α-Glucosidase Inhibitor Isolated from Blechum pyramidatum

Abstract

Blechum pyramidatum (Lam.) Urb. is a species of extensive medicinal use in the American continent. In fact, antidiabetic and anticancer preparations from this plant have been patented in Mexico, even though their active constituents are not yet known. It was recently discovered that B. pyramidatum inhibits the action of the -glucosidase enzyme, thereby corroborating the antidiabetic properties attributed to this plant. The primary purpose of this study was to identify and characterize the -glucosidase inhibitors from this species. Bioassay-guided fractionation of a crude extract of B. pyramidatum led to the isolation of a main - glucosidase inhibitor, Palmitic acid (IC50 237.5). This compound was identified by both spectroscopic and spectrometric analysis. Its inhibitory activity was similar to that of the antidiabetic drug acarbose (IC50 241.6 µM), which was used as a positive control in our bioassay. Kinetic analysis established that palmitic acid acted as a competitive inhibitor. Docking analysis predicted that this compound binds to the same site as acarbose does in the human intestinal α‑glucosidase (PDB: 3TOP). The presence of palmitic acid in B. pyramidatum and its potent inhibitory activity against α‑glucosidase enzyme provides solid evidence to support the antidiabetic use of this plant in traditional medicine.

Full text

α-Glucosidase Inhibitor Isolated from Blechum pyramidatum
Lilia Cherigoa and Sergio Martínez-Luisb,*
aDepartamento de Química Orgánica, Facultad de Ciencias Exactas y Tecnología, Universidad de Panamá,
P.O. Box 3366, Panamá, República de Panamá
bCentro de Biodiversidad y Descubrimiento de Drogas, Instituto de Investigaciones Científicas y Servicios
de Alta Tecnología (INDICASAT AIP), Edificio 219, Ciudad del Saber, Apartado 0843-01103, Panamá,
República de Panamá
smartinez@indicasat.org.pa
Received: January 25th, 2018; Accepted: February 28th, 2018
Blechum pyramidatum (Lam.) Urb. is a species of extensive medicinal use in the American continent. In fact, antidiabetic and anticancer preparations from this
plant have been patented in Mexico, even though their active constituents are not yet known. It was recently discovered that B. pyramidatum inhibits the action
of the -glucosidase enzyme, thereby corroborating the antidiabetic properties attributed to this plant. The primary purpose of this study was to identify and
characterize the -glucosidase inhibitors from this species. Bioassay-guided fractionation of a crude extract of B. pyramidatum led to the isolation of a main -
glucosidase inhibitor, Palmitic acid (IC50 237.5). This compound was identified by both spectroscopic and spectrometric analysis. Its inhibitory activity was
similar to that of the antidiabetic drug acarbose (IC50 241.6 µM), which was used as a positive control in our bioassay. Kinetic analysis established that
palmitic acid acted as a competitive inhibitor. Docking analysis predicted that this compound binds to the same site as acarbose does in the human intestinal
α‑glucosidase (PDB: 3TOP). The presence of palmitic acid in B. pyramidatum and its potent inhibitory activity against α‑glucosidase enzyme provides solid
evidence to support the antidiabetic use of this plant in traditional medicine.
Keywords: Blechum pyramidatum, -Glucosidase inhibition, Kinetic analysis, Docking analysis.
Medicinal plants have been systematically used since ancient times,
especially in countries where significant ancestral cultures
flourished [1]. Despite today’s technological advances, medicinal
plants continue to be a widely used resource to prevent and treat
human diseases. Recently, the World Health Organization estimated
that approximately three-quarters of the world’s population trust
and utilize plants for their health care needs [2]. Faced with this
panorama, scientists from all over the world must continue to
generate better information to develop recommendations for
effective and safe uses of many medicinal plants.
Blechum pyramidatum (Lam.) Urb. is a species of wide medicinal
use in different countries of the American continent. Cooking of the
leaves is prescribed to treat skin problems [3]. A decoction of this
plant can be administered as a diuretic [4] and as an antiemetic [5].
In some places, this plant is applied to treat snakebites [6]. In
Mexico, the antidiabetic and anticancer uses of this plant have been
patented, although the active compounds have never been isolated
nor identified [7]. Therefore, there is an urgent need to identify the
active components of this plant to guarantee its quality and safe use.
In a preliminary study, we detected that the organic extract from
this plant showed significant inhibitory activity against the α-
glucosidase enzyme, although it was not possible for us to fully
identify the component(s) responsible for that activity [8]. The latter
was partly due to the presence of large amounts of chlorophyll and
other related compounds, which interfered with the bioactivity
results we obtained in the initial extraction of this plant. Still,
obtained results suggests that inhibition of -glucosidase enzyme is
part of antidiabetic mechanisms from this plant.
It is important to point out that conventional bioassays performed to
detect -glucosidase activity typically quantify the enzymatic
activity by measuring the absorbance of the reaction mixture at 400
nm [9a-c]. Chlorophyll and related compounds also exhibit intense
absorption peaks at this same wavelength. Therefore, the removal of
these types of compounds prior to chemical studies is essential to
obtain more accurate results in detecting -glucosidase bioactivity.
For this reason, we proceeded with the removal of chlorophyll and
related compounds from the initial crude extract by using activated
carbon to avoid interferences in the enzymatic readings.
The aerial parts of B. pyramidatum were macerated with a mixture
of CHCl3-MeOH (1:1). Subsequently, this mixture was blended
with activated carbon, which was filtered and concentrated to obtain
the crude extract. This extract inhibited 69.4 % of the -glucosidase
activity at a final concentration of 6.25 mg/mL. Afterward, the
organic extract was dissolved in a hydro- methanol mixture (20:80)
and successively partitioned by two solvents, hexane and ethyl
acetate, to obtain three major fractions with increasing polarity. Of
these three fractions, only the hexane fraction showed promising
activity (80.1 % of enzyme inhibition at 6.25 mg/mL). Bioassay-
guided fractionation of the hexane fraction allowed the isolation of
palmitic acid (compound 1), as the main active compound from B.
pyramidatum. This compound was identified by spectroscopic
analyses including ESI-HR-MS and NMR (1H, 13C, DEPT 135,
DEPT 90, COSY, NOESY, HMBC and HMQC) [8, 10].
Compound 1 inhibited the α-glucosidase enzyme in a concentration-
dependent manner with an IC50 value of 237.5 µM therefore
showing similar potency to acarbose (positive control, IC50 241.6
µM). During -glucosidase evaluations, we initially found that
palmitic acid only showed moderate activity, which did not
correlate with the activity detected in the hexane fraction through
which this compound was isolated. This fact allowed us to
recognize that palmitic acid had poor solubility in DMSO. There
have been previous reports about -glucosidase inhibition by fatty
acids, including palmitic and oleic acids, with DMSO being
regularly used to solubilize samples as part of standard protocols to
NPC Natural Product Communications 2018
Vol. 13
No. 4
461 - 464

