A STUDY ON GREEN SYNTHESIS AND SPECTRAL CHARACTERISATION OF SILVER AND IRON NANOPARTICLES FROM AMARANTHUS VIRIDS AND THEIR ANTIMIDROBIAL ACTIVITY

Biotechnology 0 Comment 32

                                                 

Abstract

There is an increasing commercial demand for nanoparticles due to their wide applicability in various areas such as electronics chemistry, catalysis, energy and medicine metallic nanoparticles are traditionally synthesized by wet chemical techniques where the chemicals used are quite often toxic and flammable. in this paper we described a cost effective and environment friendly technique for green synthesis of silver and iron nanoparticles from 1mM silver nitrate,1mM ferric chloride solutions through the extract of leafy vegetable amaranths viridis as a reducing agent. Nanoparticles were characterized by UV-VIS absorption spectroscopy, the surface plasmoressonance spectrum of silver and iron nanoparticles was obtained at 422nm and 261nm.SEM&EDAX data reveals silver and iron nanoparticles are spherical in shape. EDAX indicates the reduction of silver ions to elemental silver and iron ions to elemental iron.SEM determine the morphology and size of nanoparticles. zetapotencial of silver and iron nanoparticles are determined in water as a dispercant. Zetapotencial of silver and iron nanoparticles is found to -24.6mV and 28,8mV.the synthesized silver and iron nanoparticles have very good antimicrobial activity. This is the for the first time that any leafy vegetable was used for synthesis of nanoparticles.

Keywords: Green synthesis, SEM&EDAX , FTIR ,AFM, Zetapotencial, Antimicrobial activity

 

Introduction

The field of nanotechnology is one of the active areas of research in modern material sciences. Nanoparticles exhibit completely improved properties based on specific characteristics such as morphology ,size, and distribution, new applications of nanoparticles and nanomaterials are emerging rapidly[1,2,3].silver nanoparticles are wide used in industry and have  an inhibitory effect on no. of microorganisms .they have also been used in manufacture of ointment and creams to prevent infection of bones and wounds. synthesized of nanoparticles can take place by using various methods especially silver by chemical, electrochemical[4],gamma radiation[5],photochemical[6],laser ablations[7]and het vaporization[8.9] etc…However other metals and metal oxide nanoparticles can also prepared synthetically. Synthesis of different nanoparticles by microorganisms such as fungus, yeast, bacteria, algae,etc. Ha been reported. Iron nanoparticles are the most ubiquitous of the transition metals used for treating industrial sites contaminated with chlorinated organic compounds and to treat many types polychlorinated bi phenyls (pcBs) organ chlorine pesticides and chlorinated organic solvents .iron oxides are used extensively due to the development of preparation technology of nanometer powders. synthesis of iron nanoparticles by micro emulsion technique[10],hydro thermal synthesis[11],sonochemical approach[12],non aqueous route[13],and and thermal composition of organic iron precursor [14],sol-gel technique[15],thermal decomposition of organic metal[16],microbial plasma synthesis[17],etc. and recently  via green chemistry route[18][19][20].

The use of  environmental benign materials like plant leaf extracts[53-55] , bacteria, fungi, and enzymes [21][22][23][24].for the synthesis of silver and iron nanoparticles officers numerous benefits of eco-friendliness and compatibility for pharmaceutical and other bio medical applications as they do not use toxic chemicals for the synthesis of protocol.

Chemical synthesis methods lead to presence of some toxic chemical absorbed on the surface that may have adverse affect in the medical applications. Green synthesis provides advancement over chemical and physical method as it is cost effective environment friendly, easily scaled up for large scale synthesis and in this method there is no need to use high pressure energy, temperature , and toxic chemicals. The most important applications of silver nanoparticles is in medical industry such as tropical ointments to prevent infection against burn and open wounds and iron nanoparticles is in medical and laboratory applications. It has applications in plastic, nanowires, coatings, nanofibers and textiles.

