The antibacterial effect of phyto-mediated silver nanoparticles produced from Ocimum sanctum L . ( Lamiaceae ) leaf extract on textile fabrics

The Ocimum sanctum (holy thulsi) leaves extract perform as a reducing and capping agent in the formation of silver nanoparticles. A UV-VIS spectrum of the silver nanoparticles showed a peak at 421 nm. The morphology and size of the silver nanoparticles were carried out by the transmission electron microscope (TEM) and scanning electron microscope (SEM). The sizes of the synthesized silver nanoparticles were found to be in the range of 7 to 45 nm. The structural characteristics of biomolecules hosted silver nanoparticles were studied by X-ray diffraction. The chemical composition of elements present in the solution was determined by energy dispersive spectrum. The FTIR analysis of the nanoparticles indicated the presence of proteins, which may be acting as capping agents around the silver nanoparticles. This study reports that green synthesis is medicinally useful nanoparticles to avoid toxic chemicals with adverse effects in medical applications rather than physical and chemical methods. The antibacterial activity of phyto mediated silver nanoparticles was assessed by the paper disc method against Klebsiella pneumonia and 12 mm clear zone was observed. The antibacterial activity of the finished fabrics was assessed quantitatively by reduction test. The topographical analysis of the treated fabric and untreated fabric was studied and compared. The results show that the finished fabric demonstrated significant antibacterial activity against Escherichia coli.


INTRODUCTION
Remarkable advances are made in the field of nanobiotechnology to harness the benefit of life sciences (Huang et al., 2007), healthcare (Ahmad et al., 2010) and Industrial biotechnology (Elechiguerra et al., 2005).Nanomaterials especially silver nanoparticles may provide solutions to technological and environmental challenges in the areas of solar energy conversion, catalysis, medicine, nano-biosensor, targeted drug delivery system and water treatment (Bao et al., 2007).Silver has long recognized as an inhibitory effect on microbes present in medical and industrial process (Reda et al., 2011) and have antimicrobial properties with low toxicity (Jain et al., 2009).
This increasing demand must be accompanied by ''green'' synthesis procedures.A number of approaches are existing by the use of chemical and photochemical reactions for syntheses of silver nanoparticles such as reverse micelles, thermal decomposition of silver compounds, radiation assisted, electrochemical, sonochemical and microwave assisted process (Parashar et al., 2009).Chemical synthesis methods lead to presence of toxic chemical engrossed on the surface that nanoparticles may have adverse consequence in the medical applications (Jain et al., 2009).Now a day biosynthesis of nanoparticles are used by most researchers to overcome the above problems (Udayasoorian et al., 2011).The green synthesis is more advantageous over chemical and physical method as it is cost effective and environment friendly (Kowshik et al., 2003;Nabhikha et al., 2009).
Concerning the biological application of nanoparticles, it has been emphasized that methods of synthesis through biological systems namely; microorganisms including bacteria, yeasts, fungi and diatoms synthesizing inorganic materials either intra or extracellular would make the nanoparticles more biocompatible (Thirumurugan et al., 2011).One of them is the synthesis using plant extracts eliminating the elaborate process of maintaining the microbial culture and often found to be kinetically favorable than other bioprocesses.Bio-molecules as reducing agents are found to have a significant advantage over their counterparts as protecting agents.
With the growth in world population and the spread of disease, the number of antibiotic resistant microorganisms is rising along with the occurrence of infections from these microorganisms.With this increase in health awareness, many people focused their attention on educating and protecting themselves against harmful pathogens.It soon became more important for antimicrobially finished textiles to protect the wearer from bacteria than it was to simply protect the garment from fiber degradation (Rajendran et al., 2010).
In these aspects synthetic methods based on naturally occurring biomaterials provide an alternative means for obtaining these nanoparticles.During recent times several groups have achieved success in the synthesis of Ag nanoparticles using extracts obtained from unicellular organisms like bacteria and fungi as well as extracts from plant parts, for example, geranium leaves, lemon grass, neem leaves, Aloe vera, and several others (Ahmad et al., 2003;Nair and Pradeep, 2002;Shahverdi et al., 2007;Ahmad et al., 2010;Shiv Shankar et al., 2005;Chandran et al., 2006).The spectacular success in this field has opened up the prospect of developing bioinspired methods of synthesis of metal nanoparticles with tailor-made structural properties.Among the various bioreductants, Ocimum sanctum (Labiatae), a sweet basil leaf extract was chosen for the present study since they have essential oils and synthesized Ag nanoparticles are used for preparation of antimicrobial fabrics.

