Pain is an unpleasant sensory and emotional experience that generally arises from actual or potential tissue injury. The International Association for the Study of Pain defines pain as “an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage” (Raja et al., 2020). Pain acts as an important protective response by enabling organisms to avoid harmful stimuli, protect injured tissues during healing and prevent further damage (Cervero, 2012). Both nociception and pain trigger several physiological and behavioural reactions that help protect the body against injury and contribute to survival in animals (Bayne, 2000; Ohnesorge et al., 2021).
       
Medicinal plants have been utilized since ancient times for the prevention and treatment of diseases because of their therapeutic properties. Numerous herbs contain a wide range of phytoconstituents possessing diverse chemical structures and pharmacological activities effective against several pathogenic conditions (Gangatharan et al., 2025).
       
Annona squamosa, belonging to the family Annonaceae and commonly referred to as custard apple or sugar apple, is widely distributed in deciduous forest regions and cultivated throughout India. Besides its ornamental and nutritional importance, the plant is traditionally recognized for several medicinal properties such as analgesic, anti-inflammatory, antipyretic, antiulcer, antiseptic and abortifacient activities (Pandey and Barve, 2011). In Ayurvedic practices, the ripe fruit is used to facilitate abscess maturation, while preparations containing powdered unripe fruits are traditionally applied externally to promote suppuration in infected lesions and tumours (Varadharajan et al., 2012).
       
The zebrafish, (Danio rerio), is a small freshwater teleost belonging to the family Cyprinidae under the order Cypriniformes. Native to South Asia, zebrafish are commonly maintained as ornamental aquarium fish and are also extensively used as vertebrate model organisms in biomedical research (Van et al., 2017). Owing to their exceptional regenerative ability and availability of transgenic strains, zebrafish have become increasingly important in pharmacological and toxicological investigations (White et al., 2008).
       
Zebrafish are particularly valuable for genetic and biological studies because of their genetic similarity to humans, ease of maintenance and suitability for large-scale experimental applications (Porkodi et al., 2025). As a bony teleost species, they are frequently employed in anti-inflammatory and analgesic research. Their transparent developmental stages and suitability for real-time monitoring of cellular responses make them highly useful in experimental pharmacology. Moreover, zebrafish possess inflammatory mediators, receptors and signalling pathways that closely resemble those of mammals, including humans (Zanandrea et al., 2020). Therefore, the present investigation was undertaken to evaluate the anti-nociceptive potential of Annona squamosa using different chemically induced nociception models in zebrafish.

Collection and processing of plant material
 
Fresh leaves of Annona squamosa (Fig 1) were collected from the Department of Plant Physiology, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh. The leaves were shade dried and pulverized into fine powder using a mechanical grinder. Ethanolic extraction was carried out according to the procedure described by Bernatoniene et al., (2016).


 
Preparation of ethanolic extract by Soxhlet extraction
 
The crude extract was prepared using 90% ethanol as solvent. Approximately 20 g of coarsely powdered leaves of Annona squamosa were packed into a thimble prepared from Whatman filter paper no. 1 and placed in a Soxhlet apparatus. Extraction was performed using 400 mL ethanol at 80±5°C for nearly 12 hours. The obtained extract was transferred into petri dishes and concentrated on a water bath maintained at 90°C for removal of excess solvent. The percentage yield was calculated and the final extract (Fig 2) was preserved in airtight containers at 4°C until further use.

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Technical programme of work
 
Location and place of work
 
The experiment was conducted in the Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science and Animal Husbandry, N.D.V.S.U., Jabalpur, Madhya Pradesh.
 
Duration of work
 
The experimental study was carried out over a period of six months during the year 2022.
    
Experimental animals
 
Healthy adult zebrafish (Danio rerio) of either sex were used in the present investigation. The fish were procured from a local commercial breeder and maintained according to CPCSEA recommendations.
 
Housing and acclimatization
 
The zebrafish were housed in standard glass containers containing RO water with continuous aeration. The fish were acclimatized to laboratory conditions for 15 days before initiation of the experiment under regular observation.
 
Environmental conditions
 
Water temperature was maintained between 26°C and 28°C with pH ranging from 6.8 to 7.4. Proper oxygen supply was ensured using aerators. Tanks were cleaned at three-day intervals to maintain hygienic conditions. A controlled photoperiod of 14 hours light and 10 hours dark was maintained throughout the study.
 
Feeding management
 
All fish were fed ad libitum with commercially available fish feed pellets (Tetra Bits Complete and Optimum Micro-pellet) during the experimental period.
 
Evaluation of anti-nociceptive activity of Annona squamosa
 
The anti-nociceptive potential of Annona squamosa was investigated using different nociceptive models in zebrafish. A total of 90 zebrafish were divided into three groups. Except for Group III, each group was further subdivided into four subgroups designated as A, B, C and D. Experimental procedures were conducted according to the protocol described by Magalhaes et al., (2017).
 
