**3. Results**

172 Toxicity and Drug Testing

The intraperitoneal (i.p) acute toxicity of the extract was evaluated in Swiss albino mice using a slightly modified Lorke's method (Lorke, 1998). Briefly, this method involved the determination of LD50 value in biphasic manner. The animals were starved of feed but allowed access to water 24 h prior to the study. In the initial investigatory step (phase 1), a range of doses of the extract producing the toxic effects was established. This was done by intraperitoneal administration of geometric doses of the extract (10, 100, 1000, 1500 mg/kg) to four groups of mice (n = 4). Based on the results obtained, a phase 2 investigatory step was done by giving more specific doses (200, 400, 600, 800 mg/kg i.p) to four other groups

The mice were observed for 24 h for such behavioral signs as, excitement, dullness, ataxia or death. The LD50 was estimated from the geometric mean of the dose that caused 100%

The same procedure was used in rats which received (1000, 2000, 3000, 5000 mg/kg oral) in

Twenty eight Wistar rats divided into four weight-matched groups, of seven rats each, (both sexes) were used for the study. Three test groups received 500, 1000, 2000 mg/kg *V. amygdalina* by gavage with biomedical needle (G 16, Length 76.2mm, diameter 3mm,

Food and water intake were measured daily while the animal's body weights were taken every other day. All animals were observed at least once daily for clinical signs (behavior such as lethargy, hyperactivity, depression and diarrhea). On day 14, immediately prior to euthanasia, all animals were anesthetized with chloroform and bled via the descending aorta for hematology and clinical chemistry determination. Organs were dissected and weighed to determine absolute and relative weight. The blood for clinical chemistry was allowed to clot in microtainer separator tubes, centrifuged and sera collected and stored at - 200C till ready for biochemical analyses. Commercial kits for Biosystem BTS-310, (Biosystem S.A Costa Brava 30, Barcelona, Spain) and Vitrous DT systems, Orthoclinical Diagnostics Johnson Company (US17) were used to analyze liver function, renal function and the

The hematological tests were carried out in an ethylene diamine tetra-acetic acid (EDTA) – anticoagulated blood. Hemoglobin (Hb) concentration was analysed by the cyanmethaemoglobin method, packed cell volume (PCV) by the micro-method, and white blood cell (WBC, total and differential) and platelet counts by visual methods Dacia et al, 1991). The mean cell hemoglobin concentration (MCHC) was calculated by dividing Hb by

For the six-week exposure studies, twenty eight male Wistar rats were divided into four weight-matched groups of seven rats each. Three of the four test groups received 750 1500, 3000 mg/kg *V. amygdalina* by gavage with biomedical needle (G 16, Length 76.2mm, diameter 3mm, Straight Harvard Apparatus) consecutively for 43 days. The control group

Straight Harvard Apparatus) for 14 days. The control group received normal saline.

**2.6 Acute and sub-chronic toxicity of the herbs** 

mortality and the dose which caused no lethality.

**2.6.2 Two weeks sub-chronic toxicity test** 

phase 1 and (1500, 3000, 4000, 5000 mg/kg oral) in the second phase.

**2.6.1 Acute – toxicity test** 

of mice.

electrolyte test.

PCV ( Dioka et al, 2002).

**2.6.3 Six weeks sub-chronic toxicity test** 

received distilled water vehicle only, via gavage.

In the study, a total of 90 healers from - sub-county from the three districts were interviewed. Most of them were members of traditional healers association in their district. Men dominate the practices of traditional medicine.

Most of the traditional healers interviewed (81 out of 90), indicated to have passed through a several routes to become healers. All of the 81 were raised in families with traditional healers, who had involved them in the healing process and they were familiar with the profession. After some time they started to practice on their own. All reported on the importance of the family environment of a traditional healer in the context of acquiring knowledge and experience by members of the family. Furthermore it was reported that, the entrance into practice through these routes is facilitated through training by an experienced healer or a family member, who decides when the apprentice is ready to become an independent healer or to take up the practice. Eight out of 81 of the traditional healers indicated to have been instructed through dreams by their ancestral spirits to take up the traditional healing practice. They were required to learn and observe traditional healing procedures as dictated by the spirits. Indeed, the ancestral spirits are considered to be supernaturally powerful and ignoring them is to invite punishment to an individual and or her family. Of the 90 healers interviewed, two indicated to be self-taught healers.

