Research Article
Synergistic Activity of a Mixture of Lantana camara and Ocimum gratissimum Leaves Extracts against Aedes aegypti Larvae (Diptera: Culicidae) 
,
Elias Nchiwan Nukenine2 ,
Simon Pierre Yingyang Danga2 
,
Charles Okechukwu Esimon1 2 Department of Biological Sciences, Faculty of Science, University of Ngaoundere, PO Box 454, Ngaoundere, Cameroon
Author Correspondence author
Journal of Mosquito Research, 2016, Vol. 6, No. 23 doi: 10.5376/jmr.2016.06.0023
Received: 21 Mar., 2016 Accepted: 25 Apr., 2016 Published: 10 Jun., 2016
Keziah E.A., Nukenine E.N., Yinyang Danga S.P., and Esimone C.O., 2016, Larvicidal effect of Lantana camara and Ocimum gratissimum leaves extracts and their isolates against Aedes aegypti Larvae (Diptera: Culicidae), 6(23): 1-10 (doi: 10.5376/jmr.2016.06.0023)
Introduction
Mosquitoes are insect vectors which transmit diseases such as malaria, dengue, yellow fever, etc. to humans worldwide, causing them economic loss and social disruption (Becker et al., 2003; Okigbo et al., 2010). Supratik et al. (2010) stated that Aedes aegypti alone is the very well known transmitter of several arboviral diseases viz. chikungunya, yellow fever and dengue fever.
The best way to interrupt the disease transmission is to kill the biting vector, especially at the larval stage because the larvae can be easily managed (Kishore et al., 2011). Synthetic larvicides (DDT, Pyrethruim, Heptachlor, Diedrin and Lindane) have been used in the past to achieve this control measure (Okigbo et al., 2010). Although they are still the most effective, their repeated use caused environmental pollution, health concern and development of resistance in mosquito populations (Devine and Furlong, 2007). With these problems in focus, it becomes increasingly necessary to search for an alternative in the development of environmentally safe, biodegradable, low cost, target specific insecticides for mosquito control (Dua et al., 2010) which can be used with minimum care by individuals and communities in specific situations. Plants may be a source of alternative agent to replace the synthetic insecticides for mosquito control (Ghosh et al., 2012). Plant products can be obtained either from the whole plant or from a specific part by extraction and evaporation with different types of solvent such as aqueous, methanol, acetone, petroleum ether, chloroform, hexane etc. depending on the polarity of the photochemicals (Essam et al., 2005). Many plant species are known to possess biological activity that is frequently assigned to the secondary metabolites (Rameshwar, 2010). Mixing of plant extracts may produce a synergistic effect which enhances their toxicity even at very low concentrations (Poonia et al., 2013). Two of these plants are Lantana camara and Ocimum gratissimum.
L. camara L. is a flowering ornamental plant belonging to family Verbenaceae. In Nigeria, L. camara has as local names “Ewonadele” in Yoruba, “Kimbamahalba” in Hausa and “Anya nnunu” in Igbo (Gabi et al., 2011). According to Sanjeeb et al. (2012), the plant is said to have antibacterial, antifungal, wound healing, antimotility, antiulcerogenic, hemolytic, antihyperglycemic, antifilarial, antiinflammatory, antifertility, antiurolithiatic, anticancer and antiproliferative, antimutagenic, antioxidant, mosquitoes controlling activities. Recent scientific studies have emphasized the possible use of L. camara in modern medicine (Sanjeeb et al., 2012).
O. gratissimum L. (Lamiaceae) is an aromatic perennial herb wildly grown in Nigeria. It is commonly known as Scent leaf or “Nchuanwa” by the Igbos, “Effirin” by the Yorubas and "Dai doyatagida" by the Hausas (Orwa et al., 2009; Okoli et al., 2010). The plant is used as food spice (Okwu, 2006) and for the treatment of ailments such as malaria, diabetes, respiratory and urinary tract infections, cough, fever, diarrhoea, abdominal pains, pneumonia, conjunctivitis, oral wounds, tooth infection (Ilori et al., 1996; Aguiyi et al., 2000; Rabelo et al., 2003). In Nigeria, Esimone et al. (2011) findings showed the repellent activity of ointments formulated with O. gratissimum oil against Ae. aegypti and Cx. quinquefasciatus mosquitoes.
