ArticlePDF Available

Abstract and Figures

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.
This content is subject to copyright. Terms and conditions apply.
Lopézetal. BMC Chemistry (2019) 13:22
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*
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
© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/
publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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
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,
Full list of author information is available at the end of the article
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 2 of 11
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.
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)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 3 of 11
Lopézetal. BMC Chemistry (2019) 13:22
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
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
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 4 of 11
Lopézetal. BMC Chemistry (2019) 13:22
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
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
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 5 of 11
Lopézetal. BMC Chemistry (2019) 13:22
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
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
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
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 6 of 11
Lopézetal. BMC Chemistry (2019) 13:22
(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
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 7 of 11
Lopézetal. BMC Chemistry (2019) 13:22
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 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
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 8 of 11
Lopézetal. BMC Chemistry (2019) 13:22
site, and this could explain the lack of correlation with
experimental results.
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
erefore, this fungus represents a new source of
α-glucosidase inhibitors, which could possess beneficial
properties for human health.
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
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 9 of 11
Lopézetal. BMC Chemistry (2019) 13:22
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.
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
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.
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
1. Spalding M, Kainuma M, Collins L (2010) World atlas of mangroves. Earth‑
scan Ltd, London
2. Patra JK, Mohanta YK (2014) Antimicrobial compounds from mangrove
plants: a pharmaceutical prospective. Chin J Integr Med 20:311–320
3. Sithranga Boopathy N, Kathiresan K (2010) Anticancer drugs from marine
flora: an overview. J Oncol 2010:214186
4. Zhou Z, Guo Y (2012) Bioactive natural products from Chinese marine
Flora and Fauna. Acta Pharmacol Sin 33:1159–1169
5. Debbab A, Aly AH, Proksch P (2013) Mangrove derived fungal endo‑
phytes—a chemical and biological perception. Fungal Divers 61:1–27
6. Wang K‑W, Wang S‑W, Wu B, Wei J‑G (2014) Bioactive natural compounds
from the mangrove endophytic fungi. Mini Rev Med Chem 14:370–391
7. Bischoff H (1994) Pharmacology of glucosidase inhibitor. Eur J Clin Invest
8. Huang HB, Feng XJ, Liu L, Chen B, Lu YJ, Ma L, She ZG, Lin YC (2010) Three
dimeric naphtho‑γ‑pyrones from the mangrove endophytic fungus
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 11 of 11
Lopézetal. BMC Chemistry (2019) 13:22
fast, convenient online submission
thorough peer review by experienced researchers in your field
rapid publication on acceptance
support for research data, including large and complex data types
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
At BMC, research is always in progress.
Learn more
Ready to submit your research
? Choose BMC and benefit from:
Aspergillus tubingensis isolated from Pongamia pinnata. Planta Med
9. Huang H, Feng X, Xiao Z, Liu L, Li H, Ma L, Lu Y, Ju J, She Z, Lin Y (2011)
Azaphilones and p‑Terphenyls from the mangrove endophytic fungus
Penicillium chermesinum (ZH4‑E2) isolated from the South China Sea. J
Nat Prod 74:997–1002
