1 Introduction

Nanotechnology has become a revolutionary field of research, offering exciting opportunities to design and engineer materials with unique properties at the nanoscale [1, 2]. Nanoparticles possess distinctive qualities due to their size, shape, morphology, and large surface area which contribute to their potential applications [3, 4]. Conventional methods of synthesizing nanoparticles involve costly chemical and physical processes with the risk of causing environmental pollution, cellular toxicity, carcinogenicity, and ecological harm [5, 6]. Therefore, current scientific research has been geared towards less expensive and more eco-friendly synthesis approaches [7, 8]. Green nanoparticle synthesis has recently gained interest as a promising and sustainable alternative to conventional methods, to reduce the environmental impact of nanoparticle manufacturing [9, 10]. To synthesize nanoparticles, three essential requirements must be met: the selection of an environmentally friendly solvent, a suitable reducing agent, and a safe stabilizing substance [7, 11]. These factors are crucial for ensuring efficient and sustainable nanoparticle synthesis [12]. Green synthesis is generally carried out using medicinal plants, bacteria, fungi, herbs, spices, and hybrid materials [5, 13, 14]. These methods are more affordable, environmentally benign, and widely accessible [15].

In recent years, there has been a growing interest in using plant-based methods [16] to synthesize metal nanoparticles due to their wide range of potential applications. The characteristics of these nanomaterials are influenced by the biological and physicochemical properties of the plant-based synthesis process, which relies on specific phytoconstituents acting as reductants or stabilizers [17, 18]. Various plant extracts have been employed for the fabrication of metal nanoparticles such as gold, silver, copper, zinc, iron, nickel, and magnesium [19,20,21,22,23]. These nanoparticles have exhibited notable traits such as anti-diabetic, antibacterial, antiviral, anti-inflammatory, and antioxidant properties [21, 24,25,26]. Furthermore, such nanoparticles have shown catalytic activity because of their different nanostructure and large surface area, with potential application for effluent treatment of industrial dyes [14]. Among these, copper oxide nanoparticles (CuO-NPs) have gained substantial attention due to their versatile applications in medicine, environmental cleanup, and energy utilization [14, 27]. Psidium guajava, commonly known as the guava tree, is prevalent across Africa, Asia, Eastern Europe, and other regions. It belongs to the Magnoliophyta phylum, Magnoliopsida class, and Myrtaceae family, and is cultivated for its juicy fruits, medicinal attributes, and ornamental value. Guava leaves, in particular, contain a rich assortment of bioactive components, including phenolics, flavonoids, tannins, minerals, essential oils, ascorbic acid, proteins, and polysaccharides [28]. These constituents can serve effectively as both reductants and stabilizers in producing metal nanoparticles [29,30,31]. It is hypothesized that guava leaves can be used as a valuable material for the green synthesis of CuO-NPs with potential antibacterial, antidiabetic, antioxidation, and photocatalytic capabilities [32, 33]. The abundance and widespread availability of guava leaves make them an economical and sustainable resource for the eco-friendly synthesis of metal nanoparticles [34]. Incorporating plant extracts during the nanoparticle synthesis process not only enhances the physicochemical properties of the nanoparticles but also adds valuable bioactive components with potential health benefits [35, 36]. The increasing prevalence of chronic diseases, such as diabetes, has prompted the exploration of novel therapeutic strategies [37, 38]. In this context, nanoparticles have shown promise as potential therapeutic agents due to their unique physicochemical properties [24]. The nanoparticles derived from guava extract could offer a new avenue for addressing this global health challenge. Alongside their therapeutic potential, the antibacterial potential of copper oxide nanoparticles is of utmost importance in combating the growing threat of antibiotic resistance. By evaluating the efficacy of these nanoparticles against pathogenic microorganisms, we aim to shed light on their potential as an alternative antimicrobial agent. Additionally, the antioxidant capacity of copper oxide nanoparticles derived from guava leaf extracts is an essential aspect to explore, considering the rising interest in natural antioxidants for various health benefits [25, 30]. Moreover, the ability of these nanoparticles to scavenge free radicals and protect cells from oxidative stress-related diseases is assessed. Furthermore, the field of environmental remediation has benefitted from nanotechnology, with photocatalytic materials being employed to degrade harmful pollutants in water and air. CuO-NPs have demonstrated remarkable photocatalytic activity, especially in the degradation of organic dyes, which are persistent environmental contaminants [39, 40]. The objectives of this research were to synthesize copper oxide nanoparticles by the utilization of leaf extracts from two Psidium guajava varieties (Allahabad Safeda, Hisar Safeda), and to evaluate their capabilities in antibacterial properties, antioxidant activity, anti-diabetic effects, and photocatalytic degradation potential. By harnessing the green synthesis approach, this research contributes to the sustainable fabrication of CuO-NPs, while also exploring their multifaceted applications in healthcare and environmental protection.

