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Studies on the acute and sub-chronic toxicity of the essential oil of Ocimum gratissimum L. leaf.

Summary

Oral and intra-peritoneal acute toxicity and the sub-chronic intra-peritoneal toxicity of the essential oil of Ocimum gratissimum Linn, Lamiaceae (Ocimum oil), was investigated. The acute toxicity test involved the oral and intra-peritoneal administration of graded doses of Ocimum oil prepared as a 4% v/v emulsion to 2 groups each of 30 rats and mice. L[D.sub.50] and L[D.sub.100] were determined for both routes and species. In the sub-chronic toxicity study, 25 male Sprague-Dawley rats were randomized into 4 test groups (treated with three graded sub-lethal doses of Ocimum oil prepared as a 4% v/v emulsion) and a control. Organs and blood samples were taken for analyses after a 30 day treatment period. A dose-dependent sedative effect of Ocimum oil was observed during the acute toxicity study in mice and rats and in the sub-chronic test in rats. Evidence of treatment, route, and dose-dependent toxicity were detected in both studies. Changes in weight of the testes, hearts, kidneys, intestines and lungs of the rats were statistically insignificant (ANOVAP < 0.05). Data analyses of blood biochemical, haematological and histopathological findings showed significant differences between control and treated groups and revealed that Ocimum oil is capable of invoking an inflammatory response that transits from acute to chronic on persistent administration. While the study revealed that Ocimum oil might be better tolerated when administered orally for systemic delivery, the oil has toxic potentialities that should not be overlooked.

Key words: Ocimum gratissimum Linn, Lamiaceae, common names-Mosquito plant, Fever plant-Leaf essential oil, Acute toxicity, Sub-chronic toxicity, Sedative effect, Histopathology, Haematology, Enzyme assay

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Introduction

The genus Ocimum belongs to the economically important group of herbaceous plants used extensively in flavouring and perfumery. Ocimum gratissimum Linn., Family Lamiaceae, is indigenous to Persia, East Bengal, Nepal, and Daccan peninsula, often cultivated in various parts of West Africa, widely distributed in tropical Africa and grows in all parts of Nigeria (Ekejuiba, 1984). A chemical survey of O. gratissimum leaf reveals that it contains mainly essential oil (Ocimum oil) and non-phlobatannins. The main constituent of the essential oil is thymol (48.1%), followed by p-cymene (12.5%) with trace amounts of 40 other constituents (Martins et al. 1999). O. gratissimum leaves collected in south western Nigeria and used in this experiment have been shown to contain 47.0%, 16.2% and 6.2% thymol, p-cymene and [alpha]-terpinene, respectively (Ekejuiba, 1984). The leaves have been put into a number of ethno-botanical uses, which include treatment of seminal weakness, as aromatic baths, tonic expectorants and anti-spasmodic. The crude extract is used as febrifuge and as an ingredient in many malaria remedies (Hocking, 1985; El Said et al. 1969). Reports suggesting that the antibacterial and antifungal activities of the plant lie in the essential oil of the leaf are numerous (El Said et al. 1969). Ocimum oil has been shown to calm overactive gastro-intestinal tracts in diarrhoea, with an efficacy comparable to loperamide (Orafidiya et al. 2000), thus explaining its folkloric use to relieve abdominal colic in children (Offia and Chukwendu, 1999). Its usefulness in the management of Acne vulgaris has been recently demonstrated (Orafidiya et al. 2002).

With such a spread of folkloric use, it has become imperative that preparations of Ocimum oil be standardized and its toxicity profile determined. This study attempts to evaluate the oral and intra-peritoneal acute toxicity and the sub-chronic intra-peritoneal toxicity of Ocimum oil. Consistent with the goals of toxicology (Doull 1996), this study aims to identify and characterize the adverse effects that can be produced in biological systems by exposure to Ocimum oil and later to use this information and that from further studies to predict the type and severity of responses in man under other exposure situations.

Experimental

The "principles of laboratory animal care" (NIH Publication No. 85-23) were followed in this study.

