Acute and Subacute Toxicity Studies of Eurycoma longifolia in Male Rats

MATERIALS AND METHODS

The study was conducted from March, 2009 to May, 2010. Eurycoma longifolia extract was supplied by Phytes Biotek Sdn. Bhd. (Selangor, Malaysia) in the form of freeze dried standardized extract (Batch No: TA 071210). It is extracted from the root of the plant using a patented high pressure water extraction (US 7, 132, 117 B2), filtered at 1-4 micron and freeze dried without maltodextrin or lactose. The extract is in brownish powder form. It contains bioactive eurypeptide (>22%), glycosaponin (>40%) and eurycomanone (>1%). Male Sprague-Dawley rats (2-3 months of age) weighing between 250g-300g were obtained from the UKM Animal House. The rats were housed in a plastic cage at 29±3°C and daily dark/light cycle. They were fed standard food pellets and tap water ad libitum. They were allowed to adjust to the new environment for a week before the study was started. The study was approved by the UKM Animal Ethic Committee (PP/FAR/2008/NAZRUN/21-AUGUST/241-SEPT-2008-MAY, 2009).

Acute toxicity study was done according to the Organization for Economic Cooperation and Development (OECD) Acute Oral Toxicity-Fixed Dose Procedure 420 Guideline (OECD, 2001). Five rats were used for the acute toxicity study. The starting dose for the main study was set at 5000 mg kg^-1 body weight based on the sighting study. The crude aqueous extract powder was dissolved into normal saline to get 25% w/v EL crude extract solution. The dose was given within 24 h by oral gavage and the rats were observed for 14 days. Daily observation was done to check for mortality and abnormal clinical manifestations such as piloerection, salivation and lacrimation. The rats were weighed on day 1, 7 and 14. The rats were sacrificed after 14 days and the liver, kidneys and testes were taken out and fixed in neutral buffered formalin for 72 h. They were then processed and stained with hematoxylin and eosin stain for histopathology examination. The slides were examined single-blindly by a qualified pathologist.

Subacute toxicity study was conducted according to OECD Test Guideline 407 (OECD, 1995). Thirty two Sprague-Dawley rats were randomly divided into four groups. The first group served as the control group while the rest of the groups were given crude aqueous extract of EL at doses of 600, 1200 and 2400 mg kg^-1 body weight, respectively via oral gavages for 28 days. The crude aqueous extract powder was dissolved into normal saline to get 7.5, 15 and 30% w/v for the 600, 1200 and 2400 mg kg^-1 dose, respectively. The body weight, mortality and clinical observations were recorded daily. At the end of the treatment, rats were fastened overnight and blood was taken via the orbital sinus under anaesthesia. Part of the blood samples was centrifuged at 825x g for 10 min to separate the sera in order to avoid haemolysis. The serum samples were used for measurements of Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Alkaline Phosphatase (ALP), total bilirubin, total protein and serum creatinine using an autoanalyser (Siemens Advia Chemistry 2400, US). Blood was also collected in EDTA tubes for measurements of Red Blood Cell count (RBC), Haemoglobin concentration (Hb), Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin Concentration (MCHC), total White blood Cell Count (WCC), differential white blood cell percentage, platelet count, bleeding time and clotting time using haematology analyser (Siemens Advia 2120, US).

Blood were also collected into EDTA tubes by cardiac puncture for peripheral blood smear. Wright stain was used to stain all blood smear samples. Finally rats were sacrificed and the brain, liver, kidneys and testes were taken out and weighed. Liver, kidneys and testes were fixed in neutral buffered formalin for 72 h and stained with hematoxylin and eosin stain for histopathology examination. The slides were examined single-blindly by a qualified pathologist.

Statistical analysis: The results were expressed as Mean±SEM. The statistical significance of the data has been determined using one-way Analysis of Variance (ANOVA) and post hoc Tukey test. The level of significance was taken as p<0.05.

RESULTS

In the acute toxicity study, there were no significant differences in the body weight for all the rats (Fig. 1). Toxicity signs such as piloerection, salivation and lacrimation were not observed.

Fig. 1:	Weekly mean body weight change in acute toxicity study. Data were expressed as Mean±SEM. The level of significance was taken at p<0.05

The oral lethal dose 50 (LD₅₀) for crude aqueous extract of EL was more than 5000 mg kg^-1 and cannot be categorized under the GHS (Globally Harmonised System) category. Histopathological examination of the liver, testes and kidneys tissues did not show any pathological changes.

