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Acute and subacute toxicity studies of Aegle marmelos Corr., an Indian medicinal plant
ARTICLE IN PRESS
Phytomedicine 14 (2007) 209–215 www.elsevier.de/phymed
Acute and subacute toxicity studies of Aegle marmelos Corr., an Indian medicinal plant A. Veerappana,b,, S. Miyazakib, M. Kadarkaraisamyc, D. Ranganathana a Department of Pharmacology and Environmental Toxicology, Dr. A.L.M. Post-Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600 113, Tamil Nadu, India b Toxico-Biochemistry Laboratory, Department of Safety Research, National Institute of Animal Health, Kannondai, Tsukuba Science City, Ibaraki, Japan c National Institute of Advanced Industrial Science and Technology, Tsukuba Central 5-2, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8565, Japan
Abstract This study was designed to elucidate the toxicity of the widely used plant Aegle marmelos in rats. We have taken total alcoholic, total aqueous, whole aqueous and methanolic extracts isolated from the leaves of A. marmelos and studied their toxic effects. Acute, subacute and LD50 values were determined in experimental rats. The dead animals were obtained from primary screening studies, LD50 value determination experiments and acute studies subjected to postmortem studies. The external appearance of the dead animals, the appearance of the viscera, heart, lungs, stomach, intestine, liver, kidney, spleen and brain were carefully noted and any apparent and significant features or differences from the norm were recorded. Following the chronic administration of A. marmelos for 14 days, the vital organs such as heart, liver, kidney, testis, spleen and brain were carefully evaluated by histopathological studies and any apparent and significant changes or differences from the norm were studied. From the acute administration of A. marmelos, the LD50 values were determined using graphical method. The hearts stopped in systolic stand-still in the acute experiments. There were no remarkable changes noticed in the histopathological studies after 50 mg/kg body wt of the extracts of A. marmelos when administered intraperitoneally for 14 days successively. Pathologically, neither gross abnormalities nor histopathological changes were observed. After calculation of LD50 values using graphical methods, we found a broad therapeutic window and a high therapeutic index value for A. marmelos extracts. Intraperitoneal administration of the extracts of the leaves of A. marmelos at doses of 50, 70, 90 and 100 mg/kg body wt for 14 consecutive days to male and female Wistar rats did not induce any short-term toxicity. Collectively, these data demonstrate that the extracts of the leaves of A. marmelos have a high margin of drug safety. r 2006 Elsevier GmbH. All rights reserved. Keywords: Aegle marmelos; Indian bael; Acute toxicity; Subacute toxicity; LD50 values; Therapeutic index; Drug margin of safety
Introduction Corresponding author. Department of Physiology (Box 75), Weill
Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA. Tel.: +1 212 832 9708; fax: +1 212 746 6226. E-mail address: [email protected] (A. Veerappan). 0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2006.05.004
Recent trends have shown a dramatic rise among Americans in use of complementary and integrative medicine approaches to health. Forty-two percent of Americans reported using alternative therapies in 1997;
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A. Veerappan et al. / Phytomedicine 14 (2007) 209–215
40% for treatment of chronic illness and 60% for disease prevention. Despite this rapid growth, there is limited evidence about the effectiveness and toxicity of alternative medicine. Much more needs to be done to develop the evidence base for herbals, botanicals and dietary supplements. Since ancient times, plants have been a source of drugs; but scientific medicine tends to ignore the importance of herbal medicine (Sofowora, 1982). The WHO suggested in 1997 that effective, locally available plants be used as substitutes for drugs. Research work on medicinal plants has intensified and information on these plants has been exchanged. This research will go a long way in the scientific exploration of medicinal plants for the benefit of man and is likely to decrease the dependence on imported drugs (Amadou, 1998). The plant Aegle marmelos Corr. (AM) belongs to the family Rutaceae and is known as vilvam in Tamil, bael in Hindi, bilwa or sripal in Sanskrit and bael tree in English (Nadkarni, 1986). The various parts of this plant (mainly leaves and fruits) are widely used in traditional medicine for the treatment of various disorders. A decoction of plant leaves and fruit is used in remedies for dysentery, diarrhea, upper respiratory tract infections and heart ailments (Dymock William et al., 1890; Kirtikar et al., 1935; Murugesa Mudaliar, 1988). Fresh aqueous and alcoholic extracts of this plant have been reported to have a stimulant effect on the heart and to decrease the requirement of circulatory stimulants (Haravey, 1968). We have also proven its effects on myocardium (Arul et al., 1999). Earlier studies have shown that the alcoholic extract of AM leaves protects against histamine-induced contractions in guinea pig ileum and tracheal chain (Arul et al., 2004) and serial extracts show antiinflammatory, anti-pyretic and analgesic properties (Arul et al., 2005). Although several pharmacological studies have been carried out with this plant, there is no experimental evidence on its toxicity. Hence, in the present study, we planned to evaluate its toxicity effects.
Materials and methods Plant materials The leaves of A. marmelos Corr. were freshly collected from the Medicinal Plants Farm, Arignar Anna Hospital for Indian Medicine, Chennai and authenticated by Prof. R. Rengasamy, Centre for Advanced Studies in Botany, University of Madras, Chennai, India. A voucher specimen (No. 012) has been deposited in the herbarium of the Unit of Phytopharmacology, University of Madras, Chennai, India.
