Introduction

Anxiety is a prominent mental disorder that affects a large number of populations globally. The incidence is higher among women compared to men. Studies have shown that the global pandemic such as covid-19 has contributed to the rise of the disorder among the population [1,2,3]. This condition induced autonomic and neuroendocrine activation which is shown in behavioural activities such as change in feeding habit, fight or flight for defensive purposes. [2, 4, 5]. The brain area responsible for this adaptive function such as expression of fear, defensive behaviour and aggression are the hippocampus and amygdala [2]. The roles of various neurotransmitters in the pathophysiology of anxiety disorders are significant. For example, the decrease in the inhibitory signalling by γ-aminobutyric acid (GABA) or increased excitatory neurotransmission by glutamate has been shown to cause an increase in activity of emotional processing in brain regions of patients with anxiety disorders [6, 7]. This pathological state affects adaptive function, quality of life and productivity at work [1]. Anxiety disorder being a chronic disease requires management for life and most clinically available drugs come with some untoward effects that make adherence to medication regimen difficult. Hence, this study was aimed at searching for new molecules with more tolerable profile [8, 9].

Medicinal plants has been used locally in the time past in many Nations to manage these behavioural disorders such as anxiety, depression, and amnesia [10]. The folkloric uses of the plant V. unguiculata spp dekindtiana have been investigated and documented in literature, for example, the antidepressant effect, the sedative and other behavioural potentials of the extract from this plant have been investigated and reported [11, 12]. The analgesic potential of the plant has been investigated and documented to support the local use of the plant in the management of pain and migraine [13]. The reports from literature indicated that methanol extract and fractions of the plant may contain some active principles that may interfere with the transmission of various neurotransmitters modulating these behaviours.

Material and Methods

Chemicals and Reagents

The chemical agents used in this study included: diazepam (Roche, Basel, Switzerland); cyproheptadine, yohimbine, (Sigma Chemicals Co, St. Louis, Missouri, U.S.A.); flumazenil (Hikma Pharmaceuticals, Portugal), normal saline (Unique pharmaceuticals, Lagos, Nigeria).

Extract Preparation

The process of extraction has previously been reported (2). Briefly, the leaves were air-dried and later pulverised into powder which was extracted with methanol and later fractionated into n-Hexane, butanol, ethylacetate and aqueous fractions. The residue of the extract and fractions were obtained by evaporation to dryness in rotary evaporator. The dry extract and the fractions were stored in the refrigerator until use.

Animals

A total of 80 Swiss mice (20.6 ± 1.5 g) were obtained and were maintained under natural day and night, fed with standard pellets from UAC Foods, Nigeria Limited and were allowed free access to water. The ethical guidelines for the use of animals in research given by the National Committee for Research Ethics in Science and Technology (NENT), 2018, were followed. The approval number obtained from Obafemi Awolowo University, Ile-Ife, Nigeria, for this work was PHP11/12/H/2765. The animals were randomised into five groups (n = 5) for extract, n-Hexane, butanol, ethylacetate and aqueous fractions. Group 1 received 2% Tween 20 in 0.9% saline solution orally, and groups two to four were treated orally with extracts and fractions while group 5 received diazepam (DPM), (1 mg/kg) intraperitoneally.

Effects of the Extract and Fractions of V. unguiculata on Mice Using Elevated Plus Maze

