Antibacterial Activity of Green Synthesized Silver Nanoparticle using Banana Flower Extract

In recent years, due to unique physical and chemical properties of nanoparticles, its uses to disrupt biofilm of bacteria are revolutionary steps in antimicrobial research. To use in bioprocess the silver nanoparticles should be biocompatible and free from toxic chemicals. In the present study we report a cost effective and environment friendly route for green synthesis of silver nanoparticles using banana flower extract (BFE) as reducing as well as capping agent. This plant has been opted for the present study for its known medicinal properties and it is easily available. The biosynthesized silver nanoparticles are characterized by UV–Vis spectroscopy which showed a broad peak at around 450 nm, indicated the stability of synthesized silver nanoparticles. Antimicrobial potential of silver nanoparticles synthesized were tested against both Gram positive and Gram-negative bacteria. It was found to be effective against both of them and it showed maximum activity against Gram positive bacteria S. aureus. Silver nanoparticles (AgNPs) have been imposed as an excellent antimicrobial agent being able to combat bacteria in vitro and in vivo causing infections. The results show green synthesized silver nanoparticles, using BFE extract, have a potential to inhibit the growth of bacteria.


Introduction
Silver nanoparticles refer to particles of silver with a size ranging from 1 to 100 nanometers (nm).These particles exhibit unique properties because of their small size, including high surface area, increased reactivity, and distinctive optical, thermal, and electrical behavior [1].Silver nanoparticles have variety of applications, including electronics, medical treatments, water purification, and as catalysts in chemical reactions.For example, in electronics, silver nanoparticles are used in conductive inks for printed electronics and in anti-microbial device coating to prevent the growth of bacteria [2,3].In the medical field, silver nanoparticles have been shown to have antimicrobial properties and are being studied for use in wound healing and in the development of antimicrobial drugs.These silver nano particles can penetrate to cell membrane and they can destroy affected cells in human body [4].It is crucial to comprehend the possible hazardous effects of silver nanoparticles on human health and the environment due to their distinctive features and ubiquitous use.Therefore, research is being conducted to assess the safety and potential risks associated with the use of silver nanoparticles [5,6].Now a days green synthesis methods are most popular method because of its non-toxicity, low cost and their ease of availability.Many research works are established with this green method nano particle synthesis [7][8][9].In this work we are using banana flower extract as a reducing agent.Banana flower is chosen because of its ease of availability.The antibacterial activities of silver nanoparticles could be improved by using banana flower extract as a reducing agent [10].A prominent fruit grown in India, the banana (Musa spp.) is regarded as a good source of biologically active substances like dopamine, N-acetyl serotonin, nor-adrenaline, derivatives of isochronal-4-one, and polyphenols with high antioxidant characteristics [11].In addition to their many health advantages over synthetic antioxidant compounds, the isolation and identification of new antioxidants from natural sources, such as phenolic compounds, nitrogen compounds, carotenoids, and other phytochemicals, is attracting particular interest for its potential to prevent diseases [12].Spathes are boat-shaped bracts that are brightly colored, larger and spirally arranged structures that protect the flowers.They roll back and fall when flowers open-up.The water extract of Banana spathes found to contain wide verity of phytochemicals and showed good antioxidant potential [13,14].Thus, it has been used for the reduction of silver nitrate for nanoparticle synthesis.

Material and Methods
The chemicals used were of superior grade and glass wares were washed with detergents and rinsed twice with double distilled water before use.For nano synthesis, silver nitrate (Merck) was used as the initial component

Banana Flower Banana Flower Extract AgNO3
Ag Nanoparticles

Extract Preparation:
On a hot plate with magnetic stirrer, about 10g of dried banana spathe were extracted with 100 ml distilled water for 1 hour at 60ºC.It was then filtered and centrifuged at 4000 resolutions per minute for 10min.The synthesis and analysis of nanoparticles were done using the supernatant.Phytochemical analysis conducted.

Synthesis of Silver nanoparticle
A magnetic stirrer set to 45℃ was used to continuously mix 5 ml of the extract with 45 ml of silver nitrate solution (0.001M).UV-visible spectral scanning was used to periodically monitor the synthesis of silver nanoparticles.On Systronics UV-Visible spectrophotometer 117, a UV-Visible spectrograph of the colloidal solution of silver nanoparticles was recorded as a function of time [15].

Evaluation of antimicrobial potential of silver nanoparticles
Method: Kirby-Bauer test: Agar with bacteria growing on it is covered with antibiotic-containing discs, and the antibiotics diffuse into the agar.If an antibiotic stops the bacteria from growing, one can see circular areas around the wafers where they have not developed.The diameter of the inhibitory zone was measured using the well diffusion method using nanoparticles that had been resuspended in DMSO (10 and 15 ul).
Preparation of culture plates for antibacterial assay: Antibacterial assay was conducted on cultures plates prepared on Mueller Hinton Agar.The composition of the medium is given below.All the chemicals used were from HIMEDIA.

