Green synthesis of selenium nanoparticles conjugated Clausena dentata plant leaf extract and their insecticidal potential against mosquito vectors

Abstract

Mosquitoes are major vectors for the transmission of many diseases like chikungunya, malaria, dengue, zika, etc. worldwide. In the present study, selenium nanoparticles (SeNPs) were synthesized from Clausena dentata and were tested for their larvicidal efficacy against the fourth-instar larvae of Anopheles stephensi, Aedes Aegypti, and Culex quinquefasciatus. The synthesized nanoparticles were characterized using UV-Vis spectroscopy, Fourier Transform Infrared Radiation (FTIR) spectroscopy, EDaX, and SEM. The results recorded from UV-Vis spectroscopy show the peak absorption spectrum at 420 nm. In FTIR, the maximum peak value is 2922.25 cm⁻¹ assigned to N-H group (amide group). In EDaX analysis shows peak around 72.64 which confirm the binding intensity of selenium. In SEM analysis, the synthesized SeNPs sizes were ranging from 46.32 nm to 78.88 nm. The synthesized SeNPs produced high mortality with very low concentration (LC₅₀) were 240.714 mg/L; 104.13 mg/L, and 99.602 mg/L for A. stephensi, A. Aegypti, and C. quinquefasciatus, respectively. These results suggest that the C. dentata leaf extract-mediated biosynthesis of SeNPs has the potential to be used as an ideal ecofriendly approach toward the control of mosquito vectors at early stages.

Keywords:

• Selenium
• Clausena dentata
• SEM
• larvicidal activity
• eco-friendly

Introduction

Mosquito-borne diseases are prevalent in more than 100 countries across the world, infecting over 70 million people every year globally and 4 million of the Indian population (Ghosh et al. 2012). Mosquitoes act as a vector for most of the life-threatening diseases like malaria, yellow fever, dengue fever, chikungunya fever, filariasis, encephalitis, zika virus infection, etc. Effective vector control generally relies on the use of chemical insecticides targeting adults or larvae mosquitoes (Killeen et al. 2002). The repeated use of the chemical insecticides fosters many environmental hazards including development of resistance to insecticides.

Clausena dentata is a flowering plant in the citrus family, Rutaceae. It was first defined by the Dutch botanist Nicolaas Laurens Burman in 1768 (Clausena 1768). Genus Clausena is an evergreen tree, occurs in tropical belt of Asia, easily grown by farmers since it is pest and disease free and can withstand heavy lopping (Burkill 1966). Secondary metabolites of this plant were identified for number of biological activities like anti-cancer, antimicrobial, antioxidant, and antidiabetic activity C. dentata (Willd) is a small tree plant, found in many countries such as India, Sri Lanka, and China (Agarwal 1981). This plant is popularly known as “Anai chedi” in Tamil; Susitra et al. (2014) previously reported that C. dentata crude extract is more toxic against mosquitoes.

Nanotechnology is one of the most active research areas in the modern material science. Based upon their specific characteristics such as size, distribution, and morphology, nanoparticles (NPs) have distinct properties compared with the bulk form of the same material. NPs are being viewed as fundamental building blocks of nanotechnology. Nanotechnologies have the potential to revolutionize a great array of applications in the fields of catalysis, sensors, optoelectronics, magnetic devices, drug delivery, antimicrobials, and parasitology (Haverkamp 2010).

