International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

Eco Freinfly Synthesis And Characterization Of Silver Nanoparticles Using Aquous Leaf Extract Of Sclerocarpa And Apply It As Antimicrobial

Hassanain Hataf Jaber Altallal

M.Sc. Nanobiotechnology, Department of Biotechnology

Acharya Nagarjuna University, Guntur

Abstract:

To develop a reliable, eco- Green synthesized silver friendly and easy process for the nanoparticles are confirmed by synthesis of silver nanoparticles color change which was monitored using leave extracts of medicinal quantitatively by UV-Vis “Alphonsea sclerocarpa”. spectroscopy at 440nm (Fig.4). There has been an exponentially Further characterization with SEM, increasing interest in biological TEM and AFM analysis shows the synthesis of AgNPs. In this study, spherical, polydisperse AgNPs of AgNPs were synthesized by an particle size ranging from 5 to eco-friendly and convenient method 50nm with an average size of using Alphonsea sclerocarpa leaf 28.5nm (Fig.7,8,9). FTIR showed extract at ambient temperature. the structure, the respective bands Alphonsea sclerocarpa leaf extract of the synthesized nanoparticles, has been used as a reducing and the stretch of bonds. The agent for the synthesis of silver cytotoxicity analysis of the green nitrate into silver nanoparticles. synthesized silver nanoparticles International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

was observed that showed highest [2-6] changes in primary ways for antimicrobial activity against gram both individual molecules/atoms positive bacteria Staphylococcus and their relating bulk. Novel aureus (MTCC-6908) (Fig.11) purposes of nanoparticles and showed average zone of inhibition nanomaterials are becoming quickly of (26 mm), the Gram negative on different fronts because of their bacteria Pseudomonas citronellolis totally new or improved properties (MTCC-1191) (Fig.12) showed based on scale, their distribution average zone of inhibition of (18.3 and morphology [7,8]. mm) and the Fungi Candida albicans (MTCC-4748) (Fig.13) Nanobiotechnology is a showed average zone of inhibition branch of science that dealing of (22.2 mm) (Fig.10). However, between nanoscience and further investigations were needed biotechnology involving the to identify the scaling-up usage of application of biological systems for this extract on metallic nanoparticle the production, manipulation and synthesis and its applications as design of new functional nano- antimicrobial. scale materials [9]. It combines biological methods with physical and chemical procedures to INTRODUCTION: generate nano-scale particles with Nanotechnology is a vital unique functions. The synthesis of field of cutting edge research nanoparticles using green managing synthesis, procedure and technology is advantageous over manipulation of molecule's, sub chemical agents owing to their molecule's and atoms structure environmental anxieties. There is extending from roughly 1-100 nm significant to produce inorganic in size [1]. Inside this size range nanoparticles as they supply every one of the properties greater material properties with (chemical, physical and biological) effective resourcefulness [10]. International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

Nanobiotechnology is optics, chemical industries, presently one of the most dynamic electronics, space industries, disciplines of research in energy science, catalysis, light contemporary material science emitters, single electron transistors, whereby and different plant nonlinear optical devices, photo- products are finding an imperative electrochemical applications and use in the synthesis of many more to count on [12]. nanoparticles (NPs) [5,6]. MATERIALS AND Nanomedicine is the medical application of nanotechnology [11]. METHODS: Nanomedicine ranges from the medical applications of Alphonsea sclerocarpa plant: nanomaterials and biological devices, to nanoelectronic Scientific classification: biosensors, and even possible Kingdom: Plantae future applications of molecular nanotechnology such as biological (Unranked): Angiosperms machines [11]. (Unranked): Nanotechnology is swiftly Order: gaining renovation in a large number of fields such as health Family: care, cosmetics, biomedical, food Genus: Alphonsea and feed, drug-gene delivery, Species: A. Sclerocarpa [13] environment, health, mechanics,

International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

Fig. 1 . Alphonsea sclerocarpa plant.

