Mikania glomerata: Phytochemical, Pharmacological, and Neurochemical Study

The 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 effects 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 field, elevated plus-maze, light dark, and rotarod tests. Moreover, in the behavioral tests diazepam (GABAergic anxiolytic, 2 mg/kg) as positive control and flumazenil (GABA antagonist, 2.5 mg/kg) were used to identify mechanism of sedative and anxiolytic action produced by EPMG. The 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. This extract demonstrated anxiolytic effects, which may be mediated by GABAergic system, and was able to increase GABA levels and reduce of glutamate and aspartate concentrations in mice hippocampus, which can directly and/or indirectly assist in their anxiolytic effect. Although more studies are needed, the EPMG could represent an interesting therapeutical strategy in the treatment of anxiety.

In spite of its pharmacological properties previously discussed, studies with implications on the central nervous system for M. glomerata are quite scarce in the scientific literature, with only studies for different species of the same genus being found [15], which reinforces the need for further studies for this plant species. Thus, the present study aims to determine neuropharmacological activity of the standardized ethanol extract of leaves of M. glomerata (EPMG) by evaluation of anxiolytic effect and their possible action mechanism in animal models. The 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 effects of M. glomerata will be important to establish a new pharmacological strategy as medicinal herb.  The botanical material was collected manually and then rinsed in tap water, followed by distilled water. After the search for foreign materials, the plant material was dried in the shade.

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. The 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. After vacuum filtration, the filtrate was concentrated using a rotary evaporator and then lyophilized.

Determination of Relative Density of Standardized Ethanol
Extract of Leaves from M. glomerata. The determination of the relative density of the extract was performed using the pycnometer according to the Brazilian Pharmacopoeia in duplicate [17].

Chemical Profile of Methyl Esters Present in Standardized
Ethanol Extract of Leaves of M. glomerata. The fatty acid methyl esters were obtained by the method described by Hartman [18] with modifications. A sample of 500 mg of standardized ethanolic extract of leaves of M. glomerata was added to a 50 mL flask to which 5 mL of NaOH 0.5 mol/L dissolved in methanol was added. The mixture was refluxed for 5 minutes and added to 15 mL of esterifying reagent (a mixture prepared at reflux for 15 minutes, 2 g of NH 4 Cl, 60 mL of MeOH, and 3 mL of concentrated H 2 SO 4 ). The esterification reaction was held at reflux for 10 minutes and transferred to a separatory funnel, followed by addition of 25 mL of hexane and 50 mL of distilled water. After 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).

HPLC Profile and Chemical Marks of M. glomerata
Ethanolic Extract. The HPLC profile of the ethanolic extract of M. glomerata was obtained in a Dionex DAD liquid chromatograph model UltiMate 3000. The 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. The best conditions for the mobile phase were a 0-100% gradient of MeOH : H 2 O (0.1% acetic acid) and a flow rate of 0.8 mL/min. The MeOH (Tedia, Fairfield, USA) and acetic acid (Merck, Darmstadt, Germany) used were of HPLC grade. Water was supplied by a Milli-Q water purifier system from Millipore (Bedford, MA, USA) and was used after filtration through a 0.2 m pore size membrane filter. Coumarin and 2-hydroxycinnamic acid standards were obtained from Sigma (St. Louis, MO, USA). This analytical method described has been developed by our research group for the detection and quantification of their main constituents and involved the optimization of several stages such as the preparation of the sample, chromatographic separation, and quantification. The 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 efficiency of the method.
For the DPPH • test, the methodology described by Silva et al. [19] was used with minor modifications. A stock 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. The concentrations used were 12.5, 25, 50, 75, and 100 g/mL, all being prepared by dilution from the highest concentration. The 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 (in the dark) for 30 minutes and the absorbance was measured in a spectrophotometer at 517 nm. The antioxidant evaluation 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. The 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 modifications. 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 •+ .
The 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. The inhibitory concentration (IC 50 ) of the standardized ethanol extract of leaves of M. glomerata required to reduce the absorbance of ABTS •+ to 50% at 734 nm was determined.

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 first 24 h of the study. The 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. These experiments were approved by Ethic Committee in Animal Experimentation of Federal University of Piaui (#0071/10).
The lyophilized residue from the standardized ethanol extract of M. glomerata was diluted in distilled water to obtain a final concentration of 10, 20, and 40 mg/mL to carry out the treatment of animals.
To evaluate the possible sedative, anxiolytic, and muscle relaxant effects, 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 (vehiclecontrol, p.o.), diazepam 2 mg/kg (positive control-DZP 2, i.p.), flumazenil 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. The doses used were based on a preliminary pharmacological screening protocol [21] with different doses of the standardized 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 flumazenil at a dose of 2.5 mg/kg (i.p.) and 30 min after 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. After 30 minutes of the last administration, the animals were evaluated in experimental protocols described below.

Evaluation of Locomotor Activity and Motor Coordination in Mice Treated with Standardized Ethanol Extract of Leaves of M. glomerata.
The motor activity of the animals was checked by means of an open field with dimensions of 30 × 30 × 15 cm, made of transparent acrylic walls, black floor and divided into 09 equal quadrants [22]. After 30 minutes of administration of the last dose related to treatment of animals, one at a time was placed in the center of the field 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. After each test, the equipment was cleaned with a solution of 70% ethanol. After this procedure the device was again cleaned with a sponge soaked in water.
The effect 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 after the last dose for the treatment of animals, one at a time was placed on four legs with a bar of 2.5 cm in diameter, 25 cm high from the floor 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. After each test, the equipment was cleaned with a solution of 70% ethanol. After this procedure the device was again cleaned with a sponge soaked in water.