462 Natural Product Communications Vol. 13 (4) 2018 Cherigo & Martínez-Luis et al.
detect bioactivity [11a-c]. Our evidence suggested that a re-
evaluation of the inhibition of the α-glucosidase enzyme by palmitic
acid in other solvents was necessary to both determine whether
there was an effect by the reduced solubility of palmitic acid in
DMSO and to compare our findings to previously reported
enzymatic activity. To find a solvent which would allow us to get
more reliable results for palmitic acid activity - and, in general, for
low polar compounds - we proceeded to evaluate the inhibition of
-glucosidase by some common solvents in different quantities,
including ethanol, methanol, propanol, and isopropanol. Through
these tests, we detected that ethanol in small volumes only slightly
decreased the α-glucosidase activity and that palmitic acid was
solubilized better than with DMSO.This finding allowed us to
obtain more real inhibition results for low polarity compounds.
We proceeded to evaluate the activity of palmitic acid using ethanol
to compare to the results we obtained with DMSO. We also
assessed oleic acid since this compound has similar solubility
properties to those of palmitic acid, and we also re-evaluated the
positive control, acarbose (IC
50
217.7 µM in DMSO). As a result,
oleic acid showed potent α-glucosidase inhibitory activity (IC
50
38.9
µM), even better than palmitic acid, and acarbose showed similar
potency to that displayed using DMSO (IC
50
241.6 µM in ethanol).
This evidence suggests that reported data about -glucosidase
inhibition by certain low polar compounds could be inaccurate due
to their poor solubility DMSO which is used in most standard
protocols.
To obtain further evidence of the nature of the interaction of
palmitic (1) and oleic acids (2) with α-glucosidase, we carried out
kinetic analyses. Lineweaver–Burk plots [12a] were constructed
using different concentrations of substrate and fatty acids 1–2
(Figure 1). The results in Figure 2 and Figure 3 indicated that
compounds 1–2 showed typical reversible competitive plots, with
series of lines having the same y-intercept as the enzyme without
inhibitors. These results suggested that compounds 1–2 bind to α-
glucosidase or to the substrate-enzyme complex. Acarbose also
behaved as a competitive inhibitor [12a]. These results show that
both fatty acids are potent competitive inhibitors of the α-
glucosidase enzyme.
Figure 1: (1) Palmitic acid and (2) Oleic acid.
Taking into consideration the results of the kinetic analysis
performed, we conducted a molecular docking study to evaluate the
putative binding mode of fatty acids 1–2 into the human intestinal
α-glucosidase (PDB: 3TOP). Results indicate that fatty acids bind
mainly through hydrophobic interactions. Figure 4 shows the
superposition of docking poses of compounds 1–2 and acarbose in
the binding site. It is interesting to note that despite the analyzed
fatty acids being mainly hydrophobic they bind to the same site as
acarbose, which is a more polar compound compared to fatty acids
1–2. As expected, acarbose interacts with the binding site through
many hydrogen bonds (Figure 5a) and compounds 1–2 interact
mainly via hydrophobic interactions (Figure 5b). However, palmitic
acid interacts with Asp 1526 and Arg 1582 through the formation of
hydrogen bonds while oleic acid weakly interacts with Lys 1460 by
the formation of hydrogen bonds with hydroxyl groups (Figures 6).
Finally, in the results of the docking analysis, it is clearly observed
that the oleic acid pose fits better than the palmitic acid pose in the
Figure 2: Lineweaver–Burk plot of -glucosidase inhibition at different concentrations
of substrate and palmitic acid.
Figure 3: Lineweaver–Burk plot of -glucosidase inhibition at different
concentrations of substrate and oleic acid.
Figure 4: Superposition of docking poses of compounds 1(in green), 2 (in blue) and
acarbose (in red).
-glucosidase receptor site. This finding suggests that the three-
dimensional arrangement due to the presence of the double bond is
responsible for the better activity displayed by oleic acid.
In summary, palmitic acid was isolated from the B. pyramidatum
active fraction against -glucosidase. Even though this compound
was previously reported as a weak inhibitor of -glucosidase, our
data suggests that the use of a polar solvent (DMSO) in the bioassay
conditions has produced inaccurate results, especially for low
polarity compounds. Oleic acid showed higher activity than both
palmitic acid and acarbose. These compounds exhibited a
competitive type of inhibition against S. cerevisiae α-glucosidase.
Therefore, this fatty acid-rich plant might also be an interesting
alternative for reducing blood sugar level in people affected by DM.

