REPORT doi: 10.2306/scienceasia1513-1874.2008.34.

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Antidiabetic activity of leaf and callus extracts of Aegle marmelos in rabbit

National Taiwan University, Taipei 106, Taiwan Mother Theresa Institute of Health Science, Pondicherry, India Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, India Department of Plant Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, India Received 1 Nov 2007 Accepted 7 Mar 2008

* Corresponding author, e-mail: [email protected]

ABSTRACT: Aegle marmelos (the bael tree) is a popular medicinal plant in the Ayurveda and Siddha systems of medicine and folk medicines used to treat diabetes. The present study was undertaken to find the extent to which calluses obtained from leaf explant of A. marmelos has a potential for application in diabetes management compared to the ordinary plant material. Treatment using extracts from both leaf and callus produced significant decreases in blood sugar level in streptozotozin diabetic rabbits. Among the various extracts, the methanol extracts of the leaf and callus brought about the maximum anti-diabetic effect. The study revealed that the in vitro callus culture of A. marmelos has as much potential in diabetes management as the original leaf extract. KEYWORDS: Aegle marmelos, callus culture, methanolic extraction, anti-diabetic activity

INTRODUCTION Aegle marmelos (L.) Corr., (Rutaceae) is a popular medicinal plant in the Ayurvedic and Siddha systems of medicine and folk medicines used to treat a wide variety of ailments. The plant, popularly known as the bael tree, is native to the Indo-Malayan region1 and is currently cultivated in India, Pakistan, Bangladesh, Sri Lanka, Burma, and Thailand2. The tree is a slender, aromatic perennial, 6.07.5 m tall and 90120 cm in girth. It flowers from May to July and yields an annual average of 300400 fruits (200250 kg) per tree. Various parts of the tree, including the fruit, possess medicinal properties. The roots are useful for treating diarrhoea, dysentery, and dyspepsia3. The leaf is used for opthalmia, diabetes, and asthmatic complaints. Unripe fruit is useful for treating diarrhoea, dysentery and stomachalgia. The aqueous extracts of the stem and root bark are used to treat malaria, fever, jaundice, and skin diseases such as ulcers, urticaria, and eczema4. In pharmacological trials, both the fruit and root showed antiamoebic and hypoglycaemic activities5,6. The plant is rich in alkaloids, among which aegline, marmesin, marmin, and marmelosin are the major ones. Aqueous leaf extract and methanolic extract of the root bark of A. marmelos showed preventive effects on myocardial diseases7,8. The compounds luvangetin

and pyranocoumarin, isolated from the seeds of A. marmelos, showed significant antiulcer activity9. Essential oil isolated from the leaf has antifungal activity10. The aqueous extract of leaf possesses a hypoglycaemic effect11. The incidence of diabetes is increasing. Worldwide, it affects 230 million people of which 30 million are in India. It has been estimated that by the year 2025, the global incidence of diabetes would increase to 350 million12. Management of diabetes is a huge burden. While therapeutic insulin production is not adequate to meet demands, the recombinant DNA approach to diabetes management originally considered as a panacea has faced several problems13. It is hypothesized that the ultimate therapy for type I and type II diabetes lies in the herbal approach14. However, herbs are not inexhaustible natural resources, and the demand for herbal medicines cannot be met by cultivation only15. Plant tissue culture is a boon and can help produce large quantities of the herbal material. However, it is speculated that plant materials produced through tissue culture are deficient in secondary chemicals of therapeutic importance16. This study was therefore undertaken to find out whether in the case of A. marmelos, extracts from calluses are more effective than those from ordinary leaf material in the management of diabetes.