The maze was made up of two arms (uncovered) with dimensions of 35 × 6 cm and two arms (covered) with dimensions of 35 × 6 × 15 cm in a plus sign arrangement and was raised to 40 cm above the floor. This model was used for the assessment of the anxiolytic potential of the methanol extract and fractions. Each mouse was positioned in the middle of the maze to face the uncovered arm. The mouse was permitted to move around inside the maze for 5 min and the number of visits into both arms as well as the time spent in each arm were documented [14, 15, 16]. The animals were administered with the vehicle (10 ml/kg, p.o.) or methanol extract of V. unguiculata (50, 100 and 200 mg/kg, p.o.) 60 min before their placement at the centre of the maze. The standard anxiolytic agent was administered intraperitoneally (i.p.) thirty minutes before placing the mouse on the maze. The preference of visits to the uncovered arm compared to enclosed part of the maze was taken as an indication of anxiolytic activity of the extract (VUM) [17]. The same process was repeated for each of the fractions at 50, 100 and 200 mg/kg (N-hexane fraction (VUH), ethylacetate fraction (VUE) and butanol (VUB)). 10 ml/kg of the vehicle (2% Tween 20 in 0.9% saline solution) served as negative control while 10 ml/kg of 0.9% saline solution served as control for aqueous fraction (VUA). Diazepam (1 mg/kg i.p.) served as standard reference drug. The mechanisms of the effects exhibited by extract and fractions were delineated by intraperitoneal injection of flumazenil (3 mg/kg), cyproheptadine (0.5 mg/kg) and yohimbine (1 mg/kg).

The open arm avoidance index (OAAI) was calculated using the formula: OAAI = 100–(% period stayed on the open arm + frequency of visit to the open arm)/2 [18, 19].

Effects of the Extract and Fractions of V. unguiculata on Mice Using Hole-Board Model

The model was a flat chamber made of wood with dimensions of 40 by 40 by 25 cm) (l × b × h) containing sixteen equidistant cavities of 1 cm in diameter and 2 cm in depth. One mouse was positioned at one end of the board and permitted to move unhindered from one end of the board to the other. The head dip into holes was an indication of exploratory behaviour of the mice [20]. The extract was administered orally to the mouse for 1 h before observing the frequency of head dip for 5 min. The doses employed in this experiment were 50, 100 and 200 mg/kg. The same protocol was employed for each of the fractions. The DPM (1 mg/kg, i.p.) was employed as standard reference anxiolytic agent while 2% Tween 20 in 0.9% saline solution served as control for extract and fractions except the aqueous fraction which had 0.9% saline solution as vehicle. Yohimbine (1 mg/kg, i.p.), cyproheptadine (0.5 mg/kg,i.p.) and flumazenil (3 mg/kg, i.p.) were antagonists administered, respectively, to investigate the possible mechanism of action.

Statistical Analysis

The data were expressed as mean ± S.E.M and analysed using one-way analysis of variance. Student Newman–Keuls post hoc test determined the level of significance (p < 0.05).

