PLANT MATERIAL
Fresh leaves of Hyptis suaveolens were collected from a field along Maryam Babangida Girls Secondary School Bosso Road, Minna (long 13° 18ʹ 37.08ʺ E and lat 13° 4141ʹ 48.84ʺN). Niger State, in July, 2014. The plant was identified as Hyptis suaveolens by two independent ethnobotanists (Mrs Jemilat Ibrahim and Mallam Muazam Ibrahim) of the Herbarium Department, National Institute for Pharmaceutical Research and Development, NIPRD, Idu, Abuja. The specimen was assigned Voucher number NIPRD/H/7020 at the Institute by Mrs Jemilat Ibrahim.
PRELIMINARY PHYTOCHEMICAL SCREENING
Qualitative phytochemical screening was conducted to detect the presence or absence of various secondary metabolites (steroids, terpenoids, flavonoids, alkaloids, tannins, carbohydrates, etc.) in “B” extract using the standard methods of Harborne (1984), Trease and Evans (1996) and Medicinal Plants Research and Traditional Medicine Practice (MPR-TMP, 2006).
FRACTIONATION OF ETHYLACETATE CRUDE EXTRACT (B)
Crude ethyl acetate leaf extract of H. suaveolens (extract B) was selected for fractionation because it displayed marked antimicrobial and larvicidal activities in previous studies (Babayi et al., 2015).
Bioactivity-guided fractionation was performed as described by NIPRID (2014) using flash column chromatography (FCC) and thin layer chromatography (TLC).
Extract B (5.8 g in weight) was fractionated using flash column chromatography. The column was packed by the slurry method using Silica gel mesh size 60–120 (150 g) and n-hexane (100ml) as the stationary and mobile phases respectively. Elution was carried out with varying proportions of hexane: chloroform and chloroform: ethylacetate. Eluents were monitored by TLC, so that identical fractions were pooled and fractions B1 to B5 were obtained. The fractions were collected in sterile bottles and were stored at 4°C until required for use.
PHYTOCHEMICAL SCREENING OF FRACTIONS B1-B5
The methodology employed in phytochemical-screening of the crude extract was adopted and the phytochemicals present in B1–B5 were determined in the five fractions obtained above.
LARVAE COLLECTION AND IDENTIFICATION
The fourth instar larvae of Culex quinquefasciatus were collected at three different locations from Bosso Market Drainage in September 2014. The larvae were identified by an Entomologist, Prof. Israel Olayemi Kayode from the Department of Biological Sciences, Federal University of Technology, Minna.
The biological assay was performed under controlled conditions having kept the larvae at 25 ± 2°C, a relative humidity of 75 ± 5%, and a 12h light/dark cycle.
PREPARATION OF STOCK SOLUTION: Stock solution was prepared according to WHO (2005) recommended standard by dissolving the dried extract in the solvent of extraction in ratio 1:9 (2 g of extract in 18 ml of solvent) to obtain a final concentration of 0.11 g/ml. This was done for all the fractions in separate test tubes and all were covered and labeled accordingly. A control plate was prepared containing ethyl acetate only.
PREPARATION OF TEST CONCENTRATION
Test concentration was prepared by adding 1.0ml of the stock solution into 10 ml of sterile distilled water to obtain a final concentration of 0.011 g/ml. The procedure was repeated for all the the fractions separately and labeled accordingly. The test concentration was further increased to 2.0 ml,3.0 ml, 4.0 ml,5.0 ml and 6.0 ml to obtain the final concentration of 0.022 g/ml, 0.033 g/ml, 0.044 g/ml, 0.055 g/ml and 0.066 g/ml for the crude and all the fractions and were all labeled accordingly (Olayemi, 2013).
LARVICIDAL ACTIVITY OF THE MEDICINAL PLANT AND FRACTIONS
The method of WHO (2005) was employed in the determination of the larvicidal activity of the medicinal plant extract and fractions. The larvae were standardized by measuring one hundred milliliters (100 mL) of sterile distilled water which was transferred to 12 transparent small bowls that were already labeled with different concentrations in two replicates (0.011R1, 0.011R2, 0.022R1, 0.022R2, 0.033R1, 0.033R2, 0.044R1, 0.044R2, 0.055R1, 0.055R2, 0.066R1, and 0.066R2), while 100 ml of sterile distilled water was also measured for the Control in replicates (Control R1 and Control R2). Twenty-five (25) larvae were then counted into each bowl and were ready for the larvicidal bioassay (this was done for the crude extract and its fractions respectively).
One point zero millilitre (1.0 mL) of ethyl acetate was added to controlR1 and controlR2 that had just 100 ml of sterile distilled water and 25 larvae, while 0.11 g/ml, 0.22 g/ml, 0.33 g/ml, 0.44 g/ml, 0.55 g/ml, and 0.66 g/ml of the test concentration was added to the bowls and their replicate bowls according to the labeling. The bowls were then covered with untreated net held tight to the bowls with rubber ring to prevent the emerging adults from escaping. Observation for larval mortality pupal development or mortality and emergence of adults were carried out at different intervals (15 min, 30 min, 1 hour, 2 hours 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 48 hours and 72 hours). After treatment, dead adult mosquitoes were counted and preserved in a 50% ethanol solution. Control mortality was corrected using Abbott’s (1925) formula, when larvae mortality exceeded 5.00 percent.
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DATA ANALYSIS
Mortality in the larval bioassay was subjected to the probit analysis to determine LC50, LC90 at 95% confidence limits. Mortality in control was corrected using Abbott’s formula. Associations between the tested extract concentrations, larvae mortality and time mortality were assessed using multiple regression correlation analysis.