In Vivo Sedative and Anxiolytic Activities of Thunbergia erecta ( Acanthaceae ) Leaves Activate Gamma-Aminobutyric Acid ( GABA ) Mediated Hyperpolarization in Swiss Albino Mice

Background: Thunbergia erecta (Acanthaceae) is the most abundant medicinal plant in different parts of Bangladesh where it is known as “nilghonta”. It has been used as traditional medicine for insomnia, depression and anxiety management. However, no scientific evidence of T. erecta belonging to neuropharmacological activity has been reported. The aim of present study was to investigate in vivo sedative and anxiolytic activities of methanol extract from the leaves of T. erecta in Swiss Albino mice. Methods: Sedative activity of METE was investigated using open field, hole cross and thiopental sodium-induced sleeping time test model whereas anxiolytic activity was screened by elevated-plus maze, light-dark box, hole-board and marble-burying test method in mice at 200 and 400 mg/kg doses. The acute toxicity study and phytochemical analysis of METE also carried out. Diazepam used as the positive control for the following behavioral pharmacology test. Results: METE exhibited significant (p < 0.001) sedative effect by decreasing the number of (square and hole) crossed by mice in open field and hole cross tests in a dose-dependent (200 & 400 mg/kg) manner. In thiopental sodium-induced sleeping time test METE significantly (p < 0.001) shortened the latency period and prolonged the sleeping time in a dose dependent (200 How to cite 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 Received: February 27, 2019 Accepted: April 15, 2019 Published: April 18, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access

of METE was investigated using open field, hole cross and thiopental sodium-induced sleeping time test model whereas anxiolytic activity was screened by elevated-plus maze, light-dark box, hole-board and marble-burying test method in mice at 200 and 400 mg/kg doses.The acute toxicity study and phytochemical analysis of METE also carried out.Diazepam used as the positive control for the following behavioral pharmacology test.Results: METE exhibited significant (p < 0.001) sedative effect by decreasing the number of (square and hole) crossed by mice in open field and hole cross tests in a dose-dependent (200 & 400 mg/kg) manner.In thiopental sodium-induced sleeping time test METE significantly (p < 0.001) shortened the latency period and prolonged the sleeping time in a dose dependent (200

Introduction
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]  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.

Plant Material Collection and Extraction
Plant material (leaves of T. erecta) was procured from Dhaka (Gazipur region), Bangladesh during 21 st July 2018.Responsible scientific officer in National Harberium of Bangladesh accomplished identification with a DACB no.45803 and a specimen was kept for future reference.
The stem and other adulterants were removed from the leaves of T. erecta.
The fresh leaves were separated, rinsed and allowed for air drying at ambient temperature (25˚C ± 2˚C) until the leaves became dry for grinding.The dried leaves were grounded to the coarse powder by dint of blender equipment and before grinding of the sample, the grinder was completely cleaned to restrict contamination with any other materials grounded beforehand.Maceration of 300 g fresh powdered leaves in 3 liters methanol was performed for seven days under occasional stirring.Seven days later, filtration of the mixture followed by concentrating using rotary evaporator (<40˚C) yielded 30.8 g (yield 10.2%) of semisolid methanol extract of Thunbergis erecta (METE).

Drugs and Treatments
The Mice were separated into four different groups (n = 10) designated as con-

Phytochemical Screening
Freshly ready crude extract of T. erecta was used for the quantitative analysis of presence of flavonoids, glycosides, tannins, carbohydrates and alkaloids following the standard procedure [12].

Acute Toxicity Study
Animals were separated into several groups having (n = 10) mice in each group.
Treatment was given orally to the mice at the doses of 1000, 2000, 3000 and 4000 mg/kg.After gavages, mice were preserved in different cages and given food as well as water as per need.Those mice were placed under observations for the post 72 hours of oral gavage to find out anomalies in behaviors, allergic reactions, and mortality [13].

Sedative Activity Test 1) Open Field Test
Special apparatus made of plywood field with black and white colored squares This experiment was conducted at ambient temperature under the illumining condition.In this test, after treatment (described in Drugs and Treatment section) a mouse was kept in the middle of cage.The number of explorations by the mice was enumerated for 3 mins at 0, 30, 60, 90 and 120 mins afterwards the treatments [14].

