Research Article

Mikania glomerata: Phytochemical, Pharmacological, and

Neurochemical Study
Lorena C. L. R. Santana,
1,2
Maria R. M. Brito,
1
George L. S. Oliveira,
1
Antnia M. G. L. Cit,
1,3
Clayton Q. Alves,
4
Juceni P. David,
5
Jorge M. David,
6
and Rivelilson M. de Freitas
1,2
1
Departamento de Farm acia, Laborat orio de Pesquisa em Neuroqumica Experimental do Programa de
P os-Graduac ao em Ci encias Farmac euticas, Universidade Federal do Piau, 64049-550 Teresina, PI, Brazil
2
Programa de P os-Graduac ao em Biotecnologia (RENORBIO), Universidade Federal do Piau, 64049-550 Teresina, PI, Brazil
3
Departamento de Qumica, Centro de Ci encias da Natureza, Universidade Federal do Piau, Campus Universit ario Ministro
Petr onio Portella, Bairro Ininga, 64049-550 Teresina, PI, Brazil
4
Departamento de Ci encias Exatas, Universidade Estadual de Feira de Santana, 44031-460 Feira de Santana, BA, Brazil
5
Faculdade de Farm acia, Universidade Federal da Bahia, 40170-290 Salvador, BA, Brazil
6
Instituto de Qumica ,Universidade Federal da Bahia, 40170-290 Salvador, BA, Brazil
Correspondence should be addressed to Rivelilson M. de Freitas; [email protected]
Received 9 March 2014; Accepted 1 July 2014; Published 18 August 2014
Academic Editor: MinKyun Na
Copyright 2014 Lorena C. L. R. Santana et al. Tis is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Te present study primarily aims to identify the relative density and the fatty acids (methyl esters) content present in the
standardized ethanol extract of leaves of M. glomerata (EPMG). Meanwhile, in a second moment, this study evaluated the efects
of the EPMG on the levels of amino acids in the hippocampus, and the mechanism of sedative and anxiolytic action. Adult mice
were treated with doses of 200, 300, and 400 mg/kg and evaluated in open feld, elevated plus-maze, light dark, and rotarod tests.
Moreover, in the behavioral tests diazepam (GABAergic anxiolytic, 2 mg/kg) as positive control and fumazenil (GABA antagonist,
2.5 mg/kg) were used to identify mechanism of sedative and anxiolytic action produced by EPMG. Te EPMG is constituted by
the following compounds: methyl cinnamate, 2H-1-benzopyran-2-one, (2-hydroxyphenyl)methyl propionate, (Z)-methyl-hexadec-
7-enoate, methyl hexadecanoate, hexadecanoic acid, (Z)-methyl-octadec-9-enoate, octadecanoic acid, and squalene. Tis extract
demonstrated anxiolytic efects, which may be mediated by GABAergic system, and was able to increase GABAlevels and reduce of
glutamate and aspartate concentrations in mice hippocampus, which can directly and/or indirectly assist in their anxiolytic efect.
Although more studies are needed, the EPMG could represent an interesting therapeutical strategy in the treatment of anxiety.
1. Introduction
Mikania glomerata Sprengel (M. glomerata, Asteraceae) is a
species belonging to the genus Mikania (tribo Eupatorieae,
subtribo Miikaniinae) and popularly known as guaco,
which became ofcial in the frst Brazilian Pharmacopoeia
because of its therapeutic properties as bronchodilator, antial-
lergic, and antiasthmatic [13]. In addition to the popular use,
the M. glomerata presents other therapeutic properties such
as antihemorrhagic [4], antiophidian [5], antiviral [6], and
antimicrobial [7].
Phytochemical studies identifed in the genus Mikania
the presence of coumarin, diterpenes, lactones, and sesquiter-
penes [8, 9]. Te constituents of the species M. glomerata were
identifed as coumarin, lupeol acetate o-hydroxy-cinnamic
acid [10], kaurenoic acid [11], cinnamoylgrandiforic acid, and
stigmasterol [12]. Most of these compounds were proven to
have pharmacological activity: lupeol has anti-infammatory
activity [13]; kaurenoic acid is a potential antimicrobial,
hypotensive, and anti-infammatory; coumarin is an anti-
infammatory, immunosuppressive, antihypertensive, and
Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2014, Article ID 710410, 11 pages
http://dx.doi.org/10.1155/2014/710410
2 Evidence-Based Complementary and Alternative Medicine
antioxidant [3]; and stigmasterol has antinociceptive, anti-
infammatory, and hypocholesterolemic activity [14].
In spite of its pharmacological properties previously
discussed, studies with implications on the central nervous
system for M. glomerata are quite scarce in the scientifc
literature, with only studies for diferent species of the
same genus being found [15], which reinforces the need
for further studies for this plant species. Tus, the present
study aims to determine neuropharmacological activity of
the standardized ethanol extract of leaves of M. glomerata
(EPMG) by evaluation of anxiolytic efect and their possible
action mechanism in animal models. Te amino acids levels
(-aminobutyric acid (GABA), glutamate, glutamine, and
aspartate) in mice hippocampus, antioxidant capacity, and
chemical constituents of fatty acids were also determined. So,
the evaluation of neuropharmacological efects of M. glom-
erata will be important to establish a new pharmacological
strategy as medicinal herb.
2. Material and Methods
2.1. Reagents. Diazepam (DZP) 2 mg/kg (Sigma Chem. Co.,
St. Louis, MO, USA) and fumazenil (FLU) 2.5 mg/kg (Sigma
Chem. Co., St. Louis, MO, USA) were used as standards. 2,2-
Diphenyl-1-picrylhydrazyl (DPPH) radical, 2,2

