Fabrication and validation of silver nanoparticles from cocoon extract of silk worm Bombyx mori. L

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Fabrication and validation of silver nanoparticles from cocoon extract of silk worm Bombyx mori. L ⁎

Jeyaraj Pandiarajan , Sundaramahalingam Balaji, Kannan Revathy, Selvam Palanikumar Post Graduate Department of Biotechnology, Ayya Nadar Janaki Ammal College, (Autonomous Institution, Affiliated to Madurai Kamaraj University, Madurai), Virudhunagar District, Sivakasi 626124, Tamil Nadu, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Silk cocoon Bombyx mori Silver nanoparticles Cocoon extract Defense role Antimicrobial activity Antioxidant activity Anticancer activity

Silver nanoparticles pave the attention of researchers for the last two decades due its wide applications. At the same time silk cocoon is renewed for its defense role, hence the present investigation was coined with silk cocoon extract as the source for deriving silver nanoparticles. The silver nanoparticles obtained from cocoon extract of silk worm Bombyx mori was faster synthesis and also it was safer and eco friendly. Thus obtained nanostructures were subjected to UV–Vis, FTIR spectroscopy, SEM, EDX, DLS, particle size analysis and XRD to confirm the fine circular particles synthesis at nano-scale level. The obtained silver nanoparticles from cocoon extract of silk worm Bombyx mori showed potential anti-bacterial and anti-fungal activities against virulent strains. The same has elucidated the better anti-oxidant role by DPPH and Hydrogen peroxide assay. The cytotoxic potential of silver nanoparticles from cocoon extract against human breast adenocarinoma cancer cell line (MCF7) proved it as an abled drug for future research.

1. Introduction The silkworm Bombyx mori is a monophagous insect that mainly feeds on fresh mulberry leaves. Artificial diet for the silkworm is studied and applied extensively in Japan, China and some other countries as it contains essential nutrients for supporting the normal growth of the larva. Although artificial diet can obviate the serious drawbacks of mulberry leaves such as the seasonal limitation on supply of fresh leaves, possible harm from parasites or pesticides and high labor cost, the silkworms reared on artificial diet during all instars are not as good as those fed on fresh mulberry leaves, which are reflected in many aspects such as the filament quality and resistance of cocoons (Pandiarajan et al., 2011). Silkworm, Bombyx mori was domesticated more than 5000 years ago, is an economically imperative insect for silk production and also a fine model for lepidopteron (Goldsmith et al., 2005). In India the prime focus of the sericulture industry is to improve silk production with high quality silk. Naturally, the silk worm builds its cocoon not only with silk proteins, but also with antimicrobial proteins to avoid infection since the cocoon is non-motile and non-feeding (Pandiarajan et al., 2011). At the final instar of larva by a wandering support the insect spins the cocoon due to which pupation occurs, this instance occurs only in very few lepidopteron. The silk cocoon is composed of firm, elastic core which is enveloped by sticky materials gluing the filaments. The

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inbound proteins molecules of silk make the filament core secreted from posterior section of the paired silk-producing glands. Moreover cocoon is also enriched with flavanoids and isoquercitrin compound makes it as multifaceted role performer (Sehnal and Akai, 1990). The cocoon shell persist silk fibroin fibre (70%) bounded by a layer of sericin (25%) and non-protein compounds. The non-sericin is constituted by carbohydrate, wax, flavonoids and pigments (Kaskoos et al., 2012). Cocoon shells are one kind of natural polymeric composite materials which constitutes coarse silk at its outer region and floss silk at the inner side to protects the pupa against microbial degradation and desiccation during metamorphosis, and also protects against potential predators (Ishay et al., 2002; Das et al., 2016). Every year, severe economic losses occur in the sericulture industry, due to the attack of variety pathogens it leads to the productivity of both quantity and quality of the cocoon (Pandiarajan et al., 2011). Nanoparticles are being employed in different fields including electrical, medical, biological, textile and chemistry in which the shape and size of colloidal metal particles play crucial role in different application including preparation of magnetic and electronic devices, wound healing, antimicrobial gene expression and in the preparation of bio composites. Noble metal colloids have the optical and catalytically electromagnetic properties that dependent on the size and shape of the particles (Pandiarajan et al., 2016a, 2016b; Pandiarajan and Krishnan, 2017). Silver enters various components of cellular membrane, which

Corresponding author. E-mail addresses: [email protected], [email protected] (J. Pandiarajan).

https://doi.org/10.1016/j.bcab.2018.05.017 Received 24 January 2018; Received in revised form 5 May 2018; Accepted 23 May 2018 1878-8181/ © 2018 Elsevier Ltd. All rights reserved.

