Pharmacological Evaluation of Benincasa Hispida Cogn. Fruit on Chronic Foot Shock Induced Stress in Mice


Anjali M. Wankhade1, Manish M. Wanjari2, Rupali Dhuldhar1, Umair Akhtar1

1Department of Pharmacology, Vidyabharati College of Pharmacy,

C.K. Naidu Road, Camp, Amravati -444602 (Maharashtra) India.

2Central Research Institute (Ayurveda), Opposite Jayarog Hospital,

Aamkho, Lashkar, Gwalior - 474 009 (Madhya Pradesh), India.

*Corresponding Author E-mail:



Stress can be defined as a brain-body reaction towards stimuli arising from the environment or from internal cues that are interpreted as a disruption of homeostasis. A number of reports indicated that treatment with herbal plant extracts could lead to a significant reduction in stress. The aim of this study was to evaluate the antistress activity through Inescapable electric chronic foot shock model in mice and to assess the effect of extract on stress induced behavioral changes in animals and to determine the efficiency of Benincasa hispida Cogn. Extract on Motor activity and Elevated plus maze to assess anxiety. In this study, mice were divided into five groups with five animals each. The first group received ip dose of the vehicle saline (2ml/kg). The second group received diazepam 1mg/kg ip along with foot shock. Third, fourth, and fifth groups received oral dose of 100, 200, and 400 mg /kg of fruit extract of Benincasa hispida along with foot shock. Inescapable electricFoot-shocks (intensity 0.8mA, interval: 10sec, duration:10sec) were delivered through a grid floor once daily for 21 days. The results showed that Benincasa hispida extract (200 and 400mg/kg) was found to have an antistress effect. The dose of Benincasa hispida extract (200 and 400mg/kg), increased entries into open arm and the time spend in open arm on the elevated plus maze test indicate that extract reduced the stress level and it significantly reduced the spontaneous motor activity and decreased the anxiety. The results suggest that the ethanolic extract of Benincasa hispida has potential antistress effect that can be explored for therapeutic advantage as an alternative treatment in medical conditions.



KEYWORDS: Benincasa hispida Cogn., Chronic inescapable footshock stress, antistress activity, kushmanda.




The Benincasa hispida (Cogn) cucurbitaceae family, also known as winter melon, wax gourd, and kushmanda, is used in Ayurvedic medicine1. The plant has been used medicinally to treat a variety of conditions, including urinary diseases, heart disease, diabetes, and respiratory illnesses.


Fruits have historically been used for laxative, diuretic, tonic, aphrodisiac, cardiotonic, urinary calculi, blood disease, sanity, epilepsy, schizophrenia, and other psychological disorders, as well as for jaundice, dyspepsia, fever, and menstrual disorders.


The pharmacological study showed that the plant had a wide range of pharmacological effects, including antimicrobial, antioxidant, anti-inflammatory, analgesic, antiasthmatic, diuretic, nephroprotective, and central nervous effects (anxiolytic, muscle relaxant, antidepressant, and in the treatment of Alzheimer's disease and to minimise opiates withdrawal signs)2, which suggests that Benincasa hispida influences different neurotransmitter systems3.


A complex range of physiological and behavioural reactions that restore homeostasis are used to combat stress, which is characterised as a state of threatened homeostasis or dysharmony (adaptive stress response). Anxiety in the body or mind can be brought on by stress, which can be either physical, mental, or emotional. Stress may be internal or result from a medical procedure. It may also be external (caused by the environment, psychological issues, or social situations). Strokes, heart attacks, ulcers, and mental disorders like depression and cognitive deficit can all be made worse by stress4, 5.


The current pharmacological treatments for stress primarily target symptom inhibition, but their effectiveness was constrained by the presence of a number of negative side effects (dependence, tolerance). Focus has shifted to natural products as the new sources of antistress agents due to side effects associated with the available anti-stress medications6. Numerous plants have been studied based on the traditional knowledge of their pharmacological properties and have been found to be effective in treating and managing stress as a result of the growing interest in natural medicine6, 7.


