Acute and sub-acute toxicity studies of methanol leaf extracts of Annona squamosa linn. in mice

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1 Sky Journal of Biochemistry Research Vol. 3(7), pp , October, 2014 Available online ISSN Sky Journals Full Length Research Paper Acute and sub-acute toxicity studies of methanol leaf extracts of Annona squamosa linn. in mice Onwusonye J. C. 1 *, UWAKWE A. A. 2 and Patrick Iwuanyanwu K.C. 2 1 Department of Biochemistry Federal Polytechnic Nekede, Imo State, Nigeria. 2 Department of Biochemistry, University of Port Harcourt, Rivers State, Nigeria. Accepted 23 September, 2014 This study was conducted to determine the acute and sub-acute toxicity profiles of Annona squamosa, a herb widely used in the south-east area of Nigeria for the treatment of among other conditions, fever associated with malaria. In the acute toxicity study, methanol extract of A. squamosa was given to mice by oral gavages, up to a dose of 5,000mg/kg body weight as a single dose within 24 h. The animals were however monitored for up to 30 days period. In the sub-acute toxicity study, six groups of mice were used. The first group served as control while the other five groups were given A. squamosa extract at doses of 200, 400, 600, 800, and 1000 mg/kg body weight respectively via daily oral gavages for 30 days. Histological examinations were done on the liver of the animals for both acute and sub-acute studies. Biochemical and haematological analyses were done for subacute toxicity study. The oral LD 50 for the methanol extract of A. Squamosa leaves was observed to be more than 5000mg/kg body weight. No outstanding pathological changes were found in the livers of the treated animals compared with the control. For the sub-acute toxicity study, at the highest dose of 1000mg/kg, one animal showed signs of reduced food intake. However, no significant change was observed in the biochemical and haematological parameters. This study shows that the extract of A. squamosa is well tolerated in mice within the dose limits studied. Keywords: Acute, sub-acute, toxicity, Annona squamosa. INTRODUCTION Annona squamosa commonly known as custard apple or sweet sop is a semi-evergreen shrub or small tree reaching 6 8 m (20 26ft) tall. The plant is a native of tropical America and the West-Indies, but its original home is uncertain. It is said to have been introduced to Brazil in 1626, planted and naturalized in Southern Florida, including Florida Keys and throughout the tropics in Asia and South Pacific. It has run wild, particularly near old inhabited sites, in several parts of the central and Western India and in the Decan Peninsula. A. Squamosa has a wide array of ethno botanical uses. Fruits are normally eaten fresh. The roots are cathartic. Green fruits, seeds and leaves have effective vermicide and insecticidal properties. The plant is also traditionally used for the treatment of epilepsy, dysentery, cardiac problems, worm infestation, constipation, hemorrhage, *Corresponding author. jconwusonye@gmail.com. Tel.: bacterial infection, fever and ulcer (Kirtika et al., 1957). A. squamosa has been reported to exhibit antibacterial activity against Bacillus subtilis and Staphylococcus aureus. A number of antimalarial compounds have been isolated from A. squamosa which is traditionally used in diseases including infections associated with malarial parasites (Johns et al., 2011). Acute toxicity (Lethal toxicity) is the ability of a chemical to cause ill effect relatively soon after one oral administration or a 4 h exposure of a chemical in air (Senin, 2006). According to Senin (2006), relatively soon is usually defined as a period of minutes, hours (24) or days (up to about 2weeks), but rarely longer. LD 50 is an abbreviation for Lethal Dose 50%. It is sometimes also referred to as the Median Lethal Dose. The LD 50 for a particular substance is essentially the amount that can be expected to cause death in half (i.e. 50%) of a group of some particular animal species, usually rats or mice, when entering the animals body by a particular route.

