Green synthesis of copper oxide nanoparticles from Algerian propolis: Exploring biochemical, structural, antimicrobial, and anti-diabetic properties

2.1 Chemicals and reagents

The chemicals and reagents used in this study were sourced from Biochem Chemopharma. They included copper(ii) sulfate pentahydrate (CuSO₄·5H₂O, 99.5%), sodium hydroxide (NaOH, 97%), deionized water (DIW), absolute ethanol (C₂H₅OH, 99%), dimethyl sulfoxide (DMSO, 99.9%), 2,2,2-diphenyl-1-picrylhydrazyl (DPPH, C₁₈H₁₂N₅O₆), methanol (CH₃OH, 99.9%), TPTZ (2,4,6-tris(2-pyridyl)-1,3,5-triazine), hydrochloric acid (HCl, 37%), acetic acid (CH₃COOH, 99.5%), sodium acetate (CH₃COONa·3 H₂O, 99%), iron chloride (FeCl₃, 99%), and iron sulfate (FeSO₄).

The propolis sample used in this study was collected from the Relizane region of Algeria during the summer of 2022. Relizane, located in the northwest of Algeria, boasts a rich and diverse flora, contributing to this region’s unique composition of propolis. The collection followed ethical guidelines, ensuring minimal disruption to the bee colonies and adherence to sustainable harvesting practices.

2.2 Propolis extract preparation

Propolis was extracted using a modified version of an established technique, incorporating minor adjustments to enhance the efficiency and quality of the extraction process [24,25]. To prepare the propolis extract, 10 g of finely chopped propolis is combined with 100 mL of 70% ethanol and 30% water solvent solution in a glass container. The mixture is left to soak for precisely 24 h at room temperature in the dark, with continuous agitation. After the soaking period, the solvent solution containing the extracted propolis is collected. This extraction process is repeated three times consecutively to ensure thorough extraction of the propolis components. The solvent solution is collected and concentrated using a rotary evaporator to remove the solvent. Finally, the resulting concentrated extract is stored in a refrigerator at 4°C until further use.

2.3 Green synthesis of copper oxide NPs (CuO NPs)

Green CuO NPs were synthesized using a previously established method, with slight modifications to optimize the process [26]. CuO NPs were synthesized by reducing copper sulfate using phenolic compounds extracted from propolis. The reaction mixture was prepared by combining 100 mL of 0.1 M CuSO₄·5 H₂O solution in a 250 mL flask. The pH of the solution was adjusted to 9 using a suitable base. During the synthesis process, the propolis extract solution (1 mg·mL⁻¹) was added slowly in batches using a burette until the desired amount of extract was reached (80 mL). This slow addition allowed for precise control over the reaction and facilitated optimal NP formation.

The biosynthesis reaction was conducted at 40°C for 24 h under controlled conditions. CuO formation was verified by observing a distinct color transition from green to dark brown in the reaction mixture. The resultant brown solid was harvested through centrifugation at room temperature, followed by multiple washes with distilled water and absolute ethanol at 4,500 rpm to remove any unreacted species or impurities. Subsequently, the product was subjected to overnight drying at 60°C to remove residual moisture. Half of the obtained quantity underwent further treatment in an oven at 500°C for 4 h to enhance crystallinity and remove organic residues (Figure 1).

Figure 1

Mechanism of formation of CuO NPs.

2.4 Characterization of CuO NPs

2.5 Antioxidant properties of the synthesized NPs

2.6 Antibacterial activity of the NPs

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CuO A NPs and CuO B NPs were determined using the broth microdilution method in sterile 96-well flat-bottom microplates (Nunc, USA), with a final volume of 150 µL per well, as described in the study of Balouiri et al. [33] with minor modifications. Bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis) and the fungal strain (Candida albicans) were cultured in Mueller–Hinton broth (MHB) and Sabouraud dextrose broth (SDB), respectively. The bacterial inoculum was prepared by diluting the bacterial culture in MHB to achieve a final concentration of approximately 5 × 10⁵ CFU·mL⁻¹ (McFarland standard 0.5), which was further diluted 1:100 with broth and added (75 µL) to each well along with 75 µL of the corresponding nanoparticle suspension. The fungal inoculum was diluted to a final concentration of 10⁶ CFU·mL⁻¹ in SDB. Serial dilutions of CuO NPs were prepared in each well using sterile MHB for bacterial strains and SDB for fungal strains. The following concentrations were tested: 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.031 mg·mL⁻¹. Control wells included a growth control (no NPs), a sterile control (broth only), and a microbial control (bacterial or fungal suspension without NPs). The plates were incubated at 37°C for 18–24 h for bacterial strains and 48 h for fungal strains. After incubation, microbial viability was assessed by adding 10 µL of resazurin dye (337.5 mg of resazurin powder in 50 mL of sterile distilled water; Serva, Germany) to each well. The plates were then incubated at 37°C for 3 h to observe color changes, indicating microbial growth or inhibition. The MIC was determined as the lowest concentration of CuO NPs that completely inhibited visible microbial growth (the last well to remain purple before turning pink).

