Cytotoxicity and Characterization of Zinc Oxide Nanoparticles Synthesized using Saraca Asoca Bark Extract

Cytotoxicity and Characterization of Zinc Oxide Nanoparticles Synthesized using Saraca Asoca Bark Extract | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cytotoxicity and Characterization of Zinc Oxide Nanoparticles Synthesized using Saraca Asoca Bark Extract Aishwarya Jain, Kiran Bhise This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5393373/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract ZnO NPs have of late garnered interest for their biomedical and theranostic applications, however, all such applications would involve exhaustive toxicological testing for their safe use. This paper reports on the green synthesis of ZnO NPs by the reduction of cytotoxicity during production from Saraca Asoca bark extract. The ZnO NPs obtained were characterized through UV visible spectroscopy by observing the peak at 326 nm. The scanning electron microscopy is done to assess the particle size range, which ranges from 50 to 100 nm. The LC50 of the toxin, formula was established through an in-vivo Brine Shrimp Lethality Assay. Lethal effects at various concentrations of ZnO NPs including 5, 10, 20, 40, 80, and 100 µg/mL were tested on nauplii of brine shrimp while maintaining time constant at 24 hours. All nauplii survived at 5, 10, 20, and 40 µg/mL; at 80 µg/mL nine survived, while eight survived at 100 µg/mL, indicating a high level of nontoxicity at higher concentrations. LC50 estimation provides evidence that the ZnO NPs synthesized through the bark extract of Saraca Asoca reveal very low cytotoxicity and, therefore, would act as a promising candidate for biomedical applications. The green synthesis route here may find an alternative, non-toxic nanoparticle production technique instead of the toxic ones shown here. Zinc Oxide Nanoparticles Saraca Asoca bark Green synthesis Brine Shrimp cytotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Nanotechnology designs materials at the atomic and molecular levels. This is between 1 and 100 nanometers. Nanotechnology has enormous potential, particularly in biomedical applications. With size control, highly efficient and better therapy could be developed for all types of disease conditions. These nanoscale characteristics make the nanoparticles more reactive and enable some new mechanisms of targeted drug delivery in the medical field. (Applications of Nanotechnology | National Nanotechnology Initiative. (2024). A solvent-free methodology, otherwise known as an eco-friendly method, for the synthesis of nanoparticles called green synthesis, utilizes natural resources such as plants and microbes. This process eliminates the use of harmful chemicals in the process that in turn helps to decrease the whole pollution effect from the environment toward the conventional chemical synthesis techniques (Aishwarya Jain and Kiran Bhise. (2024) The green synthesis method of zinc oxide nanoparticles was found to be inexpensive and simple in nature, as the raw material involved was biodegradable and consisted of less number of processing steps. The method, such as plant-mediated synthesis, reduces toxic waste and taps into the natural properties of biological materials to promote the synthesis of nanoparticles (Abuzeid, H. M., Julien, C. M., Zhu, L., & Hashem, A. M. (2023). Mechanism of formation of ZnO NPs Formation: The general synthesis process involves the reduction of zinc salts in the presence of biological reducing agents from the extract of Saraca Asoca . During the synthesis process, phytochemicals like quercetin, gallic acid, ellagic acid, flavonoids, and terpenes interact with the zinc ions such that it leads to the reduction and nucleation of nanoparticles. Other parameters that may vary the size and shape of ZnO Nps include temperature, pH, and concentration of reducing agents. (Aishwarya Jain and Kiran Bhise. 2022;4(2) The main objective of using ZnO Nps as it gives a huge array of applications due to its unique properties. ZnO Nps are widely known for their antibacterial and antineoplastic properties which make them valuable for targeted drug delivery systems and also in cancer therapies (Mendes, C. R., Dilarri, G., Forsan, C. F., Sapata, V. de M. R., Lopes, P. R. M., de Moraes, P. B., Montagnolli, R. N., Ferreira, H., & Bidoia, E. D. (2022). Due to their desired size and morphology, they have a high surface area-to-volume ratio which enhances their interaction with biological systems, allowing for improved drug efficacy and reduced side effects (Jha, S., Rani, R., & Singh, S. (2023). Therefore the integration of nanotechnology with green synthesis not only addresses environmental concerns but also unlocks immense innovation potential, especially with ZnO Nps as they exemplify the synergy between environmental responsibility and technological advancement (Vijayaram, S., Razafindralambo, H., Sun, Y.-Z., Vasantharaj, S., Ghafarifarsani, H., Hoseinifar, S. H., & Raeeszadeh, M. (2023). The primary purpose of this research is the synthesis and characterization of ZnO Nps using Saraca Asoca extract. Several analyses were conducted to characterize the synthesized nanoparticles, including UV-visible spectra analysis, scanning electron microscopy, and Fourier-transform infrared spectroscopy. In addition, the cytotoxic effects of the prepared nanoparticles will be assessed through the brine shrimp lethality assay. This study gives better insight into the potential of medicinal plants for the green synthesis of nanoparticles and their wide application in several disciplines (Vijayakumar, N., Bhuvaneshwari, V. K., Ayyadurai, G. K., Jayaprakash, R., Gopinath, K., Nicoletti, M., Alarifi, S., & Govindarajan, M. (2022). Materials and Method Extract Process The maceration process is one of the most applied techniques in the extraction of the bioactive constituents from the bark of Saraca Asoca . In this process, the powdered barks are immersed in a suitable solvent-water in this case. 200 gms of powdered barks were soaked in 60 ml of water, thereby allowing the extraction to take place through passive diffusion that effectively releases the desired phytochemicals to the solvent. It favours