The few studies on the mosquitocidal activity of binary mixtures investigated the combined effects of phytochemicals with conventional synthetic insecticides or microbial control agents. Yet in the present project, we carried out the mosquitocidal activity of Lantana camara and Ocimum gratissimum extracts and fractions, individually and in binary mixtures of phytochemicals in order to check out the synergistic effect of both plants against 4th instar larvae of Aedes aegypti. The result of the present study would be useful in promoting research aiming to develop new agents for mosquito control based on bioactive chemical compounds from indigenous plant sources.
1 Materials and Methods
1.1 Test organisms
The larvae of Ae. aegypti were collected in April 2011 from WHO/National Arbovirus and Vector Research Centre Enugu, Enugu state, Nigeria. The larvae were then brought into the insectarium of the Faculty of Pharmaceutical Sciences, Agulu; Nnamdi Azikiwe University, Awka, Anambra State, Nigeria where they were reared. The larvae were fed with chicken feed (ground soya bean and maize grains mixture) mixed with fish feed in 3:1 ratio. On every alternate day, the water from the culture bowl was changed carefully until they turned into 4th instar. The larvae were maintained at room (temperature of 34±3°C, relative humidity of 81±2% and under 12L: 12D photoperiod cycles).
1.2 Collection of plant materials, extraction and fractionation
The new generated fresh leaves of Lantana camara and Ocimum gratissimum were collected from farms around Oko, Anambra State, Nigeria in august 2012. The plants were identified by Dr S. I. Okeke (PhD), a botanist at Federal Polytechnic, Oko, Anambra State, Nigeria. The plant materials were air dried at room temperature and ground until the powder passed through a 0.20 mm sieve. The extraction experiment started immediately as soon as the plant powder was ready.
The extraction and fractionation processes were performed in accordance with the method adopted by Okoye and Osadede (2009). From the collection of plant material powder, 500 g of L. camara and 325 g of O. gratissimum were extracted for 3 days by cold maceration in methanol shaking it thrice per day (morning, noon and afternoon) in the laboratory of Pharmaceutical and Medicinal Chemistry. The maceration process was then repeated twice by adding fresh methanol in the same residue for maximal extraction. The suspension was filtered through Whatman® No 1 filter paper (size: 24 cm of diameter, England) using a Buchner funnel. The methanol crude extracts (MCE) were concentrated to dryness in rotary vacuum evaporator RE300 (ROTAFLO, England). The MCE of both plant materials were first adsorbed on silica gel (60-200 mesh) and sequentially partitioned in succession with hexane, chloroform, ethyl acetate and methanol to obtain hexane fraction (HF), chloroform fraction (CF), ethyl acetate fraction (EAF) and methanol fraction (MF) based on their polarities. Their weight and percentage yields are shown in Table 1.
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Table 1 Yields of Lantana camara and Ocimum gratissimum extraction and fractionation |
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All the extracts and fractions were stored in the refrigerator (-4°C) until used.Mosquitoes are insect vectors which transmit diseases such as malaria, dengue, yellow fever, etc. to humans worldwide, causing them economic loss and social disruption (Becker et al., 2003; Okigbo et al., 2010). Supratik et al. (2010) stated that Aedes aegypti alone is the very well known transmitter of several arboviral diseases viz. chikungunya, yellow fever and dengue fever.
1.3 Larvicidal bioassay
The larvicidal activity was assessed as per the protocol previously described by WHO (2005). Stock solutions of the extracts and fractions were made using Tween 80 as emulsifier to facilitate the dissolving of material in water. The stock solutions were further diluted up to 100 mL by adding tap water. From these stocks, various concentrations from 0.025 g/100 mL (0.25 g/L) to 0.2 g/100 mL (2 g/L) were performed by serial dilutions after preliminary tests. 1 mL of Tween 80 was used for each replicate, extract or fraction as control. A commercial formulation of WARRIOR® 1000 EC (100 % DDVP: 2,2- dichlorovinyl dimethyl phosphate) larvicide (2000 ppm, recommended concentration), bought from a chemicals shop from Awka market (Anambra State, Nigeria), was used as positive control. Twenty-five 4th instar larvae of Ae. aegypti were transferred into each 250 mL beaker containing 100 mL of the aliquot and the mortality was recorded after 24 h of exposure. No food was provided either to the test or control solutions during the experiments. The larvae were considered as dead, if they were not responsive to a gentle prodding with a fine needle. All bioassays were carried out at room (temperature of 27±2°C and relative humidity of 79±3%). Experiments were set in four replicates along with controls.