2 54 Sky J. Biochem. Res. Figure 1. Leaves and fruit of A. squamosa Linn. Table 1. Record of mortality in phase 1. Extract Dose(mg/kg body weight) No. of mice No. of deaths From available literature, little or no work has been done on the toxicity profiles of alcohol extracts of A. Squamosa leaves. The objective of the present study is to evaluate the acute and sub-acute toxicity profiles of methanol extract of A. Squamosa leaves in mice. This is aimed at generating informed data on the level of safety of use of the herb in the management of the above mentioned health conditions in man. MATERIALS AND METHODS Sample collection Fresh leaves of A. squamosa (Figure 1) were collected from Nekede in Owerri West Area of Imo State, Nigeria. Identification was done by a taxonomist in the department of Biotechnology, Federal University of Technology Owerri. Sample preparation and extraction The fresh leaves of A. squamosa were air-dried for one week and later milled to powder using a mechanical blender. 500g of the ground sample was soaked in 1500ml of 95% methanol and allowed to stand for 72 h. This was subsequently filtered using filter paper. The resultant filtrate was later concentrated by rotary evaporation at a temperature of C. Animal models A total of 42 male albino mice were used in the study. 24 mice were used for the acute toxicity study while the remaining animals were used for the sub-acute toxicity study. The animals were sourced from the animal holdings of Biochemistry Department, University of Port Harcourt. All animals were acclimatized to laboratory condition for one week prior to commencement of study. The animals were grouped and housed in plastic cages. They were fed with standard pelleted commercial feed and allowed access to water ad-libitum. In all experiments, the United States National Institute of Health Principles of Laboratory Animals Care (NIH, 1978) was adhered to. Acute toxicity study (LD 50 ) The acute toxicity study was done according to the method described by Lorke (1983). The study was conducted in two phases using a total of 24 mice. In the first phase, three groups of mice (4 per group) weighing 24-28g were administered with the extract at respective oral doses of 10 mg, 100 mg and 1000 mg per kg body weight (Table 1). The mice were observed for signs of toxicity and possible mortality for 24 h, 72 h, 2weeks and 4weeks. In the second phase, another three groups of 4 mice each were administered respective doses of 1600, 2900 and 5000mg per kg body weight of the extract

3 Onwusonye et al. 55 Table 2. Record of mortality in phase 2. Extract Dose(mg/kg body weight) No. of mice No. of deaths Oral LD 50 of the extract was observed to be >5000mg/kg body weight. (Table 2). The animals were equally monitored as in phase one for toxicity signs and possible mortality. From data obtained, LD 50 was estimated. Sub-acute toxicity study The mice were divided into six groups of 3 mice per group. Group 1 was treated with vehicle (Normal saline) and kept as control. Groups II to VI were respectively treated with 200, 400, 600, 800 and 1000mg/kg body weight of the extract for 30 days. Treatments were administered once daily for 30 days. At the end of treatment, overnight fasted animals were sacrificed and blood and liver samples were collected on the 31 st day. Biochemical, haematological and histological parameters were measured in all animals both treated and control groups. Physical parameters Body weight as well as mortality was recorded during treatment in all the animal groups. Biochemical parameters Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total protein were estimated in blood samples. AST and ALT were determined according to the method of Reitman and Frankel (1957) while total protein was estimated by the Biuret method (Tietz, 1983). Haematological parameters The following haematological parameters were estimated: Haemoglobin (Hb), Packed Cell Volume (PCV), Total white blood cell count and platelets. Hb was estimated by the cyano-methaemoglobin method (Jain, 1986). PCV, Total WBC and platelets were estimated according to the bulk dilution method (Dacie and Lewis, 1991). Histological examinations Liver samples were carefully excised from the sacrificed animals and fixed in 4% formaldehyde. They were subsequently processed and embedded in paraffin wax. Tissue blocks were sectioned 5µm thick and stained with Haematoxylin and Eosin (H & E) for detailed observation. Statistical analysis Data collected were subjected to analysis of variance (Steel and Torries, 1980) implemented in SPSS statistics 17.0 (SPSS Inc. 2008). The means were separated using Duncan Multiple Range Test at the 0.05 level of significance. RESULTS Acute toxicity study This is a study that describes the adverse effects of a substance that results either from a single exposure or from multiple exposures in a short space of time (usually less than 24 h). To be described as acute toxicity, the adverse effects should occur within 14 days of administration of the substance in question. Sub-acute toxicity study This is the study of results produced in test animals by long term exposure to repeated doses or concentrations of a substance. The period of monitoring or manifestation of toxicity effects usually takes longer than 14 days. It may run into a few weeks, months or even a bit longer. Sub-acute toxicity studies are usually done in attempt to design possible dosing protocols for drugs and new substances. Discussion Our previous studies have revealed the presence of various phytochemicals like alkaloids, glycosides, saponins, flavonoids, tannins etc., occurring in various amounts in A. squamosa. These compounds elicit a wide array of pharmacological actions in living systems. The oral LD 50 for methanol extract of A. squamosa was observed to be more than 5000mg/kg (Tables 1 and 2). The herb is unlikely to cause fatality in humans because any substance with LD 50 above this observed level is considered to be practically harmless (Hodge and

