Depressant effects of Agastache mexicana methanol extract and one of major metabolites tilianin

María Eva González-Trujano, Hilda Ponce-Mu?oz, Sergio Hidalgo-Figueroa, Gabriel Navarrete-Vázquez, Samuel Estrada-Soto*

1Laboratorio de Neurofarmacología de Productos Naturales de la Dirección de Investigaciones en Neurociencias. Instituto Nacional de Psiquiatría“Ramón de la Fuente Mu?iz”, México, D.F.14370, México

2Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, México

Depressant effects of Agastache mexicana methanol extract and one of major metabolites tilianin

María Eva González-Trujano1, Hilda Ponce-Mu?oz2, Sergio Hidalgo-Figueroa2, Gabriel Navarrete-Vázquez2, Samuel Estrada-Soto2*

1Laboratorio de Neurofarmacología de Productos Naturales de la Dirección de Investigaciones en Neurociencias. Instituto Nacional de Psiquiatría“Ramón de la Fuente Mu?iz”, México, D.F.14370, México

2Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, México

ARTICLE INFO

Article history:

Received 26 December 2014

Received in revised form 10 January 2015

Accepted 22 February 2015

Available online 20 March 2015

Agastache mexicana

Anxiety

Benzodiazepine

Central nervous system

Sedative

Tilianin

Objective: To determine the depressant-like effects and the possible mechanism of action of tilianin isolated from active methanol extract of Agastache mexicana (A. mexicana). Also, to establish the pharmacophoric requirements of tilianin, as a possible ligand of GABAA/ BZD receptor, by the alignment of diazepam, CGS-9896 and diindole, using a previously described pharmacophoric model. Methods: Tilianin (30 to 300 mg/kg, ip. and 300 mg/kg, po.) and methanol crude extract (10 to 300 mg/kg, ip. and 300 mg/kg po.) from A. mexicana were evaluated for potential sedative and anxiolytic-like response drugs by using open-field, hole-board, cylinder of exploration, plus-maze and sodium pentobarbital-induced hypnosis mice methods. Results: Methanol extract and tilianin showed anxiolytic-like activity from a dosage of 30 mg/kg, ip. or 300 mg/kg, po. and were less potent than diazepam 0.1 mg/kg, a reference anxiolytic drug used. Moreover, depressant activity of both potentiates sodium pentobarbital (SP)-induced sleeping time. The anxiolytic-like effect of 30 mg/kg ip. observed for the extract and tilianin, by using the plus-maze model, was partially prevented in the presence of flumazenil (a GABAA/BZD antagonist, 5 mg/kg ip.) but not in the presence of WAY 100635 (a selective 5-HT1Areceptor antagonist, 0.32 mg/kg, ip.). Pharmacophoric modeling alignments of three agonist of GABAA/BZD allow identify seven chemical features. Tilianin contains six of the seven features previously determined. Conclusions: Results indicate that tilianin is one of the bioactive metabolites in the anxiolytic-like activity of A. mexicana, reinforcing its central nervous system uses, where GABAA/BZD, but not 5-HT1A, receptors are partially involved.

1. Introduction

Agastache mexicana (A. mexicana) is a native herb from the Asian and North American regions[1,2]. In Mexico, it is commonly called“toronjil” and due to its large demand, it is cultivated in various regions such as Mexico City and the states of Hidalgo, Mexico, Morelos, Puebla and Veracruz. It is used to treat anxiety, insomnia and cardiovascular disorders, as well as rheumatism, stomach pain, and gastrointestinal affections[3-6]. Inflorescences are preferred to alleviate pain and aerial parts to produce sedative activity[7].

Pharmacological properties such as anxiogenic[8] or anxiolyticlike[9] behaviors, vasorelaxant[10], antioxidant[11], antinociceptive and anti-inflammatory effects[12] have been recently showed in extracts from A. mexicana. It has also been reported the presence of flavonoids as the main secondary metabolites that characterize the Mexican species of Agastache genus[13]. However, scientific studies have not been undertaken to identify some active metabolite and the possible involved mechanism of action forthe tranquilizing activity of A. mexicana. In the present study, systemic administration (intraperitoneal and esophagi) of a polar extract and the compound tilianin were evaluated in different experimental models in order to analyze its sedative and/or anxiolytic-like responses and to know the tilianin's mechanism of action.

2. Materials and methods

2.1. Plant material, extraction and isolation of tilianin

A. mexicana (Kunth) E.F. Linton & Epling (toronjil) was collected in September 2008 in Morelos State, Mexico, and identified by Dr. Patricia Castillo-Espa?a. A voucher specimen (26336) was deposited at the HUMO-Herbarium of the Centro de Estudios Ambientales e Investigación “Sierra de Huautla” (CEAMISH) of the Universidad Autónoma del Estado de Morelos, México. Briefly, plant material was dried at room temperature (22 ℃) and subjected to successive macerations with hexane, dichloromethane and methanol (3 times for 72 h at room temperature). After filtration, extracts were concentrated in vacuo at 40 ℃. Tilianin (Figure 1) was obtained by precipitation from liquid methanol extract adding ethyl acetate[10]. Purity of tilianin (＞95%) was confirmed by HPLC method previously developed[14] (Supporting information).