-azinobis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS), and Trolox
(Sigma Chem. Co., St. Louis, MO, USA) were used to evaluate
the antioxidant capacity. All chemicals used were of highest
available purity grade.
2.2. Vegetable Material and Collecting the Leaves from M.
glomerata. Te aerial parts of M. glomerata were collected
in the Garden of Medicinal Plants of Phytotherapy Center
(NUFITO), which is part of Center for Pharmaceutical Care
(NUASF) of the Health Secretariat of the State of Cear a
(SESA). A voucher specimen (#27,041) was deposited in the
Herbarium Graziella Barroso at Federal University of Piaui.
Te botanical material was collected manually and then
rinsed in tap water, followed by distilled water. Afer the
search for foreign materials, the plant material was dried in
the shade.
2.3. Preparation of Standardized Ethanol Extract of Leaves
of M. glomerata. According to the method described by
Santana et al. [16], the extract was prepared by percolation
with previous soaking for 24 h, using 70% hydroethanolic
solution, with the ratio of 15 mL solution per 1 g of vegetable
drug. Te extraction solution was then concentrated in an
oven with forced air circulation to eliminate the ethanol
content and increase the solids content, which corresponded
to a reduction of 75% of the initial volume. Afer vacuum fl-
tration, the fltrate was concentratedusing a rotary evaporator
and then lyophilized.
2.4. Determination of Relative Density of Standardized Ethanol
Extract of Leaves from M. glomerata. Te determination of
the relative density of the extract was performed using the
pycnometer according to the Brazilian Pharmacopoeia in
duplicate [17].
2.5. Chemical Profle of Methyl Esters Present in Standardized
Ethanol Extract of Leaves of M. glomerata. Te fatty acid
methyl esters were obtained by the method described by
Hartman [18] with modifcations. A sample of 500 mg of
standardized ethanolic extract of leaves of M. glomerata was
added to a 50 mL fask to which 5 mL of NaOH 0.5 mol/L
dissolved in methanol was added. Te mixture was refuxed
for 5 minutes and added to 15 mL of esterifying reagent (a
mixture prepared at refux for 15 minutes, 2 g of NH
4
Cl,
60 mL of MeOH, and 3 mL of concentrated H
2
SO
4
). Te
esterifcation reaction was held at refux for 10 minutes and
transferred to a separatory funnel, followed by addition
of 25 mL of hexane and 50 mL of distilled water. Afer
careful stirring and phase separation, the aqueous phase
was discarded and the hexane phase analyzed by injection
into a gas chromatograph coupled with a mass spectrometer
(GC/MS).
2.6. HPLC Profle and Chemical Marks of M. glomerata
Ethanolic Extract. Te HPLC profle of the ethanolic extract
of M. glomerata was obtained in a Dionex DAD liquid chro-
matograph model UltiMate 3000. Te chromatograms were
registered employing a 5 m, 2.1 100 mm RP-18 column
(Dionex Acclaim), the oven temperature was set at 30

C,
and the injection volume was set as 10 L in the autosampler
and = 330 nm. Te best conditions for the mobile phase
were a 0100% gradient of MeOH: H
2
O (0.1% acetic acid)
and a fow rate of 0.8 mL/min. Te MeOH (Tedia, Fairfeld,
USA) and acetic acid (Merck, Darmstadt, Germany) used
were of HPLC grade. Water was supplied by a Milli-Q water
purifer system from Millipore (Bedford, MA, USA) and was
used afer fltration through a 0.2 m pore size membrane
flter. Coumarin and 2-hydroxycinnamic acid standards were
obtained from Sigma (St. Louis, MO, USA). Tis analytical
method described has been developed by our research group
for the detectionandquantifcationof their mainconstituents
and involved the optimization of several stages such as
the preparation of the sample, chromatographic separation,
and quantifcation. Te validation of the analytical method
was performed to ensure the success of the use of our
methodology, in addition to detecting errors of analytical
procedure, and provide proven evidence of efciency of the
method.
2.7. Antioxidant Potential of Standardized Ethanolic Extract
of Leaves of M. glomerata. Te antioxidant study was
determined by scavenging the stable free radical DPPH

(1,1-diphenyl-2-picrylhydrazyl) and ABTS

+
(2,2

-azinobis-3-
ethylbenzothiazoline-6-sulfonic acid) by compounds present
in standardized ethanolic extract of leaves of M. glomerata.
For the DPPH

test, the methodology described by Silva

et al. [19] was usedwithminor modifcations. Astock solution
of the standardized ethanol extract of leaves of M. glomerata
(150 mg/mL), of DPPH

(40 g/mL), and of standard Trolox

(94 g/mL) was prepared. Te concentrations used were 12.5,
25, 50, 75, and 100 g/mL, all being prepared by dilution
fromthe highest concentration. Te reaction mixture (0.3 mL
of extract plus 2.7 mL of a stock solution of DPPH

) was
incubated at room temperature in the absence of light
Evidence-Based Complementary and Alternative Medicine 3
(in the dark) for 30 minutes and the absorbance was mea-
sured ina spectrophotometer at 517 nm. Te antioxidant eval-
uation was performed in triplicate and the absorbance values
were expressed as percentage of inhibition of absorbance of
DPPH

solution by the following equation: % of inhibition of

radical DPPH

= {(
control

reaction mixture
)100}/
control
in
which
control
is the initial absorbance of the ethanol solution
of DPPH

and
reaction mixture
is the absorbance of the reaction
mixture containing the DPPH

and the concentrations of

ethanol extract under study. Te inhibitory concentration
(IC
50
) of the standardized ethanol extract of leaves of M.
glomerata required to reduce the absorbance of DPPH

to
50% at 517 nm was determined.
For determination of antioxidant capacity by method of
ABTS
+
the method described by Re et al. [20] was used with
modifcations. Initially the radical cation ABTS
+
from the
reaction of 5 mL of a 7 mM ABTS
+
with 88 L of a solution
2.45 mM potassium persulphate (K
2
S
2
O
8
) was formed and
incubated at room temperature in the absence of light for 16
hours. Elapsed this time, a solution of ABTS
+
was diluted in
ethanol to obtain a solution with absorbance of 0.70 (0.05),
at 734 nm. Final concentrations used were 12.5, 25, 50, 75, and
100 g/mL of the standardized ethanolic extract of leaves of
M. glomerata. In the dark, at room temperature, an aliquot
of 40 mL of each concentration was transferred to test tubes
with 1960 L of radical ABTS
+
.
Te reading of absorbance was performed at room
temperature, at time of 6 minutes in a spectrophotometer,
at 734 nm, and the results were expressed as percentage
inhibition of the absorbance of the solution ABTS
+
by the
following equation: % of inhibition of radical ABTS
+
=
{(
control

reaction mixture
) 100}/
control
, in which
control
is the initial absorbance of the ethanolic solution of ABTS
+
and
reaction mixture
is the absorbance of the reaction mixture
containing ABTS
+
and at concentrations of ethanolic extract
under study. Te inhibitory concentration (IC
50
) of the stan-
dardized ethanol extract of leaves of M. glomerata required
to reduce the absorbance of ABTS
+
to 50% at 734 nm was
determined.
2.8. Animals and Experimental Protocols. Adult male (2
months old) mice of the albino Swiss strain, weighing 25
30 g, were provided by Central Animal Facility of Center
for Agricultural Sciences, Federal University of Piaui (UFPI).
During all experiments, the animals were acclimated to 26
1