Chemicals
All analytical reagents and media components were purchased from Hi-Media (India).

Collection of plant leaves and preparation of extracts
O. sanctum plant leaves were collected from a university campus itself and thoroughly washed with distilled water and kept it at room temperature.The plant leaf broth solution was prepared by taking 20 g of leaves, cut into small pieces and ground in a mortar and pestle with 100 ml of sterile distilled water and centrifuged at 3000 RPM to get the leaf extract.

Synthesis of silver nanoparticles
Two flasks were taken, in the first flask 25 mL of leaf broth was added to 200 ml of 1 mM aqueous AgNO3 solution for reduction of Ag + ions and no leaf broth was added to the second flask and it considered as control.Both flasks were kept at room temperature on shaker for 24 h.

Extraction of silver nanoparticles
After the incubation period the silver nanoparticle solution thus obtained was purified by repeated centrifugation at 15,000 rpm for 20 min followed by re-dispersion of the pellet in deionized water.

Ultraviolet-visible spectroscopy (UV-VIS) spectra analysis
UV-Vis spectroscopy measurements (Shimadzu, Japan) were carried out as a function of time of the reaction at room temperature operated at a resolution of 1 nm.The reduction of silver ions was confirmed by qualitative testing of supernatant obtained after centrifugation with a pinch of NaCl.The reduction of silver ions was monitored by measuring the absorbance of the reaction mixture in a range of wavelength from 200 to 800 nm to find the absorbance peak different intervals (0, 30 and 60; 2, 4, 8, 16 and 24 h).

Fourier transform infrared (FTIR) analysis
For Fourier transform infrared (FTIR) spectroscopy measurement, the following method was adopted.The silver nanoparticles were synthesized after 24 h of incubation with the leaf extracts, centrifuged at 10,000 rpm for 15 min, and their pellets were then dried and the powder was subjected to FTIR spectroscopy measurement (Perkin Elmer spectrophotometer) in the reflectance mode at a resolution of 4 cm -1 in KBr pellets.

Scanning electron microscopy and energy dispersive spectroscopy (EDS) analysis
After 24 h of incubation leaf extracts were analyzed under a scanning electron microscopic (JOEL) at a voltage of 120 kV.EDS analysis was carried out on the JEOL Analysis Station at an accelerating voltage of 20 keV.

Transmission electron analysis
Samples for transmission electron microscopy (TEM) analysis were prepared by drop coating biologically synthesized silver nanoparticles solution (24 h reaction of the silver nitrate solution with the O. sanctum leaf extract) on carbon-coated copper TEM grids.The films on the TEM grid were allowed to stand for 2 min, following which extra solution was removed using a blotting paper and grid allowed to dry prior to measurement.TEM measurements were performed on a Phillips EM-CM-12 model instrument operated at an accelerating voltage of 100 KV.

Potentiometric study
The change in the oxidation -reduction potential of the nanoparticles containing solution with time was studied using a potentiometer (Digital potentiometer, 318, Systronics, India) in which saturated calomel electrode and platinum electrode were used.

Zeta potential measurement
The zeta potential of the synthesized nanoparticles was determined   by means of the zeta potential analyzer at pH around 5.5 by suspending the nanoparticles containing solution in potassium chloride solution with ionic strength 10 -3 M. The measurement of zeta potential is based on the direction and velocity of particles under influence known electric field.

Antimicrobial assay for silver nanoparticles
The antimicrobial assay was performed by the disc -diffusion technique.In this technique, 50 µl of silver nanoparticle prepared from leaf extract, applied to sterile paper discs of 5 mm diameter.
The discs were then placed on Nutrient Agar inoculated with clinical strains of bacteria (Klebsiella pneumoniae).The plates were incubited at 37 0 C for overnight.The zone of inhibition was measured in millimeter after the 24 h of incubation and recorded.