the overnight extraction time, method for the simplicity and ease of retaining compound efficacy without degradation, suitable for further applications, like green synthesis nanoparticles (Bu, N., Jamil, A., Hussain, L., Alshammari, A., Albekairi, T. H., Alharbi, M., Jamshed, A., Bazmi, R. R., & Younas, A. (2023). Green Synthesis of Zinc Oxide Nanoparticles The green synthesis of ZnO Nps is thus framed by using the aqueous extract of the Saraca Asoca as a reducing, capping, and stabilizing agent. Here, for this purpose, 5 g of the powder of the bark of the Saraca Asoca was kept for maceration in 50 ml of distilled water. Then the extract was mixed with 50 ml of 0.1 M Zinc Acetate dihydrate solution followed by a dropwise addition of Sodium hydroxide (NaOH). The pH was kept at 8. The mixture is continuously stirred by a heated magnetic stirrer at 60 0 C for 30 minutes. In this process, a white or buff colour precipitate forms. This indicates ZnO Nps. The precipitate was collected by centrifugation at 5000 RPM. The material was dried overnight at 60 0 C. The synthesized ZnO NPs were further characterized (Al-darwesh, M. Y., Ibrahim, S. S., & Mohammed, M. A. (2024) Brine Shrimp Assay Brine shrimps were cultured in the laboratory. Artificial seawater was prepared by dissolving 36gm of sea salt in 1 litre of distilled water to be used for hatching. The seawater was kept in a fish tank (hatching chamber) with a partition for dark and light areas. A lamp was fixed above the tank which will attract the hatched shrimp. Two days were allowed for the eggs to hatch and the mature brine shrimps (Nauplii) were used for the cytotoxicity assay (Waghulde S, Kale MK, Patil V. Brine Shrimp Lethality Assay of the Aqueous and Ethanolic Extracts of the Selected Species of Medicinal Plants. Proceedings. 2019) Brine shrimp lethality assay was performed using green synthesized Zinc Oxide Nanoparticles. After the brine shrimp eggs were hatched the nauplii were collected, and the stock solution was prepared and diluted to concentrations 5,10,20,40,80 and 100 ug/ml. In the 7 well plates, 10 nauplii were exposed to each Nanoparticle concentration and one was of the control group and kept it for 24 hours. After 24 hours the live nauplii were counted and the percentage of the survived nauplii was determined. The LC50 the concentration at which 50% of nauplii were killed was calculated. This brine shrimp lethality assay (BSLA) is a simple and inexpensive bioassay used for testing the efficacy of green synthesised nanoparticles offering insights into their potential application (Varghese RM, S AK, Cureus. 2024) Significance of Brine Shrimp Lethality Assay of the Green Synthesised Zinc Oxide Nanoparticles using Saraca Asoca Extract To understand the toxic action of the ZnO Nps it is indispensable to consider the treatment to be safe; this study enables the definition of the intrinsic toxicity of the plant, and the effects of acute overdose, a cost-friendly and general bioassay that appears capable of detecting a spectrum of bioactivity present in ZnO Nps is the brine shrimp (Artemia salina LEACH) has been utilised by many researchers and has proven to be useful for screening various chemical compounds found in various bioactivities (Apu, A. S., Muhit, M. A., Tareq, S. M., Pathan, A. H., Jamaluddin, A. T. M., & Ahmed, M. (2010). After 24 hours the number of survival nauplii was counted and the percentage of mortality was determined using the following equation: % Mortality= (Number of dead Nauplii / Initial Number of live Nauplii) x 100 Characterization of ZnO Nps Ultraviolet-visible (UV-Vis) analysis The bandgap energy of ZnO Nps was determined by testing UV-Vis absorbance, and it also proved that the NPs were synthesized. Briefly, 0.01 gm of ZnO Nps was dispersed in double-distilled water (DDW) by Sonicator, then 2 ml in a quartz cuvette was taken and analyzed using a UV-Vis spectrophotometer over the wavelength of 200–700 nm. (Junaid, M., Ghulam Hussain, S., Abbas, N., & khan, W. Q. (2023). Fourier Transform Infrared (FTIR) analysis Analyzing with Fourier Transform Infrared (FTIR) is crucial in the identification of the functional groups that are implicated in the biosynthesis of ZnO Nps by the spectra found in the range of 4000 − 400 cm-1. The mentioned method will be able to detect specific molecular vibrations within compounds present in extracts from plants that contribute towards zinc ion stabilization and reduction ultimately to nanoparticle formation. Characteristic peaks in the FTIR spectrum indicate the various functional groups interacting with ZnO Nps, which provides significant information regarding the potential capping agents that might avoid agglomeration and stability. Further, FTIR analysis confirms both the synthesis of ZnO NPs and the complex interaction between the nanoparticles and the biomolecules present in the extracts. In general, this approach of analysis is crucial to understanding the mechanisms involved in the green synthesis of ZnO Nps as well as gives the potential application for multiple fields. (Alamdari, S., Sasani Ghamsari, M., Lee, C., Han, W., Park, H.-H., Tafreshi, M. J., Afarideh, H., & Ara, M. H. M. (2020). SEM SEM image of the biosynthesized ZnO Nps was carried out at 200 kV accelerating voltage. The shape, size, and distribution of the nanoparticles can be obtained from the SEM images, therefore important for the functional properties. From the SEM images, the essential structural features are granularity and uniformity, and the agglomeration or dispersion state of nanoparticles. It is important to know these properties to estimate the possibility of the nanoparticle for applications in drug delivery, catalysis, as well as environmental remediation. SEM analysis helps other characterization methods to optimize the synthesized ZnO Nps for specific use in the above fields (Rajiv gandhi, G., Mythili Gnanamangai, B., Heela Prabha, T., Poornima, S., Maruthupandy, M., Alharbi, N. S., Kadaikunnan, S., & Li, W.