Aegle marmelos Extract Can Enhance Memory in Rats

Document Type: Research Paper


1 Department of Physiology, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran Department of Pharmacoeconomics and Pharmaceutical Management, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

2 Department of Environment, Faculty of Environmental Sciences, Islamic Azad University, Arak Branch, Arak, Iran


      Diabetes mellitus is associated with disturbances of learning and memory and cognitive functioning. Aegle marmelos Corr. from Rutaceae family, is widely used in Iranian folk medicine for the treatment of diabetes mellitus. It decreases blood glucose level by improving glucose tolerance and also has lipid-lowering and antioxidant properties. Considering the beneficial antidiabetic potential of A. marmelos, this study was conducted to evaluate the effect of chronic oral administration of A. marmelos as cognitive enhancer, on learning and spatial memory in diabetic rats using Morris water maze test. Male Wistar rats were randomly divided into normal-control, diabetic-control, and A. marmelos-treated diabetic groups (100, 250 and 500 mg/kg, p.o., 4 weeks). Diabetes was induced by a single dose i.p. injection of streptozotocin (45 mg/kg). In each group of animals, spatial learning and memory parameters were analyzed. A. marmelos showed dose dependent improvement in spatial learning and memory parameters. Swimming time (Escape Latency) in normal-control and A. marmelos-treated diabetic animals rats was significantly lower than diabetic-control, while swimming speed was significantly higher. The study demonstrated that A. marmelos has a significant protective effect against diabetes-induced spatial learning and memory deficits. This effect can be attributed to hypoglycemic, hypolipidemic and antioxidant activity of A. marmelos.


1. Intorduction


     Diabetes mellitus is a common metabolic disorder, characterized by hyperglycemia due to an absolute or relative insulin deficiency. Diabetes is associated with functional and structural alterations in the peripheral, as well as the central nervous system [1, 2]. Moderate disturbances of learning and memory and complex information processing have been reported in both type 1 and 2 diabetic patients [3-5]. Aegle marmelos Corr. is commonly

 used as a folk medical plant in south of Iran, but it is indigenous to India. It is a medium sized, armed deciduous tree found wild, especially in dry forests and is also cultivated throughout Iranian subcontinent for its fruit. The fruit are globose with smooth, hard and aromatic rind. The ripe fruit is used for digestive and stomachic complications. Leaves, fruits, stem and roots of A. marmelos have been used in ethno-medicine for several medicinal properties such as astringent, antidiarrheal, antidysenteric, demulcent, antipyretic, antiscourbutic, haemostatic, aphrodisiac and as an antidote to snake venom [6-9]. A. marmelos is also known as herbal medicine for the treatment of diabetes mellitus [10, 11]. Preliminary reports indicated hypoglycemic effect of leaves, seeds and fruits of A. marmelos [12-15]. Ponnachan et al. [16, 17] have observed that the alkaloid extract prepared from leaves and crude aqueous leaf extract (1 g/kg for 30 days) exhibit hypoglycemic effect in alloxanized diabetic rats. Aqueous leaf extract reversed the increase in Km values of liver malate dehydrogenase enzyme [18] and improved histopathological alterations in the pancreatic and kidney tissues of streptozotocin (STZ) induced diabetic rats [19]. Moreover, change in glucose utilization, balance of cerebral lipid metabolism and oxidative stress that occur in diabetes are main reasons for cognitive dysfunction [20, 21]. Antioxidant and anti-dyslipidemic effects of A. marmelos also have been reported [22-25] and that is suggestive of its potential as cognitive enhancer.


      Thus, treatment with herbal medicine that improve glucose utilization, decrease oxidative stress and modify lipid metabolism, may help to improve learning and memory impairment induced by diabetes. There are no available reports on the action of A. marmelos seeds on cognitive tests, therefore, the effect of aqueous extract of A. marmelos seeds on spatial learning and memory in streptozotocin induced diabetic rats has been investigated.


