European Journal of Medicinal Plants 2(2): 132-139, 2012 SCIENCEDOMAIN international www.sciencedomain.org Larvicidal and Antifungal Properties of Picralima nitida (Apocynaceae) Leaf Extracts Peace M. E.Ubulom 1, N. G. Imandeh 2, Chinweizu E. Udobi 3* and Ibrahim IIya 4 1Faculty of Pharmacy, University of Uyo, Nigeria. 2Department of Zoology, University of Jos, Nigeria. 3College of Science & Technology, Kaduna Polytechnic, Kaduna, Nigeria. 4Department of Medicinal Plant Research and Traditional Medicine, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria. Received 20 th September 2011 Research Article Accepted 29 th December 2011 Online Ready 28 th February 2012 ABSTRACT The larvicidal and antifungal activities of ethanolic and aqueous leaf extracts of Picralima nitida were evaluated in static bioassays on 4 th instar larvae of Anopheles gambiae and three fungal species: Aspergillus flavus, Candida albicans and Microsporum canis. All extractions were done using distilled water and 50% ethanol. Larvicidal assays were carried out at extract concentrations of 0.15, 0.30, 0.45, 0.60 and 0.75% w/v, for 72h. For the antifungal studies extract concentrations used were 200, 100, 50 and 25mg/ml. At the end of larvicidal assay the highest concentration recorded mortality of 57.60% and 38.40% for ethanolic and aqueous leaf extracts respectively. 72h LC 50 values obtained from Probit analysis, using SPSS version 17 were 0.660% and 1.057% w/v for ethanolic and aqueous leaf extracts respectively. Larvae in the control experiments registered no death throughout the period of experiment, rather they were actively wriggling and some even metamorphosed into pupae. For the antifungal studies the agar well diffusion technique was employed. Antifungal effects were determined using measurements of inhibition zone diameter (IZD). Results obtained revealed that both the aqueous and ethanolic leaf extracts exerted antifungal effect on A. flavus and C. albicans, but no antifungal effect was exhibited against M. canis, at the extract concentrations used in this study, rather a steady growth in the test plates seeded with M. canis was observed. The same was applicable with the negative controls. The drug, ketoconazole exerted antifungal effect on all test organisms. Phytochemical screening of the leaf revealed the presence of ____________________________________________________________________________________________ *Corresponding author: Email: [email protected]; European Journal of Medicinal Plants, 2(2): 132-139, 2012 alkaloids, cardiac glycosides, saponins and terpenes. The leaf of P. nitida possesses larvicidal and antifungal potential and therefore warrants a more thorough exploitation. Keywords: Larvicidal; antifungal; ethanolic; aqueous; leaf extracts; Picralima nitida. 1. INTRODUCTION All over the world especially in Africa South of the Sahara, vector-borne diseases remains a cause for serious concern. Mosquito vectors for instance constitute a major public health menace. The mosquito, Anopheles gambiae, has been incriminated with several disease- causing organisms such as Plasmodium spp, responsible for the notorious malaria scourge. According to Alabi (2010), “malaria fever is one of the deadliest diseases ravaging Africa. Annually, millions of people and man-hours with attendant economic implications are lost to this pandemic”. A.gambiae is also a vector of the filarial nematode, responsible for filariasis. Different strategies have been devised to curb disease transmission by these vectors but these have suffered certain limitations. These limitations have necessitated the search for environmentally safe, degradable, affordable and target-specific compounds against these insect-vectors. The search for such compounds has been directed to the plant kingdom (Mathur, 2003). Larviciding is a preferred option in vector control because larvae occur in specific areas and can thus be more easily controlled. Treatments provide control before the biting adults appear and disperse from the breeding sites. Phytomedicines have also shown great promises in the treatment of intractable infectious diseases (Firenzuoli and Gori, 2007). Several medicinal plants have been screened for their activity on different species of microorganisms. The antimicrobial activity of ethanolic and aqueous extracts of Sida acuta on microorganisms from skin infections has been documented by Ekpo and Etim (2009). Ubulom et al. (2011) have reported the antifungal property of aqueous and ethanolic seed extracts of P. nitida on A. flavus, C. albicans and M. canis. P. nitida (Gentianales: Apocynaceae) is a medicinal plant, commonly referred to as Akuamma plant. Many herbalists have claimed to use the leaves, roots, seeds or stem bark for the treatment of various fevers, hypertension, jaundice, gastrointestinal disorders and malaria (Iwu, 1993 & Etukudo, 2003). Its biological activities have also been reported by researchers such as Iroegbu and Nkere (2005) and Inya-Agha et al., (2006). However, no scientific document has been encountered on the larvicidal as well as the antifungal effect of aqueous and ethanolic leaf extracts of P. nitida on the mosquito and fungal species used in this study. This study was thus aimed at investigating the lethality/toxicity of the aqueous and ethanolic leaf extracts of P. nitida to fourth instar larvae of A. gambiae as well as the antifungal effect of these extracts on A. flavus, C. albicans and M. canis. 