-J. (2022). Results The visual examination of the reaction indicated the successful formation of zinc oxide nanoparticles (ZnO Nps) when Saraca Asoca extract was added to a zinc acetate dihydrate solution, evidenced by a colour change from dark brown to a buff-coloured precipitate. This colour transition signifies the biosynthesis of ZnO Nps and can be categorized into three main stages: Activation Stage : In this initial stage, zinc ions are generated in the salt solution, which then bind to the reducing metabolites and stabilizing agents present in the Saraca Asoca extract, such as quercetin, gallic acid, and ellagic acid. These compounds facilitate the reduction of zinc ions to zinc atoms, leading to the nucleation of the reduced zinc atoms. Growth Stage : This stage is also known as Ostwald ripening. At this stage, spontaneous coalescence occurs between nearer smaller nanoparticles and larger ones. This process contains heterogeneous nucleation, growth, and further reduction of zinc ions which contribute to the increase in thermodynamic stability of the nanoparticles. Termination Stage : The final stage of nanoparticle formation produces the definitive shape of the ZnO Nps. Following this, the nanoparticles undergo oxidation, resulting in well-dispersed and stable ZnO Nps. The visual and sequential examination of these stages underscores the effectiveness of Saraca Asoca extract in facilitating the biosynthesis of ZnO Nps, highlighting their potential for various applications in nanotechnology and biomedicine (Khan, M., Naqvi, A. H., & Ahmad, M. (2015). UV-visible Spectroscopy UV-visible spectroscopy was highly essential to confirm that the green synthesis of ZnO Nps was successful. There are generally noticeable peaks in the absorption spectrum resulting from significant absorption bands at wavelengths of about 326 nm, which would confirm the formation of ZnO Nps; other peaks were observed at 358 nm and are associated with various zinc precursors, such as zinc acetate. These results indicate an intense absorption in the UV region that suggests the synthesized nanoparticles exhibit favourable optical properties. In summary, UV-Vis analysis focuses on the effectiveness of Saraca Asoca in synthesizing functional ZnO Nps toward different applications, such as antibacterial and antibiofilm efficacy (Nandhini, J., Karthikeyan, E., Sheela, M., Bellarmin, M., Gokula Kannan, B., Pavithra, A., Sowmya Sri, D., Siva Prakash, S., & Rajesh Kumar, S. (2024). FTIR Analysis The FTIR analysis for zinc oxide nanoparticles synthesized using an extract from Saraca Asoca bark indicates that they carry different functional groups very essential for the synthesis and stabilization of the nanoparticles. Among those, the significant one is that obtained at 3457 cm-1 which occurs due to hydroxyl (–OH) groups that can be assigned to the phenolic compounds in the plant extract. Other major peaks at 2088 cm-1 for stretching of the C-N bond and the series of peaks between 1600 and 1760 cm-1 confirmed that aldehydes, ketones, and esters exist. The obtained ZnO Nps showed spectra with peaks at 3462 cm-1 and 1509 cm-1 respective to the O-H groups and amide functionalities, which thereby indicated the capping as well as reducing behaviour of phytochemicals during synthesis. In addition, FTIR further proved the existence of stretching Zn–O vibrations that justified the fact that the bark extract of Saraca Asoca not only allowed the formation of ZnO Nps but also acted as a stabilizing agent for the enhancement of the potential applications of such nanoparticles (Bekele, S. G., Ganta, D. D., & Endashaw, M. (2024). SEM Analysis SEM analysis of ZnO Nps synthesized using Saraca Asoca bark extract provides plenty of insight information regarding the characteristics of its morphology, thus signifying the successful green synthesis. The major spherical-shaped morphologies are between about 40 nm to 100 nm in size. These morphologies will be favourably suitable for use in many applications, especially those involving antimicrobial treatments. This morphological homogeneity indicates that synthesis has been stabilized properly, thus raising the possibility of the potential reactivity and interactions between the nanoparticles and bacterial cells. The SEM images also show the lack of considerable agglomeration and this suggests that the utilization of phytochemicals in Saraca Asoca mainly as capping agents led to the stabilization of nanoparticles. Summary The SEM results help underpin the utility of the biosynthesized ZnO Nps as antimicrobial agents and tap into their unique structural attributes to make them practically useful in medicine and biotechnology (Sharma, R., Agrawal, A., Sharma, A., Kumar, S., Sharma, P. K., Awasthi, K. K., Pandey, K., & Awasthi, A. (2022). Cytotoxic Effect The cytotoxicity of the ZnO Nps that were synthesized from Saraca Asoca bark extract was evaluated using an in vivo Brine Shrimp Lethality Assay, BSLA, for the derivation of the LC50 (lethal concentration) for its safety appraisal before direct biomedical applications. The nauplii were exposed to six serial concentrations of ZnO Nps at 5, 10, 20, 40, 80, and 100 µg/ml. The nauplii survival rate was recorded after 24 h incubation. Nauplii survived entirely at all the concentrations of 5, 10, 20, and 40 µg/ml. However, survival rates were recorded to be decreased with increased concentration at 80 µg/ml and 100 µg/ml as 9 and 8 nauplii. (Varghese, R. M., S, A. K., & Shanmugam, R. (2024). Thus, LC50, calculated for the synthesized ZnO Nps, proves that the synthesized particles are low cytotoxic in nature and further validates the reasoning that these nanoparticles have the potential to be used safely in biomedical applications. Besides, the accompanying UV-visible spectroscopy showed a peak at 356 nm and the SEM analysis proved that the particle sizes of the ZnO Nps ranged between 50 and 100 nm, characterizing the synthesized nanoparticle further for therapeutic use. Overall, the results show that the ZnO Nps prepared from the Saraca Asoca bark extract are non-toxic and, therefore, promising candidates for further potential applications in biomedical fields (Casiano-Muñiz, I. M., Ortiz-Román, M. I., Lorenzana-Vázquez, G., & Román-Velázquez, F. R. (2024). The X-axis shows the concentration of ZnO Nps, Y-axis shows the Percentage number of brine shrimps Nauplii that survived in each concentration. Conclusion