Animals

Eighty 8-10 weeks old male Swiss albino mice weighing 22-24 g and 100 male Sprague-Dawley rats weighing 135-150 g were maintained at standard conditions of 12 h light/darkness, humidity and temperature in the Department of Pharmacology Animal House, Obafemi Awolowo University, Ile-Ife. They were kept in cages, fed on standard diet with free access to water throughout the period of experimentation. The animals were used for the acute and sub-chronic tests.

Plant material and extraction of ocimum oil

The leaves of O. gratissimum were collected from various sites at Ile-Ife, Osun State, Nigeria. Dr H.C Illoh of the Department of Botany, Obafemi Awolowo University, Ile-Ife identified and authenticated the plant. It was identical with the voucher specimens (Ife 1930 and Ife 1819) previously deposited at the Department of Botany herbarium. Ocimum oil was extracted from the fresh leaves by hydro-distillation using the British Pharmacopoeia method (British Pharmacopoeia, 1988). The oil was then stored in glass containers in the refrigerator until needed.

Preparation of test samples

The extracted Ocimum oil was prepared into a 4% v/v oil-in-water emulsion using 1% Polysorbate 80 as emulsifier and refrigerated until needed.

Acute intra-peritoneal toxicity and behavioural activity tests

Pilot tests were conducted to determine the dose range of the oil to be administered in mice and rats. The maximum dose producing 0% death and the minimum dose that produced 100% death were obtained. From these, appropriate volumes of the 4% v/v Ocimum oil emulsion containing 0.16, 0.20, 0.26, 0.33, 0.39 and 0.46 g/kg of Ocimum oil were given intraperitoneally (IP) to 6 groups of 5 mice each. Similarly, 6 graded doses of 0.32, 0.37, 0.48, 0.59, 0.72 and 0.93 g/kg of Ocimum oil were administered IP to 30 rats randomized into 6 groups, respectively. The animals were observed for symptoms of toxicity and mortality within 24-72 h. L[D.sub.50] and L[D.sub.100] were calculated using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxin, 1949). The behavioural and CNS profiles scored were: spontaneous rearing and grooming, evidence of calmness and sedation, loss of writhing reflex and duration of sleep.

Estimation of oral acute toxicity

To 30 male Swiss albino mice and 30 male Sprague-Dawley rats in 6 groups each, were administered volumes of 4% v/v Ocimum oil emulsion corresponding to 0.66, 0.20, 1.31, 1.64, 1.96 & 2.62 g/kg and 1.07, 1.60, 2.13, 2.67, 3.20 & 3.73 g/kg orally, respectively. Symptoms of toxicity and mortality were observed within 24-72 h. L[D.sub.50] and L[D.sub.100] values were determined as above. Behavioural and CNS profiles described above were also scored.

Sub-chronic toxicity test

Twenty-five male Sprague-Dawley rats were randomly selected and grouped into 4 test groups and a control. Preliminary test to determine range of sub-lethal doses was carried out. Of these, 3 graded doses of Ocimum oil: 80, 133 and 213 mg/kg, were selected and administered intraperitoneally to 3 test groups (groups II, III, IV) respectively, with the 4th (group I) receiving 133 mg/kg orally for 30 days. The control group received no test substance. At the end of treatment period, surviving animals were anaesthetized with ether and blood was obtained for sera preparation by cardiac puncture. The livers, testes, kidneys, lungs, hearts, brains and intestines of the rats were removed, weighed and portions fixed for histopathology tests. Liver portions were stored frozen for enzyme assay.

Preparation of liver homogenates

Liver samples (0.5 g) were separately homogenized in 10ml of ice cold 0.1 M Tris-HCL buffer, pH 7.4, to produce 5% homogenate samples. The homogenates were centrifuged at 5000 rpm for 10 min. Supernatants were collected and stored; aliquots were subsequently taken for protein and enzyme assays.

Preparation of sera samples

Portions of blood drawn from the animals were allowed to clot at room temperature. The clotted blood samples were centrifuged at 2500 rpm for 15 min and clear serum samples were aspirated off and stored frozen.

