Synthesis of Cu₄O₃ nanoparticles using pumpkin seed extract: Optimization, antimicrobial, and cytotoxicity studies

2.1 Materials

Pumpkin seeds were purchased near the JNU Market (Jaipur, Rajasthan, India). Copper sulfate was acquired from Merck and Sigma-Aldrich (India).

2.2 Preparation of pumpkin seed extract

The collected seeds were properly washed and dried. The dried seeds were then ground into fine powder using a suitable grinder. Subsequently, 150 mL of double-distilled water was added to 10 g of crushed seed powder in a 250 mL round-bottom flask. The solution was refluxed for 45 min. After cooling to room temperature, the extract was filtered using Whatman No. 1 filter paper for further processing.

2.3 Biogenic synthesis of Cu₄O₃ NPs

Copper oxide nanoparticles (Cu₄O₃ NPs) were produced using pumpkin seed extract by biomimetic synthesis. At room temperature, 20 mL of pumpkin seed extract was added to 90 mL of a 5 mM copper sulfate (CuSO₄) solution. To ensure proper mixing, the mixture was continually swirled for 20 min with a magnetic stirrer. The mixture was then refluxed at 97°C for 8 h, stirring constantly throughout. After reflux, the reaction mixture was allowed to come down to room temperature for 30–45 min. Finally, the mixture was dried in a hot air oven set to 60–90°C for 24 h before being stored in a sealed glass bottle (Scheme 1). To optimize the yield of NPs using the BBD, we carried out additional experiments focusing on three key factors: the concentration of CuSO₄ (in mM), the reaction temperature (in °C), and the volume of plant extract (in mL). For each type of plant extract, we prepared 15 conical flasks, labeled from 1 to 15. The yield of Cu₄O₃ NPs was then calculated by measuring the absorbance at a wavelength of 332 nm.

Scheme 1

Formation of Cu₄O₃ NPs from pumpkin seed extract.

2.4 Characterization of biogenic Cu₄O₃ NPs

The synthesis of Cu₄O₃ NPs was verified using various physicochemical characterization techniques. UV–visible spectroscopy analysis was performed using a UV–Vis spectrophotometer (UV-1800, Shimadzu, Japan) to obtain the absorption spectra. Fourier-transform infrared (FTIR) spectroscopy was carried out using a Bruker Alpha FTIR spectrometer (Bruker Optik GmbH, Germany) to identify the functional groups based on the characteristic stretching frequencies in the range of 4,000–400 cm⁻¹. The KBr pellet method was used for sample preparation. FTIR spectroscopy was utilized to identify the functional groups in the bioactive constituents that contributed to the reduction, capping, and stabilization of Cu₄O₃ NPs synthesized using pumpkin seed extract.

To determine phase purity and the crystallite size, X-ray diffraction (XRD) analysis was conducted using a PANalytical X’Pert PRO diffractometer (Malvern Panalytical, Netherlands) with Cu Kα radiation (λ = 1.54 Å). The diffraction peaks were matched against standard JCPDS reference patterns. The XRD profile exhibited sharp peaks, confirming the crystalline nature of the NPs. Thermogravimetric analysis (TGA) was performed on a TGA 8000 (PerkinElmer, USA) instrument to assess thermal stability. A 2 mg sample was heated from 30 to 600°C at a rate of 10°C min⁻¹ under a nitrogen atmosphere.

The morphology and elemental composition of the NPs were analyzed using a field-emission scanning electron microscope (FE-SEM; Nova NanoSEM 450, FEI, USA) equipped with energy-dispersive X-ray spectroscopy (EDX). High-resolution transmission electron microscopy (HR-TEM) was performed using a JEOL JEM-2100 (JEOL Ltd., Japan), operated at an accelerating voltage of 200 kV. The samples were drop-cast on carbon-coated copper grids for analysis.

2.5 Experimental design and data analysis

The BBD, a well-established statistical experimental design within the framework of RSM [73], was utilized to evaluate the effects of three key input variables – CuSO₄ concentration (mM), reaction temperature (°C), and extract volume (mL) – on the yield (%) of Cu₄O₃ NPs. BBD was selected for its efficiency and cost-effectiveness, requiring only 15 experimental runs to systematically explore the interactions between the variables. The experimental data were analyzed and fitted into a second-order polynomial equation model using Minitab software (Minitab^® LLC, Pennsylvania, USA; Version 22.2.2.0, Academic Free Trial) [74], enabling the assessment of both individual and interactive effects of the variables on the NP yield. The BBD is known for its unique arrangement of experimental points, with three levels assigned to each factor, coded as −1, 0, and +1. This design is particularly advantageous for multiple regression analysis because it allows for efficient exploration of complex interactions using a minimal number of experiments. The model is well suited to approximating quadratic relationships and provides reliable coefficient estimates near the center of the experimental space. However, it is important to note that the precision of these estimates may decrease in the corners of the experimental domain, where there are no design points. Table 1 outlines the factors and levels employed in the BBD for optimizing Cu₄O₃ NP biosynthesis. This design aids in determining optimal conditions for maximizing NP yield and elucidates the interactions between variables, thereby improving the efficiency of NP synthesis.