Animals Wistar albino rats (150–200 g each) of both sexes were maintained in the Animal House of the Department of Pharmacology, under standard conditions (temperature 2572 1C, relative humidity 7575% and 12-h light and dark cycle). The animals had access to standard laboratory feed (Gold Mohur, Hindustan Lever Ltd., Mumbai, India) and water ad libitum. All procedures involving animals were performed in accordance with the guidelines of the National Institutes of Health on the use and care of laboratory animals. Experimental procedures were also examined and approved by internal ethical committee for animal welfare.
Drugs and chemicals Drugs and fine chemicals were purchased from Sigma Chemical Co., St. Louis, MO, USA. All other chemicals and solvents were obtained from local firms and were of the highest purity and analytical grade.
Preparation and characterization of AM leaf extracts Air-dried and coarsely powdered leaves (4 kg) were extracted with 70% ethyl alcohol in cold percolation method for 72 h. The total alcoholic extract (yield: 5.453%) was concentrated at 55 1C and kept at room temperature. From the residue of the leaves, the total aqueous extract was obtained by distillation method and the whole aqueous extract was obtained from the fresh leaves. The methanolic extract was obtained from the total alcoholic extract using a vacuum rota evaporator. The yield was 8.07%, 10.37%, 12.43% and 0.525% respectively. These extracts were prepared either as a fine suspension in 5% gum acacia (TAL) or dissolved in normal saline (TAQ, WAQ and MET) and were used for the following toxicological studies. NMR spectra were taken with a Varian 400 MHz NMR system for the characterization of extract fractions. CDCl3 was used as a solvent and the experiment was performed at room temperature. The NMR fingerprint revealed six prominent peaks for TAL, five prominent peaks for TAQ, four prominent peaks for WAQ and three prominent peaks for MET.
Acute toxicity studies The acute toxicity study of the extracts of A. marmelos was performed as described by Turner (1965). The dead animals obtained from primary screening studies, LD50 value determination experiments, and the acute studies were subjected to postmortem studies. The external appearance of the dead animals, the appearance of the
ARTICLE IN PRESS A. Veerappan et al. / Phytomedicine 14 (2007) 209–215
viscera, heart, lungs, stomach, intestine, liver, kidney, spleen and brain were carefully noted and any apparent and significant features or differences from the norm were recorded.
Determination of LD50 values by graphical method Wistar albino rats of both sex and of approximately the same weights were divided into four groups, each containing four animals for the purpose of determining the LD50 value of a single extract. Each group was caged separately. Four different doses of 50, 70, 90 and 100 mg/kg body wt were employed for each test drug. Each animal in every group was administered with an extract of a pre-determined dose intraperitoneally. About 24 h later, the number of dead animals in a group was recorded. The data were tabulated. The toxicological effect was assessed on the basis of mortality, which was expressed as an LD50 value. In the groups with no dead animals and in the groups with only dead animals, the obtained percentages were corrected using the following formulae: Correction formula for 0% dead group ¼ 100ð0:25=nÞ, (1) Correction formula for 100% dead group ¼ 100½ðn 0:25Þ=n.
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wax at 50 1C. Finally, they were prepared as wax blocks with proper tissue-morphological orientations. The wax blocks were sectioned using a microtome suitable for microscopic studies. The suitable sections so obtained were then taken on glass slides. The wax was cleared with xylol; xylol was cleared with absolute alcohol, then progressively rehydrated with gradually increasing dilutions of alcohol and then stained with eosin and hematoxylin. They were then once again dehydrated, cleared with xylol, mounted with DPX and covered with coverslips. The sections were examined carefully under the microscope at low as well as high power. Any histopathological changes deviant from the norm were carefully recorded.
Statistical analysis To calculate the LD50 values, we used the graphical method described under Methods. Other results are calculated as mean7s.e.m. and all the statistical comparisons were made by means of the Student’s t test.
Results Acute toxicity studies
ð2Þ
where n represents the number of animals in the group. After correction, the percentages were converted into probits. The values thus obtained were plotted against log dose. The LD50 value was determined by finding the dose that was intersected by probit 5.
The dead animals obtained from the acute experiments usually presented with their hearts stopped in systolic stand-still. Apart from this characteristic observation, no other significant observation deviant from the norm was seen in these dead animals.
Determination of LD50 value Subacute toxicity studies Wistar albino rats were divided into groups of five animals each. Each group was treated with an extract derived from A. marmelos, one injection per day, for 14 consecutive days. On the fifteenth day, the animals were sacrificed with approximately 100 mg sodium phenobarbital/kg body wt administered intraperitoneally and organ tissues such as heart, liver, adrenal gland, brain, kidney, uterus and testis were surgically removed for histopathological studies. First, the organs were carefully examined macroscopically for any abnormal, pathological signs of toxicity. All animals were subjected to gross necropsy, which included an external examination of all body orifices and surfaces, and an examination of all cranial, thoracic and abdominal organs. Gross pathology findings were recorded. The tissues were fixed with formal saline solution for 48 h, dehydrated with progressively increasing concentrations of alcohol and finally with absolute alcohol, cleaned with xylol and infiltrated with molten paraffin
1. The LD50 value for the total alcoholic extract 1660.00 mg/kg body wt. 2. The LD50 value for the total aqueous extract 1445.00 mg/kg body wt. 3. The LD50 value for the whole aqueous extract 1549.00 mg/kg body wt. 4. The LD50 value for the methanolic extract 1318.00 mg/kg body wt.
is is is is
The values revealed that there was a similar range in the doses of LD50 values for the extracts, ranging from 1300 mg to 1700 mg/kg body wt of the animals (Tables 1–4).
