There is a significant dimension in the development of novel anxiolytic and anti-depressant constituents that possess fewer side effects with fast onset of action and a comprehensive safety margin. In the current work, we investigated the neurobehavioral effects of oral treatment using well-validated animal models with the ethanolic extract of Psidium guajava Linn leaves at two different distinct doses as 200 mg/kg and 400 mg/kg of BW.
The phytochemical screening evaluates the presence of carbohydrates, proteins, steroids, terpenoids, flavonoids, alkaloids, glycosides, and tannins in Psidium guajava Linn leaves extract (Table 1). According to previous studies [22, 23], the presence of many biologically active phytochemicals such as flavonoids, triterpenes, alkaloids, steroids, tannins, and glycosides in different plant extracts may be accountable for their corresponding pharmacological activities. It is stated by several experimental investigations on different plant extracts that flavonoids, alkaloids, and terpenoids have been testified to be accountable for anxiolytic and sedative effects [24]. According to the study performed by Arcos-Martínez [25] and Graham [26] demonstrated that existing alkaloids might have the role to show the anxiolytic-like effects of the plant extract which was probably mediated through gamma-aminobutyric acid (GABA) receptor [27].
To assess anxiolytic properties of the extract, we use the elevated plus maze model, a most frequently employed test for screening the effect of anxiolytics on behavioral parameters of animals, and it is based on the fact that exposure of animals to an elevated plus maze evokes conflict of approach-avoidance stronger than revealed by the exposure to an enclosed arm [28,29,30]. In EPMT, normal mice will habitually prefer to spend much of their assigned time in the closed arms. This preference seems to reveal an aversion in the direction of open arms that is generated by the fears of open zone. Anxiolytic effect is indicated by reduction in aversion headed for open arms by the way of increase in time spent and number of entries into the open arms [30], whereas, anxiogenics lessen the value of these parameters [31]. In our study, it is seen that Psidium guajava Linn leaves extract (200 mg/kg and 400 mg/kg of BW) increases the time spent in the open arms, and number of entries into open arms (Table 2). These results are similar to the effects observed after administration of the reference anxiolytic drug diazepam. Therefore, according to the result, it is evident that the extract shows dose dependent anxiolytic effect as higher dose shows better effect than lower, and these findings are supported by the previously published studies conducted with different plant extracts [32, 33]. Also, these treatments reduce the anxiety index (Fig. 1), a parameter that unifies the results of the number of entries to the open arms and the time spent in these spaces correspondingly to diazepam, and the findings are also supported by the previously conducted studies [34]. The anxiolytic effect of the extract is also prefaced through an experimental model principally designed as a potency predicting model of clinically available anxiolytic agents called light/dark test. It is taken into consideration that time spent by the mice on the illuminated part is mostly fruitful and consistent parameter of anxiety. The basic principle of the light/dark test is that the inherent aversion of rodents to intensely illuminated zone and the natural exploratory behavior of rodents responding to mild stressors [35]. In that point of view, the experiment may have efficiency for the prediction of anxiolytic or anxiogenic-like effect of plant extract in mice. In the light/dark test, anxiety is engendered by the conflict between the tendency to explore and the preliminary tendency to avoid the unfamiliar [36], and can be evaluated by taking into consideration the number of transition into and the time spent in the light chamber, wherein increasing pattern of these parameters is regarded to reveal the anxiolytic-like features. The outcome obtained from our study indicates that the extract gives a statistically significant rise in time spending into bright illuminated part of the light-dark apparatus while comparing with group I, and the effect is comparable with the effect observed in group II (Fig. 2). For detecting anxiolytic effects, another advantageous experiment due mainly to its methodological simplicity and remarkable behavioral responses of the rodents when unveiled to an unacquainted environment is the hole board model, which is used to analyze head dipping behavior. This type of behavior is sensorial to the alterations in the emotional state of the animal, and indicates that the expression of an anxiolytic-like state may be mirrored by the enhanced behavior of head dipping [37, 38]. In our study, both strengths of the extract and also the reference drug increase the number of head dips into the hole, in comparison with group I (Fig. 3). So, these findings may indicate the presence of the anxiolytic effect of the extract [39]. It has mostly been suggested that for the evaluation of drugs regarding anti-obsessive-compulsive disorder, using marble-burying behavior model would be beneficial and this is due to that no alteration related to the intensity of marble-burying behavior arisen during repeated testing, called as compulsive behavior [40]. Psidium guajava Linn extract of dose 200 mg/kg BW shows statistically non-significant decrease in the number of marble buried, but extract at 400 mg/kg BW dose shows a statistically significant decrease in the number of marble buried compared with group I. Group II taking diazepam produces similar pattern of results (Fig. 4).
In hole cross test, any agent having anxiolytic property will reduce the number of locomotion, assumed as of deficiency in interest on new environment [41]. Locomotor activity works as an indicator of mental wakefulness or alertness and thus, reduction of which signifies the tranquility and sedation elucidating as decreased CNS excitability [42].