5-10 pairs, slender, pinnate, General description: prominent; intercostae reticulate as Alphonsea sclerocarpa is showen in (Fig. 1) [14-18]. small trees in size 10-15 m, braches rugose, glabrous; Culture Media: branchlets drooping. Leaves simple, Growth Media NO. 3 (Nutrient alternate, distichous, estipulate; Agar Medium): petiole 6-8 mm long, slender, To prepare Growth Media glabrous; lamina 3.5-10 x 1.5-4 NO. 3 (Nutrient Agar Medium) by cm, oblong, oblong-lanceolate, dissolving 1.0 g of Beet extract elliptic ovate; base cuneate or and 2.0 g of Yeast extract and 5.0 attenuate; apex obtuse, entire, g of Peptone and 5.0 g sodium coriaceous, glabrous; lateral nerves chloride NaCL powder and 15.0 g International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

of Agar in 1.0 L of distilled water dissolving 10.0g of Beet extract and mix it properly till homogeneity powder and 5.0 g of Yeast extract of the liquid and finally sterilizes it and 10.0g of Peptone and 20.0 g in Autoclave for 15 min in 15 lb of Glucose and 2.0g of Na2HPO4 and 121 Co [19]. and 5.0g of Sodium Acetate and

Growth Media NO. 5 (YRPO 2.0g of Triammonium citrate and

0.2g MgSO4.7H2O and 0.2g of Medium):

To prepare Growth Media MgSO4.4H2O and 15.0 g Agar and

NO. 5 (YRPO Medium) by 1.0 ml of Tween 80 liquid in 1.0 L

of distilled water and Adjust the dissolving 3.0 g of Yeast extract powder and 10.0g of Peptone and PH at 6.2-6.6 and mix it properly

till homogeneity of the liquid and 20.0g Dextrose and 15.0 g of Agar in 1.0 L of distilled water and mix finally sterilizes it in Autoclave for

15 min in 15 lb and 121 Co [21]. it properly till homogeneity of the liquid and finally sterilizes it in Growth Media NO. 65 (Malt

Autoclave for 15 min in 15 lb and Extract Agar Medium):

121 C[20]. To prepare Growth Media NO. 65 (Malt Extract Agar Medium) Growth Media NO. 11 (MRS by dissolving 3.0g of Malt extract Medium): powder and 3.0 g of Yeast extract and 5.0g of Peptone and 10.0 g of To prepare Growth Media Glucose and 20.0 g Agar in 1.0 L NO. 11 (MRS Medium) by of distilled water and Adjust the International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

PH at 6.2 and mix it properly till As mentioned bellow: homogeneity of the liquid and 3.3.1 Bacillus subtilis – (MTCC No. finally sterilizes it in Autoclave for 15 min in 15 lb and 121 Co [22]. 10407)

3.3.2 Staphylococcus aureus – Microorganisms Collection: (MTCC No. 6908)

Provided us with bacterial 3.3.3 Pseudomonas citronellolis – and fungal cultures from Microbial (MTCC No. 1191) Type Culture Collection Institute of Microbial technology Sector 39- A, 3.3.4 Klebsiella pneumoniae – Chandigarh-160036, by Mineral oil (MTCC No. 9024) Preservation method at room temperature. Mineral oil 3.3.5 Lactobacillus acidophilus –

Preservation method by prepare (MTCC No. 10307) tubes of heart infusion agar with a 3.3.6 Enterobacter aerogenes – short slant. For fastidious organisms, add fresh native or (MTCC No. 111) heated blood. Sterilize mineral oil 3.3.7 Candida albicans – (MTCC (liquid petrolatum) in hot air (170 Co for 1 hour). Grow a pure No. 4748) culture on the agar slant. When 3.3.8 Aspergillus niger – (MTCC good growth is seen, add sterile No. 9687) mineral oil to about 1 cm above the tip of the slant. Subculture when needed by scraping growth Preparation of leaf Extracts: from under the oil. Store at room Prepare Aqueous Extracts temperature. Transfer after 6–12 months. (Water): International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

The fresh and tender leaves distilled water in Broun bottle of Alphonsea sclerocarpa were (because it is sensitive to the light) collected from the South India. in room temperature. These were thoroughly washed Silver Nanoparticle (Ag NPs) with tap water and kept under shade at room temperature for Synthesis: about (20) days till they dried The aqueous solution of (1 completely. The dried leaves were mM) silver nitrate (AgNO3) was mechanically grinded and sieved to prepared to synthesize AgNPs. get fine powder. The (5 g) of (190 mL) of aqueous solution of (1 powdered leaves was mixed with mM) AgNO3 was slowly added to (100) ml of distilled water, the (10 mL) of Alphonsea sclerocarpa mixture were boiled in the stirrer aqueous leaf extract while stirring, heat for 10 min in order to for reduction into Ag ions and kept dismantle tearing the wall of plant at room temperature, the emulsion cells, and then nominate the turned to dark brown after 30 min. mixture through suppress and bowl This confirmed the synthesis of funnel using the nomination papers AgNPs in the mixture solution. It is (Whitman No.1). And this is well known that silver nanoparticles prepared crude aqueous extract exhibit dark brown color in and stored in a refrigerator at 4 °C aqueous solution due to excitation [23]. of surface Plasmon vibrations in

Preparation 1/1000 M of Silver silver nanoparticles.