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MATERIALS AND METHODS Aegle marmelos callus cultures were initiated from leaf explants. Explants were collected from a 40-year old plant, sterilized, and then cultured on a modified Murashige and Skoog medium17, i.e., a B5 medium18 supplemented with 2,4-dichlorophenoxy acetic acid (0.5 mg/l) and benzyl adenine (0.2 mg/l). The cultures were maintained under sterile control conditions at 25 2 C. A 16:8 h (light:dark) photoperiod and a light intensity of 25 mol/m2s were used19. About 5 g each of fresh as well as oven-dried (at 40 C) leaf, immature leaflet, root and callus (1, 2, 3, 4 month-old) were placed separately in 100 ml distilled water and were allowed to stand for 24 h with occasional shaking. After filtration followed by evaporation on a water bath, the extract was finally concentrated in an oven at 40 C. Fresh leaf and callus (90 days old) were dried in an oven at 40 C and powdered. Using a Soxhlet apparatus 500 g of the powder was extracted serially in petroleum ether, benzene, chloroform, and methanol. The extracts were concentrated in a rotary evaporator. Swiss albino rabbits (1 year old) of either sex, weighing 1.52.5 kg, were used as the test animal. The rabbits were fed on a standard pellet diet (Hindustan Lever Ltd., Bangalore, India) and water ad libitum and maintained at 2830 C. After laboratory acclimation for 7 days, the rabbits were starved for 48 h and divided into groups of five. The animals were induced into a diabetic state by intraperitoneal injection of a freshly prepared solution of streptozotozin (STZ) (Sigma Chemical Co., St. Louis, MO, USA) in 0.05 mM citrate buffer (pH 4.5) at a dose of 45 mg/kg body weight per day for 3 days20. The control rabbits received citrate buffer alone. Rabbits with a blood sugar level of more than 300 mg/dl were selected for the study. The rabbits were divided into 3 treatments, and the common control group in each case received physiological saline. In the first treatment, aqueous extracts of dry leaf powder, fresh leaf, immature leaflet, callus powder (1, 2, 3 and 4 month-old) and root powder were suspended in physiological saline and fed to STZ-diabetic rabbits in separate groups at a daily dose of 1.0 g/kg body weight using an oral gavage. Each rabbit received 3 doses of the preparation on consecutive days. In the second treatment, petroleum ether, benzene, chloroform, and methanol extracts of dry leaf/callus were suspended in physiological saline and fed to STZ-diabetic rabbits daily at a dose of 1.0 g/kg
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body weight per day. Each rabbit received 3 doses on consecutive days. Finally, in the third treatment, methanol extracts of leaf and callus suspended in physiological saline were fed at a daily dose of 1.0 g/kg body weight. Therapeutic human mixtard insulin (Torrent Pharmaceutical Ltd, Mehsana, India) (1 unit/kg body weight) was administered through the intraperitoneal. route to a separate subgroup of STZ-diabetic rabbits for 3 days in order to compare the antidiabetic potency of the extracts with that of insulin. Blood was collected on day 1, 5, 10, and 15 after the last dose. The rabbits were fasted for 12 h and blood was collected directly from a pinna venule using a syringe carrying a #26 needle on day 1, 5, 10, and 15 after the last dose of treatment. Blood sugar was determined using a glucometer (Johnson & Johnson Co., USA). RESULTS AND DISCUSSION Treatment of crude aqueous extract of the leaf and the three month-old callus brought about a significant decrease in blood sugar (Table 1). All organic solvent extracts proved to be anti-diabetic. Among the various extracts, the methanol extract of leaf and callus brought about the maximum antidiabetic effect (Table 2). The effect of methanol extracts of leaf and callus in the diabetic rabbits was studied for different durations (Table 3). It can be seen that there is an immediate marked drop in blood sugar levels for the leaf and callus extract treatments. Among the various
Table 1 Effect of an aqueous extract of Aegle marmelos on blood sugar levels in STZ-diabetic rabbits.

