|Year : 2013 | Volume
| Issue : 1 | Page : 11-16
The effects of aqueous leaf extract of Aegle marmelos on immobilization-induced stress in male albino Wistar rats
Chidambaram Anusha1, Arumugam Sarumathi1, Sakkaravarthy Shanmugapriya1, Singaravelu Anbu1, Rather Shabir Ahmad1, Nadanam Saravanan2
1 Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, India
2 Division of Biochemistry, Rani Meyyammai College of Nursing, Faculty of Medicine, Tamil Nadu, India
|Date of Submission||08-Mar-2012|
|Date of Acceptance||30-Mar-2012|
|Date of Web Publication||6-Feb-2013|
Division of Biochemistry, Rani Meyyammai College of Nursing, Faculty of Medicine, Annamalai University, Annamalainagar - 608 002, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: Stress is the major problem in the modern world. Stress is one of the basic factors in the development of many diseases and has been exposed to be associated with altered homeostasis that may lead to antioxidant imbalance. Our aim was to evaluate the anti-stress activities of aqueous leaf extract of Aegle marmelos (AM) in immobilization-induced stress in rats. The anti-stress activities of AM were assessed by monitoring the change in the status of stress hormone, glucose, and non-enzymic antioxidants in immobilization-induced stress in rats. Materials and Methods: Immobilization stress was induced in rats by placing in 20 cm × 7 cm plastic tubes for 2 h/day for 21 days. Aqueous leaf extract of AM was given by gavage to the experimental rats at a dose of 100 mg/kg body weight daily for 21 days. Results: Immobilization stress induced in rats for 21 days caused increased corticosterone, glucose levels and decreased levels of non-enzymic antioxidant such as Vitamin C, vitamin E, and glutathione in plasma, liver, and kidney. AM significantly increased the non-enzymic antioxidants and decreased the levels of corticosterone at the end of experimental period as compared to control. Conclusion: Natural antioxidants strengthen the endogenous antioxidant defenses from reactive oxygen species and restore an optimal balance by neutralizing the reactive species. They are gaining immense importance by virtue of their critical role in disease prevention. In this context, AM can rightly be mentioned as a plant of considerable interest. In the present study, treatment with AM was found to reverse the oxidative stress in the hypothalamus caused by immobilization stress. So, it is concluded that AM is having high antioxidant properties and anti-stress activity.
Keywords: Aegle marmelos , corticosterone, immobilization stress, non-enzymic antioxidants
|How to cite this article:|
Anusha C, Sarumathi A, Shanmugapriya S, Anbu S, Ahmad RS, Saravanan N. The effects of aqueous leaf extract of Aegle marmelos on immobilization-induced stress in male albino Wistar rats. Int J Nutr Pharmacol Neurol Dis 2013;3:11-6
|How to cite this URL:|
Anusha C, Sarumathi A, Shanmugapriya S, Anbu S, Ahmad RS, Saravanan N. The effects of aqueous leaf extract of Aegle marmelos on immobilization-induced stress in male albino Wistar rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2019 Oct 17];3:11-6. Available from: http://www.ijnpnd.com/text.asp?2013/3/1/11/106974
| Introduction|| |
Stress response is known to lead to behavioral and metabolic changes, in an effort to maintain body homeostasis and increase chances of survival. , Exposure to stress, such as restraint session, induces the release of glucocorticoids (GC), which together with the rapidly acting adrenomedullary catecholamines, alter metabolic, cardiovascular, immune, and nervous activity in response to the stressor.  In stress situations, corticotropin-releasing hormone, adrenocorticotropic hormone, and GC are released by the hypothalamic-pituitary-adrenocortical (HPA) axis  and these hormones are known to influence food ingestion. , In human beings, studies have shown that stress may affect food ingestion by decreasing it (around 30% of the population report decreasing food intake consumption during stress situations); however, most people report increased consumption during periods of stress.  The main factors implicated in stress-induced effects are central neurotransmitters, particularly linked with pituitary-adrenal axis and free radical generation. ,, Free radical generation leads to oxidative stress and tissue damage. 