UV-vis spectra analysis Nitrate (NaNO3): Samples (1 mL) of the suspension Prepared (1 Mm) of Silver were collected periodically to Nitrate (NaNO3) by waited monitor the completion of (0.16987 g) of Silver Nitrate bioreduction of Ag+ in aqueous (NaNO3) and dissolved it with 1 L solution, followed by dilution of the International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

samples with 2 ml of deionized spread it on pretty dishes which water and subsequent scan in UV- have sterilized nutrient media agar visible (vis) spectra, between wave type (3). lengths of 200 to 700 nm in a spectrophotometer. UV-vis spectra Sterilization of Aqueous were recorded at intervals of 1 hr, Extract: 24 hr and 72 hr.

It was prepared water Cultivation of extract, Hexane Extracts, Microorganisms: Chloroform Extracts and Methanol Extracts and Sterilized the mixture Cultivation of Bacteria: by pasteurization method (temperature (62 oC) for a period Preparation of subculture by of (10 minutes) and it was getting broke the head of the tube that on the benchmark center for provided us and remove the cotton alcoholic extracts. and mix the contains with I ml distilled water and taken amount of Prepare Silver Nanoparticles the sample by sterilized loop and spread it on pretty dishes which Disks: have sterilized nutrient media agar types (3,5,11 and 65). By using filter paper (Whitman No. 1) and make disks Cultivation of Fungus: from it by using piercer papers and Preparation of subculture by concentrations from the liquid of broke the head of the tube that silver nanoparticles and submersing provided us and remove the cotton the disks inside each concentration and mix the contains with I ml for 1 hr and then the disks taken distilled water and taken amount of out to dry and to using it as the sample by sterilized loop and International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

antimicrobial against the bacteria the diameter circle inhibition and fungi. resulting measurement unit.

Determination of antimicrobial Characterization of Silver activity: Nanoparticles:

Has been relying on the way UV-Vis Analysis: researcher Bauer et al (1966) to The optical property of test the sensitivity of the silver AgNPs was determined by UV-Vis nanoparticles as antimicrobial spectrophotometer. After the against both of the bacteria and addition of AgNO3 to the plant fungi, and then inoculating the extract, the spectras were taken in surface of nutrient broth (4-5) pure different time intervals up to 24 hr colonies of bacteria under study, between 350 nm to 500 nm. Then and incubated germ in degree (37 the spectra was taken after 24 hr oC) for (16-14) hr, then dilute the of AgNO3 addition. broth bacteria by using serial dilution, and then from the nine FTIR analysis: tube taken 1 ml of broth bacteria The chemical composition and brush on the surface using a of the synthesized silver cotton swab, the dishes are lefted nanoparticles was studied by at room temperature for 30 using FTIR spectrometer. The minutes, then fix disks with silver solutions were dried at 75 oC nanoparticles (which prepared) by and the dried powders were sterile forceps, the dishes were characterized in the range incubated at a temperature 37 ° C 4000–400 cm-1 using KBr pellet for 24 hours for bacterial and 7 method. days for fungal, then measuring XRD Analysis: International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

The phase variety and grain the SEM at an accelerating size of synthesized Silver voltage of 20 KV. nanoparticles was determined by TEM Analysis: X-ray diffraction spectroscopy (Philips PAN analytical). The The transmission electron synthesized silver nanoparticles microscope (TEM) forms an image were studies with CUK radiation by accelerating a beam of at voltage of 30 kV and current of electrons that pass through the 20 MA with scan rate of 0.030/s. specimen. In TEM, electrons are Different phases present in the accelerated to 100 KeV or higher synthesized samples were (up to 1 MeV), projected onto a determined by X pert high thin specimen (less than 200 nm) score software with search and by means of the condenser lens match facility. system, and penetrate the sample thickness either undeflected or SEM Analysis: deflected. The greatest advantages The morphological features that TEM offers are the high of synthesized silver magnification ranging from 50 to nanoparticles from neem plant 106 and its ability to provide both extract were studied by image and diffraction information Scanning Electron Microscope from a single sample. (JSM-6480 LV). After 24 hr of AFM Analysis: the addition of AgNO3 the SEM slides were prepared by making a The atomic force microscope smear of the solutions on (AFM) is one kind of scanning slides. A thin layer of platinum probe microscopes (SPM). SPMs was coated to make the are designed to measure local samples conductive. Then the properties, such as height, friction, samples were characterized in magnetism, with a probe. To acquire an image, the SPM raster- International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

