Evaluation of monoaminergic levels after acute administration of standardized extract of Justicia pectoralis in mice

Edith Teles Venâncioa; Alana Gomes de Souza; Klistenes Alves de Limaa; Michele Albuquerque Jales de Carvalhoa; Denia Alves Albuquerque de Souzaa; João Victor Souza Oliveiraa; Luzia Kalyne Almeida Moreira Leala; Marta Maria de França Fontelesa

Evaluation of monoaminergic levels after acute administration of standardized extract of Justicia pectoralis in mice

ARTÍCULO ORIGINAL

Evaluation of monoaminergic levels after acute administration of standardized extract of Justicia pectoralis in mice

Evaluación de los niveles monoaminérgicos después de la administración aguda de extracto estandarizado de Justicia pectoralis en ratones

Edith Teles Venâncio¹
Alana Gomes de Souza¹
Klistenes Alves de Lima¹
Michele Albuquerque Jales de Carvalho¹
Luzia Kalyne Almeida Moreira Leal^1,2
Marta Maria de França Fonteles^1,2

¹Federal University of Ceara,Faculty of Medicine, Department of Physiology and Pharmacology. Fortaleza - Ceará, Brazil.
²Federal University of Ceara,Faculty of Pharmacy, Odontology and Nursing Department of Pharmacy. Fortaleza - Ceará, Brazil.

ABSTRACT

Introduction: Justicia pectoralis is used as antiinflamatory, antimicrobial, bronchodilator, and sedative, and some studies have shown that it also acts in the Central Nervous System.
Objective: To evaluate effects of aqueous standardized extract of Justicia pectoralis (SEJP) in the monoamine levels of animals to elucidate the underlying neurochemical mechanisms associated.
Methods: Extract was administered intragastrically to male mice at single dose of 200 mg/kg at a volume of (10 mL/kg). One hour after the drug administration, the animals were sacrificed by decapitation, and the prefrontal cortex (PFC), hippocampus (HC) and striatum (ST) were dissected. The High-Performance Liquid Chromatography (HPLC) method was used for determining levels of noradrenaline (NA), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA).
Results: The work showed that SEJP decreased DA, DOPAC, NE (p< 0.01) and 5-HT (p< 0,05) levels in the prefrontal cortex. The basal level of NE in the hippocampus was significantly reduced (p< 0.05) when compared to control group. The treatment also decreased DA (p< 0.001), DOPAC (p< 0.01) and HVA (p< 0,001) levels in the striatum.
Conclusion: The findings showed that SEJP decreased the monoamines levels in the brain areas observed and suggest that Justicia pectoralis has potential clinical applications in the management of psychiatric disorders.

Keywords: plants, medicinal; Justicia pectoralis; biogenic monoamines; neuropharmacology.

RESUMEN

Introducción: Justicia pectoralis se usa como antiinflamatorio, antimicrobiano, broncodilatador y sedante y algunos estudios han demostrado que actúa también en el Sistema Nervioso Central.
Objetivo: Evaluar los efectos de extracto estandarizado de Justicia pectoral (SEJP) en los niveles de monoamina de animales para elucidar los mecanismos neuroquímicos subyacentes asociados.
Métodos: El extracto se administró intragástricamente a ratones machos a dosis única de 200 mg / kg a un volumen de (10 mL / kg). Una hora después de la administración del fármaco, los animales fueron sacrificados por decapitación, y la corteza prefrontal (PFC), hipocampo (HC) y estriado (ST). La cromatografía líquida de alta resolución se utilizó el método de (HPLC) para determinar los niveles de noradrenalina (NA), dopamina (DA), ácido 3,4 - dihidroxifenilacético (DOPAC), el ácido homovanílico (HVA), la serotonina (5 - HT) y el ácido 5 - hidroxiindolacético (5-HIAA).
Resultados: El trabajo mostró que SEJP disminuyó DA, DOPAC, NE (p< 0,01) y 5-HT (p< 0,05) en la corteza prefrontal. El nivel basal de NE en el hipocampo fue significativamente reducido (p< 0,05) en comparación con el grupo control. El tratamiento también disminuyó la concentración de DA (p< 0,001), DOPAC ( p< 0,01) y HVA (p< 0,001) en el cuerpo estriado.
Conclusiones: Los hallazgos mostraron que la SEJP disminuyó los niveles de monoaminas en las áreas cerebrales y sugieren que Justicia pectoralis tiene potenciales aplicaciones clínicas en la gestión de los trastornos psiquiátricos.

