Silver nanoparticles from insect wing extract: Biosynthesis and evaluation for antioxidant and antimicrobial potential

Silver nanoparticles (AgNPs) are among the most widely synthesized and used nanoparticles (NPs). AgNPs have been traditionally synthesized from plant extracts, cobwebs, microorganisms, etc. However, their synthesis from wing extracts of common insect; Mang mao which is abundantly available in most of the Asian countries has not been explored yet. We report the synthesis of AgNPs from M. mao wings extract and its antioxidant and antimicrobial activity. The synthesized AgNPs were spherical, 40–60 nm in size and revealed strong absorption plasmon band around at 430 nm. Highly crystalline nature of these particles as determined by Energy-dispersive X-ray analysis and X-ray diffraction further confirmed the presence of AgNPs. Hydrodynamic size and zeta potential of AgNPs were observed to be 43.9 nm and -7.12 mV, respectively. Fourier-transform infrared spectroscopy analysis revealed the presence of characteristic amide proteins and aromatic functional groups. Thin-layer chromatography (TLC) and Gas chromatography-mass spectroscopy (GC-MS) analysis revealed the presence of fatty acids in the wings extract that may be responsible for biosynthesis and stabilization of AgNPs. Further, SDS-PAGE of the insect wing extract protein showed the molecular weight of 49 kDa. M. mao silver nanoparticles (MMAgNPs) exhibit strong antioxidant, broad-range antibacterial and antifungal activities, (66.8 to 87.0%), broad-range antibacterial and antifungal activities was found with maximum zone of inhibition against Staphylococcus aureus MTCC 96 (35±0.4 mm) and Fusarium oxysporum f. sp. ricini (86.6±0.4) which signifies their biomedical and agricultural potential.


Introduction
Recent past has witnessed a significant dominance of nanotechnology in every field of human life like biomedical and engineering because it is efficient, bio-friendly, safe and economical

Synthesis of silver nanoparticles (AgNPs)
Biosynthesis of AgNPs from Mang Mao wings extract was carried out, following methodology given by Lateef et al. [8] M. mao wings (0.1 g) were hydrolyzed using 20 mL of 0.1 M NaOH at 90 o C for 1 hour, cooled and the hydrolyzed solution was centrifuged at 8000 rpm for 10 minutes. Supernatant was collected, pH adjusted to 7 from which 1 mL of wing extract was added to 49 mL of 1 mM silver nitrate (AgNO 3 ) solution taken in 100 mL beaker and incubated at 28 ±1˚C for 30 minutes under static conditions for synthesis of AgNPs. Absorption maxima was measured at 200 to 700 nm using UV-Vis spectrophotometer (HITACHI U-2900, Japan) to characterize the Mang Mao silver nanoparticles (MMAgNPs) and later on the sample was centrifuged at 8000 rpm for 10 min followed by pellet wash with acetone and air dried for further studies.

Optimization of MMAgNPs synthesis
Effect of various process parameters on MMAgNPs synthesis was optimized using one variable at a time (OVAT) approach [9] where, the effect of a single parameter was evaluated initially and concentration obtained was used as a standard for all the subsequent steps. Parameters optimized using OVAT included AgNO 3 (1 to 3 mM), the concentration ratio of silver nitrate and M. mao wings extract (1:1, 1:3, 1:5), pH (3 to 11) and reaction time (0 to 30 minutes). The presence of AgNPs in the resultant solution was detected by the absorbance maxima as mentioned above. Stability study. Above optimized MMAgNPs were incubated at 28±1˚C in dark conditions for 119 days and absorbance maxima was measured weekly once to determine its stability.