Subacute toxicity studies Gross necropsy findings did not show any adverse effects in any organ. No statistically significant differences in organ weights were present in any of the male and female rats receiving the extracts of AM as
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Table 1.
A. Veerappan et al. / Phytomedicine 14 (2007) 209–215
Determination of LD50 values for total alcoholic extract of the leaves of A. marmelos
Sl. no.
Dose (mg/kg body wt.)
Log dose
Percent mortality (after 24 h)
Corrected mortality (%)
Probit
1 2 3 4
1000 1500 2000 2500
3.0000 3.1761 3.3010 3.3979
0 25 50 100
6.25 25.00 50.00 93.75
3.45 4.33 5.00 6.55
LD50 value of total alcoholic extract ¼ 1660.00 mg/kg body wt.
Table 2.
Determination of LD50 value for total aqueous extract of the leaves of A. marmelos
Sl. no.
Dose (mg/kg body wt.)
Log dose
Percent mortality (after 24 h)
Corrected mortality (%)
Probit
1 2 3 4
1000 1250 1500 2000
3.0000 3.0969 3.1761 3.3010
0 25 50 100
6.25 25.00 50.00 93.75
3.45 4.33 5.00 6.55
LD50 value of total aqueous extract ¼ 1445.00 mg/kg body wt.
Table 3.
Determination of LD50 value for whole aqueous extract of the leaves of A. marmelos
Sl. no.
Dose (mg/kg body wt.)
Log dose
Percent mortality (after 24 h)
Corrected mortality (%)
Probit
1 2 3 4
1000 1500 1750 2000
3.0000 3.1761 3.2430 3.3010
0 25 50 100
6.25 25.00 50.00 93.75
3.45 4.33 5.00 6.55
LD50 value of whole aqueous extract ¼ 1549.00 mg/kg body wt.
Table 4.
Determination of LD50 value for methanolic extract of the leaves of A. marmelos
Sl. no.
Dose (mg/kg body wt.)
Log dose
Percent mortality (after 24 h)
Corrected mortality (%)
Probit
1 2 3 4
1000 1250 1500 1750
3.0000 3.0969 3.1761 3.2430
0 50 75 100
6.25 50.00 75.00 93.75
3.45 5.00 5.67 6.55
LD50 of methanolic extract ¼ 1318.00 mg/kg body wt.
compared to the control group (data not shown). Moreover, no lethality was recorded for any dose up to the maximum of 100 mg/kg body wt of all the extracts of AM during the 14 days of treatment. No target organs were identified by gross pathological examination in animals of the high-dose group and histopathological examination was therefore performed on animals in the control and high-dose groups only. No histopathological change was noted in the high-dose groups as compared to control.
Discussion In the toxicity studies, including the acute, LD50 value and subacute toxicities were elucidated in experimental
rats. Although poisonous plants are ubiquitous (Kingsbury, 1964), herbal medicine is used by up to 80% of the population in developing countries. Despite widespread use, few scientific studies have been undertaken to ascertain the safety and efficacy of traditional remedies. The present investigation shows that the extract of the leaves of A. marmelos is non-toxic via intraperitoneal route in rats, at least up to maximum doses of 1000 mg/ kg body wt acutely and 100 mg/kg body wt subacutely. Since the AM extract has previously been shown to be pharmacologically active (hypoglycemic) when orally administered to rats (Karunanayake et al., 1984; Ponnachan et al., 1993; Seema et al., 1996; Das et al., 1996) at the minimum active dose of mg/kg body wt, one may conclude that the active compounds present in the AM extract exhibit a rather low acute oral toxicity
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profile. In the present study, mortality and symptoms of systolic stand-still in the heart were noted only after the intraperitoneal injection of relatively high doses of the AM extract (the LD50 value is about 1500 mg/kg body wt) in rats. The active principles of the leaf extract responsible for the toxic manifestations after the intraperitoneal dose are not known. The toxicity and the lethality of the AM extract may be due to any one or more of the phytochemicals present in the crude extracts, some of which have been isolated and identified (Karawya et al., 1980; Banerji and Kumar, 1980; Pattnaik et al., 1996; Rana et al., 1997). In the chronic toxicity study in rats given the extract intraperitoneally at doses of 50–100 mg/kg body wt, we did not notice any abnormal behavior, and body weight gains were not significantly different in the treated rats when compared to controls. Since changes in body weight have been used as an indicator of adverse effects of drugs and chemicals (Tofovic and Jackson, 1999; Raza et al., 2002; Teo et al., 2002), the present results
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suggest that at the doses administered intraperitoneally, the AM extracts are non-toxic in rats. In the previous histopathological studies (Arseculeratne et al., 1985), although hepatic lesions were noted in fruit extracts of A. marmelos treated Sprague–Dawley rats, we did not notice any adverse effects with leafextracts-treated rats in our study. This difference may be due to the different types of phytochemicals present in leaves and fruit extracts of A. marmelos. Further, none noticed any adverse hepatic lesions or other toxic effects with respect to the leaf extract of A. marmelos. Nevertheless, since the LD50 value for the intraperitoneal dose is also relatively high, and human exposure to crude extracts of the leaves of A. marmelos is very unlikely to occur by the parenteral route, it can be concluded that AM is non-toxic. At the acute doses of 2000 mg/kg body wt total alcoholic extract, 1500 mg/kg body wt total aqueous extract, 1750 mg/kg body wt whole aqueous extract and 1500 mg/kg body wt methanolic extract, given intraperitoneally, two of four rats died within 24 h in each
Fig. 1. Determination of LD50 value for the extracts of the leaves of Aegle marmelos Corr. administered to rats for 24 h, using a graphical method.
