|Year : 2014 | Volume
| Issue : 2 | Page : 88-94
Adaptogenic studies of acetone extract of Musa paradisiaca L. fruit peels in albino Wistar rats
Sibi Ittiyavirah, DA Anurenj
Department of Pharmacology, University College of Pharmacy, Ettumanoor, Kottayam, Kerala, India
|Date of Submission||28-Nov-2013|
|Date of Acceptance||09-Feb-2014|
|Date of Web Publication||29-Mar-2014|
Department of Pharmacology, University College of Pharmacy, Ettumanoor, Kottayam, Kerala
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Rationale: The present study deals with the evaluation of antistress activity in acetone extracts of M.paradisiaca unripe fruit peels and acetone extracts of M.paradisiaca ripe fruit peels (AURMP, ARMP) with special reference to stress induced depression in animal models. The presence of high amounts of plant sterols and triterpenoids in the peels are expected to reduce the cholesterol level and the modulating effect of cholesterol in the stress and chronic depression. The potentially high antioxidant activity in peel extracts is hypothesized to produce the adaptogenic activity. Materials and Methods: The anoxia stress was evaluated on the 7 th , 14 th , and 21 st day, one hour after the AURMP and ARMP treatment in the anoxia stress model. The swimming endurance stress was assessed as the mean swimming time on 8 th day following AURMP and ARMP treatment. The antidepressant activity was measured using open field and forced swim test (FST) following chronic variable stress (CVS) model. The level of tryptophan in brain was estimated ex vivo by HPLC technique. Results: The mean endurance time was increased significantly by AURMP (39.17 ± 1.88) treatment (P < 0.01) alone in comparison with the control in swimming endurance test. The anoxia stress model also showed significant (P < 0.001) antidepressant activity for AURMP and ARMP. Both AURMP and ARMP had suppressed the stress induced by CVS model and this was evidenced from the results of FST and open-field test confirming antidepressant activity. It is concluded that the AURMP and ARMP had shown promising antistress activity.
Keywords: Antistress, chronic variable stress, Musa paradisiaca, tryptophan
|How to cite this article:|
Ittiyavirah S, Anurenj D A. Adaptogenic studies of acetone extract of Musa paradisiaca L. fruit peels in albino Wistar rats. Int J Nutr Pharmacol Neurol Dis 2014;4:88-94
|How to cite this URL:|
Ittiyavirah S, Anurenj D A. Adaptogenic studies of acetone extract of Musa paradisiaca L. fruit peels in albino Wistar rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2014 [cited 2019 Jan 16];4:88-94. Available from: http://www.ijnpnd.com/text.asp?2014/4/2/88/129592
| Background|| |
Stress has become an integral part of human life and organisms are constantly subjected to stressful stimuli that affect numerous physiological processes. Stress can be defined as the sum total of all the reactions of the body, which disturb the normal physiological condition and results in a state of threatened homeostasis. These stress-induced changes are compensatory, self limiting and adaptive. However, when stress events cross over certain threshold limits, the changes become rather irreversible. It leads to altered homeostasis and exhaustion, manifesting itself in the pathologic form of stress-induced disease and maladjustment.  Chronic stress may cause physical, behavioral and/or neuropsychiatric manifestations: Anxiety, depression, and/or cognitive dysfunction; cardiovascular phenomena, such as hypertension; metabolic disorders, such as obesity, the metabolic syndrome, and type II diabetes mellitus; atherosclerotic cardiovascular disease; neurovascular degenerative disease and sleep disorders, such as insomnia or excessive daytime sleepiness.  Depression results from a combination of genetic, biochemical, environmental, and psychological factors. Corticotropin releasing hormone (CRH) is released from the hypothalamus in response to the perception of psychological stress by cortical brain regions. This hormone induces the secretion of pituitary corticotropin, which stimulates the adrenal gland to release cortisol into the plasma.  Elevated cortisol may act as a mediator between major depression.  Neural changes mirror the pattern seen in other body systems, that is, short-term adaptation vs. long-term damage. 
