Natural products found from plants have been conveying a vital role among human being since ancient times. An enormous number of scientific reports evidenced of using the medicinal plants as natural remedies. Nowadays, the medicinal plant is used as an alternative to synthetic drugs [1] .

Thunbergia erecta is vigorous, a woody shrub which belongs to Acanthaceae family and 100 species in the genus of Thunbergia found in several places in Bangladesh. In most of the places T. erecta is known as bush clockvine and king’s-mantle [2] . This shrub has small, ovate leaves with entire margins borne opposite on thin, brown stems [3] . The purple flowers have a yellow throat and may appear singly or in small clusters. This plant produces rounded seed capsules that end in a beak. Thunbergia genus has ornamental value and is native to tropical regions of Africa, Western Africa, Madagascar, Australia and South Asia [4] .

Thunbergia species have some bioactive compounds regarding their pharmacological properties already reported. The member of Thunbergia genus is reported to contain alkaloids, glycosides and phenolic compounds such as flavonoids, tannins, phenolic acids, rosmaric acid, feruloylmalic and coumaroylmalic acid, naphthalene, iroidglucosides, benzyl beta glucopyranoside, grandifloric acids, delphinidin and apigenin [5] .

Traditionally, Thunbergia species leaves, stems, and roots used as anti-inflammatory and antipyretics agents [6] . It has been also reported to possess antibacterial activities against gram positive as well as gram negative bacteria such as Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Bacillus cereus, Proteus mirabilis and Streptococcus pyogenes [7] . Thunbergia species also exhibit antinociceptive and antitumor [8] , cytotoxic and antioxidant [9] , carminative [10] , antidiarrheal [11] activities.

Regarding the previous reports and our phytochemical analysis, it has been revealed that T. erecta contains several bioactive compounds which have sedative and anxiolytic activities. The scientific basis of Thunbergia protrudes us to investigate the neuropharmacological activity of T. erecta in the management of central nervous system disorder.

The therapeutic benefits of medicinal plants provide us with traditional remedies from the ancient periods [22] . Thunbergia genus were used traditionally for managing anxiety and numerous behavior disorders. Our rudimentary screening for phytochemicals suggests the availability of alkaloids, glycosides, flavonoids, tannins which may exert neuropharmacological activity. Several phytochemical studies proved that tannins exert sedative activity. Saponins also have potent sedative activity [23] . Moreover, it is already reported that flavonoids have potent sedative and anxiolytic effects [24] .

The median lethal dose (LD₅₀) of METE was found as nontoxic at 4000 mg/kg dose of body weight in mice. Our pharmacological evaluation of sedative and anxiolytic activities proposed that METE exerts as GABA-benzodiazepine receptor interaction in the brain. Several models of sedative and anxiolytic test predominately expressed that METE dose-dependently effects in recent studies. We evaluated open field, hole cross and thiopental sodium-induce sleeping test as the sedative activity. On the contrary, we considered elevated-plus maze, light-dark box, hole-board and marble burying test as anxiolytic effects.

In open field test (Table 2 & Figure 1), the locomotion effect of METE showed that decreased number of squares crossed by mice was dose-dependent activity at second (30 min) to fifth (120 min) time point of observation. According to hole cross test (Table 3 & Figure 2), the behavioral state of mice diminished the number of hole cross (from second to fifth observation) convinced us about the sedative activity of METE. These study also demonstrated that the sedative response of METE effect almost similar to diazepam. The dose-dependent sedative effects also observed in the thiopental sodium-induce sleeping test which is reflected in chronological data in same dose and also when compared between two doses (200 and 400 mg/kg) (Table 4 & Figure 3). This validated model suggests that the activity of TS on gamma-aminobutyric acid (GABA) mediate hyperpolarization of CNS [25] . TS injection dose-dependently reduced the latency period conjointly, prolonging the duration of sleeping time. Typically, diazepam effect demonstrated similar manner as well METE.

