**2.1 Research design**

Ninety traditional healers were identified through community and healers association leaders. Once identified, study staff members approached the individuals to determine eligibility. Eligibility criteria included 30 years of age and older, recognition as a traditional healer by the local community council, and having established an active practice in the community. Three districts (Kanungu/Bwindi area, Bushenyi and Mbarara) south-western Uganda were identified. With consent a taxonomist samples of antimalarial herb were obtained for this research study.

 To evaluate consistency, interviews were conducted by a person specially trained in interview administration and who is fluent in the language of the participants.

The ethnographic interview included questions about common plant names, sources of products, method of preparation, purpose of use, quantity of herbs use and perceived benefit of herbs in ameliorating malaria symptoms and improving overall health. Ethical forms were used in order to assure them of the defense of their knowledge and intellectual property right was applied. Traditional healer's name, age, gender, and ethnicity tribe, were asked. We relied on the knowledge of healers and the taxonomist to select the products of greatest importance. This enabled us target products that have a high likelihood of possessing significant pharmacological activity.

A strategy was developed that respected the healers' rights to maintain propriety of unique blends of herbal medicine. Also, a memorandum of understanding was developed that disclosed our study objective, which is to characterize the pharmacologic activity and to elucidate the toxicity of these remedies in order to determine any potential adverse effect. We emphasized that we were interested in general knowledge about the remedy and not in specific formulation, and that it was not our intention to use the knowledge gained from this

Herbal Medicine in the Treatment of Malaria:

fields, and mean survival time (within 30 days) was recorded.

every 5 min for the period between 15 and 60min (late phase).

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

mean) 100.

**2.5.2 Formalin test** 

**2.5.3 Tail-flick test** 

(Sanchez-Mateo et al, 2006).

**2.5 Evaluate the antinociceptive activity of the selected antimalarial** 

*Vernonia amygdalina*: An Overview of Evidence and Pharmacology 171

was anaesthetized with chloroform and the abdomen opened. Blood was collected through cardiac puncture with a sterile needle and syringes in such a way that 0.2 ml of the blood containing about 1 x 107 infected red cells. Twenty-five mice were inoculated i.p. with 0.2 mL each. The mice were then randomized and grouped into five (n = 5) and treated as follows on day 3 (D3). Group 1 received normal saline, group 2 received chloroquine (CQ, 5mg/kg, i.p.); while groups 3-5 received (50, 100, and 200 mg/kg, i.p.). The treatment continued daily until day 7. Thick and thin blood smear were collected daily from tail blood, fixed with methanol, stained with 4% Giemsa at pH 7.2 for 45 min and examined microscopically (Nikon YS2-H Japan). The increase/decrease in parasitaemia, defined as the number of infected and uninfected red blood cell RBCs, were counted on five different

Analgesia was assessed according to the method of Siegmund et al. (1957), as was modified by Koster et al, 1959). The mice were divided into different groups (of five mice each). They were differently pre-treated with the extract (50, 100, 200mg/kg i.p), aspirin (100mg/kg i.p) and normal saline (10ml/kg i.p). 30, 60, 90 and 120min after the treatment, 0.7% acetylsalicylic acid (ASA) (Sigma Chemicals Co) 10ml/kg i.p was administered to the mice. They were placed in a transparent cage, 5mins after administration of acetic acid, the number of abdominal constrictions (writhes) made within 10min of every mouse was counted. The results of the treatment groups were compared with those of normal saline pre-treated control. The percentage of the writhes was calculated as (test mean/control

For the formalin studies, rats were injected with 0.05 ml of formalin (2.5% formaldehyde) into the sub-plantar surface of the left hind paw 30min after treatment with saline, extract or ASA. Severity of pain (for both control and test groups (n = 5)) were simultaneously observed and rated as scores using (Dubuisson et al, 1977) pain measurements. This was rated as follows: (0) rat can bear weight on injected paw; (1) light resting of the paw on floor; (2) partial elevation of the injected paw, and (3) total elevation, licking and biting of paw. These observation were recorded every minute for the first 10 min (early phase) and at