The work on green synthesis of ZnO Nps using Saraca Asoca extract resulted in a successful process marked by well-defined stages. Visual confirmation of nanoparticle formation was marked by the change in colour of the solution from dark brown to a buff precipitate, which indicated biosynthesis of ZnO Nps. The three stages, activation, growth and termination, can be differentiated; the roles of phytochemicals such as quercetin and gallic acid in reducing zinc ions to stabilize the produced nanoparticles can be illustrated. The results of UV-visible spectroscopy were indicated by strong absorption peaks, confirming the presence of ZnO Nps. However, an important peak was found at 356 nm with optical properties, which are desirable for various applications. Also, from SEM analysis, it was concluded that the nanoparticles exist in spherical shapes and range in size from 40 nm to 100 nm, which means stabilization at the synthesis process and may pose antimicrobial application. FTIR analysis further revealed additional functional groups involved in the synthesis, which supported the stabilization roles played by the biomolecules from the Saraca Asoca extract. More importantly, the cytotoxicity evaluation through the Brine Shrimp Lethality Assay established that the synthesized ZnO Nps are of low toxicity, establishing them as safe for potential biomedical applications. Therefore, results indicate that ZnO Nps prepared from the extract of Saraca Asoca bark are promising candidates in the therapeutic and biotechnological domains. They combine efficacy and safety in applications. Declarations Ethics Approval and Consent to Participate: Not applicable. Consent for Publication: Not applicable. Funding The authors did not receive support from any organization for the submitted work. Conflicts of interest/Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study. Data availability The data will be made available upon request. Credit taxonomy: Conceptualization: Ms Aishwarya Jain; Methodology: Ms Aishwarya Jain, Dr (Mrs) Kiran Bhise; Formal analysis and investigation: Ms Aishwarya Jain, Dr (Mrs) Kiran Bhise; Writing -original draft preparation: Ms Aishwarya Jain References Applications of Nanotechnology | National Nanotechnology Initiative. (2024). nano.gov. https://www.nano.gov/about-nanotechnology/applications-nanotechnology Abuzeid, H. M., Julien, C. M., Zhu, L., & Hashem, A. M. (2023). Green Synthesis of Nanoparticles and Their Energy Storage, Environmental, and Biomedical Applications. 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Cytotoxicity and Characterization of Zinc Oxide and Silver Nanoparticles Synthesized Using Ocimum tenuiflorum and Ocimum gratissimum Herbal Formulation. Cureus . https://doi.org/10.7759/cureus.53481 Casiano-Muñiz, I. M., Ortiz-Román, M. I., Lorenzana-Vázquez, G., & Román-Velázquez, F. R. (2024). Synthesis, Characterization, and Ecotoxicology Assessment of Zinc Oxide Nanoparticles by In Vivo Models. Nanomaterials. https://doi.org/10.3390/nano14030255 Additional Declarations No competing interests reported. Supplementary Files graphicalabstract.jpeg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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2","display":"","copyAsset":false,"role":"figure","size":657325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFully grown Brine Shrimp (Nauplii)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5393373/v1/58bf5502f76f91af272474e7.png"},{"id":69070504,"identity":"c94bb31c-d1c3-451f-9e41-b9e8b2cae877","added_by":"auto","created_at":"2024-11-15 09:56:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV-Spectra of Green synthesised ZnO Nps\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5393373/v1/87dc8e07a24ea9d7edfa470a.png"},{"id":69071847,"identity":"a1937d0c-cbf4-4925-ae26-7638d590f411","added_by":"auto","created_at":"2024-11-15 10:12:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":198896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR analysis of ZnO Nps\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5393373/v1/be75573f3cbb5c10f890d66c.png"},{"id":69070758,"identity":"3e27de87-ae1b-4e8a-b6bc-8b1f6adc7725","added_by":"auto","created_at":"2024-11-15 10:04:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":555709,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM images of Green synthesised ZnO Nps\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5393373/v1/d2d2e8f983a6b9a26ea3d93f.png"},{"id":69070505,"identity":"3bfe0be0-8d2d-48b6-a0e3-e9d2624a44a4","added_by":"auto","created_at":"2024-11-15 09:56:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":21756,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBar Graph illustrating the cytotoxic effect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSaraca Asoca\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emediated ZnO Nps\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5393373/v1/942c947a3c60d6eaf09ce83e.png"},{"id":69255408,"identity":"ed091d33-9087-4b2c-a9c2-cd4e88a00cbb","added_by":"auto","created_at":"2024-11-18 12:24:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2279601,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5393373/v1/fb39cc28-bca0-41fa-8f8c-6f38891e0968.pdf"},{"id":69070757,"identity":"b47f0527-1ef9-490f-9cef-802c5edf8d3a","added_by":"auto","created_at":"2024-11-15 10:04:01","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":53504,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5393373/v1/cd874118ab23b91402a4f611.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cytotoxicity and Characterization of Zinc Oxide Nanoparticles Synthesized using Saraca Asoca Bark Extract","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanotechnology designs materials at the atomic and molecular levels. This is between 1 and 100 nanometers. Nanotechnology has enormous potential, particularly in biomedical applications. With size control, highly efficient and better therapy could be developed for all types of disease conditions. These nanoscale characteristics make the nanoparticles more reactive and enable some new mechanisms of targeted drug delivery in the medical field. (Applications of Nanotechnology | National Nanotechnology Initiative. (2024).