Natural Green Synthesis of Silver Nanoparticles using Leaf Extract of Guazuma Ulmifolia Lam and Analysis of its Antimicrobial Property

Abstract

In the field of life science, nanoparticles are one of the leading adverse technologies. Here, the particles are seen in nanometers of 1–100 nm. In small-size particles, many useful applications are seen in the fields of medicine, pharma, agriculture, and many more industry sectors. In the present study, Guazuma ulmifolia, a member of the Malvavceae family, was a test plant. In this study, silver nanoparticles were synthesised from plant extract. Characterization using SEM, XRD, FTIR, and UV spectra confirmed the presence of nanoparticles. Silver nanoparticle sizes were measured using SEM, and the sizes ranged from 132nm to 352 nm. The XRD size of silver nanoparticles calculated using Debye-Scherrer’s equation was found to be 15.31 nm. Four drop-down FT-IR peaks were observed, which are 3369.60, 2162.44, 1643.53, and 635.04. N-H and C-I bonds were observed, and UV-Vis nanoparticles synthesised showed maximum absorptions between 300 nm to 700 nm. The sharp bands of silver nitrate nanoparticles were around 535nm for fresh leaves. The antimicrobial activity of synthesised nanoparticles against bacterial test pathogens, viz., Escherichia coli, Salmonella typhi, Bacillus subtilis, and Staphylococcus aureus, showed significant antibacterial activity. The average percentage of inhibition of bacteria is 38.83%, 0%, 10%, and 18.5%. Antifungal activity was also observed against Aspergillus niger, Fusarium oxysporum, Penicillium atramentosum, Aspergillus flavus, and Alternaria solani. The average percentage of inhibition in fungi is 15.68%, 58.53%, 0%, 32%, and 22.2%.

Introduction

I. INTRODUCTION

In 1974, professor Norie Taniguchi of the University of Tokyo in Japan is credited with coining the term "nanotechnology." [1].  The manipulation of particles with one of their size dimensions less than 100 nm and possessing particular properties that can be used in specific applications is one definition for it[2]. The Greek prefix "nano" designates a "dwarf" or extremely small object, and it represents one thousand millionth of a metre (109 m). Nanoscience and nanotechnology should be distinguished from one another [3]. The reason nanoparticles are so interesting is that they serve as a link between the bulk material and the atomic/molecular structure by exhibiting entirely new or improved properties based on particular attributes like size, shape, distribution, ionic strength, capping agent, and morphology [2],[4].

Many Greeks, Romans, Persians, and Egyptians stored food items in silver containers some 5000 years ago[9]. Because of its known antimicrobial properties, cutlery was widely used throughout ancient times by different dynasties to store and consume a wide variety of food and drink items. As early as 300 BC, there are documented cases of silver being used therapeutically in literature. In Hinduism, silver utensils are still favoured for preparing "panchamrit," a dish that includes curd, ocimum sanctum, and other ingredients. The ancient Indian Aurvedic medicine book known as the "Charak Samhita" contains references to the medicinal properties of different metals. Silver was widely used as an antimicrobial until Alexzander Flemming's discovery of antibiotics. [10],[11]

A reducing biological agent and a silver metal ion solution are the two main ingredients needed for the green synthesis of AgNPs. There is typically no need to add external capping and stabilising agents because reducing agents or other components already present in the cells function as these substances' stabilising and capping agents.[14]

Utilising plant extracts, microbial cell biomass, cell free growth medium, and biopolymers, green syntheses of AgNPs have been carried out. Algae and angiosperms are among the plants used to synthesise AgNps; however, there are few reports on lower plants, so angiosperms are the best option. AgNP synthesis has been carried out using parts such as leaves, bark, roots, and stems.[14]

The presence of numerous organic compounds, such as carbohydrates, fats, proteins, enzymes and coenzymes, phenols, flavanoids, terpenoids, alkaloids, gum, etc., capable of donating electron for the reduction of Ag+ ions to Ag0, is what allows biological entities to synthesise AgNP. The active component that lowers Ag+ ions varies based on the organism or extract that is utilised. The dehydrogenation of acids (ascorbic acid) and alcohols (catechol) in hydrophytes, keto to enol conversions (cyperaquinone, dietchequinone, remirin) in mesophytes, or both mechanisms in xerophytes plants are thought to provide the electrons needed for the AgNPs' nano-transformation.[12],[13].

AgNPs have found widespread application as anti-bacterial agents in various environmental applications, food storage, textile coatings, and the health industry. It is significant to remember that there is still uncertainty regarding the toxicity of silver, even after decades of use. Numerous recognised organisations, such as the US FDA, US EPA, SIAA of Japan, Korea's Testing and Research Institute for Chemical Industry, and FITI Testing and Research Institute, have approved products made with AgNPs. [8],[3]. AgNPs electrochemical qualities allowed for their incorporation into nanoscale sensors, which have a lower detection limit and quicker response times[20]Because of their numerous uses in a range of industries, including drug delivery, cancer therapy, carbon nanotube (CNT), carbon quantum dots (CQD), epoxy resin-coated CNTs, biomarkers, cell labelling, diagnostics, and antimicrobial agents, nanoparticles have drawn a lot of attention in recent years.[16]Nanoparticles can be toxic by either transferring electrons from molecules of oxygen or by obstructing the electron transport chain via an unidentified mechanism[17],[18].

