Research Article
Synthesis of copper nanoparticles (CuNPs) from petal extracts of marigold (Tagetes sp.) and sunflower (Helianthus sp.) and their effective use as a control tool against mosquito vectors 
,
Amita Hajra 
Author Correspondence author
Journal of Mosquito Research, 2016, Vol. 6, No. 19 doi: 10.5376/jmr.2016.06.0019
Received: 07 May, 2016 Accepted: 25 Jun., 2016 Published: 23 Jul., 2016
N.K. Mondal, and A. Hajra, 2016, Synthesis of copper nanoparticles (CuNPs) from petal extracts of marigold (Tagetes sp.) and sunflower (Helianthus sp.) and their effective use as a control tool against mosquito vectors, 6(19): 1-9 (doi: 10.5376/jmr.2016.06.0019)
Synthesis and characterization of CuNPs using petal extract of marigold (Tagetes sp.) and sunflower (Helianthus sp.) and mosquito larval mortality was studied by synthesized CuNPs against Culex quinquefasciatus. The CuNPs was synthesized by petal extract of marigold and sulflower. The characterization of CuNPs was done with visual colour change, UV-Vis spectrum, Scanning Electron Micrograph (SEM), Fourier transform infrared (FTIR) spectroscopy and phase contrast micrograph. Larvaecidal study was conducted as per standard method. It was recorded that blue colour of copper sulphate changes to reddish colour in aqueous medium. The Surface Plusmon Resonance (SPR) for marigold and sunflower mediated CuNPs shows absorption at 374 nm and 315 nm, respectively. SEM and phase contrast micrograph picture showed that CuNPs are spherical in nature. Moreover, FTIR spectra indicate the different functional groups of biomoleculs involved in the formation and stabilization of CuNPs. Results also revealed that synthesized nanoparticles showed mosquito larval mortality rate 98.8 % and 70 % with marigold and sunflower mediated CuNPs, respectively. Present results suggest that green synthesis of CuNPs has immense potentiality to kill the mosquito larvae of Culex quinquefasciatus. The novelty of this work is that for the first time marigold and sunflower petal extract was used for the synthesis of CuNPs.
1 Introduction
Nanoparticles are defined as the materials in the nano range with at least one external dimension in the range of 1–100 nm. Copper nanoparticles (CuNPs) have drawn special attention among the scientific community due to its versatile application such as optical, mechanical, catalytic, electrical and thermal conduction properties and these properties are entirely different from their bulk materials. There are many methods available in the literature for the synthesis of CuNPs like precipitation, decomposition, plasma method, sol-gel, vapour deposition, electrochemical and so on (Suleiman and Mausa, 2013). However, most of these methods have serious draw backs like extensive use of hazardous organic solvents, toxic by product, difficulty of separation, preparation time etc. To overcome the above mentioned problems, nanotechnology had been introduced. The methodology is rapidly increasing because of their size which brings a change of physicochemical and optical properties of metals (Angrasan and Subbaiya, 2014). In nanotechnology, many approaches have been used for synthesis of CuNPs such as physical, chemical and biological. Among these, biological synthesis of CuNPs has been always a challenge for researcher due to their easy oxidation (Shende et al., 2015). However, there are several reports (Angrasan and Subbaiya, 2014; Shobha et al., 2014; Mittal et al., 2016) available for heavy metal nanoparticles synthesized by using biological agents such as plants, bacteria, fungi etc. Lee et al., (2013) reported in their paper that stable CuNPs can be synthesized from the aqueous solution of copper sulphate pentahydrate solution with Magnoliakobus leaf extract. Kulkarni and Kulkarni (2013) developed stable CuNPs by green route using Ocimum sanctum leaf extract. Very recently author published their cadmium nanoparticles synthesized by flower extract (Hajra et al., 2015). Similarly, Mittal et al. (2014) published a review work and suggested that plant can be used as nano-factories for synthesis of heavy metal nanoparticles like CuNPs. Pertinent literature also highlighted that plant materials can extensively utilized for the synthesis of CuNPs such as Capparis zeylanica (Saranyaadevi et al., 2014), Gloriosa subbaiya L. (Naika et al., 2015), Vitisvinifera (Angrasan and Subbaiya, 2014), Nerium oleander (Gopinath et al., 2014), Artabotrys odoratissimus (Khathad and Gajera, 2014) etc.