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA solvent-free methodology, otherwise known as an eco-friendly method, for the synthesis of nanoparticles called green synthesis, utilizes natural resources such as plants and microbes. This process eliminates the use of harmful chemicals in the process that in turn helps to decrease the whole pollution effect from the environment toward the conventional chemical synthesis techniques (Aishwarya Jain and Kiran Bhise. (2024) The green synthesis method of zinc oxide nanoparticles was found to be inexpensive and simple in nature, as the raw material involved was biodegradable and consisted of less number of processing steps. The method, such as plant-mediated synthesis, reduces toxic waste and taps into the natural properties of biological materials to promote the synthesis of nanoparticles (Abuzeid, H. M., Julien, C. M., Zhu, L., \u0026amp; Hashem, A. M. (2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMechanism of formation of ZnO NPs Formation: The general synthesis process involves the reduction of zinc salts in the presence of biological reducing agents from the extract of \u003cem\u003eSaraca Asoca\u003c/em\u003e. During the synthesis process, phytochemicals like quercetin, gallic acid, ellagic acid, flavonoids, and terpenes interact with the zinc ions such that it leads to the reduction and nucleation of nanoparticles. Other parameters that may vary the size and shape of ZnO Nps include temperature, pH, and concentration of reducing agents. (Aishwarya Jain and Kiran Bhise. \u0026nbsp;2022;4(2)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe main objective of using ZnO Nps as it gives a huge array of applications due to its unique properties. ZnO Nps are widely known for their antibacterial and antineoplastic properties which make them valuable for targeted drug delivery systems and also in cancer therapies (Mendes, C. R., Dilarri, G., Forsan, C. F., Sapata, V. de M. R., Lopes, P. R. M., de Moraes, P. B., Montagnolli, R. N., Ferreira, H., \u0026amp; Bidoia, E. D. (2022). Due to their desired size and morphology, they have a high surface area-to-volume ratio which enhances their interaction with biological systems, allowing for improved drug efficacy and reduced side effects (Jha, S., Rani, R., \u0026amp; Singh, S. (2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherefore the integration of nanotechnology with green synthesis not only addresses environmental concerns but also unlocks immense innovation potential, especially with ZnO Nps as they exemplify the synergy between environmental responsibility and technological advancement (Vijayaram, S., Razafindralambo, H., Sun, Y.-Z., Vasantharaj, S., Ghafarifarsani, H., Hoseinifar, S. H., \u0026amp; Raeeszadeh, M. (2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe primary purpose of this research is the synthesis and characterization of ZnO Nps using \u003cem\u003eSaraca Asoca\u003c/em\u003e extract. Several analyses were conducted to characterize the synthesized nanoparticles, including UV-visible spectra analysis, scanning electron microscopy, and Fourier-transform infrared spectroscopy. In addition, the cytotoxic effects of the prepared nanoparticles will be assessed through the brine shrimp lethality assay. This study gives better insight into the potential of medicinal plants for the green synthesis of nanoparticles and their wide application in several disciplines (Vijayakumar, N., Bhuvaneshwari, V. K., Ayyadurai, G. K., Jayaprakash, R., Gopinath, K., Nicoletti, M., Alarifi, S., \u0026amp; Govindarajan, M. (2022).\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Method","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\n\u003ch2\u003eExtract Process\u003c/h2\u003e\n\u003cp\u003eThe maceration process is one of the most applied techniques in the extraction of the bioactive constituents from the bark of \u003cem\u003eSaraca Asoca\u003c/em\u003e. In this process, the powdered barks are immersed in a suitable solvent-water in this case. 200 gms of powdered barks were soaked in 60 ml of water, thereby allowing the extraction to take place through passive diffusion that effectively releases the desired phytochemicals to the solvent. It favours the overnight extraction time, method for the simplicity and ease of retaining compound efficacy without degradation, suitable for further applications, like green synthesis nanoparticles (Bu, N., Jamil, A., Hussain, L., Alshammari, A., Albekairi, T. H., Alharbi, M., Jamshed, A., Bazmi, R. R., \u0026amp; Younas, A. (2023).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eGreen Synthesis of Zinc Oxide Nanoparticles\u003c/h2\u003e\n\u003cp\u003eThe green synthesis of ZnO Nps is thus framed by using the aqueous extract of the \u003cem\u003eSaraca Asoca\u003c/em\u003e as a reducing, capping, and stabilizing agent. Here, for this purpose, 5 g of the powder of the bark of the \u003cem\u003eSaraca Asoca\u003c/em\u003e was kept for maceration in 50 ml of distilled water. Then the extract was mixed with 50 ml of 0.1 M Zinc Acetate dihydrate solution followed by a dropwise addition of Sodium hydroxide (NaOH). The pH was kept at 8. The mixture is continuously stirred by a heated magnetic stirrer at 60\u003csup\u003e0\u003c/sup\u003e C for 30 minutes. In this process, a white or buff colour precipitate forms. This indicates ZnO Nps. The precipitate was collected by centrifugation at 5000 RPM. The material was dried overnight at 60\u003csup\u003e0\u003c/sup\u003e C. The synthesized ZnO NPs were further characterized (Al-darwesh, M. Y., Ibrahim, S. S., \u0026amp; Mohammed, M. A. (2024)\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eBrine Shrimp Assay\u003c/h3\u003e\n\u003cp\u003eBrine shrimps were cultured in the laboratory. Artificial seawater was prepared by dissolving 36gm of sea salt in 1 litre of distilled water to be used for hatching. The seawater was kept in a fish tank (hatching chamber) with a partition for dark and light areas. A lamp was fixed above the tank which will attract the hatched shrimp. Two days were allowed for the eggs to hatch and the mature brine shrimps (Nauplii) were used for the cytotoxicity assay (Waghulde S, Kale MK, Patil V. Brine Shrimp Lethality Assay of the Aqueous and Ethanolic Extracts of the Selected Species of Medicinal Plants. Proceedings. 