Metal NPs are widely used in drug delivery and insect control owing to their distinctive physical and chemical properties (Shaklee 2013). The utilization of nontoxic solvents, biodegradable materials, and low-cost green chemicals is central to resources synthesis and processing. The stabilizer, reaction medium, and green reducing agent are three key factors in the synthesis and stabilization of metallic NPs, and biosynthesized Ag NPs are used in antimicrobial (Duran et al. 2005), anti-viral, and anti-human immunodeficiency virus (anti-HIV) studies (Elechiguerra et al. 2005). Using plants for NP synthesis can be advantageous over other biological processes because it eliminates the elaborate process of maintaining cell cultures and can also be suitably scaled up for large-scale NP synthesis (Shankar et al. 2004). A reproducible but simple method of preparation of stable selenium nanoparticles (SeNPs) with biomedical application is still a challenge (Ramamurthy et al. 2012). Both reduction and oxidation techniques can be employed to prepare SeNPs. The main synthetic approach for preparing SeNPs is by chemical reduction, employing reducing agent, and stabilizer (Sauvaire et al. 1996). However, the use of stabilizer may hinder the normal utilization of synthesized NPs in biological applications and further stabilizer may have toxic potential due to its chemical nature. In the present study, green synthesis of SeNPs with C. dentata leaf extract was done. Further, bioefficacy of this conjugated NP in mosquito control was studied.

Materials and methods

Collection of mosquito larvae

The Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti larvae were procured from CRME, Madurai, India. The larvae were kept in plastic trays containing tap water and were maintained in laboratory, and all the experiments were carried out at 27 + 2 °C and 75–85% relative humidity under 14:10 light and dark cycles; the larvae were fed a diet of yeast and dog biscuits.

Collection of plant materials

Fresh medicinal plant C. dentata leaves were collected from Kalrayan hills, Salem District, Tamil Nadu, India. All the collected plant leafs are washed with tap water to remove unwanted solid dust particle and shade dried at room temperature. Dried leaves were powdered by using commercial electrical stainless steel blender and the powdered leaves.

Preparation of Clausena dentata leaf aqueous extract

About 10 g of C. dentata plant powder has to be taken in 250-ml Erlenmeyer flask along with 100 ml of distilled water and then boil the mixture at 60 °C for 5 min; after boiling, the solvent was decanted and 12 ml of this broth was added to 88 ml of 1 mM aqueous selenium powder, and the resulting solution become brown in color. This extract was filtered through No 1 Whatman filter paper followed by further experiment. A control setup was also maintained without plant extract, and color intensity of the extract was measured at 420 nm.

UV-visible spectrophotometer

The bioreduction SeNPs were monitored by sampling the reaction mixture at regular intervals, and the absorption maxima was scanned by UV-Vis spectra at wavelength of 200–700 nm in spectrophotometer operated at a resolution of 1 nm. The reduction was monitored by measuring the UV-visible spectra of the solution after diluting the small aliquot (0.2 ml) of the sample. The solution mixture was subjected to centrifugation at10,000 rpm for 15 min; resulting pellet was dissolved in distilled water and filtered through filter.

Characterization of selenium nanoparticles

X-ray diffraction (XRD) analysis was carried out on PAN analytical q X-Pert PRO. Diffractometer operating at 40 kV with a current of 30 mA using Cu Ka was used for the determination of purity and crystalline size of the NPs. Size and surface morphology of the SeNPs were examined by JEOL JSM 6390 Scanning Electron Microscope (SEM) instrument operated at an accelerating voltage at 15 kV. For Energy Dispersive X-ray (EDaX) analysis, the particles were dried on a carbon-coated copper grid and performed on SEM instrument with thermo EDaX attachment. The UV-visible spectrum was recorded using JASCO UV-Vis.530 spectrophotometer. The powder sample of green synthesis SeNPs was analyzed by Fourier transform infrared spectroscopy (FT-IR) using a Shimadzu infrared spectrometer (Model 400) with KBr as background over the range of 400–4000 cm⁻¹.

Larvicidal bioassay

The larvicidal activity was carried out as per the methods recommended by World Health Organization (2006). Twenty-five third-instar larvae were transferred to a small disposable paper cup in 100 ml of water and 1.0 ml of the desired SeNPs from Different concentration ranges from (100, 200, 300, 400, and 500 ppm), and controls were set up simultaneously using tap water. The number of dead larvae was counted after 24 h of exposure, and the percentage mortality was reported from average of three replicates. The LC₅₀ (lethal concentration that kill 50% of the exposure larvae) values were calculated after 24 h by probit analysis.