2. Materials and methods


2.1. Animals


      Male Wistar rats, weighing 200-250 g, were provided by the Iranian Razi Institute and were housed in standard cages with free access to food (standard laboratory rodent's chow) and water. The animal house temperature was maintained at 23±3 °C with a 12-h light/dark cycle (light on from 06:00 to 18:00 h). The study was approved by animal research committee of Kermanshah University of Medical Science and the principles of laboratory animal care (National Institutes of Health publication No. 86-23, revised 1985) were followed in this study. The ethical guidelines for the investigation of experimental animals were followed in all tests. All efforts were made to minimize animal suffering and to reduce the number of animals used.


2.2. Plant material and extraction


     Seeds of A. marmelos from fresh fruits were collected in august 2007 from south of Iran (Bushehr) and authenticated by the School of Agricultural Sciences, Razi University, Kermanshah, Iran. The fruits were macerated in large amount of water and passed through large pore size sieve to separate seeds. The seeds were shade dried and rubbed vigorously to remove the last traces of fibre attached to it. Powdered seeds were extracted with boiling water for 10 h. The resulting extract was cooled and filtered using Whatman No. 1 filter paper. The filtrate was evaporated to dryness in an oven set at 40 °C. The dried extract was weighed and dissolved in normal saline to a concentration of 200 mg/ml. The extract was maintained at 4 °C throughout experiments.


2.3. Treatments


     A freshly prepared solution of streptozotocin (Sigma Chemical Co.; St. Louis, USA; 45 mg/kg) in 0.1 M citrate buffer, pH 4.5 was injected intraperitoneally to overnight fasted rats [26]. Fasting blood glucose level (FBG) was estimated at the time of induction of diabetes and FBG was checked regularly up to stable hyperglycemia, one week after streptozotocin injection. Depending on their FBG level the severe diabetic animals showing FBG above 250 mg/dl, were studied [27]. Diabetic animals were treated with A. marmelos extract (100, 250, 500 mg/kg, p.o.) or normal saline (diabetic-control), and normal-control group received the same volume of normal saline orally for 4 weeks. At the beginning and end of study, blood samples were collected and centrifuged. FBG level was measured by glucose oxidase method [28]. After 4 weeks, learning and spatial memory tests in normal and diabetic rats were carried out using Morris water maze (MWM) test. During the MWM test, animals fed as same as before. MWM was constructed from a circular black colored water tank, 140 cm in diameter and 80 cm in height that was located in the center of small room and was surrounded by numerous extramaze cues on the wall in the room. The tank was divided into four quadrants (N, E, W and S) was filled with water till it has reached 40 cm in depth. The experimenter stood in the southwest corner of the room. Invisible round disk platform (made of Plexiglas, 10 cm in diameter) were used and located 1cm beneath the surface of the water. In the first 4 days of experiment, location of platform was constant throughout the sessions (see below). An automated infrared tracking system (CCTV B/W camera, SBC-300 (P), Samsung Electronic Co., Ltd, Korea) recorded the position of the rat in the tank. The camera was mounted 2.5 m above the surface of the water [29].


2.3.1. Handling


       Each rats received once daily, 10 min. handling period for three days, after which the animals were trained for two days to stand on the platform. On the first day, rats were placed on the platform which was at the center of the tank without water for 60 s, and on the second day, the rats were placed again on the platform under the same conditions but the tank was filled with normal saline, room temperature (20±2 ºC). When the rat climbed off the platform, the experimenter guided the rat to go back onto the platform [30].




Figure 1. Escape latency in diabetic-control, A. marmelos and normal-control groups in the training days. Values are means±SEM.


Table 1. Blood glucose levels in diabetic-control, A. marmelos and normal-control groups at the beginning and end of experiment.


Dose (mg/kg)

Blood glucose levels (mg/dl)




Post treatment












A. marmelos extract















Values are means±SEM. *p < 0.05,  **p < 0.01, *** p < 0.001 vs. Diabetic-control group.