Here in, we report for the first time of silver and iron nanoparticles, reducing the silver and iron ions present in the solution of silver nitrate and ferric chloride by the leafy vegetable extract of Amaranthus viridis these biologically synthesized nanoparticles were found to be extremely  effective against different bacterial pathogens. Common name of Amantharus viridis is totakura, Chinese spinach etc… it belongs to family amaranthaceous members of this family has simple leaves that are opposite or alternate it is cosmopolitan taxon or herbs. Amaranth levees are very good source of vitamins like vitamin A, B6, C and K. Riboflavin etc. And minerals including calcium, iron.mg, potassium, zinc, manganese  ,leaves are high in proteins including amino acid lysine. It highly nutritious food. Seeds and leaves of amaranth are used as herbal remedies, leaves have been to be very effective in stopping diarrhea and hemorrhagic problems like excessive menstruation and also a wonderful astringent and make a great wash for skin problems like eczema and wonderful acne remedy, a good remedy for hair loss and premature graying, Amaranth also makes effective mouth wash for treating mouth sores, swollen gums. In this present study, Amaranthus viridis has been used for the synthesis of silver and iron nanoparticles by green route and characterized by spectral analysis.

Fig.1:A photo graph of  Amaranthus viridis (totakura) leafy vegetable used in the silver and iron nanoparticles synthesis.

Materials and Methods

Synthesis of nanoparticles from Amaranthus viridis extracts

Preparation of the Extract

Fresh leafy vegetable are collected, cut into fine pieces and dried at room temperature. Dried leaves are powdered and 3g of powder was weighed into 60 ml of Ro water and boiled for 10 min at 100oC.the extract was filtered through WhatmanNo.1 filter paper. The extract was stored at 40 C for further experiments.

 Synthesis of Silver nanoparticles from Amaranthus viridis extract

The aqueous solution of 1mM silver nitrate (AgNO3) was prepared and used for the synthesis of silver nanoparticles. 5 ml of Amaranthus viridis extract was added into 5ml of aqueous solution of 1 mM silver nitrate for reduction into Ag+ ions. Here the filtrate acts as reducing and stabilizing agent for 1mM of AgNO3.

 

 Synthesis of iron nanoparticles from Amaranthus viridis extract

The aqueous solution of 1mM ferric chloride (FeCl3) was prepared and used for the synthesis of iron nanoparticles. 5 ml of leaf extract was added into 5ml of aqueous solution of 1 mM ferric chloride for reduction into iron ions. Here the filtrate act as reducing and stabilizing agent for 1mM of FeCl3.

 

Characterization techniques:

 

 UV-Vis Spectroscopy

The Iron and Ag nanoparticles were characterized in a Nanodrop 8000 UV-VIS spectrophotometer, to know the kinetic behavior of Iron and Ag nanoparticles. The scanning range for the samples was 200-800 nm at a scan speed of 480nm/min. The spectrophotometer was equipped with “UVWinlab” software to record and analyze data. Baseline correction of the spectrophotometer was carried out by using a blank reference. The UV-Vis absorption spectra of all the samples were recorded and numerical data were plotted in the fig-3.

 Scanning electron microscope (SEM)

In this present work Scanning Electron Microscopy (SEM)and EDX was performed by oxford Inca penta Fetx3 EDS instrument attached to Carl Zeiss EVO MA 15 Scanning electron Microscope (200kV) machine with a line resolution 2.32(in A0 ). These images were taken by drop coating AgNPs and iron nanoparticles on an aluminum foil. Energy dispersive Absorption Spectroscopy photograph of AgNPs were carried out by the SEM equipment, as mentioned above.

   Zeta potential measurement.

 

Particle size and Zeta potential measurement experiments were carried out by using a Nanopartica (HORIBA).