2 Materials and methods

2.1 Chemicals, plants, and microbial culture

Leaf samples of Allahabad Safeda and Hisar Safeda were collected from the Agriculture field of Lovely Professional University, Phagwara, Punjab. Chemicals of AR grade such as copper sulfate (CuSO4·5H2O), iodine crystal, sodium hydroxide, hydrochloric acid, nitric acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and lipase were obtained from Sigma Aldrich, St. Louis, MO, USA. The antibiotic ampicillin, muller hinton agar, and nutrient broth were procured from Hi-Media Pvt. Ltd., Mumbai, India. Bacterial cultures including Staphylococcus aureus (MTCC 3160), Streptococcus lactis (MTCC 5461), Escherichia coli (MTCC 443), and Pseudomonas aeruginosa (MTCC 424) were procured from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. Methylene blue (MB) and crystal violet (CV) were purchased from LOBA Chemie Pvt. Ltd. Mumbai, India, and Qualikems Fine Chem Pvt. Ltd. Vadodara, India, respectively. All chemicals used were of analytical reagent grade.

2.2 Preparation of plant leaf extracts

Leaf samples of Psidium guajava specifically Allahabad and Hisar Safeda varieties were collected and dried in an oven at 50 °C. Once dried, the samples were ground into a fine powder and stored in an airtight container for further use. To isolate the compounds from the dried Psidium guajava (varieties) leaf, a modified version of the method by Nagaraja et al. [41] was employed. In the extraction process, 5.0 g of the dried samples was mixed with 100 mL of double-distilled water. The mixture was then gently heated in a water bath at temperatures between 60 and 70 °C for 15 min. Then the mixture was cooled and thoroughly stirred with a magnetic stirrer for 20–30 min to enhance the extraction efficiency. To further separate the components, the resulting solutions were subjected to centrifugation at 10,000 × g rpm for 10 min. Then, the liquid extracts were filtered using Whatman No. 1 filter paper, with the filtrates being stored at 4 °C for further use.

2.3 Synthesis of copper oxide nanoparticles

A 10-mM solution of copper sulfate pentahydrate (CuSO4·5H2O) was prepared, from which 25 mL was used to obtain the same concentration of copper sulfate in all experiments. To optimize the stabilization of copper oxide nanoparticles, different concentrations of Allahabad Safeda extract (1%, 3%, 5%, 7%, and 9%) and Hisar Safeda extract (3%, 5%, 7%, 9%, and 11%) were examined. The synthesis involved mixing the plant extracts with the copper sulfate solution while maintaining constant stirring for 20 min at 500 rpm and a temperature of 80 °C. During this process, a noticeable color change indicated the successful formation of copper nanoparticles (CuO-NPs) as described in the research by Ghosh et al. [13] and Gaber et al. [37].

2.4 Characterization of synthesized copper oxide nanoparticles

Initially, the synthesized copper oxide nanoparticles (CuO-NPs) were characterized for surface plasmon resonance using UV–visible spectroscopy (Evolution 201, Thermo Fischer Scientific India Pvt. Ltd, Mumbai). The UV–visible spectra of CuO-NPs were recorded in the scan mode within a wavelength range of 800–200 nm. Dynamic light scattering (DLS) was employed to determine the particle size of CuO-NPs dispersed in water. The zeta potential of the CuO-NPs with smaller particle sizes was evaluated using a Zeta potential analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, WR14 1XZ, UK). To investigate the morphology of selected CuO-NPs, scanning electron microscopy (JEOL JSM-6510LV instrument from INCA, USA) was used. The elemental analysis of the nanoparticles was performed using energy-dispersive X-ray spectroscopy (RONTEC’s EDS system Model QuanTax 200, USA.) Additionally, Fourier transform infrared spectroscopy (FTIR) analysis was conducted (Cary 630 Agilent) to identify the functional groups present in the CuO-NPs.