Enzymes catalyzed the removal of hydroxyl radical or singlet oxygen and these dangerous species are removed by quenchers and interceptors. These molecules are termed "non enzymic antioxidants" and include ascorbic acid, α-tocopherol, and glutathione (GSH). Non-enzymic antioxidants form a second line of defense to limit the interaction of tissue oxidants with essential tissue components. Vitamin is the most important free radical scavenger in extracellular fluids, trapping radicals in the aqueous phase and protecting biomembranes from peroxidative damage. , Vitamin E is the major lipophilic, chain-breaking antioxidant that resides in the lipid bilayer of the cell membrane. , GSH is a well-established metabolic regulator, has many important roles as a redox buffer for the prevention of oxidative damage, maintenance of protein thiol groups, removal of hydroperoxides, transport of amino acids, stabilization of cell membranes, and detoxification. GSH scavenges H 2 O 2 in the reaction catalyzed by GSH peroxidase. 
The physiological reaction to stress involves alteration in the autonomic nervous system, the endocrine system, and the immune system. The secretion of GC is a classic endocrine response to stress.  Stressful stimulation influences antigen-specific as well as non-specific reactions. Many herbs reported in ancient literature have potent anti-stress activity and their utilities in current scenario need to be unveiled.
Aegle marmelos (L.) Corr., (AM) belongs to Rutaceae family, a popular medicinal plant in the Ayurvedic and Siddha systems of medicine and in folk medicines used to treat a wide variety of ailments. The plant is popularly known as the bael tree, native to the Indo-Malayan region and is currently cultivated in India, Pakistan, Bangladesh, Sri Lanka, Burma, and Thailand.  The tree is a slender, aromatic perennial, 6.0-7.5 m tall, and 90-120 cm in girth. It flowers from May to July and yields an annual average of 300-400 fruits (200-250 kg) per tree. The leaf is used for ophthalmia, diabetes, and asthmatic complaints.  This study was therefore undertaken to find out whether in the case of AM, extracts from leaf material will be useful in the management of stress.
| Materials and Methods|| |
Fresh leaves of AM were collected from Shiva temple at Chidambaram, authenticated by Prof. V. Venketasvaralu, Department of Botany, Annamalai University, Annamalai Nagar, Tamil Nadu, India, and the specimen was deposited in the departmental herbarium.
Preparation of Aegle marmelos leaf extract
The plant leaves were cleaned with distilled water and shade dried at room temperature and powdered by using electric blender.  About 500 g of powdered leaves of AM was mixed with 5 l of distilled water and boiled for 5 hours to get 50 g of the paste. 
Adult male Albino Wistar rats weighing around 160- 180 g were purchased from Rajah Muthaiah Medical College and Hospital, Annamalai University, and were maintained in Central Animal House, Rajah Muthaiah Medical College and Hospital, Annamalai University. The animals were housed in polypropylene cages and provided with commercially available rat chow (Hindustan Lever Ltd., Bangalore, India) and water ad libitum. The animals were maintained under controlled conditions of temperature (20 ± 5°C) and humidity (55 ± 5%) with a 12 ± 1 h light/dark cycle. Institutional animal ethics committee (Register number 166/1999/CPCSEA), Annamalai University, Annamalai Nagar, India, approved the experimental design.
Animals were randomly separated into four groups of six rats each and housed in individual ventilated cages. Feed and water were provided ad libitum to the animals. AM leaf extract was administered orally once daily for 21 days.
Group I: Control
Group II: Stress
Group III: Control + Aqueous leaf extract of AM (100 mg/kg body weight)
Group IV: Stress + Aqueous leaf extract of AM (100 mg/kg body weight)
After 21 days of exposure to stress, the rats were fasted overnight, anesthetized by injecting ketamine hydrochloride (30 mg/kg body weight), sacrificed by cervical decapitation, and the blood samples were collected in separate tubes containing heparin as the anticoagulant for the estimation of various parameters in plasma. Liver and kidney tissues were dissected out and collected in ice cold saline for various estimations.
Administration of Aegle marmelos
The paste of AM leaf extract was dissolved in distilled water and administered at a dose of 100 mg/kg body weight per day to the experimental rats for 21 days. A gavage tube was used to deliver the substance by the oral route, which is a clinically accepted route of administration of AM. 
