Parasitol Res DOI 10.1007/s00436-014-3811-2
ORIGINAL PAPER
Larvicidal potential of silver nanoparticles synthesized from Leucas aspera leaf extracts against dengue vector Aedes aegypti Ganesan Suganya & Sengodan Karthi & Muthugounder S. Shivakumar
Received: 5 November 2013 / Accepted: 2 February 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Vector-borne diseases caused by mosquitoes are one of the major economic and health problems in many countries. The Aedes aegypti mosquito is a vector of several diseases in humans like yellow fever and dengue. Vector control methods involving the use of chemical insecticides are becoming less effective due to development of insecticides resistance, biological magnification of toxic substances through the food chain, and adverse effects on environmental quality and non-target organisms including human health. Application of active toxic agents from plant extracts as an alternative mosquito control strategy was available from ancient times. These are nontoxic, easily available at affordable prices, biodegradable, and show broad-spectrum targetspecific activities against different species of vector mosquitoes. Today, nanotechnology is a promising research domain which has wide-ranging application vector control programs. The present study investigates the larvicidal potential of solvent leaf extracts of Leucas aspera and synthesized silver nanoparticles using aqueous leaf extract against fourth instar larvae of Aedes aegypti. Larvae were exposed to varying concentrations of plant extracts and synthesized AgNPs for 24 h. The results were recorded from UV–Vis spectra, x-ray diffraction (XRD), Fourier transform infrared (FT-IR), and scanning electron microscopy (SEM), and were used to characterize and support the biosynthesis of silver nanoparticles. The formation of the AgNPs synthesized from the XRD spectrum compared with Bragg reflections can be indexed to the (111) orientations, respectively, confirmed the presence of AgNPs. The FT-IR spectra of AgNPs exhibited prominent peaks at 3,447.77; 2,923.30; and 1,618.66 cm−1. The spectra showed sharp and strong absorption band at 1,618.66 cm−1 G. Suganya : S. Karthi : M. S. Shivakumar (*) Molecular Entomology Laboratory, Department of Biotechnology, Periyar University, Salem, Tamil Nadu, India e-mail: [email protected]
assigned to the stretching vibration of (NH) C═O group. The band 1,383 developed for C═C and C═N stretching, respectively, and was commonly found in the proteins. SEM analysis of the synthesized AgNPs clearly showed the clustered and irregular shapes, mostly aggregated, and having the size of 25–80 nm. Energy-dispersive x-ray spectroscopy showed the complete chemical composition of the synthesized AgNPs. In larvicidal activity, the results showed that the maximum efficacy was observed in synthesized AgNPs leaf extracts against the fourth instar larvae of A. aegypti (LC50 values of 8.5632, 10.0361, 14.4689, 13.4579, 17.4108, and 27.4936 mg/l) and (LC90 values of 21.5685, 93.03928, 39.6485, 42.2029, 31.3009, and 53.2576 mg/l), respectively. These results suggest that the synthesized AgNPs leaf extracts have a higher larvicidal potential as compared to crude solvent extracts thus making them an effective combination for controlling A. aegypti. Keywords Aedes aegypti . Nanoparticles . Leucas aspera . Plant extracts . AgNPs
Introduction Mosquitoes serve as a vector of numerous diseases throughout the world such as malaria, dengue, chikungunya, filariasis, Japanese encephalitis, and leishmaniasis (WHO 2009). Mosquito-transmitted diseases kill about one million people every year. Aedes aegypti is a vector of dengue, yellow fever, and chikungunya. The Aedes aegypti mosquito is an urbandwelling mosquito, which is a leading cause for transmitting dengue in Southeast Asia (Gubler 1998). Insecticide application although highly effective against Aedes aegypti, as vector control still faces a threat due to the development of resistance to chemical insecticides, resulting in rebounding vectorial