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extract-treated group. The dead animals obtained from these acute experiments presented with their hearts stopped in systolic stand-still, the characteristic toxic response for cardiotonic drugs (Arul et al., 1999). Apart from this characteristic observation, no other significant observation deviant from the norm was observed in these dead animals. Although these relatively short-term studies document no gross organ toxicity, longer-term use could result in serious toxicity. Further studies are therefore required to study long-term toxicity. Based on the results of the acute toxicity studies, it was concluded that a dose of 1000 mg/kg body wt of all the extracts of AM given interperitoneally appeared to be non-toxic (Fig. 1). However, there must be a point at which it can be concluded that a test substance is practically non-toxic or non-lethal after an acute exposure. The dose of 1000 mg/kg body wt for acute intraperitoneal toxicity is generally considered to be very high and the LD50 values of all the AM extracts showed a broad therapeutic window and high therapeutic index value. Thus AM leaf extracts have a high margin of safety.
Conclusion It is therefore concluded that the intraperitoneal administration of the extracts of A. marmelos at 50, 70, 90 and 100 mg/kg body wt for 14 consecutive days to male and female Wistar rats did not induce any short-term toxicological effects. Also, doses of up to 1000 mg/kg body wt(i.p.) did not produce any mortality, indicating that the leaf extract of AM has no short-term harmful effect. Collectively, these data demonstrate that the extracts of the leaves of A. marmelos have a high margin of drug safety. However, further long-term studies with graded doses of AM extract are required in order to rule out any long-term adverse effects and more phytochemical analysis should also be performed.
Acknowledgements One of the authors (A.V.) gratefully acknowledges the financial assistance rendered by the University of Madras, Chennai, India, in the form of a University Research Fellowship award. The Science and Technology Agency, Government of Japan, supported part of this work.
References Amadou, C.K., 1998. Promoting alternative medicine. Africa Health J. 2, 20.
Arseculeratne, S.N., Gunatilaka, A.A., Panabokke, R.G., 1985. Studies on medicinal plants of Sri Lanka. Part 14: toxicity of some traditional medicinal herbs. J. Ethnopharmacol. 13, 323–335. Arul, V., Kumaraguru, S., Dhananjayan, R., 1999. Effects of aegeline and lupeol, the two cardioactive principles isolated from the leaves of Aegle marmelos Corr. J. Pharm. Pharmacol. 51 (S), 252. Arul, V., Miyazaki, S., Dhananjayan, R., 2004. Mechanisms of the contractile effect of the alcoholic extract of Aegle marmelos Corr on the isolated guinea pig ileum and tracheal chain. Phytomedicine 11 (7–8), 679–683. Arul, V., Miyazaki, S., Dhananjayan, R., 2005. Studies on the anti-inflammatory, antipyretic and analgesic properties of the leaves of Aegle marmelos Corr. J. Ethnopharmacol. 96 (1–2), 159–163. Banerji, N., Kumar, R., 1980. Studies on the seed oil of Aegle marmelos and its effects on some bacterial species. J. Inst. Chem. 52, 59–60. Das, A.V., Padayatti, P.S., Paulose, C.S., 1996. Effect of leaf extract of Aegle marmelos (L.) Correa ex Roxb. on histological and ultrastructural changes in tissues of streptozotocin induced diabetic rats. Indian J. Exp. Biol. 34, 341–345. Dymock William, C.J., Warden, H., David, H., 1890. Pharmacographia Indica, A History of the Principal Drugs of Vegetable Origin Met within British India, vol. I. Kegan Paul, Trench Trubner and Co. Ltd., London, pp. 277–281. Haravey, S.K., 1968. A preliminary communication of the action of Aegle marmelos (Bael) on heart. Indian J. Med. Res. 56, 327–331. Karawya, M.S., Mirhom, Y.W., Shehata, I.A., 1980. Sterols, triterpenes, coumarins and alkaloids of Aegle marmelos Correa, cultivated in Egypt. Egypt. J. Pharm. Sci. 21, 239–248. Karunanayake, E.H., Welihinda, J., Sirimanne, S.R., et al., 1984. Oral hypoglycaemic activity of some medicinal plants of Sri Lanka. J. Ethnopharmacol. 11, 223–231. Kingsbury, J.M., 1964. Poisonous Plants of the United States and Canada. Prentice-Hall, Inc., Englewood Cliffs, NJ, USA. Kirtikar, K.R., Basu, B.D., ICS, 1935. Indian Medicinal Plants, second ed. International Book Distributors, Dehradun, India, pp. 448–502. Murugesa Mudaliar, K.S., 1988. Materia Medica of Tamil Systems of Medicine, Part I. Tamil Nadu Siddha Medical Board, Govt. of Tamil Nadu, India, pp. 638–642. Nadkarni, A.K., 1986. Dr. K.M. Nadkarni’s Indian Materia Medica, vol. 1. Popular Prakashan Company, Bombay, India, p. 45. Pattnaik, S., Subramanyam, V.R., Kale, C., 1996. Antibacterial and antifungal activity of ten essential oils in vitro. Microbios 86, 237–246. Ponnachan, P.T.C., Paulose, C.S., Panikkar, K.R., 1993. Effect of leaf extract of Aegle marmelos in diabetic rats. Indian J. Exp. Biol. 31, 345–347. Rana, B.K., Singh, U.P., Taneja, V., 1997. Anti-fungal activity and kinetics of inhibition by essential oil isolated from leaves of Aegle marmelos. J. Ethnopharmacol. 57, 29–34. Raza, M., Al-Shabanah, O.A., El-Hadiyah, T.M., Al-Majed, A.A., 2002. Effect of prolonged vigabatrin treatment on