Please cite this article as: Pandiarajan, J., Biocatalysis and Agricultural Biotechnology (2018), https://doi.org/10.1016/j.bcab.2018.05.017

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are most important than nucleic acids. The binding affinity reveals toxicity or detoxification remains unclear, few sensitive bacterial strains have been reported as accumulating more silver called than the consequent resistant strain, in others the reaction is overturn proportionate. In resistance case has been exposed to be plasmid mediated. The plasmids are quiet difficult to transfer and to maintain, dissimilarities between resistant and sensitive strains were with narrow success such as increased cell surface hydrophobicity in a resistant Escherichia coli (Julia and Penelope, 1994) Silver nanomaterials are fine particles of metallic silver that have at least one dimension less than 100 nm. Nano silver or suspensions of Nano silver refers to colloidal silver. To produce colloidal silver, a positive electrical current is applied through pure silver bars suspended in water, resulting in colloidal silver particles with the size range of 15–500 nm (Pandiarajan et al., 2016a, 2016b; Pandiarajan and Krishnan, 2017). Silver nanoparticles are actually used in several industrial sectors and end up in the environment and human health attributes. Silver nanoparticles are renewed for its antimicrobial property; antioxidant property and anticancer activity other than these now it has scaled new heights (Pandiarajan and Krishnan, 2017). During the antimicrobial activity, silver nanoparticles get attached to the cell membrane and also penetrate inside the host. The nanoparticles preferably attack the respiratory chain, cell division finally cell necrosis. The release of silver ions in the bacterial cells, enhance their bactericidal activity (Rai et al., 2009). Silver nanoparticle has anti-inflammatory properties as well. Silver nanoparticle has anti-inflammatory effects and improved the healing process significantly (Wong, 2012). Many efforts have been made to use silver nanoparticles as an anticancer agent and all turned up successful. As like the silver nanoparticle, silk own numerous properties such as microfabrication, photonics, tissue engineering, drug delivery and therapeutics for various diseases. The combination of silk and nano will pave a new dimension in the drug delivery and therapeutics in nanomedicine (Philipp Seib, 2017). 2. Materials and method 2.1. Sample collection of cocoons The Bombyx mori eggs were collected from Central Silk Board [CSB]. Extension Centre, Vaniyampatti, Srivilliputtur, Virudhunagar District, Tamil Nadu. It was reared in the laboratory under disease free environment for the collection healthy cocoon shells (Rajathi et al., 2010; Pandiarajan et al., 2011).

Fig. 1. A. White cocoon shell collected from Silkworm Bombyx mori without pupa. B. Sliced cocoon pieces for the preparation of Bombyx mori cocoon shell extract.

2.2. Preparation of Cocoon extract Fresh and healthy white cocoons were collected and cut into small pieces and dried at room temperature. Fig. 1 About 10 g of these finely incised cocoons were weighed separately and transferred into 250 ml beakers containing 100 ml distilled water and boiled for about 10 mins. The extracts were then filtered thrice through Whatman No. 1 filter paper to remove particulate matter and to get clear solutions which were then refrigerated (4 °C) in 250 ml Erlenmeyer flasks for further experiments.Fig. 2A 2.3. Synthesis of silver nanoparticles Accurate concentration of 1 mM silver nitrate was prepared by dissolving 0.016 g of AgNO3 in 100 ml of double distilled water and stored in brown colored bottle to prevent auto oxidation of silver (Pandiarajan et al., 2016a). 10 ml of cocoon extract was added into 90 ml of aqueous solution of 1Mm silver nitrate (AgNo3) for the reduction of silver nitrate into Ag2+ ions and kept in dark room at 37 °C for 24 h. After 24 h, colour of the solution changed from colourless to dark brown indicating the formation of silver nanoparticles. The

Fig. 2. Silver nanoparticles synthesized using Bombyx mori cocoon shell extract.

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values of zeta potential ranged from higher than + 30 mV to lower than − 30 mV. Surface zeta potentials were measured using the laser zeta meter (Malvern zeta seizer 2000, Malvern). Liquid samples of the nanoparticles (5 ml) were diluted with double distilled water (50 ml) using NaCl as suspending electrolyte solution (2 × 10–2 M NaCl). The pH was then adjusted to the required value. The samples were shaken for 30 mins. After shaking, the equilibrium pH was recorded and the zeta potential of the metallic particles was measured. A zeta potential was used to determine the surface potential of the silver nanoparticles. In each case, an average of three separate measurements was reported. The criteria of stability of NPs are measured when the values of zeta potential ranged from higher than + 30 mV to lower than − 30 mV (Pandiarajan et al., 2016a).

bioreduced silver nanoparticles solution was measured using UVVisible absorbance. The silver nanoparticles solution thus obtained was purified by repeated centrifugation at 7000 rpm for 20 mins. It is followed by redispersion of the pellet in deionized water to get rid of any uncoordinated biological molecules. 2.4. Characterization of synthesized silver nanoparticles 2.4.1. UV- Visible spectrophotometer Initial characterization of silver nanoparticles was carried out using UV-Visible spectroscopy. Change in colour was visually observed in the silver nitrate solution incubated with cocoon extract of Bombyx mori. The bioreduction of precursor silver ions was monitored by sampling of aliquots (Silver nanoparticles diluted with distilled water) at different time intervals. Absorption measurements were carried out on UVVisible Spectrophotometer at a resolution 1 nm between 200 and 800 nm. Distilled water was used as a blank. The spectrum recorded was then plotted (Pandiarajan et al., 2016a).

2.10. Particle size analyser The PSA analysis was carried out for the sample which is lyophilized and dispersed by ultrasonicator for the determination of size.