Palmitic acid as -glucosidase inhibitor Natural Product Communications Vol. 13 (4) 2018 463
Figure 6: Comparison between the interaction of Palmitic acid (a), Oleic acid (b) and acarbose (c) with a-glucosidase active site.
Experimental
General Experimental Procedures: NMR spectra were acquired on
Jeol Eclipse 400 MHz spectrometer (referenced to 
H
7.26, 
C
77.0
for CDCl
3
). APCIHR-MS were acquired on a JEOL LC-mate mass
spectrometer. The purification of the compounds was carried out on
Agilent 1100 HPLC system equipped with a quaternary pump, a
diode array detector, and a normal phase silica gel column
(Phenomenex Luna, 4.6 mm × 100 mm, 5 μm) at a flow rate of 1
mL/min. Column chromatography was used with silica gel 60 (70-
230 mesh, Merck). TLC (analytical) was performed on pre-coated
silica gel 60 F254 plates (Merck). All solvents were HPLC grade
and used without further purification.
Plant material and extract preparation: B. pyramidatum
(Acanthaceae) was collected in Santa Clara, Chiriquí, in the
Republic of Panama. This plant was identified by Jorge Lezcano
and was deposited to the Herbarium at the University of Panama.
The aerial plant parts were dried at room temperature for a week
and then were grounded for further study. This material (50.0 g)
was extracted by maceration at room temperature with a mixture of
CHCl
3
-MeOH (1:1). The solvent mixture was mixed with activated
carbon (charcoal; 5 g) and stirred for 20 minutes at room
temperature. The solution was filtered through celite in a Büchner
funnel. The resulting solution was evaporated under reduced
pressure to remove the solvent to leave a semi-solid paste (4.6 g).
Chemical studies: The crude extract was subjected to bioassay-
guided fractionation in which the extract was re-suspended in a
mixture of water-MeOH 70:30 and sequential partitions were made
with Hexanes [Hex] (5×200 mL) and ethyl acetate [AcoEt] (5×200
mL). Each obtained fraction, including the final hydromethanol
fraction, was evaporated to dryness and then subjected to enzymatic
assay. Hex fraction (active, 1.61 g) was fractionated by column
chromatography on silica gel (35 g). The column was eluted with
Hex, followed by a gradient of Hex:EtOAc (1:0→0:1) and finally
with a gradient of EtOAc:MeOH (1:0→1:1). Altogether, 109
fractions (25 ml each) were collected and combined according to
their TLC profiles to yield nine primary fractions (FA to FI), which
were re-evaluated against -glucosidase and the activity was
presented in fraction FA [eluted with 100% Hex]. Fraction FA (0.31
g) was further subjected to silica gel column chromatography and
eluted with a gradient of Hex:EtOAc (1:0→0:1). This process led to
six fractions (FA-1 to FA-6), which were also evaluated. Fraction
FA-2 was eluted with Hex:EtOAc (9:1) to afford 3.1 mg of
compound 1, which was the compound responsible for the activity.
The other fractions were also evaluated but did not show an
inhibitory effect in vitro against the enzyme -glucosidase.
Alpha-glucosidase inhibitory assay: The -glucosidase inhibitory
assay was performed according to Chan and collaborators [9a], with
modifications [12a and b]. α-Glucosidase from baker’s yeast was
purchased from Sigma Chemical Co. The inhibition was measured
spectrophotometrically at pH 7.0 and 37°C employing 2 mM p-
nitrophenyl α-
D
-glucopyranoside (PNP-G) as a substrate and 32
mU/mL of the enzyme, in 100 mM potassium phosphate buffer
(enzyme stock). Acarbose was dissolved in phosphate buffer, and
serial dilutions were prepared (in order to obtain the IC
50
) and
employed as positive control. The 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, plant extract or isolated compound solution (in DMSO or
Etanol) were incubated for 7 min with 150 µL of enzyme stock at
37°C. After incubating, 150 µL of substrate was added and further
incubated for 20 min at 37°C. All assays were performed in 96-well
microplates (Greiner bio-one 655101) in triplicate. The activity of
samples was calculated as a percentage in comparison to a control
according to the following equation:
Fi
g
ure 5: Dockin
g
p
oses of com
p
ound 1
(
a
)
and com
p
ound 2
(
b
)
. H-
b
ond interactions are hi
g
hli
g
hted.
(b)
(a)