10. Song Y, Wang J, Huang H, Ma L, Wang J, Gu Y, Liu L, Lin Y (2012) Four
eremophilane sesquiterpenes from the mangrove endophytic fungus
Xylaria sp. BL321. Mar Drugs 10:340–348
11. Liu Y, Yang Q, Xia G, Huang H, Li H, Ma L, Lu Y, He L, Xia X, She Z (2015)
Polyketides with α‑glucosidase inhibitory activity from a mangrove
endophytic fungus, Penicillium sp. HN29‑3B1. J Nat Prod 78:1816–1822
12. Chen S, Liu Y, Liu Z, Cai R, Lu Y, Huang X, She Z (2016) Isocoumarins and
benzofurans from the mangrove endophytic fungus Talaromyces ames-
tolkiae possess α‑glucosidase inhibitory and antibacterial activities. RSC
Adv 6:26412–26420
13. Cui H, Liu Y, Nie Y, Liu Z, Chen S, Zhang Z, Lu Y, He L, Huang X, She Z
(2016) Polyketides from the mangrove‑derived endophytic fungus Nec-
tria sp. HN001 and their α‑glucosidase inhibitory activity. Mar Drugs 14:86
14. Lopez D, Cherigo L, de Sedas A, Spadafora C, Martínez‑Luis S (2018)
Evaluation of antiparasitic, anticancer, antimicrobial and hypoglycemic
properties of organic extracts from Panamanian mangrove plants. Asian
Pac J Trop Med 11:32–39
15. Braun U, Crous PW, Schubert K, Shin HD (2010) Some reallocations of
Stenella species in Zasmidium. Schlechtendalia 20:99–104
16. Vlahov G (1999) Application of NMR to the study of olive oils. Prog Nucl
Magn Reson Spectrosc 35:341–357
17. Simova S, Ivanova G, Spassov SL (2003) Alternative NMR method for
quantitative determination of acyl positional distribution in triacylglycer‑
ols and related compounds. Chem Phys Lipids 126:167–176
18. Javaid K, Saad SM, Rasheed S, Moin ST, Syed N, Fatima I, Salar U, Khan KM,
Perveen S, Choudhary MI (2015) 2‑Arylquinazolin‑4(3H)‑ones: a new class
of α‑glucosidase inhibitors. Bioorg Med Chem 23:7417–7421
19. Paul VJ, Arthur KE, Ritson‑Williams R, Ross C, Sharp K (2007) Chemical
defenses: from compounds to communities. Biol Bull 213:226–251
20. Alvarez HM, Steinbüchel A (2002) Triacylglycerols in prokaryotic microor‑
ganisms. Appl Microbiol Biotechnol 60:367–376
21. Miyazawa M, Yagi N, Taguchi K (2005) Inhibitory compounds of
α‑glucosidase activity from Arctium lappa L. J Oleo Sci 54:589–594
22. Nguyen TH, Kim SM (2015) α‑glucosidase inhibitory activities of fatty
acids purified from the internal organ of sea cucumber Stichopus japoni-
cas. J Food Sci 80:H841–H847
23. Liu B, Kongstad KT, Wiese S, Jäger AK, Staerk D (2016) Edible seaweed
as future functional food: identification of α‑glucosidase inhibitors by
combined use of high‑resolution α‑glucosidase inhibition profiling and
HPLC–HRMS–SPE–NMR. Food Chem 203:16–22
24. Erickson JA, Jalaie M, Robertson DH, Lewis RA, Vieth M (2004) Lessons
in molecular recognition: the effects of ligand and protein flexibility on
molecular docking accuracy. J Med Chem 47:45–55
25. Mobley DL, Dill KA (2009) Binding of small‑molecule ligands to proteins:
“what you see” is not always “what you get”. Structure 17:489–498
26. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local
alignment search tool. J Mol Biol 215:403–410
27. Mejía LC, Castlebury LA, Rossman AY, Sogonov MV, White JF Jr (2011)
A systematic account of the genus Plagiostoma (Gnomoniaceae,
Diaporthales) based on morphology, host‑associations, and a four‑gene
phylogeny. Stud Mycol 68:211–235
28. Kato A, Ando K, Kodama K, Tamura G, Arima K (1969) Identification and
chemical properties of antitumor active monoglycerides from fungal
mycelia. Studies on antiviral and antitumor antibiotics. X. J Antibiot
29. Ichihara K, Yamaguchi C, Araya Y, Sakamoto A, Yoneda K (2010) Prepara‑
tion of fatty acid methyl esters by selective methanolysis of polar
glycerolipids. Lipids 45:367–374
30. Ichihara K, Shibahara A, Yamamoto K, Nakayama T (1996) An improved
method for rapid analysis of the fatty acids of glycerolipids. Lipids
31. López D, Cherigo L, Spadafora C, Loza‑Mejía MA, Martínez‑Luis S (2015)
Phytochemical composition, antiparasitic and α‑glucosidase inhibition
activities from Pelliciera rhizophorae. Chem Cent J 9:53
32. Copeland RA (2000) Enzymes: a practical introduction to structure,
mechanisms and data analysis. Wiley‑VCH, New York
33. Spartan’10 for Windows. Wavefunction Inc., Irvine, CA, USA
34. Ren L, Qin X, Cao X, Wang L, Bai F, Bai G, Shen Y (2011) Structural insight
into substrate specificity of human intestinal maltase‑glucoamylase.