Percentage test mortality (%) = (number of larvae dead / total number of larvae used) * 100
Corrected Mortality (%) = [(% test mortality - % control mortality) / (100 - control mortality)] x 100 (Abbott, 1925).
1.4 Determination of synergistic factors (SF)
In combination studies, tested plant extracts and fractions were mixed in 1:1 ratios at different concentrations as described by Someshwar et al. (2011). Each mixture was tested in four replicates alongside with untreated controls. The tests were carried out as mentioned above. The synergistic factor (SF) was calculated after calculating the LC50 values for each combination test (Kalayanasundaram and Das, 1985).
Synergistic factor = LC50 value of the individual plant extract / LC50 value of the combined plant extracts or assumed synergist (Someshwar et al., 2011).
The value of SF > 1 indicates synergism, SF < 1 indicates antagonism and SF = 1 means no obvious effect (additive).
1.5 Statistical analysis
The corrected mortality was determined using Abbott’s (1925) formula whenever required. Data on % mortality were tested for normality and heterogeneity of variance and then subjected to ANOVA procedure using Statistical Package for Social Science (SPSS 17.0). Duncan test (P = 0.05) was applied for mean separation. Probit analysis (Finney, 1971) was applied to determine lethal dosages causing 50% (LC50) and 95% (LC95) mortality of larvae 24 h post exposure. The SF values were calculated using Microsoft Office Excel 2007.
2 Results
The larvicidal activity of leaf extracts and fractions of L. camara and O. gratissimum were found toxic Ae. aegypti. With L. camara, the MCE, HF and EAF were found to be more effective than the CF and MF (Table 2).
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Table 2 Larvicidal activity of Lantana camara leaf extract and fractions against 4th instar larvae of Aedes aegypti after 24 h of exposure Means within a product followed by the same letter do not differ significantly at P= 0.05 (Duncan’s test); ***: p<0.001; LC50 and LC95: Lethal Concentrations able to kill 50% and 95% of larvae, respectively; LCL: Lower Confidence Limit; UCL: Upper Confidence Limit; (-): no Confidence Limit estimated; χ2: Chi-squared; Number of replicates: 4. |
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The mortality increased with ascending concentrations like all other plant extracts or fractions. With the lowest concentration of 0.25 g/L, the mortality was 20.00, 13.33 and 10.66% for MCE, HF and EAF, respectively. At the same concentration, CF and MF registered no mortality. In addition, at the highest concentration of 2 g/L, the MCE, HF and EAF exhibited 100% mortality as observed in WARRIOR each while this mortality rate was 41.33 and 21.33 % for CF and MF, respectively. The EAF recorded better LC50 value of 0.62 g/L followed by those of HF (0.72 g/L), MCE (0.96 g/L), CF (2.20 g/L) and MF (3.36 g/L). Thus, the EAF was found to be more effective than the other three solvent fractions. The results of the larval toxicity of O. gratissimum leaf revealed that all the extract and fractions except MF were effective at different degrees of effectiveness against 4th instar larvae of Ae. aegypti (Table 3)
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Table 3 Larvicidal activity of Ocimum gratissimum leaf extract and fractions against 4th instar larvae of Aedes aegypti after 24 h of exposure Means within a product followed by the same letter do not differ significantly at P= 0.05 (Duncan’s test); ***: p<0.001; LC50 and LC95: Lethal Concentrations able to kill 50% and 95% of larvae, respectively; LCL: Lower Confidence Limit; UCL: Upper Confidence Limit; (-): no Confidence Limit estimated; χ2: Chi-squared; Number of replicates: 4. |
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At 0.25 g/L, 11 and 66.33% mortality were observed in MCE and HF, respectively. Although no mortality was registered at the same concentration with the other solvent fractions, yet 72 and 90.66% mortality were obtained at 2 g/L with CF and EAF, respectively. The LC50 values were 0.37, 0.60, 0.97, 1.60 and 3.23 g/L for HF, MCE, EAF, CF and MF, respectively. This confirmed that HF from O. gratissimum leaf was more effective than the other solvent fractions against 4th instar larvae of Ae. aegypti.