For MBC determination, 3 µL of samples were taken from wells with no visible growth and spread onto Mueller–Hinton agar (for bacteria) or Sabouraud dextrose agar (for fungi). The plates were incubated at 37°C for 18–24 h for bacterial strains and 48 h for fungal strains. The MBC was defined as the lowest concentration of CuO NPs, resulting in ≥99.9% reduction in microbial growth.

The MIC and MBC values were expressed as the arithmetic mean of triplicate tests with the standard deviation. Bactericidal activity was classified based on the MBC/MIC ratio: if the ratio was ≤4, the effect was considered bactericidal; if the ratio was >4, the effect was considered bacteriostatic.

2.7 Anti-diabetic activity assessed by glucose uptake in yeast cells

Commercial baker’s yeast (Saccharomyces cerevisiae) was washed by repeated centrifugation at 3,000 × g for 5 min with distilled water until the supernatant became clear. A 10% yeast suspension (v/v) was then prepared in distilled water. Various concentrations of CuO NPs (CuONPs A and CuONPs B), ranging from 1.5 to 48 mg·mL⁻¹, were added to 1 mL of a glucose solution (25 mM or 4.5039 g·L⁻¹), followed by incubation for 10 min at 37°C. The reaction was initiated by adding 100 µL of the prepared yeast suspension to the glucose solution. The mixture was vortexed and further incubated at 37°C for two time points: 30 min (T30) and 120 min (T120). After incubation, the reaction mixture was centrifuged at 2,500 × g for 5 min, and the glucose concentration in the supernatant was measured. Glucose levels were determined by absorbance at 540 nm. All experiments were performed in triplicate to ensure the accuracy and reproducibility of the results [34].

3.1 UV-visible spectroscopy

This study focuses on the rapid and environmentally friendly synthesis of CuO NPs using propolis extract. Propolis is widely recognized for its rich composition of bioactive compounds, including phenolic acids, flavonoids, terpenes, bioelements, vitamins, proteins, and amino acids. Among these, gallic acid has been identified as a major phenolic compound within propolis extract [35], playing a potentially critical role as a catalyst in the reduction of Cu(OH)₂ to nanoscale CuO.

As depicted in Figure 2, the prominent absorption peaks at 259, 284, 301, and 325 nm in the UV-Vis spectrum of the crude Relizane propolis extract indicate the presence of polyphenols and flavonoids [36]. The observed color shift from blue to brown can be attributed to the excitation of surface plasmon resonance coupled with the antioxidant activity of polyphenols. This color transition, driven by π → π* electronic transitions, confirms the successful synthesis of CuO NPs [37].

Figure 2

UV-visible spectra of CuO NPs and propolis extract.

The UV-Vis spectrum of CuO NPs synthesized from propolis (CuO NPs A) reveals two absorption bands at 314 and 370 nm, whereas calcined CuO NPs (CuO NPs B) exhibit a broader absorption peak centered at 355 nm, corresponding to the surface plasmon absorption of the metal oxide, as illustrated in Figure 2. Surface plasmon absorption in metal oxide NPs arises from the collective oscillation of free conduction band electrons induced by incident electromagnetic radiation. This resonance effect becomes pronounced when the wavelength of the incident light significantly exceeds the particle diameter [38].

3.2 FTIR analysis

FTIR analysis was employed to identify the potential biomolecules present in propolis extract responsible for reducing Cu ions to cuprous oxide NPs. The analysis was conducted within the spectral range of 400–4,000 cm⁻¹. This allowed us to gain insights into how the propolis extract functions as both a reducing and capping agent, influencing the formation of new functional groups and biomolecules attached to the surface of CuO NPs.

