Depression is a common mental illness that affects the lives of millions of individuals worldwide, impacting their emotional, cognitive, and physical well-being in a considerable way. It is defined by persistent sadness, loss of interest in activities, and a wide range of related symptoms such as sleep disturbance, fatigue, and feelings of worthlessness. The incidence of depression has been increasing over the years, with studies indicating increasing prevalence due to reasons such as globalization, social stress, and lifestyle changes. According to a WHO report, depression is expected to be the first leading cause of the global burden of disease by 2030[1].

The disorder is not age-specific but can be seen from childhood to late older age. But it is common in some groups, and its prevalence is greater in women than in men [2]. The ill effects of depression do not stop at emotional distress but lead to the development of a range of physical illnesses like cardiovascular diseases, diabetes, and neurodegenerative diseases. Additionally, its effect on maternal and child health is enormous, Depressed mothers often cease breastfeeding early which result in poor growth of her baby. Also evidence suggests that maternal depression may be linked with low birth weight baby [3]. Depressed adolescents are likely to experience difficulties in academic performance, interpersonal relationships, and a greater likelihood of drug abuse or self-destructive behaviors and even lead to teenage pregnancy, increased hospitalization and antisocial behaviors [4]. Depressed adolescents get also often dragged to psychoactive substance and alcohol abuse in order to improve their moods and to get distracted from emotional instabilities [5]. Among the worst impacts of depression, the link of depression with suicide is a cause of concern, as the majority of people who commit suicide have a history of depressive disorders or mental disorder [3]. There are mainly three modalities of treatment available for depression viz, psychotherapy, electroconvulsive therapy (ECT), and drug therapy (antidepressants) [6]. But none of these treatments are free from disadvantages. Majority of the drugs used in depression have to be given for an extended duration before they show any therapeutic activity, and they also cause serious side effects that further complicate the patients' health. Other treatment modalities like ECT) and psychotherapy are also not free from disadvantages [7-10]. The limitations of the conventional treatment modalities have prompted the researchers to explore other therapies, particularly those of plant origin. Plant-based medicinal compounds have been employed for thousands of years in conventional systems of medicine due to their easy availability, cost effectiveness and minimal side effects.

Azadirachta indica, or “neem” in common language is known in India and its neighboring countries for more than 2000 years for its wide spectrum of biological activity [11]. It has widespread recognition throughout the world for its varied pharmacological activities, ranging from anti-inflammatory, antimicrobial, to immunomodulatory activities [12]. Studies have also paved the way for the consideration of its role in neurological and psychiatric disorders, and studies have established that neem extracts can prove to have neuroprotective as well as antidepressive effects [13-14]. The occurrence of bioactive flavonoids with antioxidant activity provides further basis to the hypothesis that neem may have a role in reversing oxidative stress, which has been reported to be of pathophysiological importance in depression [15-16].

While initial findings are encouraging, extensive pharmacological studies are required to establish the antidepressant activity of neem. All the studies conducted so far have been on pure compounds and pure extracts, with no data on its overall therapeutic action. The current study, therefore, seeks to investigate the antidepressant activity of methanolic extract of Azadirachta indica leaves in a depression animal model. The current study seeks to add to the existing literature validating the medicinal use of plants in the management of mental illness and gain insight into future alternative therapy for depression.

The present research study, entitled "EVALUATION OF THE ANTIDEPRESSANT ACTIVITY OF THE METHANOLIC EXTRACT OF LEAVES OF AZADIRACHTA INDICA (NEEM) ON ALBINO MICE," was carried out under the Department of Pharmacology of Assam Medical College & Hospital, Dibrugarh, during the period from July 1, 2017, to June 30, 2018. Ethical consent was acquired from the Institutional Animal Ethics Committee (IAEC) of Assam Medical College, Dibrugarh, as per guidelines framed by the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals).

 Laboratory Animals

The experiment used young adults of Swiss Albino mice (Mus musculus) of both genders, provided that female animals were nulliparous and non-pregnant. The body weight of each mouse was 25-35 grams. Twenty-five specimens were procured from the Central Animal House of the Department of Pharmacology, Assam Medical College, Dibrugarh (Registration no. 634/02/a/CPCSEA, dated 19.05.2002). The animals were kept in normal environmental conditions, with the temperature controlled at 25 ± 2°C and relative humidity at 55 ± 2%, according to a 12-hour light/dark cycle. The polypropylene cages accommodated the mice and were provided with a nutritionally balanced diet of Bengal gram, peas, beans, dried bananas, cabbage, and coconut shell, with free access to drinking water. All ethical standards of animal care were strictly followed.