Silver nanoparticles loading on cotton fabrics
Cotton fabrics were washed, sterilized and dried before use.Experiments were performed on samples with maximum dimensions of 5 × 5 cm.In order to impregnate cotton fabrics (5 × 5 cm), these were submersed in an Erlenmeyer (50 ml) flask containing silver nanoparticles solution and shaking at 600 rpm for 24 h and dried at 70°C.

Antibacterial activity of nanoparticles loaded on cotton fabrics
To examine the bacterial growth or death kinetics in the presence of silver nanoparticles loaded fabric, E. coli cells were grown in continuously stirred 100 ml LB medium at 37°C supplemented by a pre-weighed piece of fabric and agitated at 200 RPM.Growth kinetics rates and bacterial concentrations were determined by measuring the optical density (OD) at 600 nm.The OD values were converted into concentrations of E. coli colony forming units (CFU) per ml) using the approximation that an OD value of 0.1 corresponded to a concentration of 108 cells per ml (Pal et al., 2007).

RESULTS AND DISCUSSION
The color change was noted by virtual observation in O. sanctum leaf extract incubated with an aqueous solution of AgNO 3 .It started to change color from watery to yellowish brown at 4 th h and dark pink color at the 24 th h after incubation (Figure 1).It is due to the reduction of silver ions, this exhibit the formation of silver nanoparticles (Table 1).The color of the extract changed to intense brown after 24 h of incubation and there was no significant change afterwards (Figure 1).The presence of silver nanoparticles was confirmed by obtaining a range of 200 to 600 nm.A typical peak of λ max at 421 nm was obtained due to the surface plasmon resonance of silver nanoparticles.The surface plasmon absorption peaks depends on the size and shape of the metal nanoparticles as well as on the dielectric constant of the metal itself and the surrounding medium (Mukherjee et al., 2002) (Figure 2).
FTIR measurements were carried out to identify the possible bio-molecules responsible for capping and efficient stabilization of the metal nanoparticles synthesized by leaf extract.The silver nanoparticle sample shows peaks at 3313.48, 3193, 2976.90, 2883, 1670, 1452, 1338, 1196.78, and 1112.75 cm -1 (Figure 3).The peaks corresponding to protein and silver nanoparticles were found commonly present in the nanoparticles synthesized by leaf extract.The peaks observed for silver nanoparticles at 1678 cm -1 (C=C groups or from aromatic rings), 1338 cm -1 (germinal methyls), and 1112 cm -1 (ether linkages), 3400 to 3200 cm -1 and 3000 to 2850 cm and 1450 to 1375 cm -1 correspond to N-H (bend) of primary and secondary amides and C-H (-CH3 -bend) of alkanes, respectively (Kannan and Subbalaxmi, 2011;Sathyavathi et al., 2010).
The peaks in the region of 1350 to 1000 cm -1 correspond to -C-N-stretching vibration of the amine or -C-Ostretching of alcohols, ethers, carboxylic acids, esters and anhydrides.FT-IR analysis reveals that the carbonyl group from amino acid residues and proteins has the strong ability to bend metal indicating that the proteins could possibly form a layer covering the metal nanoparticles (that is, capping of silver nanoparticles) to prevent agglomeration and thereby stabilize the medium (Khabat Vahabi et al., 2011;Udayasooriyan et al, 2011).
The crystalline nature of silver nanoparticles was studied with the aid of X-ray diffraction as shown in Figure 4.A number of strong Bragg's diffracted peaks observed at 27.82, 32.25, 46.22 and 76.63 corresponding to the 126, 199, 131 and 24 height of the face centered cubic pattern of silver were obtained.It suggests that the synthesized silver nanoparticles are crystalline in nature.The size of the silver nanoparticles was found to be 26 nm; and it was through by using the width of the (126) Bragg's reflection.In addition, yet some unassigned peaks were also observed suggesting the crystallization of biophase occurs on the surface of silver nanoparticles.X-ray diffraction (XRD) pattern thus clearly shows that the silver nanoparticles formed from phytomediated synthesis is crystalline in nature (Harekrishna et al., 2009).
Figure 5 shows fluorescence emission spectrum from silver nanoparticles, dispersed in double distilled water.A strong maximum at 431 nm wavelength and a quantum yield was 666.450 mV appeared in the fluorescence emission spectrum of O. sanctum leaf extract mediated silver nanoparticles.The pattern of the emission spectrum revealed that the visible emission from a silver particle is due to a transition of a photo generation electron from the conduction band to a deeply trapped hole (Liu et al., 2004).
The reduction of silver ions to form nanoparticles was also monitored using a potentiometer.A sharp reduction in the potential could be observed upon 4 h of interacttion further indicating the formation of nanoparticles at this stage.The potential down from an initial value of 0.436 V for silver ions to 0.153 V at the end of 11 h (Figure 6) after which the fall in potential was gradual, falling up to 0.048 V at the end of 24 h.The result obtained further corroborated the result from UV-visible spectroscopic studies that the Ag reduced at 11 h.Silver nanoparticle solutions will always have a residual charge unless the redox potential of the reductant used is  identical to -0.7 NHE (Mulvaney, 1996).