The possible anxiolytic activity recognized in-vivo system due to interaction with the GABA_A-BZD receptor. Elevated-plus maze experiment is a reliable and widely most conventional method to determine the anxiolytic behavior in mice model. Recent data recommended that mice treated with METE preferred to spend the most of the time in the open arm rather than the close arm. Anxiolytic effect of METE in Elevated plus-maze test (Table 5 & Figure 4), confirmed the similar manner of diazepam. Light-dark box test (Table 6 & Figure 5), is another popular evaluation approach due to its methodological simplicity. METE treated mice selected light part to spend more time instead of dark part of the light-dark box in a dose-dependent manner. Time spent in light part express the anxiolytic-like behavior of mice. Diazepam treated mice also showed same effects when compared with control. The hole-board test (Table 7 & Figure 6), has used as measure exploratory behavior of mice. Anxiolytic activity upraised to decrease the number of head dipping activity in the hole-board test. METE remarkably diminished the head dipping activity in a dose-dependent manner, thereby suggesting the anxiolytic agent. Marble burying test (Table 8 & Figure 7), is another validated method to investigate the anxiolytic activity in mice. The number of marble burying decreased when treated with METE likewise diazepam in a dose-dependent manner, regarded as an anxiolytic action.

Gamma-amino butyric acid (GABA) is the major inhibitory amino acid neurotransmitter in the mammalian central nervous system. GABA is synthesized from glutamic acid by the enzyme glutamic acid decarboxylase (GAD) and catabolized by the enzyme GABA-transaminase (GABA-T) into succinic semi aldehyde [26] . GABA helps to regulate movement control, sight, anxiety, and many other brain functions. Benzodiazepines enhance responses to the inhibitory neurotransmitter GABA by opening GABA (GABA_A receptors as the main site of action of ligands with anxiolytic activity) activated chloride channels and allowing chloride ions to enter the neuron. This action allows the neuron to become negatively charged and resistant to excitation, which leads to the various anti-anxiety, sedative, or anti-seizure activity seen with these drugs.

However, some plant may contain GABA-T inhibitor which ultimately raise brain GABA level and reduce anxiety. In addition, GABA released into the synapse cleft can be reuptaken into both presynaptic terminals and surrounding glial cells for different purposes, relying on membrane GABA transporters (GAT), GABA taken back up into presynaptic nerve terminal. Hence, Inhibition of the re-uptake of GABA by potent and selective inhibitors of the GABA transporter enhances GABA activity thus reduce anxiety [27] [28] [29] .

In current study benzodiazepine used as standard drug and the exerted neuropharmacological (sedative and anxiolytic) effect of METE convinced us to assume that the METE might follow the mechanism like diazepam on GABA_A subunit.

Our experimental findings represent that several phytoconstituents present in METE demonstrate sedative and anxiolytic activity. These results support that the METE possesses sedative and anxiolytic properties like diazepam which act through binding to benzodiazepines site on GABA-BDZ receptor complex. However, the plasma GABA level measurement and the further investigation of associated physiological indexes (GAD, GABA-T) are suggested to pin-point the mechanism of sedative and anxiolytic action of METE. Additional advance studies are required to identify the active phytoconstituents associated with observed bioactivities in animal behavioral models.

The authors are thankful to Professor Dr. Bidyut Kanti Datta, Chairman, Department of Pharmacy, Stamford University Bangladesh to permit to use the facilities of the Pharmacology and Phytochemistry Laboratory.

All the experimental mice were treated following the Ethical Principles and Guidelines for Scientific Experiments on Animals (1995) postulated by the Swiss Academy of Medical Sciences and the Swiss Academy of Sciences. The Institutional Animal Ethical Committee (SUB/IAEC/17.02) of Stamford University Bangladesh approved all experimental rules.

Not applicable.

This research was not funded by any funding agencies.

AB conceived, planned and supervised the experiment. AB contributed to data analysis and manuscript writing. AH and AAM co-ordinated experimental studies. MSAM examined the data, explained the results and drafted the manuscript. MMRB contributed to manuscript writing and AB finally edited the manuscript. Manuscript was perused and approved by all authors.

The authors declare no conflicts of interest regarding the publication of this paper.

Begum, A., Hossen, A., Moly, A.A., Bhuiyan, Md.M.R. and Shahed-Al-Mahmud, Md. (2019) In Vivo Sedative and Anxiolytic Activities of Thunbergia erecta (Acanthaceae) Leaves Activate Gamma-Aminobutyric Acid (GABA) Mediated Hyperpolarization in Swiss Albino Mice. Pharmacology & Pharmacy, 10, 177-193. https://doi.org/10.4236/pp.2019.104016

META = Methanolic extract of Thunbergia erecta;

ICDDR, B = International Center for Diarrhoeal Disease and Research, Bangladesh;

GABA = Gamma-Amino Butyric Acid;

TS = Thiopental sodium;

CNS = Central Nervous System;

BZN = Benzodiazepine;

GAD = Glutamic acid decarboxylase;

GABA-T = GABA-transaminase;

GAT = GABA transporters.