In subtropical and tropical countries, it is a great challenge to control the vector-borne disease (Klemper et al., 2007). Mosquitoes are the major vectors causing several diseases among humans and animals worldwide (Marimuthu et al., 2013). Many substances like mosquito coils, chemical insecticides(themiphos, phenthions, methoprenes) are available in the market to overcome the mosquito bite. However, almost all substances are tremendous toxic for both infant and adult. In view of the increasing resistance of mosquitoes to chemical insecticides, a new biocontrol measure has been undertaken. The use of green synthesized nanoparticles to control the mosquito larvae is well documented (Hajra et al., 2015; Mondal et al., 2014; Marimuthu et al., 2011).
Therefore, the development of a novel method for the preparation of CuNPs is inevitable. Moreover, CuNPs is also considered safe for human beings for application such as food package and in water treatment (Subhankari and Nayak, 2013; Saranyaadevi et al., 2014). CuNPs are attractive to many researchers due to its low cost compared to novel metals such as Ag, Au and Pt (Kulkarni et al., 2015). With this idea, synthesis of CuNP by flower petal extracts, their characterization and application as mosquito larvicide was carried on.
2 Materials and Methods
2.1 Collection of plant material
Flowers of marigold (Tagetes sp.) and sunflower (Helianthus sp.) were collected from garden of Department of Environmental Science, Burdwan University (23.2383 ˚N, 87.8608 ˚E), Burdwan, West Bengal, India.
2.2 Preparation of copper sulphate solution
The required amount of copper sulphate pentahydrate (Merck Private Limited) was dissolved in double distilled water and stored in Borosil made glass container.
2.3 Preparation of flower petal extract
About 5 g dried flower petal of sunflower and marigold petal were crushed and mixed with distilled water (45 mL) and boiled for 10~15 min. The hot extract was cooled and filtered with wh atman No.1. The filtered extract was stored at 4˚C for further used. The sterility of the prepared extracts was strictly maintained without any contamination (Awwad et al., 2013). The extract was then kept in refrigerator to use within 1 week.
2.4 Green synthesis of CuNPs
For the synthesis of CuNPs, heated CuSO4 solution (1 mM) was added to the heated flower petal extract at 1:1 ratio and stirred with glass rod for 15 min and heated the mixture in heating mantle for 50 min at 80˚C. The color of the solution changes to reddish yellow in color indicating the formation of CuNPs.
2.5 Characterization of CuNPs
The synthesized CuNPs was primarily detected by visual observation of colour change. CuNPs were detected from the spectra by UV-Vis spectrometer (Perkin Elmer, Lamda 35) where reaction mixture was subjected to optical analysis from 300 to 700 nm at the optical path of 1nm. FT-IR (Tensor-27 Bruker) analysis was carried out which is responsible for the reduction of copper ions with the spectral range of 400~4000/cm.The CuNPs were subjected to scanning electron microscopic (SEM) study. The SEM characterization was carried out using a scanning electron microscope (Hitachi, S-530) at an accelerating voltage of 20.0 kV.
2.6 Collection of mosquito larvae and maintenance of mosquito culture
Larvae of Culex quinquifasciatus were cultured in laboratory conditions. The eggs of A. albopictus were collected from different locations (waste tires, earthen pots, discarded jars). These were brought to the laboratory and transferred (in approximately the same aliquot numbers of eggs) to (90 cm L x 90 cm H x 90 cm W) enamel trays containing 500 ml of water where they were allowed to hatch. Mosquito larvae were reared (and adult mosquitoes held). Larvae were fed ground dog biscuit and brewer’s yeast daily in 3:1 ratio. Pupae were collected and transferred to plastic containers with 500 mLof water. The container was placed inside a screened cage (90 cm L x 90 cm H x 90 W) to retain emerging adults, for which 10% sucrose in water solution were given. After emergence, the mosquitoes were provided blood feeding. The adults were identified using standard identification keys following Service (1976) and Laird (1988). Glass Petri dishes lined with filter paper and containing 50 mL of water were subsequently placed inside the cage for ovipositing (Arjunan et al., 2012). After emergence of larva from the eggs of fourth instar stage of A. albopictus larva were taken for mortality test. The larvicidal activity was assessed by the procedure of WHO (1996) with some modifications Rahuman et al. (2000).
2.6 Mosquitocidal activity of green synthesized copper nanoparticles
Twenty larvae (fourth instars) was placed in 249 mL of de-chlorinated water in a 500 mL glass beaker, and CuNPs was added in different beakers according to the dose. Tests of each concentration (1, 5 and 10 ppm) against each instar were replicated thrice. In each case, the control comprised 20 larvae in 250 mLof distilled water (Arjunan et al. 2012). Control mortality was corrected by using Abbott’s formula (Abbott 1925), and percentage mortality was calculated as follows:
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2.7 Statistical analysis
The entire experimental results were analysed by using MINITAB 16 software. The mean, standard deviation, one way ANOVA and Tukey’s honestly significant difference test were introduced in this study.