2.5. Scanning Electron Microscopy (SEM)

2.11. Antimicrobial activity assay

Scanning Electron Microscopy (SEM) analysis was performed at different magnifications (Hitachi S-4500, Japan). Thin films of synthesized and stabilized silver nanoparticles were prepared on a carbon coated copper grid by just dropping a very small amount of the sample on the grid, extra solution was removed using a blotting paper and then the film on the SEM grid were allowed to dry by putting it under a mercury lamp for 5 mins and the sample was analyzed for morphology and size of the silver nanoparticles (Pandiarajan et al., 2016a).

Bacterial and fungicidal activity was using standard agar well diffusion method against human pathogenic bacteria (Pseudomonas aeruginosa, Alkaligenes enterobacteriaceae, Salmonella typhi, Vibrio cholerae, Serratia marcescens, Escherichia coli, and klebsiella pneumoniae) and fungi (Aspergillus niger, Aspergillus clodosporium, Aspergillus nodulans, Clodo spirillum, Aspergillus flavus, Fusarium oxysporum, Aspergillus oryzae). Nutrient agar (NA) and Potato Dextrose Agar (PDA) were prepared for cultivation of the bacteria and fungi respectively. Approximately 20 ml of molten and cooled media was poured in sterilized petridishes (Balaji et al., 2015). The plates were left overnight at room temperature to check for any contamination to appear. Then under an aseptic condition, placed a sterile swab into the broth culture of a fresh overnight grown cultures of the bacteria and fungi then gently removed the excess liquid by gently pressing or rotating the swab against the inside of the tube and spread it on nutrient agar and potato dextrose agar containing Petri plates respectively. With a help of sterile borer, 5 mm. of 4 discs was placed on the solid agar medium. The disc was dried different solution 1 disc was dried with normal distilled water. 2nd disc was dried with silver nitrate and 3rd disc was dried with cocoon extract, 4th disc dried with synthesized nanoparticles and the disc was placed on the plates were incubated at 37 °C for 12–24 h and 2 or 3 days for bacteria and fungi respectively. Further, the plates were examined for evidence of zone of inhibition, which appear as a clear area around the wells surrounding bacteria and fungi growth. The diameter of such zones of inhibition was measured using a meter ruler and the mean value for each organism was recorded and expressed in millimetre (Balaji et al., 2015).

2.6. Energy dispersive X-ray spectroscopy (EDX) Cocoon extract reduced silver solutions were dried; drop coated on to carbon film, and tested using Hitachi S-attachments. Energy dispersive X-ray spectroscopy (EDAX) analysis for the conformation of elemental silver was carried out for the detection of elemental silver. EDAX was sample composition of the analyzed for the sample composition of synthesized nanoparticles (Pandiarajan et al., 2016a). 2.7. Fourier Transfrom Infrared spectroscopy (FTIR) For FTIR measurements, the Ag nanoparticle solution was centrifuged at 10,000 rpm for 30 min. The pellet was washed three times with 20 ml de-ionized water to get rid of the free proteins/ enzymes that are not capping the silver nanoparticles. The samples were dried and grinded with KBr pellets and analyzed (Pandiarajan et al., 2016a). 2.8. X-ray powder diffraction (XRD) A thin film of the silver nanoparticle was made by dipping a glass plate in a solution and carried out for X-ray diffraction studies. The crystalline silver nanoparticle was calculated from the width of the XRD peaks, using the Debye-Scherrer formula,

2.12. Antioxidant activity 2.12.1. DPPH radical scavenging assay The antioxidant activity of synthesized nanoparticles was determined on the basis of their scavenging activity of the stable 1, 1diphenyl-2-picryl hydrazyl (DPPH) free radical. The sample and ascorbic acid were mixed with 95% ethanol to prepare the stock solution (5 mg/ml). Here ascorbic acid was taken as standard. At first, 5 tubes were taken to make aliquots of 5 concentrations (20–100 µl) with the samples. DPHH was weighed and dissolved in ethanol to make 0.004% (w/v) solution and to dissolve homogeneously magnetic stirrer was used. After making the desired concentrations 3 ml 0.004% DPPH solution was applied on each test tubes by pipette. The room temperature was recorded and kept the test tubes for 30 mins to complete the reactions. DPPH was also applied on the blank test tubes the same where only ethanol was taken as blank. After 30 mins, the absorbance of each test tube was taken by a UV spectrometer at 517 nm (Paramasivam

D = 0.94λ/β cos θ Where, D is the average crystallite domain perpendicular to the reflecting planes, λ is the X-ray wave length and β is the full width at half maximum and θ is the diffraction angle (Paramasivam et al., 2017). 2.9. Zeta potential Measurements Zeta potential is a physical property which is given the net surface charge of the nanoparticles, when these particles inside the solution repelling each other's since produced Coulomb explosion between the charges of the nanoparticles giving rise to no tendency for the particles to agglomerate. The criteria of stability of NPs are measured when the 3

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et al., 2017).

