GREEN SYNTHESIS AND ANTIBACTERIAL EVALUATION OF SILVER NANOPARTICLES USING NEEM (AZADIRACHTA INDICA) LEAF EXTRACT

Syeda Farheen ANDERABI; Jahanzeb SHEIKH; Sania TANVIR; Rehana KOUSER; Iftikhar Ahmed CHANNA; Tanzeela IKRAM; Sidra Abid SYED; Ali Dad CHANDIO; Tan Tian SWEE; Jose-Javier SERRANO OLMEDO; Rashid KHAN; Muhammad FAIZAN; Madeeha SADIA

doi:10.24193/subbchem.2025.3.06

Authors

Syeda Farheen ANDERABI Department of Biomedical Engineering, NED University of Engineering and Technology, Karachi, Pakistan.
Jahanzeb SHEIKH Department of Biomedical Engineering and Health Sciences, Universiti Teknologi Malaysia, Johor, Malaysia; Department of Biomedical Engineering, Sir Syed University of Engineering and Technology, Karachi, Pakistan. https://orcid.org/0000-0002-3712-802X
Sania TANVIR Department of Biomedical Engineering, Sir Syed University of Engineering and Technology, Karachi, Pakistan. https://orcid.org/0000-0002-3094-0277
Rehana KOUSER Department of Biomedical Engineering, NED University of Engineering and Technology, Karachi, Pakistan.
Iftikhar Ahmed CHANNA Department of Metallurgy, NED University of Engineering and Technology, Karachi, Pakistan.
Tanzeela IKRAM Department of Biomedical Engineering, NED University of Engineering and Technology, Karachi, Pakistan.
Sidra Abid SYED Department of Biomedical Engineering, Sir Syed University of Engineering and Technology, Karachi, Pakistan. https://orcid.org/0000-0002-0647-8270
Ali Dad CHANDIO Department of Metallurgy, NED University of Engineering and Technology, Karachi, Pakistan. https://orcid.org/0000-0003-2212-0372
Tan Tian SWEE Department of Mechanical Engineering, College of Engineering, Al Imam Muhammad Ibn Saud Islamic University, Riyadh, Saudi Arabia. https://orcid.org/0000-0001-5826-6467
Jose-Javier SERRANO OLMEDO Centre for Biomedical Technology Madrid, Universidad Politécnica de Madrid, Madrid, Spain. https://orcid.org/0000-0002-8544-8933
Rashid KHAN Department of Mechanical Engineering, College of Engineering, Al Imam Muhammad Ibn Saud Islamic University, Riyadh, Saudi Arabia. https://orcid.org/0000-0002-5134-0289
Muhammad FAIZAN Department of Metallurgy, NED University of Engineering & Technology, Karachi, Pakistan.
Madeeha SADIA Department of Biomedical Engineering, NED University of Engineering and Technology, Karachi, Pakistan; Department of Biomedical Engineering and Health Sciences, Universiti Teknologi Malaysia, Johor, Malaysia. Corresponding author: madeehaoz@neduet.edu.pk https://orcid.org/0000-0002-5957-6229

DOI:

https://doi.org/10.24193/subbchem.2025.3.06

Keywords:

Silver nanoparticles, Green synthesis, Azadirachta indica (Neem), Antibacterial activity, SEM characterization, Plant-based nanomaterials

Abstract

The synthesis of silver nanoparticles (AgNPs) using traditional physical and chemical methods often involves toxic reagents, high energy consumption, and poor biocompatibility, making them unsuitable for many biomedical applications. Moreover, existing green synthesis approaches frequently lack control over nanoparticle size, shape, and stability, limiting their reproducibility and scalability. To address these limitations, this study employed a green synthesis route using Azadirachta indica (Neem) leaf extract as a natural reducing and stabilizing agent. Silver nitrate (AgNO₃) solutions of varying concentrations (1 mM, 5 mM, and 10 mM) were reacted with the Neem extract under ambient conditions. UV–Visible spectroscopy confirmed the formation of AgNPs with a characteristic surface plasmon resonance peak at 402 nm. Scanning Electron Microscopy (SEM) showed that the 5 mM AgNO₃ concentration produced the most desirable morphology, uniformly spherical nanoparticles with an average size of 98 nm. Energy Dispersive X-ray (EDX) analysis further confirmed the presence of pure elemental silver with no silver compounds. Moreover, antibacterial testing, conducted against Total Coliform bacteria and Propionibacterium acnes, revealed that the synthesized AgNPs, particularly in powder form, effectively inhibited bacterial growth over extended incubation periods. In conclusion, this study demonstrates that Neem-mediated synthesis is a viable, sustainable, and efficient approach for producing biologically active silver nanoparticles.

