Characterisations, Antimicrobial activities and Biological synthesis of silver (Ag) nanoparticles using the leaf extract of Urtica dioica

 

Naveen Chandra Joshi*¹, Vivek Kumar², Ajay Singh³, Rajendra Singh⁴

¹Assistant Professor, Department of Chemistry, Uttaranchal University, Dehradun (India)

²Project Fellow, Department of Chemistry, Uttaranchal University, Dehradun (India)

³Professor, Department of Chemistry, Uttaranchal University, Dehradun (India)

Assistant Professor, Department of Chemistry, P. G. College, Gopeshwar (Uttarakhand)

 

ABSTRACT:

Synthesis of metal and metal oxide nanoparticles using the extracts of biomaterials has many advantages over physical and chemical synthetic methods. Such biological or greener methods used to synthesise metal nanoparticles are highly efficient, low cost, environmentally friendly and convenient. In the present study, we have utilized the leaf extract of Urtica dioicaas reducing agent to synthesise silver nanoparticles (AgNPs) and the formation of nanoparticles confirmed by visual detection in which the colourless solution of silver nitrate (AgNO₃) get changed into brownish colored solution. The biologically synthesised silver nanoparticles have been characterised by using different analytical methods such as UV-Visible, FTIR, XRD, FESEM and EDX. Further, the biologically synthesised AgNPs were found potential antibacterial agents against S. aureus, S. mutans, P. vulgaris and E. coli. The maximum zone of inhibitions has been observed for S. aureus and P. vulgaris at 12 mg/ml of AgNPs dosage.

 

 

INTRODUCTION:

Nanotechnogy plays an important role in biologically inspired synthesis of metal and metal oxide nanoparticles in the fields of chemical, biological and physical sciences. Recently, biological or green synthetic approaches of nanomaterials by using phytochemicals, animal derived waste materials and biomolecules of microbial origin have emerged a new field of research in nanotechnology^1-5. The conventional methods are used to synthesise different nanoparticles suffering with low efficiency, high cost and use of harmful chemicals. The main goals of adopting the greener synthetic methods are to minimize hazardous chemicals, prevention of chemical wastes, low cost and high yield of products^6-10.

 

The commonly synthesised metals are gold (Au), silver (Ag), copper (Cu), platinum (Pt), manganese (Mn) and palladium (Pd) in nanostructured form. Among these nano metal particles, silver nanoparticles have gained more attention due to applications in various fields such as pharmaceuticals, air filtrations, water detoxification, catalysts, agriculture and textiles^11-17. The biologically synthesised AgNPs show variable sizes and shapes and have unique electrical, structural and mechanical properties^18-21.

 

The plants derived extracts contain various natural antioxidants and these constituents behave as reducing and capping agents for biogenic synthesis of metal and metal oxide nanomaterial²². After successful synthesis of AgNPs, characterisation methods are compulsory to explore the physical and chemical properties of silver nanoparticles. The commonly used methods have been applied by researchers included FTIR, UV-Visible, powder XRD, FESEM and EDX. It necessary to analyse AgNPs by pharmaceutical and health care industries by using above mentioned methods^23-24. The plant Urtica dioica (Fig. 1A) is also known as scorpion grass, kandali, bichoghas or siyon in Uttarakhand (India). It is herbaceous perennial flowering plant and member of family Urticaceae. Urtica dioica grass is fully comprised with thorns which contain histamine, acetylcholine and formic acid and its touch feels unbearable irritation and itching. Medicinally this plant is very important and locally, peoples used this plant for pain relief^25-27. In mountainous areas, this plant is used for the treatment of gall blotting, arthritis, sprain, stiffness and malaria.The wastes leaves of Urtica dioica were collected from Kumaun hills of Uttarakhand (India).

 

MATERIAL AND METHODS:

The chemicals used in experimental works were of analytical grades and double distilled water used to make all necessary solutions. The glassware used in the process has been washed with 10% nitric acid (HNO₃) solution and double distilled water then dried in tray dryer.

