Introduction

One of the major public health problems in developing countries is vector-borne diseases. Especially, mosquito vectors are solely responsible for transmitting diseases such as malaria, dengue, Chikungunya, Japanese encephalitis, and lymphatic filariasis. These diseases pose a major problem in disease-prevalent countries and are presently rampant owing to amplify globalization, urbanization, and global warming (Joseph et al. 2011; Simonsen and Mwakitalu 2013). Culex mosquitoes are painful and persistent biters and are responsible for filariasis. Lymphatic filariasis is a neglected tropical disease. More than 1.3 billion people in 72 countries worldwide are threatened by lymphatic filariasis, commonly known as elephantiasis. Over 120 million people are currently infected, with about 40 million disfigured and incapacitated by the disease (WHO 2012). Anopheles stephensi is responsible for the transmission of malaria in urban regions of India (Rahman et al. 1989). In India, malaria is still the most important cause of morbidity and mortality with approximately two to three million new cases rising every year (Sharma et al. 2009). Biological control is used as an alternative to currently employed larvicides for minimizing the mosquito population which provides an effective and environmentally friendly approach to bring down mosquitoes’ population under bottom level. Unfortunately, the mosquitoes developed resistance against the chemical larvicides (Cadavid-Restrepo et al. 2012; Chenniappan and Ayyadurai 2012).

Nanotechnology is a branch of science that is growing day by day and building an impact in various aspects of human life and manufacturing a new material at a nanolevel (Ahluwalia et al. 2014; Albrecht et al. 2006). The nanotechnology areas include the development and the study of materials with unique properties arising from their nanoscale dimensions (Bhattacharya and Gupta 2005). In recent years, nanotechnology is gaining profoundness owing to its increase of vital role in most dynamic areas of research in modern science (Sharma et al. 2009). The progress in this area largely depends on the ability to synthesize nanoparticles from various materials in different sizes and shapes, as well as to their effective inclusion into complex structures (Yu 2007). The metal nanoparticles have been synthesized by using several methods, including chemical, physical, electrochemical, irradiative, photochemical, and biological techniques (Kathiravan et al. 2014). Metal nanoparticles have attractive optical and electrical properties associated to the quantum size effect and their areas such as optics, optoelectronics, catalysis, nanostructure fabrication, and chemical or biochemical sensing (Qi et al. 2004). Numerous scientists reported that chemical methods and synthesis of nanoparticles employ toxic chemicals as reducing agents, organic solvents, or non-biodegradable agents. It is dangerous to the environment and biological systems (Lee et al. 2008; Njagi et al. 2011).

Nowadays, biological resources such as bacteria, fungi, and plants could be used to synthesize nanoparticles (Ramamurthy et al. 2012). The potentials and promises of plant system in the biologically assisted synthesis of metal nanoparticles called “green synthesis” have now become a key in nanotechnology research (Jha et al. 2009). In green synthesis approach, use of the plant has offered a reliable, simple, nontoxic, and ecofriendly approach (Narayanan and Sakthivel 2010; Satishkumar et al. 2009; Suman et al. 2013a, b). Overview of silver nanoparticle (AgNP) preparation by green synthesis approaches, including mixed valance polyoxometallates, polysaccharides, tollens, and irradiation methods was reported by (Sharma et al. 2009). The green synthesis methods are low cost and efficient and generally lead to the formation of crystalline nanoparticles with a variety of sizes. This depends on nature of plant extract concentration, pH, and temperature and incubation time of synthesis reaction (Noruzi et al. 2014). The effectiveness of AgNPs obtained via green synthesis is synergistically better in therapeutic activity (Iravani 2011). The synthesis of nanoparticles using green synthesis is potentially advantageous over microorganisms due to the ease of biohazards and culture of microbes (Nalawade et al. 2014).