2.5 Application of synthesized copper oxide nanoparticles

2.5.1 Antimicrobial efficacy

The CuO-NPs were tested for their antimicrobial properties against four bacterial strains, two Gram-positive strains (Staphylococcus aureus and Streptococcus lactis) and two Gram-negative strains (Escherichia coli and Pseudomonas aeruginosa). The antibacterial activity of CuO-NPs was assessed using an agar well diffusion method [39]. The bacterial cultures were adjusted to a standard concentration of 0.5 McFarland, which is equivalent to a microbial suspension of 1.5 × 108 cfu/mL. For each experiment, Petri plates were filled with 25 mL of muller hinton agar medium and inoculated with the selected microorganisms using the spread plate method. After 30 min, the wells of 6 mm in diameter were carefully drilled into the agar plates and then filled with 100 µL of CuO-NPs reconstituted in DMSO. Negative control experiments were performed using DMSO, while chloramphenicol was used as a standard antibacterial agent (as positive control). Subsequently, all the plates were incubated at 37 °C for 24 h. To ensure accuracy, each experiment was repeated in triplicate. This methodology was followed to minimize errors and obtain reliable results regarding the antimicrobial activity of CuO-NPs against the tested bacterial strains.

2.5.2 Minimum inhibitory concentration and minimum bactericidal concentration

The broth dilution method was utilized to determine the minimum inhibitory concentration (MIC) values of CuO-NPs. In each test tube, 5 mL of nutrient broth was mixed with 50 µL of microorganisms and 500 µL of the CuO-NPs. The control contained only inoculated broth. Then the tubes were incubated at 37 °C for 24 h, and the presence or absence of visible growth was noted. The MIC value was determined as the lowest concentration of the microalgae extract where no visible growth of bacteria was observed in the tubes. The turbidity of the tubes was visually examined both before and after incubation to confirm the MIC value. Once the MIC value was determined, 50-µL samples were taken from the tubes where no visible bacterial growth was observed. These samples were plated onto Muller Hinton agar and incubated at 37 °C for 24 h. After incubation, the agar plates were examined to assess the presence or absence of bacteria.

2.5.3 Antioxidant activity by DPPH scavenging assay

The DPPH radical scavenging assay was conducted to evaluate the antioxidant activity of green-synthesized CuO-NPs by using the protocol by Zafar et al. [42]. To prepare DPPH solution, 2.36 mg of DPPH was mixed with 100 mL of 95% methanol and various concentrations of nanoparticles (20–140 μg/mL) were prepared. In each of the test tubes, 3 mL of the prepared DPPH solution was taken, and then 100 µL respective nanoparticle concentration was added into the test tube. The reaction mixture in each test tube was vigorously shaken and then incubated for 30 min. After incubation, the resulting solutions were measured by UV–visible spectrophotometry at 517-nm wavelength. To establish a comparison, a control sample was prepared containing the same concentration of standard ascorbic acid as used for the test samples. Additionally, a blank sample was prepared using 95% methanol. The inhibition property of the nanoparticles was calculated using Eq. (1):

$$\mathrm{\%\; inhibition}=\frac{{{\text{A}}}_{{\text{control}}-}{{\text{A}}}_{{\text{test}}}}{{{\text{A}}}_{{\text{control}}}}\times 100$$
(1)

where Acontrol is the absorbance of the control reaction, and Atest is the absorbance in the presence of CuO-NPs derived from Allahabad and Hisar Safeda extract.

2.5.4 Antidiabetic assay

The anti-diabetic potential of CuO-NPs was evaluated using α-amylase and α-glucosidase inhibition assay reported by Zafar et al. [42].

α-Amylase inhibition assay

The amylase inhibition activity was assessed using a method described by Khan et al. [43] with slight modification, in a 96-well microplate. Various concentrations of CuO-NPs (10–100 μg/mL) were prepared in DMSO. The synthesized CuO-NPs (20 μL) of different concentrations were added to the test well, followed by the addition of 25 μL of α-amylase solution (50 μg/mL), phosphate buffer (15 μL), and starch (40 μL). Then the mixture was incubated at 50℃ for 30 min. Next, 20 µL of 1 M HCl and 90 µL of iodine solution were loaded into each well. The mixture was then subjected to centrifugation at 3000 rpm for 5 min at 4 °C, from which the supernatant was collected for the optical density measurement at 540 nm in a spectrophotometer. In addition, acarbose was used as a positive control, which is known as an α-amylase inhibitor. Next, 20 µL of 1 M HCl and 90 µL of iodine solution were loaded into each well. The experiments were repeated three times for each concentration to ensure the accuracy. Amylase inhibitory activity was calculated by Eq. (2):

$$Amylase\; inhibition \left(\mathrm{\%}\right)= \frac{{Sample}_{Abs}-{Negative \;control}_{Abs}}{{Blank}_{Abs}-{Negantive \;control}_{Abs}}\times 100$$
(2)

where Abs represents the absorbance, and the concentration required to inhibit 50% of α-amylase (IC50) was determined for acarbose and CuO-NPs derived from Allahabad Safeda and Hisar Safeda extracts.