C and kept in acrylic cages of 30 30 cm

2
with a maximum
of six animals for observation of parameters related to
acute toxicity initially during the frst 24 h of the study. Te
animals were kept in similar environmental conditions, with
light/dark cycle of alternating 12 h, being fed at a basal type
Purina diet and water ad libitum. Tese experiments were
approved by Ethic Committee in Animal Experimentation of
Federal University of Piaui (#0071/10).
Te lyophilized residue from the standardized ethanol
extract of M. glomerata was diluted indistilled water to obtain
a fnal concentration of 10, 20, and 40 mg/mL to carry out the
treatment of animals.
To evaluate the possible sedative, anxiolytic, and muscle
relaxant efects, the animals were divided into six groups of
twelve animals and were subsequently treated for a period
of 30 consecutive days with 0.25 mL of 0.9% saline (vehicle-
control, p.o.), diazepam 2 mg/kg (positive control-DZP 2,
i.p.), fumazenil at a dose of 2.5 mg/kg (group FLU 2.5, i.p.),
and standardized ethanolic extract of leaves of M. glomerata
at doses 200, 300, and 400 mg/kg (p.o.) corresponding to
groups EPMG 200, EPMG 300, and EPMG 400, respectively.
Te doses used were based on a preliminary pharmacological
screening protocol [21] with diferent doses of the standard-
ized ethanol extract of M. glomerata administered to mice.
At all doses used, no signs of acute toxicity or behaviors
suggestive of neurotoxicity were observed (data not shown).
To clarify the mechanism of action, four groups of twelve
animals were treated for a period of 30 consecutive days with
fumazenil at a dose of 2.5 mg/kg (i.p.) and 30 min afer the
last dose of the treatment were administered with diazepam
2 mg/kg (FLU 2.5 + DZP 2) and standardized ethanol extract
of leaves of M. glomerata at doses of 200, 300, and 400 mg/kg
corresponding to groups FLU 2.5 + EPMG 200, FLU 2.5 +
EPMG 300, and FLU 2.5 + EPMG 400, respectively. Afer 30
minutes of the last administration, the animals were evaluated
in experimental protocols described below.
2.9. Evaluation of Locomotor Activity and Motor Coordination
in Mice Treated with Standardized Ethanol Extract of Leaves
of M. glomerata. Te motor activity of the animals was
checked by means of an open feld with dimensions of 30
30 15 cm, made of transparent acrylic walls, black foor
and divided into 09 equal quadrants [22]. Afer 30 minutes
of administration of the last dose related to treatment of
animals, one at a time was placed in the center of the feld
where the number of intersections with four legs, the number
of self-grooming behaviors (grooming), and the number of
vertical explorations (rearing), not lean against the wall, were
recorded during the time of 5 minutes. Afer each test, the
equipment was cleaned with a solution of 70% ethanol. Afer
this procedure the device was again cleaned with a sponge
soaked in water.
Te efect of muscle relaxant and motor coordination of
the animals treated as experimental protocol was observed in
the rotarod test [23]. For this test, 30 minutes afer the last
dose for the treatment of animals, one at a time was placed
on four legs with a bar of 2.5 cmin diameter, 25 cmhigh from
the foor in a rotation of 16 rpm for a period of 3 minutes.
In this test, residence time in the rotating bar, in seconds,
and the number of falls, with three renewals maximum, were
recorded. Afer each test, the equipment was cleaned with a
solution of 70% ethanol. Afer this procedure the device was
again cleaned with a sponge soaked in water.
2.10. Evaluation of Anxiolytic Activity in Mice Treated with
Standardized Ethanol Extract of Leaves of M. glomerata. In
evaluating the anxiolytic activity, the elevated plus maze test
(EPM) was used, consisting of two open and opposed arms,
and two other opposed closed arms of equal size (50 10 cm
each), made of wood, with side walls measuring 50 cm high.
At the edges of the open arms, a small wooden ledge (0.5 cm)
is fxed in order to reduce the number of falls of the animals.
Te arms perpendicularly intersect to form a cross, bounded
4 Evidence-Based Complementary and Alternative Medicine
by a central 10 10 cm area and 50 cm high from the ground.
Te maze is in a soundproof room, partially illuminated by
an incandescent lamp (60 W), and is placed vertically, 150 cm
above the apparatus [24].
In this experiment, 30 minutes afer the last dose, each
animal was placed in the center of the maze facing one of
the closed arms. We recorded the number of entries and time
spent in the open arms for a period of fve minutes, recording
the number of entries (NEOA) and total time spent in open
arms (TTOA) in seconds. Afer each test, the equipment was
cleaned with a solution of 70% ethanol. Afer this procedure
the device was cleaned again with a sponge soaked in water.
Complementing these data in the light/dark box test, 30
minutes afer the last dose, each mouse was placed in the
center of the illuminated part facing the opening that leads
to the dark side of the box, one at a time; the equipment
is described below. Te apparatus used is made of acrylic
and is divided into two compartments (light compartment
and dark compartment) that communicate [25]. Te dark
compartment (black acrylic, 27 18 29 cm) is poorly lit. Te
light compartment (transparent acrylic, 27 18 29 cm) in
all tests was directly illuminated by a fuorescent lamp (cold)
of 20 W light power, while the dark part was ftted with a
black cap. Each animal was intensively observed and, for fve
minutes afer the frst entry in the dark side, we recorded
behavioral parameters and afer each test the equipment was
cleaned with a solution of 70% ethanol. Afer this procedure,
the device was again cleaned with a sponge soaked in water.
Te parameter used in this test was the time spent in the light
compartment and is expressed in seconds.
2.11. Evaluation of Amino Acids Levels in Mice Hippocampus
Treated with Standardized Ethanol Extract of Leaves of M.
glomerata. To evaluate the amino acid levels (GABA, glu-
tamate, glutamine, and aspartate) the animals were divided
into four groups of ten, and they were subsequently treated
for a period of 30 consecutive days with 0.25 mL saline 0.9%
(vehicle-control, p.o.) and standardized ethanol extract of
leaves of M. glomerata at doses 200, 300, and 400 mg/kg
corresponding to groups EPMG 200, EPMG 300, and EPMG
400, respectively.
Afer behavioral testing the animals were sacrifced by
administering sodium pentobarbital (40 mg/kg, i.p.). Ten,
their brains were dissected on ice to remove the hippocampus
on both sides to determine amino acid levels (GABA, gluta-
mate, aspartate, and glutamine). Te hippocampus was used
to prepare 10%homogenates. Te braintissues were sonicated
in perchloric acid (HClO
4
) for 30 seconds and centrifuged
for 15 minutes in a refrigerated centrifuge at 21500 g. Te
supernatant was separated and fltered through a membrane
flter (Millipore 0.2 mM) and then combined with a solution
of precolumn derivatization to obtain fuorescence in a ratio
of 1 : 1. One minute afer the start of this association, analiquot
of 20 L was removed and injected into the HPLCequipment
for chemical analysis.
For amino acid analysis, a CLC-ODS column (M),
with a length of 15 cm in diameter, 4.6 mm gauge, and
particle diameter of 3 m, (Shimadzu, Japan) was used.
Te mobile phase used was in gradient using two phases:
(A) NaH
2
PO
4
(50 mM) and methanol (20% v/v) at pH 5.5
and (B) pure methanol (100%). Aspartate (ASP), glutamate
(Glu), glutamine (GLN), and -aminobutyric acid (GABA)
were detected using a fuorescence detector (Model RF-
535 from Shimadzu, Japan) with wavelengths EX-wavelength
(370 nm) and MS-wavelength (450 nm). Te chromatograms
were recorded and quantifed by a computer using sofware
from Shimadzu. Te amount of amino acid was calculated by
comparing the peak height obtained from the average of the
patterns, and the results were expressed in mol/g of tissue.
2.12. Statistical Analysis. Results were expressed as the mean
the standard error of the mean (SEM) of number of animals
that were used in the experiments. Te values that followed
a parametric distribution were analyzed by the analysis of
variance (ANOVA) and the -Student-Newman-Keuls test as
post hoc test, using the program GraphPad Prism version
5.00 for Windows. Diferences were considered statistically
signifcant from < 0.05.
3. Results
3.1. Relative Density and Chemical Profle. Te relative den-
sity found for the EPMG was 1.0142 g/cm
3
, indicating that
this extract is slightly denser than water. Fatty acids such
as omega-3, particularly eicosapentaenoic acid (EPA) and
docosahexanoic (DHA), develop an important role in the
central nervous system. However, for the fatty acids identifed
as methyl esters in the extract of M. glomerata (Table 1)
reports in the literature on their activity in central nervous
system were not found.
Te main chemical constituents (Figure 1) identifed in
chromatogram (Figure 2) of the EPMG were methyl cin-
namate, 2H-1-benzopyran-2-one, (2-hydroxyphenyl)methyl
propionate, (Z)-methyl-hexadec-7-enoate, methyl hexade-
canoate, hexadecanoic acid, (Z)-methyl-octadec-9-enoate,
octadecanoic acid, and squalene (Table 1).
Te HPLC/DADprofle of EPMGindicates very fewphe-
nolic compounds present inthis extract (Figure 3). Coumarin
and 2-hydroxycinnamic acids are present and these com-
pounds are well established in this species. So, they could
be considered chemical markers of standardized extracts of
this plant once they present specifc ultraviolet spectra and
are easily separated by this technique. Quantitative analyses
of both compounds in this extract indicate the presence of
1.340.014% of 2-hydroxycinnamic acid and 0.1510.001%
of coumarin in samples analysed.
3.2. Antioxidant Potential In Vitro. In order to evaluate the
in vitro antioxidant capacity of EPMG, the use of DPPH