Hybanthus enneaspermus belongs to family Violaceae. The plant is used medicinally in Ayurveda, Siddha, and other traditional system. The plant is commonly named as “Orithal thaamarai” in Tamil and Rathnapurusha in Sanskrit. In Ayurveda, it is known as Sthalakamala. The fruit is used to treat scorpion stings (Narayanan and Sakthivel 2010; Raveendra Retnam and John De Britto 2007). It is an herbaceous plant or shrub distributed in the tropical and subtropical areas of the globe. The plant is used as an aphrodisiac, demulcent, cardiotonic, diuretic, anti-convulsant, and anti-malarial. It is applied to treat urinary infections, diarrhea, leucorrhoea, dysuria, inflammation, and male sterility (Boominathan et al. 2003; Hemalatha et al.,2003) Pharmacologically, the plant is reported to possess anti-microbial, anti-inflammatory, anti-tussive, anti-plasmodial, anti-convulsant, and free scavenging activities (Weniger et al. 2004). The components of the plant dipeptide alkaloid (aurantiamide acetate), triterpene (isoarborinol), and β-sitosterol are previously isolated from H. enneaspermus (Tripathy et al. 2009).

Hence, the present study was aimed to investigate the bioactive components present in the H. enneaspermus and biosynthesis of the AgNPs and to test the larvicidal effect.

Material and methods

Collection and identification of plant material

Fresh plant material was collected from Paradarami Village, Gudiyattam Taluk, and Vellore District of Tamil Nadu, India, during 2013. The taxonomic identification was made by Dr. Mujeera Fatima, Department of Plant Biology and Biotechnology, Nandanam Government Arts College, Chennai, India. The voucher specimen was numbered and maintained in our research laboratory and deposited in our university botany herbarium.

Preparation of plant extract for GC-MS analysis

The newly collected plant material of H. enneaspermus was washed thoroughly in tap water, shade dried, and powdered in a blender. The respective plant powder 70 g was filled in the thimble and extracted successively with methanol using a Soxhlet extractor for 10 h. The extracts were concentrated using a rotary flash evaporator and preserved at 5 °C in an airtight bottle until further use.

GC-MS analysis

GC-MS analysis was carried out with an SHIMADZU QP 2010T which composed of an auto sampler and gas chromatography interfaced to a mass spectrometer (GC-MS) instrument employing the following condition: capillary column −624 ms (30 m × 0.32 mm × 1.8 m) operating in an electronic mode at 70 eV; helium (99.99 %) was used as the carrier gas at a constant flow of 1.491 mL/min and injection volume of 1.0 mL, injector temperature of 140 °C, and ion source temperature of 200 °C. The oven temperature was programmed from 45 °C. Mass spectra were taken at 70 eV.

Synthesis of AgNPs

The fresh part of H. enneaspermus broth solution was prepared by taking 250 mg of thoroughly washed and finely cut parts of H. enneaspermus in a 250-mL Erlenmeyer flask along with 100 mL of sterilized double-distilled water, and the mixture was boiled at 60 °C for 5 min. The extract was filtered with Whatman filter paper no. 1 and stored at −10 °C for further experiments. The 3 mL of freshly prepared plant extract was added to 40 mL of 1 mM AgNO3 aqueous solution (6.79 mg of AgNO3 was added in 40 mL of Milli-Q water) at room temperature for 30 min.

Characterization of AgNPs

AgNP synthesis using H. enneaspermus extract was confirmed by UV–visible spectrophotometer (Schimadzu 1600). Further, the reaction mixture was centrifuged at 60,000×g for 40 min and the resulting pellet was dissolved in deionized water and filtered through Whatman filters (0.45 μm). An aliquot of this filtrate containing AgNPs was used for further characterization techniques. X-ray diffraction (XRD) measurement of H. enneaspermus of reduced AgNPs was carried out on films of the respective solution drop coated onto a glass substrate on a Philip PW 1830 instrument operating at a voltage 40 kV and a current of 30 mA with CuK 1 radiation. For Fourier transform infrared (FTIR) measurement, dry powder of the nanoparticles obtained in the following manner was prepared by centrifuging the synthesized AgNP solution at 10,000 rpm for 20 min. The solid residue containing AgNP solution was dispersed in sterile deionized water for three times to remove the unattached biological impurities. The pure residue was then dried perfectly in an oven overnight at 70 °C. The powder obtained was subjected to FTIR measurement carried out on a PerkinElmer Spectrum one instrumental resolution of 4 cm−1 in KBr pellets. The surface charge in terms of zeta potential and particle size distribution of the colloidal nanoparticles was determined using Malvern Zetasizer Nano Series (Malvern). Final concentration after reduction of Ag+ ions was determined using inductively coupled plasma atomic emission spectroscopy (ICP-MS) using Agilent 7700, Japan. For transmission electron microscopic studies, 25 μL of sample was sputter on carbon-coated copper transmission electron microscopy (TEM) grids. TEM measurements were performed on a JEOL model 1200EX instrument operated at an accelerating voltage of 120 kV and later with an XDL 3000 powder.

