5.1. Structural Analysis
Figure 1 displays the XRD results of NPs within 2θ range of 20° to 70°. The spectrum reveals distinct diffraction peaks at 2θ values of 37.48°, 43.49°, and 62.94°. These prominent peaks correspond to the (111), (200), and (220) Brags planes, respectively. These diffraction peaks, corresponding to their respective hkl planes, match the reference data from JCPDS card no. 01-073-1519, confirming the synthesized NiO NPs exhibit a cubic crystal structure, as shown in the inset of Fig. 1. The observed sharpness of the peaks indicates high crystallinity and well-defined crystal planes [15]. The absence of any additional impurity peaks further supports the purity of the synthesized material, suggesting that the synthesis process was successful, and the NPs are free from contamination. The crystallite size of the NiO NPs yielded 22.37 nm calculated by Debye-Scherrer equation. This relatively small crystallite size indicates that the nanoparticles have a high degree of crystallinity and a well-defined structure. The small size of the crystallites is likely to enhance the biological activity of the NiO NPs due to the increased surface area and reactivity [16]. All lattice parameters are tabulated in Table 1. Overall, these results indicate the successful preparation of high-quality NiO NPs with a cubic structure, which is expected to positively impact their biological activity.
Table 1
Lattice constant, crystallite size, and X-ray density of NiO
Sample | Lattice Constant (Å) a = b = c | Crystallite Size (nm) | X-ray Density (g/ cm3) |
NiO | 4.18 | 22.37 | 6.82 |
5.2. Morphological Study
The morphology of the prepared sample was extensively studied by FESEM at 20,000x as illustrated in Fig. 2(a). These visuals revealed that the NiO NPs exhibit significant agglomeration, with small particles forming spherical shapes with pronounced porosity within the sample, and numerous visible voids, indicating a high degree of porosity. To quantify the particle size, ImageJ software was employed, yielding an average grain size of 65 nm. This size estimation supports the observed morphology and confirms the presence of small, spherical particles with notable agglomeration and porosity. Further confirmation of the particle morphology was obtained through Scanning Transmission Electron Microscopy (STEM). The STEM analysis provided a clearer view of the spherical grains, confirming an average grain size of 40 nm as illustrated in Fig. 2(b). The STEM images corroborated the FESEM findings, showing a high level of porosity and spherical particle shapes, enhancing the overall understanding of the material’s morphology.
The EDX spectra of NiO NPs is depicted Fig. 2(c). The analysis reveals that the primary elements are nickel and oxygen, confirming the nickel oxide composition. Prominent peaks for nickel and oxygen validate the material's identity. Small peaks at 0.25 and 0.5 keV are due to C and O, while the main peaks at 0.90, 7.5, and 8.3 keV correspond to nickel. Minor peaks at 1.1, 1.8, and 2.6 keV indicate the presence of sodium, silicon, and chlorine. The elemental composition is summarized in Table 2. Carbon, chlorine, silicon, and sodium are likely residuals from the synthesis using plant materials.
Table 2
Elemental composition of prepared NPs
Sample | Wt.% Ni | Wt.% O | Wt.% C | Wt.% Cl | Wt.% Si | Wt.% Na |
NiO | 72.5 | 21.2 | 3.4 | 2.2 | 0.6 | 0.6 |
5.3. FTIR Analysis
FTIR works by using IR photons to vibrate atoms within chemical bonds, causing vibrational transitions at specific energy levels. Molecules absorb infrared light at particular wavelengths, and the resulting absorption spectrum identifies functional groups and compounds. Contaminants also emit unique IR bands, allowing for impurity detection. Fourier transformation is used to decode individual frequencies, and a computer processes the data to provide spectral information for analysis [17]. The FTIR spectrum of NiO NPs as illustrated in Fig. 3 exhibits multiple distinctive peaks. The prominent peak observed at 3701 cm− 1 is ascribed to the presence of water molecules. A broad absorbance band spanning the range of 3565 − 3398 cm− 1 indicating the stretching vibrations of (O-H) groups [18]. The peak around 1655.81 cm− 1 is attributed to the bending vibration of O-H, indicating the existence of H2O molecules in the sample. The peak at 1383.31 cm− 1 is due to the nitro group (NO₃), which originate from the precursor used in the synthesis process. Two prominent peaks at 1104.24 cm− 1 and 1068.89 cm− 1 are designated to the robust stretching vibrations of the Ni–O bond, signifying the formation of nickel oxide. Additionally, the peak at 898.85 cm− 1 represents the stretching vibrations of Ni–O [19]. Further, the bands at 545.2 cm− 1 and 447.64 cm− 1 correspond to the bending and wagging vibrations of Ni–O, confirming the presence of NiO in the sample [20].