Immobilization stress procedure
Immobilization was performed daily by placing animals in 20 cm × 7 cm plastic tubes for 2 hours per day for 21 days.  There were several 3-mm holes at the far end of the tubes which allowed ample air for breathing and the animals were unable to move.
Estimation of plasma corticosterone
The fluorimetric method was used to estimate the plasma corticosterone as an index of HPA function. Plasma stored at -70°C overnight was used for the estimation. A reaction mixture consisting of 1 ml of plasma and 7.5 ml of dichloromethane was shaken for 2 minutes, centrifuged (in order to separate the phases), and then the plasma layer was removed. At zero time, 2.5 ml of fluorescent reagent (7:3 v/v concentrated sulfuric acid and ethanol) was added and shaken for 2 s. The supernatant was discarded and exactly after 12 minutes, the acid extracts were read at 530 nm emission with 470 nm excitation. 
Estimation of blood glucose
Glucose was estimated by the method of Trinder using reagent kit (1969). 
Estimation of ascorbic acid (vitamin C)
Ascorbic acid in the plasma, erythrocytes, and tissues was estimated by the method of Roe and Kuether (1943). 
Estimation of α-tocopherol (vitamin E)
α-Tocopherol in the plasma, erythrocytes, and tissues was estimated by the method of Baker et al. (1980). 
Estimation of reduced glutathione
Reduced GSH in the plasma, erythrocytes, and tissues was estimated by the method of Ellman (1959). 
| Results|| |
[Figure 1] shows the levels of plasma corticosterone. Immobilization stress significantly increased the plasma corticosterone levels. Treatment with aqueous leaf extract of AM significantly decreased the corticosterone level when compared to control groups.
[Table 1] shows the levels of glucose in the plasma of immobilization stress-induced rats. Immobilization stress rats have increased levels of glucose. AM alone-treated rats show a normal level compared to control rats. Co-treatment of immobilization stress and aqueous leaf extract of AM significantly reduced these levels toward normal when compared to control rats.
Non-enzymic antioxidant changes
The levels of non-enzymic antioxidants such as vitamin C, vitamin E, and GSH in the plasma and tissues of control and immobilization stress rats are given in [Table 2], [Table 3], [Table 4]. The levels of non-enzymic antioxidants in immobilization stress rats were significantly decreased. On the other hand, treatment with aqueous leaf extract of AM significantly improved the non-enzymic antioxidant levels when compared to immobilization stress-induced rats.
|Table 4: Levels of reduced glutathione (GSH) in control and experimental animals |
Click here to view
| Discussion|| |
Effect of Aegle marmelos on corticosterone
The increased plasma corticosterone level in immobilization stress group is in accordance with previous studies, ,,, which shows that plasma corticosterone is an important indicator of stress. It is also reported that exposure to GC or to stress may lead to oxidative injury in various tissues.  Plasma corticosterone levels in all stress groups were found to be significantly elevated with respect to the control group. In the present study, results are in accordance with the previous studies that the increased level of plasma corticosterone has been one of the most important indicators of stress. , There are several studies which showed an increased level of plasma corticosterone depending on stress.  In accordance with the previous researchers, in this study also there was an increased corticosterone level in immobilization stress animals. After AM treatment, it decreased the plasma corticosterone levels. Upon treatment with AM, the decreased level of corticosterone in the stressed group indicates that the AM may have the anti-stress activity.
Effect of Aegle marmelos on glucose level
Increased levels of glucose are seen in the immobilization stress rats due to the decreased secretion of insulin level.  Aqueous extract of AM leaf was evaluated for hypoglycemic and antioxidant effect  by using alloxon-induced diabetes in male albino rats and proposed AM may be useful in the long-term management of glucose.  The administration of aqueous leaf extract of AM to immobilization stress-induced rats showed a normal level of plasma glucose when compared to stress-loaded animals, which shows that aqueous leaf extract of AM may have the hypoglycemic effect in stress-induced glycemic change. The aqueous leaf extract significantly controlled blood glucose. , AM leaf extract has previously been shown to be antihyperglycemic when orally administered to rats. , The glucose-lowering activity of AM leaf extract confirmed the previous reports.  Recent reports showed that Scopoletin (7-hydroxy-6-methoxycoumarin), isolated from the leaves of AM, has potential to regulate hyperglycemia in levothyroxine-induced hyperthyroid rats.  Liver has an important function in maintaining blood glucose homeostasis and the hormone insulin regulates the glucose metabolism in liver. 