% of free radical inhibition Absorb. Of blank − Absorb. Of scavenging activity sample = × 100 Absorb. Of blank 2.13. Hydrogen peroxide assay The antioxidant of the synthesized nanoparticles was determined on the basis of their scavenging activity of the stable hydrogen peroxide free radicals. The samples and hydrogen peroxide were mixed with phosphate buffer (pH-7.4) to prepare stock solution. Here ascorbic acid was taken as standard. At first, 5 test tubes were taken with aliquots of 5 concentrations (20–100 µl) of the synthesized silver nanoparticles. To that, 0.6 ml of H2O2 in phosphate buffer was added. The reaction mixture was incubated at room temperature for 10 mins. Absorbance was read at 230 nm against the blank. Then the percentage of inhibition was calculated by the following equation (Paramasivam et al., 2017).

% of free radical inhibition =

Control OD–Sample OD × 100 Control OD

2.14. Anti-cancer activity The human breast adenocarcinoma cell line (MCF7) was obtained from National Centre for Cell Science (NCCS), Pune and grown in Eagles Minimum Essential Medium (EMEM) containing 10% fetal bovine serum (FBS). All cells were maintained at 37 °C, 5% CO2, 95% air and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week. 2.15. Cell treatment procedure

Fig. 3. A. UV- Visible Spectra of Bombyx mori cocoon shell extract (control). B. UV - UV–VisSpectra of silver nanoparticle synthesized from Bombyx mori cocoon shell extract at 60 °C.

The monolayer cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) to make single cell suspensions and viable cells were counted by tryphan blue exclusion assay using a hemocytometer. The cell suspension was diluted with medium containing 5% FBS to give final density of 1 × 105 cells/ml. one hundred microlitres per well of cell suspension were seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 37 °C, 5% CO2, 95% air and 100% relative humidity. After 24 h the cells were treated with serial concentrations of the test samples. It was initially dispersed in phosphate buffered saline (PBS) and diluted to twice the desired final maximum test concentration with serum free medium. Additional four, 2 fold serial dilutions were made to provide a total of five sample concentrations. Aliquots of 100 µl of these different dilutions of AgNPs from cocoon extract were added to the appropriate wells already containing 100 µl of medium, resulted the required final sample concentrations. Following drug addition the plates were incubated for an additional 48 h at 37 °C, 5% CO2, 95% air and 100% relative humidity. The medium containing without samples were served as control and triplicate was maintained for all concentrations.

% Cell Inhibition = 100 − Abs(sample)/Abs (control) × 100. Nonlinear regression graph was plotted between % Cell inhibition and Log concentration and IC50 was determined using GraphPad Prism software. 3. Result Numerous physical, chemical and biological methods were adapted for silver nanoparticle synthesis. Among these methods, biological synthesis is impacts at very higher rate. The present study gives the evidence that cocoon extract was found to be a successful agent for the synthesis of silver nanoparticles. The white cocoons were selected for the experimentation, it was sliced manually (Fig. 1A & B). The cocoons was treated with Milli Q water and subjected to heat treatment at different temperature (30 °C–100 °C) and standardized at 60 °C which exposed the perfect amber brown colour change (Fig. 2). The colour development indicated the silver nanoparticle synthesis with respect to comparison of control and standardized temperature of heat treatment (Fig. 2). The synthesized silver nanoparticle at different temperature where analyzed for spectrum peak. The spectrum analysis revealed the peak at the range of 420–430 nm, which indicated the presence of nano silver. The SEM Analysis of silver nanoparticles synthesized through cocoon extract showed a range of 50–500 nm in different magnification at (5 ×, 20 ×, 40 ×). Zeta potential exposed the presence of single peak which indicates the synthesis of fine nanoparticles from cocoon extract. Higher intensity (200–250 nm) is 50%

2.16. MTT assay 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Therefore, the amount of formazan produced is directly proportional to the number of viable cells. After 48 h of incubation, 15 µl of MTT (5 mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 37 °C for 4 h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100 µl of DMSO and then measured the absorbance at 570 nm using micro plate reader. The % cell inhibition was determined using the following formula.

3.1. Visible observation of silver nanoparticles synthesis The silver nanoparticles were synthesized using cocoon extract of 4

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Fig. 4. A. FTIR Spectra of Bombyx mori cocoon shell extract (Control). B FTIR Spectra of silver nanoparticle synthesized from Bombyx mori cocoon shell extract at 60 °C.

The synthesis of silver nanoparticles in the mixture of solution was further analyzed and confirmed by UV-visible spectroscopy. The UV spectra peaks of cocoon extract showed only one peak at the range of 600–650 nm (Fig. 3A). The cocoon extract with silver nitrate treated at 60 °C showed three peaks at the range of 420–450 nm (Fig. 3A & B).