2.6 Cytotoxicity study

2.7 Antibacterial activity

The antimicrobial efficacy of Cu₄O₃ nanoparticles (Cu₄O₃NPs) produced through biofabrication was assessed against a strain of Bacillus subtilis using the disc diffusion method. The bacterial inoculum was standardized to approximately 1 × 10⁶ CFU/mL using the 0.5 McFarland standard. Nutrient agar plates were prepared, sterilized, and allowed to solidify. Following solidification, the bacterial culture was uniformly swabbed onto the plates. Cu₄O₃NPs were introduced into agar wells at concentrations ranging from 1 to 4 ppm. A control using only solvent (water) was also included. The plates were incubated at 37°C for 24 h without disturbance. After incubation, the diameters of the zones of inhibition were measured using digital calipers. The results were recorded as mean values from three independent replicates. Furthermore, the dimensions, morphology, and surface characteristics of Cu₄O₃NPs were also analyzed to understand their influence on antimicrobial activity.

2.8 Molecular index study

The geometry optimization of the Cu₄O₃ cluster was carried out using DFT as implemented in the Gaussian program [76]. The hybrid RB3LYP functional was employed, known for its good performance with transition metal oxides. A GenECP basis set (specifically, LANL2DZ for Cu to account for relativistic effects and 6-31G(d) for O) was used. The optimization was performed without any symmetry constraints, and the spin multiplicity was set to singlet (S = 0). The final optimized structure exhibited no imaginary frequencies, confirming that a true minimum was obtained. The convergence criteria were satisfied with a very small RMS gradient norm of 0.000011 a.u., and the total electronic energy at the optimized geometry was found to be −1009.95358203 atomic units. The Cu₄O₃ cluster optimized in the D₄h point group was then utilized for frontier molecular orbital (HOMO–LUMO) analysis and the calculation of global reactivity descriptors, including electronegativity, chemical hardness, and chemical softness, to provide insights into its potential biological interactions.

2.9 Statistical design and data processing

The experimental design and statistical analysis were conducted using Minitab^® Statistical Software (Minitab LLC, Pennsylvania, USA; Version 22.2.2.0, Academic Free Trial) [74]. A full quadratic regression model was used to evaluate the influence of independent variables on the response. Analysis of variance (ANOVA) was performed with a 95% confidence level to determine the significance of the model and individual terms. The final regression equation, representing the relationship between variables and response, is presented in Section 3.

3.1 UV–Vis spectroscopy

The color of the solution changed from brownish (Figure 1a) to green during the reaction, indicating the production of Cu₄O₃ NPs, allowing the bioreduction of CuSO₄ ions in the solution to be monitored. A 1 nm resolution UV–Vis spectroscopy at 200–800 nm further validated this transition. Analyzing the UV–Vis spectrum of the pumpkin seed extract reveals characteristic absorption peaks in the ultraviolet range, specifically between 220 and 380 nm. These absorptions are indicative of the presence of phenolic compounds and flavonoids, which are frequently found in plant extracts [77,78]. The UV–Vis spectra of Cu₄O₃NPs showed a distinctive surface plasmon resonance peak at 332 nm, which is indicative of the inherent band gap absorption of Cu₄O₃ brought on by electron transitions from the valence to the conduction band [79,80]. This absorption peak is significantly different from the typical absorption peaks of CuO (300–400 nm) and Cu₂O (250–350 nm). This peak is consistent with the reported interband transition of parahexachloride (Cu₄O₃, a mixed-valence Cu⁺/ Cu 2 + oxide) [81]. The narrow peak width further confirms the formation of Cu₄O₃ NPs without CuO/Cu₂O impurities. This absorption peak is also notably distinct from those typically observed for CuO or metallic Cu, thereby supporting the identification of the product as Cu₄O₃. The sharp peaks also indicate that the particles are nanosized and have a narrow size distribution (Figure 1b). Notably, the UV–Vis spectra of both the freshly prepared and long-term stored Cu₄O₃ NPs were identical, further confirming that no significant changes occurred in their optical properties over time [32]. Briefly, based on the above discussion, the absorption peak in our work was observed at a longer wavelength of 332 nm. This red shift (shift to a longer wavelength) may be attributed to the formation of other metastable and stable copper oxide NPs with different electronic band structures.

