Abies webbiana Mediated Zinc Oxide Nanoparticles and Its Anti-inflammatory Activity

Introduction: Green nanoparticle synthesis provides a number of advantages, including being environmentally friendly, taking less time, being less expensive, being more stable, and, most significantly, not requiring the use of harmful chemicals. Aim: The aim of the present study is to evaluate the anti-inflammatory activity of zinc oxide [ZnO] nanoparticles prepared using Abies webbiana extract. Materials and Methods: In this study, ZnO nanoparticles were characterised using ultravioletvisible spectroscopy and inhibition of albumin denaturation assay using A. webbiana extract. Results: The biosynthesised ZnO particles exhibited potent anti-inflammatory activity to inhibit COX activity. ZnO nanoparticles can be developed as a novel medicine and can be used as an alternative to commercially available anti-inflammatory agents, thus reducing the major health problems. Original Research Article Priyadharshini et al.; JPRI, 33(62B): 85-93, 2021; Article no.JPRI.78095 86 Conclusion: Using A.webbiana extract, the findings suggest a cost-effective and environmentally friendly production of ZnO nanoparticles. ZnO nanoparticles mediated by A. webbiana showed promising properties. More investigations are required to understand the properties of these nanoparticles, which have a wide range of medical and dental applications.


INTRODUCTION
Cells of living organisms are typical across 10 μm. However, the parts of the cells are much smaller, with typical size of just 5 nm. The smaller size contributes to the idea of using nanoparticles to spy on cellular machinery without much interference [1]. The driving force behind the development of nanotechnology was to understand the biological processes on the nanoscale level [2]. Nanotechnology deals with the manufacture and application of materials with a size of up to 100 nm. The use of nanoparticles in various fields such as material science, agriculture, food industry, cosmetic, medical has emerged [3].
Zinc is an essential trace element for the human system. Body metabolism is maintained by zinc through hematopoiesis, enzyme regulation, maintaining cell redox balance and DNA and protein synthesis machinery regulation [4]. Zinc oxide nanoparticles exhibit various biomedical applications such as tremendous wound healing, catalytic, bio-imaging, anti-bacterial, antiinflammatory properties [5]. Zinc oxide nanoparticles act by inhibiting the activation of NF-kB [nuclear factor kappa B cells] in mRNA expression of inflammatory cytokines [6]. Owing to the excellent bio-medical properties of zinc oxide nanoparticles, they are used as potential drug delivery vehicles for standard drugs as it provides synergistic effects for the treatment [7].
Abies webbiana tree most commonly seen in the Himalayan region from Kashmir to Assam states in India also found in neighbouring areas of Nepal, Tibet and Afghanistan. A. webbiana is a tall, large, evergreen tree seen normally at an altitude of 2500-4000 m. A. webbiana also known as Talispatra in Bengali and Hindi, Talispatram in Sanskrit and Indian Silver Fir in English [8].

Preparation of Abies webbiana Extract
1gm of powdered A.webbiana extract was mixed with 100 mL of distilled water [ Fig. 1]. The solution was boiled under 60-70 degrees celsius in the heating mantle for 10-15 minutes. And the solution was filtered using Whatman No. 1 filter paper. The filtered extract was collected and stored at 4° C for further use.

Characterization of ZnO Nanoparticles
The synthesized zinc oxide nanoparticles solution was preliminarily characterized using ultraviolet [UV]-visible spectroscopy; 3 mL of the solution is taken in the cuvette and scanned in a double-beam UV-visible spectrophotometer from 300 nm to 700 nm wavelength. The results were recorded for the graphical analysis.

Preparation of Nanoparticles Powder
The nanoparticles solution is centrifuged using Lark refrigerated centrifuge. The ZnO nanoparticles solution was centrifuged at 8000 rpm for 10 mins and the pellet was collected and washed with distilled water twice. The final purified pellet is collected and dried at 100-150°C for 24 h and finally, the nanoparticles powder was collected and stored in an airtight Eppendorf tube.