2358.94 cm−1 were assigned to the Alkenes C˭C Stretch, alkenes C˭C stretch, alkenes C˭C stretch, alkynes C≡C stretch, alkynes C≡C stretch, alkynes C≡C stretch, alkynes C≡C stretch, Silane Si-H, Silane -H. (Fig. 4A) The absorption bands for 80° C seen at 1637.56 cm−1 1917.24 cm−1 1967.39 cm−1 2023.33 cm−1 2152.56 cm−1 −1 −1 −1 2204.64 cm 2339.65 cm 2358.94 cm were assigned to the Alkenes C˭C Stretch, alkenes C˭C stretch, alkenes C˭C stretch, alkynes C≡C stretch, alkynes C≡C stretch, alkynes C≡C stretch, Silane Si-H, Silane -H. (Fig. 4B) The absorption bands for 100° C seen at 1637.56 cm−1 1951.96 cm−1 1967.39 cm−1 2031.04 cm−1 −1 −1 −1 2181.49 cm 2204.64 cm 2337.72 cm 2358.94 cm−1 were assigned to the Alkenes C˭C Stretch, alkenes C˭C stretch, alkenes C˭C stretch, alkynes C≡C stretch, Isocyanates –N = C˭O stretch, Isocyanates –N = C˭O stretch, Silane Si-H, Silane -H. (Fig. 4B).

3.3. FTIR spectroscopic analysis

3.4. SEM analysis of silver nanoparticles

FTIR spectrum absorption bands 1635.64 cm−1 21.81.49 cm−1

SEM analysis showed that the about the morphology and size of the synthesized silver nanoparticles of cocoon extract at different magnifications such as 5 × , 20 × and 40 × showed circular dot shaped particles ranges from 29.52 to 295.2 nm. (Fig. 5A, B & C).

Bombyx mori. The reduction of silver ions into silver particles during exposure to the cocoon extract is followed by colour change. As the cocoon was mixed in the aqueous solution of silver ion complex, it started to change the colour from yellowish brown to dark brown. Almost all the nanosolutions after incubation time were showed the colour change from light to dark colour. Fig. 2B. 3.2. UV-Visible spectroscopy analysis

of produced silver nanoparticles were showed many and the absorption bands for control seen at 1965.46 cm−1 1982.82 cm−1 2162.20 cm−1 −1 −1 2204.64 cm 2227.78 cm 2337.72 cm−1 5

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Fig. 7. Zeta potential value of silver nanoparticle synthesized from Bombyx mori cocoon shell extract.

Fig. 8. Particle size distribution of silver nanoparticle synthesized from Bombyx mori cocoon shell extract.

Fig. 5. A. SEM imaging of silver nanoparticle synthesized from Bombyx mori cocoon shell extract (5X). B. SEM imaging of silver nanoparticle synthesized from Bombyx mori cocoon shell extract (20X). C. SEM imaging of silver nanoparticle synthesized from Bombyx mori cocoon shell extract (40X).

Fig. 9. XRD pattern of silver nanoparticle synthesized from Bombyx mori cocoon shell extract.

3.5. EDAX analysis of silver nanoparticles

Fig. 6. EDX Spectrum of silver nanoparticle synthesized from Bombyx mori cocoon shell extract at 60 °C Strong signal of silver (Ag) at 3 KeV.

Energy Dispersive Absorption Spectroscopy (EDAX) of synthesized nanoparticles of cocoons was shown in the Fig. 6A&B. The presence of expected element (Ag) in the final products was observed. The present 6

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be positive or negative; the negative potential value shown Fig. 7 by AgNps could be due to the possible capping of the bioorganic components present in the extract. 3.7. Particle size measurements The particle size of the synthesized silver nanoparticles was determined using dynamic light scattering measurement technique. Dynamic light scattering (DLS) is a technique for characterizing the size which utilizes the illumination of a suspension of particles or molecules undergoing Brownian motion by a laser beam. The time-dependent fluctuations in the intensity of scattered light that occur are analyzed using an autocorrelator which determines the autocorrelation function of the signal. The size distribution of the synthesized AgNps was depicted in Fig. 6. From the figure, it was observed that the particles obtained are polydisperse mixtures in the range 190–290 nm. The average size of the synthesized silver nanoparticles using cocoon extract is around 222 nm. Sizes and shapes of metal nanoparticles are influenced by a number of factors including pH, precursor concentration, reluctant concentration, time of incubation, temperature as well as method of preparation. Fig. 8

Fig. 10. Representation of antimicrobial activity of silver nanoparticle synthesized from Bombyx mori cocoon shell extract.

analysis revealed that the silver nano structures were formed as peak at 3 keV solely of silver nanoparticles for cocoons. 3.6. Zeta potential measurements

3.8. X-ray diffraction analysis The zeta potential of the synthesized AgNps was determined in water as dispersant. The zeta potential was found to be − 2.10 mV. The high value confirms the repulsion among the particles and there by increases in stability of the formulation. The zeta potential value could

XRD pattern is characterized by the interplanar d -spacing and the relative intensities (I/I°) of the strongest peaks. It was found out the position of values of product crystallinity or amorphic nature. Data

Fig. 11. Antibacterial activity of silver nanoparticle synthesized from Bombyx mori cocoon shell extract. 7

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D. Vibrio cholerae

A. Pseudomonas aeruginosa B. Alkaligenes enterobacteriaceae

E. Serratia marcescens F. Escherichia coli

C. Salmonella typhi

G. Klebsiella pneumonia Fig. 11. (continued) Table 1 Zone of diameter obtained through antibacterial activity of silver nanoparticle synthesized from Bombyx mori cocoon shell extract. Organism

AgNO3(mm)

Cocoon extract (mm)

Nanoparticles synthesis(mm)

Pseudomonas aeruginosa Alkaligenes enterobacteriaceae Salomonela typhi Vibrio cholerae Serratia marcesens E.coli Klebsiella pneumoniae

11 7

5 5

9 9

9 8 7 11 8

5 5 5 5 5

8 8 8 9 6

Fig. 12. Representation of antifungal activity of silver nanoparticle synthesized from Bombyx mori cocoon shell extract.