As shown in Figure 3, the propolis extract exhibited a spectral profile characterized by 14 distinct peaks at specific wavenumbers (3,403.9, 2,912.9, 2,752.6, 2,092.9, 1,906.5, 1,707.1, 1,623.3, 1,442.5, 1,351.2, 1,246.8, 1,172.2, 1,021.3, 713.8, and 475.2 cm⁻¹). However, it is noteworthy that these peaks underwent shifts in their positions and alterations in their intensities following the formation of CuO NPs.

Figure 3

FTIR spectral characterization of propolis extract and synthesized CuO NPs (A and B).

The IR spectrum of CuO NPs A using propolis extract showed a peak corresponding to O–H stretching frequencies of 3,559.6 cm⁻¹, confirming the presence of OH groups from alcohols and phenol. Nonetheless, evidence suggests that the peak at 3,384.4 cm⁻¹ can be attributed to the stretching vibration of the N–H group [39]. The peaks at 2,930–2,850 cm⁻¹ (at approximately 2,912.9 cm⁻¹) are assigned to the asymmetric stretching of methylene, and the peaks at approximately 2,752.6, 2,092.9, and 1,906.5 cm⁻¹ are assigned to the symmetric stretching of the hydrocarbons [40]. This peak was shifted to wavenumbers of 2,294.2 and 2,085.4 cm^–1 after CuO NPs A synthesis (Figure 3). Absorptions at 1,707.1, 1,623.3, and 1,442.5 cm⁻¹ corresponding to the carbonyl C═O stretching vibration of flavonoids and lipids are found in the propolis. This peak was shifted to a wavenumber of 1,613.9 cm⁻¹ and was assigned to the bending vibrations of the carbonyl group, indicating the presence of flavonoids adsorbed on the surface of the biosynthesized CuO NPs A. The bands at 1,351.2 and 1,246.8 cm⁻¹ were due to the C–O–H stretching vibration, and the band at 1,172.2 cm⁻¹ was considered to occur due to the presence of lipids and alcohol groups (stretching of C–O and bending of C–OH), and the band at 1,021.3 cm⁻¹ was attributed to the stretching of the aromatic ether C–O–C bonds [41,42]. This peak was shifted to a wavenumber of 1,099.6 cm⁻¹ and was due to the C–O stretching vibrations of the carboxylic group and flavanones. The peak at 713.8 cm⁻¹ may be related to the vibration of the aromatic rings; after the synthesis of CuO NPs, this peak undergoes a noticeable shift to wavenumbers of 872.2 and 756.6 cm⁻¹. Moreover, a pronounced and intense peak at 603.8 cm⁻¹ can be attributed to the stretching vibration of Cu–O bonds. This peak strongly supports the formation of CuO A NPs [43].

Moreover, the IR spectrum of calcined CuONPs B displayed broad peaks corresponding to the O–H stretching frequencies at 3,570.8 and 3,488.8 cm⁻¹, confirming the presence of the OH group derived from alcohols and phenols. The peaks at 1,110.7 and 1,073.5 cm⁻¹ were attributed to C–O stretching vibrations of the carboxyl group and flavanones, consistent with previous reports [39,44]. Five IR absorption peaks in calcined CuO nanostructures show the 700–400 cm⁻¹ vibrational mode. The bands at 633.6, 596.4, and 484.6 cm⁻¹ corresponded to the vibrations of the Cu–O band, confirming the synthesis of CuO NPs B [45].

The FT-IR spectrum confirmed the role of different functional groups in propolis extract, such as phenolic acids, flavonoids, terpenes, bioelements, and other components, such as vitamins, proteins, amino acids, and sugars, in reducing Cu²⁺ to form CuO NPs, followed by capping the surface of the final product to increase its stability.

3.3 XRD analysis

Figure 4a presents the XRD pattern of CuO NPs A. A weak diffraction peak at 41.3° is observed, originating from the copper foil substrate (JCPDS 04-0836). Additionally, distinct peaks were identified at angles 22.9°, 30.9°, 33.6°, 38.5°, 48.9°, 58°, 61.7°, and 63.4°, corresponding to the (021), (110), (002), (200), (020), (202), (-113), and (200) Miller planes, respectively. The peaks at 52.9° and 66.6° are attributed to crystalline and amorphous organic phases. These diffraction peaks’ positions and relative intensities align well with standard XRD data for Cu(OH)₂ NPs (JCPDS file No. 35-0505). Additionally, distinct peaks are identified at angles 35.7°, 56.5°, and 68.1°, corresponding to the (111), (151), and (220) Miller planes, respectively, which were consistent with the standard XRD data for CuO NPs (JCPDS 48-1548) and Cu₂O NPs (JCPDS file No. 05-0667).