3 Results
3.1 Colour change of CuNPs
The sky blue color of copper sulphate (Figure 1a) gradually changes to light yellow to reddish yellow colour after addition of both marigold and sunflower petal extract. However, most prominent colour change was recorded by marigold petal extracts than sunflower petal extracts (Figure 1b, c). The formation of reddish colour after immediate addition of flower petal clearly indicates the formation of CuNPs.
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Figure 1 (a) aqueous solution of CuSO4 (b) mixture of CuSO4 solution and marigold petal extract (c) mixture of CuSO4 solution and sunflower petal extract |
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3.2 UV-Vis spectroscopy
The reduction of Cu2+ ions to copper nanoparticles was monitored by UV-Vis spectroscopy. The Surface Plasmon Resonance showed a distinct peak as 374 nm and 315 nm for marigold and sunflower mediated CuNPs, respectively (Figure 2a, b).
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Figure 2 UV-Vis spectrum of the synthesized CuNPs from a) marigold (Tagetes sp.) and b) sunflower (Helianthus sp.) |
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3.3 Scanning Electron Micrograph (SEM) and phase contrast microscopy
The surface morphology of synthesized CuNPs from both marigold and sunflower petal extracts showed spherical nature and their size ranges from 5-20 nm. Moreover, nanoparticles are distinctly separated from each other i.e., the synthesized CuNPs are not agglomerated for nanoparticles from marigold (Figure 3a). However, sunflower mediated CuNPs showed moderate agglomeration (Figure 3b). The spherical nature of CuNPs also confirmed from the phase contrast microscopic study (Figure 4a, b).
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Figure 3 (a) SEM image of Cu NPs from marigold petal extract (b) SEM image of Cu NPs from sunflower petal extract |
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Figure 4 (a) Phase contrast photograph of CuNPs obtained from (b) Phase contrast photograph of CuNPs obtained from marigold extractSunflower extract |
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3.4 FT-IR study
FTIR study is basically used to identify the biomolecules involved for reduction of metal ions and its stabilization through capping action. From the FT-IR study it is clear that both marigold and sunflower mediated CuNPs showed distinct peaks at 3340/cm and 3265/cm represent the functional groups such as alcoholic or phenolic –OH stretching (Figure 5a, b).
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Figure 5 (a) IR spectra of Cu NPs produced by Marigold extract (b) IR spectra of Cu NPs produced by Sunflower extract |
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3.5 Study of mosquito larvicidal activity
The larvicidal activity of CuNPs synthesized from marigold petal extracts (Table 1) showed highest (98.9%) at 72 h of incubation than the CuNPs synthesized from sunflower petal extract (70%) (Table 2). Results also revealed that both marigold and sunflower mediated CuNPs showed increasing mosquito larval mortality with increasing concentration of CuNPs from 1 mg/L to 10 mg/L. However, maximum significant (p<0.05) mortality was recorded at higher concentration (10 mg/L) for both petal extract of marigold and sunflower mediated CuNPs at 24 h incubation. Again, during 72 h of incubation, marigold mediated CuNPs showed almost 80% enhancement of mortality. However, when concentration increased to 10 mg/L, only 34.48% mortality increased with respect to 1 mg/L CuNPs. On the other hand, mortality rate with sunflower mediated CuNPs showed only 15% and 23% during 24 h and 72 h of incubation, respectively.
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Table 1 Mean percentage Mortality of Culex quinquifasciatus by copper nanoparticles synthesized by marigold (Tagetes erecta) petal extract |
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Table 2 Mean percentage Mortality of Culex quinquifasciatusby copper nanoparticles synthesized by sunflower (Helianthus) petal extract |
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3.6 Comparative study
The larvicidal activity of heavy metal nanoparticles are presented in Table 4. From Table 4, it is clear that almost all heavy metals including Zn, Cu, Cd, Co, Ag, Au and Ni showed significant larvicidal effect. Among the heavy metals only gold nanoparticles showed very low dose of LC50 value (1.08 mg/L) followed by NiNPs (5.84 mg/L). However, other nanoparticles such as ZnNPs, CdNPs, AgNPs and CoNPs showed moderate larvicidal effect. On the other hand, SiNPs showed very low efficacy towards larvicidal activity with LC50 value 112.5 mg/L (Table 3).