Results

The result showed that there was an increase in both the frequency of entry and the time spent at the uncovered arm of the maze. The VUM at 50 and 100 mg/kg (Fig. 1) induced an increase in the frequency of the number of entries/visits into and period of stay at open arm of the maze compared to the control. The anxiolytic effect of the extract (VUM) at 50 and 100 mg/kg was significant compared to control [VEH: vehicle 10 ml/kg (2% Tween 20 in 0.9% saline solution)], F(4.20) = 21.3, p < 0.05; F(4.20) = 27.9, p < 0.05. This anti-anxiety effect induced by the extract at 100 mg/kg was similar of diazepam (DPM 1 mg/kg, a standard anxiolytic agent) (Fig. 1). The butanol fraction at 50 mg/kg of VUH fraction revealed an increase in the frequency of entries and time spent in the open arm compared to control (Fig. 2) (F(4,20) = 8.0, p < 0.05; F(4,20) = 14.3, p < 0.05). The VUH at 100 mg/kg in hole-board model revealed an increase in exploration behaviour of the animal (Fig. 5C) indication of anxiolytic-like effect (F(4,20) = 12.1, p < 0.05). The ethylacetate fraction (VUE), 50 and 100 mg per kg (Fig. 3) raised the mean number of visits into open arm in mice and were found to be significant compared to the control F(4, 20) = 13.6. In hole-board model (Fig. 5C), VUE 50, 100 and DPM 1 mg/kg raised the mean number of head dips significantly (F(4, 20) = 21.1, p < 0.05). In Fig. 4 panel A and C, doses of 50 and 100 mg/kg of VUA enhanced the frequency of visits and period of stay in the open arms but100 mg/kg of this fraction increased the frequency of the number of head dips in this model (Fig. 5 panel D). The possible mechanism of action of anxiolytic effect of these fractions was evaluated using flumazenil, cyproheptadine and yohimbine as shown in Figs. 6, 7 and 8. Flumazenil reversed the anxiolytic effect of VUM while the effect induced by VUE 100 mg/kg was reversed by cyproheptadine in the hole-board model. Cyproheptadine and Yohimbine reversed the increase in the mean number of entries and the time spent in the open arms in both VUM 100 and VUE at 100 and 200 mg/kg in the EPM model. The reversal of the anxiolytic effect of the VUE showed an anxiety index of 51.8 ± 1.7, 43.6 ± 2.8 and 72.2 ± 1.8, respectively. The anxiety index due to VEH (control) was 63.4 ± 0.7 while that of DPM (the standard anxiolytic drug) was 43.0 ± 1.5. The anxiety index for VUH at 50, 100 and 200 mg/kg were 52.4 ± 1.4, 57.8 ± 3.0 and 58.4 ± 1.9, respectively. The aqueous fraction showed 54.4 ± 2.1, 43.6 ± 1.5, 67.4 ± 4.0, respectively, at similar dosage levels. The higher the anxiety index, the less the anxiolytic value of the agent. It has been postulated that if any group is at least10 points less than the control’s (vehicle), then the sample is said to be anxiolytic. Conversely, if the sample is at least 10 point greater than the control, then such agent is said to show anxiogenic effect. The result, therefore, showed that the VUM at 50 and100, VUH at 50; VUE at 50 and 100; VUA at 50 and 100 mg/kg and DPM possessed anxiolytic effect.

Fig. 1
figure 1

Anxiolytic effect of VUM and DPM showing notable difference compared to VEH. Each bar represents mean ± standard error of mean. *significant p < 0.05 compared to VEH. A Time spent on open arms; B Number of entries into open arms; C Open arm avoidance index (IOAA) in mice

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Fig. 2
figure 2

VUH showed anxiolytic effect at 50 mg/kg. Each bar represents mean ± S.E.M.VUH: *significant p < 0.05 compared to VEH; #significant compared to DPM. A Time spent in open arms; B frequency of visit into the open arms; C Effect of VUH on IOAA in mice

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Fig. 3
figure 3

Anxiolytic effect of VUE. Each bar represents mean ± standard error of mean.VUE at 50 and 100 mg/kg exhibited. *significant p < 0.05 compared to VEH; #significant compared to DPM. A Period of stay in the open arms; B Frequency of visit into the open arms; C Effect of VUE on OAAI in mice

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Fig. 4
figure 4

Anxiolytic effect of VUA. Each bar represents mean ± standard error of mean. VUA at 50 and 100 mg/kg exhibited anxiolytic effect. *significant p < 0.05 compared to VEH. #significant compared to DPM p < 0.05. A Period of stay in open arms; B Frequency of visit in open arms; C Effect of VUA on OAAI in mice

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Fig. 5
figure 5

Effects of VUM, VUH, VUE and VUA showing significant difference in mouse head dips in hole-board model compared to VEH. Each bar represents mean ± standard error of mean. *significant p < 0.05 compared to VEH, #significant compared to DPM p < 0.0

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Fig. 6
figure 6

Mechanism of action of anxiolytic effect of VUM and VUE using flumazenil. #significant p < 0.05: antagonist versus treatment alone

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Fig. 7
figure 7

Mechanism of action of anxiolytic effect of VUM and VUE using cyproheptadine. #significant p < 0.05: antagonist versus treatment alone

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Fig. 8
figure 8

Mechanism of action of anxiolytic effect of VUM and VUE using yohimbine. #significant p < 0.05: antagonist versus treatment alone