References

1. Bayda, S., Adeel, M., Tuccinardi, T., Cordani, M., & Rizzolio, F. (2019). The history of nanoscience and nanotechnology: from chemical–physical applications to nanomedicine. Molecules, 25(1), 112.

2. Sati, A., Ranade, T. N., Mali, S. N., Ahmad Yasin, H. K., & Pratap, A. (2025). Silver nanoparticles (AgNPs): comprehensive insights into bio/synthesis, key influencing factors, multifaceted applications, and toxicity─ a 2024 update. ACS omega, 10(8), 7549-7582.

3. Aswathy Waliaveettil, F., & Anila, E. I. (2024). A Comprehensive Review on Antibacterial, Anti‐Inflammatory and Analgesic Properties of Noble Metal Nanoparticles. Particle & Particle Systems Characterization, 41(5), 2300162.

4. Sadasivuni, K. K., Rattan, S., Waseem, S., Brahme, S. K., Kondawar, S. B., Ghosh, S., & Mazumdar, P. (2019). Silver nanoparticles and its polymer nanocomposites—Synthesis, optimization, biomedical usage, and its various applications. Polymer nanocomposites in biomedical engineering, 331-373.

5. Nguyen, N. P. U., Dang, N. T., Doan, L., & Nguyen, T. T. H. (2023). Synthesis of silver nanoparticles: from conventional to ‘modern’methods—a review. Processes, 11(9), 2617.

6. Abbas, R., Luo, J., Qi, X., Naz, A., Khan, I. A., Liu, H., ... & Wei, J. (2024). Silver nanoparticles: Synthesis, structure, properties and applications. Nanomaterials, 14(17), 1425.

7. Zahoor, M., Nazir, N., Iftikhar, M., Naz, S., Zekker, I., Burlakovs, J., & Ali Khan, F. dsynthesis, and their potential roles in biomedical applications and water treatment. Water, 13(16), 2216.

8. Tourinho, P. S., Silva, A. R. R., Santos, C. S., Prodana, M., Ferreira, V., Habibullah, G., & Loureiro, S. (2022). Microplastic fibers increase sublethal effects of AgNP and AgNO3 in Daphnia magna by changing cellular energy allocation. Environmental toxicology and chemistry, 41(4), 896-904.

9. Xu, L., Wang, Y. Y., Huang, J., Chen, C. Y., Wang, Z. X., & Xie, H. (2020). Silver nanoparticles: Synthesis, medical applications and biosafety. Theranostics, 10(20), 8996.

10. David, L., & Moldovan, B. (2020). Green synthesis of biogenic silver nanoparticles for efficient catalytic removal of harmful organic dyes. Nanomaterials, 10(2), 2020.

11. Zhao, J., Ma, L., Zayed, M. E., Elsheikh, A. H., Li, W., Yan, Q., & Wang, J. (2021). Industrial reheating furnaces: A review of energy efficiency assessments, waste heat recovery potentials, heating process characteristics and perspectives for steel industry. Process Safety and Environmental Protection, 147, 1209-1228.

12. Szczyglewska, P., Feliczak-Guzik, A., & Nowak, I. (2023). Nanotechnology–general aspects: A chemical reduction approach to the synthesis of nanoparticles. Molecules, 28(13), 4932.

13. Sun, H., Jiang, C., Wu, L., Bai, X., & Zhai, S. (2019). Cytotoxicity-related bioeffects induced by nanoparticles: the role of surface chemistry. Frontiers in Bioengineering and Biotechnology, 7, 414.

14. Tsekhmistrenko, S. I., Bityutskyy, V. S., Tsekhmistrenko, O. S., Horalskyi, L. P., Tymoshok, N. O., & Spivak, M. Y. (2020). Bacterial synthesis of nanoparticles: A green approach. Biosystems Diversity, 28(1), 9-17.

15. Salem, S. S., & Fouda, A. (2021). Green synthesis of metallic nanoparticles and their prospective biotechnological applications: an overview. Biological trace element research, 199(1), 344-370.