Treatment Control Immature leaflet Callus (1 month old) Root Fresh leaf Callus (2 month old) (3 month old) (4 month old) Dry leaf

Blood sugar level (mg/dl) (Mean SD) 310.2 1.1 262.5 1.5 255.5 0.5 251.1 1.0 246.6 2.5 236.1 1.0 209.1 1.0 226.0 1.1 217.4 1.2

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Table 2 Effect of different organic solvent extracts of leaf and callus of Aegle marmelos on blood sugar levels of STZ-diabetic rabbits.
Treatment Control Petroleum ether Ex Benzene Ex Chloroform Ex Methanol Ex Blood sugar level (mg/dl) (Mean SD) Leaf extract treated 361.6 2.8 236.5 2.5 222.5 2.6 226.1 2.0 207.1 2.0 Callus extract treated 360.9 3.1 256.6 1.5 251.6 1.6 255.0 1.8 210.1 2.0

solvent extracts, methanol extract produced the maximum reduction of sugar level, and the highest reduction was observed on the tenth day. However, in the case of the insulin treatment, the blood sugar level was decreased to less than 100 mg/dl, irrespective of the duration. The results of this study indicate that the biosynthesis of the various secondary metabolites of therapeutic value in diabetes in the callus cultures of A. marmelos is similar to that found in vivo mature leaves. The callus cultures of Cassia tora produce ten times more anthraquinone derivatives than in vivo plant material21. STZ-induced -cell death in the pancreas is due to the alkylation of DNA thereby producing hyperglycemia22. The immediate action of STZ is on -cells, even at relatively moderate doses of 45 mg/kg of body weight in a single dose. In the present study STZ treatment of rabbits for 15 days increased blood sugar to a significant level20. The extracts have the potential of correcting diabetes by acting as an anti-hyperglycemic agent rather than by inducing hypoglycemia. This idea is

supported by the demonstrated therapeutic importance of solvent extracts of A. marmelos23. The present findings confirm that the methanol extract can bring about greater reduction of sugar level in induced diabetic rabbits than other solvent extracts. The present study shows the hypoglycemic effect of extracts of leaf and callus against STZinduced diabetes. It appears that the leaf and callus extracts possess the ability to stimulate the insulin secreting cells of pancreas. This would, in turn, decrease the blood sugar as would do the leaf extract of A. marmelos11. All these findings suggest that both the leaf and callus extracts may be acting, through some mechanism, to improve the receptor-responsiveness to insulin causing increased sugar uptake by the tissue. In the present study both the leaf and callus extracts brought about a significant hypoglycemic effect in rabbits. The hypoglycemic effect of D-400, a herbal formulation, on fasting blood sugar level in rat was attained on day 20, and thereafter the blood sugar level did not decrease even after 90 days24. However, those animals were not induced to be in a

Table 3 Effect of methanol extract of leaf and callus of Aegle marmelos and insulin on blood sugar level in STZ-diabetic rabbits.
Treatment Control Leaf-extract Callus-extract Insulin Blood sugar level (mg/dl) (Mean SD) (Days) 1 317.56 2.04 208.51 2.02 200.72 5.25 95.22 2.04 5 345.52 3.62 195.54 2.05 187.72 2.36 96.26 1.42 10 349.04 1.26 123.76 2.53 106.21 1.05 97.07 1.52 15 350.04 2.25 128.76 3.71 109.05 1.52 97.52 2.24