and ABTS
+
radical was considered as a simple and rapid
procedure. Te results for the antioxidant capacity at diferent
concentrations are shown in Figure 4. Te values of the
antioxidant activity against DPPH

radical at concentrations
of 12.5, 25, 50, 75, and 100 g/mL were 22.73 0.64, 28.06
0.90, 34.16 0.94, 38.3 0.96, and 43.03 0.81%, which
decreased signifcantly ( < 0.05) the concentration of
the solution of DPPH

. Te Trolox (94 g/mL) also reduced

Evidence-Based Complementary and Alternative Medicine 5
Table 1: Fatty acids methyl esters and other constituents identifed by GC/MS, afer hydrolysis and methylation of the standardized ethanol
extract of leaves of M. glomerata Sprengel.
Compound
number
Compound name R.T. Area (%) Main fragments (%) M
+
/base peak
1 Methyl cinnamate 8.39 2.60 131 (100); 162 (53); 103 (52); 77 (37); 51 (30) 162/131
2 2H-1-Benzopyran-2-one 9.49 20.47 118 (100); 146 (70); 90 (41); 89 (40); 63 (28) 146/118
3 (2-Hydroxyphenyl)methyl propionate 10.65 2.66 120 (100); 148 (90); 91 (70); 44 (44); 180 (10) 180/120
4 (Z)-Methyl-hexadec-7-enoate 17.49 2.64 43 (100); 55 (80); 41 (72); 74 (60); 268 (>10) 268/43
5 Methyl hexadecanoate 17.77 36.00 74 (100); 87 (65); 43 (30); 55 (22); 270 (>10) 270/74
6 Hexadecanoic acid 18.12 5.77 73 (100); 43 (96); 60 (73); 57 (65); 256 (>10) 256/73
7 (Z)-Methyl-octadec-9-enoate 19.44 15.90 55 (100); 41 (76); 69 (75); 74 (100); 296 (<10) 296/55
8 Octadecanoic acid 19.63 7.40 74 (100); 87 (61); 43 (38); 55 (24); 298 298/74
9 Squalene 26.96 3.89 69 (100); 81 (69); 41 (28); 95 (20) /69
R.T.: retention time; M
+
: ion molecule.
OCH
3
OCH
3
OCH
3
OCH
3
OCH
3
O OH
O O
O
O O
O O
OH
OH
O
(2-Hidroxiphenyl)methyl propionate 2H-1-Benzopyran-2-one Methyl cinnamate
(Z)-Methyl-octadec-9-enoate
Octadecanoic acid Squalene
Hexadecanoic acid
Methyl hexadecanoate (Z)-Methyl-hexadec-7-enoate
Figure 1: Chemical structure of fatty acids methyl esters and other constituents identifed by GC/MS afer hydrolysis and methylation of
standardized ethanol extract of leaves of M. glomerata Sprengel.
the DPPH