Characterization of MMAgNPs
Scanning electron microscopy (SEM) of the sample was performed by dispersion of the sample in aluminum foil at 5.0 kV operating voltage using JSM-7500F and images were recorded at different magnifications. Energy-dispersive X-ray (EDX) analysis was performed by using Hitachi S-3400 NSEM instrument equipped with Thermo EDX for which the synthesized MMAgNPs were dehydrated and coated on carbon film. X-ray diffraction (XRD) was carried out by drop-coating MMAgNPs solution onto a glass substrate and diffraction was measured using Philips X'Pert Pro X-Ray diffractometer. The average crystal size of nanoparticles was estimated using Scherrer equation i.e., D = K λ/β cosθ [10]. The particle size distribution and zeta potential of MMAgNPs was analyzed in triplicate by electrophoretic light scattering at 25˚C, 150 V (DelsaMax PRO Light Scattering Analyzer, Beckman Coulter, United States) in distilled water. Functional groups were determined by FTIR spectroscopy (Bruker Tensor 27) and spectra were measured in the range 4000-400 cm −1 wavelength using KBr pellet as background reference.
Size and zeta potential of the silver nanoparticles were determined by Malvern Zetasizer ZEN 3600 (United Kingdom). This instrument allows the measurement of particle sized distribution in the range 2 nm-3 nm [12].
Thin layer chromatography and Gas chromatography mass spectroscopy. Reducing compounds of M. mao wings extract was analyzed by TLC on silica gel plates (silica gel 60 F254, Merck, Germany) with mobile phase (chloroform: methanol {97:3}), and chromatogram was examined under UV fluorescence (11). GC-MS analysis of Mang Mao wings extract was performed according to method as described by Ha et al. [12].
Extraction and purification of protein from M.mao wings. The protein from Mang Mao wings were extracted as per Zhang et al. [13] and precipitated using ammonium sulfate till approximately 30% saturation and incubated overnight at 4˚C followed by centrifugation. Pellet obtained was suspended in 0.05 M Tris-HCl buffer (pH-7), with 0.1 M NaCl, and dialyzed against the same buffer for 24 h. The crude protein was filtered using a 0.22 μm membrane filter and then subjected to column chromatography using silica gel and used for synthesis of AgNPs. Crude and purified proteins were further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and its molecular weight (Broad range 11 to 245 kDa, BioLabs, England) was determined [14].

Antioxidant and antimicrobial activity of MMAgNPs
DPPH free radical scavenging activity. Antioxidant activity of MMAgNPs was evaluated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay [11]. Reaction mixture consisted of equal volumes (1:1 w/v) of different concentrations of the synthesized MMAgNPs (100 to 500 μg mL -1 in water), to which 1 mL of 0.5 mM DPPH in ethanol solution was added. The reaction mixture was incubated in dark for 30 min at 28±2˚C. The absorbance is measured at 517 nm by UV-Vis spectrophotometer. Standard used was ascorbic acid to determine scavenging activity which was calculated by following equation.
Reducing power assay. Ferric reducing power assay was determined with 1 mL of the synthesized MMAgNPs, 2.5 mL of 0.2 M phosphate buffer (pH-6.6) and 2.5 mL of 1% potassium ferricyanide incubated at 50 o C for 20 minutes followed by cooling at room temperature. Then 2.5 mL of 10% trichloroacetic acid was added to the above mix and centrifuged at 8000 rpm for 20 min. Supernatant was mixed with distilled water in 1:1 v/v ratio and 1 mL of 0.1% ferric chloride was added and further incubated at 28± 2˚C for 10 min. Spectrophotometric absorbance of the resultant solution was measured at 700 nm. Increase in absorbance of reaction mixture indicated reducing activity of sample.

Statistical analysis
All the experiments were performed in triplicates, repeated twice and data was expressed as means ± standard deviation using IBM Corporation. 2012 Statistics, and graphs were drawn using Origin Pro 2015 [17,18].

Synthesis of MMAgNPs
M. mao wings extract, rich in proteins, chitin and lipids was used for the reduction of AgNO 3 into AgNPs. The change in color of reaction mixture from yellow to dark brown after 30 minutes incubation (Fig 1) indicated synthesis of MMAgNPs. The intensity of dark brown colour change indicated AgNPs formation from silver salt which is due to excitation of surface plasmon resonance (SPR) effect [19,20].