Biochemical assay

Serum and liver alanine (ALT) and aspertate amino (AST) transferases were assayed by the method described by Reitman and Frankel (Reitman and Frankel, 1957).

Estimation of protein concentration

Protein concentration was estimated in the serum and liver homogenates using the Biuret method (Chawla, 1999) with bovine serum albumin as standard.

Hematology

Blood samples obtained at the end of the 30 day treatment period were analyzed using BAYER ADVIA 60, CT TUBE SYSTEM, for erythrocyte count, total leukocyte count, differential leukocyte count, haemoglobin concentration, haematocrit value and blood platelet count.

Histopathology

Tissue biopsies from the brains, kidneys, livers, myocardia, lungs and the testes were fixed overnight in 10% buffered formol saline. The tissue biopsies were processed with automated tissue processor (Shandon Citadel, 2000). Sections were cut at 4 microns with the rotary microtome and stained with haematoxylin and eosin. Additional thin sections of the kidney cut at 3 microns were stained with periodic acid (Schiff). Histological sections were examined using Leica light microscope.

[FIGURE 1 OMITTED]

Statistical analysis

Group data difference was evaluated by a modified t-test (n < 20) (Gardiner and Gettinby, 1998) using a 1-way analysis of variance at p = 5%. ANOVA and F-test computations were further employed to determine the significance of variations within and between groups.

Results and Discussion

There was no change in the character of the stool, urine and eye colour of all the animals in the test and control groups for both the acute and sub-chronic studies. However, evidence of treatment, route, and dose-dependent toxicity were detected.

Acute toxicity study in mice and rats revealed a dose-dependent sedative effect of Ocimum oil, an effect that wore out after 6 days of repeated administration in sub-chronic studies. These observations suggest that Ocimum oil could have sedative and central nervous system depressant activities. Similar activities have been reported for the essential oil of Lanvandula angustifolia Mill. (Guillemain et al. 1989).

The L[D.sub.50] and L[D.sub.100] values of Ocimum oil by the intra-peritoneal route were calculated to be 0.27 g/kg and 0.59 g/kg in mice and 0.43 and 0.74 g/kg in rats, respectively as shown in Fig. 1. Similarly the L[D.sub.50] and L[D.sub.100] values of Ocimum oil by the oral route were determined to be 1.41 and 2.50 g/kg in mice and 2.29 and 4.07 g/kg in rats, respectively as shown in Fig. 1. These values indicate that the oil has toxic potentials according to the classification of Lorke (Lorke, 1983); though studies classifying essential oils of comparable L[D.sub.50] as least toxic to rats have been made (Skramlik, 1959). It is likely that the toxic potential of the Ocimum oil is due to thymol, which is the major component with a p.o. L[D.sub.50] of 3.75-5.67 g/kg body wt. (Opdyke, 1974).

The Ocimum oil at the given doses produced marked weakness and fatigability with increasing dose and treatment period as well as dose-dependent gross changes in the behavioural activity of the animals. A dose related reduction in spontaneous explorative and stereotaxic activity, calmness, sedation and sleep duration was recorded. Earlier study on the anti-diarrhoeal activity of the oil revealed its ability to produce a reduction in GIT motility due probably to its stabilizing parasympathetic innervations of the GIT (Orafidiya et al. 2000).

A general dose related increase in mean organ weights relative to control was observed for virtually all the organs and groups in the sub-chronic study. However, statistical analysis of the variance revealed that only the enlargement observed in the liver and brain for the oil at 80 mg/kg, 133 mg/kg, 213 mg/kg intraperitoneally in groups II, III, IV, respectively, and 133 mg/kg orally in group I were significant. Enlargement by 103%, 121%, 121%, and 82% for the liver and 95%, 43% 25% and 60% for the brain, respectively, were recorded. For the liver, the enlargement might be an indication of hepatic injury as liver cell damage, which often varies in degree and localization depending on the mechanism involved, it may be reflected in hepatic tenderness and enlargement (Crawford, 1999).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