16. Ullah, A., & Lim, S. I. (2022). Plant extract‐based synthesis of metallic nanomaterials, their applications, and safety concerns. Biotechnology and Bioengineering, 119(9), 2273-2304.

17. Hano, C., & Abbasi, B. H. (2021). Plant-based green synthesis of nanoparticles: Production, characterization and applications. Biomolecules, 12(1), 31.

18. Alsaiari, N. S., Alzahrani, F. M., Amari, A., Osman, H., Harharah, H. N., Elboughdiri, N., & Tahoon, M. A. (2023). Plant and microbial approaches as green methods for the synthesis of nanomaterials: synthesis, applications, and future perspectives. Molecules, 28(1), 463.

19. Wehrs, M., Tanjore, D., Eng, T., Lievense, J., Pray, T. R., & Mukhopadhyay, A. (2019). Engineering robust production microbes for large-scale cultivation. Trends in microbiology, 27(6), 524-537.

20. Sardella, D., Gatt, R., & Valdramidis, V. P. (2019). Metal nanoparticles for controlling fungal proliferation: quantitative analysis and applications. Current Opinion in Food Science, 30, 49-59.

21. Mateo, E. M., Mateo, F., Tarazona, A., & Jiménez, M. (2025). Engineered Metal Nanoparticles: A Possible Small Solution to Big Problems Associated with Toxigenic Fungi and Mycotoxins. Toxins, 17(8), 378.

22. Kumari, P., Devi, L., Kadian, R., Waziri, A., & Alam, M. S. (2025). Eco-friendly Synthesis of Azadirachta indica-based Metallic Nanoparticles for Biomedical Application & Future Prospective. Pharmaceutical Nanotechnology, 13(3), 448-464.

23. Kumari, P., Devi, L., Kadian, R., Waziri, A., & Alam, M. S. (2025). Eco-friendly synthesis of Azadirachta indica-based metallic nanoparticles for biomedical application & future prospective. Pharmaceutical Nanotechnology, 13(3), 448- 464.

24. Singh, V., Roy, M., Garg, N., Kumar, A., Arora, S., & Malik, D. S. (2021). An insight into the dermatological applications of neem: a review on traditional and modern aspect. Recent Advances in Anti-Infective Drug Discovery (Formerly Recent Patents on Anti-Infective Drug Discovery), 16(2), 94-121.

25. Lu, X. F., Lin, P. C., Zi, J. C., and Fan, X. N. (2019). Limonoids from Seeds of Azadirachta indica and Their Antibacterial Activity. Zhongguo Zhong Yao Za Zhi 44 (22), 4864–4873. doi:10.19540/j.cnki.cjcmm.20190813.202

26. Zanjage, A., & Khan, S. A. (2021). Ultra-fast synthesis of antibacterial and photo catalyst silver nanoparticles using neem leaves. JCIS Open, 3, 100015.

27. Ghazali, S. Z., Mohamed Noor, N. R., & Mustaffa, K. M. F. (2022). Anti-plasmodial activity of aqueous neem leaf extract mediated green synthesis-based silver nitrate nanoparticles. Preparative Biochemistry & Biotechnology, 52(1), 99-107.

28. Noor, A., Pant, K. K., Malik, A., Moyle, P. M., & Ziora, Z. M. (2025). Green Encapsulation of Metal Oxide and Noble Metal ZnO@ Ag for Efficient Antibacterial and Catalytic Performance. Industrial & Engineering Chemistry Research, 64(21), 10360-10372.

29. Suresh, N., Thomas, N. G., Mauramo, M., Waltimo, T., Sorsa, T., & Anil, S. (2025). Phytonanoparticles as novel drug carriers for enhanced osteogenesis and osseointegration. Discover Nano, 20(1), 11.

30. Sejali, S. N. F., & Anuar, M. S. (2011). Effect of drying methods on phenolic contents of neem (Azadirachta indica) leaf powder. Journal of Herbs, Spices & Medicinal Plants, 17(2), 119-131.

31. Ofongo, R. T., Ohimain, E. I., & Iyayi, E. A. (2021). Qualitative and quantitative phytochemical screening of bitter and neem leaves and their potential as antimicrobial growth promoter in poultry feed. European Journal of Medicinal Plants, 38-49.