Parasitol Res
capacity (Hidayati et al. 2011). Plants have a rich source of secondary metabolites, many of which have been thought to possess mosquitocidal properties; more over, due to their biodegradability, they are considered good candidates for controlling mosquitoes (Amer and Mehlhorn 2009; Rahuman et al. 2008). Secondary metabolites from angiosperms have been shown to possess good larvicidal efficacy against mosquitoes viz Culex quinquefasciatus, Aedes aegypti, and Anopheles stephensi (Govindarajan 2010; Elango et al. 2009; Bansal et al. 2009). Leucas aspera is a small, herbaceous erect plant with a free blooming nature and flowers in the months of August to September. It is pungently aromatic and commonly used as an antipyretic herb in South India. The juice from the leaves is used as an external application for psoriasis and painful swellings. The flowers are given with honey to treat coughs and cold in children. The leaves are useful for the treatment of chronic rheumatism. Bruised leaves are applied to the bites of serpents, poisonous insects, and scorpion sting. The plant extract with honey is a good remedy for stomach pain and indigestion. L. aspera leaves are used as an insecticide and mosquito repellent in rural areas (Maheswaran et al. 2008a, b). The preliminary chemical examination of L. aspera shows the presence of triterpenoids (Kamat and Singh 1994), oleanolic acid, ursolic acid, and 3-itosterol (Chaudhury and Ghosh 1969); aerial parts contains nicotine (Mangathayaru et al. 2006), sterols (Khaleque et al. 1970), two new alkaloids (compound A m.p. 61–2 °, α-sitosterol, and β-sitosterol) (m.p. 183–4 °), reducing sugars (galactose), glucoside (230–1 °) (Chatterjee and Majumdar 1969), diterpenes (leucasperones A and B, leucasperols A and B, isopimarane glycosides (leucasperosides A, B, and C), together with other compounds like asperphenamate, maslinic acid, (−)-isololiolide, linifolioside, nectandrin B, meso-dihydroguaiaretic acid, macelignan, acacetin, apigenin 7-O-[6′-O-(p-coumaroyl)-3-Dglucoside], chrysoeriol, apigenin, erythro-2-(4-allyl-2,6dimethoxyphenoxy)-1-(4-ydroxy-3-methoxyphenyl)propan-1ol, myristargenol B, and machilin C, (−)-chicanine, (7R,8R), and (7S,8S)-licarin A (Sadhu et al. 2003). Among the 25 compounds identified from the leaf volatiles, u-farnesene (26.4 %), x-thujene (12.6 %), and menthol (11.3 %) were the major constituents. The flower is reported to contain ten compounds; among them, amyl propionate (15.2 %) and isoamyl propionate (14.4 %) were dominant (Kalachaveedu et al. 2006). L. aspera leaves are used as an insecticide and mosquito repellent in rural areas (Reddy et al. 1993; Sadhu et al. 2003). The hexane crude extracts of L. aspera showed highest larvicidal activity against Culex quinquefasciatus and Aedes aegypti (Maheswaran et al. 2008a, b; Kovendan et al. 2011). Developments in nanotechnology have opened new avenues for integrating nanotechnology with plants molecules for effective delivery of bioactive molecules for mosquito control. Nanoparticles can be synthesized rapidly at low cost and are
eco-friendly, by a single-step method for biosynthesis process (Kovendan et al. 2012). Silver nanoparticles (AgNPs) are considered ideal for the study, as silver is safe for humans and non target animals. Plant-mediated nanoparticle synthesis is preferred as it is cost-effective, environmentally friendly, and safe for human therapeutic use (Kumar and Yadav 2009). Few of the studies done using green AgNPs have been synthesized using various natural products like Azadirachta indica (Tripathi et al. 2009), Glycine max (Vivekanandhan et al. 2009), and Cinnamon zeylanicum (Sathishkumar et al. 2009), such studies could prove to have an enormous impact in the immediate future of pest control. In the present study, we compared the bioefficacy of AgNPs-synthesized aqueous leaf extracts of L. aspera with the solvent extract against Aedes aegypti larvae.