Figure 4

XRD patterns of NPs synthesized from propolis extract: (a) CuO NPs and (b) calcined CuO NPs.

Figure 4b shows the XRD pattern of CuO NPs B. Notably, many peaks corresponding to crystalline and amorphous organic phases and peaks of CuO NPs A are absent, while others become clearer and more prominent. The peaks observed at angles 31.8°, 36.1°, 47.5°, 56.6°, 62.8°, 66.2°, and 68.8° correspond to the (110), (111), (−202), (151), (200), (-311), and (220) Miller planes, respectively. Moreover, the peaks at 34.4°, 38.7°, and 61.2° are associated with the (002), (022), and (−113) Miller planes, respectively, consistent with standard XRD data for CuO NPs (JCPDS 48-1548) and Cu₂O NPs (JCPDS file No. 05-0667).

The crystalline size was determined using the Debye–Scherrer formula:

where k = 0.9, β represents the full width at half-maximum of the XRD peak, and θ is the Bragg’s angle. The average crystalline sizes of CuO NPs A and CuO NPs B were calculated to be 68.5 and 74.82 nm, respectively.

3.4 SEM and energy-dispersive spectroscopy (EDS)

The surface properties of CuO NPs synthesized using different methods were analyzed via SEM. Figure 5 depicts the SEM analysis of CuO A NPs synthesized from propolis extract at a weight-to-volume ratio of 1:4. The SEM image reveals agglomerated CuO NPs A with spherical shapes. The observed high agglomeration could be attributed to the low percentage of propolis extract used as a stabilizing agent and the polarity of CuO NPs due to the high concentration of precursors. The particles exhibit a spherical shape, forming clumps that appear to be interconnected.

Figure 5

SEM and EDS analysis of CuONPs A synthesized from propolis extract.

EDS was employed to investigate the chemical composition of CuO NPs A. The SEM-EDS spectra reveal that the sample comprises 64.82% copper, 23.57% oxygen, and 11.61% carbon, consistent with XRD results.

Figure 6 illustrates the SEM analysis of CuO NPs B synthesized from propolis extract at a weight-to-volume ratio of 1:4. The SEM image shows agglomerated CuO NPs B with star shapes. Unlike CuO NPs A, where all particles were interconnected, CuO NPs B form sub-agglomerates, each resembling a star shape with formal symmetry.

Figure 6

SEM and EDS analysis of calcined CuO NPs B synthesized from propolis extract.

Similarly, EDS was employed to determine the chemical composition of CuO NPs B. The SEM-EDS spectra indicate that the sample comprises 88.26% copper, 9.17% oxygen, and 2.56% carbon, consistent with the XRD results. This analysis provides valuable insights into the morphology and elemental composition of the synthesized NPs, shedding light on their structural properties and potential applications.

3.5 DPPH assay

The DPPH assay, depicted in Figure 7, is a crucial method for assessing the antioxidant activity of various compounds by evaluating their capability to scavenge DPPH radicals. The assay results reveal differing antioxidant effects among the tested samples, as shown in Table 1.

Figure 7

DPPH radical scavenging activity of CuO NPs synthesized from propolis extract.

Starting with ascorbic acid, which emerges as the most active antioxidant component with the lowest IC₅₀ value of 0.021, it functions primarily as a hydrogen donor. Ascorbic acid donates hydrogen atoms from its hydroxyl groups to free radicals, thereby initiating a process that ultimately reduces free radicals to stable non-radical species. This mechanism highlights the effectiveness of ascorbic acid, even against highly potent free radicals.

Following closely, CuO NPs A exhibit an IC₅₀ value of 0.027, indicating significant antioxidant potential. This notable effect can be attributed to a synergistic interaction between the copper hydroxide NPs and the bioactive compounds present in propolis. The combined action of these components enhances the overall antioxidant activity observed.

