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Author(s): Papiya Chatterjee1, Nisha Gupta2, Jai Shankar Paul*3

Email(s): 1, 2, 3jaishankar_paul@yahoo.com

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    1Amity Institute of Biotechnology, Amity University Raipur, Chhattisgarh, India
    2School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    3School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    *Corresponding Author Email- jaishankar_paul@yahoo.com

Published In:   Volume - 3,      Issue - 2,     Year - 2021


Cite this article:
Papiya Chatterjee, Nisha Gupta, Jai Shankar Paul (2021) Synthesized Iron Nanoparticle via Green Approach and Evaluating its Antibacterial Potential. NewBioWorld A Journal of Alumni Association of Biotechnology, 3(2):26-36.

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 NewBioWorld A Journal of Alumni Association of Biotechnology (2021) 3(2):26-36            

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

ARTICLE INFORMATION

 

ABSTRACT

Article history:

Received

15 October 2021

Received in revised form

18 November 2021

Accepted

24 December 2021

Keywords:

Antibacterial activity; Green synthesis;

Metallic nanoparticles; Nanotechnology

 

Nanotechnology has found a wide range of application in almost every field such as food technology, biological remediation, agriculture, antibacterial activity, industrial and medicinal. This report mainly concerns with the work of green synthesis process used for the synthesis of FeNPs (metal salt– ferric chloride heptahydrate used as precursor) using plant leaf extract.  Metal nanoparticles that are synthesized by using plants extract usually have non-toxic and eco-friendly impact. The plants used were Nirgundi (Vitex negundo) and Bael (Aegle marmelos) which acted as reducing and capping agent and also used for stabilizing the synthesised nanoparticle. The antibacterial study was investigated against two gram positive (Staphylococcus aureus and Bacillus subtilis) and two gram negative (Klebsiella oxytoca and Escherichia. coli). Characterization of iron nanoparticle was done by UV-vis spectrophotometer. This work provides a strong basis of further research and development regarding antibacterial therapeutics using plants.

 


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).  

DOI: 10.52228/NBW-JAAB.2021-3-2-7

A perfect example of a medicinal plant credited with innumerable medicinal qualities validated by modern science and used since ancient times is Vitex negundo. It is commonly known as Nirgundi. It is a woody, aromatic and medicinal shrub. Nirgundi is a Sanskrit word used for plant or any substance which protects the body from disease (Bameta et al. 2019). V. negundo of family Lamiaceae and order Lamiales is a medicinal shrub which is known to exhibit curative activity against several ailments like cataract, headache, reduce inflammatory swellings of joints in rheumatic attacks etc. The V. negundo is an erect shrub or small tree growing from 2 to 8 m (6.6 to 26.2 feet) in height. The bark is reddish brown. Its leaves are digitate, with five lanceolate leaflets, sometimes three. The genus Vitex belongs to the family of flowering plants with 236 genera and more than 7000 species. It is commonly found in Eastern Africa, Madagascar to Iran, Burma, Pakistan, Srilanka, China, Taiwan, Afghanistan, India, Philippines, Thailand, throughout Malaysian region, Caroline Island and Moriana Islands. It is also widely cultivated in Europe, North America and West indies. It is found in various places of India, like Andhra Pradesh, Assam, Bihar, Delhi, Gujarat, Haryana, Himachal Pradesh, Jammu & Kashmir, Karnataka, Kerala, Madhya Pradesh, Uttar Pradesh, West Bengal and Uttarakhand. V. negundo contains a number of phytochemicals which find a lot of use in the pharmaceutical industry. Leaves contain an alkaloid nishindine, flavonoids like flavones, luteolin 7 glucoside, casticin, iridoidglucosides, an important oil and other constituents like vitamin C, carotene, gluconanitol, carboxylic acid and C–glucoside. Bark contains fatty acids, beta sitosterol, phydroxy carboxylic acid and luteolin. Stem bark contains leucoanthocyanidins. Leaves are antiparasitic and used as alternative vermifuge and anodyne. They are also very effective to reduce inflammatory swellings of joints in rheumatic attacks, and headache. The root is used as tonic, expectorant and diuretic. It regulates hormones, increases breast-milk production and possesses progestogenic properties. Flowers are cool, astringent, carminative, hepatoprotective, digestive, vermifuge and are useful in haemorrhages and cardiac disorders. Fruit is nervine, cephalic, aphrodisiac, emmenagogue and vermifuge (Bameta et al. 2019). Leaf extracts of V. negundo possess anti-oxidant potential and antifungal activities anthelminthic dysmenorrhoeal medication and pain suppressing activity, anti-hyperglycaemic activity, Anti-filarial, Anti-bacterial and opposed plant activity.

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 (λmax) (Fig 4a and b). The obtained peak (λmax) of all the synthesized NP was satisfactory and comparable to the previously available reports (Table 1).

λmax 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 (λmax 350-380nm)

Fig. 4(b): UV-Vis spectra of 1mM FeNPs (λmax 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)


Conclusion

Rich biodiversity of plants results in high potential of biological/green synthesis of noble nanoparticles. Green synthesis results in non-toxic, eco-friendly, and economically feasible as compared to physical or chemical synthesis. They have also developed a successful implementation in areas the of medicine and environment remediation. Iron nanoparticle synthesis with different plant extract has feasible production due to its pharmaceutical, medicinal characteristics and economic values which brought us to the need of exploring more phytochemistry behind FeNPs synthesis through biological means.

The present study was concerned with the synthesis of metallic nanoparticles (FeNPs) by using two different plant extracts viz. Aegle marmelos (AM), Vitex negundo (VN). The synthesized nanoparticles showed satisfactory antibacterial activity against B. subtilis, S. aureus, K. oxytoca and E. coli. Out of the synthesized NPs (FeNPs_VN and FeNPs_AM), best result was shown by FeNPs_VN. Green synthesis is an emerging area in the field of bio nanotechnology as it is cost effective and eco-friendly, but there are still things which are under development stage and need to be taken care of such as its stability, purification, yield which needs to be further explored.

Future prospects

Plants are nature’s “chemical factories” and vast repertoires of secondary metabolites that can be utilized as redox mediator and stabilizer for the NPs. NPs synthesized using plant products/extracts are more stable and the rate of synthesis is easy as compared to conventional techniques. Green synthesis of nanoparticle possess loads of positive applications with regards to its non-toxic, eco-friendly approach. However, there square measure sure limitations too once it involves environmental effects. The synthesized nanoparticles in the current study showed good antibacterial activity and provides a preliminary idea towards development of antibacterial drug using plant as a strong bio-reducing agent. Currently, controlling the size and shape of nanoparticles, and finding the exact mechanism of nanoparticles preparation by biological reducing agents need much more attention and experimentation. Since green approaches are eco-benign, cost effective, simple, and easy to perform and no toxic agent is involved, it can be utilized for development of potential drugs.

Acknowledgement

Authors are deeply acknowledge to School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur for providing lab facilities to conduct this research work. Authors are also acknowledge to Amity Institute of Biotechnology, Amity University Raipur for providing support to complete this work.

Conflict of Interest

The authors declare that there is no conflict of interest.

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