Larvicidal bioassay

One gram of aqueous aerial extract was first dissolved in 100 mL of distilled water (stock solution). From the stock solution, 100 mg/L was prepared with dechlorinated tap water for a bioassay test of plant extract. The larvicidal activity was assessed following as per the method of Rahuman et al. (2000). For the bioassay test, larvae were taken in five batches of 20 in 249 mL of water and 1.0 mL of aqueous plant extract concentration. Control was set up with dechlorinated tap water. The numbers of dead larvae were counted after 24 h of exposure, and the percentage of mortality was reported from the average of five replicates. The experimental media in which 100 % mortality of larvae occurs alone were selected for dose–response bioassay. Synthesized AgNP toxicity tests were conducted using a multiconcentration test, consisting of a control and different concentrations of nanoparticles. Each test was performed by placing 20 mosquito larvae into 200 mL of sterilized double-distilled water with nanoparticles into a 250-mL beaker (Borosil). The nanoparticle solutions were diluted using double-distilled water as a solvent according to the desired concentrations (50, 40, 30, 20, and 10 mg/L).

Dose–response bioassay

Based on the preliminary screening results, crude extract of H. enneaspermus and synthesized AgNPs were subjected to dose–response bioassay for larvicidal activity against the larvae of Anopheles subpictus and Culex quinquefasciatus. Different concentrations ranging from 50 to 250 mg/L (aqueous plant extracts) and 10 to 50 mg/L (for synthesized AgNPs) were prepared for larvicidal activity. The numbers of dead larvae were counted after 24 h of exposure, and the percentage of mortality was reported from the average of five replicates.

Statistical analysis

The average larval mortality data were subjected to probit analysis (FORTRAN) for calculating LC50. Other analyses at 95 % fiducial limit of upper confidence limit and lower confidence limit values were calculated following Reddy et al. (1992). Results with p < 0.05were considered to be statistically significant.

Result and discussion

In the present study, the larvicidal activity of H. enneaspermus aqueous extract and the synthesized AgNPs were observed against the fourth-instar larvae of Anopheles subpictus (LC50 = 117.83 and 126.59 mg/L) and against the fourth-instar larvae of C. quinquefasciatus (LC50 = 17.24 and 13.12 mg/L), respectively (Tables 1 and 2). It has been reported that leaf extracts of Ocimum canum, Ocimum sanctum, and Rhinacanthus nasutus have been found to be only moderately toxic against the larvae of Aedes aegypti with LC50 values ranging between 99.42 and 81.56 ppm (Kamaraj et al. 2008). The mosquito larvicidal activity of UV irradiation-induced AgNPs was found to decrease the survival of fourth-instar larvae of Aedes aegypti by 88 % after 24 h of exposure at 1-ppm concentration (Sap-Iam et al. 2010). Earlier authors reported that the larvicidal effect of aqueous crude leaf extracts, silver nitrate, and synthesized AgNPs of Mimosa pudica showed that the highest mortality was found in synthesized AgNPs against the larvae of Anopheles subpictus (LC50 = 08.89, 11.82, and 0.69 ppm) and against the larvae of C. quinquefasciatus (LC50 = 09.51, 13.65, and1.10 ppm) (Marimuthu et al. 2010). The synthesized AgNPs using Ammannia baccifera showed that the highest larvicidal efficacy with the LC50 values of 29.54 mg/L for Anopheles subpictus and 22.32 mg/L for C. quinquefasciatus (Suman et al. 2013a, b). The larvicidal activity of synthesized AgNPs utilizing an aqueous extract of Eclipta prostrate and the crude aqueous extract was observed against C. quinquefasciatus (LC50 = 27.49 and 4.56 mg/L; LC90 = 70.38 and 13.14 mg/L) and against Anopheles subpictus (LC50 = 27.85 and 5.14 mg/L; LC90 = 71.45 and 25.68 mg/L), respectively (Rajakumar and Rahuman 2011). Synthesized AgNPs using Tinospora cordifolia extract was tested against the larvae of Anopheles subpictus (LC50 = 6.43 mg/L) and against the larvae of C. quinquefasciatus (LC50 = 6.96 mg/L) (Jayaseelan et al. 2011).