In our present study (Fig. 5), the number of hole crossing by experimental mice of group I from one chamber to another group is slightly fluctuated from 0 to 120 min.
The group treated with the plant extract of 200 mg/kg shows statistically considerable decreasing effect at 60 min, 90 min, and 120 min interval of administration, but more decreasing effect on hole crossing is found at 120 min interval than other two intervals, and at interval 0 min and 30 min, a statistically non-significant effect is observed. On the other hand, a statistically considerable decreasing effect on hole crossing is found at all intervals except at 0 min in case of group IV, and the effect is more than that of in group III. In case of group II, the same statistically significant effect but more decrease is found as shown in group IV in all time intervals (Fig. 5). This locomotor activity suppressing capability as well as the sedative effect of EEPG demonstrates that the extract possesses CNS depressant activity. Drugs having sedative and anxiolytic agents, such as benzodiazepines, act to increase GABA mediated synaptic inhibition by facilitating the action of GABA upon the GABAA receptor. From our experimental findings, it is evident that the extract mainly at higher strength provides effect almost similar to that of the reference drug diazepam, a well-known benzodiazepine. Therefore, it could be supposed that substances of our extract able to mitigate the anxiety of rodents exposed to these paradigms could exert their effect through the mechanism of action similar to that of the benzodiazepines [43], although there are numerous anxiolytic compounds with different action mechanisms through glutamatergic, noradrenergic, and serotoninergic receptors [44]. A molecular docking study may be conducted to elucidate the binding pattern of phytochemicals against GABAA receptor.
In our study, we use two most extensively preferable animal models for anti-depressant screening, the FST and TST, which are behavioral despair tests expedient for piercing the pathological mechanism of depression and for the evaluation of anti-depressant drugs [45]. These tests are quite sensitive and comparatively specific to all principal classes of anti-depressants including tricyclics, selective serotonin reuptake inhibitors (SSRIs), and monoamine oxidase inhibitors (MAOIs) [20]. These are based on the inspection of an immobile behavior developed by the rodents, after preliminary escape-oriented movements, when kept in an inescapable stressful condition. In this study, the higher dose (400 mg/kg BW) of extract shows statistically significant reduction in the immobility time of mice in both FST (Fig. 6) and TST (Fig. 7), whereas, a statistically non-significant reduction of the immobility time of mice is shown by the lower dose of extract (200 mg/kg BW) in both tests in comparison with group I. Similar effect is observed after administration of the anti-depressant drug imipramine. The compounds are said to have anti-depressant effect, those are involved in decreasing immobility time in FST [46]. Therefore, the compounds with higher concentration (400 mg/kg BW dose) present in Psidium guajava Linn extract might have anti-depressant effect.
It has been recognized that the shortening of immobility time in the forced swimming and the tail suspension tests relies predominantly on the augmentation of central 5-HT and catecholamine neurotransmission [47]. The majority of the conventional anti-depressants directly affects serotonin turnover in the brain [48], by exerting their inhibitory effect on serotonin reuptake and also by their efficacious interaction with 5-HT1A and 5-HT2 receptors [49]. It is demonstrated by a number of studies that the mechanism of action of different groups of anti-depressant drugs, including tricyclics, selective serotonin reuptake inhibitors, and (MAOIs), is involved with 5-HT1A receptors [50]. In parallel with the serotonergic system, noradrenergic system is evidently involved depression and noradrenaline reuptake inhibitors or (MAOIs) exert their effect by enhancing the synaptic concentration of noradrenaline by interacting with α1 and α2-adrenoceptors, and also with β-adrenoceptors [51]. The standard anti-depressant drug (imipramine) used in our study averts reuptake of noradrenaline and serotonin resulting in their increased availability in the synapse and therefore, an increase in adrenergic and serotonergic neurotransmission [52]. A relationship between dopamine neurotransmission and depression is demonstrated by several preclinical and clinical studies, and it is also claimed that the intensity of depression is inversely correlated dopamine metabolite levels in CNS [53, 54]. The activation of dopamine D1 and D2 receptors is involved with the mechanism of action of different anti-depressant drugs such as SSRIs, imipramine (a tricyclic anti-depressant) and bupropion (dopamine reuptake inhibitor) in the FST [55, 56], where dopamine D2 receptors are related to the anti-immobility action of anti-depressants in mice reported by preclinical data [56].
The components present in the extract of Psidium guajava Linn leaves may show anti-depressant effect through above mechanisms. Also, many studies have demonstrated that protopine alkaloid possesses effects similar to anti-depressants in case of FST in mice by inhibiting transporters of serotonin and noradrenaline [57] and shows affinity for the receptor of GABAA [1].
Concerning the medical treatment of psychiatric disorders, the results obtained in this work has become important because not only anxiolytic effects were observed, but also anti-depressant activity was found. Developing biochemical and pharmacological studies are necessary for the purpose of establishing whether the observed effects in our study are produced by one chemical substance by itself as a result of distinct activation of nervous structures, or the several secondary metabolites of the plant are accountable for those biological activities.