α-Glucosidase inhibition assay

A glucosidase inhibition assay was conducted to assess the α-glucosidase inhibitory potential of CuO-NPs using a modified procedure from Zafar et al. [42]. To dissolve α-glucosidase, 50 mL phosphate buffer (pH 6.8) and 100 mg of bovine serum albumin were mixed simultaneously. After 5 min, 490 μL of phosphate buffer (pH 6.8) and 250 μL of p-nitrophenyl-d-glucopyranoside (5 mM) were mixed and incubated at 37 °C for 15 min. Subsequently, ZnO-NPs were incubated with 250 μL of α-glucosidase for 15 min at 37 °C. The reaction was terminated using a 2 mL solution of Na2CO3 (200 mM), and the absorbance was measured at 400 nm. The experiment was repeated three times to minimize the error, with acarbose being used as a positive control. Glucosidase inhibition was calculated using Eq. (2), for amylase inhibitory activity.

2.5.5 Photocatalytic dye degradation

The effectiveness of CuO-NPs in the photodegrading of the dyes MB (10 mg/L) and CV (10 mg/L) was evaluated using an experimental procedure based on the method proposed by Guleria et al. [39] with some modifications. The dye solution’s pH was kept at 8.5 using 0.1 N sodium hydroxide and hydrochloric acid solutions. The degradation of both dyes was examined under UV light (Philips, 150 W, wavelength = 200–280 nm), with the light source placed 20 cm away from the dye solution. To achieve absorption/desorption equilibrium, a suspension containing the dye and CuO-NPs was prepared and subjected to constant magnetic stirring at 150 rpm in dark conditions for 20–30 min. The experimental setup involved adding 25 mL of the dye solution and 1.0 mL of the CuO-NPs to a conical flask, which was continuously stirred. After the preparation of the suspensions, they were exposed to UV light irradiation for photodegradation experiments. To monitor the degradation process, 2 mL of the sample was collected at specific time intervals (10, 20, 30, 40, 50, 60, 70, and 80 min) and centrifuged at 10,000 × g for 15 min to obtain the supernatant. The dye concentration in the supernatant was analyzed using UV–visible spectroscopy (Shimadzu 1700) based on the absorption spectra. The changes in the dye concentration over time were used to assess the efficiency of CuO-NPs in photodegrading MB and CV under UV light, by using Eq. (3):

$$\mathrm{Dye\; degradation\; efficiency} \left(\%\right)=\frac{{C}_{0}-{C}_{t}}{{C}_{0}}\times 100$$
(3)

where C0 and Ct were the initial and remaining dye concentrations (mg/L) of the aqueous solution at time 0 and t.

The kinetics of the photocatalytic decolorization rate of MB and CV tended to follow the pseudo-first-order kinetics, as given in Eq. (4):

$$ln\frac{{C}_{0}}{{C}_{t}}=kt$$
(4)

where k is the first-order rate constant (min−1) and was calculated from the slope of ln (Co/Ct) versus irradiation time.

2.5.6 Role of ROS in photocatalytic dye degradation

Experimental protocols by Xu et al. [44] and Guleria et al. [39] were utilized to assess the efficiency of reactive oxygen species (ROS) in degrading the dyes MB (10 mg/L) and CV (10 mg/L). The tests were carried out using the same photocatalytic reaction procedure that had been previously employed for CuO-NPs. The photocatalytic reaction procedure involved combining dye solution (25 mL), CuO-NPs (1 mL), and scavengers such as benzoquinone (BQ), ethylenediaminetetraacetic acid (EDTA), tertiary butyl alcohol (TBA), potassium iodide (KI), and silver nitrate (AgNO3), which were then subjected to UV light irradiation for 80 min. Specifically, BQ was employed to neutralize the superoxide free radical (O2·−), while EDTA was utilized to counteract the formation of holes (h+). In addition, TBA was introduced to eliminate the hydroxyl radical (·OH), and AgNO3 was used to capture the free electrons.