2019)\u003c/p\u003e\n\u003cp\u003eBrine shrimp lethality assay was performed using green synthesized Zinc Oxide Nanoparticles. After the brine shrimp eggs were hatched the nauplii were collected, and the stock solution was prepared and diluted to concentrations 5,10,20,40,80 and 100 ug/ml. In the 7 well plates, 10 nauplii were exposed to each Nanoparticle concentration and one was of the control group and kept it for 24 hours. After 24 hours the live nauplii were counted and the percentage of the survived nauplii was determined. The LC50 the concentration at which 50% of nauplii were killed was calculated. This brine shrimp lethality assay (BSLA) is a simple and inexpensive bioassay used for testing the efficacy of green synthesised nanoparticles offering insights into their potential application (Varghese RM, S AK, Cureus. 2024)\u003c/p\u003e\n\u003ch3\u003eSignificance of Brine Shrimp Lethality Assay of the Green Synthesised Zinc Oxide\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eNanoparticles using\u003c/strong\u003e \u003cstrong\u003eSaraca Asoca\u003c/strong\u003e \u003cstrong\u003eExtract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the toxic action of the ZnO Nps it is indispensable to consider the treatment to be safe; this study enables the definition of the intrinsic toxicity of the plant, and the effects of acute overdose, a cost-friendly and general bioassay that appears capable of detecting a spectrum of bioactivity present in ZnO Nps is the brine shrimp (Artemia salina LEACH) has been utilised by many researchers and has proven to be useful for screening various chemical compounds found in various bioactivities (Apu, A. S., Muhit, M. A., Tareq, S. M., Pathan, A. H., Jamaluddin, A. T. M., \u0026amp; Ahmed, M. (2010). After 24 hours the number of survival nauplii was counted and the percentage of mortality was determined using the following equation:\u003c/p\u003e\n\u003ch3\u003e% Mortality= (Number of dead Nauplii / Initial Number of live Nauplii) x 100\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eCharacterization of ZnO Nps\u003c/h2\u003e\n\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n\u003ch2\u003eUltraviolet-visible (UV-Vis) analysis\u003c/h2\u003e\n\u003cp\u003eThe bandgap energy of ZnO Nps was determined by testing UV-Vis absorbance, and it also proved that the NPs were synthesized. Briefly, 0.01 gm of ZnO Nps was dispersed in double-distilled water (DDW) by Sonicator, then 2 ml in a quartz cuvette was taken and analyzed using a UV-Vis spectrophotometer over the wavelength of 200\u0026ndash;700 nm. (Junaid, M., Ghulam Hussain, S., Abbas, N., \u0026amp; khan, W. Q. (2023).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eFourier Transform Infrared (FTIR) analysis\u003c/h3\u003e\n\u003cp\u003eAnalyzing with Fourier Transform Infrared (FTIR) is crucial in the identification of the functional groups that are implicated in the biosynthesis of ZnO Nps by the spectra found in the range of 4000\u0026thinsp;\u0026minus;\u0026thinsp;400 cm-1. The mentioned method will be able to detect specific molecular vibrations within compounds present in extracts from plants that contribute towards zinc ion stabilization and reduction ultimately to nanoparticle formation. Characteristic peaks in the FTIR spectrum indicate the various functional groups interacting with ZnO Nps, which provides significant information regarding the potential capping agents that might avoid agglomeration and stability. Further, FTIR analysis confirms both the synthesis of ZnO NPs and the complex interaction between the nanoparticles and the biomolecules present in the extracts. In general, this approach of analysis is crucial to understanding the mechanisms involved in the green synthesis of ZnO Nps as well as gives the potential application for multiple fields. (Alamdari, S., Sasani Ghamsari, M., Lee, C., Han, W., Park, H.-H., Tafreshi, M. J., Afarideh, H., \u0026amp; Ara, M. H. M. (2020).\u003c/p\u003e\n\u003ch3\u003eSEM\u003c/h3\u003e\n\u003cp\u003eSEM image of the biosynthesized ZnO Nps was carried out at 200 kV accelerating voltage. The shape, size, and distribution of the nanoparticles can be obtained from the SEM images, therefore important for the functional properties. From the SEM images, the essential structural features are granularity and uniformity, and the agglomeration or dispersion state of nanoparticles. It is important to know these properties to estimate the possibility of the nanoparticle for applications in drug delivery, catalysis, as well as environmental remediation. SEM analysis helps other characterization methods to optimize the synthesized ZnO Nps for specific use in the above fields (Rajiv gandhi, G., Mythili Gnanamangai, B., Heela Prabha, T., Poornima, S., Maruthupandy, M., Alharbi, N. S., Kadaikunnan, S., \u0026amp; Li, W.-J. (2022).\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u0026nbsp;\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003cp\u003eThe visual examination of the reaction indicated the successful formation of zinc oxide nanoparticles (ZnO Nps) when \u003cem\u003eSaraca Asoca\u003c/em\u003e extract was added to a zinc acetate dihydrate solution, evidenced by a colour change from dark brown to a buff-coloured precipitate.\u003c/p\u003e\n\u003cp\u003eThis colour transition signifies the biosynthesis of ZnO Nps and can be categorized into three main stages:\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003e\n\u003cp\u003e\u003cstrong\u003eActivation Stage\u003c/strong\u003e: In this initial stage, zinc ions are generated in the salt solution, which then bind to the reducing metabolites and stabilizing agents present in the \u003cem\u003eSaraca Asoca\u003c/em\u003e extract, such as quercetin, gallic acid, and ellagic acid. These compounds facilitate the reduction of zinc ions to zinc atoms, leading to the nucleation of the reduced zinc atoms.