2. EXPERIMENTAL DETAILS 2.1 Collection of Plant Materials The leaves of P. nitida used in this study were collected from Anua Obio in Uyo Local Government Area of Akwa Ibom State, Nigeria. Identification was done by the Department of 133 European Journal of Medicinal Plants, 2(2): 132-139, 2012 Botany and Ecological Studies, University of Uyo, Nigeria and a voucher specimen with herbarium number: Ubulom UUH 875 (Uyo) was deposited in the herbarium of the Department. 2.2 Extraction Procedure The plant leaves were first air-dried on laboratory tables at room temperature (28 + 2°C). This was followed by pulverization using the crusher machine (Atlas exclusive, ALZICO Ltd, Type YL 112 M-4) in the pilot plant unit of National Institute for Pharmaceutical Research and Development (NIPRD) Abuja. 500g each of the pulverized leaves were macerated separately in distilled water and 50% ethanol for 72h, with periodic stirring. Each extract was filtered repeatedly using muslin cloth, non-absorbent cotton wool and Whatman No. 1 filter paper. This was done to get rid of the marc. The aqueous filtrate was concentrated using a lyophiliser (Aqua Lyovac GT2, Germany), while the ethanolic filtrate was first concentrated in vacuo at 40 °C using a rotary evaporator (Bibby Sterlin Ltd, England, RE. 200), after which it was freeze-dried using the aforementioned lyophiliser. 2.3 Phytochemical Screening The leaf extract of P. nitida was screened for its phytochemical components using the methods described by Harborne (1984), Evans (2002) and Sofowora (2006). The plant metabolites that were tested for were alkaloids, anthraquinones (free and combined), cardiac glycosides, flavonoids, saponins, phlobatannins, tannins and terpenes. 2.3.1 Test organisms Fourth instar larvae of A. gambiae used in this investigation were provided by National Arbovirus and Vectors Research Centre (NAVRC), Enugu, Nigeria. The fungal species ( A. flavus , C. albicans and M. canis) were obtained from the Department of Microbiology, University of Uyo, Nigeria. These fungal specimens were separately plated out on sterilized Sabouraud Dextrose Agar (Biomark). They were purified after isolation through repeated subculturing and characterized using the methods of Collins and Lyne (1970) and Cruickshank et al., (1975). They were subsequently stored in agar slants in the refrigerator at 4 °C, prior to experiment reported in this study. 2.3.2 Larvicidal assay The larvicidal activity of ethanolic and aqueous extracts of the leaf of P. nitida was evaluated in static bioassay, on fourth instar larvae of A. gambiae, for 72h (WHO (2005). Stock solution of each extract was prepared and in both cases (aqueous and ethanolic), the leaf extracts of P. nitida were first solubilised using the solvent dimethyl sulphoxide (DMSO). Sterile distilled water was further added to obtain a final volume of 100ml and this was mixed thoroughly and it formed the stock solution. The stock solution was heated in a water bath for 2minutes at a temperature of 40 °C, and then allowed to cool. This was done in order to reactivate phytochemicals that may have been inactivated due to excessive cooling as encountered in refrigerating and freeze-drying. From this stock solution of the extract, graded concentrations of ethanolic and aqueous leaf extracts were prepared to obtain 0.15, 0.30, 0.45, 0.60 and 0.75% w/v concentrations of each extract. Twenty five larvae were exposed to each bioassay medium in plastic assay cups, containing nutrients (a pinch each of fine quaker 134 European Journal of Medicinal Plants, 2(2): 132-139, 2012 oats). Each extract concentration had five replicates. The control which was also replicated had 25 larvae (per replicate) immersed in 100ml distilled water, to which larvae food had been introduced. Both the test and control set ups were maintained at room temperature (28 + 2°C). Observations were made at 24, 48 and 72h and larvicidal activity of each extract was determined, by counting the number of dead larvae each day, until the end of the experiment. Larvae were considered