2.3.2. Training procedure


     Extramaze landmarks (window, door, etc.) in the room were spatial cues for learning of platform's position for animals. The position of the platform was fixed throughout the experiments. The platform was located in the north-west quarter of MWM tank with 20 cm distance from the edge of the tank, and 1 cm beneath the surface of water. Each rat was tested for 5 sessions. Each session consisted of 4 trials in a day. In first sessions, a trial began by releasing the rat into the water facing the wall of the tank from one of the four quadrants (North, South, East or West). The sequence of starting location was chosen in a pseudorandom manner by computer in such a way that the starting location was different from the immediate preceding trial. The trial was concluded when the rat found the platform or at 60 s after start of the trial. If the rat could not reach the platform within 60 s, the experimenter led the rat to the platform and the rat remained on the platform for 30 s, then released into the water from the next starting location. After the last trial in each session, the rat was towel-wiped and placed in a drying chamber for 5 to 15 min. and then returned to the home cage. For evaluation of accuracy and validity of initial learning, probe trial was performed on the fifth day, in which, platform was expelled and animal during one session (consisting of 4 trials) was released into water exclusively from one of the above mentioned directions (East) that was determined by computer for all rats [29].


2.4. LD50 experiments

     For LD50 experiment, four groups of rats of both sex (six animals per group, three females and three males) and weighing about 200-250 g were administered orally a single dose of either 2.5, 5, 10 or 15 times of effective dose of aqueous extract of seeds of A. marmelos. Then rats were observed for gross behavioural, neurologic, autonomic and toxic effects at short intervals of time for 24 h. Food consumption, feces and urine were also examined at 2 h and then at 6 h intervals for 24 h [22].


2.5. Data analysis


The data were expressed as mean±SEM, subjected to analysis of variance (ANOVA) and followed by Tukey's test for multiple comparisons, and p < 0.05 was the critical criterion for statistical significance.


3. Results


3.1. Evaluation of escape latency and swimming speed during training days


     Results indicate that A. marmelos administration reduces escape latency during training days in a dose-dependent manner. Also, there were differences among experimental groups in third and fourth days of training. On these days, escape latencies in the normal-control and A. marmelos-treated groups were less than that of diabetic-control group. This difference was statistically significant in the third and fourth day of training( p < 0.001), while there wasn't any statistically significant difference between A. marmelos 500 mg/kg and normal-control group in all four training days (Figure 1). Results also indicated that there was a difference in swimming speed among experimental groups. Post-hoc analysis showed that differences between normal-control (p < 0.001). A. marmelos 250 mg/kg p < 0.01 and A. marmelos 500 mg/kg p < 0.001 in comparison with diabetic-control were significant. Differences in swimming speed between A. marmelos500 mg/kg and normal-control group wasn't significant (Figure 2).

3.2. Evaluation of the percentage of presence in target quarter in probe trial


      The percentage of the presence of animals in target quarter (quarter in which platform was located during training days) in probe trial session was investigated. This difference was significant (P< 0.05) between diabetic-control group and other groups (Figure 3).

Figure 2. Swimming speed in diabetic-control, A. marmelos and normal-control groups in the training days. Values are means±SEM. *p < 0.01, **p < 0.001 vs. diabetic-control group.


Figure 3. Percentages of time that animals spent in target quarter in probe trial in diabetic-control, A. marmelos and normal-control groups. Values are means±SEM. p < 0.05 vs other group.

3.3. Effect of A. marmelos seed extracts on FBG

       Effect of oral administration of the aqueous extract of A. marmelos seeds on FBG is shown in Table 1. A. marmelos administration, dose dependently, reduced FBG, and there were differences among experimental groups. Post-hoc analysis showed statistically significant differences between normal-control ( p < 0.001) , A. marmelos 100 mg/kg (p < 0.05) , A. marmelos 250 mg/kg (p< 0.01)  and A. marmelos 500 mg/kg (p< 0.001) in comparison with diabetic-control. Differences in FBG between A. marmelos 500 mg/kg and normal-control group wasn't significant.


