Mosquito larvicidal activity of Solanum nigrum berry extracts

Mosquitoes are vectors of many dreadful human diseases. Culex quinquefasciatus is the principal vector of filariasis and it is reported to infect more than hundred million people every year in more than 110 countries in the tropics1. New control methodologies aim at reducing mosquito breeding sites and biting activity by using a combination of chemical- biological methods to reduce the population of mosquito and to reduce the man-vector contact2. Plant products are given importance due to their biodegradable nature, easy availability and no effect on non-target organisms that share the same environmental guild with mosquito larvae. Solanum nigrum L. (Solanaceae: Solanales) is an Ayurvedic herb with multiple medicinal properties34. The objective of the present study was to examine the larvicidal activity of extracts of berries of S. nigrum against the larvae of Cx. quinquefasciatus and other non target organisms.

The present study was conducted at Burdwan, West Bengal, India, during June-August 2010. Fresh, mature, green berries of S. nigrum were randomly harvested from plants growing on the outskirts of Burdwan. Berries were initially rinsed with distilled water and dried on a paper towel. Crude extract was prepared by grinding the plant material in a mortar and pestle and passing the ground material through Whatman No. 1 filter paper. Required concentrations of crude extract were prepared by mixing the crude extract with suitable amount of sterilized distilled water. In the laboratory bioassay, ten each of 1^st to 4^th instar larvae of Cx. quinquefasciatus were separately introduced into different petri-dishes containing graded concentrations (1, 1.5, 2, 2.5 and 3%) and the mortalities were recorded after 24, 48 and 72 h of exposure periods following Abbott's correction5. The mortality rate at 3 per cent concentration was higher (P<0.05) than all other concentrations of the crude extract tested for larval mortality (Table I). For the preparation of solvent extract, 50 g of fresh, mature berries were rinsed with distilled water and dried in a shed. The dried berries were put in a Soxhlet apparatus and the plant extract was prepared using chloroform: methanol (1:1, v/v), (extraction period 72 h and the temperature was <40°C). Total yield of chloroform: methanol (1:1, v/v) solvent extract was 4.53 g. Similar types of bioassay were conducted with chloroform: methanol (1:1, v/v) solvent extract with concentrations of 30, 50, 80, 100 and 120 μg/ml on 1^st to 4^th instars larvae. Higher mortality was recorded at 120 μg/ml for all instars larvae and it was significantly (P<0.05) higher than the mortality rates at 30, 50, 80 and 100 μg/ml concentrations at 24, 48 and 72 h of exposures (Table II). Results of regression analysis revealed that the mortality rate (Y) was positively correlated with the period of exposure (X) having a regression coefficient close to one in each case (Table III). The results of Finney's log probit analysis6 (95% confidence level) revealed that LC₅₀ values gradually decreased with the exposure periods having the lowest value at 72 h of exposure to 3^rd instars larvae, followed by 1^st, 2^nd and 4^th instars larvae (Table III). From thin layer chromatography (TLC) plates of chloroform: methanol (1:1, v/v) solvent extract, an unsaturated spot developed in the iodine chamber having R_f= 0.924 and larvicidal bioassay with this compound resulted more than 45 per cent mortality to all the instars at a concentration of 15 μg/ml. The results of preliminary phytochemical analysis of the chloroform: methanol (1:1, v/v) extract of the mature berries was carried out using standard methods of Harbone and Stahl78. The phytochemicals tested in the study were saponins, steroid, terpenoids, flavonoids, alkaloid, essential oils, phenolics and amino acids, of which alkaloids, steroids, flavonoids and steroidal glycosides were present in berries. The effect of the chloroform: methanol (1:1, v/v) extract was tested against two non-target invertebrates Diplonychus annulatum (5^th instar larval forms) and Chironomus circumdatus. There was no change in the survival rate and swimming activity when these were exposed to the LC₅₀ values at 24 h of chloroform: methanol (1:1, v/v) extract. From functional group analysis, it seemed that the bioactive compound was aromatic amide in nature.

Table I Mean larval mortality of mosquito larvae of different instars of Cx. quinquefaciatus exposed to different concentrations of crude extracts of berries of S. nigrum (mean of three experiments)

Table II Result of larval mortality of different concentrations of chloroform: methanol (1:1, v/v) solvent extract of berries of S. nigrum on all instar of Cx. quinquefasciatus (mean of three experiments)

Table III Log-probit analysis and regression analysis of larvicidal activity of chloroform: methanol (1:1, v/v) solvent extracts of berries of S. nigrum against different instar larval forms of Cx. quinquefasciatus

Phytochemicals are basically the secondary metabolites from plants that serve as a means of defense mechanism of the plants against herbivore predators and other environmental factors. Applications of phytochemicals in mosquito control were in use from 19009. Roark10 described approximately 1,200 plant species having potential insecticidal value, while Sukumar et al11 listed and discussed 344 plant species that only exhibited mosquitocidal activity. Shallan et al12 reviewed the current state of knowledge on larvicidal plant species. Kishore et al13 reviewed the chemical nature of several plant derived secondary materials, having larvicidal potentiality. Changes in the mosquitocidal potentiality of plant parts according to the choice of solvent used for extraction of bioactive substances have also been described14. But, most of the studies were based on laboratory evaluation of the plant parts with no effort to determine the structure and commercial production of active ingredient for wide field application.

In the present study, at a very low concentration of 120 μg/ml, the chloroform: methanol (1:1, v/v) solvent extract of green berry of S. nigrum resulted in 80 per cent mortality of 3^rd instar larvae after 72 h of exposure which indicates its biocontrol potentiaity against Cx. quinquefaciatus. The qualitative and chromatographic study of green berries of S. nigrum revealed the presence of several secondary compounds. Infra Red (IR) spectra of the bioactive compounds indicated that any aromatic amide compound was responsible for the larval mortality. Leaves of S. nigram and S. villosum have been previously reported to have mosquito larvicidal properties1516. As no mortality occurred in the non-target organisms it can be assumed that the plant extracts are safe to use in the aquatic ecosystem. Considering its high target specific toxicity, biodegradable nature and no toxicity to non target organisms, the isolated bioactive ingredient may be a potential larvicide in the control of mosquito larvae. However, further research needs to be carried out to determine the mode of action and elucidate the actual structure of active ingredients present in the plant.