language edition.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations.
Received: 12 September 2016 Accepted: 29 January 2019
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