Effect of Aegle marmelos on non-enzymic antioxidant
Vitamin C (Ascorbic acid) is the most powerful antioxidant. Ascorbic acid is an excellent water-soluble free radical scavenger, which successfully prevents oxidative damage and therefore it would be helpful in prevention of diseases in which oxidative stress plays a causative role. The most important anti- stress nutrient is vitamin C. Stress depletes vitamin C supply very quickly, which weakens the ability to fight off disease. In addition, adequate amounts of vitamin C seem to make us bounce back from stressful situations more easily. It exists mostly in the reduced form. The hepatoprotective effect of AM leaves in alcohol-induced liver injury in Albino rats can directly scavenge superoxide, hydroxyl radicals, and single oxygen. Ascorbic acid reduces H 2 O 2 to water via ascorbate peroxidase reaction.  In the present study, the levels of vitamin C in plasma and tissue were significantly increased when immobilization stress animals were treated with AM.
Vitamin E is a chain-breaking antioxidant and it protects cell membrane against oxidative damage.  Vitamin E is a putative radical scavenger which is probably the most important inhibitor of membrane lipid peroxidation. It is lipid-soluble agent which can readily cross cell membranes and exerts its effects both intracellular and in membranes. It can repair oxidizing radicals directly, preventing the chain propagation step during lipid peroxidation.  In the present study, an increase in levels of vitamin E was observed in stress group of animals treated with AM.
Stress has been proven to rid in body's essential vitamins C, E, and B. This leads to lethargy, lower focused mental activities. Drying out and aging of the skin and in others an increase in the sebaceous gland activity which usually brings pustules, acne, and rosacea which all bring more stress as we then become more entangled in how other view our physical appearance.
GSH is a ubiquitous molecule found in all parts of the cell where it fulfills a range of functions from detoxification to protection from oxidative damage. It provides the main redox buffer for cells and as such has been implicated in the formation of native disulphide bonds.  Reactive oxygen species affects the antioxidant defense mechanism by reducing the intracellular concentration of GSH as well as superoxide dismutase, catalase and glutathione peroxidase SOD, CAT, and GPx activity. Under normal conditions, the inherent defense system, including GSH and the antioxidant enzymes, protects against oxidative damage.  GSH is a major non-protein thiol in living organisms, which plays a central role in coordinating the body's antioxidant defense processes. Perturbation of GSH status of a biological system can lead to serious consequences.  Decreased level of GSH were seen in the immobilization stress rats as compared to control rat. After administration of AM, increased level of GSH was observed when compared to control rats.
| Conclusion|| |
The present study suggests that immobilization stress induces the oxidative damage in the plasma, liver, and kidney due to increased plasma corticosterone, glucose, and depletion of non-enzymic antioxidants. There is a highly reduced capacity to scavenge free radicals produced in the tissues in response to stress. AM decreased the corticosterone, glucose level and increased non-enzymic antioxidant. Vitamin C and E are natural antioxidants found in variety of plant materials. The vitamins help to reduce both the physical and psychological effects of stress. The antioxidant vitamin C efficiently scavenges free oxygen radicals. Vitamin C should be considered an essential part of stress management.