3.4. LD50 studies


      In LD50 experiment, the behaviour of the treated rats appeared normal. No toxic effect was reported up to 10 and 15 times of effective dose of the water extract and there were no death in any of these groups. Only the consumption of food was increased by 20% in 10 and 15 times doses during 4 h but remaining normal afterwards.


4. Discussion


       Results of the present study suggest that chronic oral administration of A. marmelos has facilitating effects on spatial learning and memory in diabetic rats in Morris water maze. A. marmelos administration, during training days, leads to a decrease in escape latency and also an increase in the animals swimming speed as compared with the diabetic-control group. In this study, A. marmelos seed extract could reduce FBG that confirms hypoglycemic effect of this seed as reported earlier [22], but the most effective dose was 500 mg/kg.


      In previous studies, moderate disturbances of learning and memory and complex information processing have been reported in both type 1 and 2 diabetic patients [3-5]. Previous experimental studies into cognitive functioning in animal models of diabetes mellitus, such as STZ-induced diabetic rodents, have used several learning tasks. In more complex learning tasks, such as an active avoidance T-maze, or a Morris water maze, diabetic rodents consistently displayed performance deficits [31-33]. According to the existing data, patients with Alzheimer's disease have a relatively high frequency of diabetes mellitus [34]. It seems that induced impairment of cognitive performance and the decrease of learning ability may not be restricted to Alzheimer's disease [35]. A. marmelos is known as a herbal medicine for the treatment of diabetes mellitus [10, 11]. Previous reports have indicated hypoglycemic effects of leaves, seeds and fruits of A. marmelos [12-15]. Pharmacological effects of A. marmelos particularly antidyslipidemic effect [1] is suggestive of its potential as a cognitive enhancer.


       Patients receiving lipid-lowering drugs like statins have a reduced risk of dementia and cognitive dysfunction [36]. Lipids account for half of the dry matter of the brain and are integral to the myelin sheath and synapses. Anything that affects the balance of cerebral lipid metabolism could have profound effects on brain function. High cholesterol is also associated with elevated beta-amyloid, the hallmark of cognitive disorders [20]. Experimental studies have shown that cholesterol-fed wild-type rabbits develop memory dysfunction, which is supported by human studies, showing that statin therapy reduces the risk of memory impairment [37-39]. In addition, free radical generation and oxidative stress can affect all classes of macromolecules (sugar, lipids, proteins, and DNA), leading inevitably to neuronal dysfunction [21]. Moreover, in diabetic animals, impaired glucose utilization and insulin signaling has already been linked to increased oxidative stress and mitochondrial dysfunction in neuronal cells [40, 41]. A. marmelos has antioxidant properties and effectively reduces oxidative stress. This was evident from a significant decrease in lipid peroxidation, conjugated diene and hydroperoxide levels in serum as well as in liver in diabetic rats after oral administration of A. marmelos [2-4].


      The result of this study reveals that a regular administration of A. marmelos aqueous seed extract for 4 weeks improved learning and spatial memory in diabetic animals in Morris water maze test. The dose of 500 mg/kg was the most effective dose. The LD50 of the extract is high (no death even with 15 times of effective dose) indicating high margin of safety. The fall of FBG in diabetic groups treated with A. marmelos, after a period of study, further confirms our findings.


       From this study, we can conclusively state that A. marmelos aqueous seed extract had beneficial effects on blood glucose levels as well as improving spatial memory impairment due to diabetes. This effect can be attributed to modification of lipid metabolism and blood glucose level and attenuation of the oxidative stress enhancement of diabetes mellitus induced by streptozotocin. Further pharmaco-logical and biochemical investigations are underway to elucidate the mechanism of the learning and memory enhancement effect of A. marmelos seeds.




This work was supported by Kermanshah University of Medical Sciences, Kermanshah, Iran.


[1]      Biessels GJ, Kappelle AC, Bravenboer B, Erkelens DW, Gispen WH. Cerebral function in diabetes mellitus. Diabetologia 1994; 37: 643-50.


[2]      Vinik AI, Holland MT, Le Beau JM, Liuzzi FJ, Stansberry KB, Colen LB. Diabetic neuropathies. Diabetes Care 1992; 15: 1926-75.