Materials and methods Materials Fresh leaves of L. aspera were collected from Yercaud hills (latitude 11° 76′ E; longitude 78° 23′ E;) area, Tamil Nadu (Fig. 1). Preparation of Leucas aspera leaf aqueous extract The leaves were dried for 7–14 days in the shade at the environmental temperatures (27–37 °C daytime). The dried leaves (100 g) were powdered mechanically using a commercial electrical stainless steel blender and extracted with chloroform (450 ml), ethyl acetate (400 ml), petroleum ether (500 ml), and methanol (1,500 ml) in a Soxhlet apparatus (boiling point range 60–80 °C) for 8 h. The extract was concentrated under reduced pressure of 22–26 mmHg at 45 °C, and the residue obtained was stored at 4 °C. Aqueous extract was prepared by mixing 50 g of dried leaf powder with
Fig. 1 Leucas aspera (Wild.) Linn. (Lamiaceae)
Parasitol Res Table 1 Larvicidal efficacy of AgNP-synthesized L. aspera crude leaf extract and solvent extracts against fourth instar larvae of A. aegypti
Aqueous extract + AgNO3 Solvent extracts of Leucas aspera
Aqueous extract
Extracts name
(n)
LC50 mg/l
LC90 mg/l
LCL
UCL
χ2
df
AgNO3 Methanol Petroleum ether Chloroform Ethyl acetate Extracts
75 75 75 75 75 75
8.5632 10.0361 14.4689 13.4579 17.4108 27.4936
21.5685 93.03928 39.6485 42.2029 31.3009 53.2576
7.2411 2.0462 6.2794 9.2909 10.3498 2.4347
10.4942 22.119 5.3497 17.6214 24.4716 7.0387
5.8501 0.1375 0.8332 6.0365 0.0009 9.2672
1 1 1 1 1 1
Control—Nil mortality. Significant atp
ORIGINAL PAPER
Larvicidal potential of silver nanoparticles synthesized from Leucas aspera leaf extracts against dengue vector Aedes aegypti Ganesan Suganya & Sengodan Karthi & Muthugounder S. Shivakumar
Received: 5 November 2013 / Accepted: 2 February 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Vector-borne diseases caused by mosquitoes are one of the major economic and health problems in many countries. The Aedes aegypti mosquito is a vector of several diseases in humans like yellow fever and dengue. Vector control methods involving the use of chemical insecticides are becoming less effective due to development of insecticides resistance, biological magnification of toxic substances through the food chain, and adverse effects on environmental quality and non-target organisms including human health. Application of active toxic agents from plant extracts as an alternative mosquito control strategy was available from ancient times. These are nontoxic, easily available at affordable prices, biodegradable, and show broad-spectrum targetspecific activities against different species of vector mosquitoes. Today, nanotechnology is a promising research domain which has wide-ranging application vector control programs. The present study investigates the larvicidal potential of solvent leaf extracts of Leucas aspera and synthesized silver nanoparticles using aqueous leaf extract against fourth instar larvae of Aedes aegypti. Larvae were exposed to varying concentrations of plant extracts and synthesized AgNPs for 24 h. The results were recorded from UV–Vis spectra, x-ray diffraction (XRD), Fourier transform infrared (FT-IR), and scanning electron microscopy (SEM), and were used to characterize and support the biosynthesis of silver nanoparticles. The formation of the AgNPs synthesized from the XRD spectrum compared with Bragg reflections can be indexed to the (111) orientations, respectively, confirmed the presence of AgNPs. The FT-IR spectra of AgNPs exhibited prominent peaks at 3,447.77; 2,923.30; and 1,618.66 cm−1. The spectra showed sharp and strong absorption band at 1,618.66 cm−1 G. Suganya : S. Karthi : M. S. Shivakumar (*) Molecular Entomology Laboratory, Department of Biotechnology, Periyar University, Salem, Tamil Nadu, India e-mail: [email protected]
