Acute and Subacute Toxic Study of Aqueous Leaf Extract of Combretum Molle

Purpose: The purpose of the present study was to evaluate the acute and subacute toxicity of the aqueous leaf extract of Combretum molle. Methods: The acute toxicity of the extract was evaluated in rats. The animals were orally administered with doses ranging from 2000 to 8000 mg/kg and observed continuously for the first 4 h, then hourly for the next 24 h, and finally, 6-hourly for 72 h. Control animals received orally normal saline. The rats were observed carefully for mortality, pain as well as respiratory movements. For subacute toxicity, 6 groups of 6 rats (3 male and 3 female) each received intraperitoneally, normal saline (control), 400, 600, 800, 1000 and 1200 mg/kg of the extract, respectively, thrice daily for 15 days. At the end of the treatment period, the animals were sacrificed and their organs (liver, heart and kidney) removed for macroscopic examination. Results : For the acute toxicit test, no death and signs of poisoning were observed in the treated groups. In the subacute tstudy, LD 50 in the rats after intraperitoneal administration was 700 mg/kg (456 - 896, 95 % confidence interval). The clinical signs of poisoning (motor difficulties, decreased respiratory rate, and tremor preceding death) were observed, suggesting overt toxicity throughout the neuromuscular system. However, histological examination of vital organs showed normal architecture suggesting no morphological abnormalities in the heart, kidney and liver. Conclusion: The results show that the aqueous leaf extract of C. molle is moderately toxic when given intraperitoneally.


INTRODUCTION
Combretum molle is a shrub or small, graceful, deciduous tree, 3 -13 m high, with a crooked or lean trunk, occasionally swollen at the base, and up to 30 cm in diameter. It is found especially in savannah vegetation that cuts across from Senegal to West Cameroon, but generally exists in tropical Africa [1]. Traditional healers throughout Africa use this specie of Combretaceae for many medicinal purposes. These include treatment of fever, headaches, abdominal disorders, abdominal pains, gallstones, diarrhoea, dysentery, gastric ulcers, bilharziasis, hookworm, nosebleeds, sore throats, colds, chest coughs, pneumonia, conjunctivitis, dysmenorrhoea, infertility in women, venereal diseases including syphilis, and earache. It has also been used for the treatment of leprosy, scorpion and snake bitesas well as toothache, heart diseases, backache, jaundice, stomach, gastric problems, constipation and general weakness. Other uses include treatment of swelling caused by mumps and cleansing of the urinary system. Species of Combretaceae have also been used as food supplement for babies [2][3][4][5].
Some studies have been performed on the biological activities of C. molle. Its leaf has anti-asthmatic and anti-tussive activities [6]. The plant induces considerable reduction in the variation of breathing amplitudes. Furthermore, the extract probably contains anticholinesterase substances as it exerted non-competitive inhibition of acetylcholinesterase with a Michaelis-Menten constant (KM) of 192 µM and velocity at maximal concentration of substrate (Vmax) of 4444 µM/min [7]. McGaw et al [8] found C. molle to have both anti-inflammatory and antischistosomal activities which may justify the traditional use of the plant for the treatment of malaria and pain.
Despite its long-time and varied uses, there are no data on the safety profile of the plant. This study was undertaken to determine the acute and subacute toxicity of the aqueous leaf extract of Combretum molle given orally and intraperitoneally to Sprague Dawley (SD) rats.

EXPERIMENTAL Preparation of aqueous extract
The plant material (C. molle leaves) was collected in December 2009 from Korhogo, in northern Ivory Coast, and authenticated by Professor Assi L Aké of the National Floristic Garden (University of Cocody-Abidjan). A voucher specimen (no. 6129) was deposited at the National Floristic Garden (University of Cocody-Abidjan). The dried leaves were powdered in a mortar and about 100 g of the powder extracted with 2 L of distilled water for 48 h on a hot plate. The mixture was sequentially filtered through a cheese cloth, cotton wool and Whatman filter paper no.1, respectively. The filtrate obtained was concentrated under reduced pressure, using a rotary evaporator, to the desired consistency.

Animals
Adult Sprague Dawley (SD) rats used for the study (36 males and females each), weighing 180 -200 g, were obtained from the animal house of the Physiology Department, University of Cocody-Abidjan, Ivory Coast.
The experimental procedures and protocols used in this study were approved by Ethical Committee of Health Sciences of the university [9,10]. The animals were housed in plastic cages (47×34×18 cm) in an airconditioned environment with 6 rats in each cage. The temperature of the environment was 25 ± 2 ºC with a 12 h light-dark cycle. Food and water were freely available to them.

Acute toxicity
The animals were divided into a control group and four treatment groups (2000, 4000, 6000 and 8000 mg/kg of extract), each group consisting of six animals. The repeat-dose oral toxicity study was carried out according to OECD guideline 407 [11]. The control group (Group 1) received orally, normal saline (0.9% NaCl)) while Groups 2 to 5 received 2000, 4000, 6000 and 8000 mg/kg body weight, respectively. Each treated group received the aqueous extract orally and clinical signs and symptoms were observed continuously for the first 4 h, then hourly for the next 24 h, and finally, 6-hourly for 72 h [12]. All the surviving animals were sacrificed after being anesthesized with sodium entobarbital.