Statistical analysis

The larval mortality data were subjected to probit analysis for calculating LC50, LC90, and other statistics using the SPSS16.0 (Statistical Package of Social Sciences software version 16.0).

Results

Selenium synthesis and UV-spectroscopy

Green synthesis of SeNPs from selenium was confirmed by UV-Vis spectra studies, based on color change from black color to yellowish color which confirm the synthesis of NP (SeNPs), and SeNPs were characterized by UV-Vis spectroscopy; high absorption spectrum was observed 420 nm (Figure 1).

Figure 1. UV-visible absorbance spectrum of SeNPs. (A) Visible color change it indicates that synthesized selnium nanoparticle in aqueous plant extract. (B) SeNPs producing an peak at 420 nm.

FT-IR analysis

FT-IR measurements were carried out to identify the possible band present in the biomolecule responsible for peaks near capping and efficient stabilization of the metal NPs synthesized by leaf extract. The 2922.25 cm⁻¹ assigned to N-H stretching binding of 2554.45 with hydroxyl groups and 1647.24 cm⁻¹ assigned to O-H stretching carboxylic acids. The weaker band at 555.41 cm⁻¹ corresponds to C=C stretch in alkenes group (Figure 2).

Figure 2. FT-IR spectrums of SeNPs synthesized from plant extract.

EDaX analysis

Energy-dispersive micro analysis gives us further insights into the feature of the SeNPs, analysis of the sample was performed using EDaX techniques. The element analysis of the SeNPs was performed using EDaX, based on this bioreduction method. The peaks around 72.64 to the binding energies of selenium were observed. The result indicates that the reaction product is present in the pure form of SeNPs (Figure 3; Table 1).

Figure 3. Energy-dispersive spectroscope of prepared selenium nanoparticles.

Table 1. The element analysis of the SeNPs was performed using EDX. The EDX spectrum prepared by bioreduction methods.

Element	Weight %	Atomic %
C K	23.89	63.63
O K	3.47	6.94
Se L	72.64	29.43
Total	100.00

SEM analysis

The SEM image of SeNPs were synthesized plant extracts, and were assembled on to the surface due to the interaction such as hydrogen bond and electrostatic interaction between the bio-organic capping molecules bound to the SeNPs. The synthesized SeNPs were formed with size ranging from 46.32 nm to 78.88 nm (Figure 4).

Figure 4. SEM micrograph of selenium nanoparticles synthesized by aqueous extracts of Clausena dentata.

Larvicidal bioassay

The mortality rate was observed in C. dentata plant-mediated synthesized SeNPs against in larvae of A. stephensi, A. aegypti, C. quinquefasciatus mosquitoes. Green synthesized-mediated SeNPs show high mortality with very low concentration compared with aqueous solution were 240.714 mg/L; 104.13 mg/L and 99.602 mg/L for A. stephensi, A. aegypti, and C. quinquefasciatus_, respectively (Table 2).

Table 2. LC₅₀, LC₉₀, and chi-square analysis of selenium synthesized nanoparticle from Clausena dentata leaf extract against larvicidal activity of major three mosquito vectors.

Species	Sample	n^a	LC50 LCL-UCL (95% confidence limit), mg/L	LC90 LCL-UCL (95% confidence limit), mg/L	χ^2	df
Anopheles stephensi	SeNPs	375	240.714 (235.18–241.61)	367.006 (361.182–370.012)	0.579	3
	Aqueous	375	313.116 (309.28–316.82)	417.820 (414.162–421.82)	0.703	3
Aedes aegypti	SeNPs	375	104.13 (99.811–106.161)	158.13 (151.032–160.82)	0.426	3
	Aqueous	375	296.73 (291.11–298.12)	450.08 (441.18–453.16)	0.499	3
Culex quinquefasciatus	SeNPs	375	99.602 (95.116–103.82)	153.59 (148.26–159.12)	0.453	3
	Aqueous	375	293.126 (289.72–298.11)	451.754 (443.16–455.16)	0.597	3

LC₅₀: lethal concentration 50% mortality; LC₉₀: lethal concentration 90% mortality; LCL: lower confidence limits; UCL: upper confidence limits; χ²: chi square; df: degrees of freedom.