 

Preparation of leaf extract:

The collected leaves of Urtica dioica were washed with double distilled water for removing water soluble impurities and then dried for 3 hours at room temperature. The dried leaves cut into possible small pieces and 5 g of these leaves were added with 100 ml of double distilled water in a 250 ml Erlenmeyer flask. Now, contents were boiled on temperature controlled magnetic stirrer for 30-35 minutes. After boiling, these contents have been filtered and the filtrate i.e. leaf extract (Fig. 1B) preserved at 4°C for further studies.

 

Synthesis of silver nanoparticles:

Take an adequate amount ofsilver nitrate (AgNO₃) in 100 ml of double distilled water for making of 0.01 M silver nitrate solution.Then added 10 ml of leaf extract in this solution and the colour of this solution turns colourless to dark brawn (Fig. 1C and 1D).The formation of AgNPs indicated by this color change and further it was confirmed by UV-Visible spectrophotometer. Now, this final solution was centrifuged at rpm 10,000 for 15-20 minutes and silver nanoparticles settled dawn. The pellets of AgNPs were collected and washed 2-3 times with double distilled water, filtered and again centrifuged. Finally, the synthesised AgNPs were dried at 65⁰C for 1 hour under controlled conditions at hot air oven. After that, the dried nano-powder of silver was preserved for characterisations and antimicrobial activity in air tight sample bottles.

 

Characterisations:

The biologically synthesised silver nanoparticles using the extract of Urtica dioica have been characterised by different analytical methods such as FTIR, powder XRD, FESEM, UV-Visible and EDX.

 

Antimicrobial activity:

The antibacterial activity of AgNPs against Escherichia coli, Streptococcus mutans, Proteus vulgaris and Staphylococcus aureus was tested by using well diffusion method. The petri dishes or plates containing Muller Hinton Agar (MHA) have been prepared by poured liquid media over these plates and then solidified. Now, silver nanoparticles were filled in the wells on plates and incubated for 24 hours at 37⁰C and significant zone of inhibitions observed around the nanoparticles for all bacterial species.

 

RESULTS AND DISCUSSION:

UV-Visible and FTIR:

UV-Visible spectroscopy is a very common analytical method used for qualitative as well as quantitative analysis. It is very simple, economical and rapid technique and associated with the transition of electrons from lower energy levels to higher energy levels in atoms or molecules. The UV-Visible spectra of silver nanoparticles have been recorded in the range of 350 to 800 nm (Fig. 2). The visual peaks obtained at 370nm, 400 nm and 410 nm; the formation of AgNPs takes place at 400 nm.

 

Fig. 1 (A) Urtica dioica (B) Leaf extract of Urtica dioca (C) AgNO₃ solution (D) Color change of AgNO₃ solution after adding the extract

Fourier Transform Infrared spectroscopy (FTIR) is very essential and interference based analytical tool to observe the presence of some important bonds or functional groups present on surface of silver nano-powder.This technique is very common for organic species but can effectively be applied to analyse inorganic as well polymeric species. FTIR is also very low cost, rapid and non-destructive technique with high precisions and high sensitivity²⁸. The broad peaks are obtained at 3282 cm^-1, 2922 cm^-1, 1646 cm^-1, 1386 cm^-1, 1073 cm^-1 and 634 cm^-1(Fig. 3). These peaks indicate the presence of –OH, -NH, -C-H, C=O (Amide), C-O, C-Ag etc on the surface of AgNPs.