 Antimicrobial activity

The antimicrobial activity of silver and iron nanoparticles was evaluated against Gram positive: Staphylococcus aureus, Bacillus megaterium, Gram negative Escherichia coli (M&D), Pseudomonas aeruginosa, Klebsilla pneumonia by disc method. The 24h old cultures were prepared in nutrient broth (composition (g/l) peptone, yeast extract, sodium chloride, and D(+)-glucose).two replicas of respective microorganisms were prepared by spreading 100ul of revived culture on the nutrient agar plate (composition(g/l) peptone, yeast extract .sodium chloride(+)-glucose and agar-agar),with the help of spreader. Discs were prepared by using what man No.1 filter paper. The discs were placed on agar plates and sample of synthesized silver and iron nanoparticles were added on the disc with the help of micropipette. The plates were incubated at 370c overnight. Amoxyclav (Himedia SD063) disc was used as reference drug. The Bacterial strains of Microorganisms used for the determination of antibacterial activities of silver and iron nanoparticles synthesized were obtained from Department of Microbiology, S.V.University, Tirupathi .Different bacterial strains maintained on nutrient agar and subcultures were freshly prepared before use. Bacterial cultures were prepared by transferring two to three colonies into a tube containing 20 ml nutrient broth and grown overnight at 370c.

Results and Discussion

Amaranthus viridis Linn. (Amaranthaceae) is an annual herb, erect, 10 to 75 (-100) cm stem; slender, branched, angular, glabrous leaves. Commonly called as ‘Cholai’ in Hindi, which is grown in all regions of India, has been used in Indian and Nepalese traditional system to reduce labor pain and act an antipyretic [25]. Other traditional uses range from an anti-inflammatory agent of the urinary tract, in venereal diseases, vermifuge, diuretic, antirheumatic, antidiabetic, antiulcer, analgesic, antiemetic, laxative, improvement of appetite, antileprotic, treatment of respiratory and eye problems and treatment of asthma [26-28] .

Furthermore, Amaranthus viridis contains antiproliferative and antifungal lactin properties as well as ribosome inactivating protein, β-carotene [29-30] and antiviral potential [31]. Experimentally the plant evaluated for analgesic and antipyretic activities [32], in vitro anthelmintic [33] anti-inflammatory[34],antidiabetic, antihyperlipidaemic and antioxidant properties[35], Pharmacognostic study [36],antinociceptive [37], antioxident & nutrient[38], heptaprotective activity[39].

 

 Synthesis and characterization of nanoparticles

 Synthesis of Ag and iron nanoparticles from plant extract.

            Nanoparticles are synthesized according to the protocol discussed in “methods and materials” (section3). On mixing the extract with aqueous solution of the Ag ion complex for silver synthesis and FeCl3 for iron, a change in the color within 2min was observed for both the Nanoparticles. for silver nanoparticles color changes from colourless to yellowish brown colour where as for iron color changes from colorless to yellowish . It was due to the reduction of Ag+ and iron ions which indicates the formation of Ag and iron nanoparticles.

 Characterization of nanoparticles                                                   

 UV–Vis spectral analysis

UV-visible spectroscopy is important technique for analyzing the formation of silver and iron nanoparticles in aqueous solution AgNPs and iron has free electron, which gives rise to plasma resonance absorption band, due combined vibration of metal nanoparticles in resonance with the light wave. A surface plasma resonance spectrum of AgNPs and iron nanoparticles was obtained at 426nm and 259 nm after 2-3min color changing to light yellowish color in figure 2 the surface plasma AgNPs and iron nanoparticles at increasing concentration was taken and the color changes were observed for both nanoparticles .For silver color changes from colorless to yellowish brown color and for iron colorless to  light yellowish brown color respectively. Metal nanoparticles can be synthesized by reducing metal ions using some chemical molecules .in green synthesis ,it is observed that natural material extract act as reducing agent for generation of metal nanoparticles. The uv visible spectrum of silver and iron nanoparticles its excitation also depending upon particle size [40], the earlier reports showed that for silver and iron nanoparticles the absorption peaks around 410-450nm and for iron 216-276 and above can be attributed to in size range 25-50nm [41].previous reports also reveals that particles in SPR region of around 410-450 nm and for iron 216-268nm cam attributed to spherical nanoparticles[42,43]. In present study SPR of silver and iron nanoparticles are 426 and 259nm, which are spherical in shape further confirmed by SEM ,particle size analysis and AFM studies.