2.2. Drugs

All pure compounds were purchased from Sigma-Aldrich Co. Sodium pentobarbital, WAY-100635, tilianin and methanol extract were dissolved in saline solution (s.s.); flumazenil (GABAA/ BZD receptor antagonist) and diazepam (reference drug) were resuspended in the vehicle (0.2% tween 80 in s.s.). All drugs and the extract were freshly prepared the day of the experiments and administered via esophagi (po.) and/or intraperitoneal (ip.) in a volume of 10 mL/kg body weight. Control animals received the same volume of the vehicle and in the same route of administration.

2.3. Animals

Male Swiss albino mice (25-30 g, Instituto Nacional de Psiquiatría Ramón de la Fuente Mu?íz) were used in pharmacological tests. The animals were kept at constant room temperature [(22±1) ℃] and maintained in a 12 h/12 h light/dark cycle. Experiments were carried out in accordance with the Ethical Committee Guidelines laid down by the local committee regarding the care and use of animals for experimental procedures. The animals were fed ad libitum with standard food and water during the course of the study.

2.4. Pharmacological evaluations

All mice were adapted to manipulation through a daily s.s. injection or esophagi administration for 5 days before the treatments were administered. Anxiolytic or sedative-like effects of several dosages of the methanol extract (10, 30, 100 and 300 mg/kg, ip. or 300 mg/kg po.) and tilianin (30, 100 and 300 mg/kg, ip. or 300 mg/ kg, po.) were compared to that obtained with the reference anxiolytic drug diazepam ED50=0.1 mg/kg (as previously determined)[15]. For analyzing mechanism of action, administration of the GABAA/BZD receptor antagonist (flumazenil, 5 mg/kg, ip.) or 5-HT1Aantagonist (WAY100635, 0.32 mg/kg, ip.) was done 15 min before treatment. For each experimental procedure, animal groups consisted of at least 6 mice. The extract and pure compounds (tilianin or diazepam) were evaluated 60 min and 30 min after administration, respectively, as follows:

2.4.1. Open-field

Sixty minutes after the administration of the extract or tilianin and 30 min after diazepam (previous to the anti-anxiety tests), each mouse was placed into a cage divided in 12 squares (4 cm × 4 cm). The number of squares explored by each mouse in a 2 min interval was registered as ambulatory activity.

2.4.2. Hole board test

Mice were individually placed in the centre of a perforated board and the number of head-dips was registered during a 3 min trial. The perforated board test was made by using a plastic floorboard, 40 cm ×40 cm×25 cm, in which 16 evenly spaced holes (3 cm in diameter) were made; the board was delimited by a glass box having 30 cm in height[16]. The number of explored holes provides a measure of the number of head-dips. Reduced number of head-dips reveals an anxiolytic-like effect.

2.4.3. Exploratory rearings

Each mouse was individually placed on a filter paper-covered floor of a glass cylinder (16 cm in height, 11 cm in diameter). The number of spontaneous rearings on its posterior limbs is counted during the first 5 min. Reduced exploratory rearing showed by naive mice after placement in an unfamiliar environment reveals a sedative effect[17]. Control animals received vehicle. Diazepam was used as positive control in all the tests.

2.4.4. Elevated plus-maze

The method used in this study was modified from Lister (1987)[18]. The apparatus consists of two open arms (25 cm× 5 cm), facing each other, and two closed arms (25 cm×5 cm×15 cm) with an open roof and walls having 40 cm in height. Mice were placed individuallyonto the centre of the apparatus facing an open arm, and the time spent on and entries onto each arm were noted for 5 min by a trained observer who remained unaware of the treatments. The maze was wiped clean with water and dried after each trial. An arm entry was recorded when all four paws of the mouse were in the arm: the total time spent in open-sided arms provides a measure of general anxiolytic activity.

2.4.5. Sodium pentobarbital-induced hypnosis

For this test, mice received 42 mg/kg, ip. of sodium pentobarbital. Each mouse was observed for the onset of uncoordinated movements (sedative phase) and the loss of righting reflex (hypnosis), but also for duration of sleep (the criterion for sleep is defined as the loss of righting reflex). The time between loss and recovery of the righting reflex was recorded as sleeping time[19].

2.5. Pharmacophore modeling of tilianin on GABAA/BZD receptor

Tilianin was analyzed as a possible ligand of GABAA/BZD receptor using a previously described pharmacophoric model generated from the alignment of diazepam, CGS-9896 and diindole, three wellknown GABAA-BZD receptor agonists[20]. The pharmacophore analysis was generated using Discovery Studio (version 3.5, Accelrys, Inc. San Diego).

