Green Synthesized ZnO Nanoparticles: Characterization and Evaluation of Antimicrobial and Cytotoxic Potentials for Pharmaceutical Applications

Nanoparticles prepared by green synthesis  using plants, bacteria, fungi, seaweed, polysaccharides, biodegradable polymers, plant- derived materials and algae are widely used in an ecofriendly manner. Green synthesis has been considered as one of the promising method for synthesis of nanoparticles they are simple, safe, cost effective ecofriendly, and do not involve hazardous chemicals, and they have no contaminants and by-products.  Plant extracts are widely used for the synthesis of metal and metal oxide nanoparticles due to their ability to reduce and stabilize metal nanoparticles using simple synthesis methods. [1-4].  ZnO nanoparticles have great attention due to their unique optical and chemical behaviors and used in various cutting edge applications like electronics, communication, sensor, cosmetics, environmental protection, biology and medicinal industry. Recently ZnO nanoparticles has excellent potential in biological applications like biological sensing, biological labeling, gene delivery, drug delivery and nano-medicine along with its antibacterial, antifungal, acaricidal, pediculocidal, larvicidal and anti-diabetic activities [5-7]. Nanoparticles can be used as antifungal agents and can help to overcome hurdles of fungal disease management posed by the development of resistance to conventional fungicides. ZnO nanoparticles have strong activity against these microorganisms [8]. For the preparation of ZnO nanoparticle we used plant leaf extracts of Aloe vera. For biosynthesis process the aloe vera plant extract was used as a reducing agent.  The Aloe vera plant have tremendous use for its health, beauty, medicinal and skin care properties. [9-10] Aloe Barbadensis is the scientific name of Aloe Vera and contains numerous potentially active constituents: vitamins, enzymes, minerals, sugars, lignin, saponins, salicylic acids and amino acids and other bioactive compounds with emollient, purgative, antimicrobial, anti-inflammatory, anti-oxidant, aphrodisiac, anti-helminthic, antifungal and antiseptic . The plant has potential to cure sunburns, burns and minor cuts, and even skin cancer. [11-13] In the present study, zinc oxide (ZnO) nanoparticles were synthesized via an eco-friendly green synthesis approach using Aloe vera leaf extract. The structural, optical, and morphological characteristics of the synthesized nanoparticles were investigated using X-ray Diffraction (XRD), UV–Visible Spectroscopy (UV-Vis), and Scanning Electron Microscopy (SEM). Recent research highlights the potential of ZnO as a promising candidate for cancer therapy [14]. In addition, the antibacterial efficacy and cytotoxic properties of the nanoparticles were systematically evaluated to assess their pharmaceutical applicability.

1. MATERIALS AND METHODS

2.1 Materials

Zinc Nitrate (Zn (NO3)₂) were purchased from Kelvin Lab, Alappuzha, kerala, India. Aloe Barbadensis (Aloe Vera), collected from the local areas of Thiruvalla, kerala, India.

2.2 Extract preparation

Fresh Aloe vera leaves were thoroughly washed with distilled water to eliminate dust, dirt, and surface contaminants. The cleaned leaves were then air-dried at room temperature to remove residual moisture. Subsequently, the outer green rind was carefully removed, and the inner transparent gel was extracted. The obtained gel was crushed and homogenized using a sterile mortar and pestle to obtain a consistent pulp. Approximately 100 mL of deionized water was added to the gel and transferred into a 250 mL glass beaker. The resulting mixture was subjected to continuous stirring using a magnetic stirrer at 60?°C for 2 hours to facilitate the extraction of bioactive components. After the heating process, the mixture was allowed to cool to ambient temperature and was then filtered using Whatman filter paper. The clear filtrate obtained was collected and stored as the Aloe vera leaf extract, which served as the reducing and stabilizing agent for the green synthesis of zinc oxide nanoparticles.