The fruit of the Benincasa hispida has been shown to have nootropic, anti-depressant, and anxiolytic effects, which indicates that it may affect different neurotransmitter systems. The medicinal plant with central actions may offer therapeutic benefits in treating stress and its associated symptoms when compared to the currently available therapeutic approaches7. These data imply that Benincasa hispida may be effective in the management of stress-related disorders. Therefore, it was determined whether an ethanolic extract of the fruit of the Benincasa hispida could reduce chronic stress in mice caused by foot shock8, 9, 10.



1. Collection and Authentication of Plant Material:

In January 2016, the medicinal plant Benincasa hispida was procured from the neighbourhood of Amravati, Maharashtra, India. Taxonomists were able to identify the plant. The necessary plant part was separated from other unwanted material and dried in the shade11.


2. Preparation of fruit extract:

Benincasa hispida fruit ethanolic extract (EEBH) was made using maceration procedure as previously mentioned12. Fresh Benincasa hispida fruit was gathered, and after the outer skin and seeds were aseptically removed, the fruit was mash using an electric juicer to produce a soft mass. 100 ml of this juice was combined with 500 ml of ethanol to create an alcoholic preparation13. For seven days, the mixture was left at room temperature under cover while being occasionally stirred each day. Following filtration, the filtrate was heated (to below 55°C) and evaporated under reduced pressure. After the extract had fully dried, it had transformed into a brownish sticky mass (yield: 5% w/w). Then, it was shielded from sunlight and kept at a temperature of 2-4°C14.


Proteins, tryptophan, sterols, volatile oils, glycosides, and phenolic compounds were found in EEBH during the phytochemical screening process, while carbohydrates, flavonoids, and alkaloids were not present15.


3. Drugs and administration:

Diazepam 5mg (Alpazepam injection, Alpa Laboratories ltd) was the standard drug used. EEBH was dissolved in saline and diazepam was diluted with saline to reach the proper concentrations16. Standard drug or vehicle were injected i.p and EEBH was injected orally, once daily for 21 days and during the testing sessions 30 min before each experiment17.


4. Animals:

The Swiss mice (weighing 20-25g), which were obtained from the animal house of department of Pharmacology, Vidyabharti college of Pharmacy, Amravati. All the animals were acclimatized to the animal house prior to use. They were kept in cages in animal house with a 12hr light: 12hr dark cycle at temperature (25±1°C) with 50±55% of relative humidity18,19. Animals were fed on pellets and tap water ad libitum. The care and handling of mice were in accordance with the internationally accepted standard guideline for use of animals (CPCSEA). Experiments were performed in accordance with the committee for the purpose of control and supervision of experimental animals (CPCSEA) (1504/PO/Re/S/11/CPCSEA dated 9/8/16) guidelines after the approval of the experimental protocol by the Institutional animals ethical committee (IAEC) 20, 21.



A. Induction of stress:

Mice were divided into different groups (n=5). EEBH (100, 200, 400 mg/kg) po, diazepam (1 mg/kg) ip. The control group received 0.9% saline (2 mL/kg, ip). All the animals were habituated for 30 min in their home cages before the start of the experiments22, 23. Plexiglass chamber (300× 300× 350) with a stainless steel grid floor (4 mm diameter, 9 mm interval) used for foot shock. Inescapable electric Foot-shocks (intensity 0.8mA, interval:10sec, duration:10sec) were delivered through a grid floor once daily for 21days.Each animal was placed in the chamber, after a 2 min adaptation period, the inescapable electricfoot-shocks were delivered for a total 5 min. Controls were just placed in the compartment for 60 min without any foot shock24. After stress com-pletion, thetest groups of animals were subjectedto evaluate antistress activity and their behavioral paramaters was recordedas an index of stress25,26.


B. Behavioral parameters:

On day 21 post-foot shock following parameters were used to assessed stress induced anxiety27.


Motor activity:

Using an actophotometer, motor activity was measured for 10 minutes. When an animal moves, a light beam that was falling on the photocell is cut off, and a count is digitally recorded and displayed. The total number of counts of light beam interruptions in 10 minutes was used to measure motor activity28. Before evaluating motor activity, each mouse received a 5-minute acquisition period29, 30.