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haematological and biochemical parameters in plasma, liver and kidney of Swiss albino mice. Sci. Pharmaceut. 70, 135–145. Seema, P.V., Sudha, B., Padayatti, P.S., et al., 1996. Kinetic studies of purified malate dehydrogenase in liver of streptozotocin-diabetic rats and the effect of leaf extract of Aegle marmelos (L.) Correa ex Roxb. Indian J. Exp. Biol. 34, 600–602. Sofowora, A., 1982. Medicinal Plant and Traditional Medicine in Africa, second ed. Wiley, Ibadan, pp. 8–14.
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Teo, S., Stirling, D., Thomas, S., Hoberman, A., Kiorpes, A., Khetani, V., 2002. A 90-day oral gavage toxicity study of Dmethylphenidate and D,L-methylphenidate in Sprague– Dawley rats. Toxicology 179, 183–196. Tofovic, S.P., Jackson, E.K., 1999. Effects of long-term caffeine consumption on renal function in spontaneously hypertensive heart failure prone rats. J. Cardiovasc. Pharmacol. 33, 360–366. Turner, R.A., 1965. Screening Methods in Pharmacology. Academic Press, New York, pp. 302–304.
Phytomedicine 14 (2007) 209–215 www.elsevier.de/phymed
Acute and subacute toxicity studies of Aegle marmelos Corr., an Indian medicinal plant A. Veerappana,b,, S. Miyazakib, M. Kadarkaraisamyc, D. Ranganathana a Department of Pharmacology and Environmental Toxicology, Dr. A.L.M. Post-Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600 113, Tamil Nadu, India b Toxico-Biochemistry Laboratory, Department of Safety Research, National Institute of Animal Health, Kannondai, Tsukuba Science City, Ibaraki, Japan c National Institute of Advanced Industrial Science and Technology, Tsukuba Central 5-2, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8565, Japan
Abstract This study was designed to elucidate the toxicity of the widely used plant Aegle marmelos in rats. We have taken total alcoholic, total aqueous, whole aqueous and methanolic extracts isolated from the leaves of A. marmelos and studied their toxic effects. Acute, subacute and LD50 values were determined in experimental rats. The dead animals were obtained from primary screening studies, LD50 value determination experiments and acute studies subjected to postmortem studies. The external appearance of the dead animals, the appearance of the viscera, heart, lungs, stomach, intestine, liver, kidney, spleen and brain were carefully noted and any apparent and significant features or differences from the norm were recorded. Following the chronic administration of A. marmelos for 14 days, the vital organs such as heart, liver, kidney, testis, spleen and brain were carefully evaluated by histopathological studies and any apparent and significant changes or differences from the norm were studied. From the acute administration of A. marmelos, the LD50 values were determined using graphical method. The hearts stopped in systolic stand-still in the acute experiments. There were no remarkable changes noticed in the histopathological studies after 50 mg/kg body wt of the extracts of A. marmelos when administered intraperitoneally for 14 days successively. Pathologically, neither gross abnormalities nor histopathological changes were observed. After calculation of LD50 values using graphical methods, we found a broad therapeutic window and a high therapeutic index value for A. marmelos extracts. Intraperitoneal administration of the extracts of the leaves of A. marmelos at doses of 50, 70, 90 and 100 mg/kg body wt for 14 consecutive days to male and female Wistar rats did not induce any short-term toxicity. Collectively, these data demonstrate that the extracts of the leaves of A. marmelos have a high margin of drug safety. r 2006 Elsevier GmbH. All rights reserved. Keywords: Aegle marmelos; Indian bael; Acute toxicity; Subacute toxicity; LD50 values; Therapeutic index; Drug margin of safety
Introduction Corresponding author. Department of Physiology (Box 75), Weill
Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA. Tel.: +1 212 832 9708; fax: +1 212 746 6226. E-mail address: [email protected] (A. Veerappan). 0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2006.05.004
Recent trends have shown a dramatic rise among Americans in use of complementary and integrative medicine approaches to health. Forty-two percent of Americans reported using alternative therapies in 1997;
ARTICLE IN PRESS 210