scans the probe over a small area living organisms and medicine. of the sample, measuring the local Synthesis and characterization of property simultaneously. AgNPs from plant extracts may act as reducing and capping agents in AFMs operate by measuring silver nanoparticles synthesis. force between a probe and the sample. Normally, the probe is a Alphonsea sclerocarpa is a sharp tip, which is a 3-6 um tall medicinal plant. People are using pyramid with 15-40nm end radius. this plant in their day to day life all Though the lateral resolution of around the world. Green synthesis AFM is low (~30nm) due to the of silver nanoparticles using convolution, the vertical resolution aqueous leaf extract of Alphonsea can be up to 0.1nm. sclerocarpa with help of 1mM AgNO3 was proved significant in RESULTS AND this study. The fresh suspension of DISCUSSION: Alphonsea sclerocarpa was pale yellow color. However, after Nanoparticles are often addition of AgNO3 and stirring, for referred to as particles with a reduction into Ag ions and kept at maximum size of 100 nm, and room temperature, the emulsion they exhibit unique properties, turned to dark brown after 30 min which are quite different than those (Fig. 2, A). This confirmed the of larger particles. New properties synthesis of AgNPs in the mixture of nanoparticles related to variation solution. It is well known that silver in specific characteristics like size, nanoparticles exhibit dark brown shape and distribution have been color in aqueous solution due to demonstrated. Among the noble excitation of surface Plasmon metals, silver (Ag) is the metal of vibrations in silver nanoparticles. choice for potential applications in the field of biological systems, International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

The color was changed in silver ions) showed no change in the cell free extract when color of the cell filtrates when challenged with 1mM AgNO3 from incubated under same conditions. pale yellow to dark brown, attained Shoreatum buggaia and Svensonia maximum intensity after 30 min hyderobadensis aqueous extract with intensity increasing during the could synthesize silver nanopartcles period of incubation indicative of within 15 min where as Boswellia the formation of silver ovalifoliolata took 10 min to nanoparticles. Control (without synthesize nanoparticles.

A1 B1

A2 A3 B2 B3

Fig. 2: (A1). Silver Nitrate solution, (A2) Alphonsea sclerocarpaleaf aqueous extract, (A3) Silver Nitrate + Alphonseas clerocarpa emulsion after 30 min. (B1, B2 and B3) Experimental solutions after 12 hrs.

The important aspect of collective oscillation of conduction AgNPs is that their optical electrons resulting from the properties depend upon the particle interaction with electro-magnetic size and shape. These optical radiation [293]. properties are dominated by the International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

The formation of silver environmentally benign, yet nanoparticles was followed by chemically complex. Due to their measuring the surface plasmon exclusive properties, silver resonance (SPR) of the Alphonsea nanoparticles (AgNPs) may have sclerocarpa and Silver Nitrate + several applications, such as Alphonsea sclerocarpa emulsion at catalysts in chemical reactions, the wavelength range from 300–700 electrical batteries and in spectrally nm. The characteristic silver SPR selective coatings for absorption of bands were detected around 400- solar energy pharmaceutical 450 nm (Fig. 4). These absorption components and in chemical bands were assumed to sensing and biosensing. correspond to the silver nanoparticles extra fine and smaller Characterization of silver than 25 nm. UV–Vis absorption nanoparticles by UV-Visible spectra (Fig. 4) showed that the spectroscopy: broad SPR band contained two peaks. These two peaks illustrate The reduction of pure silver the presence of two broad ions was monitored by measuring distributions of hydrosol silver the UV–Vis spectrum. The change nanoparticles. in colour may be due to excitation of surface Plasmon vibrations of The reduction of Ag+ ions by silver nanoparticles (Fig.2,3). The combinations of bio molecules strong absorption observed at (440 found in these extracts (e.g. nm). enzymes/proteins, amino acids, polysaccharides, vitamins etc.) is International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

Fig. 3: Picture of flasks containing the Alphonsea sclerocarpa leaf extract after incubation in an aqueous solution of AgNO3 solution

Figure 4: UV-Vis spectrum of silver nanoparticles synthesized using Alphonsea sclerocarpa aqueous leaf extract (1,24,72) hr. time interval.