Table 1 Larvicidal activity of Hybanthus enneaspermus extract
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Table 2 Larvicidal activity of the synthesized nanoparticles
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This biological reduction of AgNPs would be a boon for the development of clean, nontoxic, and environmentally acceptable green approach to produce AgNPs involving organisms even ranging to higher plants. The formed AgNPs are highly stable and have significant mosquito larvicidal activity of Anopheles subpictus and C. quinquefasciatus. This is the first report on the larvicidal activity of synthesized AgNPs from H. enneaspermus. The use of plants can also be suitably scaled up for large-scale synthesis of nanoparticles in a controlled manner according to their size, shape, and dispersity. Moreover, the use of plants in the process of nanoparticle synthesis is more beneficial than other processes since the nanoparticles are produced extracellularly. Recently, syntheses of AgNPs by using plant extracts are getting more popular (Li et al. 2007; Song and Kim 2009).

UV–vis spectroscopy was ascertained to check the formation and stability of AgNPs in aqueous solution (Fig. 5). In the current study, AgNPs were rapidly formed after the addition of H. enneaspermus extract; the reaction mixture turned dark brown (Fig. 1a) from light green after 15 min of incubation. The appearance of dark brown indicates the formation of the AgNP formation (Fig. 1a). It is well known that the optical absorption spectra of metal nanoparticles were dominated by surface plasmon resonances (SPRs) that shift to longer wavelengths with increasing particle size due to the capping agent (Brause et al. 2002). Also, it is well recognized that the absorbance of AgNPs mainly depends upon the size and shape (Rao and Trivedi 2006). Figure 1b, c shows SPR for the sample solution to occur at the wavelength of 430 nm (Fig. 1b) in 15 min and 432 nm (Fig. 1c) after 24 h which confirms the presence of AgNPs in the prepared solution. A narrow absorption peak of UV–visible range indicates that the distribution of smaller-sized nanoparticles will be higher in the mixture (Maqusood Ahamed et al. 2011). The SPR absorbance is sensitive to the nature, size, and shapes of particles present in the solution, and also, it depends upon their inner particle distance and the surrounding media (Nazeruddin et al. 2014).

Fig. 1
figure 1

a Brown color indicates the formation of AgNPs. b UV–vis absorption spectra of synthesized AgNPs

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The XRD pattern of the H. enneaspermus plant extract AgNPs is shown in Fig. 2. Four sharp peaks are observed at 2θ values in 38.20°, 44.40°, 64.60°, and 77.50° which are indexed as (1 1 1), (2 0 0), (2 2 0), and (3 1 1) bands of face-centered cubic (fcc) structures of silver(JCPDS File No: 03-0921). The peak corresponding to (111) plane is more intense than other planes, suggesting it as a predominant orientation. No extra diffraction peaks in XRD pattern are observed, revealing that synthesized AgNPs are essentially pure. Similar values of diffraction peaks for nanosilver have been reported by others (Satishkumar et al. 2009; Vimala et al. 2009; Ashok et al. 2010). The XRD pattern clearly demonstrated that the AgNPs were crystalline in nature and the average crystal size was found to be 12.4 nm calculated by the Scherrer equation (Sheny et al. 2011).