Elevated Plus Maze:

Elevated Plus Maze is widely and universally accepted paradigm to study anxiety related behaviors in animals. The Plus Maze apparatus consist of two open arms (16 x 5 cm) and two close arms (16x 5x 12 cm) and an open roof with the entire maze elevated at a distance of 25 cm from the floor. Lamp was mounted above the apparatus to provide illumination and was kept on throughout the experiment. The drugs as well as vehicle treated mice were kept individually at the center of the Plus Maze with their head facing towards open arm. During the 10 min. test session, the number of entries into open and closed arms and time spent in open arm is recorded31,32,33.


Statastical analysis:

The data were expressed as mean ± SEM. Results were analysed stastically by one way ANOVA followed by Dunnets test. Statistical significant was considered as P< 0.05 in all cases34.



Elevated plus maze:

Effect of ethanolic extract of Benincasa hispida on Elevated Plus Maze in mice


Table no.1: Each value represents the mean±S.E.M (n=5) *p<0.01 compared with control and standard group.



 % number of entries (in 10 minutes)

 % Time spent (in 10 minutes)

Open arm

Mean ± SEM

Closed arm

Mean ± SEM

 Open arm

Mean ± SEM

Closed arm

Mean ± SEM

Saline (Control)

2ml/kg ip





Diazepam (Standard)

1mg/kg ip


21.80 ±1.8602




100mg/kg p.o.


37.00 ±0.7071




200mg/kg p.o.






400mg/kg p.o.


27.40 ±2.6782





Figure1: Effects of Benincasa hispida extract and diazepam on elevated-plus maze test performance of stressed mice. On day 21 after stress procedure, the animals received the elevated-plus maze test for 10 min. Daily administrations of Benincasa hispida extract and diazepam were started from the first day of stress procedure. The effects on the plus-maze performance were elucidated 30 min after the treatment. The number of open arm entries was recorded. Each column represents the mean with SEM p<0.01 compared with control and standard group.


Figure 2: Effects of Benincasa hispida extract and diazepam on elevated-plus maze test performance of stressed mice. On day 21 after stress procedure, the animals received the elevated-plus maze test for 10 min. Daily administrations of Benincasa hispida extract and diazepam were started from the first day of stress procedure. The effects on the plus-maze performance were elucidated 30 min after the treatment. The time spend in open arms were recorded. Each column represents the mean with SEM p<0.01 compared with control and standard group.



Motor activity:

Effect of ethanolic extract of Benincasa hispida on motor activity in mice


Table no. 2: Each value represents the mean±S.E.M (n=5). **p<0.01 Compared with control and standard group.



Locomotor activity

Mean ± SEM


2ml/kg ip

460.2 ± 1.772


1mg/kg ip

306.6 ± 1.720**


100mg/kg p.o.

429.0 ± 0.8367


200mg/kg p.o.

376.4 ± 2.731


400mg/kg p.o.

286.8 ± 4.104**


Figure 3: Effects of Benincasa hispida extract and diazepam on motor activity of mice exposed to the chronic foot shock. On day21 after chronic foot shock procedure, the locomotor activity was measured. Daily administrations of Benincasa hispida extract and diazepam were started from the first day of foot shock. The effect on motor activity was elucidated 30 min after the treatment. Each column represents the mean with SEM p<0.01 compared with control and standard group.


Elevated plus maze:

As shown in fig.1 and 2, open arm entries and time spent in open arm were significantly reduced in saline control animals. Repeated administrations of Benincasa hispida extract at doses of 200 and 400mg/kg and diazepam at dose 1mg/kg significantly increases the number of entries and the time spent into open arms.


Motor activity:

The effects of Benincasa hispida extract and diazepam on motor activity in mice with chronic foot shock were shown in fig.3. Repeated administrations of Benincasa hispida extract at doses of 200 and 400 mg/kg and diazepam at dose 1mg/kg significantly reduced the motor activity in mice.