 Drugs and Chemicals

Normal saline, reserpine (procured from Novartis (Sandoz) India Ltd., Mumbai), fluoxetine (procured from Zydus Cadila Healthcare Ltd., Ahmedabad), and methanolic extract of leaves of Azadirachta indica (MELAI) were experimented upon. Distilled water was used as the vehicle.

 Purchase of Vegetative Samples and Extract Preparation

Leaves of Azadirachta indica were collected from the vicinity of Assam Medical College & Hospital, Dibrugarh, during August 2017. The gathered leaves were treated with a thorough washing process with water to eliminate any impurities and were then air-dried at room temperature for two weeks. The leaves were powdered into fine powder after drying using an electric grinder and stored in hermetically sealed plastic containers with proper labelling. Preparation of the methanolic extract was done through the Soxhlet extraction technique. Specifically, 60 g of the powder material was treated with 300 ml of 100% methanol (analytical grade, supplied by Rankem Pharmaceutical Company, Haryana). Soxhlet apparatus was kept at 65°C temperature, and the extraction process was conducted for duration of three hours. The resulting filtrate was evaporated in a water bath at 65°C, resulting in a brownish paste-like material, which was further dried under vacuum desiccator. The resultant extract was kept in airtight glass containers and stored in a laboratory refrigerator at 4°C. The above process was done three more times with different lots of 60 g of dried powder, resulting in a yield of 42.50 g of extract (17.71% w/w compared to the original dried material).

Acute Toxicity Testing

Acute oral toxicity of methanolic extract of Azadirachta indica was assessed as per OECD guidelines 425. Female Wistar albino rats (Rattus norvegicus), 8-12 weeks old and weighing 150-200 g, were used. The test animals were fasted overnight prior to dosing. A single oral dose of MELAI at 2000 mg/kg body weight, freshly prepared in normal saline at 1 ml/100 g body weight, was given orally via a feeding tube. The animals were observed closely for 30 minutes after dosing, intermittently for the first 24 hours, and once daily for 14 days for any evidence of toxicity. Parameters observed were skin and fur condition, eyes and mucous membrane appearance, respiratory rate, circulatory signs, autonomic effects, and central nervous system changes. There was no evidence of toxicity or mortality in any of the five rats, thus determining an LD50 value > 2000 mg/kg. Two arbitrary doses for the experiment were set on this basis: a low dose (250 mg/kg body weight) and a high dose (500 mg/kg body weight).

Phytochemical Analysis

A qualitative examination of the phytochemical constituents within the extract was conducted in accordance with the methodology outlined by Prasant Tiwari and colleagues (2011) [17]. This analysis evaluated the existence of various compounds including alkaloids, glycosides, tannins, saponins, phenols, flavonoids, diterpenes, phytosterols, carbohydrates, proteins, and amino acids.

Preparation of Stock Solutions

Stock solutions of test drugs were prepared by the following method: Reserpine (2 mg/kg) was dissolved in 10 ml of distilled water to provide 0.2 mg/ml. Fluoxetine (10 mg/kg) was dissolved in 10 ml of distilled water to provide 1 mg/ml. Low-dose preparation of MELAI (250 mg/kg) was prepared by dissolving 250 mg of the extract in 10 ml of distilled water to provide 25 mg/ml, and high-dose preparation (500 mg/kg) was prepared by dissolving 500 mg of the extract in 10 ml of distilled water to provide 50 mg/ml. Dosing calculation was carried out according to the method by Shin JW et al. [18].

 Induction of Depression

Depression in all experimental groups except the normal control group was induced by reserpine at a dose of 2 mg/kg body weight given intraperitoneally 24 hours prior to testing [19].