The fruit of M.paradisiaca is used in many magical remedies, and many herbal formulations in traditional practice. They are used to exorcise evil spirits etc., by the local tribes and village doctors in India. The ripe fruits are given to cure patients suffering from mental disorders and skin diseases. The practice where the fruit is bitten by an angry cobra-snake and this poisoned fruit is given to cure leprosy patients, is still practiced in some areas.  Acyl steryl glycosides and steryl glycosides have been isolated from fruits of M. Paradisiaca.  Serotonin, nor-epinephrine, tryptophan, indole compounds, tannin, starch, iron, crystallisable and non-crystallisable sugars, vitamin C, vitamin B, albuminoids, fats, mineral salts have been found in the fruit pulp of M. paradisiaca It has been reported that the anti-ulcerative effect of banana may vary depending on different varieties of banana. It was also seen that the ethanolic extract of both M. sapientum and M. paradisiaca have significant gastroprotective effect but only M. paradisiaca promotes ulcer healing by a similar mechanism like prostaglandins.  The antihypertensive effect of ripe banana pulp in deoxycorticosterone enantate-induced hypertensive rats which may be due to the high tryptophan and carbohydrate content of banana that increases serotonin levels and gives serotonin mediated natriorexic effect. 
| Results|| |
Acute toxicity studies
The acetone extracts of the ripe and unripe fruit peels of plant M. paradisiaca was found to be safe up to 2000 mg/kg body weight by oral route. After 24 hours, animals were found well tolerated, there was no mortality and no signs of toxicity. The extract was found to be safe, so the dose of 200 mg/kg body weight was selected for the present study.
Swimming endurance test
The swimming endurance time was significantly (P < 0.05) enhanced on 8 th day in AURMP (200 mg/kg), AURMP (200 mg/kg) and diazepam (2 mg/kg) treated groups as compared to the control group. The AURMP showed an increase of (39.17 ± 1.887) in swimming endurance time in comparison to the control group (28.83 ± 1.815) [Figure 1].
Anoxia stress tolerance time
Anoxia stress tolerance time was significantly (P < 0.001) enhanced on 14 th and 21 st day in URMP (200 mg/kg) and Diazepam (2 mg/kg) treated groups. Values are expressed as Mean ± SEM compared with the control. The anoxia tolerance time was seen to be increased 14 th and 21 st day of AURMP treatment. AURMP increased the anoxia stress tolerance to (48.50 ± 2.30) in comparison to the control group (30.33 ± 2.49) on the 3 rd week of treatment. The ARMP (200 mg/kg) was also seen to increase the anoxia tolerance time on the 3 rd week of treatment [Figure 2].
|Figure 2: Effect of M.paradisiaca on anoxia stress tolerance time in rats|
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Chronic variable stress model
The chronic stress was induced by chronic variable stress (CVS). Rats in stressed groups were exposed to the following stressors once daily for 5 consecutive weeks: 2-h immobilization; 24-h food deprivation; 24-h water deprivation; 5-min cold swim at 4°C; wet bedding; 1-min tail pinch.
Forced swim test
The results obtained from the forced swim test were expressed as Mean ± SEM. The immobility time was significantly (P < 0.001) decreased in animals treated with AURMP and ARMP in comparison to the stress group. In comparison with the control group (274 ± 3.97) AURMP (307 ± 2.88) and ARMP (313 ± 1.47) showed a marked decrease in immobility time [Figure 3].
The grooming response was significantly (P < 0.001) increased in animals treated with fluoxetine (10 mg/kg) and AURMP (P < 0.01) and ARMP (P < 0.05) in the fifth week of treatment. Values are expressed as Mean ± SEM in comparison with the stress group [Figure 4].
[Figure 5] shows that the rearing response was significantly (P < 0.001) increased in animals treated with fluoxetin (10 mg/kg) and AURMP (P < 0.001) and ARMP (P < 0.05) in the fifth week of treatment in comparison with the stress group. The increase in rearing scores by the AURMP in the fifth week was significant similar to that of the standard group. While the AURMP showed a score of 24.17 ± 1.97 the stress group has a score of 11 ± 1.36. On the second week there was no significant increase in rearing scores.
