Synthetic insecticides have been widely used to control mosquito vectors of disease in various parts of the world; however, resistance to these insecticides has recently become problematic in vector control programs. Moreover, the continuous monitoring of mosquito populations may play an important role in attempting to develop management strategies that will prevent or minimize the development of resistance to pesticides, as well as to develop new products to combat insects2. Thus, the use of biological products is an alternative approach for preventing the development of resistance in mosquitoes.
Plants are a source of bioactive compounds that have insecticidal properties and therefore may be suitable for mosquito control3. Phytoextracts have previously been shown to be successful in various biocontrol programs, and essential oils of different plants are used as potential mosquito repellents. Different species of the genus Solanum have demonstrated larvicidal and pupicidal activities against C. quinquefasciatus4,5. The species Solanum lycocarpum A. St. Hil., belonging to the Solanaceae family, is popularly known as lobeira and is widely distributed in the Brazilian cerrado6. It is widely used in traditional medicine as a sedative, in the treatment of epilepsy, asthma, diabetes, obesity, for the reduction of cholesterol levels, and for abdominal and renal pain7.
To our knowledge, for the first time, we show the larvicidal activities of the extracts and fractions obtained from the green fruits of S. lycocarpum against C. quinquefasciatus.
The green fruits of S. lycocarpum A. St. Hil. were collected in the City of São Sebastião do Oeste, State of Minas Gerais, Brazil, in August 2011. The plant material was identified by Dr. Alexandre Salino, and a voucher specimen (BHCB 159397) was deposited at the Instituto de Ciências Biológicas Herbarium, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. Methanol was used as solvent to obtain the extract from 170.01g of dried and powdered green fruits, using a Soxhlet extractor. The extract was then concentrated in a rotary evaporator at 50°C under reduced pressure to yield the methanol extract (MET, 14.53g). Some of this extract (6.82g) was dissolved in MeOH/H2O (1:1) and then partitioned successively with hexane, dichloromethane, and ethyl acetate (Vetec®, São Paulo, Brazil; 100 mL, twice with each solvent), yielding 0.09, 0.23, 0.34, and 5.50g of hexane (HEX), dichloromethane (DCM), ethyl acetate (AC), and hydromethanolic (HM) fractions, respectively. The extract and fractions were screened for the presence of different phytoconstituents such as saponins, tannins, alkaloids, steroids, triterpenes, coumarins, and flavonoids8.
Culex quinquefasciatus larvae were obtained from a laboratory culture, as previously described by Gerberg9. Both the third and fourth instar larvae of C. quinquefasciatus were exposed to different concentrations (12.5, 50, and 200mg/L; dissolved in 1% dimethylsulfoxide) of the methanol extract and fractions up to the emergence of adults, to determine the optimal sub-lethal concentration. For each sample tested, the larvae were divided into test and control groups, consisting of 60 specimens each, with three replications for each treatment. The control group larvae were exposed to water dissolved in 1% dimethylsulfoxide. The temperature was maintained at 26 ± 1°C throughout all of the tests. The larvicidal bioassay was performed according to the World Health Organization standard protocols10. The larvae were exposed to these solutions, and mortality was recorded every 24h over a total period of 144h. Three replications of each treatment were performed and the larvae used were all of the first generation. Larvae were considered dead when they did not respond to the stimulus or when they did not rise to the surface of the solution. LC50 and LC90 values were calculated by Probit regression11.
In this study, phytochemical tests revealed the presence of coumarins, flavonoids, and tannins in the methanol extract. Saponins were present in the dichloromethane fraction, while alkaloids and coumarins were found in the hexane fraction. On the other hand, flavonoids, coumarins, and tannins were found to be present in the dichloromethane, ethyl acetate, and hydromethanolic fractions, and terpenes/sterols were found in the dichloromethane and hydromethanolic fractions.