In contrast, CuO NPs B demonstrate a higher IC₅₀ value of 0.052, suggesting a relatively weaker antioxidant effect compared to the other samples tested. This weaker effect may stem from the NPs undergoing electron exchange reactions, wherein they either accept or donate electrons to neutralize the radicals. However, this mechanism may not be as efficient in neutralizing the DPPH radicals as the hydrogen donation mechanism exhibited by ascorbic acid and the synergistic effect observed in CuO A NPs. These findings underscore the importance of NP composition and structure in determining their antioxidant properties.

3.6 FRAP assay

The FRAP assay, depicted in Figure 8, is a valuable method for evaluating antioxidant capacity by measuring the reduction of ferric ions (Fe³⁺) to ferrous ions (Fe²⁺) in the presence of antioxidants. The results from this assay provide insights into the antioxidant potential of the tested samples, as shown in Table 2.

Figure 8

Ferric reducing antioxidant power of CuO NPs synthesized from propolis extract.

Starting with ascorbic acid, which emerges as the most potent antioxidant in this assay, it demonstrates high antioxidant capacity by donating electrons to ferric ions (Fe³⁺), thereby reducing them to ferrous ions (Fe²⁺). This process results in the formation of dehydroascorbic acid, which effectively neutralizes harmful free radicals, thus preventing oxidative damage. Ascorbic acid’s ability to efficiently donate electrons makes it highly effective in combating oxidative stress.

Similarly, CuO NPs A exhibit a higher antioxidant capacity, as evidenced by their AEAC value of 2.01. This indicates a significant ability to donate electrons and facilitate the reduction of ferric ions. The observed antioxidant capacity of CuO A NPs may be attributed to the presence of bioactive compounds in propolis, which synergistically enhance the antioxidant activity of copper.

In contrast, CuO NPs B display a lower AEAC value of 1.76, suggesting a reduced capability to reduce ferric ions compared to CuO NPs A and ascorbic acid. Consequently, CuO NPs B exhibit lower antioxidant capacity in this assay. This difference in antioxidant capacity may stem from variations in the chemical properties and surface reactivity of NPs, affecting their ability to donate electrons and participate in the reduction of ferric ions. These findings underscore the importance of the composition and structure of NPs in determining their antioxidant properties.

3.7 TAC

Figure 9 and Table 3 present the TAC results obtained from the synthesized CuO NPs A and B. The TAC measurements reveal a notable antioxidant capacity in both CuO NPs A and B. Notably, CuO NPs A exhibited the highest antioxidant activity, with a value of 11.28 mg EAA/g NPs.

Figure 9

TAC of CuO NPs synthesized from propolis extract.

Copper deficiency adversely affects the body’s antioxidant function and predisposes individuals to various diseases. The CuO NPs prepared in this study have the potential to elevate copper levels in the blood of individuals suffering from copper deficiency, consequently enhancing antioxidant activity. Moreover, the impact of CuO NPs extends to cell membranes’ composition, thereby fortifying the integrity and functionality of cell membranes, tissues, and organs.

Min et al. [46] investigated the effects of copper oxide (Cu₂O NPs) on the antioxidant function of Cu-deficient Kazakh sheep. They observed significantly lower copper levels in the blood, wool, and liver of Cu-deficient Kazakh sheep compared to healthy animals. To address this deficiency, the authors supplemented Cu-deficient Kazakh sheep with Cu₂O NPs or CuSO₄. Consequently, the blood copper concentration notably increased. Interestingly, starting from day 5 of supplementation, the copper content in the Cu₂O groups surpassed that of the CuSO₄ group [46].

3.8 Assessing antibacterial activity mediated by NPs

CuO NPs A (Table 4 and Figure 10) demonstrate significantly higher antimicrobial efficacy compared to CuO NPs B (Table 5 and Figure 11), as evidenced by the lower MIC values across the tested strains. For B. subtilis, CuO NPs A achieved an MIC of 5 mg·mL⁻¹, while CuO NPs B required a much higher MIC (20 mg·mL⁻¹). Similarly, CuONPs A exhibited an MIC of 10 mg·mL⁻¹ for E. coli and S. aureus compared to those of 40 mg·mL⁻¹ and 80 mg·mL⁻¹, respectively, for CuO NPs B. In the case of P. aeruginosa, CuO NPs B displayed very high concentrations (VHC) to inhibit bacterial growth, whereas CuO NPs A managed to achieve inhibition with an MIC of 40 mg·mL⁻¹. Regarding the fungal strain Candida albicans, CuO NPs B were ineffective at standard concentrations, requiring VHC for both MIC and MFC, while CuO NPs A maintained their efficacy with an MIC of 40 mg·mL⁻¹. These results highlight the superior performance of CuO NPs A as an antimicrobial agent, driven by its compositional and structural advantages compared to CuO NPs B (Table 6).