radical presenting 69 0.43% of antioxidant

capacity. As for the DPPH

radical, a similar signifcant

reduction ( < 0.05) was also observed for the ABTS
+
radical by the extract at study that showed an antioxidant
capacity of 21.56 0.20, 25.56 0.72, 27.46 0.05, 30.5
1.05, and 37.83 0.96% for concentrations of 12.5, 25, 50,
75, and 100 g/mL, respectively. Te Trolox showed 67.9
0.17% of antioxidant capacity, and this result was greater
than the antioxidant capacity presented by samples by EPMG
in concentration of 100 g/ml. According to the results of
antioxidant capacity, the inhibitory concentration (IC
50
) of
EPMG required to reduce DPPH

and ABTS
+
at 50% of
its initial concentration was 138.91 g/mL and 175.68 g/mL,
respectively.
3.3. Evaluationof Locomotor Activity and Motor Coordination.
In the open feld test, the groups treated with EPMG at the
three doses did not present alterations in the number of
crossings (
10
= 1370, = 0.99, > 0.001) and rearings
(
10
= 799.5, = 0.99, > 0.001), but it had changes in
6 Evidence-Based Complementary and Alternative Medicine
Table 2: Evaluation of sedative action mechanism of standardized ethanol extract of leaves of M. glomerata in open feld test.
Groups () Number of crossings Number of groomings Number of rearings
Control (12) 98.58 1.19 4.83 0.30 45.33 0.14
DZP 2 (12) 38.58 0.96
a
2.58 0.15
a
13.33 0.14
a
EPMG 200 (12) 94.5 0.15
b
2.42 0.26 41.0 0.25
b
EPMG 300 (12) 95.5 0.15
b
2.58 0.19 46.0 0.25
b
EPMG 400 (12) 103.5 0.15
b
2.75 0.18 47.0 0.25
b
FLU 2.5 (12) 92.5 0.15 4.58 0.31 44.3 0.14
FLU 2.5 + DZP 2 (12) 89.5 0.15
b
4.67 0.39
b
45.33 0.14
b
FLU 2.5 + EPMG 200 (12) 94.5 0.15 2.58 0.29 45.5 0.55
FLU 2.5 + EPMG 300 (12) 97.5 0.15 2.67 0.26 46.67 0.67
FLU 2.5 + EPMG 400 (12) 103.5 0.15 2.75 0.22 47.5 0.78
Values represent the mean SEM of number of animals used in experiments. represents the number of animals in each group.
a
< 0.05, compared with
negative control (vehicle),
b
< 0.05, compared with diazepam group (ANOVA and -Student-Newman-Keuls as post hoc test). EPMG: standardized ethanol
extract of leaves of M. glomerata; DZP: diazepam; and FLU: fumazenil.
3.79
3.70
8.39
9.49
10.05
14.87
17.12
17.77
19.44
19.63
23.08
26.08
26.98
29.89
100
90
80
70
60
50
40
30
20
10
0
30 25 20 15 10 5 0
Time (min)
R
e
l
a
t
i
v
e

a
b
u
n
d
a
n
c
e
Figure 2: Chromatogram of standardized ethanol extract of leaves
from M. glomerata Sprengel for identifcation of fatty acid methyl
esters and other constituents by GC/MS, afer hydrolysis and
methylation.
O
O
7.743
9.190
13.918
17.428
30.0 27.5 25.0 22.5 20.0 17.5 15.0 12.5 10.0 7.5 5.0 2.5 0.0
35.0
30.0
20.0
10.0
0.0
5.0
(
m
A
U
)
(min)
WVL: 330nm
Mikania #25 (modifed by user, 4 peaks manually assigned)
OH
CO
2
H
UV VIS 5
Figure 3: HPLCprofle of the standardized ethanol extract of leaves
of M. glomerata Sprengel.
the number of groomings (
10
= 21.99, = 0.66, > 0.01).
However, the group treated with DZP reduced the number
of crossings (
10
= 1370, = 0.99, > 0.001), rearings
(
10
= 799.5, = 0.99, > 0.001), and groomings (
10
=
21.99, = 0.66, > 0.01), when compared to the group
treated with vehicle (negative control). On the other hand,
the group treated with the three doses EPMG only increased
the number of crossings (
10
= 1370, = 0.99, > 0.001)
and rearings (
10
= 799.5, = 0.99, > 0.001), compared to
DZP-treated group, suggesting that the sedative efect of the
extract is lower than that produced by diazepam, since only
one of the parameters has changed (groomings).
In the open feld test, fumazenil reversed the efect of
diazepam on the number of crossings (
10
= 1370, = 0.99,
> 0.001), groomings (
10
= 21.99, = 0.66, > 0.01), and
rearings (
10
= 799.5, = 0.99, > 0.001), when compared
to the group treated only with DZP (Table 2).
In the same test, fumazenil, in turn, did not reverse the
efect of the EPMG on the number of intersections at doses
of 400 mg/kg (
10
= 1370, = 0.99, > 0.001), 300 mg/kg
(
10
= 1370, = 0.99, < 0.001), and 200 mg/kg (
10
=
1370, = 0.99, > 0.001). In relation to the number of
groomings (
10
= 21.99, = 0.66, > 0.05) and rearings
(
10
= 799.5, = 0.99, > 0.001), no change was observed,
suggesting that the EPMG does not act by GABA
A
receptor
(Table 2).
3.4. Evaluation of Anxiolytic Activity. In the light/dark box
test, the EPMG at the dose of 400 mg/kg increased the time
spent in the light compartment of the treated animals when
compared to the negative control (
10
= 553, = 0.99, >
0.001) and the diazepam group (200 mg/kg (
10
= 553, =
0.99, > 0.001), 300 mg/kg (
10
= 553, = 0.99, > 0.001),
and 400 mg/kg (
10
= 553, = 0.99; > 0.001)) (Table 3).
Te animals treated with doses of 200 mg/kg (
10
= 553,
= 0.99, > 0.001), 300 mg/kg (
10
= 553, = 0.99,
> 0.001), and 400 mg/kg (
10
= 553, = 0.99, > 0.001)
produced an increase in this parameter only when compared
to the vehicle group (negative control), suggesting that this
extract has anxiolytic efects at the doses tested (Table 3).
In the same test, it was found that fumazenil reversed
the efect on time spent in the light compartment at doses
of 200 mg/kg (
10
= 553, = 0.99, > 0.001), 300 mg/kg
(
10
= 553, = 0.99, < 0.001), and 400 mg/kg (
10
= 553,
= 0.99, > 0.001) of the EPMG. Tese results suggest that
the anxiolytic efect can be mediated by GABA
A
receptors.
Likewise, fumazenil could reverse the DZP efect (
10
= 553,
= 0.99, > 0.001).
Evidence-Based Complementary and Alternative Medicine 7