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth Stage\u003c/strong\u003e: This stage is also known as Ostwald ripening. At this stage, spontaneous coalescence occurs between nearer smaller nanoparticles and larger ones. This process contains heterogeneous nucleation, growth, and further reduction of zinc ions which contribute to the increase in thermodynamic stability of the nanoparticles.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003e\u003cstrong\u003eTermination Stage\u003c/strong\u003e: The final stage of nanoparticle formation produces the definitive shape of the ZnO Nps. Following this, the nanoparticles undergo oxidation, resulting in well-dispersed and stable ZnO Nps.\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe visual and sequential examination of these stages underscores the effectiveness of \u003cem\u003eSaraca Asoca\u003c/em\u003e extract in facilitating the biosynthesis of ZnO Nps, highlighting their potential for various applications in nanotechnology and biomedicine (Khan, M., Naqvi, A. H., \u0026amp; Ahmad, M. (2015).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eUV-visible Spectroscopy\u003c/h2\u003e\n\u003cp\u003eUV-visible spectroscopy was highly essential to confirm that the green synthesis of ZnO Nps was successful. There are generally noticeable peaks in the absorption spectrum resulting from significant absorption bands at wavelengths of about 326 nm, which would confirm the formation of ZnO Nps; other peaks were observed at 358 nm and are associated with various zinc precursors, such as zinc acetate. These results indicate an intense absorption in the UV region that suggests the synthesized nanoparticles exhibit favourable optical properties. In summary, UV-Vis analysis focuses on the effectiveness of \u003cem\u003eSaraca Asoca\u003c/em\u003e in synthesizing functional ZnO Nps toward different applications, such as antibacterial and antibiofilm efficacy (Nandhini, J., Karthikeyan, E., Sheela, M., Bellarmin, M., Gokula Kannan, B., Pavithra, A., Sowmya Sri, D., Siva Prakash, S., \u0026amp; Rajesh Kumar, S. (2024).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eFTIR Analysis\u003c/h2\u003e\n\u003cp\u003eThe FTIR analysis for zinc oxide nanoparticles synthesized using an extract from \u003cem\u003eSaraca Asoca\u003c/em\u003e bark indicates that they carry different functional groups very essential for the synthesis and stabilization of the nanoparticles. Among those, the significant one is that obtained at 3457 cm-1 which occurs due to hydroxyl (\u0026ndash;OH) groups that can be assigned to the phenolic compounds in the plant extract. Other major peaks at 2088 cm-1 for stretching of the C-N bond and the series of peaks between 1600 and 1760 cm-1 confirmed that aldehydes, ketones, and esters exist. The obtained ZnO Nps showed spectra with peaks at 3462 cm-1 and 1509 cm-1 respective to the O-H groups and amide functionalities, which thereby indicated the capping as well as reducing behaviour of phytochemicals during synthesis. In addition, FTIR further proved the existence of stretching Zn\u0026ndash;O vibrations that justified the fact that the bark extract of \u003cem\u003eSaraca Asoca\u003c/em\u003e not only allowed the formation of ZnO Nps but also acted as a stabilizing agent for the enhancement of the potential applications of such nanoparticles (Bekele, S. G., Ganta, D. D., \u0026amp; Endashaw, M. (2024).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eSEM Analysis\u003c/h2\u003e\n\u003cp\u003eSEM analysis of ZnO Nps synthesized using \u003cem\u003eSaraca Asoca\u003c/em\u003e bark extract provides plenty of insight information regarding the characteristics of its morphology, thus signifying the successful green synthesis. The major spherical-shaped morphologies are between about 40 nm to 100 nm in size. These morphologies will be favourably suitable for use in many applications, especially those involving antimicrobial treatments. This morphological homogeneity indicates that synthesis has been stabilized properly, thus raising the possibility of the potential reactivity and interactions between the nanoparticles and bacterial cells. The SEM images also show the lack of considerable agglomeration and this suggests that the utilization of phytochemicals in \u003cem\u003eSaraca Asoca\u003c/em\u003e mainly as capping agents led to the stabilization of nanoparticles. Summary The SEM results help underpin the utility of the biosynthesized ZnO Nps as antimicrobial agents and tap into their unique structural attributes to make them practically useful in medicine and biotechnology (Sharma, R., Agrawal, A., Sharma, A., Kumar, S., Sharma, P. K., Awasthi, K. K., Pandey, K., \u0026amp; Awasthi, A. (2022).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eCytotoxic Effect\u003c/h2\u003e\n\u003cp\u003eThe cytotoxicity of the ZnO Nps that were synthesized from \u003cem\u003eSaraca Asoca\u003c/em\u003e bark extract was evaluated using an in vivo Brine Shrimp Lethality Assay, BSLA, for the derivation of the LC50 (lethal concentration) for its safety appraisal before direct biomedical applications. The nauplii were exposed to six serial concentrations of ZnO Nps at 5, 10, 20, 40, 80, and 100 \u0026micro;g/ml.\u003c/p\u003e\n\u003cp\u003eThe nauplii survival rate was recorded after 24 h incubation. Nauplii survived entirely at all the concentrations of 5, 10, 20, and 40 \u0026micro;g/ml. However, survival rates were recorded to be decreased with increased concentration at 80 \u0026micro;g/ml and 100 \u0026micro;g/ml as 9 and 8 nauplii. (Varghese, R. M., S, A. K., \u0026amp; Shanmugam, R. (2024).\u003c/p\u003e\n\u003cp\u003eThus, LC50, calculated for the synthesized ZnO Nps, proves that the synthesized particles are low cytotoxic in nature and further validates the reasoning that these nanoparticles have the potential to be used safely in biomedical applications. Besides, the accompanying UV-visible spectroscopy showed a peak at 356 nm and the SEM analysis proved that the particle sizes of the ZnO Nps ranged between 50 and 100 nm, characterizing the synthesized nanoparticle further for therapeutic use. Overall, the results show that the ZnO Nps prepared from the \u003cem\u003eSaraca Asoca\u003c/em\u003e bark extract are non-toxic and, therefore, promising candidates for further potential applications in biomedical fields (Casiano-Mu\u0026ntilde;iz, I. M., Ortiz-Rom\u0026aacute;n, M. I., Lorenzana-V\u0026aacute;zquez, G., \u0026amp; Rom\u0026aacute;n-Vel\u0026aacute;zquez, F. R. (2024).