RESEARCH ARTICLE

Synthesized Iron Nanoparticle via Green Approach and Evaluating its Antibacterial Potential

Papiya Chatterjee¹, Nisha Gupta², Jai Shankar Paul²*

¹Amity Institute of Biotechnology, Amity University Raipur, Chhattisgarh, India

²School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India

*Corresponding Author Email- jaishankar_paul@yahoo.com

Introduction

There is increased prevalence of antibiotic resistant bacteria emerging from the extensive use of antibiotics. This renders current antimicrobial agents insufficient to control at least some bacterial infections (Riffel et al. 2002). Due to such concerns, efforts have been focused on developing a potentially effective, safer approach through antimicrobial activity by the help of plant extracts which is safe and easily degradable. Medicinal plants are a precious natural resource, as they supply staple for pharmaceutical industry, modern and traditional sorts of medicine and generate employment and income additionally to conservation of bio-diversity and traditional knowledge. Screening of various plants has disclosed various bioactive compounds as well as alkaloids, tannins, flavonoids, glycosides and saponins. These plant secondary metabolite’s function defence mechanisms against several microorganisms, insects and herbivores (Amor et al. 2009).  

Aegle marmelos (Bael) has been proverbial to be one amongst the foremost vital healthful plants of Asian nation. Over one hundred phytochemical compounds are isolated from numerous components of the plant, specifically phenols, flavonoids, alkaloids, internal organ glycosides, saponins, terpenoids, steroids and tannins. These compounds possess antimicrobial activity against numerous gram positive and gram negative bacteria. Other therapeutic effects of this plant are anti-cancerous, vas and epithelial duct disorders, antiulcer, anti-hyperlipidemic, and anti-spermatogenic effects which is proved by animal models by the crude and pure extracts of this plant. Each part of A. marmelos plant like its fruits, stem, bark, and leaves possess healthful property and is employed for treating numerous eye and skin infections. Leaf is taken into account to be one amongst the best accumulatory components of potent bioactive compounds (Jassal et al. 2016; Karumaran et al. 2016). A. marmelos tree grows up to approximately 13-15m tall with slender drooping branches. The bark is pale brown or grey in colour, swish or finely fissured, and flaking armed with long straight spines. The stem components oozing out is slimed sap or gum from the cut components. The leaves are pale green, or yellow. The flowers are little pale inexperienced or yellow, sweet scented, bisexual develop in clusters at the tip of twigs or leaf axils. The fruit is spherical or oval formed and is 5–20 cm in diameter could have a skinny, exhausting woody shell, with soft rind, and is grey-green till the fruit is totally ripe. Fruit has little minute glands with aroma. The pulp is pale orange in colour, pasty, sweet, and pitchy (Orwa et al. 2009). The stem barks, leaves, pulp, and seeds of A. marmelos fruits is widely employed in preparations of ancient medication (Mujeeb et al. 2014; Murthy et al. 2020; Rahman and Parvin 2014).

Nanotechnology is among the foremost widely used technologies in translational research. The development of metal nanoparticles through biological materials by an eco-friendly approach has developed a significant interest in the research field. Nanotechnology deals with particles of a size starting from 1-100 nm (Ali et al. 2016). This field of research naturally commingles all the fields of natural sciences alongside chemistry, physics, biological sciences, engineering, materials science, and computational sciences for the formulation of nanostructures and nanomaterial. Nanotechnology might be a strong tool in handling pollution remediation and is also eco-friendly. Several studies indicate that combining nanoparticles with conventional treatment could increase the efficiency of contaminants removal, like organic materials (El-Refai et al. 2018; Rickerby and Morrison, 2007; Herlekar et al. 2014; Ijaz et al. 2020). FeNPs are very effective for the transformation and detoxification of common environmental contaminants, such as chlorinated organic solvents, organochlorine pesticides, and PCBs (Kuppusamy et al. 2016; Makarov et al. 2014; Mittal et al. 2013; Singh et al. 2018). Focused on the above benefits and importance, current research based on synthesize metallic nanoparticle (FeNPs) via green synthesis and evaluation of the antibacterial potential of the synthesized metallic nanoparticles against gram positive (Bacillus subtilis, Staphylococcus aureus) and gram negative bacteria (Escherichia coli, Klebsiella oxytoca) via agar well diffusion method.

Materials and Methods

Plants selection and collection

In the current study, leaves of two plants viz. Aegle marmelos (AM) (Bael) and Vitex negundo (VN) (Nirgundi) (Fig. 1) was used for the green synthesis of iron nanoparticle (FeNPs). The leaves were procured from the campus of School of Studies in Biotechnology, Pt. Ravishankar Shukla University Raipur CG. The collected leaves were washed thoroughly with tap water to remove dirt and dust. Further washing was done via distilled water and then subjected to sun-drying for few hours followed by drying in oven at 40°C for 48-72 hours.

Fig. 1: Leaf of (a) Vitex negundo (Nirgundi) (VN) (b) Aegle marmelos (Bael) (AM)

Pulverization of plant leaves

The dried leaves were then grounded to make fine powder using mortar and pestle and stored in sterile packets for further use (Fig. 2b).

Fig. 2: The schematic representation of plant extract and NP preparation (a) leaves of V. negundo (Nirgundi) and A. marmelos (Bael) (b) leaf powder (c) plant extract preparation via decoction (d) filtration of extracts (e) filtered extracts (f) FeNPs synthesis

Preparation of leaf extract

The leaves extract of the selected plants were prepared via decoction method in the ratio 1:20 (plant leaf powder: distilled water) (Fig. 2a-e). The decoction was performed for about 15-20 minutes followed by filtration via Whatmann filter paper. The prepared extracts were stored at 4°C for further use in NP synthesis (Fig. 2f).