0
25
50
75
100
125
I
n
h
i
b
i
t
i
o
n

o
f

D
P
P
H

r
a
d
i
c
a
l

(
%
)
Ethanol extract of M. glomerata
System
12.5g/mL
25 g/mL
50g/mL
75g/mL
100 g/mL
Trolox
(94g/mL)
(a)

0
25
50
75
100
125
I
n
h
i
b
i
t
i
o
n

o
f

A
B
T
S

r
a
d
i
c
a
l

(
%
)
System
Ethanol extract of M. glomerata
12.5g/mL
25 g/mL
50g/mL
75g/mL
100 g/mL
Trolox
(94g/mL)
(b)
Figure 4: Antioxidant capacity of standardized ethanol extract of leaves from M. glomerata by inhibition of DPPH

and ABTS
+
radicals.
Values represent the mean SEM ( = 3).

< 0.05 compared with system (ethanolic solution of DPPH

and ABTS
+
).
Table 3: Evaluation of anxiolytic action mechanismof standardized
ethanol extract of leaves from M. glomerata in light dark box test.
Groups ()
Residence time in the light
compartment (seconds)
Control (12) 104.3 0.33
DZP 2 (12) 158.3 0.48
a
EPMG 200 (12) 133.3 0.38
a
EPMG 300 (12) 157.3 0.41
a
EPMG 400 (12) 169.3 0.38
a,b,c,d
FLU 2.5 (12) 107.3 0.38
FLU 2.5 + DZP 2 (12) 102.7 0.38
b
FLU 2.5 + EPMG 200 (12) 102.3 0.38
FLU 2.5 + EPMG300 (12) 104.7 0.51
FLU 2.5 + EPMG400 (12) 108.7 0.62
e
Values represent the mean SEM of the number of animals used in
experiments. represents the number of animals in each group.
a
<
0.05 compared with vehicle (negative control),
b
< 0.05, compared with
diazepam group,
c
< 0.05, compared with group EPMG 200,
d
<
0.05 compared with EPMG 300 group,
e
< 0.05, compared with group
EPMG400 (ANOVAand -Student-Newman-Keuls as post hoc test). EPMG:
standardized ethanol extract of leaves of M. glomerata; DZP: diazepam; and
FLU: fumazenil.
In rotarod test, the EPMG increased the number of falls
(
10
= 3.31, = 0.23, > 0.05) and reduced the time spent
on the rotating bar (
10
= 6.91, = 0.39, > 0.001), only at
the dose of 400 mg/kg when compared to the negative control
group. Te doses, in turn, produced no muscle relaxant efect
superior to DZP ( > 0.05). However, the efects on motor
coordination of group DZP (number of falls (
10
= 3.31,
Table 4: Evaluation of anxiolytic action mechanismof standardized
ethanol extract of leaves of M. glomerata on motor coordination in
rotarod test.
Groups () Number of falls
Residence time on the
rotating bar (seconds)
Control (12) 1.67 0.14 176.1 1.02
DZP 2 (12) 2.83 0.17
a
167.4 3.18
a
EPMG 200 (12) 1.33 0.31 177.3 0.81
EPMG 300 (12) 1.50 0.19 176.3 0.60
EPMG 400 (12) 2.08 0.47
a
174.1 1.29
a
FLU 2.5 (12) 1.58 0.19 177.5 0.36
b
FLU 2.5 + DZP 2 (12) 1.67 0.31
b
176.5 0.36
FLU 2.5 + EPMG 200 (12) 1.33 0.33 177.8 0.27
FLU 2.5 + EPMG300 (12) 1.58 0.23 177.4 0.47
FLU 2.5 + EPMG400 (12) 1.58 0.26
c
177.7 0.79
c
Values represent the mean SEM of the number of animals used in
experiments. represents the number of animals in each group.
a
<
0.05 compared with negative control (vehicle),
b
< 0.05, compared with
diazepam group, and
c
< 0.05, compared with FLU 2.5 + EPMG 400
group (ANOVA and -Student-Newman-Keuls as post hoc test). EPMG:
standardized ethanol extract of leaves of M. glomerata; DZP: diazepam; and
FLU: fumazenil.
= 0.23, > 0.01) and time spent on the rotating bar
(
10
= 6.91, = 0.39, < 0.001)) and the dose of 400 mg/kg
of the EPMG(number of falls (
10
= 3.31, = 0.23, > 0.05)
and time spent on the rotating bar (
10
= 6.91, = 0.39,
> 0.05)) were reversed by fumazenil, suggesting that the
EPMGproduces incoordination, and only at the highest dose
similar to DZP by GABA receptors (Table 4).
8 Evidence-Based Complementary and Alternative Medicine
Table 5: Evaluation of anxiolytic action mechanismof standardized
ethanol extract of leaves from M. glomerata in elevated plus-maze
test.
Groups () NEOA TTOA (seconds)
Control (12) 11.67 0.5 123.8 0.41
DZP 2 (12) 15.83 0.24
a
207.3 0.70
a
EPMG 200 (12) 11.33 0.38 123.6 0.40
EPMG 300 (12) 11.67 0.75 124.6 0.40
EPMG 400 (12) 17.08 0.78
a,b,c,d
226.3 0.62
a,b,c,d
FLU 2.5 (12) 10.92 0.68 123.5 0.56
FLU 2.5 + DZP 2 (12) 10.58 0.48
b
124.6 0.40
b
FLU 2.5 + EPMG 200 (12) 10.25 0.46 122.8 0.28
FLU 2.5 + EPMG 300 (12) 10.58 0.48 123.6 0.40
FLU 2.5 + EPMG 400 (12) 11.5 0.47
e
123.8 0.55
e
