Larvicidal Activities of Hyptis suaveolens and Ocimum sanctum against Anopheles gambiae

Aim: To determine the larvicidal activities of Hyptis suaveolens and Ocimum sanctum against Anopheles gambiae. Study Design: A 24 h LC50 concentration-mortality static non-renewal bioassay was carried out. Place and Duration of Study: The study was carried out at Rohi Biotechnologies Ltd’s Toxicity Laboratory, Port-Harcourt, Rivers State Nigeria, between January and March 2015. Methodology: The solvent extracts were assessed against the mosquito larva at varying concentrations in a 2-phased rapid and final screening test. Results: Results show that the methanol extract of H. suaveolens induced the highest mortality with LC50 values of 73.25 ppm, while the chloroform and hexane extracts induced mortalities with LC50 values of 76.25 and 97.25 ppm respectively. On the other hand, the O. sanctum extracts induced mortalities of 125.00, 150.00 and 194.08 ppm for methanol, chloroform and hexane extracts respectively. Conclusion: Based on our findings, we recommend solvent extracts of H. suaveolens and O. sanctum as prospective larvicides in the integrated management of malaria. Original Research Article Ohimain et al.; JALSI, 3(3): 131-137, 2015; Article no.JALSI.2015.034 132


INTRODUCTION
In the past decades, the morbidity burden and incident rate of malaria have become of global concern. Statistics in literature reveal that malaria is the most prevalent insect-borne disease, globally affecting about 3.5 billion persons per annum [1]. Report of the World Health Organization also stated that malaria is the most devastating parasitic disease [2,3]. Malaria is endemic in many developing countries (especially in Africa). For instance, there are over 100 countries, with an annual global incident rate of 700 million [4]. There are several species of mosquitoes of which only about 30-40 species transmit malaria [1,4]. However, in Africa the predominant transmitter is Anopheles gambiae [1,3].
Hyptis suaveolens is a medium aromatic weedy shrub found in the tropics and subtropical region [5]. In Nigeria, it is abundantly distributed as aggressive annual weedy species (especially in the northern and western Nigeria). The genus Hyptis is well known for its traditional application as an anticancer [5,6], antibacterial [7], antifungal [8,9], and anticonvulsant agent [10].
Plants generally have curative application due to the metabolites and secondary compounds they possess. Ocimum species has been widely reported to possess repellent properties against mosquito [16], elephantiasis [17] and some parasitic pathogen [18]. Multifaceted combat against malaria has witnessed global remarkable success. Some challenges associated with malaria control include; rapid mosquito prolificacy and disease re-emergence as chemotherapy only proves to be abatement [19,20]. Furthermore, the toxicity of synthetic pesticides to non-targeted species, high cost and abatement of morbidity burden observed in chemotherapeutic techniques have become sources of concern to indigent people in endemic areas [19]. A result, integrated exploration of eco-friendly pesticides, plant-derived pesticides have emerged as basic panacea in the control of most insect-borne public health diseases. The objective of the research is to investigate the larvicidal activities of H. suaveolens and O. sanctum against Anopheles gambiae mosquito vectors.

Mosquito Larvae Culture and Identification
The larvae were cultured in-vivo in the wild, close to a bush in a fenced compound with plantain vegetation, located at Uyo street in Port Harcourt, Rivers State, Nigeria (Latitude 4° 49 ! 45.2 !! N and Longitude 007° 01 ! 27.9 !! E). The larva culture was carried out using standard methods as described by Ohimain et al. [1] and Angaye et al. [20]. The emerging larvae were identified using the identification protocol of Ahmed and Ahmed [21].

Plant Collection and Extraction
The leaf of H. Suaveolens and O. Sanctum were collected from Edo State, Nigeria in August 2014. The taxonomic identification of both plants was carried out using identification keys as described by Ogunkunle [22]. The leaves were shade-dried for 7 days at ambient environmental temperatures (31±2°C). About 300 g of the leaves were powdered and macerated in 500 ml of the respective solvents (hexane, Fisher Scientific international Company; chloroform and methanol, BHD Chemical Ltd. Poole England) for 72 hours. The filtrates of the concoctions were respectively extracted in a rotary evaporator (60°C). The obtained residues (i.e. extracted active ingredients) were preserved at 4°C.