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diabetic state and therefore carbohydrate metabolism was already in homeostasis and hence no further fall could occur. A. marmelos would act like insulin in the restoration of blood sugar and body weight to normal levels in rat and was therefore recommended as a potential hypoglycemic agent5,23. A similar result was obtained in the present study with the callus extract. Furthermore, the callus extract-treated animals appeared healthier and less prone to fluctuation in the extent of the hypoglycemic condition than was observed in their insulin-treated counterparts. These results suggest that both the leaf and callus materials contain anti-diabetic active principles, which would reduce the sugar level in STZ-diabetic rabbits. It is also inferred that the crude solvent extracts of leaf and callus powder may have some compounds in addition to the active anti-diabetic principles. Also, the solvents may affect the action of the principal compound responsible for the anti-diabetic effect. Further study using the purified active principle from the leaf and callus extracts may reveal the role of the respective preparations as hypoglycaemic agents in diabetes management. In conclusion, the present findings indicate that the methanol extract of the callus powder of Aegle marmelos is as potent as the in vivo leaf extract in the management of diabetes. ACKNOWLEDGEMENTS S. Arumugam gratefully acknowledges The Junior Research Fellowship from the University Grants Commission, New Delhi. REFERENCES 1. Hooker JD (1975) The flora of British India. Vol. 1, pp 5167 Reeve, United Kingdom. 2. Islam R, Hossain M, Karim MR, Joarder OI (1995) Regeneration of Aegle marmelos (L.) Corr., plantlets in vitro from callus cultures of embryonic tissues. Curr Sci 69, 4945. 3. Mazumder R, Bhattacharya S, Mazumder A, Pattnaik AK, Tiwary PM, Chaudhary S (2006) Antidiarrhoeal evaluation of Aegle marmelos (Correa) Linn. root extract. Phytother Res 20, 824. 4. Nadkarni K, Nadkarni KM (1954) Indian material Medica, 3rd edn, Popular Book Depot, Bombay, India. 5. Ponnachan PTC, Paulose CS, Panikar KR (1993) Effect of the leaf extract of Aegle marmelos (L.) Corr. in diabetic rats. Indian J Exp Biol 31,
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3457. 6. Kamalakkannan N, Prince PS (2005) The effect of Aegle marmelos fruit extract in streptozotocin diabetes: a histopathological study. J Herb Pharmacother 5, 8796. 7. Kakiuchi N, Senaratne LR, Huang SL,Yang XW, Hattori M, Pilapitiya U, Namba T (1991) Effect of constituents of Belli [Aegle marmelos (L.) Corr.] on spontaneous beating and calcium-paradox of myocardial cells. Planta Med 57, 436. 8. Prince PS, Rajadurai M (2005) Preventive effect of Aegle marmelos leaf extract on isoprenalineinduced myocardial infarction in rats: biochemical evidence. J Pharm Pharmacol 57, 13537. 9. Goel RK, Maiti RN, Manickam M, Ray AB (1997) Antiulcer activity of naturally occurring pyrano-coumarin and isocoumarins and their effect on prostanoid synthesis using human colonic mucosa. Indian J Exp Biol 35, 10803. 10. Rana BK, Sing UP, Taneja V (1997) Antifungal activity and kinetics of inhibition by essential oil isolated from leaves of Aegle marmelos (L.) Corr. J Ethnopharmacol 57, 2934. 11. Das AV, Padayatti PS, Paulose CS (1996) Effect of leaf extract of Aegle marmelos (L.) on histological and ultrastructural changes in tissues of streptozotozin induced diabetic rats. Indian J Exp Biol 34, 3415. 12. International Diabetes Federation (2006) (www. idf.org) 13. Laurotzen T, Frost LK, Larsen HW, Dekert T (1983) Effect of 1 year of near-normal blood sugar levels on retinopathy in insulin-dependent diabetics. Lancet 1, 2004. 14. Keen H (1984) Normoglycaemic re-entry and diabetic complications. Diabetic Med 1, 857. 15. Phillipson JD (1990) Plants as source of valuable products. In: Secondary products from plant tissue culture (Charlwood BV, Rhodes MJC, eds), pp 121, Oxford Clarendon Press. 16. Ravishankar GA, Venkataraman LV (1993) Role of plant cell culture in food biotechnology: current trends, limitations and future prospects. In: Plant biotechnology: commercial prospects and problems, (Prakash J, Pierik RLM, eds), pp 25574, Oxford IBH Press, New Delhi. 17. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant 15, 47397. 18. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirement of suspension cultures of soybean root cultures. Exp Cell Res 50, 1518. 19. Arumugam S, Rao AS, Rao MV (2003)

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