Values represent the mean SEM of the number of animals used in
experiments. represents the number of animals in each group.
a
< 0.05
compared with negative control,
b
< 0.05, compared with diazepam
group,
c
< 0.05, compared with group EPMG 200,
d
< 0.05 compared
with EPMG 300 group, and
e
< 0.05, compared with FLU 2.5 + EPMG
400 group (ANOVA and -Student-Newman-Keuls as post hoc test). EPMG:
standardized ethanol extract of leaves of M. glomerata; DZP: diazepam; and
FLU: fumazenil.
In the rotarod test fumazenil did not alter the efect of the
EPMG on motor coordination of the mice in the number of
falls at doses of 200 mg/kg (
10
= 3.31, = 0.23, > 0.05)
and 300 mg/kg (
10
= 3.31, = 0.23, > 0.05, Table 4).
Additionally, it produced no change on the residence time
on the rotating bar, in the animals treated with doses of
200 mg/kg (
10
= 6.91, = 0.39, > 0.05) and 300 mg/kg
(
10
= 6.91, = 0.39, > 0.05), when compared to the
groups treated with EPMG 400 mg/kg (Table 4).
In EPMtest, we found that the EPMG, only at the highest
dose, increased the parameter values NEOA(
10
= 18.51, =
0.63, > 0.001) and TTOA EPMG 400 mg/kg (
10
= 1442,
= 1.0, < 0.001), compared to negative control. Likewise,
only that dose produced an increase in NEOA (
10
= 18.51,
= 0.63, > 0.05) and TTOA (
10
= 1442, = 1.0, <
0.001) when compared to DZP group. In the group treated
with DZP, an increase in NEOA (
10
= 18.51, = 0.63, >
0.001) and TTOA (
10
= 1442, = 1.0, < 0.001) was also
observed, when compared with negative control (Table 5).
In this test, fumazenil blocked the efects of diazepam on
NEOA parameters FLU 2.5 + DZP 2 (
10
= 18.51, = 0.63,
> 0.001) and TTOA FLU 2.5 + DZP 2 (
10
= 14420,
= 1.0, > 0.001). Moreover, fumazenil reversed the
efect of the extract at 400 mg/kg on the NEOA parameters
FLU 2.5 + EPMG 400 (
10
= 18.51, = 0.63, > 0.001)
and TTOA FLU 2.5 + EPMG 400 (
10
= 1442, = 1.0,
< 0.001), when compared with mice treated only with
EPMG 400 mg/kg (Table 5).
3.5. Evaluation of Amino Acids Levels. In this study it was
found that the EPMGonly at the highest dose increased levels
of GABA compared to doses of 200 (
4
= 5.40, = 0.47,
< 0.1) and 300 mg/kg (
4
= 5.40, = 0.47, < 0.1)
and increased the concentration of this amino acid when
compared to the negative control (Figure 5).
Moreover, the three tested doses of extract reduced the
aspartate concentration (200 mg/kg (
4
= 6.72, = 0.53,
< 0.05), 300 mg/kg (
4
= 6.72, = 0.53, < 0.05), and
400 mg/kg (
4
= 6.72, = 0.53, < 0.01)), as compared to
negative control. Te group treated with the highest dose of
EPMG, in turn, produced a decrease in glutamate content as
compared to doses of 200 mg/kg (
4
= 17.20, = 0.74, <
0.001) and 300 mg/kg (
4
= 17.20, = 0.74, < 0.001) and
negative control (
4
= 17.20, = 0.74, < 0.001) (Figure 5).
Te doses of 200 mg/kg and 300 mg/kg produced no change
in glutamate content when compared with negative control
( > 0.05, Figure 5).
Te EPMG, in turn, at any test did not produce changes
in glutamine levels ( > 0.05) when compared with negative
control and no change was seenbetweenthe doses tested ( >
0.05, Figure 5).
4. Discussion
Based on those results, it can be verifed that standardized
ethanol extract of leaves of M. glomerata contains the follow-
ing fatty acids: methyl cinnamate, 2H-1-benzopyran-2-one,
(2-hydroxyphenyl)methyl propionate, (Z)-Methyl-hexadec-
7-enoate, methyl hexadecanoate, hexadecanoic acid, (Z)-
methyl-octadec-9-enoate, octadecanoic acid, and squalene.
In other studies with M. glomerata, it was shown that this
plant has chemical constituents such as coumarin, lupeol,
ent-15b-isobutyryloxykaur-16 (17)-en-19-oic acid, sesquiter-
penes, diterpenes, kaurenoic acids, cinnamoylgrandiforic
acid, stigmasterol, and tannins [26]. Among the chemical
constituents cited and identifed in this study, coumarin 2H-
1-benzopyran-2-one can be considered as a major constituent
of M. glomerata and therefore can contribute signifcantly to
several of its pharmacological properties [2729].
Te antioxidant study of M. glomerata by DPPH