2.6. Statistical analysis

Results are expressed as mean±SEM. Statistical differences were analyzed using a one-way analysis of variance (ANOVA) followed by Dunnett's test. A value of P ＜ 0.05 was considered significant. A SIGMA STAT? version 2.3 software program was used.

3. Results

In the experimental models, methanol extract and tilianin did not modify the ambulatory activity of mice when tested in the open-field test (Figure 2A). In contrast, the extract and the active metabolite tilianin induced a significant reduction in the number of head-dips from the 30 mg/kg dosage ip. and 300 mg/kg po. (Figure 2B), this effect was observed in a dose-dependent manner in both cases. In a similar manner, a significant diminution in the rearing behavior was observed in mice treated with the extract and tilianin (Figure 2C). These significant anti-anxiety responses resembled the effect observed with diazepam at 0.1 mg/kg (Figure 2).

As shown in Figure 3, the methanol extract of A.mexicana aerial parts and tilianin did not modify the latency to the onset of sedation and hypnosis induced by sodium pentobarbital (Figure 3A and 3B), however the sleeping time was significantly increased in mice receiving 30, 100 and 300 mg/kg ip. and 300 mg/kg po., respectively, of both test samples (Figure 3C). Finally, in order to corroborate the anxiolytic-like effect of test samples, plus-maze model were used. Thus, both extract and tilianin induced significant anxiolytic effect at 30 mg/kg ip. and 300 mg/kg po., compared to control group (Figure 4). Also, it was evaluated the anxiolytic-like effect of the flavonoid tilianin in the presence of flumazenil (a GABAA/BZD antagonist, 5 mg/kg, ip.) and WAY100635 (a 5-HT1Areceptor antagonist, 0.32 mg/kg, ip.) in the elevated plus-maze model. As observed in Figure 4, anxiolytic-like response of tilianin is prevented in the presence of flumazenil but not in the presence of WAY100635, suggesting a GABAergic involvement.

On the other hand, tilianin was analyzed as a possible ligand of GABAA/BZD receptor using a previously described pharmacophoric model[20]. Pharmacophore modeling was performed by alignment of diazepam, CGS-9896 and diindole, three GABAA/BZD receptor agonists (Figure 5A). This model contains seven chemical features: four hydrophobic sites H1-H4 (cyan color), two hydrogen bond acceptors A1 and A2 (green color) and a donor of hydrogen bonds D1 (magenta color). According to the analysis of the structural requirements in the pharmacophoric model for GABAA/BZD receptor agonists, we found that tilianin contains six of the seven chemical features (Figure 5B). The gray sphere (H3, Figure 5B) represents a feature not found in the pharmacophore mapping with tilianin. However, this hydrophobic fragment belongs exclusively to diazepam structure (Figure 5C) and it was not common between the other two compared GABAA/BZD receptors agonists.

4. Discussion

In the present study, it was investigated the anxiolytic-like activity of a polar extract obtained from the A. mexicana aerial parts and one of its major active metabolites identified as tilianin, from which it was also explored the feasible mechanism of action.

A. mexicana is popular in markets of Mexico because of its use as a tranquilizer. As a medicinal remedy, it is common that both allthe aerial parts and only the flowers, fresh or dried, are prepared in boiling water as an infusion or decoction or even as maceration in ethanol to treat anxiety and insomnia[3,4]. In our study, a methanol extract of aerial parts of A. mexicana produced a tranquilizer effect that was not associated to sedative-like response as observed in the open-field test in mice. These results were resembled with an active metabolite that was identified as tilianin obtained as a white-yellowish amorphous solid by spontaneously precipitation of the methanol extract of A. mexicana. The spectroscopic and spectrometric data (IR, NMR1H,13C and GC-MS) of this metabolite were compared with values previously reported[10].