This test as first described [28], and subsequently modified (Janssen et al, 1969; Asongalem et al 2004a) was used. Briefly, before treatment, the terminal (2 cm) of each rat tail was immersed in hot water contained in a 500ml beaker and maintained at 55 ± 1oC using a thermo-regulated hot plate (Ugo Basile, Socrel DS-35) and the time (in seconds) between the onset of stimulation and tail withdrawal was measured as the tail-flick latency. Twenty five rats that shows response within 0 – 4 s were selected and grouped into five (n = 5) for the study. Immediately after basal latency assessment, normal saline, reference drug (Pethidine hydrochloride; Bayer, England) or the plant extract were administered and the reaction time again recorded at 30 and 60min. after administration the extract. (50 – 200 mg/kg p.o)

study for commercial profit. Rather we would report back to them in a workshop the remedy's indication and contraindication after completion of the study. We requested that all parties sign the agreement and copies were kept in a secure file. The result of the study was used to determine the most common botanical/herbal products used by the healers to treat malaria. Among the herbal products, *Vernonia amygdalina* which appeared in 80% in the interview was chosen for studies.

**Fidelity level:** The fidelity level (Fl) (Alexiades et al, 2000) among the healers from the same district was calculated according to the following formula:

$$\text{Fl (\%)} = \text{(Np/N)} \times 100$$

Np is the number of healers from one given district that claim a use of a plant species to treat a particular disease, and N is the number of healers from the same district that use the plants as a medicine to treat any given disease. The formula was applied in order to compare data from different district where the survey was performed.

#### **2.2 Extract preparation**

Leaves of *V. amygdalina* were collected from the botanical garden of the Rukararwe Traditional Medicine Health Center, a division of Rukararwe Partnership Workshop for Rural Development (RPWRD) in Bushenyi district. The plant was authenticated by Dominic Byarugaba, a botanist with the Department of Plant Biology, Mbarara University of Science and Technology (MUST), Uganda. A voucher specimen is kept in the department. The plant material was air-dried and grounded into a coarse powder. About (350g) of this powder were macerated in 2 L of distilled water for 24 h with occasional shaking (GFL 3017 Germany) and then extracted using a soxhlet extractor (Gallemkamp, England). The resultant extract was evaporated in a water bath, under controlled temperature not exceeding the one used by the healers in their plant preparation (80oC) to yield a 32.3 g of semi solid residue.

#### **2.3 Animals**

Adult Wistar rats (130 – 150g) and Swiss albino mice (18 – 26g) of either sex, maintained at Animal Facility Centre were used for the acute toxicity and 14 days sub-chronic experiments. The animals were kept in plastic cages at room temperature and moisture, under a naturally illuminated environment of 12:12 h dark/light cycle. Animals were fed the standard diet and had access to tap water *ad libitum*.

Male Wistar rats were used for the 6 week exposure studies.. At dosing, animals were 8 to 12 weeks old. All animals were clinically monitored at the time of delivery and during acclimation period, and were maintained at the Animal Facility Centre. Animals found unsuitable were excluded from the experiment. Animals were housed in plastic cages under same conditions described above; they were fed with standard diet (Mice pallet), and had access to tap water *ad libitum.* The animal experiments were conducted according to the NIH Guide on Laboratory Animals for Biomedical Research (NIH, 1978) and ethical guidelines for investigation of experimental pain in conscious animals (Zimmermann et al, 1983).

#### **2.4 Antiplasmodial activity**

The test was conducted according to the curative procedure described earlier (Saidu et al, 2000; Adzu et al, 2003). A donor mouse infected with rodent malaria (*Plasmodium berghei*) was anaesthetized with chloroform and the abdomen opened. Blood was collected through cardiac puncture with a sterile needle and syringes in such a way that 0.2 ml of the blood containing about 1 x 107 infected red cells. Twenty-five mice were inoculated i.p. with 0.2 mL each. The mice were then randomized and grouped into five (n = 5) and treated as follows on day 3 (D3). Group 1 received normal saline, group 2 received chloroquine (CQ, 5mg/kg, i.p.); while groups 3-5 received (50, 100, and 200 mg/kg, i.p.). The treatment continued daily until day 7. Thick and thin blood smear were collected daily from tail blood, fixed with methanol, stained with 4% Giemsa at pH 7.2 for 45 min and examined microscopically (Nikon YS2-H Japan). The increase/decrease in parasitaemia, defined as the number of infected and uninfected red blood cell RBCs, were counted on five different fields, and mean survival time (within 30 days) was recorded.