Figure 10

Antimicrobial efficacy of CuO NPs A. Broth microdilution assay shows (a) antibacterial activity against E. coli, P. aeruginosa, S. aureus, and B. subtilis, and (b) anti-candida activity against C. albicans at concentrations of 80–2.5 mg·mL⁻¹.

Figure 11

Antimicrobial efficacy of CuO NPs B. Broth microdilution assay showed (a) antibacterial activity against E. coli, P. aeruginosa, S. aureus, and B. subtilis and (b) anti-candida activity against C. albicans at concentrations of 80–2.5 mg·mL⁻¹.

The observed differences in antimicrobial activity between CuO NPs A and CuO NPs B can be attributed to fundamental variations in their chemical composition and preparation methods. CuO NPs A, synthesized using propolis, incorporate bioactive compounds such as flavonoids and phenolic acids, which synergistically enhance their antimicrobial properties. These compounds promote increased production of ROS, which directly attacks microbial cells by damaging their membranes and interfering with essential intracellular processes [48]. Additionally, CuO NPs A possess superior surface properties, allowing for enhanced interaction with microbial cells. In contrast, CuO NPs B, prepared through calcination, lack these bioactive components, limiting their ability to interact effectively with microbial membranes. This limitation is particularly evident with resistant strains such as P. aeruginosa and C. albicans, which have thicker and more complex cell walls [49]. Furthermore, the crystallinity and surface structure of CuO NPs B reduce their efficiency in penetrating microbial cells and disrupting their integrity, explaining the higher MIC and MFC values observed.

The antimicrobial activity of CuO NPs is primarily driven by their ability to generate ROS, including hydroxyl radicals and hydrogen peroxide, which play a pivotal role in microbial cell destruction [50]. ROS oxidize membrane lipids and proteins, compromising the structural integrity of microbial cells. Additionally, ROS inflict direct damage on microbial DNA and intracellular proteins, disrupting key processes such as cellular respiration and energy production [51]. CuO NPs A demonstrated superior ROS generation, largely due to the presence of flavonoids and phenolic compounds, which act as chemical and physical catalysts to enhance ROS production. Upon interaction with microbial cell surfaces, CuO NPs induce changes in membrane permeability, facilitating the internalization of NPs into the cytoplasm, where they disrupt essential cellular functions (Figure 12) [52].

Figure 12

Mechanism of antibacterial activity of CuO NPs.

In contrast, CuO NPs B exhibited lower efficacy due to their lack of active compounds and limited surface properties. Resistant microorganisms like P. aeruginosa and C. albicans possess thick and complex cell walls composed of components such as chitin and glucans, making them less susceptible to penetration by less potent NPs like CuO NPs B. However, CuO NPs A, with their enhanced properties, effectively penetrate these barriers and exert antimicrobial action [48].

3.9 Glucose uptake in yeast cells

The mechanism of glucose transport across the yeast cell membrane has garnered significant attention as an in vitro screening method for the hypoglycemic effects of various compounds. The findings of this study revealed that calcined CuO NPs A and non-calcined CuO NPs B effectively enhanced glucose uptake in yeast cells. At T30 (Figure 13), the use of CuO NPs A resulted in a significant reduction in extracellular glucose concentration, demonstrating their efficiency in stimulating immediate glucose uptake by yeast cells. Glucose concentrations at CuO NP concentrations (1.5, 3, 6, 12, 24, and 48) were 325, 301, 285, 244, 198, and 172 mg·mL⁻¹, respectively. In contrast, glucose concentrations for CuO NPs B at the same doses were 334, 326, 319, 285, 274, and 258 mg·mL⁻¹, respectively. Both results showed superior performance compared to the negative control, where the glucose concentration was 453 mg·mL⁻¹. The positive control, based on metformin and the drug (MTZ) reported in studies by Pitchaipillai and Ponniah [53], and Pulivarthi et al. [54] demonstrated 95% and 88% efficacy, respectively (Table 7).

Figure 13

In vitro antidiabetic activity of CuO NPs A and CuO NPs B using a yeast cell model represented by extracellular glucose concentration (mg·mL⁻¹) uptake by yeast cells by different concentrations of NPs at 30 min; Sc = yeast of S. cerevisiae.