Figure 1

(a) Pumpkin seed extract showing a brownish color before reaction; (b) green coloration indicating the biosynthesis of Cu₄O₃ NPs after reaction with CuSO₄ solution. The UV–Vis spectrum (bottom left) of the plant extract shows no characteristic peak in the visible range, while the spectrum of the synthesized Cu₄O₃NPs (bottom right) displays a distinct absorption peak at 332 nm, confirming NP formation.

3.2 FTIR spectroscopy analysis

FTIR spectra were recorded to identify the functional groups involved in the reduction and stabilization of Cu₄O₃ NPs synthesized using pumpkin seed extract (Figure 2a and b). The FTIR spectrum of pumpkin seed extract (Figure 2a) shows a broad band at 3363.49 cm⁻¹, corresponding to the O–H stretching vibration of hydroxyl groups, which are commonly found in alcohols and phenolic compounds. The peaks at 2923.15 and 2853.19 cm⁻¹ are attributed to aliphatic C–H stretching vibrations, indicating the presence of –CH₂ and –CH₃ groups. The peak at 1668.96 cm⁻¹ is attributed to the C═O stretching vibration (amide I), indicating the presence of amide compounds. The peaks at 1543.98 and 1461.84 cm⁻¹ are associated with aromatic C═C stretching vibrations, while the peak at 1406.23 cm⁻¹ is associated with O–H bending vibrations. The prominent band at 1156.40 cm⁻¹ represents the C–O–C stretching vibrations within the polysaccharide. The peaks at 1078.09 and 1023.95 cm⁻¹ are attributed to the C–O stretching vibrations of alcohols and ethers. The bands in the fingerprint region, such as those observed at 859.61 and 576.07 cm⁻¹, are typical features of out-of-plane bending modes and metal–oxygen bonds, respectively. In the FTIR spectrum of the synthesized Cu₄O₃ NPs (Figure 2b), similar functional groups were observed, but the peak positions were significantly shifted, indicating the interaction between the bioactive compounds in the extract and the NP surface. The O–H stretching vibration band appeared at 3328.58 cm⁻¹, and the aliphatic C–H stretching vibration bands were observed at 2959.02 and 2928.00 cm⁻¹, respectively. The C═O stretching vibration (amide I) shifted to 1648.40 cm⁻¹, while the aromatic C═C stretching vibration and O–H bending vibration bands were observed at 1547.83 and 1401.61 cm⁻¹, respectively. The peaks at 1082.30 and 1023.30 cm⁻¹ confirmed the presence of C–O stretching vibrations associated with alcohols, ethers, esters, and carboxylic acids. Importantly, new peaks appeared in the lower wavenumber region at 553.92, 615.51, and 688.03 cm⁻¹, which correspond to Cu–O stretching vibrations, confirming the successful formation of Cu₄O₃ NPs [68]. The FTIR spectra of both the freshly prepared and long-term stored Cu₄O₃ NPs showed similar peak positions and intensities, indicating that no significant chemical changes occurred during storage. This suggests that the stability and surface composition of the NPs were maintained over time [32]. Together, these results suggest that the phytochemicals in the pumpkin seed extract not only play a key role in reducing copper ions but also act as stabilizers, forming a capping layer around the NPs. Additionally, by combining gas chromatography and mass spectrometry to confirm the presence of chemicals in the sample, eight compounds were found in the extract from pumpkin seeds [82], as detailed in Table 2. The NIST database was used for compound identification. These compounds include ethyl hexadecanoate, oleic acid, 9-octadecenoic acid, cis-octadecenoic acid, 9,12-octadecadienoic acid, 1-chloro-7-heptadecene, and 6,11-dimethyl-2,6,10-dodecatriene-1-ol.

Figure 2

(a) FTIR spectrum of pumpkin seed extract, and (b) FTIR spectrum of Cu₄O₃NPs synthesized using pumpkin seed extract.