#### **2.5 Evaluate the antinociceptive activity of the selected antimalarial 2.5.1 Acetic acid-induced writhing in mice**

Analgesia was assessed according to the method of Siegmund et al. (1957), as was modified by Koster et al, 1959). The mice were divided into different groups (of five mice each). They were differently pre-treated with the extract (50, 100, 200mg/kg i.p), aspirin (100mg/kg i.p) and normal saline (10ml/kg i.p). 30, 60, 90 and 120min after the treatment, 0.7% acetylsalicylic acid (ASA) (Sigma Chemicals Co) 10ml/kg i.p was administered to the mice. They were placed in a transparent cage, 5mins after administration of acetic acid, the number of abdominal constrictions (writhes) made within 10min of every mouse was counted. The results of the treatment groups were compared with those of normal saline pre-treated control. The percentage of the writhes was calculated as (test mean/control mean) 100.

#### **2.5.2 Formalin test**

170 Toxicity and Drug Testing

study for commercial profit. Rather we would report back to them in a workshop the remedy's indication and contraindication after completion of the study. We requested that all parties sign the agreement and copies were kept in a secure file. The result of the study was used to determine the most common botanical/herbal products used by the healers to treat malaria. Among the herbal products, *Vernonia amygdalina* which appeared in 80% in

**Fidelity level:** The fidelity level (Fl) (Alexiades et al, 2000) among the healers from the same

Fl (%) = (Np/N) × 100 Np is the number of healers from one given district that claim a use of a plant species to treat a particular disease, and N is the number of healers from the same district that use the plants as a medicine to treat any given disease. The formula was applied in order to

Leaves of *V. amygdalina* were collected from the botanical garden of the Rukararwe Traditional Medicine Health Center, a division of Rukararwe Partnership Workshop for Rural Development (RPWRD) in Bushenyi district. The plant was authenticated by Dominic Byarugaba, a botanist with the Department of Plant Biology, Mbarara University of Science and Technology (MUST), Uganda. A voucher specimen is kept in the department. The plant material was air-dried and grounded into a coarse powder. About (350g) of this powder were macerated in 2 L of distilled water for 24 h with occasional shaking (GFL 3017 Germany) and then extracted using a soxhlet extractor (Gallemkamp, England). The resultant extract was evaporated in a water bath, under controlled temperature not exceeding the one used by the healers in their plant preparation (80oC) to yield a 32.3 g of

Adult Wistar rats (130 – 150g) and Swiss albino mice (18 – 26g) of either sex, maintained at Animal Facility Centre were used for the acute toxicity and 14 days sub-chronic experiments. The animals were kept in plastic cages at room temperature and moisture, under a naturally illuminated environment of 12:12 h dark/light cycle. Animals were fed

Male Wistar rats were used for the 6 week exposure studies.. At dosing, animals were 8 to 12 weeks old. All animals were clinically monitored at the time of delivery and during acclimation period, and were maintained at the Animal Facility Centre. Animals found unsuitable were excluded from the experiment. Animals were housed in plastic cages under same conditions described above; they were fed with standard diet (Mice pallet), and had access to tap water *ad libitum.* The animal experiments were conducted according to the NIH Guide on Laboratory Animals for Biomedical Research (NIH, 1978) and ethical guidelines for investigation of experimental pain in conscious animals (Zimmermann et al, 1983).

The test was conducted according to the curative procedure described earlier (Saidu et al, 2000; Adzu et al, 2003). A donor mouse infected with rodent malaria (*Plasmodium berghei*)

the interview was chosen for studies.

**2.2 Extract preparation** 

semi solid residue.

**2.4 Antiplasmodial activity** 

**2.3 Animals** 

district was calculated according to the following formula:

the standard diet and had access to tap water *ad libitum*.

compare data from different district where the survey was performed.

For the formalin studies, rats were injected with 0.05 ml of formalin (2.5% formaldehyde) into the sub-plantar surface of the left hind paw 30min after treatment with saline, extract or ASA. Severity of pain (for both control and test groups (n = 5)) were simultaneously observed and rated as scores using (Dubuisson et al, 1977) pain measurements. This was rated as follows: (0) rat can bear weight on injected paw; (1) light resting of the paw on floor; (2) partial elevation of the injected paw, and (3) total elevation, licking and biting of paw. These observation were recorded every minute for the first 10 min (early phase) and at every 5 min for the period between 15 and 60min (late phase).