Characterization of silver nanoparticles by FTIR:

The FTIR measurements of the aqueous samples were carried out to identify the possible interactions between silver and bioactive molecules, which may be responsible for the synthesis and stabilization of silver International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

nanoparticles with the capping agent available in the leaf extract filtrate. The silver nanoparticles are stable and well dispersed. The FTIR spectrum of silver nanoparticles showed three distinct peaks, 462.86, 1643.82 and 3360.72cm−1 (Fig. 5).

Fig. 5 . FTIR spectrum of silver nanoparticles.

Characterization of silver Nanoparticles by XRD:

The XRD pattern shows four peaks in the whole spectrum of 2 values, ranging from 20 to 80. XRD spectra of pure crystalline silver structures was matched with by the Joint Committee on Powder Diffraction Standards (file nos. 04-0783 and 84-0713). A comparison of our XRD spectrum with standard confirmed that the silver nanoparticles had been formed in the form of nanocrystals. It was evidenced by the peaks at 2 values of 36.25°, 59.37°, 62.60° and 67.62° corresponding to 111, 200, 220 and 311 planes for silver, respectively (Fig.6). International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

Fig.6. X-ray diffraction pattern of silver nanoparticles

Characterization of silver nanoparticles by SEM:

SEM analysis shows high-density AgNPs synthesized by Alphonsea sclerocarpa leaf extract. It was shown that relatively spherical and uniform AgNPs were formed with diameter of (5- 50)nm. The SEM image of silver nanoparticles was due to interactions of hydrogen bond and electrostatic interactions between the bioorganic capping molecules bound to the AgNPs. The nanoparticles were not in direct contact even within the aggregates, indicating stabilization of the nanoparticles by a capping agent. The larger silver particles may be due to the aggregation of the smaller ones, due to the SEM measurements (Fig. 7). International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

Fig.7 .SEM micrograph showing silver nanoparticles of various size ranges 5–50 nm.

Characterization of silver nanoparticles by TEM:

TEM are most powerful method to determine the morphology and size of nanostructures. TEM micrographs of the synthesized silver nanoparticles confirmed the formation of spherical nanoparticles with a size range of 5–50 nm (Figure 8).

International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

Fig.8.TEM micrograph showing silver nanoparticles of various size ranges 5–50 nm

AFM images of synthesized silver nanoparticles:

The biosynthesized silver nanoparticles were further confirmed by AFM micrographic images (Fig.9 a). It has been reported that the silver nanoparticles size was measured using line profile determination of individual particles in the range of 24.631 nm (Fig.9 b). The fabricated silver nanoparticles were imaged by AFM to understand the exact configuration of the fabricated silver nanoparticles and also used to verify that the silver nanoparticles were more or less homogenous in size and were spherical in shape. The particle size was measured using line profile determination of individual spherical shaped particles in the range of 18-48 nm which was previously confirmed by FE SEM micrographic images. The 3-dimensional structure image of the synthesized AgNPs was shown in (Fig. 9 c). International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

Fig.9 AFM images of synthesized AgNPs showed the spherical shaped particle size of 5-50 nm (a), particle size determination profile of the AFM image (b), the 3- dimensional structure of AgNPs (c).

Determination of antimicrobial activity:

The synthesized silver nanoparticles shows significant antimicrobial activity against bacterial pathogens Bacillus subtilis, Staphylococcus aureus, Pseudomonas citronellolis, Klebsiella pneumonia, Lactobacillus acidophilus, Enterobacter aerogenes, Candida albicans and Aspergillus niger ( Fig. 10) using agar well diffusion methods. The synthesized silver nanoparticles showed highest antimicrobial activity against gram positive bacteria Staphylococcus aureus (MTCC-6908) (Fig.11) showed average zone of inhibition of (26 mm). The Gram negative bacteria Pseudomonas citronellolis (MTCC-1191) (Fig.12) showed average zone of inhibition of (18.3 mm). The Fungi Candida albicans (MTCC-4748) (Fig.13) showed average zone of inhibition of (22.2 mm). The silver nanoparticles causes an increase in cell International Journal of Research p-ISSN: 2348-6848 e-ISSN: 2348-795X Available at https://edupediapublications.org/journals Volume 03 Issue 12 August 2016

membrane permeability and results finally in cell death [298]. The present study showed a simple, rapid and economical route to synthesized Silver nanoparticles.

Zone of Inhibition in mm

30

25

20

15

10

5 Zone of Inhibition in mm Inhibitionof Zone 0

Microorganisms

Fig.10. Antimicrobial activities of silver nanoparticles against bacteria and fungi.

All values represented are average of results of three separately conducted experiments.