Experimental Design

Mice were divided into five groups (n=5), each of five mice. They were acclimatized for seven days before the experiment. After overnight fasting, drugs were administered intraperitoneally as follows: Group I (Normal Control) was administered with normal saline (10 ml/kg), Group II (Disease Control) was administered with reserpine (2 mg/kg) without further treatment, Group III (Standard) was administered with reserpine and followed by fluoxetine (10 mg/kg), Group IV (Test drug-1) was administered with reserpine and followed by MELAI at 250 mg/kg, and Group V (Test drug-2) was administered with reserpine and followed by MELAI at 500 mg/kg.

Assessment of Antidepressant Effectiveness

Antidepressant activity of MELAI was evaluated by Forced Swimming Test (FST) and Tail Suspension Test (TST).The test animals, mice, were challenged by these tests after a period of 30 and 60 minutes of drug administration, respectively [19]. The behavioral activity was recorded by the "ANY-maze" animal tracking software (version 6.04) instead of visual observation.

Forced Swimming Test (FST)

This behavioral despair paradigm, as used by Porsolt et al. (1977, 1978), consisted of putting mice into a glass open-chamber (25 cm × 15 cm × 25 cm) with 15 cm depth water at 26 ± 1°C. At this water height the mouse was not be able to support its own posture by touching the bottom or side walls of the chamber and assume a characteristic behaviour of immobility and floating position.This reflects a state of despair, which cannot be reduced by several clinically effective antidepressants Four minutes of immobility was measured after two-minute of vigorous acclimatization. Mice were said to be immobile when they had stopped struggling and floated passively, moving their heads only minimally in an attempt to remain afloat [20][21].

Tail Suspension Test (TST)                  

Mice were hung by the tail 50 cm from the floor with adhesive tape 1 cm from the terminal tail end. Six minutes of immobility time were observed and immobility was considered as  absolute absence of movement, which is equivalent to behavioral despair. The test was conducted in dim-lighted room and one animal was used only once at a time [21][22].

Quantitative Analysis

All values are given as Mean ± SEM. Statistical significance was evaluated by one-way ANOVA supported by Bonferroni's Multiple Comparison Test. A p-value of less than 0.05 was considered statistically significant.

Phytochemical Analysis of MELAI

The phytochemical qualitative analysis of the Methanolic Extract of Leaves of Azadirachta indica (MELAI) was conducted to identify the existence of bioactive compounds. The findings, as reflected in Table 1, signify the existence of alkaloids, glycosides, tannins, saponins, phenols, flavonoids, diterpenes, phytosterols, proteins, and amino acids, with the absence of carbohydrates.

Table 1: Phytochemical Composition of MELAI

Evaluation of Antidepressant Activity of MELAI in Albino Mice

Forced Swimming Test (FST)

Forced Swimming Test (FST) was employed to evaluate the antidepressant activity of MELAI in albino mice. Immobility time was measured for each group, and a reduction in immobility time was considered an antidepressant-like effect.

The findings, as indicated in Table 2, reveal that the disease control group (Reserpine 2 mg/kg) had a greatly enhanced immobility time (238.902 ± 1.927 sec) over that of the normal control group (172.036 ± 0.8138 sec), ascertaining induction of depression-like behavior. Treated with the conventional antidepressant drug fluoxetine (10 mg/kg), the immobility time was substantially decreased (89.944 ± 0.7637 sec), pointing toward its expected antidepressant effect.

MELAI in both administered doses (250 mg/kg and 500 mg/kg) also significantly lowered the duration of immobility to 128.12 ± 1.578 sec and 106.656 ± 2.450 sec, respectively, showing its efficacy as an antidepressant. The dose of 500 mg/kg was more effective, although statistically significant effects (p < 0.001) for both doses were observed compared to the disease control and normal groups.

Table 2: Effect of MELAI on Immobility Duration in the Forced Swimming Test

One-way ANOVA: F = 1330.0, Df = 24, p < 0.05

Significance: a p < 0.001 compared to normal and disease controls.

The findings are further illustrated in Figure 1, which represents a graphical comparison of the immobility duration across different treatment groups.

Figure 1: Bar Diagram Representing the Effects of MELAI on the Forced Swimming Test

(Figure 1 displaying immobility duration across different groups, with statistical significance indicated.)

Tail Suspension Test (TST)

To further confirm the antidepressant activity of MELAI, the Tail Suspension Test (TST) was performed. The immobility time was measured, and the shorter the immobility, the more evidence of an antidepressant effect.