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 fixed in order to reduce the number of falls of the animals. The arms perpendicularly intersect to form a cross, bounded by a central 10 × 10 cm area and 50 cm high from the ground. The 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 after 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 five minutes, recording the number of entries (NEOA) and total time spent in open arms (TTOA) in seconds. After each test, the equipment was cleaned with a solution of 70% ethanol. After this procedure the device was cleaned again with a sponge soaked in water.
Complementing these data in the light/dark box test, 30 minutes after 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. The apparatus used is made of acrylic and is divided into two compartments (light compartment and dark compartment) that communicate [25]. The dark compartment (black acrylic, 27 × 18 × 29 cm) is poorly lit. The light compartment (transparent acrylic, 27 × 18 × 29 cm) in all tests was directly illuminated by a fluorescent lamp (cold) of 20 W light power, while the dark part was fitted with a black cap. Each animal was intensively observed and, for five minutes after the first entry in the dark side, we recorded behavioral parameters and after each test the equipment was cleaned with a solution of 70% ethanol. After this procedure, the device was again cleaned with a sponge soaked in water. The parameter used in this test was the time spent in the light compartment and is expressed in seconds.

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, glutamate, 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.
After behavioral testing the animals were sacrificed by administering sodium pentobarbital (40 mg/kg, i.p.). Then, their brains were dissected on ice to remove the hippocampus on both sides to determine amino acid levels (GABA, glutamate, aspartate, and glutamine). The hippocampus was used to prepare 10% homogenates. The brain tissues were sonicated in perchloric acid (HClO 4 ) for 30 seconds and centrifuged for 15 minutes in a refrigerated centrifuge at 21500 ×g. The supernatant was separated and filtered through a membrane filter (Millipore 0.2 mM) and then combined with a solution of precolumn derivatization to obtain fluorescence in a ratio of 1 : 1. One minute after the start of this association, an aliquot of 20 L was removed and injected into the HPLC equipment 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. The 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 fluorescence detector (Model RF-535 from Shimadzu, Japan) with wavelengths EX-wavelength (370 nm) and MS-wavelength (450 nm). The chromatograms were recorded and quantified by a computer using software from Shimadzu. The 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. The 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. Differences were considered statistically significant from < 0.05.

Relative Density and Chemical
Profile. The relative density 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 identified 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.
The HPLC/DAD profile of EPMG indicates very few phenolic compounds present in this extract (Figure 3). Coumarin and 2-hydroxycinnamic acids are present and these compounds are well established in this species. So, they could be considered chemical markers of standardized extracts of this plant once they present specific ultraviolet spectra and are easily separated by this technique. Quantitative analyses of both compounds in this extract indicate the presence of 1.34 ± 0.014% of 2-hydroxycinnamic acid and 0.151 ± 0.001% of coumarin in samples analysed.   the DPPH • radical presenting 69 ± 0.43% of antioxidant capacity. As for the DPPH • radical, a similar significant 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. The 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.

Evaluation of Locomotor Activity and Motor Coordination.
In the open field 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).
In the same test, it was found that  Table 3: Evaluation of anxiolytic action mechanism of standardized ethanol extract of leaves from M. glomerata in light dark box test.
The EPMG, in turn, at any test did not produce changes in glutamine levels ( > 0.05) when compared with negative control and no change was seen between the doses tested ( > 0.05, Figure 5).
The antioxidant study of M. glomerata by DPPH • method has been reported by Vicentino and Menezes [30], and antioxidant studies by ABTS •+ method in scientific articles were not found. The antioxidant property of a substance is often linked to their ability to donate electron or hydrogen atom to the radical [31]. Thus, 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 field 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. The evaluation of the EPMG motor effects in mice in the rotarod test, a model widely used to assess the peripheral motor coordination and neuromuscular blockade, showed that the EPMG (200-300 mg/kg), unlike diazepam (2 mg/kg), had no significant effect 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 flocking change in motor coordination, performance in anxiolytic tests (light/dark and EPM tests) was not affected 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 different genus as Passiflora edulis [35]. The anxiolytic effect of EPMG was evaluated using several models of anxiety in mice. In the light-dark test, the anxiety generated by the conflict 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 effects [36]. Thus, the results of this study showed that treatment with EPMG increased residence time in the light compartment (Table 3), demonstrating the anxiolytic effect at doses of 200, 300, and 400 mg/kg. The same effect 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 effect 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].
Thus, animals treated with antianxiety drugs such as diazepam increase the number of entries and time spent on open arms. Thus, 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 effect to this test; since the light-dark test, anxiolytic effects were observed at the three doses tested.
In the maze test in high light and dark cross, flumazenil (benzodiazepine receptor antagonist) was used to demonstrate the specific antagonism of GABA A receptors in the anxiolytic action of EPMG mechanism. In this work, the use of EPMG with flumazenil caused a significant decrease in exploration of the open arms of the elevated plus maze, indicating that the anxiolytic effect of EPMG occurs by interaction with GABA A receptors. The same mechanism of anxiolytic action is also attributed to EPMG in light-dark test.
The 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][39][40]. Thus, the present study aimed to investigate the effects of amino acids levels (GABA, aspartate, glutamate, and glutamine) on mice hippocampus, in order to correlate it with behavioral studies, investigating the anxiolytic potential and protocols to clarify its possible action mechanism. The results suggest that the anxiolytic effect of this extract may be mediated by decreased glutamate levels (400 mg/kg), as well as by the increased concentration of GABA in mice hippocampus ( Figure 5). These 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 first report on its neurochemical effect, 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].

Conclusions
In conclusion, the present study provides important results with respect to the pharmacological activity of an important medicinal plant. The EPMG demonstrated anxiolytic effects, 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 effect. Although more studies are needed [44], the EPMG could represent an interesting therapeutical strategy in the treatment of anxiety.