Experimental Setup
The experimental setup of this bioassay to verify the larvicidal activity of the solvent extracts against the larvae was carried out following standard protocol [23,24], with slight modifications incorporating rapid and final screening as described by several authors [1,2,20]. The positive control was adjusted with 1 ppm pyrethrum pesticide, while the negative control was set up with water from the breeding site.

Rapid and Final Screening
The rapid screening was set up (in replicates), in order to determine the range of activity as per the solvent extracts. A wide range of concentrations (ranging from 500-1000 ppm) were first used to deselect extract(s) whose minimal total mortality rate (MTMrt) exceeds 500 ppm after 24 hours. This implies that, only extract(s) with MTMrt at 500 ppm within 24 hours were selected for the final screening.

Statistical Analysis
The mean mortality and standard deviation of data from the bioassay were calculated, furthermore, in order to estimate the median lethal concentration; the primary mean mortality data were statistically subjected to concentrationmortality assessment using the 2013 version of Microsoft Excel package with 5% error.

RESULTS AND DISCUSSION
The result of the rapid screening (not presented) indicated that all extracts of H. suaveolens and O. sanctum scaled the final screening phase (i.e. MTMrt≤500 ppm). Results of the final screening ( Table 1), shows that the solvent extracts of H. suaveolens had minimal total mortality rates (MTMrt), at the following concentrations; 200-250 ppm for chloroform extract, 150-200 ppm for methanol extract and 250-300 ppm for hexane extract. On the other hand, the solvent extracts of O. sanctum were toxic to the larvae with MTMrt of 250-300 ppm for both chloroform and methanolic extracts, while the hexane extract had MTMrt at a range of 300-350 ppm. Furthermore, it was worthy of note that while the positive control was lethal at 1 ppm, the negative control induced no mortality throughout the bioassay.
Result of the median lethal dose (LC 50 ) as presented in Fig. 1 In another study, a lower LC 50 value (88.78 ppm) was obtained using O. sanctum [30]. Also, the ethyl acetate leaf extract of Ocimum tenuiflorum induced LC 50 value of 44 ppm against anopheles mosquito [26]. The termiticidal activities of crude and some solvent extracts of O. sanctum was also reported showing varying toxic activities between 1-11 days, however the minimum and maximum mortalities were recorded as 43.89±39.97% for the stem water extract and 84.45±27.21% for the ethyl acetate leaves extract [31]. The phytochemical properties of the leaves, stems, and root of Hyptis suaveolens have been established in literatures from the foregoing by several authors [32][33][34].Furthermore, their results indicated the absence of saponin in the root [34], compared to the stem (10.50±0.79%), and the leaves (6.10±0.42%) [32,33]. Alkaloids in the leaves was 2.80±0.28%, flavonoids was 1.90±0.14%, and tannins 5.50±0.074%; alkaloids was 1.60±0.00%, flavonoids in the leaves and tannins was 0.30±0.14 and 0.23±0.07% in the stem [32]. Also the biocidal activities of essential oil from both H. suaveolens and O. sanctum have been reported for repellence against malaria and filariasis vectors [7][8][9]11,17].  [36][37][38][39]. Monoterpene has the ability to stimulate the cellular leakage of potassium [40], which results to membrane disruption and mortality [39]. Although, the metabolites of the plant were largely responsible for the mortality. However, the applied solvents (chloroform, methanol and hexane), being medium for extraction, are similar as alcoholic solvents, but dissimilar polarity wise. The dissimilarity in polarity of the solvents can affect the enrichment of envisaged metabolites of the plant [19] and possibly induce varying degree of toxicity as indicated in the results.

CONCLUSION
Malaria is a hyper-endemic vector-borne disease both in tropical and subtropical region. In malaria control, adoption of several techniques will best abate the morbidity burden. Targeting the stationary larvae phase with eco-friendly pesticides (especially plant-derived pesticides) is desirable as this will supress their fecundity and is environmentally friendly. Solvent extracts of both plants induced moderate activities against the larvae. This study justifies the application of H. suaveolens and O. sanctum in integrated pest management. We recommend the field application of these plants for the control of the vector.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable. Comparative toxicity effect of bush tea leaves (Hyptis suaveolens) and orange peel (Citrus sinensis) oil extract on larvae of the yellow fever mosquito Aedes