In the present study the identification and isolation of key phytochemicals, such as Quercetin and ß-sitosterol, from fresh neem leaves marked a significant advancement in understanding the therapeutic potential of neem17.
These polyphenolic flavonoids have been recognized for their notable antifungal and antibacterial activities. In the aqueous neem leaf extracts analyzed by Dash et al., saponins were found to be the predominant constituents, followed by moderate levels of tannins and glycosides, and lower amounts of alkaloids, flavonoids, and reducing sugars. However present study recorded that alkaloids found to be predominant, followed by flavanoids, terpenes and phenolic compounds with the absence of saponins18.
The multifaceted nature of these bioactive compounds positions neem as a valuable resource in traditional medicine, with alkaloids, flavonoids, glycosides, and saponins exhibiting a spectrum of biological activities. These include anti-inflammatory, anti-allergic, antioxidant, anti-diabetic, anti-viral, anti-cancer, anti-leprosy, and antimicrobial effects19. These compounds collectively contribute to the pharmacological versatility of neem. Similarly, Gupta et al. have extensively documented the biological and pharmacological activities of neem compounds, showcasing their antioxidant, anti-inflammatory, antiarthritic, antipyretic, antiviral, spermicidal, hypoglycemic, anthelminthic, antigastric ulcer, and antitumor properties20.
Due to the Surface Plasmon Resonance phenomena in silver nanoparticles, this colour change has occurred as a result of the excitation of free electrons in nanoparticles21. No further color changes were observed after 24 hrs, which indicated the completion of the reduction process. An absorption peak of 450nm in silver nanoparticle spectra had been reported22. A peak near 400 nm corresponds to smaller size nanoparticles whereas peak at longer wavelength represents larger sizes shown in previous reports23. The peaks at the base widened, demonstrating polydispersity of the nanoparticles24. The present study showed that the exposure of secondary metabolites present in extracellular extract of A. indica reduces the metallic ions into nanosized metallic particles. The result confirms the nanoparticle nature of the synthesized crystals and supports the observation obtained from the UV-Vis spectroscopy, regarding the phytofabrication of colloidal particles from silver ions in the presence of aqueous leaf extracts of A. Indica. Using an aqueous extract of C. papaya leaves, AgNPs were synthesized within the size range of 12–83 nm25.
FTIR spectrum of AI extract with a band at 3437 cm-1 corresponds to O-H stretching hydrogen bonded phenols and alcohols. The absorption band 2929 and 2918 cm-1 in AI extract and AI-AgNPs corresponds to C-H stretching vibrations of aromatic alkyl functional groups due to the presence of the polyphenolic compound. The peaks near 1619.30, 1420.61 in AI and 1627.04, 1441.14 cm − 1 in AI-AgNPs depict aromatic compounds, proteins, and metabolites that play role for capping and stability of the AgNPs. The strong bands at 1049 in AI and 1075 cm − 1 in AI-AgNP are due to ether linkages and suggest the presence of flavanones adsorbed on the surface of metal nanoparticles. The peak at 618.90 in AI extract 670.80 cm-1 represents C-H stretching vibrations of alkyl functional groups. Similar findings were recorded by using of natural products as reducing agents in the synthesis of silver nanoparticles26. FT–IR results show that some of the bioorganics from the A. indica extract strongly coated or capped the nanoparticles.
The IR spectra of the sample unequivocally demonstrate that the leaf extract serves as both a reducing and a stabilising agent. The results are in good correlation with that of the other reports27. Certain flavonoid compounds are known to have hydroxyl groups (-OH). The group is negatively charged so that it can attach to the Ag + surface and reduce the ion. It is also known that the –OH group in flavonoids is also involved in the reduction of the Ag + ion 28. This confirmed that various functional groups and moieties of the organic compounds present in the A. indica leaf extract contributed to the reduction of the Ag(I) ions (leading to the AgNPs synthesis) and the capping of the resultant nanostructures (providing their stability in time and functionality, likely related to their biocompatibility and biological activity.
The XRD spectra of the nanoparticles derived from A. indica leaf extract independently verified the synthesis of AgNPs. The strong, distinct peaks clearly showed that the AgNPs made from the reduction of silver ions with the aqueous extract of A.indica were extremely crystalline (Table 1, Fig. 5a). Remarkably, the XRD pattern also shows a few more unidentified peaks. Even though the findings show that silver is the major phase, there may be other crystalline phases from the leaf extract's inorganic moiety that are present and contribute to the XRD pattern as contaminants. As an illustration, several recent studies on the environmentally friendly synthesis of AgNPs utilising plant extracts have shown the existence of similar additional peaks in the AgNPs' XRD pattern29. According to Awwad et al. (2013), these peaks were caused by crystalline impurities on the surface of the nanoparticles created from the leftover leaf extracts30.