The combinations were made with MCE, HF and EAF because the CF and MF appeared to be less effective in single bioassay (Tables 2 and 3). From the different combinations, LC50 values of HF decreased from 0.72 and 0.37 g/L for L. camara and O. gratissimum, respectively when alone to 0.36 g/L in mixture (Table 4).
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Table 4 Larvicidal activity of Lantana camara + Ocimum gratissimum (1:1/v:v ratio) leaf extracts and fractions against 4th instar larvae of Aedes aegypti 24 h post exposure Means within a product followed by the same letter do not differ significantly at P= 0.05 (Duncan’s test); ***: p<0.001; LC50 and LC95: Lethal Concentrations able to kill 50% and 95% of larvae, respectively; LCL: Lower Confidence Limit; UCL: Upper Confidence Limit; (-): no Confidence Limit estimated; χ2: Chi-squared; Number of replicates: 4. |
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The same decreasing LC50 value was observed in HF of L. camara combined with EAF of O. gratissimum from 0.72 and 0.97 g/L, respectively to 0.70 g/L in combination. HF of (L. camara + O. gratissimum) showed assumed synergistic action. In addition, the mixture HF of L. camara + EAF of O. gratissimum showed the same effect. However, EAF of (L. camara + O. gratissimum) on the one hand and the combination EAF of L. camara + HF of O. gratissimum on the other hand exhibited assumed antagonistic effects. The values of synergistic factor (SF) values are presented in Table 5.
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Table 5 Estimation of SF of Lantana camara and Ocimum gratissimum leaves solvent extracts and fractions (1:1/v: v) 24 h post exposure against 4th instar larvae of Aedes aegypti. *: synergism; **: Antagonism; SF: Synergistic factor; V: volume; SF > 1: synergism; SF < 1: antagonism; Number of replicates: 4; SF = LC50 individual plant extract / LC50 value of the combined plant extracts. |
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3 Discussion
Several secondary metabolites produced by some plants for their own defense from their enemies have good mosquito larvicidal activity, such as titerpenes, isoflavonoids, tannins, saponines, phenolics etc. (Someshwar et al., 2011). So far in Nigeria, the results obtained by Adamou et al. (2008) showed that the aqueous leaf extract of O. gratissimum contained varying concentration of the following active principles viz. reducing sugars (free and combined), tannins, saponins, cardiac glycosides, terpenes, steroids, flavonoids and alkaloids. In addition, the findings using the aerial parts of O. gratissimum showed that carbohydrates, anthraquinones, phenolics, tannins and saponins were present in alcohol fractions. sterols were found in ethanol crude extract as well as hexane and chloroform soluble fractions (Abdullahi, 2012). Besides, other reports showed that the methanol crude extract of leaf and stem parts of the same plant contained high percentage of phenol, tannins and flavonoids (Omale et al., 2008). The phytochemical analysis of the leaf ethanol crude extract of the plant revealed the presence of tannins, cyanogenic glycosides, saponins, terpenes, alkaloids, reducing sugar, fats and oil, steroidal aglycone (Emeka, 2009); the presence of alkaloids, tannins, flavonoids, phenol, saponins (Omodamiro et al., 2012).
Still in Nigeria but this time with L. camara, the results showed the presence of alkaloids, cardiac glycosides, and saponins in all the extracts (petroleum ether, ethanol and aqueous extracts). Carbohydrates were detected in both ethanol and aqueous extracts, while flavonoids and steroids were present in both petroleum ether and ethanol extracts. Tannins and terpenoids were detected in aqueous and petroleum ether extracts, respectively (Gabi et al., 2011).
In the present study, MCE, HF and EAF of L. camara leaf showed higher mortality when applied separately. The same rate of mortality was observed in MCE and HF of O. gratissimum leaf. Kishore et al. (2011) reviewed the efficacy of phytochemicals against mosquito larvae according to their chemical nature and described the mosquito larvicidal potentiality of several plant derived secondary materials, such as, alkanes, alkenes, alkynes and simple aromatics, lactones, essential oils and fatty acids, terpenes, alkaloids, steroids, isoflavonoids, pterocarpans and lignans. The effectiveness of the MCE from the two plant materials could be explained by the broad spectrum of phytochemicals cited above. It is evident that the MCE empowered all the phytochemicals which were partitioned in serial extraction by different solvents according to their polarity.