464 Natural Product Communications Vol. 13 (4) 2018 Cherigo & Martínez-Luis et al.
%

󰇛∆ ∆󰇜∆


100%
The concentration required to inhibit activity of the enzyme by 50%
(IC50) was calculated by regression analysis [13].
Kinetics of α-glucosidase inhibition: Fixed amounts of -
glucosidase were incubated with increasing concentrations of PNPG
at 37C for 15 min, in the absence or presence of inhibitors
(concentration equivalent to their IC50). Reactions were terminated,
and absorption was measured and analyzed by Lineweaver–Burk
plot. All the determinations were performed in triplicate.
Statistical analysis: The data were expressed as the mean ± SD of
three replicates. The analysis was performed using Excel 2013.
One-way analysis of variance (ANOVA) and Tukey posttest were
used to evaluate the possible differences among the means. p values
≤ 0.05 were considered as significant differences.
Docking Study: Ligands were constructed in Spartan’10 [14], and
their geometry was optimized using MMFF force field. A protein-
ligand docking study was carried out based on the crystal structures
for C- terminal domain of human intestinal α-glucosidase (PDB:
3TOP) [15], which was retrieved from the Protein Data Bank [16].
Before docking, all of the solvent molecules and the co-crystallized
ligand were removed. Molecular docking calculations were
performed using Molegro Virtual Docker v. 6.0.1 [17]. A sphere of
12 Å radius was centered in the binding site for searching.
Experimental data indicates palmitic and oleic acids are competitive
inhibitors; thus, the active site was chosen as the binding site.
Protonation states and assignments of the charges on each protein
were based on standard templates of the Molegro Virtual Docker
program, and no other charges were necessary to set. Flexible
ligand model was used in the docking and subsequent optimization
scheme. Different orientations of the ligands were searched and
ranked based on their energy scores. The RMSD threshold for
multiple cluster poses was set to <1.00 Å. The docking algorithm
was set to 5000 maximum iterations with a simplex evolution
population size of 100 and a minimum of 50 runs for each ligand.
After docking, some further scores were calculated including the
binding affinity (MolDock Score) and re-ranking score (Rerank
Score). The re-ranking score utilizes a more advanced scoring
scheme than that used during docking and is often more useful for
accurate ranking of the poses. Poses with lower score were selected
for further analysis. To assess the efficacy of this procedure for
finding low energy solutions, we docked the co-crystallized ligand
(acarbose).
Acknowledgments - We would like to thank the government of
Panama for granting permission to make plant collections. Lilia
Cherigo and Sergio Martinez-Luis were supported by funds from
the National Research System of SENACYT [SNI-147-2016 and
SNI-145-2016, respectively]; Michelle Flores for her technical
support in some -glucosidase evaluations; and Alberto E. Morales
(University of California, Irvine) for language and technical editing.
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