Table 2

Study on the active components of pumpkin seed extract

Compound	Molecular formula	Molecular formula	Figure
Octadecanoic acid or stearophanic acid	C18H36O2	284
Oleic acid	C18H34O2	282
9-Octadecenoic acid	C18H34O2	282
cis-Vaccenic acid	C18H34O2	282
9,12-Octadecadeinoic acid	C18H32O2	280
9,12-Octadecadeinoic acid	C18H32O2	280
Oleic acid	C18H34O2	282
7-Heptadecene, 1-chloro	C17H33Cl	272
6,11-Dimethyl-2,6,10-dodecatrien-1-ol	C14H24O	208
9,12-Octadecadeinoic acid	C18H32O2	280

3.3 Synthesis of copper oxide NPs using pumpkin seed extract: A mechanistic overview

The synthesis of copper oxide NPs (Cu₄O₃) using pumpkin seed extract involves a multifaceted process driven by the phytochemicals present in the extract, which function as both reducing and stabilizing agents. While the exact reaction mechanisms are complex and may involve multiple simultaneous pathways, a generalized framework of the likely chemical transformations can be proposed based on the existing literature and the bioactive compounds identified in pumpkin seed extract.

3.4 XRD pattern analysis

The XRD patterns of Cu₄O₃ NPs are shown in Figure 3a. The peak position is the same as that of standard Cu₄O_3, and the sharp peak creates a diffraction pattern that forms a crystal structure. The most intense diffraction peak at approximately 2θ ≈ 14° is indexed to the (112) plane (d ≈ 3.19 Å), indicating it is the most preferentially oriented or most abundant plane in the sample. The additional peaks at 2θ values around 28°, 33°, 44°, and 56° are attributed to the (200) (d ≈ 2.75 Å), (004) (d ≈ 2.34 Å), (220) (d ≈ 2.01 Å), and (224) (d ≈ 1.67 Å) planes, respectively. These results are supportive of the standard JCPDS card no. 33-480 and the literature [68], confirming the presence of tetragonal copper oxide paramelaconite. Thus, the well-known Debye–Scherrer equation (D = Kλ/βcosθ) was used to determine the average crystalline size of the Cu₄O₃NPs. In the above equation, D is the particle size (nm), λ is the wavelength of the X-ray source, K is a constant (0.94), β is the peak half-width (rad), and 2θ is the Bragg angle. The high-intensity peak for the analysis indicated a crystalline size of 16 nm in the peak plane of the XRD pattern.

Figure 3

(a) XRD pattern of Cu₄O₃ NPs, matching with JCPDS card no. 33-0480, confirming the crystalline nature of the Cu₄O₃ phase. (b) TGA plot of weight loss versus temperature.

To evaluate the purity of the synthesized Cu₄O₃ NPs, XRD data, EDX, alongside TGA were analyzed. The XRD results confirmed the formation of pure Cu₄O₃ with no detectable impurities, as evidenced by the absence of prominent additional peaks corresponding to other phases or contaminants. However, it is important to note that while the inorganic phase (Cu₄O₃) is highly pure, the NPs may still contain residual organic material from the pumpkin seed extract, which serves as a stabilizing agent. This organic material is not detrimental to the crystallinity of the NPs but contributes to their colloidal stability and prevents agglomeration.

3.5 TGA

TGA provided further insights into the purity of the synthesized Cu₄O₃ NPs. The TGA curve revealed distinct stages of weight loss, which can be attributed to the removal of volatile components and organic residues. In the initial stage (30–150°C), a weight loss of 9.7% was observed, primarily due to the evaporation of water and low-molecular-weight organic compounds. The second stage (150–220°C) showed an additional weight loss of 12.7%, corresponding to the decomposition of more complex organic molecules from the pumpkin seed extract. These two stages collectively indicate that approximately 22.4% of the total weight corresponds to organic material and moisture, leaving behind the inorganic Cu₄O₃ phase. Beyond 220°C, the gradual weight loss up to 450°C (total decrease of 80.17%) is attributed to the continued breakdown of residual organics and the phase transition of Cu₄O₃. Finally, in the temperature range of 450–600°C, an increase in weight of 65% was observed, likely due to oxidation processes leading to the formation of higher copper oxides. These results confirm that the NPs retain some organic content for stabilization, while the residual inorganic phase is predominantly Cu₄O₃, as supported by the XRD data. Thus, the TGA provides quantitative evidence of the high purity of the Cu₄O₃ phase, with controlled retention of organic material to ensure NP stability.