32. Kim, D. H., Park, J. C., Jeon, G. E., Kim, C. S., & Seo, J. H. (2017). Effect of the size and shape of silver nanoparticles on bacterial growth and metabolism by monitoring optical density and fluorescence intensity. Biotechnology and Bioprocess Engineering, 22, 210-217.

33. Zanjage, A., & Khan, S. A. (2021). Ultra-fast synthesis of antibacterial and photocatalyst silver nanoparticles using neem leaves. JCIS Open, 3, 100015.

34. Ghazali, S. Z., Mohamed Noor, N. R., & Mustaffa, K. M. F. (2022). Anti-plasmodial activity of aqueous neem leaf extract mediated green synthesis-based silver nitrate nanoparticles. Preparative Biochemistry & Biotechnology, 52(1), 99-107.

35. Noor, A., Pant, K. K., Malik, A., Moyle, P. M., & Ziora, Z. M. (2025). Green Encapsulation of Metal Oxide and Noble Metal ZnO@Ag for Efficient Antibacterial and Catalytic Performance. Industrial & Engineering Chemistry Research, 64(21), 10360-10372.

36. Sati, A., Ranade, T. N., Mali, S. N., Ahmad Yasin, H. K., & Pratap, A. (2025). Silver nanoparticles (AgNPs): comprehensive insights into bio/synthesis, key influencing factors, multifaceted applications, and toxicity‒ a 2024 update. ACS Omega, 10(8), 7549-7582

37. Kim, D. H., Park, J. C., Jeon, G. E., Kim, C. S., & Seo, J. H. (2017). Effect of the size and shape of silver nanoparticles on bacterial growth and metabolism by monitoring optical density and fluorescence intensity. Biotechnology and Bioprocess Engineering, 22, 210-217.

38. Restrepo, C. V., & Villa, C. C. (2021). Synthesis of silver nanoparticles, influence of capping agents, and dependence on size and shape: A review. Environmental Nanotechnology, Monitoring & Management, 15, 100428.

39. Ullah, A., & Lim, S. I. (2022). Plant extract‐based synthesis of metallic nanomaterials, their applications, and safety concerns. Biotechnology and Bioengineering, 119(9), 2273-2304.

40. Salem, S. S., & Fouda, A. (2021). Green synthesis of metallic nanoparticles and their prospective biotechnological applications: an overview. Biological Trace Element Research, 199(1), 344-370.

41. Guilger-Casagrande, M., & Lima, R. D. (2019). Synthesis of silver nanoparticles mediated by fungi: a review. Frontiers in Bioengineering and Biotechnology, 7, 87/

42. Kumari, P., Devi, L., Kadian, R., Waziri, A., & Alam, M. S. (2025). Eco-friendly synthesis of Azadirachta indica-based metallic nanoparticles for biomedical application & future prospective. Pharmaceutical Nanotechnology, 13(3), 448-464.

43. Zainurin, M. A. N., & Zainol, I. (2022). Biogenic synthesis of silver nanoparticles using neem leaf extract as reducing agent and hydrolyzed collagen as stabilizing agent. Malaysian Journal of Microscopy, 18(1).

44. Bai, V. R., Kit, A. C., Kangadharan, G., Gopinath, R., Varadarajan, P., & Hao, A. J. (2022). Experimental study on total coliform violations in the complied NH2CL, O3, and UV treated municipal water supply system. The European Physical Journal Plus, 137(6), 689.

45. Perry, A., & Lambert, P. (2011). Propionibacterium acnes: infection beyond the skin. Expert Review of Anti-Infective Therapy, 9(12), 1149–1156. https://doi.org/10.1586/eri.11.137

46. Sadia, Fazilah Abd Manan, Norjihada Izzah Ismail, Saravana Kumar Jaganathan, Syafiqah Saidin; Extraction of bioactive compounds from Moringa oleifera leaves using different organic solvents. AIP Conf. Proc. 2 June 2023; 2601 (1): 020046. https://doi.org/10.1063/5.0129684

47. Sadia, F. A. M., Ismail, N. I., Jaganathan, S. K., & Saidin, S. (2023). Extraction of bioactive compounds from Moringa oleifera leaves using different organic solvents. AIP Conference Proceedings, 2601(1), 020046. https://doi.org/10.1063/5.0129684