 SEM and EDX analysis of Ag and iron nanoparticles

The Morphology and size of nanoparticles in solution is determined by SEM images . It shows both nanoparticles are spherical in shape spectrum around .the EDX data shows very strong signal which indicate the reduction of silver ions into elemental silver and iron ions to elemental iron ions and are possibly originated from the molecules attached to the surface of iron and silver nanoparticles (figure 5). Similarly earlier report showed silver and nanoparticles showed an EDX spectrum, emission energy at 3 KeV and some of the weak signals from Cl, K ,O ,Ca ,Mg were observed[44]

 Zeta potential

The electrostatic repulsive forces between the nanoparticles depends on the charge, when they are negatively charged prevents the nanoparticles from agglomeration in the medium leading to the long term stability [45].in the present study the higher negative value of  zeta potentials confirms the repulsion among the particles and their by increases in stability of formulations .The zeta potential of the synthesized silver and iron nanoparticles is determined in water as a dispercant .The zeta potential is found to be -24.6 mV and 28.8mV.

 Antimicrobial activity

The nanoparticles synthesis by green route was found extremely against 6 bacterial species at a concentration of 20µl Ag and iron nanoparticles, Gram positive. Bacillus megaterium, Staphylococcus aures, Gram negative..Escherichia coli (M), Escherichia coli (D), Pseudomonas aeruginosa, Klebsiella pneumonia. The results are shown in table 1 were comparable with reference drug viz. Amoxyclav (himedia SD063).

The antimicrobial activity of silver was recognized by clinicians for over 100 years[46] . It is only in large few decades the action of silver and iron nanoparticles as an antimicrobial agent has been studied. [47]. Aparna et al[56] reported the antimicrobial activity against pseudomonas florescens ,E.coli, lactobacillus ,s.aureus.Sundarm Moorthi.C et al[48] investigated the antimicrobial activity of silver  nanoparticles against staphylococcus aures, bacillus subtilis, E.coli, psuedomonus aeuroginosa. Rout Rajesha et al.[49] investigated the antibacterial activity of phytosynthesised silver nanoparticles against staphylococcus aures, E.coli ,p.aeuroginosa, and k.pneumonia. Similarly, Kim et al. [50] Reported antimicrobial activity of silver nanoparticles against E.coli and s. aures. The effect was dose dependent.

The antimicrobial activity of FeO nanoparticles on .E.coli and staphylococcus aureus  was determined  by Saba A.Mahdy[51] by MTT assay . M.Senthil et al. [52] reported the antimicrobial activity of iron nanoparticles was performed against pseudomonas aeruginosa using agar well diffusion method.  The  cultures shown zone of inhibition which was about 1.6,2.2,2.8,3.2,2.6,2.7cm for Ag nanoparticles and 1.7,1.7,1,2,2,1.4 for iron nanoparticles in diameter respectively(table 1). The culture of Escherichia (D) shows maximum zone of inhibition for both nanoparticles.

The leaf extract Amaranthus viridis is found suitable for simple and rapid extraction of Ag and iron nanoparticles by green synthesis within 5- 10min.The spectroscopy characterization from UV-Vis, SEM, and EDX support the formation and stability of the biosynthesized AG and iron nanoparticles. This is a very simple and rapid method of green synthesis of Ag and iron nanoparticles which can be useful in various biomedical and biotechnological applications.

 

Conclusion:

The iron and silver nanoparticles of average sizes have been synthesized using dried leaves of plant Amaranthus viridis. Characterizations from UV-Vis, SEM, EDX, Support the stability of biosynthesized nanoparticles. The silver and iron nanoparticles using Amaranthus proved excellent antimicrobial activity. These silver and iron nanoparticles may used in food and pharmaceutical industries. Why we are prefer this green synthesis means it provides advancement over chemical and physical method as it is cost effective, environment friendly, easily scaled up for large scale synthesis and in this method there is no need to use high pressure, energy temperature and toxic chemicals. so our ancients advice the people mainly prefer leafy vegetables for their daily food items because of these medicinal values.

 

 

 

 

 

 

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