2) Hole Cross Test
This model consists of a wooden cage with the middle partition which contains a hole of 3 cm diameter.The size of cage used was 30 × 20 × 14 cm 3 with 7.5 cm height.Mouse placed one end of the cage was allowed to cross the hole for going one chamber to another adjacent chamber.Then the number of hole cross was recorded for 3 mins at 0, 30, 60, 90 and 120 mins for each mouse [15].

3) Thiopental Sodium-Induced Sleeping Time Test
The mice were randomly grouped into three groups control, test groups and positive control where each group consisted of ten mice [16].The test groups were provided with METE orally, the control group was treated with 0.9% saline water.Mice of positive control group were treated with diazepam (1 mg/kg).
Thiopental sodium (40 mg/kg, i.p.) used as sleeping agent was given to all experimental animals thirty (30) mins later of sample or 15 mins later of standard the latent period (interval between Thiopental sodium administrations and loss of righting reflex) and duration of sleeping (interval between the loss and recovery of righting reflex).

Anxiolytic Activity Test 1) Elevated Plus-Maze Test
The elevated plus maze test (EPM) was carried out to determine the anxiolytic behavior of mice.The apparatus was constructed of two uncovered sides (16 × 5 × 12cm) and two closed sides (16 × 5 × 12 cm) which had 50 cm elevated from the floor with an open roof [17].Each mouse was put in the middle of the apparatus where the head of the mouse was positioned towards the uncovered sides.
Effects related to the behavior of the mouse were watched for the duration of 5 mins free exploration with different parameters (time period spent in uncovered arms and closed arms).

2) Light-Dark Box Test
This test was used to determine anxiolytic-like effects in mice [18].This apparatus consists of a completely automated box to be observed by the investigator.A rectangular box (46 × 27 × 30 cm high) that has opening at top and is separated into a tiny (18 × 27 cm) area and a big (27 × 27 cm) area along with an unveiling door (7.5 × 7.5 cm) situated at the middle of the divider at the ground level.One chamber was colored black and made dark but the large part was marked white and glaringly enlightened by a 60-W light source.The time spent in the illuminated and dark compartment recorded for 5 mins.The ambience was kept dark during the experiment.This test capitalized the conflict occurred between the propensity of mice to probe a new ambience and bright light phobia of mice [19].

3) Hole-Board Test
A wooden box with (40 cm × 40 cm × 25 cm) size and sixteen equally distant holes of 3 cm diameter situated upon the floor was used as appliance [20].The middle of individual cleft was 10 cm away from the surrounding wall of the box.
The basement of the box is situated 15 cm upwards the floor and separated into squares of (10 cm × 10 cm) with a water-protective marker.After 15 mins later of treatment with diazepam or 30 mins later of treatment with METE or vehicle each mouse placed at the middle of the hole-board and the number of head dipping by mouse was counted for next 5 mins.The dipping of head was considered for scoring if either eye became concealed into the hole.

4) Marble Burying Test
Mice were separately kept in cages made of glass material with the selected bedclothes for 30 min (habituation period) and afterwards same were put into different cages for anticipation [21].Twenty-five (25) glass marbles were situated at evenly maintained 7 cm aside on 4 cm strata of lying stuff in the cages.
Experimental mice previously habituated with the cage environments.The marble burying test was accomplished by putting each mouse in the cage and the count of marbles greater than two-thirds clothed with bedclothes were counted.
After the end of each trial, bedclothes and cages were changed by new sets and marbles were cleaned with water, dried by using towel of paper, and left to achieve to ambient temperature.

Statistical Analysis
The output of the work has been shown as mean ± SEM.To calculate the statistical significance one way ANOVA followed by Dunnett's post hoc test by SPSS 20 program was employed.Statistics were considered significant compared to the control group at p < 0.001, p < 0.01 and p < 0.05.

Phytochemical Screening
The preliminary phytochemical screening revealed the presence of alkaloids, glycoside, flavonoid carbohydrate, saponin and tannin in methanol extract of leaves of Thunbergia erecta (Table 1).