The leaves are the most frequently used plant part (56.3%), the root and fruits are used about 30% and 8.5% respectively, and the less used plant part is the bark (5.3%). The majority of the remedies are prepared in the form of decoction of fresh leaves. In our study area people do usually not store remedies for prolonged period of time. When needed they go out and collect the plant and prepare the remedy from fresh or sun dried material. Powders are prepared by pounding the fresh plant part or the crushed plant material after sun drying,

Decoction is the most frequent method way of remedy preparation (65%) followed by infusion (13%), which is used for the powders; the maceration (11%) is mostly used for the

Herbal Medicine in the Treatment of Malaria:

were however seen in high doses.

**3.2 Acetic acid-induced writhing in mice** 

**3.1 Acute toxicity test** 

(100mg/kg i.p; Fig 1).

Number of writhes

treated group and NS control

control (Fig 2).

**3.3 Formalin and tail-flick tests** 

*Vernonia amygdalina*: An Overview of Evidence and Pharmacology 175

The median lethal dose LD50 was established to be 560 1.21 mg/kg i.p. in mice and 3.32 0.15 g/kg oral in rats. Adverse signs of gaiting, reduction in stereotypic activities and deaths

The aqueous extract of *V. amygdalina* (50, 100, 200mg/kg i.p) exhibited a significant (P<0.05) antinocicetive activity against acetic acid-induced writhing in mice. 50 and 100 mg/kg oral doses exhibited a dose-dependent anti-nociception that progressively reduced over a period of 90 min post-treatment. However, at 120min the reduced anti-nociceptive activity increases again at these doses. The dose of 200 mg/kg on the other hand caused a total antinociception up to 120min. These results compared favorably with those of aspirin

30 60

Time (min)

The extract at doses of 100 and 200 mg/kg induced significant (p<0.05) reduction in pain response in both phases (aphasic and tonic) of pain induced by formalin in comparison with

In all cases ASA, a positive analgesic agent demonstrated significant anti-nociceptive action with a slightly stronger pharmacological intensity than *V. amygdalina* at 200 mg/kg in the late phase. The extract exerted no significant effect on nociception in tail-flick as values

Fig. 1. Effect of aqueous extract of *V.amygdalina* leaves on acetic acid-induced writhing in mice for 5 min (NS, normal saline; Va, *V. Amygdalina*; ASA acetysalicylic acid). All data are presented as means ± S.E.M., n=5. The asterisk (\*) denotes, significance (p<0.05) between

\*

\*

 NS 10 ml/kg Va 50 mg/kg Va 100 mg/kg Va 200 mg/kg ASA 100 mg/kg

\*

\*

\*

\*

\*

\*

root preparation. Some remedies are prepared from a single plant species; however, in a few cases mixtures of plants or other substances are added as noted in Table 2

Most of the remedies are taken orally and by external application as body bath, steam bath, and as ointment. Fumigation is mainly used in the treatment of headache and chest pain.

For most of the remedies, the administered dose depends on the patient's age, physical and health condition, and the duration of the illness. The doses vary from a teacup (70 ml) for adults to a handful (25 ml) for a child; a lack of agreement among the healers on doses of remedies was sometimes noted. The variation of the doses from one healer to another may show that the plants have a low degree of toxicity. For pharmacological investigation the active doses of these plants may not be high since they appear to treat the patients with low doses. The duration of treatment is not given for all remedies. According to the healers duration of treatment is difficult to determine and depends on how long the patient has been ill. The patient is supposed to take the remedy until healed. The only person able to determine the end of a treatment is the patient himself since the remedy is taken at home in the absence of the healers.

The reported adverse effects for the use of these medicinal plants are vomiting and dizziness. According to the healers these effects are generally due to an overdose of the remedy. The adverse effects are generally moderate, and disappear at the end of the treatment. Also, patients are advice to drink a lot of milk, meat soup or porridge made from sorghum to help alleviate serious side effects.


Table 1. An overview of the traditional healers interviewed.