The superior performance of CuONPs A can be attributed to their structural properties, such as smaller size and larger surface area, which enhance their interaction with glucose transporters on the cell membrane. At T120 (Figure 14), the NPs exhibited a more sustained effect on glucose absorption, indicating the simultaneous activation of high- and low-efficiency glucose transporters, accelerating glucose uptake. At T120 (Table 8), the glucose concentrations at the same doses were 302, 298, 274, 212, 102, and 73 mg·mL⁻¹ for CuO NPs A and 319, 308, 306, 285, 274, and 252 mg·mL⁻¹ for CuO NPs B. The superior physiological efficacy of CuO NPs A at this stage reflects their enhanced capability to influence cellular processes governing glucose uptake.

Figure 14

In vitro antidiabetic activity of CuO NPs A and CuO NPs B using a yeast cell model represented by extracellular glucose concentration (mg·mL⁻¹) uptake by yeast cells by different concentrations of NPs at times 120 min; Sc = yeast of Saccharomyces cerevisiae.

This mechanism aligns with recent findings suggesting that sugar transport across the yeast cell membrane is mediated by stereospecific membrane transporters [55]. Specifically, glucose transport in yeast (S. cerevisiae) is highly complex, and it is well established that glucose is transported via facilitated diffusion. Facilitated transporters are specific carriers that move solutes along their concentration gradient, crucial in enhancing efficient glucose uptake. The improved glucose uptake, as demonstrated in this study with CuO NPs A, highlights the potential of NPs to enhance glucose utilization, thus promoting the regulation of blood glucose levels.

Recent studies on sugar and non-metabolizable glycoside transport suggest that sugar transport across the yeast cell membrane is mediated by stereospecific membrane transporters [56]. Reports indicate that in yeast cells (S. cerevisiae), glucose transport occurs through a complex process, primarily facilitated diffusion. Facilitated transporters are specific carriers that move solutes along their concentration gradient, effectively improving glucose uptake [54]. The increased glucose absorption demonstrated in this study with CuO NPs A underscores the ability of NPs to enhance glucose utilization, aiding in blood glucose regulation. The superior performance of CuO NPs A, particularly in glucose uptake at T30 and T120, can be attributed to their smaller size and larger surface area, which enhance their interaction with glucose transporters on the cell membrane, contributing to more efficient glucose absorption and utilization.

The observed increase in glucose uptake in yeast cells treated with CuO NPs can be attributed to several mechanisms, as illustrated in Figure 15. CuO NPs interact with the yeast cell membrane, stimulating the activity of glucose transporters (GLUT) (Figure 15c). These NPs enhance the functionality of glucose transporters, facilitating glucose movement into the cell. This interaction leads to a reduction in extracellular glucose concentration, indicating effective glucose uptake. Enhanced glucose uptake positively contributes to cellular energy production by promoting intracellular glucose utilization [53].

Figure 15

Antidiabetic mechanism of action: (a) Glucose uptake by the cell. (b) Mechanism of inhibition of oxidative stress in the cell by CuO NPs. (c) Mechanism of inhibition of enzymes by CuO NPs. (d) Mechanism of expansion of glucose transporters by CuO NPs.

CuO NPs exhibit inhibitory effects on carbohydrate-digesting enzymes such as α-amylase and α-glucosidase (Figure 15b). By inhibiting these enzymes, CuO NPs reduce the breakdown of complex carbohydrates into glucose, thereby limiting the amount of free glucose available in the extracellular medium. This reduction improves the efficiency of glucose transporters in absorbing available glucose into cells, further enhancing glucose uptake and utilization [57].

CuO NPs interact with ROS produced during cellular metabolism (Figure 15a). By neutralizing ROS, these NPs reduce oxidative stress within the cell, which is known to impair glucose transporter activity. The antioxidant properties of CuO NPs help maintain the integrity of GLUT, enhancing their functionality and promoting efficient glucose absorption. Furthermore, reducing oxidative stress improves mitochondrial function, boosting ATP production and enhancing cellular energy metabolism [55].

The results of this study are consistent with recent reports suggesting that NPs can modulate glucose uptake through their interaction with membrane transporters and enzymatic pathways. The ability of CuO NPs to regulate glucose transport and utilization highlights their potential as therapeutic agents for diabetes management [53,54,56].