Optimization of MMAgNPs synthesis
During the optimization study using OVAT, AgNO 3 when used at 1 mM concentration showed maximum absorption at 430 nm, indicated active formation of MMAgNPs. Further increase in the concentration of AgNO 3 resulted in decreased absorption (Fig 2A). These results were in agreement with the previous investigations carried out as per Veerasamy et al. [17]. When, M. mao wings extract (1 mL) was added to AgNO 3 solution (1 mM), rapid conversion to brown color observed within 30 minutes, indicated active MMAgNPs synthesis ( Fig  2B). The increased color change of brown color was directly proportional to the incubation period which is due to reduction of AgNO 3 and excitation of SPR [19]. The absorption peak varied with different pH and it ranged between 390-470 nm (Fig 2C). The MMAgNPs formation was found to be slow at acidic pH (2)(3)(4)(5). At neutral pH (7) absorption maxima was observed at 430 nm and the reaction started as soon as AgNO 3 was added to the reaction mixture. The change in color to brown was observed within 30 minutes which indicated MMAgNPs synthesis ( Fig 2D).
Further, beyond pH 7 MMAgNPs resulted in aggregation and fall in flocculation. Previous studies revealed that at alkaline pH, Ag (I) ions in solution partly hydrolyze to form bioorganic-Ag(OH)x or bioorganic-Ag(NH 3 ) 2 complex on the surface of the particle and AgOH/Ag 2 O colloid in the medium [21]. The characteristic absorption peak at 430 nm corresponded to SPR of AgNPs previously reported by Bahrami-Teimoori et al. [22], which thereby confirmed the synthesis of MMAgNPs. This indicates the significance of M. mao wings extract in the reduction of metal salts to their respective metal nano-particles.
Stability study of MMAgNPs. The stability of MMAgNPs was monitored up to 119 days (weekly once) and there was no change in absorbance at 430 nm (S1 Fig). This indicated strong stability of biosynthesized MMAgNPs which might be attributed to presence of carboxylate group in proteins that may result in stable nanoparticles [23].

Characterization of MMAgNPs
UV-visible spectroscopic analysis of MMAgNPs. Nanoparticles synthesis is generally found to occur due to excitation of surface plasmon resonance (SPR) [24]. The strongest absorption peak observed at 430 nm ( Fig 1C) corresponds to SPR of AgNPs which is in accordance with previous reports [25,26].
SEM and EDX analysis of MMAgNPs. SEM analysis revealed the occurrence of MMAgNPs in a spherical shape and their size ranged between 40 to 60 nm. The results showed that variation of pH in reaction mixture altered the nanoparticles size (Fig 3), however, at neutral pH the nanoparticles were uniform (Fig 3C). Reddy et al. [27] reported that biosurfactants (surfactin) used for AgNPs synthesis altered the pH and decreased AgNPs size (9.7 to 4.9 nm) and at pH-9 the nano-particles were uniform. However, MMAgNPs were not uniform in size, and variations in nanoparticle's size were reported by Ahmed et al. [28] and Narayan and Dipak [29] using plant and seaweed extracts. The EDX spectra of MMAgNPs revealed the presence of characteristic signals of silver ions at 3keV (S2 Fig) similar to those observed with chemically synthesized AgNPs. The emission energy at 3 keV indicated the reduction of silver ions to elemental silver [30].
XRD analysis of MMAgNPs. The XRD pattern of MMAgNPs showed the diffraction peaks at 2θ values of 32.6˚, 46.57˚, 67.8˚and 77.04˚which further confirmed crystalline nature of MMAgNPs (Fig 4) and corresponded to standard JCPDS file No. 04-0783 [31]. Broadening in peaks occurred due to smaller particle size, which reflects the experimental conditions on nucleation and growth of crystal nuclei [32]. According to Debye Scherrer equation, the average nanoparticle size in this study was found to be 32 nm, which was relatively similar as described earlier by Sri Ramkumar et al. [33].
Zeta potential. Particle size distribution and zeta potential of MMAgNPs was depicted in S3(A) & S3(B) Fig. Average particle size of synthesized MMAgNPs was 43.9 nm and zeta potential -7.12 mV. The negative charges on AgNPs might be due to Mang Mao wings extract covering on nanoparticles. For the determination of overall surface charges on nanoparticles zeta potential analysis was applied.
Stability and prevention of aggregate formation, attributed to repulsions, due to same charge is provided by positive or negative charge on surface of nanoparticles [34]. This indicates better stability of nanoparticles and prevents agglomeration [35]. Hydrodynamic size and zeta potential of MMAgNPs in this study, corroborate with recent report of Badoei-dalfard et al. [36] hydrodynamic size (30-50 nm) of AgNPs synthesized using uricase from Alcaligenes faecalis GH3 and zeta potential (-4.6 mV) using Madhuca longifolia flower extract [34]. The characteristic FTIR peaks observed in the present study were similar to those reported by Dhanasekaran et al. [37] and Usha et al. [38]. Similar results were reported by Selvakumar et al. [39] and Soman and Ray [40] where synthesis is performed using Acalypha hispida and Ziziphus oenoplia (L.) leaf extract as reducing and stabilizing agent.
TLC of Mang Mao wings extract. TLC analysis of Mang Mao wings extract corresponded to Rf value 0.96 which is similar to insect chemicals based on previous studies [41]. These chemicals may also be responsible for biofabrication of MMAgNPs (Fig 6A).
Gas chromatography mass spectrometric analysis. GC-MS data revealed that M.mao wings extract have 30 major compounds with their molecular weight shown in Table 1. Major  Octadecynoic acid and hexadecanoic acid are fatty acids that are widely observed in insects, plants and animals [42][43][44]. These fatty acids are situated within wings membrane under the epicuticular surface [13,44]. The components present in Mang Mao wings extract corresponds with that of previous reports on nanoparticles synthesis [39,45,46].