5.4. Thermogravimetric Analysis
Thermogravimetric analysis (TGA) monitors a sample's mass change with temperature and time, providing accurate compositional data for quality and process control. However, it cannot identify volatile compounds without additional analytical tools. TGA can also determine oxidation induction time (OIT) by heating a small sample (typically < 10 mg) in oxygen at an isothermal temperature, generally around 200°C, and observing the mass gain when oxidation occurs [21]. The TGA curve of NiO NPs presented in Fig. 4 shows two significant stages of weight loss. The first sharp weight loss occurs between 160°C and 275°C, which corresponds to the evaporation of physically absorbed water molecules. This is typical for samples synthesized using plant-based methods, where moisture remains after synthesis. The second stage, occurring between 276°C and 540°C, reflects a gradual weight loss due to the removal of chemically adsorbed water and the decomposition of organic compounds. These organic compounds are likely residuals from the plant extract used in the green synthesis process. After 540°C, the weight stabilizes, indicating that all volatile and organic components have been fully decomposed, leaving behind the final, thermally stable NiO product [22]. This final stage confirms the complete formation of the NiO NPs, highlighting their purity and thermal stability.
5.5. Enzymatic Inhibition Activity of Alpha-Amylase
The inhibitory effect of NiO NPs on alpha-amylase was evaluated by pre-incubating various concentrations of NiO NPs (100–500 µg/mL) with 0.5 mL of phosphate buffer containing 500 µL of alpha-amylase enzyme (1 mg/mL) at 37°C for 10 min [23]. Subsequent to the pre-incubation phase, 1 mL of starch solution was introduced, and the resultant mixture was subjected to an additional incubation period for 5 min at 37°C. The enzymatic reaction was subsequently terminated by the addition of NaOH and DNS, followed by heating the mixture for 5 min at 100°C. Upon reaching room temperature, the absorbance of the solution was quantitatively measured at a wavelength of 540 nm as illustrated in Fig. 5(a). Furthermore, Acarbose was employed as a standard inhibitory agent, yielding an IC50 value of 0.029 µg/mL. The inhibition activity of NiO NPs was measured as the percentage of enzyme activity inhibited, with IC50 indicating the concentration required to inhibit 50% of enzyme activity, as illustrated in Fig. 5(b). The results showed that the inhibition of alpha-amylase by NiO NPs was directly proportional to their concentration: higher concentrations led to greater inhibition and lower absorbance values. The absorbance values of NiO NPs at 10, 20, 40, 60, 80, and 100 µg/mL were 0.094, 0.078, 0.044, 0.012, and 0.002, respectively. Corresponding percent inhibition activities were 67%, 72%, 84%, 95%, and 99%. All the parameters are summarized in Table 3. The IC50 value for NiO NPs, determined from the data, was 1.18 µg/mL. This indicates that NiO NPs are effective inhibitors of alpha-amylase, with their inhibition potential increasing with concentration.
Table 3
Percentage inhibition of NiO NPs
No. | Concentration(µg\mL) | Absorbance | %inhibition | IC50 |
1 | 10 | 0.094 | 67.5 | 1.18 |
2 | 20 | 0.078 | 72.6 |
3 | 40 | 0.044 | 84.5 |
4 | 60 | 0.012 | 95.3 |
5 | 80 | 0.002 | 99.1 |
5.6. Antioxidant
Antioxidants are substances capable of neutralizing free radicals and reducing their formation. The antioxidant activity of prepared NPs was evaluated using the ABTS free radical and results were compared with standard ascorbic acid as shown in Fig. 6(a-b). As various concentrations of NiO NPs were tested for antioxidant activity against ABTS free radicals [24]. A clear trend emerged, as the concentration of NiO NPs increased from 20 to 80 µL, the ABTS radical scavenging activity also increases, ranging from 44–78%, respectively. This suggests that a higher concentration of NiO NPs provides a greater number of surface binding sites for free radicals, thereby enhancing their scavenging potential. A similar trend was observed with ascorbic acid, where at 80 µL it achieved a 58.5% scavenging activity compared to the 78.4% displayed by NiO NPs at the same concentration. These results highlight the superior ABTS•+ radical scavenging activity of NiO NPs over ascorbic acid. Additionally, the lower IC50 value and higher correlation coefficient further support the enhanced antioxidant potential of NiO NPs compared to the standard. All the calculated parameters are tabulated in Table 4. Furthermore, the % of radical reduction (%RSA) was calculated based on the decolorization of the ABTS solution, which occurs due to the reaction between the radicals and the antioxidants in the nanoparticles. The IC50 value, indicating the concentration required to achieve 50% inhibition, was derived from these measurements. NiO NPs were tested at concentrations ranging from (20–100 µg/mL). Higher concentrations of NiO NPs resulted in a greater decrease in ABTS absorbance, reflecting increased radical neutralization activity [25–27]. The absorbance values for NiO NPs at different concentrations were 0.12, 0.090, 0.069, 0.046, and 0.019 for 20, 40, 60, 80, and 100 µg/mL, respectively. The corresponding percent reduction values were 44%, 58%, 68%, 78%, and 91%. The IC50 value for NiO NPs was calculated to be 28 µg/mL, indicating their effective concentration for 50% radical inhibition.