2.3 Green synthesis of zinc oxide nanoparticles

One-third of the prepared Aloe vera extract was taken, and 5 grams of zinc nitrate [Zn(NO?)?] was added to it under constant stirring. The mixture was stirred continuously at 80?°C for 2 hours using a magnetic stirrer to ensure uniform mixing and initiate the reaction. Following this, the resulting solution was transferred to a crucible and placed in a muffle furnace at 200?°C for 3 hours and 30 minutes. During the thermal treatment, the solution gradually transformed into a pale yellow powder, indicating the formation of zinc oxide nanoparticles. The resulting powder was collected and stored for further characterization and analysis.

2. RESULTS AND DISCUSSION

3.1 X-RAY Diffraction analysis

X-ray diffraction (XRD) analysis is a reliable technique for examining the structural characteristics of synthesized materials. The XRD pattern of the prepared sample is presented in Figure 1. The appearance of broad diffraction peaks indicates the formation of nanoparticles, as supported by previous studies [15]. The average crystallite size of the ZnO nanoparticles was estimated using the Debye–Scherrer equation [16]:

Here, D represents the crystallite size, K is the shape factor (typically ~0.9), λ is the X-ray wavelength (1.54 Å for Cu Kα radiation), β is the full width at half maximum (FWHM) of the peak in radians, and θ is the Bragg diffraction angle.The X-ray diffraction (XRD) pattern of the synthesized ZnO nanoparticles reveals distinct broad diffraction peaks located at approximate 2θ values of 9.05°, 13.40°, 24.75°, and 34.24°. These peaks correspond to interplanar spacings (d-values) of 9.7604 Å, 6.6012 Å, 3.5947 Å, and 2.6171 Å, respectively, as calculated using Bragg's law. The broad nature of the peaks is indicative of nanoscale crystallinity, which is a common characteristic of particles with dimensions in the nanometer range. The average crystallite size was estimated using the Debye–Scherrer equation, yielding an approximate particle size of 35.9 nm. Detailed calculations of the crystallite size corresponding to each diffraction angle are presented in Table 1, providing insight into the crystallographic characteristics of the ZnO nanoparticles.

Table 1.   Particle Size of Corresponding to Each Angle

1. 2. UV- Visible spectroscopy

UV–Visible spectroscopy serves as a powerful analytical tool for the investigation and identification of nanomaterials, particularly for evaluating their optical properties [17]. In this study, the optical behavior of the green-synthesized ZnO nanoparticles was examined using UV–Visible absorption spectroscopy, and the corresponding absorption spectrum is presented in Figure 2(a). The absorbance versus wavelength plot reveals two prominent peaks, which can be attributed to the surface plasmon resonance (SPR) phenomena typically observed in nanoscale materials. These peaks provide insight into the electronic transitions occurring within the ZnO nanoparticles.To determine the optical band gap energy, the Tauc plot method was employed by plotting (αhν)2against photon energy (hν, where α is the absorption coefficient and hν is the photon energy. The band gap energy was estimated by extrapolating the linear portion of the plot to the x-axis (where α=0)[12]. From this analysis, the optical band gap of the ZnO nanoparticles was found to be approximately 3.62 eV. This value is consistent with the known band gap range for ZnO nanostructures and reflects the quantum confinement effect associated with their nanoscale dimensions.

Figure 2 (a) UV Absorption Spectra                  Figure 2 (b) Tauc Plot

3.3 Scanning Electron Microscope

The surface morphology and particle size of the synthesized ZnO nanoparticles were investigated using Scanning Electron Microscopy (SEM). Figure 3 presents the SEM micrographs of ZnO nanoparticles prepared via green synthesis using Aloe vera extract. The images reveal that the nanoparticles predominantly exhibit a tubular or rod-like morphology, indicative of anisotropic growth facilitated by bioactive compounds present in the plant extract.Furthermore, the SEM analysis indicates the formation of secondary ZnO structures with an average size of approximately 1 μm. These larger particles appear to be agglomerates composed of primary ZnO nanocrystallites, which are estimated to be around 20 nm in size. The average crystallite size was determined using the line-intercept method applied to the high-magnification SEM images. This hierarchical structure, consisting of nanoscale building blocks forming micron-sized assemblies, is characteristic of ZnO synthesized through bio-mediated routes and has implications for enhanced surface activity in pharmaceutical and optoelectronic applications.