4 56 Sky J. Biochem. Res. Figure 2. Photomicrograph of liver section of animals treated with 1500 mg/kg body weight of extract (H & E 400). Figure 3. Photomicrograph of liver section of animals treated with 2900mg/kg body weight of extract (H&E 400). Figure 4. Photomicrograph of liver section of animals treated with 5000mg/kg body weight of extract (H&E 400).

5 Onwusonye et al. 57 Figure 5. Photomicrograph of liver section of control animals (H&E X400). Figure 6. Photomicrograph of liver section of animals treated with 200 mg/kg body weight of extract (H&Ex400). Figure 7. Photomicrograph of liver section of animals treated with 400 mg/kg body weight of extract (H & Ex400).

6 58 Sky J. Biochem. Res. Figure 8. Photomicrograph of liver section of animals treated with 600 mg/kg body weight of extract (H & E x 400). Figure 9. Photomicrograph of liver section of animals treated with 800 mg/kg body weight of extract (H&Ex400). Figure 10. Photomicrograph of liver section of animals treated with 1000 mg/kg body weight of extract (H & E x 400).

7 Onwusonye et al. 59 Table 3.Biochemical parameters in control and extract treated mice. Parameter Group I Group II Group III Group IV Group V Group VI AST (i.u/l) ± ± ± ± ± ± 0.36 ALT (i u/l) ± ± ± ± ± ± 0.96 Total protein (g/dl) ± ± ± ± ± ± 0.75 Values are Mean ± SEM of 3 determinations. Table 4.Haematological parameters in control and extract-treated mice. Parameter Group I Group II Group III Group IV Group V Group VI Hb (g/l) ± ± ± ± ± ± 1.72 PCV(%) ± ± ± ± ± ± 1.96 Total WBC x10 9 /L 5.30 ± ± ± ± ± ± 0.09 Values are Mean ± SEM of 3 determinations. Sterner, 2005).When the highest dose of 1000 mg/kg was administered, an animal presented symptoms of reduced food intake. However, histological examination of the animals both in the acute and sub-acute studies revealed no outstanding damage to the liver (Figures 2, 3 4, 5, 6, 7, 8, 9 and 10). Also in support of the safety profile of the plant is the observation that no significant differences occurred in biochemical and hematological parameters of the treated and untreated animals (Tables 3 and 4). Senin R (2006). Acute Toxicity Study. Retrieved from ( on 27/3/2010. SPSS Inc Statistical Packages of Social Science (SPSS) Statistical software version 17.0 for windows. IBM SPSS Inc. Chicago, USA. Steel RDG, Torrie JH Principles of Stastitics. MacGraw Hill, NewYork. 2 nd ed Tietz NW (1983). Textbook of Clinical Chemistry, W.B. Saunders company, Philadelphia, PA PP Conclusion From the observations made in this study, it could be concluded that the administration of the methanol extract of A. squamosa is well tolerated in mice within the limits of the doses studied. By extrapolation, this implies the safety of the plant in humans. The continued use of the herb by man is therefore not discouraged. REFERENCES Dacie JV, Lewis SM (1991).Practical Haematology, 7 th edn.elbs with Churchill Livingstone, England. pp Hodge A, Sterner B (2005). Toxicity classes in: Canadian Centre for occupational health and Safety Retrieved from ( on 3/5/2010). Jain NC (1981). Schalm s Veterinary Haematology, 4 th edn. Lea and Fabriger, Philadelphia, pp Johns T, Windust A, Jurgens T, Mansor SM(2011). Antimalarial alkaloids isolated from A. squamosa. Phytopharmacol., 1(3): Kirtikar KR, Basu BD (1957).Indian Medicinal Plants, 2nd edition, vol.1, Dehradun; pp Lorke D (1983). A New Approach to Tropical Acute Toxicity Testing. Arch. Toxicol, 53: Reitman S, Frankel AS (1957). A colometric method of determination of serum glutamic pyruvic transaminase. Am. J. Clin.Pathol., 28: 56.