Table 4
Absorbance %RSA and IC50 reading of NiO NPs
No. | Concentration (µg\mL) | Absorbance | %RSA (Å-A\Å)100 | IC50 |
1 | 20 | 0.12 | 44 | 28 |
2 | 40 | 0.090 | 58 |
3 | 60 | 0.069 | 68 |
4 | 80 | 0.046 | 78 |
5.7. Iron Chelating Activity
The iron chelating activity of NiO NPs was evaluated using a method based on the reduction of the ferrous ion-ferrozine complex [25] and shown in Fig. 7. Ferrozine forms a red-colored complex with Fe²⁺ ions and the reduction in this color measured by the absorbance at 517 nm, indicates the iron chelating activity of the NPs. The IC50 value for NiO NPs, representing the concentration required to achieve 50% inhibition of the ferrozine-Fe²⁺ complex formation, was calculated to be 23 µg/mL. All the calculated values at various conditions are given in Table 5.
Table 5
Fe+ percentage inhibition of NiO NPs
No. | Concentration (µg\mL) | Absorbance | %Fe+ 2 | IC50 |
1 | 10 | 0.170 | 40 | 23 |
2 | 20 | 0.142 | 50 |
3 | 40 | 0.110 | 61 |
4 | 60 | 0.078 | 72 |
5 | 80 | 0.044 | 84 |
5.8. Antibacterial activity
The antibacterial activity of NiO NPs was evaluated against both Gram-positive bacteria (S. pyogenes, S. aureus) and Gram-negative bacteria (P. aeruginosa, K. pneumoniae, S. marcescens). The results represented by zones of inhibition (in mm) are shown in Fig. 8, while Table 6 enlists the inhibition zone measurements for both the NiO NPs and conventional antibiotics. It was observed that the synthesized NiO NPs were particularly effective against Gram-positive bacteria, showing inhibition zones of 18 mm for S. pyogenes, and 13 mm for S. aureus which were larger compared to those seen for Gram-negative pathogens. This indicates that Gram-negative bacteria exhibited greater resistance to NiO NPs than Gram-positive strains. The antibacterial activity of the nanoparticles depends on various factors, including the specific bacterial species, particle size, stability, and the concentration within the growth medium. The nanoscale pores in the outer membranes of bacterial cells allow for greater interaction between the NPs and pathogens. In this study, NiO NPs synthesized using Piper nigrum extract had a very small particle size which plays a critical role in their antibacterial efficacy. The antibacterial mechanism of the NiO NPs is likely attributed to electrostatic interactions between the positively charged nickel ions (Ni²⁺) and the negatively charged bacterial cell membranes. This interaction facilitates the release osf Ni²⁺ ions, which penetrate the bacterial cell wall, damaging essential biomolecules such as DNA and proteins, disrupting mitochondrial function, and interfering with electron transport, ultimately leading to cell death [28–30]. The calculated parameters are tabulated in Table 6.
Table 6
Antibacterial activity and inhibition zone of NiO NPs
Bacteria | Antibiotics | Diameter(nm) of inhibition zones concentrations (5 mg/50 mL) 5 10 30 50 100 |
S.aru | 30 ± 01 | 7.33 ± 1.247 | 27 ± 2.054 | 26.3 ± 2.867 | 15.3 ± 2.054 | 13 ± 2.449 |
S.pyo | 20 ± 2.2 | 13 ± 2.054 | 14 ± 2.449 | 14 ± 2.449 | 16.3 ± 3.299 | 18.3 ± 2.867 |
S.mre | 20 ± 4.1 | 8.33 ± 0.471 | 11 ± 1.635 | 11.3 ± 1.243 | 17.3 ± 1.249 | 16 ± 1.642 |
K.pne | 28 ± 1.2 | 14 ± 1.381 | 14.3 ± 2.494 | 13 ± 2.449 | 14.6 ± 3.299 | 15.3 ± 3.681 |
P.se | 30 ± 3.2 | 9.3 ± 2.054 | 2.6 ± 1.632 | 11.6 ± 1.247 | 17 ± 1.632 | 13.6 ± 1.246 |