Extraction and purification of Mang Mao wings extract
SDS-PAGE analysis revealed the presence of different cellular proteins with molecular weights that ranged between 22-245 kDa. Protein band corresponded to 46 kDa (Fig 6B) was found to act as a capping agent for stabilization of the MMAgNPs.
Khan and Ahmad [47] reported, purified sulfite reductase enzyme with molecular weight of 43 kDa to be responsible for gold nanoparticles stability and synthesis. Kumar et al. [48] also claimed protein of 35.6 kDa is responsible for biosynthesis and capping agent of gold nanoparticles. However, further studies are required towards characterization and identification of this protein to validate this result.

Antioxidant activity of MMAgNPs
MMAgNPs exhibited DPPH scavenging activity in the range of 66.8 to 87.0% and in case of ascorbic acid it was found to be in the range of 89.7 to 95.5% (Fig 7A). Difference in the activity can be attributed to a different functional group attached to them. MMAgNPs showed good ferric ion reducing activity which was comparable to that of standard ascorbic acid (Fig 7B). Results obtained from this study suggested the use of biosynthesized MMAgNPs as natural antioxidants. Free radical scavenging activity of MMAgNPs is mainly due to the donation of hydrogen molecules, such as proteins, polyphenols and other biomolecules present in the colloidal solution of AgNPs [26]. Radical scavenging activity of AgNPs is due to presence of bioreductant molecules on surface of nanoparticles increasing the surface area for antioxidant activity [12]. Therefore, these MMAgNPs can be employed as a natural antioxidant in the pro- oxidants, antioxidants and to balance reactive oxygen species (ROS) levels. Previous study of AgNPs synthesized using Catharanthus roseus showed radical scavenging activity and prevented human cell damage and degenerative diseases [49]. This work concludes the use of MMAgNPs as potential agent of antioxidant formulations in biomedical/ pharmaceutical areas. and Colletotrichum falcatum respectively when the MMAgNPs were used at a concentration of 10 μg mL -1 . MMAgNPs served as potential antibacterial, antifungal agents and may emerge as an alternative to conventional antibiotics [50]. These AgNPs due to their small size can adhere to bacterial cell membrane, increase permeability and can cause structural changes in bacteria. In case of fungi, AgNPs disrupt the membrane integrity and fungal spores leading to cell death [51]. Some researchers claimed that AgNPs enter the microorganisms and can cause damage by interacting with DNA and proteins, resulting in apoptosis [52].

Conclusion
In this study, we explored M. mao wings extract as an eco-friendly, cost-effective and novel biomaterial for biofabrication of AgNPs. The synthesized AgNPs were spherical, 40-60 nm in size and are highly crystalline nature. This is the first report on use of M. mao (seasonal insect) wings with metal chelating potential can be used as natural reducing agents for the synthesis of nanoparticles. MMAgNPs exhibit strong antioxidant (66.8 to 87.0%) and antimicrobial activities was found maximum zone of inhibition against Staphylococcus aureus MTCC 96 (35±0.4 mm) and Fusarium oxysporum f. sp. ricini (86.6±0.4). Antioxidant and antimicrobial properties of MMAgNPs may further widen their application in the biomedical and agricultural sectors.