The alteration in zeta potential with a moment in time is shown in Figure 7.It can be observed that there was a charge stabilization from 11 to 16 h, with the charge stabilized around -57 mV.The zeta potential was -62 mV for the 14 h interacted samples which further decreased to -35 mV for the 24 h interacted samples.Solutions with zeta potential above +25 mV or below -25 mV are considered stable (Su et al., 2010).The stability is determined by the surface charge density and an increase in surface charge density decreases the tendency of aggregation and vice versa (Gast, 1977).Minor changes in the measured zeta potential sometimes may indicate significant changes in the surface charge density (El Badawyet al., 2010).
The results obtained from visible color changes, UV- visible studies, potentiometry, and zeta potential studies typically prove that silver nanoparticles started to appear in the system at the end of 4 h.Another significant conclusion drawn was that there was a notable agglomeration in the system at 12 h of interaction as observed from XRD and zeta potential analysis.
The shape and size of silver nanoparticles were analyzed after 24 h of incubation using SEM is shown in Figure 8.In general, the nanoparticles were in spherical shape with varying size ranged from 7 to 28 nm.Most of the nanoparticles were combined with only a few of them were scattered, as observed under SEM.
The energy dispersive spectrum (Figure 9) revealed the clear identification of the elemental composition profile of the synthesized nanoparticles, which suggests the presence of silver as the ingredient element.The EDS spectrum showed high for silver signals.The vertical axis shows the counts of the X-ray and the horizontal axis shows energy in keV.The strong signals of silver correspond to the peaks in the graph confirming presence of silver.
TEM technique was employed to visualize the size and shape of Ag nanoparticles. Figure 10 shows the typical TEM micrograph of the synthesized Ag nanoparticles.It is observed that most of the Ag nanoparticles were spherical in shape.A few agglomerated silver nanoparticles were also observed in some places, thereby indicating possible sedimentation at a later time.It is evident that there is variation in particle sizes and the average size estimated was 26 nm and the particle size ranged from 8 to 45 nm.The natural products, namely glycosides, flavanones, and reducing sugars are the main constituents of the O. sanctum leaf extract.The aldehydic groups and reducing sugars are responsible to the reduction of Ag+ ions into metallic Ag0 and also stabilizing the resulting nanoparticles (Zaheer Khan et al., 2012).The results of experiments may conclude that the O. sanctum leaf extract acts as reducing, stabilizing, and capping agents.
Antimicrobial activities of silver nanoparticles synthesized by leaf extracts from O sanctum are analyzed.The antibacterial activity of silver nanoparticles showed, the inhibition zone of 12 mm diameter was formed against K. pneumoniae by Ag nanoparticles synthesized by leaf extract (Figure 11).The fiber surfaces of the finished fabrics were observed by SEM.Images of the samples in Figure 12 show the deposi-tion of nanoscaled silver particles on the textile surface.The particles size varies from 12 m to 28 nm as can be seen.
The growth and death kinetics of Ag nanoparticles loaded cotton fabrics against E. coli was depicted in Figure 13.The antibacterial action of plain fabric (control), showed a dense population of bacterial colonies.The Ag-loaded fabric showed antibacterial activity it inhibits the growth of bacteria.The results of these antibacterial tests (Figure 13), clearly indicated that the fabric prepared by immersing in Ag nano-particles demon-  demonstrated greater biocidal activity.Therefore, the Ag content of the fabric is a key factor in controlling its antibacterial activity.It is clear from Figure 13 that in the initial phase bacterial growth is almost the same in the media containing plain and Ag-loaded fabrics.The killing action of Ag-loaded fabric began approximately 4 h after its incubation in nutrient broth medium.This may be attributed to the fact that when the Ag-loaded fabric was put in the bacterial suspension, the fabric began to expose to bacteria and killed the bacterial cells (Pranee et al., 2008).The killing activity of Ag-loaded fabric began to be reduced later, perhaps because nearly    all the Ag nanoparticles were consumed in binding to bacterial cells.These findings proved with the experimental data reported by Lee et al. (2003) and Yeo et al. (2003).Kim et al. (2007) and Pranee et al. (2008) suggested that the inhibitory activity of silver nanoparticles was influenced by free radical generated on the surface of silver nanoparticles.