Particle size distribution data for A. indica silver nanoparticles indicates a concentration of particles in smaller size ranges, notably within the 0–15 and 35–45 intervals, implying a prevalence of smaller nanoparticles. This observation holds significance as the antimicrobial efficacy of silver nanoparticles is intricately linked to their size, suggesting potential advantages in leveraging these smaller particles for antimicrobial applications31.
The significant antioxidant potential of AI-AgNPs was evaluated by 2, 2-Diphenyl-1-Picryl hydrazyl (DPPH) radical scavenging assay in terms of their capacity to donate hydrogen or to scavenge free radicals. It was performed to study the antioxidant potentials of AI extract and AI-AgNPs solution. Phenolic and flavonoid content have been showed to contribute significantly to antioxidant activity32. The results of the FTIR studies in the present research confirm the presence of phytoconstituents with antioxidant properties. The reactivity of the extracts prepared from A.indica was analyzed with 2, 2-Diphenyl-1-Picryl hydrazyl, a stable free radical reduced by accepting hydrogen or electron from the donor molecule. Plant polyphenolic chemicals have been found to have potent antioxidant capabilities that protect cells from the oxidative stress free radicals scavenging property33. The improved antioxidant effects of A.indica identified have been the subject of similar research34.
Antimicrobial resistance in microbes has caused the spread of microbes that are resistant to effective drugs, it inevitable to search for new antimicrobial agents with strategies to reduce emergence and rate of resistance of pathogenic organism. Small nanoparticles can interfere with the respiratory chain dehydrogenases and induce generation of intracellular ROS, which can cleave DNA35 and diminish bacterial life. The significant impact of physicochemical properties of AgNPs on bacterial cells (MRSA and E.coli) was observed36.
Proteomics, which is based on mass spectrometry (MS), serves as one of the omics techniques that is gaining increasing application in clinical practice since it enables phenotypic characterization of the dynamic functional status of the organism. The MALDI-TOF MS is the simplest form of mass spectrometry, successfully applied to effectively detect bacteria species. Using the MALDI-TOF MS approach, it was confirmed that the Staphylococcus, and Escherichia bacteria was used in the determination of MIC.
The findings are consistent with the results obtained in previous studies37,38. The results showed that the A. indica leaf extract and the phytofabricated AI-AgNPs have a good antibacterial activity against the tested bacteria strains. The phytofabricated AI-AgNPs indicated a higher antibacterial activity than that determined for AEAIL. The antibacterial effect of the produced AgNPs could be explained by considering their small size and an extremely large surface area-to-volume ratio, providing better conditions for interacting with bacterial cells39. Nano sized silver nanocomposites promoted bacterial cell wall interactions and permeability, therefore enhanced antibacterial activity by leakage of cellular contents and also disturb the cell function by interacting with amino acids and enzymes, causing generation of reactive oxygen species (ROS) and destruction of bacterial deoxyribonucleic acid40. FTIR study also revealed that particular phytoactive compounds of A. indica covered the AgNPs has inherent strong antibacterial activities. Indeed, literature reported that many phytochemicals (polyphenols, alkaloids, terpenoids, etc.) exhibit antimicrobial properties41.
Furthermore, the AgNPs can more effectively penetrate the bacteria cell membranes and interact with the intercellular biomolecules such as DNA and proteins, disturbing the replication, permeability, respiration, loss of the cell viability and, in the extreme cases, to the cell death42. Hence, the present study results revealed the efficiency of AI-AgNPs with more promising biological applications in future therapeutic approaches.
The leakage of protein represents the effect of antimicrobial compound on the bacterial cell wall lysis. Protein concentration in the supernatant at 12 h after incubation, from E. coli cells treated with AI-AgNPs was considerably increased as compared to MRSA which is potentially attributable to distinctions in their cell walls thickness43. Our results indicate that biosynthesized AgNPs exert potent effects by inducing oxidative stress on bacterial cells, disrupting protein content. The treated cultures were found to have increased protein concentrations which suggest that biogenic AgNPs alter the structure and permeability of bacterial cell, inactivate membrane-bound enzymes and influence membrane functions and membrane integrity by interacting with disulphide bonds. Nano silver composites generate free radicals, induce damage to intracellular machinery and activate the apoptosis pathway44,45. Our results of FTIR also recorded the presence of phytochemicals in plant extract which interact with bacterial enzymes to inhibit cell wall synthesis46.