3 Results and discussion

3.1 Synthesis and optimization of copper oxide nanoparticles by Psidium guajava extracts

CuO-NPs were successfully synthesized by reducing copper sulfate pentahydrate using aqueous extracts obtained from Allahabad Safeda and Hisar Safeda leaves. The nanoparticles were stabilized by varying concentrations of Allahabad Safeda extract (1%, 3%, 5%, 7%, and 9%) and Hisar Safeda extract (3%, 5%, 7%, 9%, and 11%) during the synthesis process. The completion of CuO-NP synthesis was confirmed by observing a color change from blue to brown (Fig. 1a), which indicated the occurrence of localized surface plasmon resonance in copper oxide nanoparticles [29, 30]. This phenomenon is a result of the sp-conduction band to the d-band transition of copper oxide nanoparticles [45]. The successful reduction of CuO-NPs with the plant extracts was supported by similar findings from previous studies [41, 46, 47]. The UV–visible spectra analysis was carried out in the wavelength of 800–200 nm (Fig. 1b, c).

Fig. 1
figure 1

a Visible confirmation of CuO-NPs, and UV–visible spectra of CuO-NPs derived from b Allahabad Safeda extract (1–9%) and c Hisar Safeda extract (3–11%)

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3.2 Characterization of copper oxide nanoparticles

3.2.1 Particle size and zeta potential

The particle size results of CuO-NPs derived from Allahabad Safeda and Hisar Safeda are illustrated in Fig. 2. In this analysis, specific concentrations were chosen based on UV–visible spectra. For the CuO-NPs synthesized from Allahabad Safeda extract, different concentrations (3%, 5%, and 7%) were selected for particle size analysis. For CuO-NPs from Hisar Safeda, concentrations of 5%, 7%, and 9% were selected for particle size analysis. The particle size analysis revealed that CuO-NPs from Allahabad Safeda had sizes ranging between 20 and 50 nm (Fig. 2a, c), whereas CuO-NPs from Hisar Safeda showed sizes between 14 and 50 nm (Fig. 2d, f). Based on the smallest particle sizes in Table 1, one concentration from each CuO-NPs sample was chosen for further analysis. Specifically, 3% of Allahabad Safeda with a particle size of 15.88 nm and 7% of Hisar Safeda with a particle size of 14.05 nm were chosen. Additionally, an analysis of the zeta potential was performed on the selected concentrations (Fig. 2g, h).

Fig. 2
figure 2

The average particle size of CuO-NPs synthesized by using a Allahabad Safeda extract of 3%, b 5%, and c 9%; and d Hisar Safeda extract of 5%, e 7%, and f 9%. The zeta potential of CuO-NPs with the least particle size was synthesized using g 3% of Allahabad Safeda and h 7% of Hisar Safeda extracts

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Table 1 The particle size of CuO-NPs derived from different leaf extracts
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3.2.2 Scanning electron microscopy

The morphology and structural characterization of plant-synthesized CuO-NPs nanoparticles were examined using SEM. Figure 3a and d represent the SEM images of the CuO-NPs derived from Allahabad Safeda and Hisar Safeda extracts. The SEM micrographs revealed the presence of CuO-NPs with varying diameters, indicating a polydisperse distribution. These nanoparticles exhibited a variety of shapes, irregular surfaces, and small structures with uneven edges. This diversity in morphology can be attributed to the synthesis process involving the plant extracts. As previously mentioned, the average particle size remained minimal due to the low concentration of copper sulfate, which promoted electrostatic stabilization and prevented aggregation. In addition, the composition of the CuO-NPs synthesized using the green method was analyzed through energy-dispersive X-ray (EDX) analysis and the EDS graph with KeV on the x-axis and peak intensity on the y-axis. As shown in Fig. 3b and e, the EDX spectra show the percentage composition of elements, with major elemental peaks observed at 1 keV, 8 keV, and 9 keV for CuO-NPs derived from Allahabad Safeda extract and at 0.95 keV for CuO-NPs derived from Hisar Safeda extract, which indicated the presence of copper. Additionally, minor peaks corresponding to the biomolecules used for capping the CuO-NPs were also detected.