.png) Table 3 Comparative study of larvicidal activity of different synthesized nanoparticles |
4 Discussion
In the present study, the colour of copper sulphate solution changes to reddish colour is due to reduction of Cu2+ ions to Cu0 (Shende et al., 2014) Almost similar SPR of CuNPs was reported by Shende et al. (2014). They synthesized CuNPs by Citrus media Linn (Idilimbu) juice. The reducing agent such as flavonoids, alkaloids and carotenoids which present in the petal extracts of marigold (Hajra et al., 2015) and sunflower are responsible for reduction of copper ions. However, Hajra et al. (2015) in their earlier paper also reported that marigold flower also contain 5-(3-buten-lynyl) 2,2-bithienyl and alpha terthienyl12. The FTIR study also justifies the presence of different biomolecules with active functional groups. The distinct peaks of the active biomolecules such as tannins, flavonoids, alkaloids and cartenoids which are mostly abundant in flower extract and produce CuNPs (Hajra et al., 2015). Earlier researcher (Dhyayananthprabhu et al., 2013) also reported the similar flower based gold nanoparticles. On the other hands, the bands at 1635/cm and 1636/cm are probably due to carbonyl group of flavonoids play vital role towards capping the CuNPs. From this study it is also clear that phenolic compounds have stronger ability to bind CuNPs and prevent agglomeration through subsequent stabilization of CuNPs. Moreover, this study also suggests that the biomolecules which are present in the flower petals could possibly dual functions towards formation of CuNPs and its stabilization (Kulkarni et al., 2015). The shapes of the synthesized CuNPs are spherical in nature which was proved from SEM and phase contrast microscopy picture. However, SEM picture demonstrate that both the petal extracts do not equally stabilize the CuNPs, as a matter of fact, to some extent agglomeration occur. Without presence of capping agents, the micron size metal particles which may be because of the agglomeration or formation of aggregates of CuNPs (Shende et al., 2014). Nowadays, people normally use different toxic chemicals to control mosquito. All these chemicals are different types of insecticides. These insecticides are carbamate, organophosphate and pyrethroids (Boarse et al., 2013). However, recent research highlighted the extensive use of metal nanoparticles as mosquito control agent (Hajra et al., 2015; Mondal et al., 2014; Boarse et al., 2013). Very recently Kumar et al. (2016) pointed out that biosynthesized silver nanoparticles can be effectively used to kill the larvae and pupae of Ae. Albopictus. A growing body of evidences towards synthesis of heavy metal nanoparticles such as silver nanoparticles (AgNPs) showed remarkable larvicidal and pupicidal toxicity against mosquito vectors (Benelli et al., 2016; Murugan et al 2016). Soni and Prakash (2014) reported that AgNPs and AuNPs can be effectively utilized for the control of mosquito. Similarly mosquito larvae can also be controlled by cobalt nanoparticles (Marimuthu et al., 2014). Many previous literature (Rajakumar and Rahuman, 2011; Santhoshkumar et al., 2011) reported that the biosynthesized silver nanoparticles can be effectively utilized to kill the mosquito larvae. In this study we have recorded moderate mortality with CuNPs. However, these results are not as promising as reported by earlier researcher (Mondal et al., 2014) who conducted same experiment with silver nanoparticles. Hajra et al. (2015) reported in their earlier paper that the mortality of A. albopictus is 68.9% with CdNPs (10 mg/L) at 24 h and 100% mortality was recorded after 72 h of incubation with same concentration of CdNPs. Our present results showed much better performance of mosquito larval mortality rate than the CdNPs prepared from rose petal (Table 3). On the other hand, mosquito larval mortality with only cadmium chloride salt showed almost similar mortality with aqueous copper sulphate solution (Table 4)
.png) Table 4 Mean percentage mortality of A. albopictus by copper sulphate salt (CuSO4.5H2O) |
5 Conclusion
This study report that green synthesis of CuNPs from aqueous extracts of sunflower and marigold flower petals. Finally, the application of CuNPs towards mosquito larvicidal effect in laboratory conditions clearly suggest that marigold mediated CuNPs showed better performance than sunflower mediated CuNPs. Therefore, flower petal extract mediated CuNPs could be an effective and alternative method to control the mosquito. Moreover, this method has merits over other reported methods because of easy availability of plant materials, simple reaction condition, use of aqueous medium as solvent, avoidance of use of expensive, hazardous and toxic reagent as well as it is a pollution free method.
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