method
has been reported by Vicentino and Menezes [30], and
antioxidant studies by ABTS
+
method in scientifc articles
were not found. Te antioxidant property of a substance is
ofen linked to their ability to donate electron or hydrogen
atom to the radical [31]. Tus, the antioxidant capacity of
leaves of EPMG can be attributed in part to their chemical
composition, found in substances such as cinnamic acid
or coumarin derivatives, which have antioxidant capacity
[32], and is one of major constituents responsible for the
pharmacological properties of M. glomerata [33].
In feld tests, the administration of EPMG in three
doses did not alter the number of groomings, rearings, and
crossings, indicating the absence of changes in locomotor
activity. Te evaluation of the EPMG motor efects in mice
in the rotarod test, a model widely used to assess the
peripheral motor coordination and neuromuscular blockade,
showed that the EPMG (200300 mg/kg), unlike diazepam
(2 mg/kg), had no signifcant efect on motor coordination.
However, with increasing dose of EPMG (400 mg/kg) there
were no changes on motor coordination observed by the
increased number of falls and decreased length of stay in
the rotarod test. It is important to note that despite focking
Evidence-Based Complementary and Alternative Medicine 9
3
2
1
0
(

m
o
l
/
g

t
i
s
s
u
e
)
Vehicle EPMG 200 EPMG 300 EPMG 400 Vehicle EPMG 200 EPMG 300 EPMG 400
GABA Aspartate
a, b, c
a a a
(a)
3
2
1
0
(

m
o
l
/
g

t
i
s
s
u
e
)
Vehicle EPMG 200 EPMG 300 EPMG 400
Vehicle EPMG 200 EPMG 300 EPMG 400
Glutamate Glutamine
a, b, c
(b)
Figure 5: Efect of standardized ethanol extract of leaves of M. glomerata on amino acids levels (GABA, aspartate, glutamate, and glutamine)
in mice hippocampus. Values represent the mean SEM of the number of animals used in experiments.
a
< 0.05, compared with negative
control,
b
< 0.05, compared with EPMG 200 group,
c
< 0.05, compared with group EPMG 200, and
d
< 0.05, compared with EPMG 300
group (ANOVA and -Student-Newman-Keuls as post hoc test).
change in motor coordination, performance in anxiolytic
tests (light/dark and EPM tests) was not afected at a dose of
400 mg/kg, as discussed below. Similar result in rotarod test
(500 mg/kg) was also found in plants of the same genus as M.
scandens (L.) Willd. [34] and of diferent genus as Passifora
edulis [35].
Te anxiolytic efect of EPMGwas evaluated using several
models of anxiety in mice. In the light-dark test, the anxiety
generated by the confict between the desire to explore new
environments and aversion to an unknown illuminated area
can be evaluated according to the residence time in the
lighted area, and an increase in this parameter indicates
anxiolytic efects [36]. Tus, the results of this study showed
that treatment with EPMG increased residence time in the
light compartment (Table 3), demonstrating the anxiolytic
efect at doses of 200, 300, and 400 mg/kg. Te same efect
was also seen for diazepam control (Table 3).
In addition to the light-dark test, the test of the elevated
plus maze is a widely used experimental model to evaluate the
efect of substances on anxiety in animals, and the animals
exhibit anxiety behavior when there is usually a reduction
in exploiting open arms evidenced by the reduction in the
number of entries and time spent in the open arms [37].
Tus, animals treated with antianxiety drugs such as
diazepam increase the number of entries and time spent
on open arms. Tus, the results of this study indicate that
only the 400 mg/kg dose of EPMG reduced the behavioral
symptoms of anxiety in the elevated maze test cross, which
supports the notion of a dosage selective anxiolytic efect
to this test; since the light-dark test, anxiolytic efects were
observed at the three doses tested.
In the maze test in high light and dark cross, fumazenil
(benzodiazepine receptor antagonist) was used to demon-
strate the specifc antagonism of GABA
A
receptors in the
anxiolytic action of EPMG mechanism. In this work, the
use of EPMG with fumazenil caused a signifcant decrease
in exploration of the open arms of the elevated plus maze,
indicating that the anxiolytic efect of EPMG occurs by
interaction with GABA
A
receptors. Te same mechanism of
anxiolytic action is also attributed to EPMGin light-dark test.
Te literature shows that reduction of anxiety is related
to elevated GABA levels, with a decrease in content of
this amino acid, glutamate, aspartate, and glutamine being
directly associated with increased anxiety and behavior [38
40]. Tus, the present study aimed to investigate the efects
of amino acids levels (GABA, aspartate, glutamate, and
10 Evidence-Based Complementary and Alternative Medicine
glutamine) onmice hippocampus, inorder to correlate it with
behavioral studies, investigating the anxiolytic potential and
protocols to clarify its possible action mechanism. Te results
suggest that the anxiolytic efect of this extract may be medi-
ated by decreased glutamate levels (400 mg/kg), as well as
by the increased concentration of GABA in mice hippocam-
pus (Figure 5). Tese outcomes corroborate with studies of
Pereira et al. [41] and Galal et al. [42], which demonstrated
increased GABA levels and reduced glutamate concentration
in mice treated with coumarin and nandrolone decanoate,
respectively. As discussed earlier, there are several studies
addressing the pharmacological properties of M. glomerata;
however, this is the frst report on its neurochemical efect,
whereas other studies focusing on the neuropharmacological
properties have been conducted with plants of the same
genus, such as M. scandens (L.) Willd. [15, 34] and M. cordata
(Burm.) [43].
5. Conclusions
In conclusion, the present study provides important results
with respect to the pharmacological activity of an important
medicinal plant. Te EPMG demonstrated anxiolytic efects,
which may be mediated by GABAergic system, and was able
to increase GABA levels and reduce glutamate and aspartate
concentrations in mice hippocampus, which can directly
and/or indirectly assist in their anxiolytic efect. Although
more studies are needed [44], the EPMG could represent an
interesting therapeutical strategy in the treatment of anxiety.
Conflict of Interests
Te authors declare no confict of interests.
Acknowledgments
Te authors would like to thank the National Council of
Technological and Scientifc Development (CNPq/Brazil)
and the Research Supporting Foundation of State of Piaui
(FAPEPI/Brazil) for the fnancial support.
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