[3]      Reaven GM, Thompson LW, Nahum D, Haskins E. Relationship between hyperglycemia and cognitive function in older NIDDM patients. Diabetes Care 1990; 13: 16-21.


[4]      Ryan CM. Neurobehavioral complications of type I diabetes. Examination of possible risk factors. Diabetes Care 1988; 11: 86-93.


[5]      Tun PA, Nathan DM, Perlmuter LC. Cognitive and affective disorders in elderly diabetics. Clin Geriatr Med 1990; 6: 731-46.


[6]       Nandkarni AK. Indian materia medica. Vol. I, 3rd ed. Bombay: Popular Prakshan, 1976; pp. 45-9.


[7]      Kirtikar KR, Basu BD. Indian medicinal plant. Calcutta: Lalit Mohan Publication, 1935; p. 499.


[8]      Zargari A. Medicinal Plants, vol. II. Tehran University Press, Tehran. 1989; p. 111-112.


[9]      Avesina A. The canon.Tehran: Soroosh Press. 1988; p. 95.


[10]   Alam MM, Siddiqui MB, Husain W. Treatment of diabetes through herbal drug in rural India. Fitoterapia 1990; 61: 240-2.


[11]   Prakash V. Diabetes management-some useful herbs. Vol. 4., Jeevaniya Shishir. 1992; p. 10.


[12]   Chakrabarti B, Mallick C, Battacharya S. Studies on the green leaves of Aegle marmelos and Piper nigrum on the glucose and cholesterol levels of blood in diabetes mellitus. Indian Med For 1960;9:  285-6.


[13]   Karunanyake EH, Welhinda J, Sirimanne SR, Sinnadorai G. Oral hypoglycemic activity in some medicinal plants from Sri Lanka. J Ethnopharmacol 1984; 11: 223-31.


[14]   Kamalakkannan N, Prince PSM. Hypoglycemic effect of water extract of Aegle marmelos fruits in streptozotocin diabetic rats. J Ethnopharmacol 2003; 87: 207-10.


[15]   Kamalakkannan N, Prince PSM. Antidiabetic and antioxidant activity of Aegle marmelos extract in streptozotocin induced diabetic rats. Pharm Biol 2004; 42: 125-30.


[16]   Ponnachan PTC, Paulose CS, Panikkar KR. Hypoglycemic effect of alkaloid preparation from leaves of Aegle marmelos. Amala Res Bull 1993;13:  37-41.


[17]    Ponnachan PTC, Paulose CS, Panikkar KR. Effect of leaf extract of Aegle marmelos in diabetic rats.

Indian J Exp Biol 1993; 31: 345-7.


[18]    Seema PV, Sudha B, Padayatti PS, Abraham A, Raghu KG, Paulose CS. Kinetic studies of purified malate dehydrogenase in liver of streptozotocin-diabetic rats and the effect of leaf extract of Aegle marmelose (L.) Correa ex Roxb. Indian J Exp Biol 1996; 34: 600-2.


[19]   Das AV, Padayatti PS, Paulose, CS. Effect of leaf extract of Aegle marmelose (L.) Correa ex Roxb. on histological and ultrastructural changes in tissues of streptozotocin induced diabetic rats. Indian J Exp Biol 1996; 34: 341-5.


[20]  Borek C. Garlic reduces dementia and heart disease risk. J Nutr 2006; 136: 810-2.


[21]  Polidori MC, Mecocci P. Plasma susceptibility to free radical-induced antioxidant consumption and lipid peroxidation is increased in very old subjects with Alzheimer's disease. J Alzheimers Dis 2002; 4: 517-22.


[22]  Kesari AN, Gupta RK, Singh SK, Diwakar S, Watal G. Hypoglycemic and antihyperglycemic activity of Aegle marmelos seed extract in normal and diabetic rats. J Ethnopharmacol 2006; 107: 374-9.


