Antimicrobial Activity of Zinc Oxide Nanoparticles Synthesized Using Leaves Extract of Abies webbiana

Introduction: Green synthesis of nanoparticles using plant extract, bacteria, fungi and enzymes are eco-friendly and cost effective which do need high pressure, energy, temperature and toxic chemicals for its synthesis protocol. Zinc oxide nanoparticles have various physical, chemical and biological properties which can be applied in the treatment of oral diseases. Aim: The main objective of the current study was to synthesize the zinc oxide nanoparticles (ZnO NPs) in a cost effective biological method. The extract of A. webbiana was used to synthesis Zinc oxide nanoparticles. Materials and Methods: In this report, we used the extract of A. webbiana to synthesize zinc oxide nanoparticles and were characterized using U-V visible spectrophotometer and were tested for their antimicrobial activity against oral pathogens. Original Research Article Nivethitha et al.; JPRI, 33(60B): 3702-3710, 2021; Article no.JPRI.78314 3703 Results: These biosynthesized zinc oxide nanoparticles exhibited potent antimicrobial activity against oral pathogens, Streptococcus mutans, Staphylococcus aureus and Enterococcus faecalis, Candida albicans and can be developed as a novel medicine against pathogenic oral diseases. Abies webbiana extract mediated zinc oxide nanoparticles showed effective antimicrobial activity comparable to others. Conclusion: With increasing demand in advancements in diagnosis and treatment modalities, green synthesis of zinc oxide nanoparticles using A. webbiana which was found to have potent antimicrobial action has wider applications in dentistry. This formulation needs to be further purified to isolate the component in the extract that makes it effective to be used in large scale production for targeted drug delivery against a wide array of oral microbial infections.


INTRODUCTION
Nanoparticles (NPs) are small estimated particles with improved synergist reactivity, warm conductivity, non-straight optical execution, and compound relentlessness due to their enormous surface territory to volume proportion [1,2]. NPs have begun being considered as nano antibiotics in light of their antimicrobial activities [3,4]. Nanoparticles have been included into a variety of modern health, nutrition, feed, space, chemical, and cosmetic care goods, necessitating a green and environmentally friendly approach to their synthesis [5,6]. The properties of metallic nanoparticles, for example, gold, silver, iron and zinc nanoparticles have developed interests in biomedical applications [7].
Zinc oxide is a unique material with semiconducting, piezoelectric, and pyroelectric capabilities that has a wide range of applications in transparent electronics, UV light matters, piezoelectric devices, compound sensors, spin electronics, individual consideration items, coatings, and paints [8]. Biosynthesis

Study Organisms
The test organisms used in this study were obtained from the culture collections of the Nanomedicine Laboratory, Saveetha Dental College, Chennai. The organisms used in this study were Streptococcus mutans, Staphylococcus aureus and Enterococcus faecalis, Candida albicans.

Biosynthesis of Zinc Oxide Nanoparticles (ZnO NPs) Using A. webbiana Leaf Extract
1g of A. webbiana mixed with 100 mL of distilled water and boiled in 60-70 degree celsius in the heating mantle for 10-15 minutes and filtered using Whattman no 1 filter paper. 20 milli molar (0.574g) of Zinc sulphate was dissolved in 60 mL of distilled water. 40 mL of filtered A. webbiana extract is mixed with 60 mL of metal solution to obtain ZnO NPs.

Characterization of ZnO NPs
The optical properties of the synthesis of ZnO NPs were determined using UV -Vis spectroscopy wavelengths ranging from 250 to 650 nm. They were measured at different time periods.to determine the synthesis of ZnO NPs.

Antimicrobial Activity of Zinc Oxide Nanoparticles
The ZnO nanoparticles synthesized using A. webbiana were tested for antimicrobial activity by agar well diffusion method against pathogenic organisms namely S. aureus, E.faecalis, S. mutans, C. albicans. The pure culture of the bacterium was subcultured on Mueller Hinton agar. Wells of 9 mm diameter were made on Muller Hinton agar plates using gel puncture and each strain was uniformly swabbed onto the individual plates using sterile cotton swabs. Using a sterile micropipette, a standard antibacterial agent was loaded into one well and three different concentrations of zinc oxide nanoparticles sample solutions (25 μl, 50 μl and 100 μl) were poured into three other wells on all plates. The isolates of C. albicans were subcultured onto Rose Bengal agar. A standard antifungal disc (voriconazole) was placed on the agar plate and then nanoparticle sample solutions (25 μl, 50 μl and 100 μl) were poured onto three other wells on the plate. After incubation at 37°C for 24 hours, the different levels of the zone of inhibition of bacteria were observed and measured.

