Abstract: The sub-acute toxicity study of 3,6-dimethyl-4-ethyl-0-acetyl benzene (BM-5), was carried out on Long Evan’s rats using daily administration (300 μg/rat/day) of compound for 14 consecutive days. Non-significant differences between weight of compound receiving rats and control rats (48±0.816 vs 46±0.816) were found. The change in hematological parameters was found to be nonsignificant (total count of RBC, 5.1±0.081 vs 4.63±0.094; white blood cell, 7.33±0.124 vs 5.73±0.124; platelets, 361666±10274 vs 261666±8498, (cell/ml) x106; percentage of hemoglobin 65.67±1.60 vs 56±0.816; ESR, 25±0.816 vs 19.67±0.471 for experimental and control rats, respectively) and biochemical parameters (serum glutamate pyruvate transaminase, 8.5±0.408 vs 8.33 ±0.471 IU L-1; serum glutamate oxaloacetate transaminase, 9.83±0.235 vs 9.33±0.234 IU L-1; bilirubin, 0.36±0.104 vs 0.34±0.016 μg dL-1; creatinine, 0.67 ±0.089 vs 0.61±0.009 mg dL-1; urea, 18.83±0.235 vs 18.5±0.408 mg dL-1; for experimental and control rats). Therefore, the changes in body weight, hematological and biochemical parameters were statistically non-significant. No detectable abnormalities were found in histopathology of heart, kidney, liver and lung in experimental group of rats as compared with that of the control group of rats.
Introduction
The challenge of developing modern medicine for twenty first century needs more systemic research on the branch of medicine for welfare of the humanity, because microbial drug resistance is one of the serious problems, which the human race is facing today. Therefore, the worldwide increasing demand for medicine from natural sources has motivated search for new drugs with high potential activity.
The production of antibiotic from genus Streptomyces species is well reputed (Atoni et al., 1997; Hamada et al., 1999; Anisuzzaman et al., 2001). Based on this concept, we isolated an antagonistic strain of organism and later it was identified as Streptomyces species (Holt et al., 1994) from which active metabolite 3,6-dimethyl-4-ethyl-0-acetyl benzene was isolated having Rf value 0.61 in solvent (CHCl3: MeOH, 12:1).
In order to assess the safety and efficacy level of a drug, toxicity studies are very essential. Toxicological data help to make decision whether a new drug should be clinically used or not. Therefore keeping this objective in view, we herein, report the toxicological studies of the compound 3,6-dimethyl-4-ethyl-0-acetyl benzene (BM-5) in rats.
Methods and Materials
Collection of rats: For this purpose, Long Evan’s rats of same sex (male) and age (7 weeks) were collected from International Center for Diarrhoeal Disease Research, Bangladesh (ICDDR, B).
Maintenance of the rats: The rats were kept in properly numbered iron cages individually in a clean animal house with an optimum room temperature and were given standard balanced diet (Hawk et al., 1954). The rats were maintained by this way for 15 days prior to administration of the compound and continued until completion of the experiment.
Grouping of the rats: Rats were weighed individually and divided into two groups; group-A (average body weight, 45 g) and group-B (average body weight 46.5 g), each comprising of three rats. Group A is used as a control.
Administration of the sample: The compound was dissolved in distilled water with the help of tween- 20 in such a way that 0.3 ml contained 300 μg of the compound. The rats in group A and B were injected intra-peritoneally with vehicle (300 µl) and compound 300 μg/rat/day respectively for 14 consecutive days.
Gross general observation: The body weight of each rat of both groups was taken before administration of the compound and just prior to sacrifice them. During the whole experimental period their behaviour, CNS excitation, CNS depression, reflexes, muscular weakness, salivation and diarrhoea were monitored daily.
Experimental procedure: For hematological studies, blood was drawn from the tail vein of rats of both the groups before the administration of the compound and blood smears were made on glass slides and stained with "Leishmen reagents" to perform TC, DC and Platelet count (Ghai et al., 1990). With the use of capillary tubes, blood was drawn from each rat to estimate the hemoglobin percentage by "Van Kampen-Zijlstra’s" method (Ghai et al., 1990). The test was repeated on 7th and 14th day after administration of the compound.
For biochemical study, blood was collected from the throat vein of each of the rats after sacrificing them at the end of 14 days of the administration of compound and determined SGPT, SGOT, bilirubin, creatinine, urea by using procedure and reagents as described in Enlehringer Mannheim GmbH Diagnostica (King and Armstrong, 1934; Reitman and Frankel, 1957; Coulomble and Favreau, 1963).
Histopathological studies of heart, kidney, liver and lung were performed by staining method using hematoxylin, eosin reagent and diphenyl xylene mounting fluid. The tissues were observed under microscope at the Department of Genetics and Breeding, University of Rajshahi, Bangladesh.
Results and Discussion
The structure of the compound whose toxicological studies were performed is shown below on the basis of spectral data (Bytul, 2002).
3,6- dimethyl- 4-ethyl- O-acetyl benzene |
Gross general observation: The rats of group A and B showed no signs of tremor, convulsion and reflex abnormalities. No muscular numbness of hind, salivation and diarrhoea was observed. However, the observed changes in body weight before and after drug treatment were found statistically non significant (Table 1).
| Table 1: | Effect of compound on body weight of rats |
| M1 and M2 = sample mean value; SD1 and SD2, standard deviations; n, number of rats; + , increase NS, non significant | |
Hematological profiles: No abnormalities were found in total counts of RBC and WBC, platelet count, hemoglobin percentage and ESR of the drug treated rats in comparison with that of control rats (Table 2).
Biochemical parameters of blood: The record of biochemical parameters in experimental groups of rats is non-significantly different in comparison with control group of rats (Table 3). These indicate that the compound has no adverse effects on liver and kidney functioning (Anisuzzaman et al., 2001).
Histopathological studies: Histopathological studies of the heart, kidney, liver and lung of both control and drug treated rats (Table 4) showed no detectable abnormality between the two groups of rats, indicating that the compound has no adverse effect on cellular structures of these organs (Anisuzzaman et al., 2001).
| Table 2: | Hematological profiles after i.p. administration of compound (300 μg/rat/day) for 14 days in control and compound receiving rats |
| Table 3: | Effect of compound on the biochemical parameters of rat’s blood after i.p. administration of 300 μg/rat/day for 14 consecutive days |
| M1 and M2 = sample mean value; SD1 and SD2, standard deviations; n, number of rats; + , increase and NS, non significant. | |
| Table 4: | Effect of the compound on histopathology of rat’s heart, kidney, liver and lungs tissue i.p. administration of 300 μg/rat/day for 14 consecutive days |
| NAD = No abnormality detected. | |
All drugs are toxic at higher doses and even many drugs have unavoidable toxic effects at therapeutic doses. Therefore, it is important to assess the safety and efficacy of a drug. Form the subacute study, we conclude that the compound 3,6-dimethyl-4-ethyl-0-acetyl benzene has no toxic effect in rats at the dose and duration used in this study and the compound is suitable for further clinical trial.
Acknowledgment
The authors like to thanks Professor Dr. Md. Abdur Rashid, Department of Pharmacy, University of Dhaka, Bangladesh, for producing spectral analysis and Dr. Anwar Habib, Lecturer, Department of Pharmacology, Rajshahi Medical College, Rajshahi, Bangladesh, for his useful criticisms during sub- acute toxicity studies of the compound.