In the subacute toxicity study, there were no significant differences in the body weight for all the rats (Fig. 2). Toxicity signs such as piloerection, salivation and lacrimation were not observed. The biochemistry parameters of the liver and kidneys of treated rats were not significantly different compared to the control groups (Table 1). There were also no significant differences in the hematological parameters between the control and treated groups (Table 2). Peripheral blood smear showed normal morphology of red blood cells in all the rats (data not shown). Histopathological examination did not show any pathological change in testis and kidney tissues. However, there were pathological changes in the liver tissues of two rats in the treated groups. The livers of a at from 1200 mg kg^-1-treated group and another rat from 2400 mg kg^-1-treated group have shown hydropic changes (Fig. 3).

Fig. 2:	Weekly mean body weight change in subacute toxicity study. Data were expressed as Mean±SEM. The level of significance was taken at p<0.05

Table 1:	Biochemistry parameters
Data were expressed as Mean±SEM. The level of significance was taken at p<0.05

Table 2:	Haematological parameters
Data were expressed as Mean±SEM. The level of significance was taken at p<0.05

Fig. 3 (a-d):	Hepatocytes stained with H and E. (a) The hepatocytes of control group, (b) 600 mg kg-1 EL treated group show normal histology. Hydropic changes (HC) were seen in the (c) 1200 mg kg-1 and (d) 2400 mg kg-1. EL treated groups. (H and E; X100)

DISCUSSION

Male rats were used in this toxicity study as EL is taken mainly by men for its aphrodisiac effects. Based on our toxicity study on rat, the oral LD₅₀ for aqueous extract of EL was more than 5000 mg kg^-1. It is unlikely to cause fatality in human as the equivalent dose of EL is 35 in a 70 kg^-1 man. We may be the first to report on the effects of E. longifolia aqueous extract on liver, kidneys and testes of rat model. Histopathological examination of the subacute study revealed normal histology of the testis and kidney tissues but there were hydropic changes seen in the hepatocytes of two rats, one each from the 1200 and 2400 mg kg^-1 treated groups. These degenerative changes were an early manifestation of cell injury, characterized by vacuolization of cell with the nuclei in the centre due to accumulation of water in the cells. It is reversible but if the injuries persisted, necrosis will occur. Despite of the pathological changes in the livers, the ALT levels of the affected rats were not raised. This specific enzyme for liver injury usually peaks for 1 to 2 days only and then drops back to basal level (Gabriel and Michel, 2008), thus making it difficult to rely on its level. It is evident that EL aqueous extract had caused hepatocellular injury but based on the normal levels of ALP and total bilirubin combined with the absence of bile retention in the liver tissue, it did not seem to cause liver cholestasis. In a study on the liver enzymes, EL was not found to have any effects on the human cytochrome P450 system. The ethanolic extract of EL has shown negligible inhibitory activities towards three cytochrome P450 enzymes i.e., CYP2C9, CYP2D6 and CYP3A4 (Hanapi et al., 2010).

EL extract may contain compounds which are toxic to liver. The bioactive compounds of EL are quassinoids (Guo et al., 2005) consisting of eurycomalactone (Goh et al., 1995), eurycomanone (Goh et al., 1995), eurylactone (Grieco et al., 1995) and longilactone (Rahman et al., 1997). Other compounds are saponin, sterol, terpenoids and alkaloid (Goh et al., 1995). Subacute oral intake of EL aqueous extract was hepatotoxic to rats given 1200 and 2400 mg kg^-1 treated groups. This dose was equivalent to at least 8200 mg of EL aqueous extract taken by a 70 kg^-1 man. The highest dose of EL normally recommended is about 400 mg per day which is a relatively safe dose from its hepatotoxic effects.

In conclusion, acute oral intake of aqueous extract EL does not cause acute toxicity to rats. Subacute oral intake of aqueous extract EL may cause liver toxicity in rats. Further chronic toxicity study should be carried out and the compound in EL which has the potential to cause liver toxicity should be identified. With these findings, we recommend that EL should be used with caution especially in elderly or patients with liver problems.

ACKNOWLEDGMENT

We thank the Faculty of Medicine UKM for providing the grant FF-226-2008 for this study.