In addition, the effectiveness of HF would be due to the extraction of the essential oil this particular solvent is able to extract. It was shown that the extraction of active biochemicals from plants depends on the polarity of the solvents used. Polar solvents extract polar molecules and non-polar solvents extract non-polar molecules. The hexane, the most non polar solvent (polarity index of 0.1) mainly extracts essential oil (Ghosh et al., 2012). This was confirmed by Kishore et al. (2011) who reviewed that the essential oil possessed significant larvicidal activity against 4th instar larvae of Ae. Aegypti and Ae. Albopictus. Eventually, Ae. Aegypti larvae lack a respiratory siphon, they breathe through spiracles located on the 8th abdominal segment and therefore must come to the surface frequently to breathe (Kaufmann and Briegel, 2004). The HFs used in this study were oily; hence, the oils could block the spiracles, resulting in asphyxiation and death of the larvae (Rotimi et al., 2011). Furthermore, results from this study are in accord with previous findings, where the HF of Eclipta alba more effective than CF, EAF and MF against Ae. Aegypti larvae was reported (Govindarajan and Karuppannan, 2011). Besides, according to Ghosh et al. (2012), ethyl acetate is moderately polar solvent (polarity index of 4.1) that mainly extracts steroids, alkaloids, etc. Earlier, Gabi et al. (2011) revealed the presence of these two important phytochemical in L. Camara. They should be mainly extracted in EAF which displayed good toxicity against 4th instar larvae of Ae. Aegypti in the present study.
In combination, the assumed synergistic effect showed by HFs of L. Camara and O. Gratissimum on the one hand and HF of L. Camara + EAF of O. Gratissimum on the other hand corroborate previous studies. Someshwar et al. (2011) investigated the synergistic effect of solvent extracts of Croton caudatus (fruits) and Tiliacora acuminata (flowers) against Cx. Quinquefasciatus larvae. The chloroform fraction of C. caudatus mixed with methanol fraction of T. Acuminata on the one hand and the combination of ethyl acetate fractions of the two plants on the other hand showed synergistic effect. However, the combination of benzene fractions of the two plants exhibited antagonistic action against Cx. Quinquefasciatus larvae. All the combinations were performed in 1:1 ratios as in the present study. In the same vein, Pongamia pinnata (PP) and Kigelia africana (KA) petroleum ether extracts for combined larvicidal activity against Ae. Aegypti 3rd and 4th instar larvae were evaluated in PP 50% : KA 50%, PP 75% : KA 25% and PP 25% : KA 75% ratios. The mixtures PP 50% : KA 50% and PP 75% : KA 25% showed strong synergism and the mixture PP 25% : KA 75% exhibited antagonistic effect (Poonia and Kaushik, 2013).
In conclusion, hexane and ethyl acetate fractions of L. Camara and O. Gratissimum were very effective. The high extracted yields of hexane fractions (30.05% for L. Camara and 26.11% for O. Gratissimum) and ethyl acetate fractions (24.59% for L. Camara and 27.55% for O. gratissimum) could allow them to be included in the management strategies of mosquitoes. In addition to their environmental friendliness, these plants are readily available, cheap, and would be affordable to the resource-poor persons in the fight against mosquito borne diseases in Nigeria. Identifying the synergist compounds within mixtures may lead to the development of more effective biopesticides as well as the use of smaller amounts in the mixture to achieve satisfactory levels of efficacy. Synergistic action in the present study could be exploited for Integrated Pest Management (IPM) programs with lesser toxicity to aquatic non-targets.
Aknowledgements
Authors gratefully acknowledge the guidance of Dr S. I. Okeke (PhD), a botanist at Federal Polytechnic, Oko, Anambra State, Nigeria during the harvesting of the leaves. We are also grateful to WHO/ National Arbovirus and Vector Research Centre Enugu, Enugu state, Nigeria for the provision of the mosquito larvae. We are appreciative of Dr Ken Ngwoke, Head of Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria, for his help, guidance and unlimited facilities for conducting phytochemical extraction in the laboratory.
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