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Fig. 13. Antifungal activity of silver nanoparticle synthesized from Bombyx mori cocoon shell extract.

helps in finding out the fingerprinting region of relative intensity with respect to d-spacing values. The XRD pattern showed the intense peaks in the whole spectrum of 2Ө values ranging from 10 to 90 for the silver nanoparticles. The synthesized silver nanoparticles were in the form of nanocrystals of cocoon 2Ө values were 30.2054, 36.0112, and 39.6120 corresponding to the diffraction peaks exhibited from 10 to 90 range of 2Ө (Fig. 9).

bacterial cultures were used namely (Aspergillus niger, Aspergillus flavus, Aspergillus clodosporium, Clodo spirillum, Aspergillus nodulans, Fusarium oxysporum, Aspergillus oryzae). After the incubation of 48 hrsthe zone of inhibition was assessed to evaluate the activity (Figs. 12 and 13) (Table 2).

3.9. Antibacterial activity

3.11.1. Radical scavenging activity of DPPH Assay The free radical scavenging activity of silver nanoparticles of cocoon determined by DPPH (1,1-diphenyl-2-picrylhydrazyl) method. The DPPH radical scavenging effects of nanoparticles of cocoon were showed in Fig. 14 and Table 3 and the silver nanoparticles showed the significant free radical scavenging activities when compared to with standard ascorbic acid. In various concentrations of synthesized silver nanoparticles (20–100 µl) about 36–59% of free radicals were observed in cocoon silver nanoparticles. (Fig. 14 and Table 3).

3.11. Antioxidant activity

Antibacterial activities of silver nanoparticles from extract of cocoons were performed by disc diffusion method. In this study, five bacterial cultures were used namely (Pseudomonas aeruginosa, Alkaligenes enterobacteriaceae, Salmonella typhi, Vibrio cholerae, Serratia marcescens, Escherichia coli, and klebsiella pneumoniae). In that plates the silver nanoparticles were added and incubated for 24 h. After incubation, the zone of inhibition was well was measured (Figs. 10 and 11) (Table 1).

3.12. Scavenging activity of Hydrogen peroxide 3.10. Antifungal activity The H2O2 scavenging activity of the silver nanoparticles of cocoon·H2O2 scavenging activity of silver nanoparticles compared with standard ascorbic acid. The percentage of inhibition of free radicals

Antifungal activities of cocoon extract derived silver nanoparticles were performed by Agar-well diffusion method. In this study, five 9

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A. Aspergillus niger

D. Clodo spirillum

B. Aspergillus clodosporium

E. Aspergillus flavus

C. Aspergillus nodulans

F. Fusarium oxysporum G. Aspergillus oryzae

Fig. 13. (continued) Table 2 Zone of diameter obtained through antifungal activity of silver nanoparticle synthesized from Bombyx mori cocoon shell extract. ORGANISM

AgNO3(mm)

Cocoon extract(mm)

Nanoparticle synthesis(mm)

Aspergillus niger Aspergillus clodosporium Aspergillus nodulans Clodo spirillum Aspergillus flavus Fusarium oxysporum Aspergillus oryzae

2 9

5 5

8 7

25 16 4 17 25

5 5 5 5 5

5 6 9 7 6

Fig. 14. Percentage of antioxidant activity of ascorbic acid by DPPH Assay in silver nanoparticle synthesized from Bombyx mori cocoon shell extract at 60 °C.

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The promising reagent of a pale yellow substrate of tetrazolium salts (MTT) by incubation with one hundred microlitres of cell suspension per well were seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 37 °C, 5% CO2, 95% air and 100% relative humidity for 12 h. The incubation of living human breast adenocarcinoma cell line (MCF7) at 48 hrsproduced a dark blue formazan product. The MTT formazan reaction product was only partially soluble in the medium, and so an alcohol was used to dissolve the formazan and produce a homogeneous solution suitable for measurement of optical density. The normal tissue culture medium has a variable colour due to pH changes and the red form of phenol red interfered at the wavelength most suitable for blue MTT formazan measurement. The results of anticancer activity of silver nanoparticles from cocoon extract of Bombyx mori are presented in Fig. 16. The results show that the absorbance is directly proportional to the number of viable cells. Optical density was measured on a Dynatech MR 580 plate reader, using a reference wavelength of 630 nm and a test wavelength of 570 nm. The high difference in the OD values between the test and control wells of the strains at a concentration of 1.0 × 105 cells/ml in comparison to other test groups. The increase in absorbance values between the test wells of human breast adenocarcinoma cell line (MCF7) above to the concentration (1.0 × 105 cells/ml) level was nonsignificant. In this case, the absorbance values of the test wells of human breast adenocarcinoma cell line (MCF7) were found to be less than the absorbance values of control wells. In addition, the 10 μg and 20 μg concentration of silver nanoparticles of cocoon extract from Bombyx mori displayed 100% cell inhibition compared to other concentrations (Fig. 17).