Discussion

Insecticide application, although highly effective against mosquitoes, yet faces a vector threat due to the development of resistance to chemical insecticide resulting in rebounding vectorial capacity (Li et al. 2007). Emerging and re-emerging mosquito-borne diseases are considered to be major threats to global health in both developing and developed countries. Their tendency of spreading outside their known geographic range and causing large-scale epidemics has been clearly demonstrated during the recent global epidemic of CHIKV (Ng and Hapuarachchi 2010). Zika was a neglected tropical disease, and like CHIKV, interest in ZIKV epidemiology was limited until recently, when its high epidemic potential was demonstrated during a large-scale outbreak in the Pacific Island of Yap in 2007 (Lanciotti et al. 2008). Continuous use of chemical insecticides has hampered the vector control program; successive changes in the insecticides result in multiple insecticide resistant to mosquitoes (Ramkumar and Shivakumar 2015). Although effective, repeated use of these controlling agents has fostered several environmental and health concerns, including disruption of natural biological control systems, outbreaks of other insect species, widespread development of resistance, and undesirable effects on nontarget organisms (Yang 2002). Nowadays nanoparticle synthesis is overcome by plant-mediated biological process, and this bio-route attracts a considerable interest because of its ecofriendliness and biocompatibility (Krumov et al. 2009).

UV-Vis spectroscopy is one of the most widely used techniques for structural characterization of SeNPs. Generally, UV-Vis spectroscopy can be used to examine size and shape of the controlled NPs in aqueous suspense (Shankar et al. 2004). Our result shows that SeNPs synthesized from leaf extract of C. dentata and measured through spectrophotometer ranges from 200 to 700 nm with a peak at 420 nm, indicating the production of SeNPs. FTIR analysis was carried out to identify the possible interactions between silver and bioactive molecules, which may be responsible for synthesis and stabilization (capping) of silver NP indicated the presence of hydroxyl (OH) group, benzene ring, carboxylic (C=O) group, alkyl halide group, respectively. The results of the FTIR used to identify the possible biomolecules responsible for the stabilization of the synthesized SeNPs. The prominent peaks of the FTIR results are showing the corresponding values of the amide group. The value 2922.25 cm⁻¹ assigned to N-H stretching binding of 2554.45 with hydroxyl groups and 1647.24 cm⁻¹ assigned to O-H stretching carboxylic acids. The weaker band at 555.41 cm⁻¹ corresponds to C=C stretch in alkenes group. Similar observation also found as flavonoids, triterpenoids, and polyphenols. Hence, the terpenoids are proved to have good potential activity to convert the aldehyde groups to carboxylic acids in the metal ions.

Energy-dispersive microanalysis to gain further insight into the feature of the SeNPs was performed using EDaX techniques. Our results show that EDaX peaks around 72.64 to the binding energies of selenium. The result indicates that the reaction product in present in the pure form of SeNPs. The EDaX recorded for SeNPs synthesized extracts showed strong signal of selenium from 3 keV. X-ray emission from carbohydrates/proteins/enzymes present within the leaves of C. dentata. Throughout the scanning range of binding energies, there is no peak to detect the impurity. The result of the XRD pattern indicates the presence of sharp bands of Bragg peaks and this might be due to the stabilization of the synthesized NPs by the leaf extract of the C. dentata reducing agents, and thus confirming the crystallization of the bioorganic phase occurs on the surface of the SeNPs.

