Acute and subacute toxicity studies of Aegle marmelos Corr., an Indian medicinal plant.
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 [LD.sub.50] values were determined in experimental rats. The dead animals were obtained from primary screening studies, [LD.sub.50] 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 [LD.sub.50] 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 [LD.sub.50] 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 100mg/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.
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Keywords: Aegle marmelos; Indian bael; Acute toxicity; Subacute toxicity; [LD.sub.50] values; Therapeutic index; Drug margin of safety
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; 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 anti-inflammatory, 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
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.
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 25 [+ or -] 2 [degrees]C, relative humidity 75 [+ or -] 5% 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 [degrees]C 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 400MHz NMR system for the characterization of extract fractions. CD[Cl.sub.3] 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, [LD.sub.50] value determination experiments, and the acute studies were 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.
Determination of [LD.sub.50] 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 [LD.sub.50] 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 [LD.sub.50] 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 = 100(0.25/n), (1)
Correction formula for 100% dead group = 100[(n - 0.25)/n]. (2)
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 [LD.sub.50] value was determined by finding the dose that was intersected by probit 5.
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 wax at 50 [degrees]C. 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.
To calculate the [LD.sub.50] values, we used the graphical method described under Methods. Other results are calculated as mean[+ or -]s.e.m. and all the statistical comparisons were made by means of the Student's t test.
Acute toxicity studies
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 [LD.sub.50] value
1. The [LD.sub.50] value for the total alcoholic extract is 1660.00 mg/kg body wt.
2. The [LD.sub.50] value for the total aqueous extract is 1445.00 mg/kg body wt.
3. The [LD.sub.50] value for the whole aqueous extract is 1549.00 mg/kg body wt.
4. The [LD.sub.50] value for the methanolic extract is 1318.00 mg/kg body wt.
The values revealed that there was a similar range in the doses of [LD.sub.50] values for the extracts, ranging from 1300mg 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 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.
In the toxicity studies, including the acute, [LD.sub.50] value and subacute toxicities were elucidated in experimental rats. Although poisonous plants are ubiquitous (Kings-bury, 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 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 [LD.sub.50] 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 50-100mg/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 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 leaf-extracts-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 [LD.sub.50] 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 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.
[FIGURE 1 OMITTED]
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 1000mg/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 [LD.sub.50] 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.
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.
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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A. Veerappan (a,b,*), S. Miyazaki (b), M. Kadarkaraisamy, (c) D. Ranganathan (a)
(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
*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: firstname.lastname@example.org (A. Veerappan).
Table 1. Determination of [LD.sub.50] values for total alcoholic extract of the leaves of A. marmelos Dose (mg/kg Percent mortality Corrected Sl. no. body wt.) Log dose (after 24 h) mortality (%) Probit 1 1000 3.0000 0 6.25 3.45 2 1500 3.1761 25 25.00 4.33 3 2000 3.3010 50 50.00 5.00 4 2500 3.3979 100 93.75 6.55 [LD.sub.50] value of total alcoholic extract = 1660.00 mg/kg body wt. Table 2. Determination of [LD.sub.50] value for total aqueous extract of the leaves of A. marmelos Dose (mg/kg Percent mortality Corrected Sl. no. body wt.) Log dose (after 24 h) mortality (%) Probit 1 1000 3.0000 0 6.25 3.45 2 1250 3.0969 25 25.00 4.33 3 1500 3.1761 50 50.00 5.00 4 2000 3.3010 100 93.75 6.55 [LD.sub.50] value of total aqueous extract = 1445.00 mg/kg body wt. Table 3. Determination of [LD.sub.50] value for whole aqueous extract of the leaves of A. marmelos Dose (mg/kg Percent mortality Corrected Sl. no. body wt.) Log dose (after 24 h) mortality (%) Probit 1 1000 3.0000 0 6.25 3.45 2 1500 3.1761 25 25.00 4.33 3 1750 3.2430 50 50.00 5.00 4 2000 3.3010 100 93.75 6.55 [LD.sub.50] value of whole aqueous extract = 1549.00 mg/kg body wt. Table 4. Determination of [LD.sub.50] value for methanolic extract of the leaves of A. marmelos Dose (mg/kg Percent mortality Corrected Sl. no. body wt.) Log dose (after 24h) mortality (%) Probit 1 1000 3.0000 0 6.25 3.45 2 1250 3.0969 50 50.00 5.00 3 1500 3.1761 75 75.00 5.67 4 1750 3.2430 100 93.75 6.55 [LD.sub.50] of methanolic extract = 1318.00 mg/kg body wt.
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|Author:||Veerappan, A.; Miyazaki, S.; Kadarkaraisamy, M.; Ranganathan, D.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Article Type:||Drug overview|
|Date:||Feb 1, 2007|
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