3.6 FESEM analysis

Scanning electron microscopy (SEM) was used to study the surface morphology of the synthesized Cu₄O₃ NPs at different magnifications. At lower magnification, SEM images show a uniform distribution of particles. However, the resolution is not enough to distinguish the boundaries of individual grains. The surface morphology of NPs, including the presence of monodisperse particle clusters and formed aggregates, can be observed using high-magnification imaging. These data imply that gravity causes uniformly distributed particles to initially coalesce into clusters. Based on SEM analysis, the average particle size was found to be about 1 µm (Figure 4a). The elemental composition of the synthesized NPs was analyzed using EDX spectroscopy (Figure 4b). The EDX spectrum confirmed the presence of copper (Cu) and oxygen (O) as the primary elements, with significant signals corresponding to 47.1 at% Cu and 40.6 at% O. The detection of only Cu and O in significant quantities suggests a relatively high purity of the synthesized Cu₄O₃ NPs. The atomic ratio of Cu to O is approximately 1.16:1, which is close to the theoretical ratio of 4:3 (1.33:1) for Cu₄O₃, considering potential surface oxidation or minor variations. The presence of minor peaks corresponding to other elements such as Na (1.0%), P (5.2%), S (0.7%), Cl (0.8%), and K (0.7%) indicates the presence of trace amounts of residues likely originating from the pumpkin seed extract used in the synthesis process. While these impurities are present in small quantities, the dominant signals of Cu and O confirm that the synthesized product is predominantly copper oxide.

Figure 4

(a) SEM micrograph and (b) EDX spectrum of Cu₄O₃ NPs.

3.7 HRTEM of Cu₄O₃ NPs

The fabricated Cu₄O₃ NPs were characterized using transmission electron microscopy (TEM). The scale bar in the TEM image corresponds to a field-of-view width of 100 nm. The NPs exhibited a range of morphologies, including spherical, oval, and irregular shapes. Additionally, blocky or rounded morphologies with protrusions were observed. The NPs’ diameter ranged from 20 to 100 nm, with some evidence of aggregation, as shown in Figure 5a. The TEM analysis determined the size, which roughly agreed with the Scherrer equation’s estimate. This observation supports the successful formation of Cu₄O₃ NPs using aqueous pumpkin seed extracts in the present study. High-resolution TEM analysis revealed a thin, amorphous layer surrounding the Cu₄O₃ NPs. This layer is made up of organic material from aqueous extracts, which may serve as a capping agent for the NPs. The selected area electron diffraction (SAED) pattern in Figure 5b indicates that the biofabricated Cu₄O₃ NPs have a partially crystalline structure. The SAED pattern’s diffraction rings match that of the tetragonal copper oxide paramelaconite, which is in line with the XRD results and adds to the reliability of the characterization. The particle size distribution of Cu₄O₃ NPs synthesized using pumpkin seed extract was analyzed, and the histogram (Figure 5c) reveals a mean particle size of 26.463 nm. The size range was found to be between approximately 8.92 and 55.84 nm, indicating a relatively uniform distribution with most particles concentrated between 20 and 35 nm. These data, consistent with TEM analysis, confirm the nanoscale nature of the synthesized particles and suggest effective capping and stabilization by the phytochemicals present in the extract.

Figure 5

(a) TEM image of Cu₄O₃ NPs at a field of view width of 100 nm, (b) corresponding SAED pattern, and (c) particle size distribution of Cu₄O₃ NPs with a mean size of 26.463 nm, indicating a relatively narrow range, with most particles falling between 20 and 35 nm.

3.8 Optimization of Cu₃O₄ NP synthesis from pumpkin seed extract using BBD

The optimization of Cu₄O₃ NP synthesis using pumpkin seed extract was carried out using the BBD. This approach allowed for a systematic evaluation of the impact of key process variables on the synthesis. The independent factors studied were precursor concentration (A), reaction temperature (B), and reaction time (C). By varying these factors, their influence on both the yield and properties of the NPs was thoroughly analyzed (Table 3). The experimental design matrix and the resulting response values were assessed using ANOVA to determine the statistical significance of the factors and to evaluate the adequacy of the model.