The tranquilizer effect of the extract and tilianin was observed as a significant decrease in the rearing and head-dipping behaviors. These results are in agreement to a recent report that demonstrates anxiolytic-like response in both A. mexicana subspecies mexicana and xolocotziana aerial parts by administration of an aqueous extract at doses of 0.1 to 10 mg/kg, suggesting that over 10 mg/kg is possible to induce sedation and locomotor activity diminution because of a general inhibition in the central nervous system[9]. In our study, the methanol extract and tilianin did not modify the latency to the onset of sedation and hypnosis induced by sodium pentobarbital, however, the sleeping time was significantly increased in mice receiving extract or tilianin. The synergism in the depressant response was observed in a dose-dependent manner, being more effective in the presence of the extract, suggesting that more than one compound is involved in the extract effect even though tilianin. It is important to mention that several flavonoids have been reported into the aqueous and organic extracts of A. mexicana aerial parts[9,13], which might generate synergism to produce depressant activity. This fact also might explain why lower dosage of the aqueous extract produced major sedative-like response than the alcoholic extract. Moreover, it has been described that some flavonoids might not produce anxiolysis[21]; but they may modulate motor movements and locomotion[22]. Besides, it had been reported that lower dosage (3, 9 and 12 mg/kg, ip.) of A. mexicana did not produce sedative effect; on the contrary it induced an anxiogenic-like reponse instead of anxiolytic- by using elevated plus-maze rat model[8]. In that report, authors used an aqueous extract prepared from the leaves of A. mexicana; however, it has been described that aerial parts or even inflorescences are preferred to produce tranquilizing activity[3,4,7]. In our study, the elevated plus-maze model was used to corroborate the anxiolytic-like action of the methanol extract of the A. mexicana aerial parts and its metabolite tilianin as a real alternative for the treatment of depressive issues. In this context, diazepam is a wellknown benzodiazepine used as reference drug; it is a GABAergic agonist and the most widely prescribed class of psychoactive drugs for anxiety, despite the important unwanted side-effects that they produce such as sedation, myorelaxation, ataxia, amnesia, ethanol and barbiturate potentiation and tolerance[23].

Pharmacological studies have reported that the presence of flavonoids, as major components in polar extracts of A. mexicana species, is responsible for the pharmacological properties found among tilianin[9,10,13,14,24]. Although it was described the total flavonoids contents in organic[13] and aqueous extracts of A. mexicana by an HPLC method[9], it has not been found the possible compound(s) responsible for the CNS effects and its mode of action. Also, in neither of them was reported the presence of tilianin[8,9,13]. In this framework, we evaluated the anxiolytic-like effect of the flavonoid tilianin, as one of the major constituents in A. mexicana, in the presence of flumazenil (a GABAA/BZD antagonist) and WAY100635 (a 5-HT1Areceptor antagonist). Since anxiolytic-like response of tilianin is prevented in the presence of flumazenil but not in the presence of WAY100635 suggested that a GABAergic system is involving in its mode of action. It is well known that some flavonoids isolated from plants used as tranquilizers in folk medicine have shown to possess a selective and relatively mild affinity for GABAA/BZD receptors and a pharmacological profile compatible with a partial agonistic action[20,25-28]. Tilianin was analyzed as a possible ligand of GABAA/BZD receptor using a previously described pharmacophoric model[20]. Pharmacophore modeling was performed by alignment of diazepam, CGS-9896 and diindole, three GABAA/BZD receptor agonists. According to the analysis of the structural requirements in the pharmacophoric model for GABAA/BZD receptor agonists, we found that tilianin contains six of the seven chemical features: H1, H2 and H4 (cyan color), two hydrogen bond acceptors A1 and A2 (green color) and a donor of hydrogen bonds D1 (magenta color). The gray sphere (H3, Figure 5B) represents a feature not found in the pharmacophore mapping with tilianin. However, this hydrophobic fragment belongs exclusively to diazepam structure and it was not common between the other two compared GABAA/BZD receptors agonists. This pharmacophore analysis could be a reasonable qualitative prediction of binding of the spatial arrangement of these six chemical features. Last results reinforce the participation of GABAergic system in anxiolytic-like activity of tilianin.

Chemical entities with new or known therapeutic targets are needed to control anxiety more effectively, with less adverse effects and having a neutral impact on known cardiovascular risk factors. Bioactive compounds from medicinal plants are one of the alternatives for the discovery and development of new agents for the treatment of anxiety and related diseases[28]. Thus, flavonoids represent serious candidates for the detection of hits or leads with potential uses in therapeutics due to their beneficial properties in cardiovascular disease among their pharmacological effects as anti-inflammatory, antioxidant, antiviral, neuroprotector and anticarcinogenic agents[29].

In conclusion, current results give evidence that tilianin is one of the bioactive compounds responsible for the central nervous activity of A. mexicana as anxiolytic agent, suggesting the GABAA/BZDs, but not 5-HT1A, receptor involvement.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

We are thankful to Mr. Rubén Luviano, Raúl Cardoso and José Luis Calderón for the technical assistance. This work was partiallysupported by CONACYT 80811, NC12.3280 grant and Faculty of Pharmacy Budgets (FECES 2011 and 2012).

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ent heading

10.1016/S1995-7645(14)60312-6

*Corresponding author: Dr. Samuel Estrada-Soto, Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Colonia Chamilpa, C.P. 62209. Cuernavaca, Morelos, México.

Tel/Fax: +52 777 329 7089

E-mail: enoch@uaem.mx

Foundation project: This work was partially supported by CONACYT 80811, NC12.3280 grant and Faculty of Pharmacy Budgets (FECES 2011 and 2012).