3.3 Scanning Electron Microscope

The surface morphology and particle size of the synthesized ZnO nanoparticles were investigated using Scanning Electron Microscopy (SEM). Figure 3 presents the SEM micrographs of ZnO nanoparticles prepared via green synthesis using Aloe vera extract. The images reveal that the nanoparticles predominantly exhibit a tubular or rod-like morphology, indicative of anisotropic growth facilitated by bioactive compounds present in the plant extract.Furthermore, the SEM analysis indicates the formation of secondary ZnO structures with an average size of approximately 1 μm. These larger particles appear to be agglomerates composed of primary ZnO nanocrystallites, which are estimated to be around 20 nm in size. The average crystallite size was determined using the line-intercept method applied to the high-magnification SEM images. This hierarchical structure, consisting of nanoscale building blocks forming micron-sized assemblies, is characteristic of ZnO synthesized through bio-mediated routes and has implications for enhanced surface activity in pharmaceutical and optoelectronic applications.

Figure 3. SEM Micrographs of the ZnO nanoparticles

3.4: Photoluminescence (PL) Analysis

Photoluminescence characterization was conducted to evaluate the optical emission behaviour of the synthesized ZnO nanoparticles under various excitation conditions. Specifically, the PL spectra were examined with respect to excitation wavelength dependence, particle size variation, and the influence of reducing agents used during synthesis. The corresponding emission spectra are depicted in Figure. 4. PL studies were carried out using excitation energies corresponding to below band gap, near band gap, and above band gap regimes. The emission spectra exhibited distinct peaks, with notable shifts in peak positions and intensities depending on the excitation wavelength. These spectral shifts ranging over several nanometers are attributed to variations in the defect states and recombination mechanisms associated with different excitation conditions. At lower excitation energies, a red-shift in the emission was observed, which is likely due to increased contributions from defect-related transitions, particularly those associated with oxygen vacancies and zinc interstitials. These low-energy excitations tend to promote recombination through defect states within the band gap, influenced by interstitial site occupation and lattice strain within the nanoparticles [18]. The observed excitation energy-dependent variation in both emission wavelength and intensity confirms the presence of structural defects and interstitial sites within the ZnO nanocrystals. Such defects play a crucial role in tuning the optical properties of the material, which is particularly significant for potential applications in optoelectronic and biomedical devices.

Figure 4. Emission Spectra of Sample Studied at Different Excitation Energy.

3.5 Antimicrobial Activity

The antimicrobial efficacy of the ZnO nanoparticles synthesized using Aloe vera leaf extract was investigated against selected microbial strains using the agar well diffusion method [19]. For comparative analysis, ZnO nanoparticles were also synthesized using Azadirachta indica (Neem) and Ocimum tenuiflorum (Thulsi) extracts, following the same green synthesis protocol as employed for the Aloe vera-derived sample.The antimicrobial performance of these green-synthesized ZnO nanoparticles was tested against three representative microbial strains: Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Candida albicans—representing Gram-negative, Gram-positive, and fungal pathogens respectively. The zone of inhibition values for each test organism are summarized in Table 2, and corresponding agar plate images are shown in Figure 3.Comparative analysis indicates that ZnO nanoparticles synthesized with Aloe vera extract exhibit significantly higher antimicrobial activity against all tested pathogens compared to those synthesized with Neem and Thulsi extracts. The superior inhibition effect is likely attributed to the presence of bioactive phytochemicals in Aloe vera, which may enhance the capping, stabilization, and surface reactivity of the ZnO nanoparticles. The pronounced antimicrobial response suggests that Aloe vera-mediated ZnO nanoparticles possess effective bactericidal and fungicidal properties. This makes them promising candidates for pharmaceutical and biomedical applications as broad-spectrum antimicrobial agents.

Figure 5: Antimicrobial activity of   ZnO Nanoparticle

Table 2. Antimicrobial Result of Prepared Sample