ANTIMALARIAL, IMMUNOMODULATORY AND CONTRACTILE ACTIVITIES OF ALSTONIA BOONEI (APOCYNACEAE)

Abstract

The major ethnomedical uses of Alstonia Boonei (AB) by indigenes of Tropical West African countries are in the treatment of malaria and rheumatoid arthritis. In the pathophysiology of rheumatoid arthritis, the complement and polymorphonuclear neutrophils (PMNs) have been implicated. The main purpose of this study was to isolate the antimalarial and immunomodulatory constituents of the plant using a bioassay guided fractionation technique. Samples of the stem bark of the plant (collection number. Lowe 2323; Herbarium number. U.I . H, 13134) were sun-dried, coarsely ground and subjected to soxhlet extraction successively with petroleum ether (PE), diethyl ether (DE), ethylacetate (EtOAc) and ethanol (EtOH). Subsequently, the mac was refluxed in water (AQ). Extracts were dried by evaporation under reduced pressure (PE, DE, EtOAc, and EtOH) or by freeze drying (AQ). The crude extracts and the pure anti-malarial compound isolated from the most active extract were assessed against Plasmodium yoeli nigeriensis (P. y. nigeriensis) and P. berghei ANKA infections in mice and rats, respectively. Activities of the extracts or isolated compound were investigated in early infection (4-day test) and established infection (Rane's test). Their repository activities were also investigated. Chloroquine and pyrimethamine served as reference anti-malarial drugs. The effects of the crude extracts were also investigated on the immune system in vitro using complement system and polymorphonuclear (PMN) neutrophils as immunological parameters. Anticomplementary (classical (CP) and alternative (AP) pathway) activities were assessed using human serum and antibody-sensitized sheep erythrocytes for CP and rabbit erythrocytes for AP. Effects of extracts on phagocytic activities of PMNs were assessed by using PMNs isolated from venous blood of healthy volunteers. Apocynin was used as the reference compound. In P.y. nigeriensis infection, the order of anti-malarial activity of the extracts was DE<Et0Ac < AQ < EtOH. The doses of the extract screened ranged from 100 to 800 mg/ kg and the mean suppression of parasitaemia in early infections and repository test ranged from 1.23 to 61.4% and 3.3 to 56.7%, respectively. The chemo suppressive effect of Chloroquine (5mg/kg) and pyrimethamine (1.5mg/kg) were 85.9% and 87.7% respectively. None of the extracts showed strong activity in established infection. However, mice treated with the highest dose of EtOH extract had prolonged survival over the controls. Activity guided fractionation of EtOH extract using different chromatogractic techniques yielded a pure alkaloidal constituent called AB-1, melting

point 208 - 210°C. Doses of 10 - 80 mg/kg of AB-1 produced chemosuppressive effect of 41.3 to 81.9% in early infection and 52.0 to 86.5% in repository test. In established infection, Chloroquine (5 mg/kg) and AB-1 (80 mg/kg) produced chemosuppressive effects of 98.0 and 88.5%, respectively on day 8 post infection. The mean survival periods in the control, AB-1-treated and Chloroquine-treated mice were 7.4 ± 0.77, 25.7± 1.23 and over 30 days, respectively. All doses of AB-1 produced higher the effects in P. yoehi inigeriensis infection than in P. berghei ANKA infection. The order of anticomplementary activity of the extracts was DE = EtOAc > Et0H > AQ. The anticomplementary activity of DE and EtOAc extracts was mediated through the classical pathways. None of the extracts inhibited AP-mediated hemolysis and chemiluminescence, generated by stimulated PMNs. A triterponoid, called AB-2, isolated from Et0Ac extract, showed very strong anticomplementary activity. The concentration producing 50% inhibition of CP-mediated hetemolysis was 1.4 mg/ml. The anticornplementary activity increased with increase in temperature and time of preincubation. AB-.2 significantly inhibited zymosan-induced mouse footpad swelling. AG extracts contracted both guinea pig ileum and rat stomach strip. The contraction was antagonised by methysergide in a competitive manner. The efficacy of the anti-malarial and anti-inflammatory constituents isolated from Alstonia boonei lend strong support for the ethnomedical use of the plant extracts. This is the first report on the anti-complementary activity of Alstonia boonei extracts.