\u003c/p\u003e\n\u003cp\u003eThe X-axis shows the concentration of ZnO Nps, Y-axis shows the Percentage number of brine shrimps Nauplii that survived in each concentration.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe work on green synthesis of ZnO Nps using \u003cem\u003eSaraca Asoca\u003c/em\u003e extract resulted in a successful process marked by well-defined stages. Visual confirmation of nanoparticle formation was marked by the change in colour of the solution from dark brown to a buff precipitate, which indicated biosynthesis of ZnO Nps. The three stages, activation, growth and termination, can be differentiated; the roles of phytochemicals such as quercetin and gallic acid in reducing zinc ions to stabilize the produced nanoparticles can be illustrated.\u003c/p\u003e \u003cp\u003eThe results of UV-visible spectroscopy were indicated by strong absorption peaks, confirming the presence of ZnO Nps. However, an important peak was found at 356 nm with optical properties, which are desirable for various applications. Also, from SEM analysis, it was concluded that the nanoparticles exist in spherical shapes and range in size from 40 nm to 100 nm, which means stabilization at the synthesis process and may pose antimicrobial application.\u003c/p\u003e \u003cp\u003eFTIR analysis further revealed additional functional groups involved in the synthesis, which supported the stabilization roles played by the biomolecules from the \u003cem\u003eSaraca Asoca\u003c/em\u003e extract. More importantly, the cytotoxicity evaluation through the Brine Shrimp Lethality Assay established that the synthesized ZnO Nps are of low toxicity, establishing them as safe for potential biomedical applications.\u003c/p\u003e \u003cp\u003eTherefore, results indicate that ZnO Nps prepared from the extract of \u003cem\u003eSaraca Asoca\u003c/em\u003e bark are promising candidates in the therapeutic and biotechnological domains. They combine efficacy and safety in applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors did not receive support from any organization for the submitted work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest/Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data will be made available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCredit taxonomy:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u003c/strong\u003e Ms Aishwarya Jain; \u003cstrong\u003eMethodology:\u003c/strong\u003e Ms Aishwarya Jain, Dr (Mrs) Kiran Bhise; \u003cstrong\u003eFormal analysis and investigation:\u003c/strong\u003e Ms Aishwarya Jain, Dr (Mrs) Kiran Bhise; \u003cstrong\u003eWriting -original draft preparation:\u003c/strong\u003e Ms Aishwarya Jain\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eApplications of Nanotechnology | National Nanotechnology Initiative. 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Cytotoxicity and Characterization of Zinc Oxide and Silver Nanoparticles Synthesized Using Ocimum tenuiflorum and Ocimum gratissimum Herbal Formulation. \u003cem\u003eCureus\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7759/cureus.53481\u003c/span\u003e\u003cspan address=\"10.7759/cureus.53481\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCasiano-Mu\u0026ntilde;iz, I. M., Ortiz-Rom\u0026aacute;n, M. I., Lorenzana-V\u0026aacute;zquez, G., \u0026amp; Rom\u0026aacute;n-Vel\u0026aacute;zquez, F. R. (2024). Synthesis, Characterization, and Ecotoxicology Assessment of Zinc Oxide Nanoparticles by In Vivo Models. Nanomaterials. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/nano14030255\u003c/span\u003e\u003cspan address=\"10.3390/nano14030255\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Zinc Oxide Nanoparticles, Saraca Asoca bark, Green synthesis, Brine Shrimp, cytotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-5393373/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5393373/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eZnO NPs have of late garnered interest for their biomedical and theranostic applications, however, all such applications would involve exhaustive toxicological testing for their safe use. This paper reports on the green synthesis of ZnO NPs by the reduction of cytotoxicity during production from \u003cem\u003eSaraca Asoca\u003c/em\u003e bark extract. The ZnO NPs obtained were characterized through UV visible spectroscopy by observing the peak at 326 nm. The scanning electron microscopy is done to assess the particle size range, which ranges from 50 to 100 nm.\u003c/p\u003e\n\u003cp\u003eThe LC50 of the toxin, formula was established through an in-vivo Brine Shrimp Lethality Assay. Lethal effects at various concentrations of ZnO NPs including 5, 10, 20, 40, 80, and 100 µg/mL were tested on nauplii of brine shrimp while maintaining time constant at 24 hours. All nauplii survived at 5, 10, 20, and 40 µg/mL; at 80 µg/mL nine survived, while eight survived at 100 µg/mL, indicating a high level of nontoxicity at higher concentrations.\u003c/p\u003e\n\u003cp\u003eLC50 estimation provides evidence that the ZnO NPs synthesized through the bark extract of \u003cem\u003eSaraca Asoca\u003c/em\u003e reveal very low cytotoxicity and, therefore, would act as a promising candidate for biomedical applications. The green synthesis route here may find an alternative, non-toxic nanoparticle production technique instead of the toxic ones shown here.\u003c/p\u003e","manuscriptTitle":"Cytotoxicity and Characterization of Zinc Oxide Nanoparticles Synthesized using Saraca Asoca Bark Extract","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-15 09:55:56","doi":"10.21203/rs.3.rs-5393373/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"08c32739-1dd5-44d4-beaf-4398a1e9e8f7","owner":[],"postedDate":"November 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-18T12:23:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-15 09:55:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5393373","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5393373","identity":"rs-5393373","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}