The central square activity also showed a significant (P < 0.01) increase in animals treated with fluoxetin (10 mg/kg) and AURMP (P < 0.01) in the fifth week of treatment. The ARMP treatment was not able to show any significant effect on the central square activity [Figure 6].
The square crossing response was significantly (P < 0.001) increased in animals treated with fluoxetin (10 mg/kg) and AURMP (P < 0.001) and ARMP (P < 0.01) in the fifth week of treatment in comparison to the stress group. The square crossing was 32 ± 1.15 for the AURMP treated group, whereas it was 16.83 ± 1.35 for the stress group [Figure 7].
Ex vivo quantification of tryptophan in rat brain HPLC method
The ex vivo analysis of rat brain was done for the quantification of tryptophan levels and quantification was done by HPLC technique. The tryptophan levels were quantified and expressed per gram of brain. The average brain weight was taken as 2 g. The AURMP and ARMP treated groups were shown to have an increased tryptophan level in their brain in comparison to the control as well as the stress group animals. The values were expressed as Mean ± SEM values.
| Discussion|| |
In case of swimming endurance test AURMP exhibited significant anti-stress activity as indicated by increase in swimming endurance time [Figure 1]. The AURMP showed a significant (P < 0.01) increase in mean swimming endurance time of (39.17 ± 1.88 ) in comparison with the control value of (28.83 ± 1.18). These results indicate clearly that the AURMP increases the physical endurance as well as the overall performance in rats. The anti-stress activity exhibited in swimming endurance test may be by its modulating effect on the plasma levels of catecholamines and monoamine oxidase (MAO). This can be due to increased utilization of the ATP-CP pathway, increased levels of muscle glycogen (a storage form of glucose that can provide energy for more prolonged activities), or decreased concentrations of muscle lactic acid and ammonia (two toxic by-products of muscular effort). It can be attributed to the anti-oxidant effect of plant extract, which prevent the free radical-induced damage of the vital organs.  Plasma levels of adrenaline and noradrenaline are enhanced during stress induced by swimming endurance test. In addition, MAO levels in the brain are reportedly decreased during stress. Under stressful conditions, cortisol in human and corticosterone in rats will be secreted by adrenal cortex. Hyper secretion of cortisol helps the maintenance of internal homeostasis through the process of gluconeogenesis and lipogenesis. 
In anoxia stress tolerance model, depletion of oxygen in hermetic vessel leads to convulsions in animals and pre-treatment with AURMP and ARMP had increased the duration of stress tolerance indicating their adaptogenic/anti-stress activity [Figure 2]. The anoxia stress value of AURMP and ARMP were respectively 48.50 ± 2.30 and 43.50 ± 3.64 and it was seen to be increased significantly in comparison to the positive control 30.33 ± 2.49. This effect may be due to the fact that during stress the acetone extract of M.paradisiaca was capable of increasing succinate dehydrogenase (SDH) in the brain. The enzyme that is responsible for utilization and conservation of energy in the cellular system of the organism helps the adaptive processes during stress. 
The anti-depressant activity of M.paradisiaca was estimated using chronic variable stress model. The chronic treatment of AURMP and ARMP had shown significant anti- depressant activity in open-field test, which was evidenced by the increase in rearing, grooming, central square activity and square-crossing parameters [Figure 4], [Figure 5], [Figure 6], [Figure 7]. The open-field behavioral model, studies the exploratory and locomotor activity in animals. The AURMP showed a rearing score of (2.33 ± 0.42) whereas the stress only group showed the rearing score of (0.5 ± 0.2). The positive results in the open-field test shows that the AURMP and ARMP have the ability to reverse or normalize the locomotor suppressant behavior in the experimental animals and hence may help to cope with immobility associated with depression in humans.