Protein Cell 2:827–836
35. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shin‑
dyalov IN, Bourne PE (2000) The protein data bank. Nucleic Acids Res
36. Thomsen R, Christensen MH (2006) MolDock: a new technique for high‑
accuracy molecular docking. J Med Chem 49:3315–3321
37. Krieger E, Vriend G (2015) New ways to boost molecular dynamics simula‑
tions. J Comput Chem 36:996–1007
38. Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling
C (2015) ff14SB: improving the accuracy of protein side chain and back‑
bone parameters from ff99SB. J Chem Theory Comput 11:3696–3713
39. Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin
JL (1982) Brine shrimp: a convenient general bioassay for active plant
constituents. Planta Med 45:31–34
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
... Regarding AG inhibitors, compounds 6 and 7 were the only active compounds and experimentally showed to bind to a site other than the active site. Blind docking studies of compounds 6 and 7 suggested at least three alternative binding sites to the catalytic site; this agrees with studies reported by Ding et al., where several allosteric AG sites were assessed by in silico and in vitro studies, and other authors have also reported allosteric regulation by synthetic and natural compounds [50][51][52]. However, in this work, only the binding site with the highest affinities was considered for further analysis ( Figure 5). ...
... Enantiomers 7R and 7S are slightly shifted from each other; Regarding AG inhibitors, compounds 6 and 7 were the only active compounds and experimentally showed to bind to a site other than the active site. Blind docking studies of compounds 6 and 7 suggested at least three alternative binding sites to the catalytic site; this agrees with studies reported by Ding et al., where several allosteric AG sites were assessed by in silico and in vitro studies, and other authors have also reported allosteric regulation by synthetic and natural compounds [50][51][52]. However, in this work, only the binding site with the highest affinities was considered for further analysis ( Figure 5). ...
Full-text available
Digestive enzymes are currently considered important therapeutic targets for the treatment of obesity and some associated metabolic diseases, such as type 2 diabetes. Piper cumanense is a species characterized by the presence of bioactive constituents, particularly prenylated benzoic acid derivatives. In this study, the inhibitory potential of chemical constituents from P. cumanense and some synthesized compounds was determined on digestive enzymes (pancreatic lipase (PL) and α-glucosidase (AG)). The methodology included isolating and identifying secondary metabolites from P. cumanense, synthesizing some analogs, and a molecular docking study. The chemical study allowed the isolation of four prenylated benzoic acid derivatives (1–4). Four analogs (5–8) were synthesized. Seven compounds were found to significantly inhibit the catalytic activity of PL with IC50 values between 28.32 and 55.8 µM. On the other hand, only two compounds (6 and 7) were active as inhibitors of AG with IC50 values lower than 155 µM, standing out as the potential multitarget of these chromane compounds. Enzyme kinetics and molecular docking studies showed that the bioactive compounds mainly interact with amino acids other than those of the catalytic site in both PL and AG. This work constitutes the first report on the antidiabetic and antiobesity potential of substances derived from P. cumanense.
... Saad [105] isolated endophytic fungi from root samples of Malva parviflora and leaf samples of Chenopodium album, Pelargonium graveolens and Melia azedarach. Nine fungi presented bioactivity and were identified using DNA-sequences, with five being isolated from C. album: Fusarium chlamydosporum, A. alternata saad5 MG786542, A. alternata saad8 MG786545, Fusarium oxysporum and Phoma sp. ...
Full-text available
Mangroves are ecosystems with unique characteristics due to the high salinity and amount of organic matter that house a rich biodiversity. Fungi have aroused much interest as they are an important natural source for the discovery of new bioactive compounds, with potential biotechnological and pharmacological interest. This review aims to highlight endophytic fungi isolated from mangrove plant species and the isolated bioactive compounds and their bioactivity against protozoa, bacteria and pathogenic viruses. Knowledge about this type of ecosystem is of great relevance for its preservation and as a source of new molecules for the control of pathogens that may be of importance for human, animal and environmental health.
