Laboratory and semi-field evaluation of some Vitex species for larvicidal activity against Anopheles gambiae larvae

Abstract

Background: Over two billion people in tropical countries are at risk from mosquitoborne diseases such as dengue fever, hemorrhagic fever, malaria and filariasis. It is estimated that US$ 2 billion is spent annually on malaria control and treatment programmes in sub-Saharan Africa alone, where 90% of all malaria-related deaths occur. The problem has become increasingly difficult to manage because of the emergence of drug-resistant parasites to the currently available anti-malarial drugs. Personal protection from mosquito bites with synthetic insecticides is currently the dominant measure to control the bites from mosquitoes. However, vector resistance to insecticides is a recurring problem and a threat to malaria control programmes. To address these problems, attention to insecticides of natural origin, particularly botanical products, has been the subject of current research. Objectives: The study set out to investigate the larvicidal and insect growth regulatory (IGR) potential of Vitex schiliebenii, V. payos, and V. trifolia against Anopheles gambiae larvae under laboratory and simulated semi-field conditions. Phytochemical tests of the extracts were carried out to compare their constituents and the larvicidal results. Toxicity test for the active extracts was evaluated on Brine shrimp larvae and compounds isolated from the extracts were also evaluated for their activity. Materials and methods: The present study was designed to determine the mosquito larvicidal/Insect Growth Regulatory (IGR)/adult inhibition activities of acetone, methanol and aqueous extracts from three Vitex species belonging to the family Verbenaceae viz, V. payos, V. schiliebenii and V. trifolia. Plant materials were collected from the coastal region of Kenya and tested on 3rd and early 4th instar larvae of a malaria vector Anopheles gambiae s.s. in a dose-dependent manner. Different parts of the plants (root bark, stem bark, leaves and seeds) were air-dried, ground, extracted and concentrated to dryness using a rotary evaporator at 40oC and the combined extract stored at 4oC. This procedure was repeated with methanol in the same proportion and for the same periods while the aqueous extracts were obtained using soxhlet extraction. The extracts were filtered and then freeze dried to obtain the dry powder which was then stored at 4oC for further chemical and biological analysis. The crude solvent extracts were tested for their biological activity under laboratory and simulated semi-field conditions. The extracts were then subjected to column chromatography and the fractions and pure compounds thereof obtained were also tested for their biological activity. Lethal concentrations of each test sample was calculated using probit analysis. The structural elucidation of the isolated compounds was done using physical properties, (melting point) and spectroscopic methods [infra red (IR), nuclear magnetic resonance (NMR) and mass spectroscopy (MS)]. Results: Bioassay of the extracts gave different levels of mortality of the larvae. Methanol extract of V. trifolia leaves, acetone extracts of stem bark and leaves of V. schiliebenii and acetone extract of root bark of V. payos caused 100% mortality at 100 ppm (LT50=8 h) than that of V. trifolia (LT50=14 h). At < 50 ppm, most of the larvae failed to transform to normal pupae but gave larval-pupal intermediates between 4-14 days of exposure. Some pupated normally but the adults that emerged appeared to be weak and died within 48 hours. Larvae exposed to extracts of the stem bark of V. payos were relatively hyperactive compared to those in control treatments. They later became stretched, inactive, died and floated in clusters on the surface. These observations suggest some interesting growth-disrupting constituents in the plants, with possible application in the practical control of mosquito larvae in aquatic ecosystems. The results of the simulated semi-field conditions revealed that An. gambiae larvae were susceptible to the Vitex extracts with the percentage inhibition of emergence of adult mosquitoes falling below the threshold value of 80% at concentrations ≥ 25 ppm. Phytochemical screening revealed the presence of flavonoids, terpenoids, steroids, alkaloids, saponins and tannins in the extracts.The isolation and purification of bioactive compounds resulted into four compounds two from V. payos: 20-hydroxyecdysone-20, 22-monoacetonide (166) and 20-hydroxecdysone (80); and three from V. schiliebenii: 20-hydroxyecdysone (80), stigmasterol (168) and γ-sitosterol (167).The isolated phytosteriods showed good larvicidal activity against An. gambiae s.s. larvae when evaluated individually. When tested in blends, three variants were noted. First, production of a less active blend from active constituents; secondly, enhancement of the activity of an active compound by an inactive constituent and thirdly, synergism between moderately active compounds to give a mixture that is more active than the individual activities of the constituents. The first variant was illustrated by the high lethal activity of compounds 20-hydroxyecdysone-20, 22-monacetonide (166) and 20-hydroxyecdysone (80) with LD50 value of less than 1 ppm. The second variant was illustrated by the enhancement of the activity of compound 168 in blends with 20-hydroxyecdysone-20,22-monacetonide (166) and 20-hydroxyecdysone (80) and compound 167 in blends with 20-hydroxyecdysone-20,22-monacetonide (166) and 20- hydroxyecdysone (80) (LD50 < 1 ppm). The third variant was illustrated by the combination of compounds stigmasterol (168) and γ-sitosterol (167) (LD50 = 1 ppm). These findings have important practical implication in the strategy adopted in the search for and use of plants and their phytochemicals for mosquito larvae control. Conclusion: In summation, results of this study show interesting larvicidal and/or growth-disrupting effects of Vitex extracts and the isolated compounds. Enriched extracts of the plants may have potential for controlling malaria vectors in breeding sites around human dwellings. This will also go along with a reduction in the annual entomological inoculation rate and consequently lead to a reduction in malaria incidences