As noted from Table 3, the disease control group (Reserpine 2 mg/kg) had a longer immobility time (236.672 ± 2.633 sec) than the normal control group (144.52 ± 1.904 sec). The antidepressant standard drug fluoxetine (10 mg/kg) significantly decreased immobility (73.594 ± 0.5933 sec).

MELAI at 250 mg/kg and 500 mg/kg also had a significant reduction in immobility times to 110.704 ± 2.994 sec and 100.14 ± 0.6079 sec, respectively (p < 0.001), reinforcing its antidepressant-like activity.

Table 3: Effect of MELAI on Immobility Duration in the Tail Suspension Test

One-way ANOVA: F = 987.07, df = 24, p < 0.05

Significance: a p < 0.001 compared to normal and disease controls.

A graphical representation of these results is provided in Figure 2, which illustrates the differences in immobility duration across treatment groups.

Figure 2: Bar Diagram Representing the Effects of MELAI on the Tail Suspension Test

(Figure 2 depicts immobility duration among different groups, highlighting significant reductions in depression-like behavior following MELAI administration.)

The outcomes from both behavioral tests (FST and TST) show that MELAI decreases immobility time to a significant level, implying an antidepressant-like effect. The effect was dose-dependent, as the higher dose (500 mg/kg) proved more effective.

Statistical analysis established the decreases in immobility time as significant (p < 0.001) relative to both normal and disease control groups. The results indicate MELAI has antidepressant activity similar to fluoxetine, which endorses its therapeutic application in the treatment of depression.

In addition, all the animals were healthy throughout the experiment, and no mortality was seen, which dictates the safety of MELAI at doses administered.

The current research focused on assessing the antidepressant potential of methanolic extract of leaves of Azadirachta indica (Neem) in albino mice and exploring its potential mechanism of action. Two validated behavioral models, the Forced Swimming Test (FST) and the Tail Suspension Test (TST), were used to examine antidepressant activity. Reserpine, an uptake blocker of vesicles that causes depression by depleting biogenic amines, was employed to induce an acute depression model, and fluoxetine, an SSRI, was the control antidepressant drug. The outcome revealed that the methanolic extract lowered the immobility time in both FST and TST remarkably, indicating an antidepressant-like activity.

The duration of immobility was maximum in the disease control group treated with reserpine alone (238.902 ± 1.927 sec in FST and 236.672 ± 2.633 sec in TST), as in earlier studies that revealed reserpine-induced depression resulted in increased immobility for a longer time in both models of behavior [19]. As anticipated, administration of fluoxetine substantially decreased the time of immobility (89.944 ± 0.7637 sec in FST and 73.594 ± 0.5933 sec in TST), validating its antidepressant activity through inhibition of serotonin reuptake. The same trend was manifested in earlier research where fluoxetine markedly reversed the depressive action of reserpine [19][23]. The test groups treated with MELAI in two varying doses (250 mg/kg and 500 mg/kg) also had a notable decrease in immobility time compared to the disease control group. The action was dose-dependent, with the greater dose (500 mg/kg) eliciting a more pronounced decrease in immobility (106.656 ± 2.450 sec in FST and 100.14 ± 0.6079 sec in TST) compared to the lesser dose (250 mg/kg) (128.12 ± 1.578 sec in FST and 110.704 ± 2.994 sec in TST). This dose-dependent effect indicates that the antidepressant activity of MELAI is directly proportional to its administered dose.

Comparative assessment of the outcome between FST and TST showed that there was a more significant antidepressant effect in the TST. The reduction in immobility in TST was superior to that in FST, consistent with earlier observations that TST is a more pharmacologically responsive model and less stressful for animals than FST [24]. These results suggest that MELAI has appreciable antidepressant activity, possibly mediated through the central monoaminergic system. Nonetheless, the precise mechanism is yet to be completely elucidated.

Phytochemical screening of Azadirachta indica was found to contain alkaloids, glycosides, tannins, phenols, flavonoids, diterpenes, phytosterols, proteins, and amino acids. Such bioactive entities are likely to be responsible for the reported antidepressant effects. It is proposed by the study that the antidepressant-like behavior of MELAI can be associated with its capacity to reverse reserpine-induced monoamine depletion. Given that reserpine lowers levels of brain monoamines, medications restoring or augmenting monoaminergic transmission can help prevent depressive behaviors. The demonstration that MELAI lowered immobility time significantly in both FST and TST lends support to the hypothesis that it acts on central neurotransmitter systems. Nevertheless, because fluoxetine, a selective serotonin reuptake inhibitor, had a greater effect, MELAI probably does not primarily exert its effect through serotonin reuptake inhibition but through a different or additive mechanism.