... The reduction of postprandial hyperglycemia by inhibiting carbohydrate hydrolyzing enzymes in gastrointestinal tract is one of the promising approaches for management of diabetes [4,5]. α-Amylase is involved in hydrolyzing long chain of starch and α-glucosidase release glucose into the small intestine by breaking down oligosaccharides and disaccharides [2,6]. α-Glucosidase and α-amylase inhibitors reduced postprandial blood glucose level by delaying the hydrolysis of carbohydrate by inhibiting the digestive enzymes [7]. ...
Full-text available
A series of 2-chloro-5-[(4-chlorophenyl)sulfamoyl]-N-(alkyl/aryl)-4-nitrobenzamide derivatives (5a-5v) has been synthesized and confirmed by physicochemical(Rf, melting point) and spectral means (IR, 1HNMR, 13CNMR). The results of in vitro antidiabetic study against α-glucosidase indicated that compound 5o bearing 2-CH3-5-NO2 substituent on phenyl ring was found to be the most active compound against both enzymes. The electron donating (CH3) group and electron withdrawing (NO2) group on a phenyl ring highly favoured the inhibitory activity against these enzymes. The docking simulations study revealed that these synthesized compounds displayed hydrogen bonding, electrostatic and hydrophobic interactions with active site residues. The structure activity relationship studies of these compounds were also corroborated with the help of molecular modeling studies. Molecular dynamic simulations have been done for top most active compound for validating its α-glucosidase and α-amylase inhibitory potential, RMSD analysis of ligand protein complex suggested the stability of top most active compound 5o in binding site of target proteins. In silico ADMET results showed that synthesized compounds were found to have negligible toxicity, good solubility and absorption profile as the synthesized compounds fulfilled Lipinski's rule of 5 and Veber's rule.
... strain EM5À10 and analyzed for in vitro α-glucosidase inhibitory activity and compared with standard tripalmitin. When compared to the standard drug acarbose, tripalmitin and fungal triglycerides showed a better inhibition rate (Lopéz et al., 2019). ...
This study deals with the assessment of mangrove floral diversity at three major districts of Odisha: Kendrapara, Jagatsinghpur, and Bhadrak, which constitute major parts of state mangrove vegetation. In these three districts, the study sites included Mahanadi mangrove wetland (MMW) of Kendrapara, the river mouth regions of the river Devi (DRM) and Baitarani (BRM) in Jagatsinghpur and Bhadrak district, respectively, and the Baitarani river bank (BRB) region of Bhadrak district. The total area of study included 1 ha in MMW, 0.5 ha in DRM, and 0.1 ha each in BRM and BRB. A total of 63 species of plant were found to be distributed within the study sites that included 27 trees, 13 herbs, 17 shrubs, 5 climbers, and 1 creeper. Among trees, Avicennia marina came out as the dominant species for BRM (important value index-IVI=70.33) and MMW (IVI=117.308), while Avicennia alba was dominant in DRM (IVI=108.33), and Sonneratia caseolaris was dominant in BRB (IVI=137.40). The highest diversity was found at MMW (Shannon diversity index=0.79±0.38 and Simpson’s index=0.42±0.22), and the least diversity was found at BRB (Shannon diversity index=0.76±0.08 and Simpson’s index=1.19±0.15), but the evenness was highest in BRB (0.63±0.06) followed by MMW (0.48±0.15) and lowest in DRM (0.38±0.13). The soil parameters of the study sites showed variations with a mean pH ranging from 5.1 in BRM to 7.12 BRB, electrical conductivity from 0.01 to 0.05 S/m and organic carbon from 0.51% in BRB to 1.97% in DRM. This study will act as supplement information for mangrove vegetation of Odisha, which may be implemented further for the conservation and management of mangroves on the east coast of India.
The culturable fungal endophytes of mangrove plants so far investigated appear to be not distinct from those associated with the terrestrial plants. The pattern of distribution of endophytes in a leaf and its species composition is similar in plants of mangrove and other ecosystems. This reflects the ecological success of a few fungal species to lead an endophytic life in plants of different environment and taxonomic affiliation. Despite this commonality, endophytes of mangroves are distinct in possessing certain traits which enable them to survive in the harsh mangrove environment. Their ability to produce novel bioactive compounds and enzymes make them attractive candidates for bioprospecting. Considering the endophyte-mediated improvement of performance of plants of other ecosystems, more studies are needed on mangrove endophytes addressing their role in abiotic and biotic stress tolerance of mangroves. Their functions in mangrove ecosystem including litter degradation and nutrient recycling, as well as their enzyme arsenal and secondary metabolite spectrum, need to be studied in detail in order to improve our understanding of these unique plant endosymbionts. Information gleaned on these aspects may aid in the protection and restoration of deteriorating mangrove vegetation.