Fig. 2
figure 2

XRD spectra of biosynthesized AgNPs by H. enneaspermus

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In order to determine the possible bio-reducing functional groups involved in reduction process and their unique interactions with AgNPs, FTIR analysis was performed. The band intensities of infrared spectra of AgNPs are depicted in Fig. 3. The broad spectrum peak at 3420 cm−1 corresponds to strong stretching vibrations of the hydroxyl functional group (Priyadarshini et al. 2013). Less-intense peak at 2920 and 2851 cm−1 could be assigned to the presence of secondary amines and C–H stretching vibrations (Ahluwalia et al. 2014). The bands at 1038 and 1636 cm−1 correspond to the –N–H and carbonyl (C–O–) stretching vibrations in amide linkages (amide I and amide II) of protein. The band at 1384 cm−1 was mainly attributed to the amide bands, −C–N stretching vibrations of proteins. The spectrum supports the presence of alcohol, phenols, carbonyl, and amines (both aromatic and aliphatic) and amide functional groups in the synthesis of AgNPs (Suresh et al. 2011). Hence, it may be assumed that these functional groups are responsible for the reduction, capping, and stabilization of synthesized AgNPs.

Fig. 3
figure 3

FTIR spectra of biosynthesized AgNPs by H. enneaspermus

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The zeta potential is commonly used to find out the stability of colloidal systems Jayaseelan et al. 2013; Sun et al. 2007). The zeta potential of nanoparticles was evaluated in water as dispersant. Generally, a suspension that exhibits an absolute zeta potential less than 20 mV is considered unstable and will result in precipitation of particles from solution (Sun et al. 2006), whereas the absolute zeta potential higher than 20 mV is stable (Badawy et al.,2010). In this study, zeta potentials of AgNPs were −27.1 mV indicating the stability of AgNP suspensions (Fig. 4a). Particle size determination was carried out; the particle size range was found to be 5.84 to 61.21 nm, and the Z-average was found to be 25.2 nm (Fig. 4b).

Fig. 4
figure 4

a Zeta potential of biosynthesized AgNPs by H. enneaspermus. b DLS measurement of biosynthesized AgNPs by H. enneaspermus

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The morphology and size of the synthesized AgNPs were determined by TEM image. The particles formed were spherical, hexagonal, and triangular in shape, and the size range was 16–26 nm (Fig. 5a). The presence of the elemental silver can be identified by the EDX analysis, which indicated the reduction in silver ions to elemental silver in the reaction mixture. The EDX spectrum illustrated the presence of strong metallic Ag signals along with weak oxygen and carbon peak, which may be originating from the biomolecules that are bound to the surface of nanosilver particles (Fig. 5b). ICP-MS analysis showed the final AgNP solution prepared using H. enneaspermus extract containing 96 % of Ag0.

Fig. 5
figure 5

a TEM image of biosynthesized AgNPs by H. enneaspermus. b EDX image of biosynthesized AgNPs by H. enneaspermus

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The results of the GC/MS analysis showed that 39 compounds (Table 3) were present in the methanol extract of H. enneaspermus. These compounds were identified through mass spectrometry attached with GC. The mass spectra of these compounds were matched with those found in the NIST/NBS spectral database and the data are given. The fragmentation pattern of the major compound (retention time 45.808) was found to be dodecanoic acid, 1,2,3-propa. The plant is already reported to contain propane,1,1,3-triethoxy-, phenol,4,6-di(1,1-dimethyl)-2-methyl, 1,14-tetradecanediol, phytol, 2-pepridionne, N-[4-bromo-nbutyl]-, cedran-diol, 8S, 14- and 2H-pyran, 2-(7-heptadecynyloxy)tetrahydro reported to possess anti-cancer, hepatoprotective, anti-inflammatory, and anti-microbial and inhibition of parasitic growth (Anand and Gokulakrishnan 2012).

Table 3 GC-MS analysis of H. enneaspermus
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Conclusion

In the present study, for the first time, green synthesis of AgNPs using aqueous H. enneaspermus was attempted. The physical property of synthesized nanoparticle was characterized using relevant techniques. The formed AgNPs are highly stable and had significant mosquito larvicidal activity against Anopheles subpictus and C. quinquefasciatus. These synthesized AgNPs have the potential to be used as an ideal ecofriendly approach for the vector control programs.