Fig. 3
figure 3

Characterization of CuO-NPs derived from Allahabad Safeda extract by a scanning electron microscopy, b EDX, and c FTIR analysis; and CuO-NPs from Hisar Safeda extract by d scanning electron microscopy, e EDX, and f FTIR analysis

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3.2.3 Fourier transform infrared spectroscopy

FTIR was used to identify the functional groups present in plant extracts that contribute to the stabilization of CuO-NPs. The FTIR spectra of cupric sulfate solution, extracts from Allahabad Safeda and Hisar Safeda plants, and the resulting copper oxide nanoparticles obtained from these extracts are shown in Fig. 3c and f. The FTIR spectra of cupric sulfate pentahydrate exhibited prominent peaks at 3328.55 cm−1, 2112.10 cm−1, 1639 cm−1, and 586.25 cm−1, which correspond to O–H stretching, H-bonded interactions, C = C stretching, and Cu–O stretching vibrations, respectively [46]. In contrast, the spectra of Allahabad Safeda and Hisar Safeda extracts revealed multiple peaks at 3333, 3226, 230, 1633, 1075, 589, and 2223 cm−1, which signify the presence of diverse bioactive phenolic compounds within the extracts [21], whereas CuO-NPs derived from Allahabad Safeda extract showed a band at 3272 cm−1 indicating O–H stretching and H-bonding [41]. Additional bands at 2119 cm−1 were attributed to C–H stretching and symmetric stretching, while the peaks at 1068 cm−1 and 872 cm−1 indicated C–O stretching, and C–Cl stretching, respectively [46]. In comparison, CuO-NPs synthesized from Hisar Safeda extract displayed a peak at 2115 cm−1, attributed to C = C stretching. Additional bands at 1199 cm−1, 1604 cm−1, and 1648 cm−1 also indicated C = C stretching [48]. An interesting observation was the presence of a band at 1431 cm−1, which was absent in the spectra of synthesized copper oxide nanoparticles. This band was assigned to N–H bending and stretching, possibly originating from an amide group, which may be responsible for the reduction of CuO-NPs. Additionally, a band at 698 cm−1 was identified, associated with C–Cl stretching from an alkyl halide group, which also played a role in the reduction of CuO-NPs. These functional groups are formed through interactions between the plant extracts and CuO-NPs, contributing to the production of plant-synthesized CuO-NPs. This finding is consistent with the work of Islam et al. [49], who identified hydroxyl, alkyne, and alkene groups in plants that were involved in copper nanoparticle synthesis. The elemental analysis further confirmed the presence of significant copper content alongside minor functional compounds.

3.3 Application of synthesized copper oxide nanoparticles

The efficiency of green synthesized CuO-NPs in antimicrobial activity, antioxidant potential, antidiabetic properties, and photocatalytic dye reduction was comprehensively assessed.