Larval mortality rates in each group, expressed as percentages, are shown in Figure 1. Analysis of variance tests, comparing the percentage of mortality between the various treatments, showed significant differences, depending on the concentrations and extracts or fractions used. Furthermore, a positive correlation was observed between the concentration of the methanol extract and fractions and the mortality rate (Figure 1), with the mortality rate being directly proportional to concentration. The dichloromethane and ethyl acetate fractions showed the greatest larvicidal effect at 200mg/L (83.3% and 86.7%, respectively), and was significantly different compared to the other treatments (p < 0.05). The greatest mortality was observed at 200mg/L for the methanol extract (66.7%) as well as for the hydromethanolic fraction (51.7%). The hexane fraction did not show larvicidal activity, yielding values lower than 20% (Figure 1). No significant differences in mortality were observed for the ethyl acetate and hydromethanolic fractions at 12.5mg/L, the dichloromethane fraction at 12.5mg/L, the hexane fraction at 50mg/L, the hydromethanolic fraction at 50mg/L, and the hexane fraction at 200mg/L. There was a complete absence of larval mortality in the dimethylsulfoxide controls.
The pertinent values for the methanol extract and different fractions, estimated by the LC50-LC90regression equation and chi-square tests, are presented in Table 1. The methanol extract and dichloromethane, ethyl acetate, and hydromethanolic fractions had larvicidal effects against C. quinquefasciatus, with LC50 values between 75.13 and 207.05mg/L; this represents higher larvicidal activity than that of other plant species of the Solanum genus. For instance, ethanolic extracts of the leaves of S. xanthocarpum showed activity against the third and fourth instar larvae of C. quinquefasciatus, with LC50 values of 271.12 and 377.40mg/L, respectively4. However, the larvicidal activity of the methanol extract of S. lycocarpum reported in the present study are comparable to those achieved by Sakthivadivel and Daniel5, who demonstrated the larvicidal effect of the ethanolic extract of leaves of the S. trilobatum on the fourth instar larvae of C. quinquefasciatus, with LC50 > 200mg/L.
|Samples||LC50 (95% CI) mg/L||LC90 (95% CI) mg/L||Regression equations||χ2||P value|
|MET||126.24||709.53||y = 1.568x + 1.629||2.77||0.76|
|DCM||75.13||387.18||y = 1.748x + 1.757||7.93||0.57|
|AC||83.15||271.57||y = 0.714x + 2.280||8.22||0.62|
|HM||207.05||1,743.57||y = 2.008x + 1.283||0.74||0.96|
LC50: median lethal concentration; CI: confidence interval; LC90: 90% lethal concentration; χ2: chi-square value. MET: methanol extract; DCM:dichloromethane fraction; AC: ethyl acetate fraction; HM:hydromethanolic fraction; y: probit value; x: log concentration of the methanol extract and fractions of the green fruits.
In this study, the dichloromethane and ethyl acetate fractions showed the lowest LC90 values (387.18 and 271.57mg/L, respectively), making them the most toxic fractions against C. quinquefasciatus. The methanol extract and hydromethanolic fraction showed LC90 values of 709.53 and 1,743.57mg/L. Ethanolic extracts of the leaves of S. xanthocarpum also showed activity against the third and fourth instar larvae of C. quinquefasciatus, with LC90 values of 1,011.89 and 1,058.85mg/L, respectively4. Changbunjong et al.12 also showed larvicidal activity of the ethanolic extract of green fruits of S. xanthocarpum against C. quinquefasciatus, with LC50 and LC90 values of 573.20 and 1,066.93mg/L, respectively, which were greater than those found in our study.
The effectiveness of the derived secondary compounds of a plant, such as saponins, steroids, isoflavonoids, and tannins have been well documented for their larvicidal activity13. Chowdhury et al.14 also evaluated the green berries of S. villosum, and suggested that a steroid compound was responsible for its observed larval toxicity. Thus, the higher larvicidal activity found in the dichloromethane fraction can be attributed, at least partially, to the action of saponins, steroids and tannins.
In conclusion, our preliminary results indicate that the methanol extract and dichloromethane, ethyl acetate, and hydromethanolic fractions of the green fruits of S. lycocarpum showed larvicidal activity against C. quinquefasciatus. These results form the basis for additional studies for evaluating the possibilities of using the extracts and fractions of green fruits of S. lycocarpum as natural, plant-based sources of larvicides.