The present study has been aimed to investigate the antioxidant and antidiabetic activities of Christia vespertilionis leaves extracts. The analysis of the extracts obtained using different solvents revealed that ethyl acetate: methanol extract as the most potential in 2, 2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging while hexane: ethyl acetate exhibited highest ferric reducing antioxidant power (FRAP) capacity and α-glucosidase inhibition. The infrared analysis displayed the presence of O-H, C-H, C=O, C=C, C-O, and N-H predominantly in the extracts reflecting some of the compounds reported previously; quercetin 3-O-glucoside, oleanolic acid methyl ester, β-sitosterol, stigmasterol, geraniol and 2’-hydroxygenistein. The latter two displayed competitive mode of inhibition in both the yeast and human protein receptors. Conclusively, C. vespertilionis leaves contain potent antioxidants and α-glucosidase inhibitors; thus, further studies can enhance its use in pharmaceutical applications.
Full-text available
In recent years the prevalence of diabetes has increased globally and by 2040 the number of diabetic people has been estimated to increase to 642 million. Various classes of drugs are available to treat Type ll diabetes. However, these drugs are associated with certain side effects. -Glucosidase is an intriquing target enzyme to treat Type II diabetes, and -glucosidase inhibitors (AGIs) are considered as first-line drugs for Type ll patients. Fungi, in general, produce natural products with some amazing chemical diversity and many fungal metabolites have illustrated a wide range of biological and pharmacological effects. In this review the focus is on describing the -glucosidase effects and their potential as anti-diabetic agents of various metabolites isolated from fungi.
Full-text available
Objective: To investigate 33 organic extracts of mangrove plants for: antiparasitic, anticancer, and antibacterial activities, as well as their ability to inhibit the activity of the ?-glucosidase enzyme. Methods: Leaves from all different plant mangrove species located in five mangrove zones of the Pacific coast of Panama were collected according to standard procedures. Qualitative phytochemical analysis of the organic extracts was performed by thin layer chromatography. The antiparasitic activity against Plasmodium falciparum, Trypanosoma cruzi and Leishmania donovani, toxicity against Artemia salina, anticancer activity in MCF-7 cell line, and antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa of all organic extract were investigated according protocols stablished in our institution. Finally, the ability to inhibit the enzymatic activity of ?-glucosidase was evaluated by monitoring the hydrolysis of p-nitrophenyl ?-D-glucopyranoside. Results: Thirty-three different samples belonging to nine different species of vascular plants with seeds of true mangroves were collected. Triterpenoids, phenolics, and tannins were the main groups of compounds found in the sampled mangroves. Saponins, quinones, and coumarins were found in less than 50% of the samples. Laguncularia racemosa showed moderate activity against Plasmodium falciparum. None of the extracts presented anticancer activity. Rhizophora mangle exhibited potent activity against Staphylococcus aureus and Bacillus subtilis [(90.41�7.33)% and (96.02�6.14)% of inhibition]; Avicennia germinans and Conocarpus erectus had activity against Escherichia coli [(71.17�6.15)% and (60.60�5.13)% of inhibition, respectively]. About 60% of the mangroves showed ?-glucosidase inhibitory activity. In particular, extracts from Laguncularia racemosa, Pelliciera rhizophorae, Conocarpus erectus, Mora oleifera, and Tabebuia palustris species showed ?-glucosidase inhibitory potential, with IC50 values of (29.45�0.29), (20.60�0.70), (730.06�3.74), (25.59�0.37), and (853.39�5.30) �g/mL, respectively. Conclusions: Panamanian mangroves are mainly a promising potential source of hypoglycemic compounds, specifically ?-glucosidase inhibitors. These results highlight the therapeutic virtues of extracts from American mangrove plants. � 2018 by the Asian Pacific Journal of Tropical Medicine. All rights reserved.