Table 3 Comparative percentage of antioxidant activity of ascorbic acid with silver nanoparticle synthesized from Bombyx mori cocoon shell extract by DPPH Assay. S.no

Conc. of silver nanoparticles

Scavenging activity for cocoon AgNps(%)

Scavenging activity for Ascorbic acid (%)

1 2 3 4 5

20 ul 40 ul 60 ul 80 ul 100 ul

36% 45% 50% 56% 59%

63% 72% 85% 93% 98%

Fig. 15. Percentage of antioxidant activity of ascorbic acid by Hydrogen Peroxide Assay in silver nanoparticle synthesized from Bombyx mori cocoon shell extract at 60 °C.

4. Discussion

Table 4 Comparative percentage of antioxidant activity of ascorbic acid with silver nanoparticle synthesized from Bombyx mori cocoon shell extract by Hydrogen Peroxide Assay. S.no

Conc. of silver nanoparticles

Scavenging activity for cocoon AgNps(%)

Scavenging activity for Ascorbic acid (%)

1 2 3 4 5

20 ul 40 ul 60 ul 80 ul 100 ul

22% 36% 43% 48% 55%

44% 62% 83% 89% 97%

Nanotechnology is an innovative field which influences all aspects of human's life (Mohanpuria et al., 2008). The “green” method for nanoparticles synthesis which is rapidly traditional chemical synthesis is of great interest because of eco-friendliness, economic views, feasibility and wide range of applications in several areas. Since the domain of biosynthesized nanoparticles is somewhat novel, and their use in different areas including the delivery of drug, cancer therapy, gene treatment and DNA analysis, antibacterial factors, biosensors, increasing response rates, separation science provided (Akai et al., 2003). Silver nanoparticles (AgNps) are being extensively studied because of its smaller size and its superior activity. AgNps are basically of two major types; chemically synthesized Silver nanoparticles and biologically synthesized Silver nanoparticles. Quantum and semiquantums are the best example of the chemically synthesized nanoparticles. Biological silver nanoparticles are derived from sources such as plants, bacteria, fungi and algae. Recently in sericulture, the scientists are attempting to improve the silk production by using the nanoparticles synthesized from mulberry leaves (M. alba), which may be exploited to devise an artificial diet during the scarcity of leaf production. Also the scientific community was very much interested to study the physiological and biochemical impact of nanoparticles on silkworm B. mori (Pandiarajan et al., 2016a & b). The present investigation on cocoon extract of Bombyx mori revealed its potential to reduce silver nitrate to silver nanoparticles. The colour of the reaction medium changed into dark brown colour after 24 h. In the present study the colour changes was observed within 10 mins. For, instance, the colour change in 5 mins incubation period of using cocoon extract (Raja et al., 2012). The increase in intensity of UV absorption spectra indicates increase in the number of silver nanoparticles with increasing concentration of AgNO3. This indicates that maximum synthesis of silver nanoparticles (Ag) occurred with Ag+ concentration in the range 5–15 mg/ml, which a few earlier researchers also reported in the literature (Fei et al., 2013). The incident light acts as a catalyst which initiates the reduction process by breaking ionic bonds of metal nitrates and SF protein structures, there by creation of

Fig. 16. Effect of silver nanoparticle synthesized from Bombyx mori cocoon shell extract at 60 °C on cell inhibition of human breast adenocarcinoma cell line (MCF7).

increased with increase in concentrations of substrates. The concentration from 20 µ to 100 µl of silver nanoparticles showed 22–55% for cocoon silver nanoparticles. (Fig. 15 and Table 4).

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Fig. 17. Inhibition of human adenocarcinoma (MCF7) cell line growth at different concentration by drug - silver nanoparticle synthesized from Bombyx mori cocoon shell extract at 60 °C.

The secondary metabolites in substrate which have functional groups like amines, alcohols, aldehydes and carboxylic acids. The observed peaks were characteristics of polyphenolic compounds which were very abundant in substrate cocoon. From the FTIR analysis of silver nanoparticles, it was clear that it is reduced to nano silver and being encapsulated by secondary metabolites (Pandiarajan et al., 2016a & b) In the present study, SEM images the morphological character of silver nanoparticles synthesized by using cocoon extract of Bombyx mori. The arrows in the above figure show presence of circular nanoparticles in a range of 200 − 250 nm. It has been found that circular nanoparticles can be effectively used as a medicine for treatment of various ailments. Thus the circular shaped silver nanoparticles synthesized from cocoon extract can be exploited for many purpose and

free moving ions. Light irradiation reduction of silver nanoparticles will change the optical properties of colloidal solution, when the concentration of AgNO3 is changed, which can be related to the size and shape of the reduced silver nanoparticles (Tran and Le, 2013). For the higher concentration of AgNO3 (20 mg/ml) the intensity of the absorption peak is decreased and the peak is shifted. The peak shift may be attributed to the change in particle size (Rao et al., 2010). In the present study FTIR spectrum indicates the cocoon extract silk worm Bombyx mori assisted production of silver nanoparticles by showing functional groups like Alkenes, Alkynes, Isocyanates, and silane present at different position. From this observation, the identified biomolecules present in the cocoon extract of silk worm Bombyx mori was responsible for reduction and stabilization of silver nanoparticles. 12