Palabras clave: plantas medicinales; Justicia pectoralis; monoaminas biogénicas; neurofarmacología.

INTRODUCTION

The use of natural products as medicines has been described throughout history in the form of traditional medicines, remedies, potions and oils with many of these bioactive natural products still being unidentified.^1,2 Justicia pectoralis Jacq., Acanthaceae, popularly known in northeastern Brazil as chambá, is traditionally used in the treatment of respiratory tract diseases such as asthma, cough and bronchitis.³ In Cuba the tea from the leaves ofJusticia pectoralis is used as tranquilizer and sedative.⁴

Its aerial root and leaves contain various chemical constituents such as flavonoids, saponines, tannins.^4,5 Also, coumarin (1,2-benzopyrone) and umbelliferone (7-hydroxycoumarin) were found in high concentrations.⁶ This herb has been described as having a large number of actions, for example, as antinociceptive and anti-inflammatory, ⁷ antimicrobial⁸ and bronchodilator.⁵ Some studies have shown that Justicia pectoralis acts in the Central Nervous System (CNS) such as anxiolytic.^9,10 However, the characteristic of this involvement should be determined.

In fact, some natural products including compounds and extracts from plants have been shown to interfere with monoamine levels in central nervous system.¹¹ The central monoaminergic system, including serotonin (5-hydroxytryptamine, 5-HT), noradrenaline (NA), dopamine (DA) and their metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), has been widely implicated in the pathophysiology and therapeutic strategies for emotional disorders, such as anxiety.¹²

In order to elucidate the underlying neurochemical mechanisms associated with the administration of a standardized extract of Justicia pectoralis (SEJP) on adult animals. It examined the monoamine contents (DA, NA and 5-HT) and their metabolites in the prefrontal cortex (PFC), hippocampus (HC) and striatum (ST). These are discrete brain areas related with emotion.^13,14

METHODS

Male Swiss mice (25-35 g) were obtained from the Animal House of the Federal University of Ceara, Brazil. They were kept in a room with a controlled temperature of 25±2°C, and a 12 h light/dark cycle with food and water ad libitum, except during the experiment. The project was previously approved by the Animal's Ethics Committee, of the Faculty of Medicine of the Federal University of Ceara, under the protocol number 14/09.

DRUGS

Standard Coumarin and Umbelliferone were obtained from Sigma-Aldrich. Acetonitrile, methanol and tetrahydrofuran were High-Performance Liquid Chromatography (HPLC) grade from J. T. Baker and absolute ethanol was from Santa Cruz. Triethylamine and phosphoric acid were analytical grade and from Merck and Dinâmica Química, respectively.

Aerial parts of Justicia pectoralis were collected in Fortaleza, Ceara State, Northeast of Brazil. Voucher specimens (nº 16.079) were deposited at the Prisco Bezerra Herbarium of the Federal University of Ceará (UFC), Fortaleza, Brazil, and authenticated by Prof. Edson P. Nunes, Department of Biology, UFC.

EXPERIMENTAL PROTOCOLS

Animals were treated, intragastrically, with distilled water (control) or SEJP 200 mg/kg at a volume of 10 mL/kg. The dose chosen was considered safe according to a toxicity study¹⁵and it had previously shown antinociceptive, antiinflammatory and bronchodilator activities⁵ . One hour after the drug administration, the animals were sacrificed by decapitation, and the PFC, HC, ST were dissected on ice as described below.

For the isolation of the PFC, the anterior portion of frontal lobes (about 1.5 mm from the olfactory bulb) was removed, and bilateral section was made with the aid of a micro-dissection scissor.¹⁶ After removal of the PFC by tracking the median sagital fissure, the cerebral cortical layer was removed from the leptomeninges, and cut over all its front-occipital extension. The cortex was folded to the side, exposing the hippocampus and striatum which was also dissected. After dissection, the areas were weighed and stored at -70 ^oC until analyses.

NEUROCHEMICAL ANALYSIS

The brain tissue regions were homogenized at room temperature in a solution of 0,1 M perchloric acid. The homogenates were centrifuged at 20.375 x g, for 15 min at 4^oC and supernatants were collected and filtered through a membrane (Millipore 0.2 µm) purchased from Millipore Apparatus^© (Bedfort, MA, USA) for determining the levels of monoamines.