SEM analysis of the synthesized SeNPs was clearly distinguishable owing to their size difference. SEM image of nanoparticles observed from the micrograph majority are spherical with a small percentage of elongated particles and ranged in size of 46–78 nm. Uniformly distributed SeNPs on the surface of the pellets are observed. The uniform size of selenium suggests that the particles are observed. SEM analysis of SeNPs was clearly distinguishable owing to their size difference of synthesized SeNPs.

Botanicals are basically secondary metabolites that serve as a means of defense mechanism of the plants to withstand the continuous selection pressure from herbivore predators and other environmental factors. Several groups of phytochemical such as alkaloids, steroids, terpenoids, essential oils, and phenolics from different plants have been reported previously for their insecticidal activities (Shaalan et al. 2005). Biosynthetic products or reduced cofactor play an important role in the reduction of respective metal NPs. It seems quite probable that the phenols play an important part in the reduction of ions to SeNPs as the concept of antioxidant action of phenol compounds is not new. Therefore, in combination with mosquito nets or other vector control measures, such plant synthesized SeNPs may have significant impact on malaria and filariasis incidence and can be potential candidates to be considered in integrated vector control programs. Plants synthesized SeNPs, being readily available and their application methods being simple and affordable, may be useful in protecting malaria and filariasis.

Fourth-instar larvae of A. stephensi, A. Aegypti, and C. quinquefasciatus were treated with biosynthesized SeNPs, and the percentage mortality was assessed against various concentrations. The larval mortality rate was observed in C. dentata plant-mediated synthesized SeNPs against in larvae of A. stephensi, A. aegypti, and C. quinquefasciatus mosquitoes with very low LC₅₀ values 240.714 mg/L; 104.13 mg/L, and 99.602 mg/L, respectively (Table 2). Similar studies investigated that the biosynthesized NPs from leaf extract of Leucas aspera showed potential larvicidal activity against A. aegypti larvae (Suganya et al. 2014). Nanoparticle conjugated plant extracts are highly toxic to mosquito larvae and at the same time do not show any toxicity to nontarget aquatic species (Govindarajan and Benelli 2016). Spergularia rubra and Pergularia daemia synthesized AgNPs did not exhibit any evident toxicity effect against fishes, after 48 h of exposure (Patil et al. 2012). AgNPs synthesized Barleria cristata extracts were not toxic against the nontarget organisms (Benelli 2016)

Hence, the larvicidal activity of the SeNPs might be due to the denaturation of the sulfur-containing proteins or phosphorous-containing compound like DNA that leads to the denaturation of organelles and enzymes and thus reduces the cellular membrane permeability and reduction in ATP synthesis which finally causes the loss of the cellular function and cell death (Shankar et al. 2004). Today environmental safety is a vital concern while designing of nanopesticides, in this aspect SeNP mediated plant extracts are safe and do not cause environmental pollution. An insecticide should be ecofriendly in nature and acceptable by the community to cause the desire mortality against target organisms. Phytochemicals from plants are relatively safe, inexpensive, and several plants used in traditional medicines are being explored for mosquito control properties worldwide.

Conclusion

In conclusion, SeNPs were successfully synthesized via a C. dentata leaf extracts via green synthesis. The NPs were characterized such as EDaX, SEM, UV-vis spectroscopy, and FTIR. The NPs were used at a very low concentration and were found to exhibit strong mosquito larvicidal activity in a dose-dependent manner. The results of the larvicidal studies clearly demonstrate that treatment with Se Ps is more effective in mosquito control. The importance of the present study lies in the possibility that the next-generation NPs conjugated plant bioactive molecules may be more effective control of mosquito agents. Further investigations for the mode of action of the NPs conjugated plant extract effect on nontarget organisms and field evaluation are necessary prior to commercialization.

Acknowledgements

We are grateful thank to the Department of Biotechnology, Periyar University, Salem, Tamil Nadu, India for providing funding and the infrastructural facility for carrying out this research work.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.