The histopathology results are consistent with the manifestation of chronic inflammation. The most important changes were seen in the brain. The changes in the brain suggest a global or generalized reduction in cerebral perfusion (hypotention/hypoperfusion) leading to hypoxic brain injury (Girolami et al. 1999). These changes are graded, with group I animals showing a mild change and group IV the most severe. Hypoperfusion may be responsible for the lower increases in brain weight with increasing dose earlier mentioned. The results showed infiltration of the brains. lungs and livers with mononuclear cells which includes lymphocytes; likely a reflection of persistent reaction to injury and tissue destruction (Cotran et al. 1999). The livers of groups III and IV show the presence of prominent Kuffer cells, lymphocytes within the sinusoids and few eosinophils within the portal areas which are expected morphological changes in chronic inflammation of the liver (Crawford, 1999). The presence of binucleate hepatocytes in the livers of groups II, III and IV is an evidence of liver cell unrest, probably as a reaction to a toxic agent in the oil. The changes in the lungs, kidneys, hearts and testes were of little functional significance.

The haematological results (Fig. 2) showed that white blood cell (WBC) count increased with treatment and dose. This is a common reaction when there is inflammation.

Inflammation often transit from acute to chronic when the acute inflammatory response cannot be resolved due either to interference in the normal resolution process, prolonged exposure to the potentially toxic agent or persistence of the injurious agent as is the case in this study (Aster and Kumar, 1999). The effect of the oil on mean haematocrit and haemoglobin levels (Fig. 2) is not significant indicating a possible "no effect" on the integrity of red blood cells. The mean differential WBC count in Fig. 3 does not show significant difference between groups II and III indicating that the inflammation becomes marked at a dose higher than 133 mg/kg. The marked decrease in granulocyte level in group IV (Fig. 3) could be due to accelerated removal resulting from increased peripheral use, destruction of neutrophil or the suppression of committed granulocytic precursors (Aster and Kumar, 1999). This could be due to the toxic effect of the oil and may explain the dose related increase in weakness and fatigability observed in the animals during the period of experimentation. This effect was mild in the orally treated group (group I). The blood platelet count for the control and the treatment groups in Fig. 4 revealed a dose and route dependent decrease in platelet count. The exact mechanism by which the oil produces this effect remains a subject for future studies. However, a probable mechanism is the suppression of the bone marrow, a condition that may also lead to the suppression of committed granulocytic precursors (Aster and Kumar, 1999).

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Assay to estimate the mean group protein concentration as well as liver and serum levels of glutamic oxaloacetate transaminase (AST) and glutamic pyruvate transaminase (ALT) revealed decreases (Fig. 5) in liver activity due likely to hepatic injury. The decrease in activity levels appears to correlate with the quantity of the oil reaching the liver as shown in Fig. 5. Decrease in albumin level, the serum protein most responsible for maintaining colloidal osmotic pressure, due to reduced synthesis in liver inflammation (Moshage et al. 1987) may underlie the oedematous appearance and increase in weight of the rat organs earlier observed. Usually when albumin level falls, the lowered protein level causes a decrease in the plasma osmotic pressure and water is forced into tissue spaces resulting in oedema (Chawla, 1999).

The overall effects of Ocimum oil were less severe in the orally treated groups compared with the intra-peritoneally treated groups at the same dose as revealed in the L[D.sub.50] and L[D.sub.100] values for both routes and species. Similar deduction can be made from the observation that 20% and 60% of the animals treated intra-peritoneally with 133 mg/kg (group III) and 213 mg/kg (group IV) of the Ocimum oil, respectively, died between day 20 and 30 with no death recorded in group I (133 mg/kg orally), group II (80 mg/kg IP) and the control that received no test substance during the 30 day sub-chronic test period.

Conclusion

Although not conclusive, the results of this study suggest that the essential oil of Ocimum gratissimum Linn has toxic potentialities.