3.3.1 Antimicrobial activity

The antibacterial effectiveness of CuO-NPs against various pathogenic bacteria was determined, and the diameter of the inhibition zone around each well is presented in Fig. 4a. When comparing CuO-NPs synthesized from different Psidium guajava (Allahabad Safeda and Hisar Safeda) varieties, the CuO-NPs obtained from Hisar Safeda extract exhibited a significantly lower zone of inhibition compared to CuO-NPs synthesized from Allahabad Safeda extract. Particularly, CuO-NPs derived from Allahabad Safeda extract showed a higher zone of inhibition for Staphylococcus aureus (20.5 mm), Streptococcus lactis (20.7 mm), Escherichia coli (19.5 mm), and Pseudomonas aeruginosa (19.7 mm). In addition, in the positive control group, CuO-NPs synthesized from extracts of both plants exhibited a significant zone of inhibition (24–25 mm) against all the tested bacterial strains. Based on the findings, it can be inferred that CuO-NPs derived from Allahabad Safeda extract are more promising as a potential source of antimicrobial compounds than CuO-NPs derived from Hisar Safeda extract. It is reported that Lysiloma acapulcensise extract-derived Ag-NPs showed a high antibacterial effect against E.coli with the inhibition zone 18.0 ± 1.3 mm, 16.0 ± 1.0 mm for Staphylococcus aureus [7]. Furthermore, a promising wide-spectrum antimicrobial activity was exhibited by graphene oxide from Ziziphus spinna-cristi extracts [50]. CuO-NPs derived from Mangifera indica aqueous extract showed effective antibacterial activities toward both E. coli and S. aureus strains [11]. Moreover, Curcuma longa–derived copper oxide nanoparticles exhibited effective antimicrobial potential against P. aeuroginosa and P. vulgaris [27]. Furthermore, Ullah et al. [51] demonstrated that gold nanoparticles synthesized using Euphorbia wallichii shoot extract possessed potent antibacterial activity against a range of common pathogens, including Escherichia coli, Staphylococcus aureus, Bacillus pumilus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Moreover, Al-Radadi et al. [2] demonstrated the antibacterial potential of silver nanoparticles mediated by using Spirogyra hyalina extract. In addition, according to several studies conducted by Caroling et al. [52], Ntoumba et al. [53], Sathiyavimal et al. [54], Imran et al. [16], Hassan et al. [49], and Dinh et al. [21], Psidium guajava extract contains various active metabolites such as phenolics, flavonoids, tannins, minerals, essential oils, ascorbic acid, proteins, and polysaccharides that inhibit the bacterial growth. CuO-NPs derived from Allahabad Safeda extract exhibited higher antimicrobial activity due to their well-developed chemical stability surface chemistry, allowing effective interaction with the microorganisms. Their smaller size enhances their ability to interact with microorganisms and enhance their antimicrobial properties. CuO-NPs target the outer membrane, leading to structural changes, degradation, and cell lysis [39]. They also hinder microorganisms’ DNA replication, resulting in inhibitory effects [55]. Moreover, their positive charge enables electrostatic interactions with the negatively charged cell membranes of microorganisms, leading to their death. In addition, CuO-NPs derived from herbal extracts produce ROS, which can damage the DNA and cell membranes of microbes, inhibiting their growth [56]. Furthermore, active metabolites from CuO-NPs can disrupt the quorum-sensing systems of bacteria, preventing them from causing infections. CuO-NPs release copper cations that can attach electrostatically to the cell walls of bacteria, and penetrate the surface of the bacterial membrane, further contributing to their bactericidal activity [57, 58]. In summary, CuO-NPs derived from plant extracts possess multiple antimicrobial mechanisms, including physical interactions with cell membranes, inhibition of DNA replication, generation of ROS, and interference with bacterial quorum sensing. These properties make CuO-NPs effective in defending against pathogenic microorganisms [3, 9, 13, 22].

Fig. 4
figure 4

a Zone of inhibition, b minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC), c DPPH scavenging activity, and d amylase inhibitory effects of CuO-NPs derived from Allahabad Safeda and Hisar Safeda extracts

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3.3.2 MIC and MBC values

The MIC is the lowest concentration of test compounds that inhibit the visible growth of microorganisms, whereas minimum bactericidal concentration (MBC) is defined as the lowest concentration of antimicrobial agent that causes a 99.9% reduction in the bacterial population. The results (Fig. 4b) show the MIC and MBC values of CuO-NPs synthesized from the extracts of two different Psidium guajava varieties (Allahabad Safeda, Hisar Safeda). According to the findings, CuO-NPs derived from Allahabad Safeda extract exhibited lower MIC and MBC values than those from Hisar Safeda extracts. Especially, the MIC values of CuO-NPs from Allahabad Safeda extract against S. aureus, S. lactis, E. coli, and P. aeruginosa were found to be 8, 8, 9, and 10 µg/mL respectively. Correspondingly, the MBC values for these bacteria were 10, 10, 11, and 12 µg/mL respectively. In contrast, MIC values of CuO-NPs derived from Hisar Safeda extract were found to be 10, 11, 13, and 13 µg/mL against S. aureus, S. lactis, E. coli, and P. aeruginosa, respectively. Additionally, MBC values for these bacteria were determined to be 12 µg/mL, 13 µg/mL, 15 µg/mL, and 15 µg/mL, respectively.