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and survival, the bactericidal activity of macrophages. In MTT assay, the numbers of metabolically active living cell were measured in control (1.0 × 105 cells/ml) as well as other dilutions. MTT assay was judged by conversion of MTT dye with the actively proliferating human breast adenocarcinoma cell line (MCF7) of the test and control; it required establishing optimal cell concentration for effective discrimination of the test and control. The optimal concentration of cells to microculture plate was found to be 1.0 × 105, as cell concentrations of 1.5 × 104 to 1.5 × 102 cells/ml dispensed in 100 µl total volume were found to be either dead or low in measurable formazan activity. Although it was interesting to observe that wells receiving human breast adenocarcinoma cell line (MCF7) at 1.0 × 102 − 1.0 × 104 cells/ml may have caused an overcrowded condition resulting in mostly dead cells after 24 h after incubation and with lower formazan activity in comparison to zero h cultures. The results of the MTT assay suggest that increase in formazan activity of human breast adenocarcinoma cell line (MCF7) due to incubation with low concentration of silver nanoparticles (Kaewkorn et al., 2012).

process in future. Energy Dispersive Absorption Spectroscopy photographs of derived AgNps. All the peaks of Ag are observed and are assigned. Peaks for Cu and C are from the grid used and the peaks for S, P and N correspond to the protein capping over the AgNps. Silver nanocrystallites display an optical absorption band peak at approximately 3 keV, which is typical of the absorption of metallic silver nanocrystallites due to surface (Pandiarajan et al., 2016a). Through DLS identification the larger particles, as the smaller nanoparticles undergoes scattering. The identification of large sized particles in DLS results is mainly due to increasing the possibility of formation of larger fluffy particles during reduction reaction (Esumi et al., 2000). Hence from this study it is evident that the average size of the nanoparticle increased with increasing the concentration of silver nanoparticles. This is also supported by UV–visible study. The zeta potential of synthesized silver nanoparticles is − 33.6, − 200, + 1.2, + 5 and + 14.3 mV indicates the stability of nanoparticles. A negative zeta potential was observed in the present study. Generally silver nanoparticles carry a negative charge (Gou et al., 2015). But at higher concentration silver nanoparticles carry a positive charge because SF acts as a good stabilizer. Biosynthesized silver nanoparticles from SF were stable at room temperature. The X-ray diffraction peaks were found to be broad around their bases indicating that the silver particles are in nanosizes. The peak broadening at half maximum intensity of the X-ray diffraction lines is due to a reduction in crystallite size, flattening and micro-strains within the diffracting domains. The mean particle diameter of AgNps was calculated from the XRD pattern, according to the line width of the maximum intensity reflection peak (Paramasivam et al., 2017). The mechanism of the bactericidal effect of silver colloid particles against bacteria is not very well known. The different mechanism by which the silver nanoparticle inhibits cell wall synthesis is by interference with cell wall synthesis, inhibition of protein synthesis, interference with nucleic acid synthesis and inhibition of a metabolic pathway. As the concentration of the silver nanoparticles increased the zone of inhibition increased (Guzman et al., 2012). It has been reported the greater the zone of inhibition, greater the antibacterial properties of the silver nanoparticles and that antibacterial effect was size and dose dependent (Devi et al., 2012). The inhibition of bacterial growth reported in this study is dependent on the concentration of silver nanoparticles in the medium. Antioxidant activities are attributed to the phenolic contents in plants probably due to their redox properties, which allow them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers (Chang et al., 2001). Although no available literature on the total phenolic content in C. murale, (Laghari et al., 2011) reported higher total phenolic contents (3066 mg of GAE/100 g) in Chenopodium album. In the same concern, (Imam et al., 2006) reported that plants in the family Chenopodiaceae are rich in phenolic and flavonoid compounds and induced antioxidant potentials. Similar to the current results, (Nsimba et al., 2008) reported a higher antioxidant activity to other spices of the family Chenopodiaceae i.e. chenopodium quinoa and chenopodium album. Moreover, the results also indicated that the marginal increase in antioxidant activity of plant-AgNps, compared to the plant extract suggested that the plant extract itself is responsible for the majority of the antioxidant activity and AgNps is not contributing much to the antioxidant activity. The growth characteristics of human breast adenocarcinoma cell line (MCF7) was monitored in a medium and diluted to variable concentration. The diluted cell lines poured on microculture plate and carried out MTT assay. MTT assay is based on the activity of the mitochondrial enzyme, succinate-dehydrogenase of viable cells to transform the MTT tetrazolium salt into a blue colored product, MTT formazan and is proportional to the number of living cells present. In a similar fashion, the MTT assay was used primarily in mammalian cell studies as a measure of cell activation (Devi et al., 2012), cell growth

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