Levels of NA, DA, DOPAC, HVA, 5-HT and 5-HIAA were measured by HPLC. Monoamines were separated on a 5 µm particle size column C18 with a 250-mm-long, 4.6 mm caliber (Thermo Scientific) connected to a carbon electrode set at a potential of + 0.85V relative to the Ag/AgCl reference electrode, in turn connected to an L-ECD-6A amperometric detector (from Shimadzu, Japan) in the HPLC system.

The mobile phase consisted of 0.163 M citric acid, 0.69 M octanicsufanic sodium acid (Sigma), acetonitrile 4 % v/v (JT. Baker) e tetrahydrofuran 1.7 % v/v (Carlo Erba). External standards of DA, DOPAC, HVA, 5-HT, 5-HIAA, and NA were dissolved in 0.1M perchloric acid and run simultaneously with each experiment. Tissue contents of the monoamines are presented as ng/g of tissue.

STATISTICAL ANALYSIS

Statistical analysis of differences in the levels of DA, DOPAC, HVA, 5-HT, 5-HIAA and NA was performed by t test Student. Values are expressed as mean ± S.E.M. The accepted level of significance for tests was p< 0.05.

RESULTS

JUSTICIA PECTORALIS EXTRACTION AND IDENTIFICATION OF THE MAIN CONSTITUENTS

The fresh plant material was dried and submitted to extraction in the Pharmacognosy Laboratory of the Federal University of Ceara, and to be used in this work, it was separated by HPLC .¹⁰ The identification of the coumarin and umbelliferone, main constituents, in the extract of Justicia pectoralis was performed by HPLC. Standards: umbelliferone (rt= 4.87 min) and coumarin (rt= 5.96 min) (Fig. 1).

BRAIN DOPAMINE AND METABOLITES

Figure 2 shows the effect of SEJP on dopamine levels and it metabolites (n= 7-9). It observed a significant decrease in DA [control: 776.7 ± 86.98; SEJP200: 424.6 ± 34.79] and DOPAC [control: 8.700 ± 0.99; SEJP200: 4.100 ± 0.59] levels in the prefrontal cortex, while no significant change was observed in HVA levels [cont.: 40.40 ± 7.06; SEJP200: 45.05 ± 5.03].

No significant differences were found in the levels of DA in the hippocampus [control: 659.4 ± 113.5; SEJP200: 478.7 ± 81.63] and DOPAC [control: 83.07 ± 18.11; SEJP200: 45.51 ± 9.29], but there was an increase in the level of HVA [control: 875.5 ± 84.58; SEJP200: 3190 ± 448.5].

DA, DOPAC and HVA levels in the striatum treated with SEJP 200 mg/Kg were significantly reduced when compared to control group: DA [control: 1821 ± 147.5; SEJP200: 832.9 ± 97.49]; DOPAC [control: 2658 ± 226.7; SEJP200: 1596 ± 175.8]; HVA [control: 973.6 ± 98.94; SEJP200: 472.8 ± 55.28].

BRAIN NORADRENALINE

Figure 3 shows the effect of SEJP in the noradrenaline levels (n= 7-9). The treatment also significantly decreased NE levels in the prefrontal cortex [control: 2324 ± 110.8; SEJP200: 1929 ± 36.26].

The basal level of NE in the hippocampus was (1654 ± 114.4) ng/g tissue in the control group and 1271 ± 103.6 ng/g tissue in the SEJP200 group. Student t test revealed significant differences between groups in basal level.

No significant differences were found in the levels of NE in the striatum [control: 1777 ± 115.5; SEJP200: 1969 ± 89.71].

BRAIN SEROTONIN AND THEIR METABOLITES

Figure 4 shows the effect of SEJP on the levels of serotonin and its metabolites (n= 7-9). 5-HT levels were significantly decreased in the prefrontal cortex [control: 52.67 ± 6.53; SEJP200: 35.03 ± 1.93], but there was no significant difference in the levels of the metabolite 5-HIAA [control: 282.0 ± 27.99; SEJP200: 241.3 ± 22.1].