Acute Toxicity Study
After oral administration of 1000 -4000 mg/kg MESC showed no hypersensitive symptoms or fatality in mice over the observation time period (72 hr).This nontoxic profile of METE made us certain to select the dose (200 and 400 mg/kg) for this study.

Open Field Test
The count of squares elapsed decrement by mice movements concealed significantly (p < 0.01) by fourth (90 min) and fifth investigation (120 min) time at 200 and 400 mg/kg (Figure 1).We observed that the sedative effect achieved by the diazepam (1 mg/kg) taking group, showed parallel modification with METE (Table 2).

Hole Cross Test
Reduction of the count of holes crossed was significant (p < 0.001) from third observation (60 min) and remained at fifth observation (120 min) period which was comparable to vehicle treated group (Figure 2).In addition, METE and the positive control group (diazepam 1 mg/kg) revealed a similar pattern of modification (Table 3).

Thiopental Sodium-Induced Sleeping Time Test
This test showed significant (p < 0.001) reduction in sleep onset.Moreover, significant (p < 0.001) rise in term of total sleeping period was noticed in mice at the doses of 200 and 400 mg/kg when comparison was done with the control group (Figure 3).Diazepam, used as positive control resulted an analogous effect to that of METE (

Elevated Plus-Maze Test
In this test mice opted the open side of the plus-maze when treated with METE (Figure 4).The frequency of time used in open side at 200 and 400 mg/kg were statistically significant (p < 0.001) and comparable to control.The positive control group (diazepam 1 mg/kg) showed the similar effect to that of METE (Table 5).

Light-Dark Box Test
In this test, the time stayed in light parts significantly (p < 0.001) aggravated at 200 and 400 mg/kg when compared with the control group (Figure 5).The same effects were noticed in mice treated with diazepam (1 mg/kg) (Table 6).

Hole-Board Test
In this test, the propensity for head dipping significantly (p < 0.001) decreased at 200 and 400 mg/kg of METE when compared with the control group (Figure 6).
The similar effects were viewed in animal examined with diazepam (1 mg/kg) (Table 7).

Marble-Burying Test
In the marble-burying test, the behavioral condition of animal significantly (p < 0.001) decreased by the count of burying at 200 and 400 mg/kg of METE (Figure 7) when measured with control.Diazepam, as the positive control exerted the same effects as METE (Table 8).

Discussion
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 (LD 50 ) 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.

Pharmacology & Pharmacy
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.

Conclusion
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.

(
72 cm × 72 cm × 36 cm) were used in open field method.The basement of open field appliance consists of cardboard separated into 16 squares (18 cm × 18 cm).

Figure 1 .
Figure 1.The sedative effect of METE on the open field test (number of square crossed at 0 min, 30 mins, 60 mins, 90 mins and 120 mins) in mice.

Figure 2 .
Figure 2. The sedative effect of METE on the hole cross test (number of square crossed at 0 min, 30 mins, 60 mins, 90 mins and 120 mins) in mice.

Figure 3 .
Figure 3.The sedative effect of METE on the Thiopental sodium induce sleeping test (Latency time and sleeping time) in mice.

Figure 4 .
Figure 4.The anxiolytic effect of METE on elevated plus-maze test (Time spent in open arms and Time spent in close arms) in mice.

Figure 5 .
Figure 5.The anxiolytic effect of METE on light-dark box test (Time spent in light part and Time spent in dark part) in mice.

Figure 6 .
Figure 6.The anxiolytic effect of METE on hole board test (number of head dips) in mice.

Figure 7 .
Figure 7.The anxiolytic effect of METE on marble burying test (number of marble burying) in mice.

Table 2 .
The sedative effect of METE on the open field test in mice.

Table 3 .
The sedative effect of METE on hole cross test in mice.

Table 4 .
The sedative effect of METE on thiopental sodium-induced sleeping time test in mice.

Table 5 .
The anxiolytic effect of METE on elevated plus-maze test in mice.

Table 6 .
The anxiolytic effect of METE on light-dark box test in mice.

Table 7 .
The anxiolytic effect of METE on hole-board test in mice.

Table 8 .
The anxiolytic effect of METE on marble-burying test in mice.