References

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Chawla R (1999) Serum total proteins and albumin-globulin ratio. In: Practical Clinical Biochemistry (Methods and Interpretations) (eds Chawla R): 106-118, Jaypee Brothers Medical Publ India

Cotran RS, Kumar V, and Collins T (1999) (Edits). Acute and chronic inflammation. In: Robbins' Pathologic Basis Of Disease, pp 50-88. 6th Edition, WB Saunders Company, Philladelphia

Crawford JM (1999) The liver and Biliary Track. In: Robbins' Pathologic Basis Of Disease (eds Cotran RS, Kumar V, Collins T): 845-901, 6th Edition, WB Saunders Company, Philladelphia

Doull J (1996) Specificity and dosimetry of toxicological responses. Toxicology and Pharmacology 24: 555-557

Ekejuiba EC (1984) Establishment of some pharmacopoeia standards for selected Nigerian medicinal plants. M Phil thesis, UNIFE

El Said, F, Sofowora EA, Malcolm SA, Hofer A (1969): An investigation into the efficacy of Ocimum gratissimum as used in Nigerian native medicine. Planta Med 17: 195-200

Gardiner WP and Gettinby G (1998) (Edits) Experimental design techniques in statistical practise--A practical software base approach. pp 23-26, 1st edition. Chinchester: Horwood pubishing

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Guillemain J, Rousseau A, Delaveau P (1989) Neurodepressive effect of the essential oil of Lavandular angustifolia Mill Ann Pharm Fr 47: 337-343.

Hocking GMC (1985) A dictionary of terms in pharmacognosy and other division of economic botany, 152-153

Litchfield JT, Wilcoxin F (1949) A simplified method of evaluating dose-effect of experiments. J Pharmacol and Experimental Therapeutics 96: 99-118

Lorke D (1983): A new approach to practical acute toxicity testing. Archives Of Toxicology 54: 275-287 Springer Verlag

Martins AP, Salgueiro LR, Vila R, Tomi F, Canigueral S, Casanova G, Proenca da Cunha A, Adzet, T. (1999) Composition of the Essential Oils of Ocimum canum, O. gratissimum and O. minimum. Planta Medica 65: 187-189

Moshage HJ, Janssen JA, Franssen JH, Hafkenscheid JC, Yap SH (1987) Study of the molecular mechanism of decreased liver synthesis of albumin in inflammation. J Clin Invest 79: 1635-1641

Offia VN, Chukwendu UA (1999) Antidiarrhoeal effects of Ocimum gratissimum leaf extract in experimental animals. J Ethnopharmacol 68: 327-330

Opdyke DLJ (1974) Food Cosmet Toxicol 12: 1003-1004

Orafidiya OO, Elujoba AA, Iwalewa EO, Okeke IN (2000) Evaluation of anti-diarrhoeal properties of Ocimum gratissimum volatile oil and its activity against enteroaggregative Escherichia coli. Pharm Pharmacol Lett 10: 9-12

Orafidiya LO, Agbani EO, Oyedele AO, Babalola OO, Onayemi O (2002) Preliminary clinical tests on topical preparations of Ocimum gratisimum Linn leaf essential oil for the treatment of Acne vulgaris. Clin Drug Invest 22: 313-319

Reitman S, Frankel, S (1957) A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvie transaminases. Am. J. Clin. Pathol. 28: 56-62

Von Skramlik EV (1959),,Uber die Giftigkeit und Vertraglichkeit von atherischen Olen". Pharmazie 14: 435-445

L. O. Orafidiya (1), E. O. Agbani (1), E. O. Iwalewa (2), K. A. Adelusola (3), and O. O. Oyedapo (4)

(1) Department of Pharmaceutics, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria

(2) Department of Pharmacology, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria

(3) Department of Morbid Anatomy and Forensic Medicine, College Of Health Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria

(4) Department of Biochemistry, Faculty of Science, Obafemi Awolowo University, Ile-Ife, Nigeria

Address

Lara O. Orafidiya, Department of Pharmaceutics, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria

Tel: 00234-(0)8034551845; e-mail: laraoraf@yahoo.com
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Author:Orafidiya, L.O.; Agbani, E.O.; Iwalewa, E.O.; Adelusola, K.A.; Oyedapo, O.O.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:6NIGR
Date:Jan 1, 2004
Words:3386
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