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40% for treatment of chronic illness and 60% for disease prevention. Despite this rapid growth, there is limited evidence about the effectiveness and toxicity of alternative medicine. Much more needs to be done to develop the evidence base for herbals, botanicals and dietary supplements. Since ancient times, plants have been a source of drugs; but scientific medicine tends to ignore the importance of herbal medicine (Sofowora, 1982). The WHO suggested in 1997 that effective, locally available plants be used as substitutes for drugs. Research work on medicinal plants has intensified and information on these plants has been exchanged. This research will go a long way in the scientific exploration of medicinal plants for the benefit of man and is likely to decrease the dependence on imported drugs (Amadou, 1998). The plant Aegle marmelos Corr. (AM) belongs to the family Rutaceae and is known as vilvam in Tamil, bael in Hindi, bilwa or sripal in Sanskrit and bael tree in English (Nadkarni, 1986). The various parts of this plant (mainly leaves and fruits) are widely used in traditional medicine for the treatment of various disorders. A decoction of plant leaves and fruit is used in remedies for dysentery, diarrhea, upper respiratory tract infections and heart ailments (Dymock William et al., 1890; Kirtikar et al., 1935; Murugesa Mudaliar, 1988). Fresh aqueous and alcoholic extracts of this plant have been reported to have a stimulant effect on the heart and to decrease the requirement of circulatory stimulants (Haravey, 1968). We have also proven its effects on myocardium (Arul et al., 1999). Earlier studies have shown that the alcoholic extract of AM leaves protects against histamine-induced contractions in guinea pig ileum and tracheal chain (Arul et al., 2004) and serial extracts show antiinflammatory, anti-pyretic and analgesic properties (Arul et al., 2005). Although several pharmacological studies have been carried out with this plant, there is no experimental evidence on its toxicity. Hence, in the present study, we planned to evaluate its toxicity effects.
Materials and methods Plant materials The leaves of A. marmelos Corr. were freshly collected from the Medicinal Plants Farm, Arignar Anna Hospital for Indian Medicine, Chennai and authenticated by Prof. R. Rengasamy, Centre for Advanced Studies in Botany, University of Madras, Chennai, India. A voucher specimen (No. 012) has been deposited in the herbarium of the Unit of Phytopharmacology, University of Madras, Chennai, India.
Animals Wistar albino rats (150–200 g each) of both sexes were maintained in the Animal House of the Department of Pharmacology, under standard conditions (temperature 2572 1C, relative humidity 7575% and 12-h light and dark cycle). The animals had access to standard laboratory feed (Gold Mohur, Hindustan Lever Ltd., Mumbai, India) and water ad libitum. All procedures involving animals were performed in accordance with the guidelines of the National Institutes of Health on the use and care of laboratory animals. Experimental procedures were also examined and approved by internal ethical committee for animal welfare.
Drugs and chemicals Drugs and fine chemicals were purchased from Sigma Chemical Co., St. Louis, MO, USA. All other chemicals and solvents were obtained from local firms and were of the highest purity and analytical grade.
Preparation and characterization of AM leaf extracts Air-dried and coarsely powdered leaves (4 kg) were extracted with 70% ethyl alcohol in cold percolation method for 72 h. The total alcoholic extract (yield: 5.453%) was concentrated at 55 1C and kept at room temperature. From the residue of the leaves, the total aqueous extract was obtained by distillation method and the whole aqueous extract was obtained from the fresh leaves. The methanolic extract was obtained from the total alcoholic extract using a vacuum rota evaporator. The yield was 8.07%, 10.37%, 12.43% and 0.525% respectively. These extracts were prepared either as a fine suspension in 5% gum acacia (TAL) or dissolved in normal saline (TAQ, WAQ and MET) and were used for the following toxicological studies. NMR spectra were taken with a Varian 400 MHz NMR system for the characterization of extract fractions. CDCl3 was used as a solvent and the experiment was performed at room temperature. The NMR fingerprint revealed six prominent peaks for TAL, five prominent peaks for TAQ, four prominent peaks for WAQ and three prominent peaks for MET.
Acute toxicity studies The acute toxicity study of the extracts of A. marmelos was performed as described by Turner (1965). The dead animals obtained from primary screening studies, LD50 value determination experiments, and the acute studies were subjected to postmortem studies. The external appearance of the dead animals, the appearance of the
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viscera, heart, lungs, stomach, intestine, liver, kidney, spleen and brain were carefully noted and any apparent and significant features or differences from the norm were recorded.