One of the key factors responsible for stress syndromes seems to be an imbalance of the sympathetic and parasympathetic nervous systems35. Stress is the body’s physical, mental or chemical reaction observed usually during excitation, confusion or in unsafe condition. A large proportion of all illness is believed to occur due to stress closely associated with modernization of life36.The stress response occurs via very complex and multifaceted mechanisms involving a series of physiological, behavioral, metabolic, and immunological reactions37, 38. The integrated response to stress includes a number of neuroendocrine molecules of both the hypothalamic-pituitary adrenalaxis and the sympathetic nervous system, such as catecholamines, adrenocorticotropin andglucocorticoids39.Stress is an unavoidable phenomenon that affects the body system at various levels. Any stress can induce three major changes exogenous stresses or endogenous stress initiating a disturbance in the body, the physical and chemical disturbances induced by the stressor in the body, and the body counteracts these responses40.


Despite to the availability of many pharmaceutical products for the treatment of stress in the market, their successes were limited by presence of several adverse effects41.Due to reported side effects of available antistress drugs, focused have been shifted towards natural products as the new sources of antistress agents. With the increasingly growing interest in natural medicine, various plants have been studied based on the traditional knowledge of their pharmacological properties and confirmed to be useful in treating and managing stress. Furthermore, medicinal plants have been known to be amongst the most attractive sources of new drugs, and have been shown to give promising results in the treatment of various diseases and disorders42.


This study, demonstrated that the animals submitted to foot shocks not only exhibited long-term and increased anxiety level but also induced a specific avoidance to the context associated with the aversive foot shock. The present study investigated the antistress effect of Benincasa hispida extract on foot shock induce stress in mice compared to diazepam and control group (vehicle). The Benincasa hispida extract (200 and 400mg/kg) was found to have an antistress effect on locomotor and elevated plus maze activity. In the present study, after repeated daily administration for 21 days diazepam dose of (1mg/kg) significantly decrease the motor activity, increases the open arm entry and time spend in open arm in elevated plus maze test.


Chronic stress is generally considered as a key risk factor for the development of a variety of human ailments. Specifically anxiety and depressive disorders have been frequently associated with stressful life events43. Activation of the stress system leads to behavioral and peripheral changes that adjust homeostasis and improve coping with stress situations44. On the other hand, a lack of adaptation to excessive demands can lead to the development of pathological syndromes, such as depression and anxiety45. Chronic stress affects brain areas such as hippocampus, amygdala and prefrontal cortex, involved in anxiety and affective disorder, evidenced in postmortem and brain imaging studies of depressed and anxious patient46. The neurochemical pathways in central nervous system have been reported to play a vital role in the regulation of stress responses such as diminish the serotonergic transmission in the prefrontal cortex postulated to involve in pathogenesis of depression and anxiety47.The increased depression and anxiety like behaviour may be related to the dysfunction of serotonergic neurotransmission in the prefrontal cortex and limbic system, although involvement for glutamatergic system have also been reported. Several lines of evidence suggest that, depletion of monoamines: serotonin, noradrenalin and dopamine sustained stress could be the reason for anxiety and behavioural depression48.


In animal studies, long lasting change in synaptic plasticity in the medial prefrontal cortex has been implicated in the pathophysiology of stress, in addition to the stress vulnerability of the hippocampus, possibly induced by elevated glucocorticoid levels, altered levels of neurotrophic factors, and by changes in serotonergic and noradrenergic neurotransmission. Selective serotonin reuptake inhibitors (SSRIs) have been reported to show clinical efficacy in the treatment of stress. Since after chronic administration of Benincasa hispida extract and diazepam the behavioral deficiencies in this animal model also can be reversed at some specific dose  range49, 50.



In present study it is concluded that Benincasa hispida extract may significantly have the antistress activity. It decreases the locomotor activity and it increases the open arm entry and time spend in open arm. The extract also decreases the freezing behavior in mice. The results suggest that the ethanolic extract of Benincasa hispida has potential antistress effect that can be explored for therapeutic advantage as an alternative treatment in medical conditions.


The antistress activity is probably due to the presence of bioactive compounds like triterpenoids, flavonoids, glycosides and sterols in the EEBH. Further studies are required to confirm the exact mechanism and isolation of the bioactive compounds involved in the extract.



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Received on 28.01.2023           Modified on 25.02.2023

Accepted on 16.03.2023       ©A&V Publications All right reserved

Res.  J. Pharmacology and Pharmacodynamics.2023;15(2):49-54.

DOI: 10.52711/2321-5836.2023.00010