The optimization of Cu₄O₃ NP synthesis using pumpkin seed extract was systematically investigated using the BBD. The effects of three independent variables (Tables 1 and 3) were evaluated to understand their influence on the yield and properties of the NPs. The experimental data were analyzed using ANOVA to assess the statistical significance of the factors and the adequacy of the model. Figure 6(a) presents the Pareto chart of standardized effects for yield (%), with a significance level of α = 0.05. The graph illustrates the relative importance of each term in the model, including the main factors (A, B, C), their quadratic terms (AA, BB, CC), and interaction terms (AB, AC, BC). The length of each bar corresponds to the size of the standardized effect, with a reference line indicating the threshold for statistical significance. This analysis helps identify the most influential factors and interactions that affect the yield of NPs. The normal probability plot of the residuals is shown in Figure 6b, evaluating the model’s normality assumption. Based on the plot, it appears that the residuals are roughly straight lines, indicating that the errors are normally distributed and that the assumptions of the model are valid. Figure 6c shows the histogram of residuals to illustrate their distribution visually. Using the histogram, we can see that the residuals are centered around zero with no skewness or outliers, thus confirming the validity of the model. As shown in Figure 6d, the residuals versus fitted values plot checks for homoscedasticity (constant residual variance). Across the range of fitted values, the residuals around the zero line are distributed randomly, confirming the model’s reliability. Figure 6e shows the independence of residuals over time by plotting residuals versus the order of observations. As the pattern of residuals shows no systematic trend or autocorrelation, the experimental runs were conducted under steady-state conditions. The RSM analysis revealed the individual and interactive effects of the selected parameters on performance. The contour plots (Figure 6f and g) demonstrated that elevating the concentration of CuSO₄ and the volume of pumpkin seed extract led to enhanced production, with optimal yield observed at intermediate levels of both parameters. The impact of reaction temperature was evident, exhibiting an increase in the yield until an optimal threshold was attained, after which a decline occurred, indicating possible heat degradation of bioactive compounds that stabilize the NPs. The 3D response surface plot (Figure 6h) corroborated the curvature in the response, signifying a strong interaction between the components. The quadratic model indicated that both linear and quadratic effects significantly influenced NP performance. Figure 6i shows that a maximum yield of 89.87% was obtained at 105.71°C using 16.26 mL of pumpkin seed extract and 7.0 mM CuSO₄. The optimum was determined by a BBD. The model had a desirability score of 0.9956, indicating its accuracy in predicting the optimal response. The maximum yield was achieved within the specified limits, confirming the efficacy of the biogenic synthesis under regulated conditions.

Figure 6

Statistical analysis of Cu₃O₄ NP synthesis optimization using a BBD. (a) The Pareto chart identifies significant effects, with bars exceeding the red line (α = 0.05), indicating statistical significance. (b) The normal probability plot assesses residual normality, with points expected along the diagonal line. (c) The residuals vs fitted values plot checks for constant variance. (d) The histogram further evaluates residual normality. (e) The residuals vs observation order plot examines potential time-related trends. Response surface plots showing the influence of CuSO₄ concentration, extract volume, and reaction temperature on the Cu₃O₄ NP yield. (f) CuSO₄ concentration vs extract volume. (g) CuSO₄ concentration vs reaction temperature. (h) 3D plot of CuSO₄ concentration vs extract volume. (i) Individual factor effects on Cu₃O₄ NP yield at the optimal setting.

Table 4 provides a more comprehensive description. The elevated R-squared number suggests that the model is significant overall, although this is not definitively proven. The individual linear effects of CuSO₄ concentration, extract volume, and temperature were not statistically significant. Changing any one factor alone, while holding the others constant, did not have a statistically significant effect on the yield within the range examined.

Table 4 highlights the significant influence of the interaction between CuSO₄ concentration and extract volume (p = 0.035). This indicates that the impact of CuSO₄ concentration on yield is significantly influenced by the volume of extract employed and vice versa. Enhancing one aspect without accounting for the other would be inadequate. This interaction facilitates the high projected yield depicted in Figure 6i at the precise combination of 7.0 mM CuSO₄ and 16.26 mL extract, despite the distinct linear effects being insignificant independently. The marginal p-value for the quadratic term of the extract volume (p = 0.051) further substantiates the intricate link between the extract volume and yield, indicating a non-linear influence. The lack-of-fit p-value of 0.099 in Table 4 indicates that the model, while beneficial, may not entirely represent all the intricacies of the system. This may result from a minor factor excluded from the model or slight departures from the presumed quadratic relationship. Notwithstanding this, the model effectively discerned a practically advantageous optimum, as demonstrated by the elevated desire and the recorded maximum yield. Using pumpkin seed extract, the BBD approach significantly improves the NP synthesis conditions. Collectively, these evaluation plots confirm the robustness of the BBD and the suitability of the quadratic model to optimize the synthesis of Cu₄O₃ NPs. The insights gained from this analysis provide a solid foundation for further optimization and scaling.

3.9 Statistical analysis

The experiments were designed and analyzed using MiniTab Statistical Software (Minitab LLC, Pennsylvania, USA). A full quadratic model was employed for data analysis, and ANOVA was performed with a 95% confidence level. The quadratic regression model is shown as follows: Regression Equation in Uncoded Units Yield (%) = −390 − 34.6 concentration of CuSO₄ solution + 3.58 volume of pumpkin seed extract + 10.57 reaction temperature (°C) − 0.417 concentration of CuSO₄ solution × concentration of CuSO₄ solution − 0.0617 volume of pumpkin seed extract × volume of pumpkin seed extract − 0.0635 reaction temperature (°C) × reaction temperature (°C) − 0.075 concentration of CuSO₄ solution × volume of pumpkin seed extract + 0.430 concentration of CuSO₄ solution × reaction temperature (°C) − 0.0100 volume of pumpkin seed extract × reaction temperature (°C).