The decrease of immobility time in the forced swim test in treated groups also showed the significant anti depressant activity of AURMP and ARMP. The AURMP decreased the duration of immobility to (307 ± 2.88) while the stress group showed an immobility time of (330 ± 5.03). It has been shown that stress induces a state of helpless despair condition in animals which is equivalent and claimed to a condition similar to human depression. This attribution of animal's response to development of depression process can be managed by treatment with anti depressants. In this study, significant reduction in immobility time (by increase in swimming time and climbing) was observed following oral administration of AURMP and ARMP [Figure 3]. Anti-depressant drugs with predominant noradrenaline or dopaminergic elevating effects, reduce the immobility by increasing climbing behavior, whereas, anti depressant drugs with predominant serotonin elevating effect reduce immobility time by increasing the swimming behavior. Catecholamines such as norepinephrine, serotonin, dopamine, , tryptophan, and indole compounds are present in the M.paradisiaca.  The ex vivo HPLC quantification for tryptophan has also shown the increased levels of tryptophan in the brains of animal groups treated with AURMP and ARMP in comparison to the stress group [Table 1]. Amino acid tryptophan is the precursor of serotonin. The combination of tryptophan with a monoamine oxidase inhibitor (known to raise brain 5-HT concentration) is more effective in treating depression than a MOA inhibitor alone. The explanation seems to be that tryptophan 5-hydroxylase, the first enzyme in the pathway of 5-hydroxytryptamine synthesis, is normally unsaturated with substrate so that when brain L-tryptophan concentrations are raised by administration of this amino-acid 5-HT synthesis is increased.  Hence the possible mechanism behind the anti depressant activity of AURMP and ARMP may be due to the increase in monoamine levels in the synaptic cleft.
| Conclusion|| |
Studies proved that the acetone extracts of fruit peels of Musa paradisiaca Linn possess significant anti-stress activity and the effect on chronic stress was also proved.
| Methodology|| |
Collection and identification of the plant materials
Musa paradisiaca fruit peels were collected from Aryankode village of Thiruvananthapuram district, Kerala, India during the month of March, 2013 and were authenticated by Dr. A.G Pandurangan, Scientist F, Head, Plant systematic and evoloutionary science division Jawaharlal Nehru tropical botanical garden and research Insitiute, Palode, Thiruvananthapuram. Kerala, India and the herbarium was deposited at JNTBGRI. The field number of the sample was 76810.
Preparation of extracts
Extraction of fruit peels of M.paradisiaca was carried out using acetone by hot continuous extraction. From this extraction, 1000 g of fresh ripe and unripe peels were taken, size reduced, and extracted with 2 L of acetone in the round bottom flask and the extraction was continued for few hours. The extract obtained was collected and concentrated by gentle heating. Thus the acetone extract was obtained. 
Healthy adult Wistar albino rats, weighing about 150-220 g obtained from the registered Animal house of University College of Pharmacy, MG University, Cheruvandoor campus, Kottayam, Kerala, India were used for the study. The study protocol was approved by the Institutional Animal Ethical Committee, University College of Pharmacy, Cheruvandoor Campus [001/MPH/UCP/CVR/13]. All the animals were housed individually in polypropylene cages, maintained under standard husbandry conditions (12 h of light and dark cycles, at room temperature and 45-55% relative humidity) and given standard pellet diet and water ad libitum.
Acute toxicity studies (OECD 423)
The preliminary pharmacological studies were conducted to assess the acute pharmacological effects of acetone extract of plant M. paradisiaca. The acute toxicity study was carried out in female albino rats by "acute toxic class method" (OECD 423). The animals were fasted overnight and extracts of the M.paradisiaca suspended in 0.5% sodium carboxy methyl cellulose (NaCMC) was administered starting at 2000 mg/kg; food was withheld for next 3-4 h. The animals were observed continuously for general or gross behavioral changes and for the signs of tremors, salivation, diarrhoea, lethargy, sleep, convulsions and coma every 30 min, after 3 h, for 20 hours and finally till 48 h. 
Anoxia stress tolerance test
Wistar albino rats of either sex weighing 150-220 g were selected and divided into four groups. Group I received 2 ml of 0.5% w/v CMC, p.o and group II received diazepam 2 mg/kg, p.o for 21 days. The group III and group IV received AURMP and ARMP 200 mg/kg, respectively for 21 days. Animals were treated for 3 weeks. On 7 th , 14 th and 21 st day, one h after the treatment stress was induced in all rats by placing each animal individually in the hermetic vessel of 1 L capacity to record anoxia tolerance time. The time duration from the entry of the animal into the hermetic vessel and the appearance of the first convulsion was taken as time of anoxia tolerance. 