Visual Observation
The present study shows the synthesis of Zinc oxide nanoparticles by using A. webbiana. Reduction of Zinc sulfate to zinc ion process was observed by colour change through direct visual observation of the solution. At various stages of incubation, the colour changes in the reaction mixture were examined continuously. Color change revealed that the zinc oxide had been converted into ZnO nanoparticles. After incubating for 1 hour, the colour of the solution had changed to brown. After 24 hours of incubation, the colour turned from light brown to dark brown [ Figs. 2 and 3]. After 24 hours, no colour change was observed indicating that the creation of Zinc oxide nanoparticles was complete. Bio-based synthesis of ZnO nanoparticles prepared from using amla fruits observed colour change from yellow to dark brown [32]. Similar colour change was observed in the present study.

UV-visible Spectroscopy
The UV-visible analysis of the ZnO nanoparticles was analyzed in the absorbency range of 250-500 nm. The peak was found to be maximum at 350 nm [ Fig. 4

Anti-inflammatory Activity
The anti-inflammatory activity of the A. webbiana extract was measured by the inhibition of albumin denaturation assay. The synthesized zinc oxide nanoparticles showed the quality of anti-inflammatory activity in the range of 80-90%, respectively (Fig. 5). The extract showed a greater percentage of inhibition when compared to the standard solution.

. The quality of anti-inflammatory activity ZnO nanoparticles synthesized using A. webbiana which is compared to the standard substance
Inflammation occurs as a response of our immune system to harmful stimuli which represents pathological conditions. Various chronic diseases are associated with inflammation process e.g., cancer, diabetes, inflammatory bowel disease and rheumatoid arthritis. Several cell types of neutrophils, basophils, eosinophils, and mononuclear cells are involved in the inflammatory process [33][34][35].
Inflammation can be of two types as either acute or chronic inflammation. Cyclooxygenase [COX] is the key causative factor/ enzyme in the synthesis of prostaglandins, prostacyclins and thromboxanes which are responsible for inflammation, pain, and platelet aggregation [32,36].
Non-steroidal anti-inflammatory drugs and immunosuppressants are currently most widely used in the treatment of acute inflammatory disorders. However there are many undesired side effects compared to the effectiveness of the drugs which deepens the needs and discovery of new anti-inflammatory drugs [37,38].
Some researchers have reported that ZnO nanoparticles exhibit potent anti-inflammatory action by inhibiting COX activity. ZnO nanoparticles extracted from Polygala tenuifolia root, exhibited excellent anti-inflammatory activity by dose-dependently inhibiting the LPS-induced protein expression of COX-2 and iNOS [39].
The most commonly used anti-inflammatory group of drugs includes aspirin, diclofenac, and ibuprofen. The drugs have adverse side effects on the liver and gastrointestinal tract. Based on in-vitro studies, ZnO nanoparticles showed antiinflammatory activity and can be considered as a potential candidate as an anti-inflammatory agent.

CONCLUSION
The present study has demonstrated an ecofriendly and cost-effective synthesis of ZnO nanoparticles using A.webbiana extract. ZnO synthesis was identified initially by direct visual observation of colour change to dark brown colour. UV-visible analysis of the ZnO nanoparticles was analyzed in the absorbency range of 250-500 nm. The peak was found to be maximum at 350 nm. The ZnO nanoparticles showed potent anti-inflammatory activity by inhibiting COX. Assuring its effectiveness in the therapeutic application for various diseases. The development of new classes of analgesics and anti-inflammatory drugs from A. webbiana is still under further investigation.

DISCLAIMER
The products used for this research are commonly and predominantly used products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by the personal efforts of the authors.

ACKNOWLEDGEMENT
The authors sincerely acknowledge the support from Saveetha institute of Medical and Technical sciences [SIMATS] and also the authors would like to thank Saveetha Dental College and Hospitals, Saveetha institute of Medical and Technical sciences [SIMATS], Saveetha University and Auditing solutions, Trichy for providing a platform to carry out this study.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.