[23]  Sabu MC, Kuttan R. Antidiabetic activity of Aegle marmelos and its relationship with its antioxidant properties. Indian J Physiol Pharmacol 2004; 48: 81-8.


[24]  Upadhya S, Shanbhag KK, Suneetha G, Balachandra Naidu M, Upadhya S. A study of hypoglycemic and antioxidant activity of Aegle marmelos in alloxan induced diabetic rats. Indian J Physiol Pharmacol 2004; 48: 476-80.


[25]  Kamalakkannan N, Stanely Mainzen Prince P. Effect of Aegle marmelos Correa. (Bael) fruit extract on tissue antioxidants in streptozotocin diabetic rats. Indian J Exp Biol 2003; 41: 1285-8.


[26]  Brosky G, Logothelopoulos J. Streptozotocin diabetes in the mouse and guinea pig. Diabetes 1969; 18: 606-9.


[27]  Gupta RK, Kesari AN, Murthy PS, Chandra R, Tandon V, Watal G. Hypoglycemic and antidiabetic effect of ethanolic extract of leaves of Annona squamosa L. in experimental animals.J Ethnopharmacol 2005; 99: 75-81.


[28]  Triender P. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Bio Chem 1969; 6: 24.


[29]  Farshchi A, Ghiasi G, Farshchi S, Malek Khatabi P. Effects of Boswellia papyrifera gum extract on learning and memory in mice and rat. Iran J Basic Med Sci 2010; 13: 9-15.


[30]  Kikusui T, Tonohiro T, Kaneko T. Simultaneous evaluation of spatial working memory and motivation by the allocentric place discrimination task in the water maze in rats. J Vet Med Sci 1999; 61: 673-81.


[31]  Biessels GJ, Kamal A, Ramakers GM, Urban IJ,  Spruijt BM, Erkelens DW. Place learning and hippocampal synaptic plasticity in streptozotocin-induced diabetic rats. Diabetes 1996; 45: 1259-66.


[32]  Biessels GJ, Kamal A, Urban IJ, Spruijt BM, Erkelens DW, Gispen WH. Water maze learning and hippocampal synaptic plasticity in streptozotocin-diabetic rats: Effects of insulin treatment. Brain Res 1998; 800: 125-35.


[33]  Flood JF, Mooradian AD, Morley JE. Charac-teristics of learning and memory in streptozocin-induced diabetic mice. Diabetes 1990; 39: 1391-8.


[34]  Nitta A, Mrai R, Suzuki N, Ito H, Nomoto H, Katoh G, Furukawa Y, Furukawa S. Diabetic neuropathies in brain are induced by deficiency of BDNF. Neurotoxicol Teratol 2002; 24: 695-701.


[35]  Wandell PE. Quality of life of diabetes mellitus. An overview of research in primary health care in the Nordic countries. Scand J Prim health Care 2005; 23: 68-74.


[36]  Jick H, Zornberg GL, Jick SS, Seshadri S, Drachman DA. Statins and the risk of dementia. Lancet 2000; 356: 1627-31.


[37]  Parale GP, Baheti NN, Kulkarni PM, Panchal NV. Effects of atorvastatin on higher functions. Eur J Clin Pharmacol 2006; 62: 259-65.


[38]  Raja SG, Dreyfus GD. Statins: Much more than just a lipid lowering therapy. Indian Heart J 2004; 56: 204-9.


[39]  Masse I, Bordet R, Deplanque D, Al Khedr A, Richard F, Libersa C, Pasquier F. Lipid lowering agents are associated with a slower cognitive decline in Alzheimer's disease. J Neurol Neurosur Ps 2005; 76: 1624-9.


[40]  Lannert H, Hoyer S. Intracerebroventricular administration of streptozotocin causes long-term diminutions in learning and memory abilities and in cerebral energy metabolism in adult rats. Behav Neurosci 1998; 112: 1199-208.


[41]  Chander R, Khanna AK, Pratap R. Antioxidant activity of guggulsterone, the active principle of guggulipid from Commiphora mukul. J Med Aromat Plant Sci 2002; 24: 370-4.