Many researchers have used the disc diffusion method, which is the most widely used technique to access a liquid's antimicrobial activity, to confirm the AgNPs solution's antimicrobial action. This technique involves covering the surface of the targeted microbe that has been inoculated on nutrient medium plates with a disc of adsorbent material that is uniformly sized and dipped in a solution of increasing AgNP concentration. The formation of an inhibition zone surrounding the disc is indicative of the antimicrobial activity of the nanomaterials and well diffusion [13],[5],[6],[7],[8]. Nanoparticles capacity to generate reactive oxygen species (ROS) makes them highly promising as antibacterial agents. Reactive oxygen species generation damages the cell membrane mechanically, impairs ATP synthesis, and weakens the antioxidant defence system[15].

A. Objectives

1. Synthesis of silver nanoparticles from Guazuma ulmifolia.
2. Characterization of silver nanoparticles from Guazuma ulmifolia.
3. Antibacterial activity of silver nanoparticles from Guazuma ulmifolia
4. Antifungal activity of silver nanoparticles from Guazuma ulmifoila.

II. MATERIAL AND METHOD

A. Chemicals

Analytical grade silver nitrate (AgNO3) was used in this study. With out further purification the chemicals where used in this study.

B. Collection of leaves

Fresh Leaves of Guazuma ulmifolia were collected from Yuvaraja’s College campus, University of Mysore, Mysuru.

C. Preparation of Fresh Leaves Extract

 Plucking the fresh leaves from the plant measured it for 20gm. Washed it cleanly under tap water for few minutes again washer under double distilled water for few more minutes and dried. leaves were finely chopped as small pieces then transferred into 1000ml borosil Beaker. Added 150ml of double distil water to 20gm of fresh leaves and boiled it for 20 minutes. Then filtered it through filter paper (Whatman no: 1 filter paper, pore size 25µm) and used immediately for the biosynthesis of Silver Nanoparticles.

D. Green synthesis of silver nanoparticles

Silver nitrate was provided in the department of botany, Yuvaraja’s college Mysuru. For synthesis of silver nitrate (4mg of silver nitrate in 25ml of double distil water) 1M of AgNO3. Adding of 10ml of fresh leaves extract to the 90ml Silver nitrate in 250ml of borosil conical flask stir it for few minutes, slowly it changes colourless to dark brown in colour indicates that formation of silver nanoparticle’s reaction carried out in shade inside a BOD incubator for 24 hours.

E. Extraction of Nanoparticle Samples

The incubated samples where centrifuged in 10,200 rpm for 25 minutes continued for several times. The residue was dark brown in colour as pellet, pored it in sterilized Petri plate. Dried in room temperature to observe nanoparticles powder.

F. Characterization Techniques

Ultraviolet-visible spectrum (UV-vis) was conducted and noted on the Beckman coulter DU730 UV-vis spectrophotometer. The absorption spectra of the prepared NPs were recorded All ultraviolet- visible (UV-vis) spectra were recorded on the Beckman coulter by taking the aqueous dispersion of the NPs and scanned in the range of 300- 700 nm operated at resolution 1nm at IOE, Mysore. Distilled water was taken to adjust the baseline.

Scanning electron microscope (SEM)was used to study the morphological features of synthesized nanoparticles from fresh leaf extract of Guazuma ulmifolia. SEM images were recorded using Carl Zeiss Germany, model: EVO MA 15 SEM instrument at IOE, Mysore. X-ray diffraction the particle size and nature of the silver NPs were determined using Bruker Eco D8 advance X-pert PRO operating at a voltage of 40kV, a current of 20mA with silver Kα radiation at 2θ angle ranging from 10* to 80*. A thin film of the silver nanoparticles was made by dipping a glass plate in a solution and carried out for X-ray diffraction studies. The crystalline silver nanoparticle was calculated from the width of the XRD peaks and the average size of the nanoparticles can be estimated using the Debye Scherrer D= kλ/βcosθ. FT-IR analysis. Spectrum Two FT-IR 1600 spectrophotometer was used to IR spectra of Ag. NPs in the λ range of 600 to 4000 cm−1 and 4 cm−1(%T). Spectrum Two universal disc method was followed for the analysis.