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Discussion

Herbal medicines have being in use to manage various neurological disorders such as stress and anxiety and many of the active principles responsible for this activity have been isolated. Examples of such plants include Passiflora incarnata, Cimicifuga racemosa. The evaluation of the extract from this plant was premise on the use by the natives to manage epilepsy. The results showed that the extract produced a significant rise in both the percentage of time that the mouse spent in and the frequency of entry into the uncovered portion of the EPM as well as inducing an increase in the frequency of head dips on the hole-board [23]. These indicated that the extract and fractions of this plant possessed anxiolytic property similar to diazepam (a standard anxiolytic agent) used in this experiment. Usually, rodents have natural tendency to explore an enclosed portion of the maze than the opened portion [18, 19]. Therefore, any chemical agent that induces an increase in the number of visits as well as prolongs the period that rodents spend in the exposed portion of the maze is said to exhibit anxiolytic property whereas the agent that induces a reverse effect is said to be anxiogenic agent [20]. The ethylacetate fraction produced more convicing anxiolytic effect in both models compared to other fraactions. The anxiolytic action of this fraction is comparable to DPM (Fig. 5).

The Index of open arm avoidance (IOAA) was determined to further validate anxiolytic property of the extract. The interpretation of IOAA is such that, if any group is at least10 points less than the control’s (vehicle), then the sample can be said to show anti-anxiety effect. On the other hand, if the sample is at least 10 points greater than the control’s then, such sample is said to have shown an anxiogenic activity [21, 22]. In this study, the values of IOAA for the control, VUM, VUE, VUA and diazepam were 64%, 46%, 44%, 45% and 46%, respectively. Like diazepam, the OAAI for each of the extract and fractions was less than the control’s by more than 10%, and as such they convincingly demonstrated an anxiolytic property (panel C in Figs. 1, 2, 3 and 4).

Neurotransmitters such as acetylcholine, noradrenaline, 5-HT (serotonin), dopamine, histamine and GABA that are found in hypothalamic–pituitary–adrenal axis and serotonergic systems have been shown to modulate anxiety disorders [1, 2, 5, 21]. This informed the use of some antagonists like yohimbine (alpha adrenergic antagonist), cyproheptadine (serotonin antagonist) and flumazenil (GABA antagonist) to delineate the possible mechanism of anxiolytic effect of the extract and fractions in this study (Figs. 6, 7 and 8). The reversal anxiolytic effect of VUM and VUE by flumazenil in both models was an indication that GABAergic neurotransmission may be involved in the anti-anxiety effect of extracts of this plant (Fig. 6 panel C). The reversed effect of cyproheptadine on VUE fraction may also suggest some constituents of this fraction may produce anti-anxiety effect via serotonergic pathway. In the same vein, Yohimbine reversal effect in both models may also suggest adrenergic involvement in the anti-anxiety effect since increase in cerebral noradrenergic activity has been shown to be closely related to anxiety-related behaviour [23, 24]. Thus, the result of this study therefore suggests the connection between GABAergic, serotonergic and alpha2 adrenergic neurotransmission and the anxiolytic potential of VUE while GABAergic and serotonergic is suggested to be involved in anxiolytic effect of VUM. Some studies have shown that the action of anxiolytic drugs improved the response to GABA by opening of GABAA-activated chloride channels [25,26,27]. Therefore, Gabaergic receptor stimulation may be one of the possible mechanisms of action by which V. unguiculata spp dekindtiana crude extract and its fractions exerted their anxiolytic effect. From the results, the extract and pharmacologically active fractions of V. unguiculata spp dekindtiana can be utilised to manage anxiety-related disorders. The anti-agitation properties of this plant is similar to those of the essential oil of Melissa officinalis [28]. Further studies will be required to isolate the pharmacologically active molecules in the fractions that are responsible for this anxiolytic effect.

Conclusion

This work has shown that the methanol extract, ethylacetate and aqueous fractions of V. unguiculata spp dekindtiana possess anxiolytic activity in mice, thereby justifying the ethnomedicinal use in the treatment of anxiety disorders. The obtained results suggested the involvement of GABAergic, catecholaminergic and serotonergic pathways in the anxiolytic property of these phytoconstituents in mice.