Full-text available
Four new polyketides: nectriacids A-C (1-3) and 12-epicitreoisocoumarinol (4), together with three known compounds: citreoisocoumarinol (5), citreoisocoumarin (6), and macrocarpon C (7) were isolated from the culture of the endophytic fungus Nectria sp. HN001, which was isolated from a fresh branch of the mangrove plant Sonneratia ovata collected from the South China Sea. Their structures were determined by the detailed analysis of NMR and mass spectroscopic data. The absolute configuration of the stereogenic carbons for compound 4 was further assigned by Mosher's ester method. All of the isolated compounds were tested for their α-glucosidase inhibitory activity by UV absorbance at 405 nm, and new compounds 2 and 3 exhibited potent inhibitory activity with IC50 values of 23.5 and 42.3 μM, respectively, which were more potent than positive control (acarbose, IC50, 815.3 μM).
Full-text available
Six new isocoumarins, compounds 1−4 and 14−15, two new benzofurans, 16−17, along with nine known isocoumarin analogues, 5−13 were obtained from the mangrove endophytic fungus Talaromyces amestolkiae YX1. Their structures were elucidated by analysis of spectroscopic data. The absolute configuration of compounds 4, 14 and 15 were determined by the modified Mosher’s method and comparison of their CD spectra with dihydroisocoumarins described in the literature. Structure of compound 5 was further confirmed by a single-crystal X-ray diffraction experiment using Cu Kα radiation. Compounds 2, 6, 8, and 10 showed α-glucosidase inhibitory activity with IC50 of 89.4, 17.2, 36.4, and 38.1 μM, respectively. In antibacterial assay, compounds 16 and 17 exhibited antibacterial activities with the MIC values between 25–50 μg/mL against the Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Bacillus subtilis.
Full-text available
Background: Panama has an extensive mangrove area and it is one of the countries with the highest biodiversity in America. Mangroves are widely used in traditional medicine, nevertheless, there are very few studies that validates their medicinal properties in America. Given the urgent need for therapeutic options to treat several diseases of public health importance, mangrove ecosystem could be an interesting source of new bioactive molecules. This study was designed to evaluate the potential of Pelliciera rhizophorae as a source of bioactive compounds. Results: The present investigation was undertaken to explore the possible antiparasitic potential and α-glucosidase inhibition by compounds derived from the Panamanian mangrove Pelliciera rhizophorae. Bioassay-guided fractionation of the crude extract led to the isolation of ten chemical compounds: α-amyrine (1), β-amyrine (2), ursolic acid (3), oleanolic acid (4), betulinic acid (5), brugierol (6) iso-brugierol (7), kaempferol (8), quercetin (9), and quercetrin (10). The structures of these compounds were established by spectroscopic analyses including APCI-HR-MS and NMR. Compounds 4 (IC50 = 5.3 µM), 8 (IC50 = 22.9 µM) and 10 (IC50 = 3.4 µM) showed selective antiparasitic activity against Leishmania donovani, while compounds 1 (IC50 = 19.0 µM) and 5 (IC50 = 18.0 µM) exhibited selectivity against Tripanosoma cruzi and Plasmodium falciparum, respectively. Moreover, compounds 1-5 inhibited α-glucosidase enzyme in a concentration-dependent manner with IC50 values of 1.45, 0.02, 1.08, 0.98 and 2.37 µM, respectively. Their inhibitory activity was higher than that of antidiabetic drug acarbose (IC50 217.7 µM), used as a positive control. Kinetic analysis established that the five compounds acted as competitive inhibitors. Docking analysis predicted that all triterpenes bind at the same site that acarbose in the human intestinal α-glucosidase (PDB: 3TOP). Conclusions: Three groups of compounds were isolated in this study (triterpenes, flavonols and dithiolanes). Triterpenes and flavones showed activity in at least one bioassay (antiparasitic or α-glucosidase). In addition, only the pentacyclic triterpenes exhibited a competitive type of inhibition against α-glucosidase.