3.10 MTT assay

The cytotoxicity of biogenic Cu₄O₃ NPs against human lung adenocarcinoma (A549) and colon cancer (HCT-116) cell lines was evaluated using the MTT assay (Figure 7). The assay measures the activity of mitochondrial succinate dehydrogenase, an enzyme that is an indicator of cell viability. The results showed that the cell viability of both cell lines decreased in a dose-dependent manner with increasing concentrations of Cu₄O₃ NPs (ranging from 0 to 1,280 μg/mL). The calculated IC₅₀ values, which represent the concentration required to inhibit 50% cell viability, were 402.37 μg/mL for A549 cells and 449.32 μg/mL for HCT-116 cells. The biogenic Cu₄O₃ NPs exhibited an IC₅₀ of 11 µg/mL against HCT-116 cells when freshly prepared [32]. Statistical analysis of the differences in cell viability between the two cell lines at each concentration is provided in Figure 7. However, after long-term storage, the IC₅₀ increased substantially to 449.32 µg/mL when the sample was retested, indicating a reduction in cytotoxic efficacy. These values indicate that the synthesized Cu₄O₃ NPs exhibited a higher cytotoxic effect against A549 lung cancer cells than against HCT-116 colon cancer cells. These results are consistent with other studies investigating the cytotoxic potential of Cu₄O₃ NPs, although specific IC₅₀ values may vary depending on the synthesis method, NP characteristics (size, morphology, and surface properties), and the cell line tested. For instance, Jabir et al. [83] reported a significantly lower IC₅₀ value of 9.5 µg/mL for Cu₄O₃ NPs against the PA-1 human ovarian cancer cell line. This higher efficacy in PA-1 cells could be attributed to differences in cellular uptake mechanisms or specific interactions between the NPs and the PA-1 cell line. This higher efficacy in PA-1 cells may be attributed to differences in the cellular uptake mechanism or specific interactions between the NPs and the PA-1 cell line. Similarly, another study [68] examined the effects of Cu₄O₃ NPs on PC-3 human prostate cancer cell lines and normal NIH/3T3 cell lines. While the study reported dose-dependent cell growth arrest in PC-3 cells, cytotoxicity data in normal cells were also provided, which is critical for evaluating the potential therapeutic window of these NPs. The moderate cytotoxic effect observed in our study, with IC₅₀ values in the range of hundreds of μg/mL, suggests that while the biogenic Cu₄O₃ NPs are active against these cancer cell lines, higher concentrations may be required for significant inhibition compared to other reports. The differences in IC₅₀ values between studies highlight the importance of considering the specific characteristics of the NPs and cell lines used in the evaluation. Our study focused on two cancer cell lines, and future work could explore the selectivity of our bioderived Cu₄O₃ NPs by performing analyses in normal cell lines to evaluate their potential in cancer-targeted therapy. Although the IR spectra, EDX profiles, and TGA thermograms of the stored NPs were superimposable with those of the fresh samples [32], indicating no major chemical, elemental, or thermal changes, characterization revealed significant aggregation. This aggregation likely reduced dispersion and surface area, impairing cellular uptake and leading to diminished biological activity [84]. These findings highlight the critical role of NP stability and storage conditions in preserving cytotoxic efficacy. Aggregation of NPs can decrease surface reactivity and hinder cellular internalization, which are crucial factors affecting their therapeutic potential. Therefore, strategies to prevent aggregation or improve redispersion after storage should be considered to maintain biological activity.

Figure 7

Dose-dependent cytotoxic effect of biofabricated Cu₄O₃ NPs on HCT-116 and A549 cancer cell lines assessed by MTT assay. Cells were treated with increasing concentrations (0–1,280 μg/mL) of Cu₄O₃ NPs for 24 h. Data represent mean  ±  SD of triplicate measurements. Statistical significance between HCT-116 and A549 at each concentration was evaluated (*p  <  0.05, **p  <  0.01, ***p  <  0.001; ns = not significant).