 

 

Fig. 2 UV-Visible spectra of AgNPs synthesised by using leaf extract of Urtica dioca

 

 

Fig. 3 FTIR spectra of AgNPs synthesised by using leaf extract of Urtica dioca

 

FESEM, EDX and powder XRD:

Field-emission Scanning Electron Microscope (FESEM) is based on scanning of object by electrons under high vacuum and the electrons released from a field emission source. Under high vacuum, the electrons are focussed and deflected by electronic lenses to produce a smooth and narrow beam. The FESEM is used to observe the morphological features of nanomaterials and also provides the topographical and elemental characteristics. The FESEM images (Fig. 3) of biosynthesised AgNPs are represented in figure 4; these images indicate the smaller oval shaped, agglomerated and uniformly distributed silver nanoparticles. The Energy Dispersive X-ray (EDX) technique with FESEM is used to determine the elemental composition of nanomaterials. This technique is also applicable for quantitative, qualitative and semi-qualitative analysis. The EDX peaks are related to the true compositions of constituents present with a specific nanomaterial and EDX detectors measure the relative abundance of X-rays versus energy. The EDX spectrum for AgNPs and elemental composition are shown in figure 4 and table 1. X-ray diffraction (XRD) technique is very compact, essential and non-destructive analytical tool to characterize powder and crystalline nanomaterials. It is based on the diffraction of X-ray on powder or crystals and a diffractogram is represented by plotting the curve intensity versus diffracted angle (2q) (Fig. 5). The characteristic peaks of AgNPs have been assigned at 23q, 36q, 38q, 44q and 64q and these peaks are corresponding to 012, 110, 200, 220 and 311 planes^5,29,30.

 

 

Fig. 3 FESEM images of AgNPs synthesised by using leaf extract of Urtica dioca

Table. 1 EDX (Composition)

Element	Weight%	Atomic%
C, K	2.34	6.19
O, K	36.81	73.13
Cl, K	3.83	3.43
Ca, K	0.88	0.70
Ag	56.14	16.54
Totals	100.00

 

 

Fig. 4 EDX spectrum of AgNPs synthesised by using leaf extract of Urtica dioca

 

 

Fig. 5 XRDpattern of AgNPs synthesised by using leaf extract of Urtica dioca

 

Antimicrobial activity:

Metal and metal oxide nanoparticles have been found as potential antimicrobial agents. Among other nanoparticles, AgNPs have gained more attention for the treatment of some bacterial diseases. Due to larger surface area, AgNPs provide a better contact with microorganisms. They are highly toxic to microorganisms and antimicrobial activity is greatly enhanced by reducing particle size³¹. The mechanism of bactericidal activity is based on the attachment of AgNPs to the cell wall and generation of free radicals. Finally, AgNPs disturb the permeability of membrane by penetrating to the cell membrane and causing leakage of ATPs and death of bacterial cell⁵. Different amount of AgNPs have been loaded on the agar plate and after incubation period a significant zone of inhibition observed. At initial dosage of AgNPs (3mg/ml), the zone of inhibition achieved 23 mm,18 mm, 15 mm and 22 mm for S. aureus, S. mutans, E. coli and P. vulgaris. The maximum zones of inhibition have been recorded 42 mm and 39 mm for S. aureus and P. vulgarisat 12 mg/l of AgNPs dosage (Fig. 6).

 

Fig. 6 Zone of inhibitions at different amount of AgNPs

 

CONCLUSIONS:

Biologically synthesized materials show various specific properties and comparatively safe than chemically synthesized materials. In the present work, we have synthesized AgNPs very efficiently and environmentally friendly from a localized weed and characterized by different analytical methods such as FTIR, UV-Visible, FESEM, EDX and XRD. All these methods indicate the efficiency of the selected method under laboratory conditions.The biosynthesized AgNPs were very effective for selected bacterial species and a valuable zone of inhibition obtained by using well diffusion method.

 

ACKNOWLEDGEMENT:

We are very thankful to the Department of Chemistry, Uttaranchal University Dehradun (India), for providing all facilities during the experimental works.

 

CONFLICT OF INTEREST:

Authors declare that they have no conflict of interest.

 

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Accepted on 21.05.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(9):4429-4433.