Chemicals

For synthesis of Iron (Fe) nanoparticles, ferric chloride heptahydrate (FeCl₃.7H₂O) was used as a precursor solution. All other chemicals used in the present study were of the analytical grade and were procured either from HiMedia Laboratories Pvt. Ltd. or Sigma-Aldrich.

Preparation of stock solution of FeCl₃.7H₂O

Stock solutions was prepared in two concentrations (0.1mM and 1mM) in autoclaved distilled water to avoid any kind of cross-reactions.

Nanoparticle synthesis (FeNPs)

For the synthesis of FeNPs, ferric chloride heptahydrate (FeCl₃.7H2O) was used as precursor using leaf extract of Nirgundi (Vitex negundo) and Bael (Aegle marmelos) as reducing/capping agent for the biological synthesis (green synthesis) of FeNPs (Fig. 3). The precursor solution of Fe and plant extract was mixed in 1:1 ratio and kept in an incubator at 30°C for 24 hours.

0.1mM FeCl₃ (precursor) + plant extract (AM/VN) = incubation at 30°C for 24 hours

1mM FeCl₃ (precursor) + plant extract (AM/VN) = incubation at 30°C for 24 hours

Characterization of the synthesized nanoparticles

After observing the physical changes in the synthesized nanoparticle solution it was further subjected to UV–Vis absorbance spectroscopy scan (200–800 nm) for further confirmation.

Bacteria selection for antibacterial activity

For assessing the antibacterial activity of the synthesized nanoparticles four bacteria were selected and procured from School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur (CG). Out of the four, two were gram positive (Bacillus subtilis, Staphylococcus aureus) and two were gram negative (Escherichia coli, Klebsiella oxytoca). All the selected bacteria were sub-cultured and preserved in NAM slants at 4°C for the study.

Assessing the antibacterial activity of the synthesized metallic nanoparticles

Antibacterial potential of the synthesized nanoparticles were analysed in different concentrations (150, 100, 50, 10mg/ml) via agar well diffusion method against two gram positive and two gram negative bacteria. The nutrient agar media plates that were 24 h old have been used. Wells were made using sterile well puncher/borer into which the nanoparticle solution prepared in DW at various concentration (150, 100, 50, 10mg/ml) was loaded and kept for incubation at 37°C for 24 h. The zone of inhibition was measured in mm.

Results and Discussion

Nanoparticle synthesis (FeNP synthesis)

The colour changes from pale yellow to black/dark brown indicates the formation of FeNPs. In addition to the visual changes in the nanoparticle solution, the production of FeNPs was confirmed by the characteristic surface plasmon resonance (SPR) band using UV-Vis spectrophotometry (Fig. 4a and b).

The formation of FeNPs was stimulated by various phytoconstituents available in the leaves extract of the selected plant (AM and VN). Several other researchers reported the biological FeNPs synthesis via various plant extract (Bibi et al. 2019; Devi et al. 2019). The natural compounds in the extract has played a role in the synthesis and stability of NPs.

A critical need in the field of nanotechnology is the development of eco-friendly and reliable processes for the synthesis of metal oxide nanoparticles. In the current study, Iron nanoparticles were synthesized using various plant leaves extracts and FeCl₃.7H₂O as precursor molecule. The appearance of reddish brown colour is an observable characteristics indicating FeNPs synthesis.

UV-Visible Spectroscopy of synthesized FeNPs

The UV-Visible spectrum scan of all the synthesized NP were perform to determine the surface plasmon resonance (λ_max) (Fig 4a and b). The obtained peak (λ_max) of all the synthesized NP was satisfactory and comparable to the previously available reports (Table 1).

λ_max of the synthesized FeNPs in our current study is well consistent and comparable with the range reported in previous literature and hence confirms NPs synthesis.

Antibacterial activity

Antibacterial potential of synthesized FeNPs (0.1mM and 1mM) was assessed against K. oxytoca, E. coli, S. aureus and B. cereus (Fig. 5-8). The zone of inhibition (in mm) obtained by FeNPs_VN was presented in Table 2 and Fig. 5-6 and FeNPs_AM was presented in Table 3 and Fig. 7-8.

The zone of inhibition by the FeNPs_VN and FeNPs_AM obtained in the current study was comparable with the works reported in literature including Bibi et al., 2019; Devi et al., 2019.

Fig. 3: Biological synthesis of FeNPs

Fig. 4(a): UV-Vis spectra of 0.1mM FeNPs (λ_max 350-380nm)

Fig. 4(b): UV-Vis spectra of 1mM FeNPs (λ_max 360-380nm)

Fig. 5: Antibacterial activity of 1mM FeNP_VN against (a) K. oxytoca (b) E. coli (c) B. subtilis (d) S. aureus (first lane is back view and second lane is front view)

Fig. 6: Antibacterial activity of 0.1mM FeNP_VN against (a) K. oxytoca (b) E. coli (c) B. subtilis (d) S. aureus (first lane is back view and second lane is front view)

Fig. 7: Antibacterial activity of 1mM FeNP_AM against (a) E. coli  (b) K. oxytoca (c) S. aureus (d) B. subtilis (first lane is back view and second lane is front view)

Fig. 8: Antibacterial activity of 0.1mM FeNP_AM against (a) E. coli  (b) K. oxytoca (c) S. aureus (d) B. subtilis (first lane is back view and second lane is front view)