3.11 Antibacterial activity analysis of NPs

This study examined the antibacterial effectiveness of Cu₄O₃ NPs against the Gram-positive bacterium Bacillus subtilis. Table 5 demonstrates the formation of distinct regions devoid of development around wells containing Cu₄O₃ NPs, suggesting their inhibitory effect on B. subtilis. As the concentration of NPs increased from 1 to 4 ppm, the effectiveness of NPs increased (Table 5). The absence of bacterial growth in these clear zones indicates the antibacterial impact of the NPs. The findings additionally indicate that the NPs engage with and exert a potent impact on B. subtilis cells. The Cu₄O₃ NPs exhibit a larger diameter of the zone of inhibition, which is a quantitative indicator of antibacterial activity, in comparison to tetracycline (30 mcg, standard) [85]. These findings indicate that Cu₄O₃ NPs have a more potent antibacterial effect against B. subtilis compared to the reference material. The minimum inhibitory concentration (MIC) of biosynthesized Cu₄O₃ NPs was found to be 3 ppm against B. subtilis, as this was the lowest concentration that completely inhibited visible bacterial growth in the broth dilution assay.

Table 5

Antibacterial activity of biogenic Cu₄O₃NPs, demonstrating the zone of inhibition on discs

Concentration of Cu4O3 NPs (ppm)	B. subtilis; inhibition zone diameter
	Cu4O3 NPs NZI (cm)	Standard (30 mcg)	NC NZI (cm)
1	1.7 ± 0.1	3.0 ± 0.1	0 ± 0
2	1.9 ± 0.1
3	2.4 ± 0.05
4	2.8 ± 0.06

NZI: no zone of inhibition; NC: negative control. The results are reported as mean ± SD (n = 3).

3.12 Relating to existing work

The synthesis and characterization of Cu₄O₃ NPs were reported in previous studies, as summarized in Table 6.

Our green synthesis approach utilizing pumpkin seed extract offers a potentially more sustainable and cost-effective alternative to some conventional methods. The characteristic UV–Vis absorption peak observed at 332 nm in our study aligns with the reported interband transition for Cu₄O₃, although the specific wavelength can vary slightly depending on factors such as particle size and synthesis conditions. The morphology and size of the synthesized Cu₄O₃ NPs in our work were predominantly spherical with sizes ranging from 8.92 to 55.84 nm, showing a different size profile compared to other reports. For instance, Table 6 reports a larger size of Cu₄O₃ NPs with sizes ranging from 27 nm (sponge-like morphology) to >100 nm (spherical, large, irregular, and agglomerated forms), <100 nm (spherical shape), ≈200 nm (tetragonal), and ∼200 nm (hexapod morphology). These differences in size and morphology could influence the surface area and, consequently, the observed properties. Our investigation into the cytotoxicity of the pumpkin seed extract-mediated NPs on HCT-116 and A549 cell lines provides insights into their potential biomedical applications. While some previous studies on Cu₄O₃ NPs have explored properties like antibacterial and anticancer activities, our findings specifically contribute to the understanding of their cytotoxic effects on these cancer cell lines. Further research directly comparing the antibacterial and cytotoxic activities of Cu₄O₃ NPs synthesized through different routes and exhibiting varying sizes would be beneficial. The present study contributes to the growing body of literature on copper oxide NPs by offering a green synthesis route using a plant extract and providing valuable data on their size distribution and cytotoxic potential against specific cancer cell lines. The comparative analysis with existing reports highlights both the consistency of our UV–Vis identification of the copper oxide and the unique size characteristics of the NPs produced through our method and their investigated bioactivity.

3.13 Molecular index analysis

The calculated HOMO–LUMO energy gap (ΔE = 1.23 eV) suggests a moderate chemical reactivity. While a smaller gap typically indicates higher reactivity, this value still implies a potential for interaction with biological systems through electron transfer processes. The electronegativity (χ = 4.218 eV) indicates the tendency of the Cu₄O₃ cluster to attract electrons, which could be relevant in interactions with bacterial cell walls or cancer cell membranes. The chemical hardness (η = 0.613 eV) and softness (S = 0.816 eV⁻¹) provide further insights into the cluster’s stability and polarizability. A higher hardness implies greater resistance to deformation of the electron cloud, while a higher softness indicates greater polarizability and, thus, potentially stronger interactions with biological molecules (Table 7 and Figure 8).

Figure 8

HOMO and LUMO orbitals of the optimized Cu₄O₃ cluster, showing their respective energies.

Considering the observed antimicrobial activity against B. subtilis and cytotoxicity against A549 and HCT-116 cell lines, the moderate reactivity indicated by our DFT calculations suggests that the Cu₄O₃ NPs can interact with biological targets. The specific mechanisms likely involve a combination of factors, including the release of copper ions, the generation of reactive oxygen species, and direct interactions of the NP surface with biomolecules. The electronic properties, particularly the electronegativity and softness, could influence the NP’s affinity for and interaction with cellular components. Further experimental studies are needed to fully elucidate these mechanisms, but these computational insights provide a valuable foundation for understanding the inherent reactivity of the Cu₄O₃ NPs and their potential for biological activity.