The treatment did not alter 5-HT levels in the hippocampus [27.57 ± 10.23; SEJP200: 35.94 ± 13.04], but an increase was found in 5-HIAA levels [366.1 ± 33.87; SEJP200: 1464 ± 79.21]. Also, in the animals treated with SEJP was not observed some alteration in the 5-HT levels in the striatum [control: 991.5 ± 133.8; SEJP200: 671.9 ± 94.61], but an increase was found in the 5-HIAA levels [control: 314.5 ± 22.94; SEJP200: 1467 ± 150.3].

DISCUSSION

Sources of stress or danger (called stressors) provoke fear, a basic emotion, and generate immediate responses, such as escape, freezing, or aggression. Stress can also lead to anxiety, a more complex state that is considered a secondary emotion because it occurs when the stressor is absent or not clearly identified.^17,18

In adult rats, chronic stress causes alterations in various systems, such as dopaminergic, serotonergic and noradrenergic, in different brain areas.^19,20

The monoaminergic systems play important roles in maintaining homeostasis by inducing various physiological and behavioural changes.²¹ Stress-induced alterations in the monoaminergic systems with subsequent enhancement of oxidative load are associated with a wide range of central and peripheral disorders.^19,22 The monoaminergic neurotransmitters (dopamine, norepinephrine and serotonin) and their metabolites, exert major influence on brain circuits implicated in the regulation of mood.^23,24

A main challenge in systems neuroscience is to determine the underlying neural circuitry and associated neurotransmitters and receptors involved in psychiatric disorders, such as anxiety and depression. A focus of many of these studies has been specific brainstem nuclei that modulate levels of arousal via their ascending monoaminergic projections, e.g. the serotonergic dorsal raphé, noradrenergic locus ceruleus and cholinergic laterodorsal tegmental nucleus.²⁵

In this study, we demonstrated the effects of administration of SEJP in mice with a special consideration to monoamines levels in different brain areas.

It perceives that after the SEJP acute administration, dopamine levels and its metabolite (DOPAC) decreased in the prefrontal cortex and striatum. In the hippocampus, a significant increase was observed in the HVA levels, while in the striatum, the level was reduced.

This antidopaminergic effect of SEJP could corroborate to the strong anxiolytic influence of the extract in the plus maze and light/dark tests, ¹⁰ as it is known from clinical practice that small doses of neuroleptics are effectively used as sedative and anxiolytics in the states of strong emotional hyperarousal.²⁶

Impairments in the dopamine system result from dopamine dysfunctions in the substantia nigra, ventral tegmental region, striatum, prefrontal cortex, and hippocampus.^27,28 The "original dopamine hypothesis" states that hyperactive dopamine transmission results in schizophrenic symptoms. ^29,30 Some studies have identified differences in dopamine contents in the prefrontal cortex and hippocampus between schizophrenia patients and neuropsychiatric healthy control subjects.³¹ Our study also showed that the analysis of the concentration of NE of animals treated had a reduced level of NE only in the cortex and hippocampus.

Taken together, this evidence suggests that an alteration in the noradrenergic system be involved in the regulation of energy levels, vigilance, reactivity, and executive function. The functions of NA and DA are closely linked. The effects of NA, 5-HT and DA overlap in the brain and all three transmitters are implicated in the symptoms of depression.³²

Moreover, in this work a decrease of 5-HT concentration was observed in prefrontal cortex area, while levels of 5-HIAA were markedly increased in the hippocampus and striatum.

The serotonin system can be seen to play an important role in stress and anxiety and/or fear, because evidence suggest that repeated mild stress increases hypothalamic 5-HT turnover and 5-HIAA concentration is low in the cerebrospinal fluid of animals that display aggressive behavior, this way, SEJP could be used in the reduction of those behavior symptoms.

Conflicto de interés

Los autores declaran que no existe conflicto de interés.

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Recibido: 13 de noviembre de 2018.
Aprobado: 10 de enero de 2019.

Alana Gomes de Souza
Federal University of Ceara,Faculty of Medicine, Department of Physiology and Pharmacology. Fortaleza - Ceará, Brazil.

Correo electrónico: alanagomesdesouza@gmail.com

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Copyright (c) 2019 Edith Teles Venâncioa, Alana Gomes de Souza, Klistenes Alves de Limaa, Michele Albuquerque Jales de Carvalhoa, Denia Alves Albuquerque de Souzaa, João Victor Souza Oliveiraa, Luzia Kalyne Almeida Moreira Leala, Marta Maria de França Fontelesa

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