Measurement of zone of inhibition
The test plates were held in front of a desk lamp, and the zones were measured with a ruler held against the back of the petri plate. The diameters of the zones of inhibited growth were measured to the nearest whole millimeter.

Visual Identification
The present study showed the synthesis of ZnO NPs by using A. webbiana. The colour changes in the reaction mixture was observed continuously at various periods of incubation. The zinc oxide was reduced into ZnO nanoparticles that are visually identified by colour changes (Fig. 1). The colour of the solution was changed to light brown at 1 h incubation time.
The color was changed to dark brown after 24 h of incubation. There was no color change observed after 24 h which indicated that the ZnO NPs synthesis process was completed. According to several investigations, after one hour of incubation at room temperature with Zn nitrate, the colour of the leaf extract changed from light yellow to pale brown. The appearance of nanoparticles is indicated by a change in colour [36].

UV Vis Spectrophotometer Analysis
UV -vis spectroscopy analysis depends on the arising of colour in the reaction due to excitation of the surface plasmon resonance band in a reaction mixture. Fig. 2 shows the UV absorption spectra of the synthesized ZnO NPs. The spectrum clearly shows that the absorbance steadily decreased with decrease in incubation time. The plasmon resonance band for ZnO NPs synthesized using A. webbiana showed high absorption at 370 nm. UV spectroscopy research revealed maximal absorption at around 330 nm in several experiments. A particle size analyser was used to determine the particle size. The diameter of the particles was discovered to be 853 nm. When compared to a particle size analyzer, SEM characterizations of produced Zn nanoparticles reveal a substantially smaller diameter. Various magnifications were used to analyse the nanoparticles. Individual zinc particles as well as a number of aggregates were seen in the SEM picture. The antibacterial activity of the produced Zn nanoparticles was demonstrated against Gram positive and Gram negative bacteria, as well as a fungal strain [37].

Antimicrobial Activity of Zinc Oxide Nanoparticles against Oral Pathogens
The antimicrobial activity of ZnO NPs of A. webbiana assayed by a well diffusion method against oral pathogens showed minimum inhibitory concentrations at different concentrations. In Table 1 Some researchers have reported that ZnO NPs disrupt bacterial cell membrane integrity, reduce cell surface hydrophobicity, and downregulate the transcription of oxidative stress-resistance genes in bacteria which leads to its lysis. Rajeshkumar et al. reported that the positively charged AgNPs accumulate on negatively charged bacterial cell membranes to bring about structural alteration in cell membranes. Various mechanisms contribute to its antimicrobial activity such as generation of reactive oxygen species and bacterial protein denaturation [39]. Studies have also revealed that Zinc Oxide nanoparticles, clove and cinnamon have antiinflammatory and antioxidant properties. Zinc Oxide nanoparticles are known to have antiinflammatory properties by blocking proinflammatory cytokines, inhibiting mast cell proliferation and suppressing LPS induced COX-2 expression. Antioxidant Zinc Oxide nanoparticles are due to release of hydrogen which reduces DPPH free radical easily [40,41].

Fig. 2. UV Spectroscopic analyses of Zinc oxide nanoparticles synthesized from the leaf extract of A. webbiana recorded as function of time
Anti-bacterial activity of mouthwash incorporated with silica nanoparticles was assessed against oral pathogens. Silica nanoparticles were synthesized using the sol-gel method. In sol-gel reaction, there is hydrolysis and condensation of Tetra-ethoxy silane, where it was first hydrolyzed to silicic acid, followed by a condensation reaction leading to the formation of Si-O-Si bonds. Silica nanoparticles incorporated in mouthwash showed a good potential as an antimicrobial agent that maintains control of biofilm, preventing initial colonization of bacteria [42].
Thus biologically synthesis nanoparticles have various biomedical applications owing to its antimicrobial, anti inflammatory, antioxidants properties [43-51.

CONCLUSION
This study has demonstrated that ZnO NPs synthesis was initially identified by dark brown colour and the surface plasmon resonance was peak positioned at 370 nm. These ZnO NPs showed potent antimicrobial activity against oral pathogens assuring its effective therapeutic application in several infectious oral diseases.

CONSENT
It is not applicable.

ACKNOWLEDGEMENT
This research was done under the supervision of the department of research of saveetha dental college and hospitals. We thank our colleagues who provided insight and expertise that greatly assisted the research.