Experimental design
 
Initially, zebra fish were anaesthetized using chilled water maintained at 4°C. After anaesthesia, fish were positioned on a moist sponge and administered either Annona squamosa extract or meloxicam 30 minutes before injection of algogenic substances. Except for the control group, all animals received intramuscular administration of nociceptive agents including glutamate (12.5 μM) and capsaicin (40.93 μM), as shown in Table 1. Following treatment, fish were individually transferred into beakers containing 150 mL tank water for recovery for 30 minutes.


 
Glutamate induced nociception
 
Glutamate (12.5 μM; 5.0 μL; i.m.) dissolved in distilled water was injected in tail of the fish, 30 minutes after treatment with ethanolic extract of Annona squamosa (200 and 400 μg/mL; 20 µL; i.p.). The anti-nociceptive activity of ethanolic extract of Annona squamosa was evaluated by calculating the number of line crossing by each fish during 0-15 minutes (after administration of glutamate). Further, behavioural responses to noxious stimuli were monitored by novel tank diving test.
 
Capsaicin induced nociception
 
Capsaicin (4093 μM; 5.0 μL; i.m) dissolved in ethanol, phosphate-buffered saline (pH 7.4) and distilled water (1:1:8) was injected in tail of the fish, 30 minutes after treatment with ethanolic extract of Annona squamosa (200 and 400 μg/mL; 20 µL; i.p.). The anti-nociceptive activity of ethanolic extract of Annona squamosa was evaluated by calculating the number of line crossing by each fish during 0-10 minutes (after administration of capsaicin). Further, behavioural responses to noxious stimuli were monitored by novel tank diving test.
 
Parameters of the study
 
Number of line crossing
 
For behavioural analysis, following treatment and administration of the algogenic agent, each fish was placed in a glass Petri dish measuring 10 x 15 cm as shown in Fig 3. Nociceptive responses were assessed by quantifying locomotor activity, specifically the number of line crossings within a set time period determined for each pain model. The anti-nociceptive effect was evaluated individually for each fish during the designated observation period for each nociception model, as outlined by Ohnesorge et al., (2021).


 
Novel tank diving paradigm
 
The novel tank diving test is a rapid and efficient method, typically completed within 5-10 minutes and is highly responsive to pharmacological, genetic and environmental changes. The efficacy of Annona squamosa was assessed by monitoring zebrafish behaviour in a novel tank as shown in Fig 4, with specific movements recorded as per the procedure described by Collier et al., (2017).
I. Time spent in upper portion of tank.
II. Number of entries into upper portion of tank.
III. Average entry duration.
IV. Number of erratic movements.
V. Number of freezing bouts.
VI. Freezing duration.


 
Behavioural anti-nociceptive activity
 
The concentrations of the nociceptive and antagonistic agents, along with the duration of nociceptive response analysis, were selected based on previously established parameters. Four nociception models were employed using capsaicin and glutamate. Meloxicam (0.2 mg/mL; 20 μL; i.p.), was used as the standard nociceptive antagonist.

The present investigation was designed to evaluate the anti-nociceptive activity of ethanolic extract of Annona squamosa leaves in zebrafish. Previous phytochemical analyses of A. squamosa leaves have reported the presence of several biologically active constituents including flavonoids, phenolic compounds, tannins, alkaloids, saponins, triterpenoids, proteins and organic acids, indicating the rich phytochemical composition of the plant (Varadharajan et al., 2022). Zebrafish behavioural assays are increasingly used for screening pharmacologically active compounds because of their measurable behavioural and physiological responses.
       
In the current study, anti-nociceptive efficacy was assessed by behavioural evaluation of zebrafish after administration of nociceptive agents in different pain models. Nociceptive responses were determined by assessing locomotor activity through the number of line crossings within a fixed observation period, as suggested by Ohnesorge et al., (2021). In addition, behavioural changes were assessed using the Novel Tank Diving Test, which included parameters such as time spent in the upper portion of the tank, number of entries into the upper zone, average duration of entry, erratic movements, freezing bouts and freezing duration according to Collier et al., (2017).
 
Efficacy of ethanolic extract of Annona squamosa leaves on glutamate induced nociception
 
The anti-nociceptive activity of Annona squamosa against glutamate-induced nociception is illustrated in Fig 5. The glutamate nociception model consisted of four subgroups. Subgroup A received glutamate alone (12.5 ìM; 5.0 μL; i.m.), subgroup B received glutamate along with ethanolic extract of Annona squamosa (200 μg/mL; 20 μL; i.p.), subgroup C received glutamate with extract at 400 ìg/mL and subgroup D received glutamate with meloxicam (0.2 mg/mL; 20 μL; i.p.).


 
Number of line crossings
 
The mean number of line crossings recorded in control, subgroup A, subgroup B, subgroup C and subgroup D were 203.5±54.2, 97.7±13.8, 130.5±18.3, 144.8±14.6 and 163.7±21.3, respectively. A significant decline in locomotor activity was observed in glutamate-treated fish compared with the control group. Administration of Annona squamosa extract significantly increased the number of line crossings compared with the glutamate-only group, indicating attenuation of nociceptive behaviour.
 