| References|| |
|1.||Chrousos GP, Gold PW. The concepts of stress system disorders: Overview of behavioral and physical homeostasis. JAMA 1992;267:1244-52. |
|2.||Tsigos C, Chrousos GP. Hipotalamic-pituitary-adrenal axis, neuroendocrine factors and stress.J Psychosom Res 2002;53:865-71. |
|3.||Dallman MF, Pecoraro N, La Fleur SE. Chronic stress and comfort foods: Self-medication and abdominal obesity. Brain Behav Immu 2005;19:275-80. |
|4.||Adam TC, Epel SE. Stress, eating and the reward system. Physiol Behav 2007;91:449-58. |
|5.||La Fleur SE. The effects of glucocorticoids on feeding behaviour in rats. Physiol Behav 2006;89:110-4. |
|6.||Palkovits M. Neurotransmitters in stress. In: Van Loon GR, Kvetnansky R, McCarty R, Axelrod J, editors. Stress Neurochemical and Humoral Mechanisms. New York: Gordon and Breach Science Publishers; 1989. p. 31-42. |
|7.||Herman JP, Cullinan WE. Neurocircuitary of stress: HPA axis. Trends Neurosci 1997;20:78-84. |
|8.||Akil HA, Morano MI. Stress. In: Bloom FE, Kupfer DJ, editors. Psychopharmacology: The Fourth Generation of Progress. New York: Raven Press; 1995. p. 773-84. |
|9.||Subash S, Subramanian P. Impact of morin (a bioflavonoid) on ammonium chloride-mediated oxidative damage in rat kidney. Int J Nutr Pharmacol Neurol Dis 2011;1:174-8. |
|10.||Patra R, Swarup D. Effect of antioxidant ascorbic acid, L-methionine or a tocopherol alone or along with chelator on cardiac tissue of lead-treated rats. Veterinarski Arch 2004;74:235-44. |
|11.||Rai DK, Rai PK, Rizvi SI, Watal G, Sharma B. Carbofuran-induced toxicity in rats: Protective role of vitaminC. Exp Toxicol Pathol 2009;61:531-5. |
|12.||Wardlaw GM, Kessel MW. The fat-soluble vitamins. In: Perspectives in nutrition New York, USA: McGraw-Hill Higher Education 2002. p. 322-63. |
|13.||Meydani M. Vitamin E modulation of cardiovascular diseases. Ann N Y Acad Sci 2004;1031:271-9. |
|14.||Rahman SH, Ibrahim K, Larvin M, Kingsnorth A, McMahon MJ. Association of antioxidant enzyme gene polymorphisms and glutathione status with severe acute pancreatitis. Gastroenterology 2004;126:1312-22. |
|15.||Sapolsky RM, Krey LC, McEwen BS. Stress downregulates corticosterone receptors in a site-specific manner in the brain. Endocrinology 1984;114:287-92. |
|16.||Islam R, Hossain M, Karim MR, Joarder OI. Regeneration of Aegle marmelos (L.) Corr., plantlets in vitro from callus cultures of embryonic tissues. Curr Sci 1995;69:494-5. |
|17.||Nadkarni K, Nadkarni KM. Indian material Medica. 3 rd ed. Bombay, India: Popular Book Depot;1954. |
|18.||Sathiyaraj K, Sivaraj A, Madhumitha G, Vinothkumar P, Mary Saral A, Devi K. Antifertility effect of aqueous leaf extract of Aegle marmelos on male albino rats. Int J Curr Pharma Res 2010;2:1. |
|19.||Upadhya S, Shanbhag KK, Suneetha G, Balachandra Naidu M, Upadhya S. A study of hypoglycaemic and antioxidant activity of Aegle marmelos in Alloxan induced diabetic rats. Indian J Physiol Pharmacol 2004;48:476-80. |
|20.||Marcilhac A, Dakine N, Bourhim N, Guillaume V, Grino M, Drieu K. Effect of chronic administration of Ginkgo biloba extract or Ginkgolide on the hypothalamic-pituitary axis in the rat. Life Sci 1998;62:2329-40. |
|21.||Shah ZA, Sharma P, Vohora SB. Ginkgo biloba normalizes stress-elevated alterations in brain catecholamine's, serotonin and plasma corticosterone levels. Eur Neuropsychopharmacol 2003;13:321-5. |
|22.||Trinder P. Determination of blood glucose using an oxidase peroxidase system with a non-carcinogenic chromogen. J Clin Pathol 1969;22:158- 61. |