Full-text available
We describe a set of algorithms that allow to simulate dihydrofolate reductase (DHFR, a common benchmark) with the AMBER all-atom force field at 160 nanoseconds/day on a single Intel Core i7 5960X CPU (no graphics processing unit (GPU), 23,786 atoms, particle mesh Ewald (PME), 8.0 Å cutoff, correct atom masses, reproducible trajectory, CPU with 3.6 GHz, no turbo boost, 8 AVX registers). The new features include a mixed multiple time-step algorithm (reaching 5 fs), a tuned version of LINCS to constrain bond angles, the fusion of pair list creation and force calculation, pressure coupling with a "densostat," and exploitation of new CPU instruction sets like AVX2. The impact of Intel's new transactional memory, atomic instructions, and sloppy pair lists is also analyzed. The algorithms map well to GPUs and can automatically handle most Protein Data Bank (PDB) files including ligands. An implementation is available as part of the YASARA molecular modeling and simulation program from © 2015 The Authors Journal of Computational Chemistry Published by Wiley Periodicals, Inc. © 2015 The Authors Journal of Computational Chemistry Published by Wiley Periodicals, Inc.
Crude chloroform, ethanol and acetone extracts of nineteen seaweed species were screened for their antioxidant and α-glucosidase inhibitory activity. Samples showing more than 60% α-glucosidase inhibitory activity, at a concentration of 1 mg/ml, were furthermore investigated using high-resolution α-glucosidase inhibition profiling combined with high-performance liquid chromatography - high-resolution mass spectrometry - solid-phase extraction - nuclear magnetic resonance spectroscopy (HR-bioassay/HPLC-HRMS-SPE-NMR). The results showed Ascophyllum nodosum and Fucus vesicolosus to be rich in antioxidants, equalling a Trolox equivalent antioxidant capacity of 135 and 108 mM trolox mg-1 extract, respectively. HR-bioassay/HPLC-HRMS-SPE-NMR showed the α-glucosidase inhibitory activity of A. nodosum, F. vesoculosus, Laminaria digitata, L. japonica and Undaria pinnatifida to be caused by phlorotannins as well as fatty acids - with oleic acid, linoleic acid and eicosapentaenoic acid being the most potent with IC50 values of 0.069, 0.075 and 0.10 mM, respectively, and showing a mixed-type inhibition mode.
Twenty-five derivatives of 2-arylquinazolin-4(3 H )-ones ( 1 – 25 ) were evaluated for their yeast (Saccharomyces cerevisiae) α-glucosidase inhibitory activities. All synthetic compounds, except 1 and 6 , were found to be several hundred fold more active (IC 50 values in the range of 0.3 ± 0.01–117.9 ± 1.76 μM), than the standard drug, acarbose (IC 50 = 840 ± 1.73 μM). The enzyme kinetic studies on the most active compounds 12 , 4 , 19 , and 13 were performed for the determination of their modes of inhibition and dissociation constants K i . Study of the modes of inhibition of compounds 12 , and 4 were also performed using molecular modeling techniques. In brief, current study identifies a novel class of α-glucosidase inhibitors which can be further studied for the treatment of hyperglycemia and obesity.
Molecular mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Average errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a physically motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reproduction of NMR χ1 scalar coupling measurements for proteins in solution. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.
Five new compounds, pinazaphilones A and B (1, 2), two phenolic compounds (4, 5), and penicidone D (6), together with the known Sch 1385568 (3), (±)-penifupyrone (7), 3-O-methylfunicone (8), 5-methylbenzene-1,3-diol (9), and 2,4-dihydroxy-6-methylbenzoic acid (10) were obtained from the culture of the endophytic fungus Penicillium sp. HN29-3B1, which was isolated from a fresh branch of the mangrove plant Cerbera manghas collected from the South China Sea. Their structures were determined by analysis of 1D and 2D NMR and mass spectroscopic data. Structures of compounds 4 and 7 were further confirmed by a single-crystal X-ray diffraction experiment using Cu Kα radiation. The absolute configurations of compounds 1-3 were assigned by quantum chemical calculations of the electronic circular dichroic spectra. Compounds 2, 3, 5, and 7 inhibited α-glucosidase with IC50 values of 28.0, 16.6, 2.2, and 14.4 μM, respectively, and are thus more potent than the positive control, acarbose.