Novel tank diving paradigm
 
The mean values of various behavioural parameters of zebrafish in glutamate induced nociception have been graphically depicted in Fig 6, 7, 8, 9, 10 and 11. Behavioural observations in the novel tank test demonstrated that glutamate administration significantly reduced the time spent in the upper region of the tank compared to controls. Treatment with Annona squamosa extract significantly improved this parameter and produced effects comparable to meloxicam. Similar improvements were observed in the number of entries into the upper portion and average entry duration.












       
Glutamate administration markedly increased erratic movements, freezing bouts and freezing duration, whereas treatment with Annona squamosa significantly reduced these nociceptive-associated behaviours. The extract effectively restored behavioural responses altered by glutamate exposure.
       
These findings suggest that glutamate induces nociceptive-like responses by reducing locomotion and enhancing stress-related behaviour. Pretreatment with Annona squamosa significantly alleviated these effects, indicating anti-nociceptive action possibly mediated through modulation of NMDA receptor-associated pathways. Similar observations have been reported by Batista et al., (2008) and Beirith et al., (2002), who demonstrated involvement of glutamatergic mechanisms in nociception.
 
Efficacy of ethanolic extract of Annona squamosa leaves on Capsaicin induced nociception
 
The effects of Annona squamosa on capsaicin-induced nociception are represented in Fig 12. Anti-nociceptive activity was evaluated by assessing the number of line crossings during the 0-15 minute observation period after capsaicin administration.


 
Number of line crossings
 
The mean values of line crossings in control, subgroup A, subgroup B, subgroup C and subgroup D were 187.9±11.3, 86.8±12.3, 108.8±11.1, 127.8±19.6 and 154.4±18.9, respectively. Fish receiving capsaicin alone exhibited a significant reduction in locomotor activity relative to the control group. Treatment with Annona squamosa extract significantly improved locomotor behaviour compared to capsaicin-treated fish.
 
Novel tank diving paradigm
 
The mean values of various behavioural parameters of zebrafish in capsaicin induced nociception have been graphically depicted in Fig 13, 14, 15, 16, 17 and 18. Behavioural evaluation revealed that capsaicin administration significantly reduced the time spent in the upper zone of the tank, whereas Annona squamosa treatment significantly restored this behaviour in a dose-dependent manner. Similar improvements were observed in the number of entries into the upper region and average entry duration.












       
Capsaicin treatment also produced a marked increase in erratic swimming, freezing bouts and freezing duration. Administration of Annona squamosa extract significantly reduced these abnormal behavioural responses. The extract effectively mitigated capsaicin-induced nociceptive behaviours, indicating its anti-nociceptive potential through modulation of TRPV1 receptor-mediated pathways.
       
Comparable findings were reported by Nguelefack et al., (2015), who demonstrated significant inhibition of capsaicin-induced nociception through modulation of TRPV1 receptors.
       
Adult zebrafish are increasingly recognized as valuable translational models for studying nociception induced by different noxious stimuli (Costa et al., 2019). In the present study, ethanolic extract of Annona squamosa leaves demonstrated considerable anti-nociceptive activity in both glutamate- and capsaicin-induced nociception models. Glutamate acts as an excitatory neurotransmitter in the central nervous system and induces pain responses through glutamatergic receptors, particularly NMDA receptors (Sharma et al., 2022). Therefore, modulation of glutamatergic signalling is considered a promising target for analgesic drug development. The inhibitory effects observed in the present study suggest involvement of NMDA receptor pathways in the anti-nociceptive activity of Annona squamosa.Transient receptor potential (TRP) channels, particularly TRPV1 and TRPA1, are important mediators of pain perception in sensory neurons (Batista et al., 2008). The inhibitory effects of Annona squamosa in the capsaicin-induced nociception model suggest that the extract may exert analgesic action through modulation of TRPV1 receptor activity.

The findings of the present investigation demonstrate significant anti-nociceptive activity of ethanolic extract of Annona squamosa leaves in zebrafish models of chemically induced pain. Behavioural assays using glutamate and capsaicin successfully produced nociceptive responses characterized by reduced locomotion, enhanced freezing behaviour and altered exploratory activity, indicating activation of NMDA and TRPV1 receptor pathways, respectively. Pretreatment with Annona squamosa extract significantly reduced these nociceptive behavioural alterations, suggesting that its analgesic effects may involve modulation of NMDA-and TRPV1-mediated signalling mechanisms.
       
Overall, the study indicates that bioactive constituents present in Annona squamosa contribute to its anti-nociceptive activity through receptor-mediated mechanisms. These observations highlight the therapeutic potential of Annona squamosa as a plant-derived candidate for pain management. Further studies are required to isolate specific active compounds and clarify their detailed pharmacological and molecular mechanisms of action.

The authors gratefully acknowledge the support of the Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (NDVSU), Jabalpur, MP, for providing laboratory facilities and technical assistance.
 
Disclaimer
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.