G. Antibacterial Assay

The selected bacteria are Escherichia coli, Salmonella typhi, Bacillus subtilis, Staphylococcus aureus, which were sub cultured from the pure culture in an inoculation tube containing nutrient agar media for antibacterial study. The pure culture was provided by the P.G department of Microbiology, manasagangothri campus, Mysore. The nutrient agar high medium was prepared as per the requirement according to the number of plates. A small amount of agar is added to solidify. Then the media was kept for sterilization. After sterilization the NA media was poured into sterile Petri plates under aseptic condition and allowed for solidification. Well diffusion method by agar plates was used for calculating the zone of inhibition. These 4 bacterial pathogens were then coated over a agar plate with the help of sterile swab of cotton. Then these plates were permitted to dry. After those 5 wells were bored by sterile cork borer measuring 8.0mm diameter in each agar plate. Subsequently, 25µl,50µl,75µl, positive control and negative control was taken. The antibiotic Ampicillin was taken as positive control distilled water as negative control 25µl,50µl, and 75µl synthesis silver nanoparticles. Then the plates were kept for complete diffusion followed by incubation at 37 0C for 24 hrs and measured the diameter of inhibitory zones in mm.

H. Antifungal Assay

The selected fungi are Aspergillus niger, Fusarium oxysporum, Penicillium atramentosum, Aspergillus flavus and Alternaria solani which were sub cultured from the pure culture for further study. The pure culture was provided by the P.G department of Biotechnology manasagangothri campus, Mysore. PDA high medium was prepared as per the requirement according to the number of plates. A small amount of agar is added as solidifying agent and kept for sterilization. After sterilization the PDA media was poured into sterile Petri plates under aseptic condition and allowed for solidification. The antifungal activity of the NPs was determined by well diffusion method. The fungal inoculums prepared were used to test the antifungal potential. The PDA media was poured into sterile Petri plates in aseptic condition then plates were allowed to solidify in laminar air flow chamber. The 5 fungal pathogens were then coated over a media containing plates with the help of sterile swab of cotton. Then these plates were permitted to dry. After those 5 wells were bored by sterile cork borer measuring 8.0mm diameter in each agar plate. Subsequently, 25µl,50µl,75µl, positive control and negative control was taken. The antibiotic Bavistin was taken as positive control distilled water as negative control 25µl,50µl,and 75µl synthesis silver nanoparticles. Then the plates were sealed and incubated at room temperature for 5-7 days and finally antifungal activity was calculated by measuring the diameter of inhibitory zones in mm.

III. RESULT AND DISCUSSION

A. Synthesis of Silver Nanoparticle

The fresh leaf extract which was prepared, taken10ml of sample and added to 90ml of 1M concentration of silver nitrate in the ratio of 1:9 in 250 ml borosil conical flask at room temperature. The reaction is carried out in shade inside a BOD incubator for 48 hours. The colour change was observed colourless to dark brown in colour. The colour change suggests the synthesis of silver nanoparticles.

B. UV-Vis Spectroscopy

UV-Vis analysis is one of the most important characterization methods to study nanoparticles. The surface plasmon resonances (SPR) of synthesized nitrate nanoparticles have been studied by UV-Vis Spectrophotometer. The absorption of visible radiations due to the excitation of SPR, imparts various colours to nanoparticles. As the nanoparticles size changes, colour of the solution is also supposed to change. So, UV-Vis absorption spectrum is quite sensitive to the formation of nanoparticles. All the nanoparticle samples are subjected to UV-Vis study. Fig. 2 shows the UV-Vis spectrum of the 1sample. The nanoparticles synthesized showed maximum absorptions between 300 nm to700 nm. The sharp bands of silver nitrate nanoparticles were around 535nm for fresh leaf.

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IV. ACKNOWLEDGMENT

 The authors gratefully acknowledge faculty of Post Graduate department of Botany, Yuvaraja’s College Mysore, University of Mysore, for providing their support and laboratory facility to conduct this research work.

Conclusion

Guazuma ulmifolia fresh leaf eco-Fridley silver nanoparticles are synthesised sustainably and have a wide range of industrial, medicinal, and antibacterial uses. We can determine the presence of silver ion particles by the colour changing to brown. By observing the FT-IR, SEM, XRD, and UV-vis spectroscopy characterization. With the aid of spectroscopy, the ionising agent can be observed. SEM technology was used to analyse the shape and size of the particles. There is still a lot to discover about green living. XRD was used to determine the silver NPs\' nature and particle size. FT-IR reveals flavonoids and alkaline components. Along with having antimicrobial qualities against bacteria and fungi, the plant also performed well in the zone of inhibition. This makes it simple to determine that it has inhibiting ability as well. This makes it suitable for use in pharmacological and ayurvedic medicinal applications, among many others. Since plants have a good ability to inhibit environmental harm, further research can be done to compare the effects of AgNPs with those of common medications used to treat these diseases. One of the innovations and a rapidly expanding material in a variety of fields, silver nanoparticles produced positive results in this medicinal plant.

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Copyright

Copyright © 2023 G Rohan Hugar , Dr. M. L. Pruthvi, Dr. M. K. Mahesh . This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.