Determination of LD50 values by graphical method Wistar albino rats of both sex and of approximately the same weights were divided into four groups, each containing four animals for the purpose of determining the LD50 value of a single extract. Each group was caged separately. Four different doses of 50, 70, 90 and 100 mg/kg body wt were employed for each test drug. Each animal in every group was administered with an extract of a pre-determined dose intraperitoneally. About 24 h later, the number of dead animals in a group was recorded. The data were tabulated. The toxicological effect was assessed on the basis of mortality, which was expressed as an LD50 value. In the groups with no dead animals and in the groups with only dead animals, the obtained percentages were corrected using the following formulae: Correction formula for 0% dead group ¼ 100ð0:25=nÞ, (1) Correction formula for 100% dead group ¼ 100½ðn 0:25Þ=n.
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wax at 50 1C. Finally, they were prepared as wax blocks with proper tissue-morphological orientations. The wax blocks were sectioned using a microtome suitable for microscopic studies. The suitable sections so obtained were then taken on glass slides. The wax was cleared with xylol; xylol was cleared with absolute alcohol, then progressively rehydrated with gradually increasing dilutions of alcohol and then stained with eosin and hematoxylin. They were then once again dehydrated, cleared with xylol, mounted with DPX and covered with coverslips. The sections were examined carefully under the microscope at low as well as high power. Any histopathological changes deviant from the norm were carefully recorded.
Statistical analysis To calculate the LD50 values, we used the graphical method described under Methods. Other results are calculated as mean7s.e.m. and all the statistical comparisons were made by means of the Student’s t test.
Results Acute toxicity studies
ð2Þ
where n represents the number of animals in the group. After correction, the percentages were converted into probits. The values thus obtained were plotted against log dose. The LD50 value was determined by finding the dose that was intersected by probit 5.
The dead animals obtained from the acute experiments usually presented with their hearts stopped in systolic stand-still. Apart from this characteristic observation, no other significant observation deviant from the norm was seen in these dead animals.
Determination of LD50 value Subacute toxicity studies Wistar albino rats were divided into groups of five animals each. Each group was treated with an extract derived from A. marmelos, one injection per day, for 14 consecutive days. On the fifteenth day, the animals were sacrificed with approximately 100 mg sodium phenobarbital/kg body wt administered intraperitoneally and organ tissues such as heart, liver, adrenal gland, brain, kidney, uterus and testis were surgically removed for histopathological studies. First, the organs were carefully examined macroscopically for any abnormal, pathological signs of toxicity. All animals were subjected to gross necropsy, which included an external examination of all body orifices and surfaces, and an examination of all cranial, thoracic and abdominal organs. Gross pathology findings were recorded. The tissues were fixed with formal saline solution for 48 h, dehydrated with progressively increasing concentrations of alcohol and finally with absolute alcohol, cleaned with xylol and infiltrated with molten paraffin
1. The LD50 value for the total alcoholic extract 1660.00 mg/kg body wt. 2. The LD50 value for the total aqueous extract 1445.00 mg/kg body wt. 3. The LD50 value for the whole aqueous extract 1549.00 mg/kg body wt. 4. The LD50 value for the methanolic extract 1318.00 mg/kg body wt.
is is is is
The values revealed that there was a similar range in the doses of LD50 values for the extracts, ranging from 1300 mg to 1700 mg/kg body wt of the animals (Tables 1–4).
Subacute toxicity studies Gross necropsy findings did not show any adverse effects in any organ. No statistically significant differences in organ weights were present in any of the male and female rats receiving the extracts of AM as
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Table 1.
A. Veerappan et al. / Phytomedicine 14 (2007) 209–215
Determination of LD50 values for total alcoholic extract of the leaves of A. marmelos
Sl. no.
Dose (mg/kg body wt.)
Log dose
Percent mortality (after 24 h)
Corrected mortality (%)
Probit
1 2 3 4
1000 1500 2000 2500
3.0000 3.1761 3.3010 3.3979
0 25 50 100
6.25 25.00 50.00 93.75
3.45 4.33 5.00 6.55
LD50 value of total alcoholic extract ¼ 1660.00 mg/kg body wt.
Table 2.
Determination of LD50 value for total aqueous extract of the leaves of A. marmelos
Sl. no.
Dose (mg/kg body wt.)
Log dose
Percent mortality (after 24 h)
Corrected mortality (%)
Probit
1 2 3 4
1000 1250 1500 2000
3.0000 3.0969 3.1761 3.3010
0 25 50 100
6.25 25.00 50.00 93.75
3.45 4.33 5.00 6.55
LD50 value of total aqueous extract ¼ 1445.00 mg/kg body wt.
Table 3.
Determination of LD50 value for whole aqueous extract of the leaves of A. marmelos
Sl. no.
Dose (mg/kg body wt.)
Log dose
Percent mortality (after 24 h)
Corrected mortality (%)
Probit
1 2 3 4
1000 1500 1750 2000
3.0000 3.1761 3.2430 3.3010
0 25 50 100
6.25 25.00 50.00 93.75
3.45 4.33 5.00 6.55
LD50 value of whole aqueous extract ¼ 1549.00 mg/kg body wt.
Table 4.
Determination of LD50 value for methanolic extract of the leaves of A. marmelos
Sl. no.
Dose (mg/kg body wt.)