assigned to the stretching vibration of (NH) C═O group. The band 1,383 developed for C═C and C═N stretching, respectively, and was commonly found in the proteins. SEM analysis of the synthesized AgNPs clearly showed the clustered and irregular shapes, mostly aggregated, and having the size of 25–80 nm. Energy-dispersive x-ray spectroscopy showed the complete chemical composition of the synthesized AgNPs. In larvicidal activity, the results showed that the maximum efficacy was observed in synthesized AgNPs leaf extracts against the fourth instar larvae of A. aegypti (LC50 values of 8.5632, 10.0361, 14.4689, 13.4579, 17.4108, and 27.4936 mg/l) and (LC90 values of 21.5685, 93.03928, 39.6485, 42.2029, 31.3009, and 53.2576 mg/l), respectively. These results suggest that the synthesized AgNPs leaf extracts have a higher larvicidal potential as compared to crude solvent extracts thus making them an effective combination for controlling A. aegypti. Keywords Aedes aegypti . Nanoparticles . Leucas aspera . Plant extracts . AgNPs
Introduction Mosquitoes serve as a vector of numerous diseases throughout the world such as malaria, dengue, chikungunya, filariasis, Japanese encephalitis, and leishmaniasis (WHO 2009). Mosquito-transmitted diseases kill about one million people every year. Aedes aegypti is a vector of dengue, yellow fever, and chikungunya. The Aedes aegypti mosquito is an urbandwelling mosquito, which is a leading cause for transmitting dengue in Southeast Asia (Gubler 1998). Insecticide application although highly effective against Aedes aegypti, as vector control still faces a threat due to the development of resistance to chemical insecticides, resulting in rebounding vectorial
Parasitol Res
capacity (Hidayati et al. 2011). Plants have a rich source of secondary metabolites, many of which have been thought to possess mosquitocidal properties; more over, due to their biodegradability, they are considered good candidates for controlling mosquitoes (Amer and Mehlhorn 2009; Rahuman et al. 2008). Secondary metabolites from angiosperms have been shown to possess good larvicidal efficacy against mosquitoes viz Culex quinquefasciatus, Aedes aegypti, and Anopheles stephensi (Govindarajan 2010; Elango et al. 2009; Bansal et al. 2009). Leucas aspera is a small, herbaceous erect plant with a free blooming nature and flowers in the months of August to September. It is pungently aromatic and commonly used as an antipyretic herb in South India. The juice from the leaves is used as an external application for psoriasis and painful swellings. The flowers are given with honey to treat coughs and cold in children. The leaves are useful for the treatment of chronic rheumatism. Bruised leaves are applied to the bites of serpents, poisonous insects, and scorpion sting. The plant extract with honey is a good remedy for stomach pain and indigestion. L. aspera leaves are used as an insecticide and mosquito repellent in rural areas (Maheswaran et al. 2008a, b). The preliminary chemical examination of L. aspera shows the presence of triterpenoids (Kamat and Singh 1994), oleanolic acid, ursolic acid, and 3-itosterol (Chaudhury and Ghosh 1969); aerial parts contains nicotine (Mangathayaru et al. 2006), sterols (Khaleque et al. 1970), two new alkaloids (compound A m.p. 61–2 °, α-sitosterol, and β-sitosterol) (m.p. 183–4 °), reducing sugars (galactose), glucoside (230–1 °) (Chatterjee and Majumdar 1969), diterpenes (leucasperones A and B, leucasperols A and B, isopimarane