The antidepressant action of MELAI could be due to its antioxidant activity. Oxidative stress has been implicated in depression, wherein excess oxidative damage may impair neuronal integrity and function. Research has indicated that markers of oxidative stress, including lipid peroxidation and DNA strand breaks, are higher in depressed patients [25]. MELAI is composed of a number of antioxidant constituents, including flavonoids, phenols, and tannins, which have been found to be neuroprotective in their action by neutralizing free radicals and lowering oxidative damage [26].The occurrence of flavonoids in MELAI is consistent with earlier findings establishing that flavonoid-rich plant extracts, including those of Moringa oleifera and Terminalia bellirica, have antidepressant effects due to their antioxidant and anti-inflammatory actions [27,28].

Consistent with this hypothesis, nimbolide and azadirachtin, two bioactive components of Azadirachta indica, were found to show significant free radical scavenging activity [29]. Polyphenols of the plant have been found to modulate oxidative stress and exert antidepressant effects [30]. Quercitrin, a flavonoid present in a number of medicinal plants, was found to exert antidepressant-like activity, also lending evidence towards the possibility that the phytoconstituents of MELAI play a role in its therapeutic benefits [22]. This concurs with evidence showing that extracts of polyphenol-rich plants, including Mimosa pudica Lagenaria siceraria, exert antidepressant effects through modulating oxidative stress pathways [23][31].

Besides antioxidant mechanisms, MELAI antidepressant activity might also engage other neuropharmacological pathways. Tannic acid, a constituent of Azadirachta indica, has been reported to be a non-selective monoamine oxidase (MAO) inhibitor, which may elevate monoaminergic neurotransmitter levels in the brain [32][33]. This action is of particular interest as MAO inhibitors are a well-documented class of antidepressants. In addition, Azadirachta indica is rich in nimbosterol, a beta-sitosterol, which has been reported to have neuromodulatory activity, indicating another potential mechanism for its antidepressant action [34]. The occurrence of these bioactive molecules in MELAI supports the hypothesis that its antidepressant action is mediated by multiple neurochemical mechanisms.

In conclusion, the findings of this study suggest that the methanolic extract of Azadirachta indica has potent antidepressant activity, as shown by its capacity to decrease immobility time in reserpine-induced depression models. The response is dose-dependent, with increased dose eliciting a greater response. The likely mechanisms of its antidepressant action include the central monoaminergic system, modulation of oxidative stress, and the occurrence of bioactive phytochemicals like flavonoids, tannins, and beta-sitosterol. Although the precise mechanism is yet to be elucidated, the current findings add to the body of evidence favoring the possible use of Azadirachta indica in depression treatment. Additional research, such as neurochemical studies and receptor-binding assays, will be required to validate these results and investigate the particular molecular targets that are involved in its antidepressant effect.

The results of the current study indicate that the methanolic leaf extract of Azadirachta indica (MELAI) has appreciable antidepressant activity, as evidenced by decreased immobility time in both Forced Swim Test (FST) and Tail Suspension Test (TST) in albino mice. The effect of the antidepressant was dose-dependent, such that the higher dose of 500 mg/kg induced more reduction in immobility than the lower dose of 250 mg/kg. The efficacy of MELAI, while lesser compared to that of fluoxetine, suggests participation in the central monoaminergic system and is implied to possess mechanisms other than selective serotonin reuptake inhibition. The phytochemical components present in multiple forms, mainly flavonoids, tannins, saponins, and polyphenols, having reported antioxidant properties, could also play a part in this action. Moreover, tannic acid, a non-selective monoamine oxidase inhibitor, and nimbosterol, a beta-sitosterol with neuromodulatory activity, may also contribute to the seen pharmacological activity. In conclusion, the study presents clear evidence for the antidepressant potential of Azadirachta indica leaves that necessitates further investigation to understand the exact mechanisms and determine its clinical usefulness.

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Conflict of interest: None declared

Funding sources: No funding sources