Conclusion
Silver nanoparticles have been synthesized from the O. sanctum leaf extract.Structural analysis by XRD together with the chemical composition by EDS, strongly suggests the formation of elemental silver nanoparticles instead of their oxides.From the TEM analysis, the sizes of the nanoparticles are found to be 5 to 60 nm.FTIR measurements provided strong evidence for proteins to form a coat covering the silver nanoparticles to stabilize and prevent the agglomeration of the particles.This simple procedure for the biosynthesis of silver nanoparticles has several advantages such as costeffectiveness, compatibility and eco-friendliness for biomedical and pharmaceutical applications.
The phyto mediated silver nanoparticles and Ag nanoparticles treated fabrics show durable significant antimicrobial activity against two common infectious bacteria, namely K. pneumoniae and E. coli.The synthesis of phyto-mediated silver nanoparticles from O. sanctum is useful for application of dressing materials, delicate fabrics, knitted materials etc.,

Figure 1 .
Figure 1.The leaf extract of O. sanctum on colour changes in silver nitrate at different time interval.

Figure 2 .
Figure 2. UV-Visible spectra of silver nanoparticles synthesized through leaf extract of O. sanctum at different time interval.
O-H stretching of alcohol and phenol compounds and aldehydic -C-H-stretching of alkenes, respectively.The peaks in the region of 1640 to 1550 cm -1

Figure 3 .Figure 4 .
Figure 3. FTIR spectra of silver nanoparticles synthesized from the leaf extract of O. sanctum.

Figure 5 .Figure 6 .
Figure 5. Specrofluorimetric analysis of silver nanoparticles synthesized from the leaf extract of O. sanctum.

Figure 7 .
Figure 7. Zeta potential analysis in the formation of silver nanoparticles by leaf extract of O. sanctum.

Figure 8 .
Figure 8. SEM images of silver nanoparticles by leaf extract of O. sanctum.

Figure 9 .
Figure 9. EDAX spectra of silver nanoparticles synthesized from leaf extract of O. sanctum.

Figure 10 .
Figure 10.TEM image of silver nanoparticles by leaf extract of O.sanctum.

Figure 11 .
Figure 11.The antibacterial effect of silver nanoparticles synthesized from the leaf extract of O. sanctum.

Figure 12 .
Figure 12.SEM image of nano silver coated Cotton fabric.

Figure 13 .
Figure 13.Growth kinetics of Bacterial culture in LB medium in the presence of nanoparticles coated cotton fabrics and control.

Table 1 .
Effect of leaf extract of O. sanctum on colour changes in silver nitrate solution at different time interval.