glycosides (leucasperosides A, B, and C), together with other compounds like asperphenamate, maslinic acid, (−)-isololiolide, linifolioside, nectandrin B, meso-dihydroguaiaretic acid, macelignan, acacetin, apigenin 7-O-[6′-O-(p-coumaroyl)-3-Dglucoside], chrysoeriol, apigenin, erythro-2-(4-allyl-2,6dimethoxyphenoxy)-1-(4-ydroxy-3-methoxyphenyl)propan-1ol, myristargenol B, and machilin C, (−)-chicanine, (7R,8R), and (7S,8S)-licarin A (Sadhu et al. 2003). Among the 25 compounds identified from the leaf volatiles, u-farnesene (26.4 %), x-thujene (12.6 %), and menthol (11.3 %) were the major constituents. The flower is reported to contain ten compounds; among them, amyl propionate (15.2 %) and isoamyl propionate (14.4 %) were dominant (Kalachaveedu et al. 2006). L. aspera leaves are used as an insecticide and mosquito repellent in rural areas (Reddy et al. 1993; Sadhu et al. 2003). The hexane crude extracts of L. aspera showed highest larvicidal activity against Culex quinquefasciatus and Aedes aegypti (Maheswaran et al. 2008a, b; Kovendan et al. 2011). Developments in nanotechnology have opened new avenues for integrating nanotechnology with plants molecules for effective delivery of bioactive molecules for mosquito control. Nanoparticles can be synthesized rapidly at low cost and are
eco-friendly, by a single-step method for biosynthesis process (Kovendan et al. 2012). Silver nanoparticles (AgNPs) are considered ideal for the study, as silver is safe for humans and non target animals. Plant-mediated nanoparticle synthesis is preferred as it is cost-effective, environmentally friendly, and safe for human therapeutic use (Kumar and Yadav 2009). Few of the studies done using green AgNPs have been synthesized using various natural products like Azadirachta indica (Tripathi et al. 2009), Glycine max (Vivekanandhan et al. 2009), and Cinnamon zeylanicum (Sathishkumar et al. 2009), such studies could prove to have an enormous impact in the immediate future of pest control. In the present study, we compared the bioefficacy of AgNPs-synthesized aqueous leaf extracts of L. aspera with the solvent extract against Aedes aegypti larvae.
Materials and methods Materials Fresh leaves of L. aspera were collected from Yercaud hills (latitude 11° 76′ E; longitude 78° 23′ E;) area, Tamil Nadu (Fig. 1). Preparation of Leucas aspera leaf aqueous extract The leaves were dried for 7–14 days in the shade at the environmental temperatures (27–37 °C daytime). The dried leaves (100 g) were powdered mechanically using a commercial electrical stainless steel blender and extracted with chloroform (450 ml), ethyl acetate (400 ml), petroleum ether (500 ml), and methanol (1,500 ml) in a Soxhlet apparatus (boiling point range 60–80 °C) for 8 h. The extract was concentrated under reduced pressure of 22–26 mmHg at 45 °C, and the residue obtained was stored at 4 °C. Aqueous extract was prepared by mixing 50 g of dried leaf powder with
Fig. 1 Leucas aspera (Wild.) Linn. (Lamiaceae)
Parasitol Res Table 1 Larvicidal efficacy of AgNP-synthesized L. aspera crude leaf extract and solvent extracts against fourth instar larvae of A. aegypti
Aqueous extract + AgNO3 Solvent extracts of Leucas aspera
Aqueous extract
Extracts name
(n)
LC50 mg/l
LC90 mg/l
LCL
UCL
χ2
df
AgNO3 Methanol Petroleum ether Chloroform Ethyl acetate Extracts
75 75 75 75 75 75
8.5632 10.0361 14.4689 13.4579 17.4108 27.4936
21.5685 93.03928 39.6485 42.2029 31.3009 53.2576
7.2411 2.0462 6.2794 9.2909 10.3498 2.4347
10.4942 22.119 5.3497 17.6214 24.4716 7.0387
5.8501 0.1375 0.8332 6.0365 0.0009 9.2672
1 1 1 1 1 1
Control—Nil mortality. Significant atp