48. Andreica, A.M, Vlassa, M.C, R. Carpa, I. Petean, Green Synthesis of Silver Nanoparticles Using Galium Verum L. Aqueous Extract and Evaluation of its Antimicrobial Activity, Studia UBB Chemia, 2025, 70 (1), 87-100. https://doi.org/10.24193/subbchem.2025.1.06

49. Sadia, M., Mohd Zaki, M.A., Jaganathan, S.K. et al. Blending of Moringa oleifera into Biodegradable Polycaprolactone/Silver Electrospun Membrane for Hemocompatibility Improvement. Arab J Sci Eng, 48, 7323–7336 (2023). https://doi.org/10.1007/s13369-023-07736-6

50. Wylie, M. R., & Merrell, D. S. (2022). The antimicrobial potential of the neem tree Azadirachta indica. Frontiers in Pharmacology, 13, 891535.

51. Ershov VA, Ershov BG. (2024). Oxidative Dissolution and the Aggregation of Silver Nanoparticles in Drinking and Natural Waters: The Influence of the Medium on the Process Development. Toxics, Oct 18; 12(10):757. doi: 10.3390/toxics12100757

52. Kara, I. (2024). Use of geopolymers as tunable and sustained silver ion release mediums. Sci Rep, 14, 8606. https://doi.org/10.1038/s41598-024-59310-1

53. Hochvaldová, L., Panáček, D., Válková, L. et al. (2024). E. coli and S. aureus resist silver nanoparticles via an identical mechanism, but through different pathways. Commun Biol, 7, 1552. https://doi.org/10.1038/s42003-024-07266-3

54. Roberto Vazquez-Muñoz, Nina Bogdanchikova, and Alejandro Huerta-Saquero. (2020). Beyond the Nanomaterials Approach: Influence of Culture Conditions on the Stability and Antimicrobial Activity of Silver Nanoparticles, ACS Omega, 5 (44), 28441-28451, DOI: 10.1021/acsomega.0c02007

55. Liu, J., Sonshine, D. A., Shervani, S., & Hurt, R. H. (2010). Controlled release of biologically active silver from nanosilver surfaces. ACS Nano, 4(11), 6903–6913. https://doi.org/10.1021/nn102272n

56. Dube, E., & Okuthe, G. E. (2025). Silver nanoparticle-based antimicrobial coatings: Sustainable strategies for microbial contamination control. Microbiology Research, 16(6), 110. https://doi.org/10.3390/microbiolres16060110

57. Wang, L., Periyasami, G., Aldalbahi, A., & Fogliano, V. (2021). The antimicrobial activity of silver nanoparticles biocomposite films depends on the silver ions release behaviour. Food Chemistry, 359, 129859. https://doi.org/10.1016/j.foodchem.2021.129859

58. Barik, B., Satapathy, B. S., Pattnaik, G., Bhavrao, D. V., & Shetty, K. P. (2024). Sustainable synthesis of silver nanoparticles from Azadirachta indica: Antimicrobial, antioxidant and in silico analysis for periodontal treatment. Frontiers in Chemistry, 12, 1489253. https://doi.org/10.3389/fchem.2024.1489253

59. Rodrigues, A. S., Batista, J. G., Rodrigues, M. Á., Thipe, V. C., Minarini, L. A., Lopes, P. S., & Lugão, A. B. (2024). Advances in silver nanoparticles: A comprehensive review on their potential as antimicrobial agents and their mechanisms of action elucidated by proteomics. Frontiers in Microbiology, 15, 1440065. https://doi.org/10.3389/fmicb.2024.1440065

60. Lu, X. F., Lin, P. C., Zi, J. C., & Fan, X. N. (2019). Limonoids from Seeds of Azadirachta indica and Their Antibacterial Activity. Zhongguo Zhong Yao Za Zhi, 44(22), 4864–4873.

61. Sheikh, J., Tan, T. S., Saidin, S., Ahmed, S., & Chua, L. S. (2024). Bacterial morphology and microscopic advancements: navigating from basics to breakthroughs. Microbiol Immunol Commun, 3, 03-41.

62. Swee, T., Sheikh, J., Saidin, S., Serrano Olmedo, J., Agha, S. & Salim, M. (2025). Chamber design and intensity-modulated ultraviolet-C LEDs for advanced pulsed photonic disinfection. Biomedical Engineering / Biomedizinische Technik. https://doi.org/10.1515/bmt-2025-0070.