Nanoparticle synthesis
The formation of silver nanoparticle in the reaction mixture is indicated by the appearance of a yellow colour which gradually changed to reddish brown tinge.Also, it is confirmed by comparing the absorption spectra of produced colloidal solutions with extract blanks.A broad absorption band near the wavelength of 453 nm is obtained, and it was discovered that the intensity increased over time (Figure 1).The phenomenon known as plasmon absorption is what gives silver nanoparticle colloidal solution its characteristic colour.Conduction electrons oscillate on the surface of the nanoparticles as a result of incident light.Surface plasmon resonance allows the scattering and absorption of light at a specific frequency, which gives materials their colour.By centrifuging the colloidal nanoparticle solution at 12000 rpm for 15 minutes, silver nanoparticles were obtained.The precipitate was centrifuged after being rinsed with double-distilled water.The stability of the produced silver nanoparticles was demonstrated by a fragment of the precipitate that was dispersed in water and displayed a broad peak at about 450 nm.UV-Visible spectrum of the silver nanoparticle synthesized against plant extract blank.

Antimicrobial Studies
Antimicrobial potential of the synthesized silver nanoparticles was tested against Gram positive and negative bacteria.It was found to be active against both of them and showed maximum efficiency against Gram positive bacteria S. aureus.Silver nanoparticles (AgNPs) have been recommended as a superior antibacterial agent capable of attacking bacteria that cause infections both in vitro and in vivo [16].Gram-negative and Gram-positive bacteria, including multidrug resistant strains, are all covered by AgNPs' antibacterial property.AgNPs have several, concurrent modes of action, and when combined with antibiotics or other antibacterial agents such as chemical compounds, they have a synergistic effect on pathogenic bacteria like Escherichia coli and Staphylococcus aureus.The characteristics of silver nanoparticles make them suitable for their application in medical and healthcare products where they may treat infections or prevent them efficiently [17].

Klebsiella pneumonia
Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, rod-shaped bacterium.it can lead to a range of illnesses, including pneumonia, bloodstream infections, meningitis, and urinary tract infections.Compared to silver in its bulk form, they have demonstrated increased surface (area-to-volume ratio) and capacity.This substance displays distinctive electrical, optical, and catalytic capabilities at the nanoscale, which has stimulated research and the creation of goods for targeted drug administration, diagnostics, detection, and imaging.Yet, AgNPs' excellent antibacterial activity is what has drawn researchers' and companies' attention to this nanomaterial.AgNPs have shown antimicrobial activity against a variety of infectious and pathogenic microorganisms, including multidrug-resistant bacteria [18].Currently, the literature supports principally three mechanisms that have been observed together or separately, by which AgNPs exert their antibacterial action.The first one postulates that AgNPs act at a membrane level as they are able to penetrate the outer membrane, accumulating in the inner membrane where the adhesion of the nanoparticles to the cell generates their destabilization and damage, increasing membrane permeability and inducing leakage of cellular content and subsequently its death [19].Moreover, there is evidence that AgNPs can interact with sulfur-containing proteins in bacterial cell walls, which may result in structural damage and cell wall rupture.Nanoparticles can break and cross the cell membrane, changing its permeability and structure, similar to how they can damage intracellular machinery, activate the apoptotic pathway, and alter respiration.The second mechanism suggests that nanoparticles can enter cells, where it has been suggested that AgNPs will have an affinity to interact with sulphur or phosphorus groups present in intracellular content such as DNA and proteins, altering their structure and functions.The release of silver ions from the nanoparticles, which because of their size and charge can interact with cellular components and change metabolic pathways, membranes, and even genetic material, is a third process that is hypothesised to happen concurrently with the other two [20].

Conclusion
Banana flower extract is used as a reducing agent during the green synthesis process to create silver nanoparticles.Gram positive and gramme negative bacteria were tested against the produced nanoparticles in antimicrobial investigations.Both Gram-negative and Gram-positive bacteria are resistant to AgNPs' antibacterial properties.When exposed to the Gram-positive bacteria S. aureus, both of them displayed their peak activity.AgNPs have several, concurrent modes of action, and when combined with antibiotics or other antibacterial agents such as chemical compounds, they have a synergistic effect on pathogenic bacteria like Escherichia coli and Staphylococcus aureus.Based on the investigation, it is confirmed that both gram-positive and gram-negative bacteria are resistant to the antibacterial action of the produced silver nanoparticles.