Anxiety is a fundamental emotional response that enables organisms to cope with perceived threats. However, when anxiety becomes excessive, persistent, or disproportionate to stimuli, it manifests as a pathological condition requiring medical intervention. Anxiety disorders constitute a major global health burden, affecting nearly 300 million individuals worldwide and significantly impairing quality of life [1,2].

Pharmacotherapy for anxiety primarily includes benzodiazepines, selective serotonin reuptake inhibitors, and serotonin–norepinephrine reuptake inhibitors. Despite their efficacy, benzodiazepines are associated with adverse effects such as sedation, cognitive impairment, tolerance, and dependence, limiting their long-term utility [3,4]. This has driven increasing interest in identifying safer anxiolytic agents from medicinal plants with favourable safety profiles.

Artocarpus heterophyllus Lam. (family Moraceae), commonly known as jackfruit, is widely distributed in tropical regions and has been used in traditional medicine for its anti-inflammatory, antioxidant, antidiabetic, and neuroprotective properties [5–7]. Phytochemical analyses of its seeds have revealed the presence of flavonoids, phenolic compounds, saponins, and alkaloids, many of which are known to modulate central nervous system activity [8,9].

While earlier studies have explored the antioxidant and anticonvulsant effects of A. heterophyllus, systematic evaluation of its anxiolytic potential remains limited. Therefore, the present study aimed to investigate the anxiolytic activity of the methanolic extract of Artocarpus heterophyllus seeds using validated behavioural paradigms—the Dark–Light Arena in Swiss albino mice.

Ethical Approval

The study was approved by the Institutional Animal Ethical Committee, A.J. Institute of Medical Sciences, Mangalore (IAEC No: AJIMS/IAEC/18-19/02; dated 12/11/2018) and conducted in accordance with CPCSEA guidelines.

Experimental Animals

Adult Swiss albino mice of either sex (3 months old; n = 30) were housed under controlled laboratory conditions (26–28°C, 12-h light/dark cycle) with free access to food and water. Animals were acclimatized for one week prior to experimentation.

Drugs and Chemicals

Methanolic extract of Artocarpus heterophyllus seeds (MEAH) was procured from the Department of Pharmacognosy, Srinivas College of Pharmacy, Mangalore. Diazepam (Valium®, Roche Healthcare) was used as the standard anxiolytic drug.

Preparation of Methanolic Extract

Dried jackfruit seeds were powdered and subjected to successive solvent extraction using petroleum ether, chloroform, and methanol (1:6 ratio). The methanolic extract was concentrated under reduced pressure and stored at <10°C until use.

Study Design

Animals were randomly divided into five groups (n = 6):

●             Group I: Normal saline (10 ml/kg)

●             Group II: Diazepam (1 mg/kg)

●             Group III: MEAH (50 mg/kg)

●             Group IV: MEAH (100 mg/kg)

●             Group V: MEAH (200 mg/kg)

All treatments were administered orally 60 minutes prior to behavioural testing.

Behavioural Assessment

Anxiolytic activity was evaluated using:

1.            Dark–Light Arena (DLA): Time spent in the bright chamber, number of entries, and rearing behaviour were recorded over 5 minutes.

Statistical Analysis

Data were expressed as Mean ± SEM and analysed using one-way ANOVA followed by Dunnett’s post hoc test. A p-value < 0.05 was considered statistically significant.

In the Dark–Light Arena test, mice in the control group spent a mean duration of 100.83 ± 1.04 seconds in the bright arena. Treatment with the standard anxiolytic drug diazepam (1 mg/kg) produced a significant increase in the time spent in the bright arena (108.00 ± 1.06 seconds; p < 0.001), indicating reduced anxiety levels. Similarly, administration of methanolic extract of Artocarpus heterophyllus seeds (MEAH) at doses of 50, 100, and 200 mg/kg resulted in a highly significant increase in time spent in the bright arena compared to the control group (p < 0.001). Among the test doses, MEAH at 100 mg/kg produced the maximum anxiolytic effect (123.83 ± 1.10 seconds), which was greater than both the lower and higher doses, suggesting an optimal dose-dependent response.

NUMBER OF ENTRIES INTO THE BRIGHT ARENA OF THE DLA (ACUTE STUDY):

Mean number of entries into the bright arena for the control group was observed to be 3.33 ± 0.49 and for the group that received standard drug Diazepam was 7.16 ± 0.47.  The mean number of entries for the group pre-treated with a single dose of MEAH 50mg/kg was 8.66 ± 0.42, for MEAH 100 mg/kg was 12.0 ± 0.57, for MEAH 200 mg/kg was 9.0 ± 0.36.

The differences in the time spent in the bright arena for various groups of the DLA are as shown in Table 2 & Fig 1.

The differences in the number of entries into the bright arena for various groups of the DLA are as shown in Table 3 & Fig 2.

NUMBER OF REARS IN THE BRIGHT ARENA (ACUTE STUDY):

Mean number of rears in the Bright arena for the control group was found to be 0.83 ± 0.30. The standard group treated with diazepam showed a mean number of 2.66 ± 0.33. For the group pre-treated with a single dose of MEAH 50 mg/kg, it was 2.33 ± 0.49, for MEAH 100 mg/kg was 2.83 ± 0.54, for MEAH 200 mg/kg was 2.5 ± 0.42.

The differences in the number of rears in the bright arena for all groups of the DLA are as shown in Table 4 & Fig 3.

MEAH produced a significant increase in exploratory behaviour compared to control animals.

●        Time spent in bright arena: MEAH at 100 mg/kg showed the highest increase (123.83 ± 1.10 s), surpassing diazepam and other doses (p < 0.001).

●        Number of entries: A dose-dependent increase was observed, with the 100 mg/kg dose demonstrating maximal effect (12.0 ± 0.57 entries).

●        Rearing behaviour: Significant increases were observed at 100 and 200 mg/kg doses, indicating enhanced exploratory activity.

Overall, MEAH exhibited anxiolytic activity comparable to diazepam, with optimal efficacy at 100 mg/kg.

The present study demonstrates that methanolic extract of Artocarpus heterophyllus seeds possesses significant anxiolytic activity in Swiss albino mice. Increased exploration of aversive environments in both the Dark–Light Arena is a well-established indicator of reduced anxiety levels [10,11].

The anxiolytic effect of MEAH may be attributed to its rich phytochemical composition, particularly flavonoids and phenolic compounds. Flavonoids are known to interact with the GABA_A receptor complex, producing benzodiazepine-like anxiolytic effects without pronounced sedation [12–14]. The observed dose-dependent response, with maximal activity at 100 mg/kg, suggests receptor-mediated modulation rather than nonspecific CNS stimulation.

Interestingly, the anxiolytic efficacy of MEAH was comparable to diazepam, indicating its potential as a natural alternative with possibly fewer adverse effects. These findings align with previous studies demonstrating CNS-modulatory properties of plant-derived flavonoids and phenolics [15–18].

However, the study is limited by the absence of neurochemical and molecular analyses. Further studies exploring neurotransmitter modulation, receptor binding, and chronic toxicity are warranted.

The methanolic extract of Artocarpus heterophyllus seeds exhibits significant anxiolytic activity in validated animal models of anxiety. The extract enhanced exploratory behaviour in a manner comparable to diazepam, particularly at a dose of 100 mg/kg. These findings support the traditional use of A. heterophyllus and highlight its potential as a promising natural anxiolytic agent. Further mechanistic and clinical studies are recommended to establish its therapeutic utility.

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