Subcute toxicity test
The animals were divided into six groups of six animals each. The treatments were given by intraperitoneal injection. Group 1 served as control and received normal saline, while Groups 2 to 6 received 400, 600, 800, 1000 and 1200 mg/kg body weight, respectively, of the extract. Each group received the specified treatment dose thrice daily for 15 days. The animals were observed every 2 hours for toxic symptoms, signs of poisoning and mortality over a period of 30 days. The organs of the dead animals (liver, heart and kidney) were removed for macroscopic examination.

Determination of lethal dose (LD 50 )
The mortality rate of the animals for each dose was computed [13,14]. The arithmetical method of Karber was used for the determination of LD 50 [15]. The interval mean of the dead animals in each group of animals was used as well as the difference between doses for the same interval. The product of interval mean and dose difference was obtained. The sum of the product was divided by the number of animals in a group and the resulting quotient was subtracted from the least lethal dose in order to obtain LD 50 value (Eq 1).
where LD is the apparent lethal dose of all the groups, N is the number of animals in each group, a is the dose difference and b the mean mortality.

Histopathologic examination
For post-mortem, the rats were dissected and careful examination of the organs, liver, kidneys and heart were carried out. Tissue samples were fixed in 10 % formalin and dehydrated overnight using upgraded ethanol series and embedded in paraffin blocks. Ultrathin sections were de-waxed by xylene, hydrated through a degraded ethanol series, and stained with haematoxylin and eosin. A pathologist, blinded to the treatments, performed the histopathologic examination with an optical microscope [Nikon Eclipse E600, USA (x 400). Sections were assigned grades as reported by Billingham et al [16].

Statistical analysis
The LD 50 values were computed by probit analysis with a calculator. One-way analysis of variance (ANOVA) with Tukey post hoc test was applied to evaluate significant differences between groups with Instat Statistical Package (Graph Pad Software). Values of p < 0.05 were considered significant.

Acute toxicity
Oral administration of the aqueous extract of C. molle (2000 to 8000 mg/kg) did not produce significant changes in behavior, breathing, cutaneous effects (irritation, ulceration, caustic injuries and skin rashes), sensory nervous system responses, and gastrointestinal effects in the animals. No deaths occurred in any of the groups during the entire period of treatment. Also, no mortality was observed after oral ingestion of doses ranging from 4000 to 8000 mg/kg of extract weekly.

Sub-acute toxicity
In the animals that received the extract intraperitoneally, abdominal muscle contractions and ataxia were observed, and these persisted for a few hours. At the 6 th h, the animals became drowsy and less responsive to stimuli. Some of the animals died and mortality was as well as the severity of the toxic effects were dose-related (Table 1). However, at the 24 th h, most of the survivors had recovered from these symptoms. LD 50 of C. molle in rats was determined to be 700 mg/kg (range: 456 -895 mg/kg, 95% confidence interval) after intraperitoneal injection. The results are given in Tables 2 and 3.

Histopathological results
Histopathological data on the liver, heart and kidney of the rats are shown in Figs 1 -3     leading to increasing demand. Experimental screening method is, therefore, important in order to ascertain the safety of these herbal remedies [17]. A previous study showed the antiantiasthmatic effect of C. molle aqueous extract at 7.14 mg/kg body weight in rabbit [18]. In the acute toxicity study, there was no mortality observed even at a maximum oral dose of 8000 mg/kg of the aqueous extract. Also, no changes in the behavior and in the sensory nervous system responses were observed. No death or signs of poisoning were observed after oral administration of the extract; suggesting oral doses of C. molle are not toxic. However, histological analysis showed infiltration of inflammatory cells at the level of the portal vein in the liver. Inflammation was probably due to passive immune response to the extract . The maximum tolerated dose (MTD) obtained intraperitoneally was 400 mg/kg body weight. The clinical signs of toxicity observed after intraperitoneal administration, such as motor difficulties, decreased respiratory rate, and tremor preceding death, suggest overt toxicity throughout the neuromuscular system. The LD 50 (intraperitoneal injection) of C.molle extract in rats was 700 mg/kg; indicating that the plant is moderately toxic [19]. Macroscopic analysis of the target organs of the treated animals (liver, heart, and kidney) did not show significant changes in color and texture when compared with control [14].

CONCLUSION
Our findings did not show any damage to the kidney and heart of rats following oral administration of high doses of C. molle extract. Furthermore, no mortality was observed following oral administration. However, the extract seems to be moderately toxic after intraperitoneal administration. It also induced infiltration of inflammatory cells into the portal vein of the liver; a phenomenon that could compromise the medicinal use of this plant in folk medicine. However further studies are necessary, such as hematological assay and more in-depth morphological experiments, to confirm this evidence.