|23.||Roe JH, Kuether CA. Detection of ascorbic acid in whole blood and urine through 2,4-DNPH derivative of dehydroascorbic acid. J Biol Chem 1943;47:399-407. |
|24.||Baker H, Frank O, DeAngeles B. Plasma tocopherol in man at various times after ingestion free or acetylated tocopherol. Nutr Rep Int 1980;21:531-6. |
|25.||Ellman GL. Tissue sulphydryl groups. Arch Biochem Biophys 1959;82:70-7. |
|26.||Malendowicz LK, Andreis PG, Nussdorfer GG, Markowska A. The possible role of endogenous substance P in the modulation of the response of rat pituitary-adrenal axis to stresses. Endocr Res 1996;22:311-8. |
|27.||Laugero KD, Moberg GP. Energetic response to repeated restraint stress in rapidly growing mice. Am J Physiol Endocrinol Metab 2000;279:33-43. |
|28.||Lehmann J, Russig H, Feldon J, Pryce CR. Effect of a single maternal separation at different pup ages of the corticosterone stress response in adult and aged rats. Pharmacol Biochem Behav 2002;73:141-5. |
|29.||Djordjevic J, Cvijic G, Davidovic V. Different activation of ACTH and corticosterone release in response to various stressors in rats. Physiol Res 2003;52:67-72. |
|30.||Fontella FU, Siqueira IR, Vasconcellos AP, Tabajara AS, Netto CA, Dalmaz C. Repeated restraint stress induces oxidative damage in rat hippocampus. Neurochem Res 2005;30:105-11. |
|31.||Willis L, Thomas P, Garry PJ, Goodwin JS. A prospective study of response to stressful life events in initially healthy elders. J Gerontol 1987;42:627-30. |
|32.||Ricart-Jane D, Rodriguez-Sureda V, Benavides A, Peinado-Onsurb J, Lopez-Tejero MD, Llobera M. Immobilization stress alters intermediate metabolism and circulating lipoproteins in the rat. Metabolism 2002;51:925-31. |
|33.||Zardooz H, Asl SZ, Naseri MK, Hedayati M. Effect of chronic restraint stress on carbohydrate metabolism in rat. Physiol Behav 2006;89:373-8. |
|34.||Ponnachan PT, Paulose CS, Panikkar KR. Effect of leaf extract of Aegle marmelos in diabetic rats. Indian J Exp Biol 1993;31:345-7. |
|35.||Grover JK, Yadav S, Vats V. Medicinal plants of India with antidiabetic potential. J Ethnopharmacol 2002;81:81-100. |
|36.||Das D, Bandyopadhyay M, Bhattacharjee RK, Banerjee RK. Hydroxyl radical is the major causative factor in stress-induced gastric ulceration. Free Radic Biol Med 1997;23:8-18. |
|37.||Panda S, Kar A. Evaluation of the antithyroid, antioxidative and antihyperglycemic activity of scopoletin from Aegle marmelose leaves in hyperthyroid rats. Phytother Res 2006;20:1103-5. |
|38.||Manoharan S, Umadevi S, Jayathi S, Baskaran N. Antihyperglucemic effect of Cossinium fenestratum and Catharanthus roseus in alloxan-induced diabetic rats. Int J Nutr Pharmacol Neurol Dis 2011;1:189-93. |
|39.||Noctor G, Foyer CH. Ascorbate and glutathione: Keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 1998;49:249-79. |
|40.||Amic D, Davidovic-Amic D, Beslo D, Trinajstic N. Structure radical scavenging activity relationship of ﬂavonoids. Croatia Chem Acta 2003;76:55-61. |
|41.||Serbinonva EA, Packer L. Antioxidant properties of tocopherol and tocotrienol. Methods Enzymol 1994;234:354-67. |
|42.||Bass R, Ruddock LW, Klappa P, Freedman RB. A major fraction of endoplasmic reticulum-located glutathione is present as mixed disulfides with protein. J Biol Chem 2004;279:5257-62. |
|43.||Begum N, Prasad NR, Thayalan K. Apigenin protects gamma-radiation induced oxidative stress, haematological changes and animal survival in whole body irradiated Swiss albino mice. Int J Nutr Pharmacol Neurol Dis 2012;2:45-52. |
[Table 1], [Table 2], [Table 3], [Table 4]