Swimming endurance test
Wistar albino rats of either sex weighing 150-220 g were selected and divided into four groups of six. Group I received 2 ml of 0.5% w/v CMC, p.o for 8 days and group II received diazepam 2 mg/kg, orally for 8 days. The group III and group IV received AURMP and ARMP 200 mg/kg, respectively for 8 days. The rats were subjected to swimming stress by keeping them in propylene tank filled with water to a height of 25 cm. Extracts were given to rats, once daily for a period of 7 days. On 8 th day, the rats were allowed to swim till complete exhaustion and the endpoint was taken when the animal started drowning. 
Chronic variable stress model
Albino Wistar rats of either sex weighing 150-220 g were selected and divided into five groups. Animals in the group I were treated with 2 ml of 0.5% w/v CMC, p.o and the animals did not receive any stress throughout the study period. The group II was the stress group receiving chronic stress daily, and the animals were not treated with any other agents. Group III animals received the standard drug (fluoxetin 10 mg/kg, orally) treatment along with the stress, whereas the Group IV and Group V received AURMP and ARMP (200 mg/kg), respectively for five weeks.
The chronic stress model was induced by chronic variable stress (CVS). Rats in stressed groups were exposed to the following stressors once daily for 5 consecutive weeks: 2-h immobilization; 24-h food deprivation; 24-h water deprivation; 5-min cold swim at 4°C; wet bedding; 1-min tail pinch with a clothe-pin placed 1cm distal from the base of tail. The same stressor will not be applied successively so that the rats cannot anticipate the occurring of stress. 
Forced swimming test
The test was carried out on two successive days after the last stress period. Rats were forced to swim in a vertical plastic cylinder containing 25 cm of water maintained at 25 ± 1°C. On the 1 st day of the experiment, rats were trained to swim for 15 min. On the following day, the rats were re-exposed to the forced swimming for 5 min and the immobility time was evaluated. A rat was judged to be immobile whenever it remained floating passively in the water in a slightly hunched but upright position with its head just above the surface. 
Open-field test was carried out before stress, 2 weeks after stress and thereafter 5 weeks of stress. Each rat was gently placed onto the middle square of an open-field apparatus facing away from the observer and allowed to explore freely for 5 minutes. During the test time the animal movements in the field were quantified by counting the number of crossings (atleast three paws in a square), number of rearings (wall rears and free rears), activity in the central square and also the number grooming. 
Ex vivo determination of tryptophan in brain samples
The brain samples were collected by cervical dislocation from the treatment groups and homogenized with the help of an electric homogenizer in the presence of 5 ml of 0.4 M perchlorate extraction medium per brain. Homogenates were then allowed to stand for 10-15 min for the precipitation of proteins. The supernatant was decanted in a separate Eppendorf tube and centrifuged at 2,000 rpm for 5 min. Finally, the supernatant containing tryptophan TRP was separated and stored at -20°C until analysis. Stock solution (100 mg/ml) of aqueous TRP was prepared and diluted into 5, 10, 20, 30, 40 and 50 mg/ml to prepare calibration curves. The liquid chromatographic system consisted of a Shimadzu model LC-10AT VP pump with a SPD-10AT VP, variable wavelength UV-Visible detector. Analysis was conducted on a Purospher STAR C18 (250 × 4.6 mm) analytical reversed-phase column with mobile phase of water-acetonitrile (90:10 v/v) at pH 2.7 maintained by orthophosphoric acid. Assays were performed at ambient temperature at a flow rate of 1.5 ml/min. The eluents were monitored at 270 nm. 
Graph pad Prism Version 6.0 was used for statistical analysis. Results were expressed as Mean ± S.E.M. Data was analyzed by one-way ANOVA followed by Dunnett's multiple comparison test in multiple test groups. The P < 0.05 were considered as statistically significant.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]