Log dose
Percent mortality (after 24 h)
Corrected mortality (%)
Probit
1 2 3 4
1000 1250 1500 1750
3.0000 3.0969 3.1761 3.2430
0 50 75 100
6.25 50.00 75.00 93.75
3.45 5.00 5.67 6.55
LD50 of methanolic extract ¼ 1318.00 mg/kg body wt.
compared to the control group (data not shown). Moreover, no lethality was recorded for any dose up to the maximum of 100 mg/kg body wt of all the extracts of AM during the 14 days of treatment. No target organs were identified by gross pathological examination in animals of the high-dose group and histopathological examination was therefore performed on animals in the control and high-dose groups only. No histopathological change was noted in the high-dose groups as compared to control.
Discussion In the toxicity studies, including the acute, LD50 value and subacute toxicities were elucidated in experimental
rats. Although poisonous plants are ubiquitous (Kingsbury, 1964), herbal medicine is used by up to 80% of the population in developing countries. Despite widespread use, few scientific studies have been undertaken to ascertain the safety and efficacy of traditional remedies. The present investigation shows that the extract of the leaves of A. marmelos is non-toxic via intraperitoneal route in rats, at least up to maximum doses of 1000 mg/ kg body wt acutely and 100 mg/kg body wt subacutely. Since the AM extract has previously been shown to be pharmacologically active (hypoglycemic) when orally administered to rats (Karunanayake et al., 1984; Ponnachan et al., 1993; Seema et al., 1996; Das et al., 1996) at the minimum active dose of mg/kg body wt, one may conclude that the active compounds present in the AM extract exhibit a rather low acute oral toxicity
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profile. In the present study, mortality and symptoms of systolic stand-still in the heart were noted only after the intraperitoneal injection of relatively high doses of the AM extract (the LD50 value is about 1500 mg/kg body wt) in rats. The active principles of the leaf extract responsible for the toxic manifestations after the intraperitoneal dose are not known. The toxicity and the lethality of the AM extract may be due to any one or more of the phytochemicals present in the crude extracts, some of which have been isolated and identified (Karawya et al., 1980; Banerji and Kumar, 1980; Pattnaik et al., 1996; Rana et al., 1997). In the chronic toxicity study in rats given the extract intraperitoneally at doses of 50–100 mg/kg body wt, we did not notice any abnormal behavior, and body weight gains were not significantly different in the treated rats when compared to controls. Since changes in body weight have been used as an indicator of adverse effects of drugs and chemicals (Tofovic and Jackson, 1999; Raza et al., 2002; Teo et al., 2002), the present results
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suggest that at the doses administered intraperitoneally, the AM extracts are non-toxic in rats. In the previous histopathological studies (Arseculeratne et al., 1985), although hepatic lesions were noted in fruit extracts of A. marmelos treated Sprague–Dawley rats, we did not notice any adverse effects with leafextracts-treated rats in our study. This difference may be due to the different types of phytochemicals present in leaves and fruit extracts of A. marmelos. Further, none noticed any adverse hepatic lesions or other toxic effects with respect to the leaf extract of A. marmelos. Nevertheless, since the LD50 value for the intraperitoneal dose is also relatively high, and human exposure to crude extracts of the leaves of A. marmelos is very unlikely to occur by the parenteral route, it can be concluded that AM is non-toxic. At the acute doses of 2000 mg/kg body wt total alcoholic extract, 1500 mg/kg body wt total aqueous extract, 1750 mg/kg body wt whole aqueous extract and 1500 mg/kg body wt methanolic extract, given intraperitoneally, two of four rats died within 24 h in each
Fig. 1. Determination of LD50 value for the extracts of the leaves of Aegle marmelos Corr. administered to rats for 24 h, using a graphical method.
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extract-treated group. The dead animals obtained from these acute experiments presented with their hearts stopped in systolic stand-still, the characteristic toxic response for cardiotonic drugs (Arul et al., 1999). Apart from this characteristic observation, no other significant observation deviant from the norm was observed in these dead animals. Although these relatively short-term studies document no gross organ toxicity, longer-term use could result in serious toxicity. Further studies are therefore required to study long-term toxicity. Based on the results of the acute toxicity studies, it was concluded that a dose of 1000 mg/kg body wt of all the extracts of AM given interperitoneally appeared to be non-toxic (Fig. 1). However, there must be a point at which it can be concluded that a test substance is practically non-toxic or non-lethal after an acute exposure. The dose of 1000 mg/kg body wt for acute intraperitoneal toxicity is generally considered to be very high and the LD50 values of all the AM extracts showed a broad therapeutic window and high therapeutic index value. Thus AM leaf extracts have a high margin of safety.
Conclusion It is therefore concluded that the intraperitoneal administration of the extracts of A. marmelos at 50, 70, 90 and 100 mg/kg body wt for 14 consecutive days to male and female Wistar rats did not induce any short-term toxicological effects. Also, doses of up to 1000 mg/kg body wt(i.p.) did not produce any mortality, indicating that the leaf extract of AM has no short-term harmful effect. Collectively, these data demonstrate that the extracts of the leaves of A. marmelos have a high margin of drug safety. However, further long-term studies with graded doses of AM extract are required in order to rule out any long-term adverse effects and more phytochemical analysis should also be performed.
Acknowledgements One of the authors (A.V.) gratefully acknowledges the financial assistance rendered by the University of Madras, Chennai, India, in the form of a University Research Fellowship award. The Science and Technology Agency, Government of Japan, supported part of this work.
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