3.3.3 DPPH scavenging assay

The DPPH assay was conducted to assess the scavenging properties of CuO-NPs synthesized using a green method. The ability of CuO-NPs to act as hydrogen donors is responsible for their scavenging activities. Figure 4c illustrates the results of different concentrations of CuO-NPs, along with ascorbic acid as a positive control. Both CuO-NPs and ascorbic acid demonstrated significant inhibition of DPPH radicals. In this study, CuO-NPs derived from Allahabad Safeda extract exhibited the highest inhibition of 68.23% at a concentration of 70 µg/mL, while CuO-NPs from Hisar Safeda showed inhibition of 62.32% at the same concentration. The positive control, ascorbic acid, showed the highest inhibition of 73.55% at the same concentration. These results suggest that the scavenging activity of CuO-NPs increases with higher sample concentrations, which is consistent with previous findings that higher concentrations of samples lead to increased inhibitory activity [59]. This can be attributed to the presence of phytoconstituents in the crude extract, contributing to the higher antioxidant activity of the CuO-NPs. Numerous researchers have explored the potential of nanoparticles derived from natural sources. Khan et al. [43] demonstrated that cobalt oxide and magnesium oxide nanoparticles synthesized using Hibiscus rosa sinensis extracts exhibited promising antioxidant activity. Similarly, Xu et al. [44] reported excellent antioxidant potential from nanoparticles derived from Psidium guajava. Additionally, Shah et al. [60] and Kokilananthan et al. [61] highlighted the inhibitory activity of gold nanoparticles synthesized from Sageretia thea leaf extract. These studies collectively point towards the potential of naturally derived nanoparticles as antioxidants, opening avenues for further research and development in this promising field. For scavenging the DPPH radicals, the CuO-NPs exhibit three primary mechanisms such as electron transfer, hydrogen atom transfer, and metal-ion complexation. In the electron transfer mechanism, CuO-NPs can act as electron donors for DPPH radicals, causing their inactivation and decolorization [27]. This process is often associated with the presence of Cu(I) ions on the nanoparticle surface. In the case of hydrogen atom transfer, specific surface hydroxyl groups on CuO-NPs can donate hydrogen atoms to DPPH radicals, thereby quenching their free radical character. Additionally, in metal-ion complexation, Cu(II) ions on the nanoparticle surface can form complexes with DPPH radicals, thereby stabilizing and reducing their radical activity, as reported by Peddi et al. [62], Luque et al. [63], Kaningini [45], and Abdullah et al. [1].

3.3.4 α-Amylase and α-glucosidase inhibition assay

The results of α-amylase and α-glucosidase inhibition assays for different concentrations of CuO-NPs synthesized from Allahabad Safeda, Hisar Safeda extract, and acarbose are provided in Fig. 4d and e, and Tables 2 and 3. In the α-amylase inhibition assay, acarbose of 100 μg/mL showed 77.98% inhibitory effects with an IC50 value of 55.32 μg/mL. CuO-NPs derived from Allahabad Safeda exhibited an inhibition effect of 67.32% at a concentration of 100 µg/mL, with an IC50 value of 65.21 µg/mL. Such an inhibition is more significant (P < 0.005) than by CuO-NPs from Hisar Safeda which showed 63.25% inhibition and IC50 of 70.26 µg/mL. In the α-glucosidase inhibition assay, acarbose demonstrated a notable inhibitory effect, with 80.56% inhibition at a concentration of 100 μg/mL and an IC50 value of 53.32 μg/mL. CuO-NPs from Allahabad Safeda exhibited a higher inhibition effect (75.18% inhibition at 100 µg/mL and IC50 value of 60.59 µg/mL) than CuO-NPs from Hisar Safeda (71.38% inhibition and an IC50 of 65.62 µg/mL). These results indicate the promising inhibitory potential of CuO-NPs from both Safeda extracts against α-amylase and α-glucosidase activity. Recent research suggests nanoparticles hold promise in combating diabetes. Studies by Faisal et al. [14] and Al-Radadi et al. [2] demonstrated the anti-diabetic potential of zinc oxide nanoparticles (ZnO-NPs). Radadi et al. explored ZnO-NPs specifically derived from Zingiber officinale (ginger). Zafar et al. [42] reported iron oxide nanoparticles (FeO-NPs) derived from Enterobacter biomass showing anti-diabetic activity. Recently, diabetes mellitus has become the most common endocrine disorder in India, attributed to people’s lifestyle and genetic disorders. This condition causes morbidity and mortality globally, accompanied by a range of associated complications [24, 59, 63]. The digestive enzymes like α-amylase and α-glucosidase can hydrolyze starch, which is involved in postprandial hyperglycemia [31]. The inhibition of these two enzymes by nanomaterials can reduce the release and absorption of glucose in the small intestine, leading to anti-diabetic activity [64]. The main mechanisms shown by the extracts are to decrease the digestion of glucose and carbohydrates, which targets reducing postprandial hyperglycemia, and to reduce the hydrolysis process in the enzymatic reaction of carbohydrate complexes [65].

Table 2 α-Amylase inhibitory effects of acarbose and CuO-NPs derived from Allahabad Safeda and Hisar Safeda extracts
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Table 3 α-Glucosidase inhibitory effects of acarbose and CuO-NPs derived from Allahabad Safeda and Hisar Safeda extracts
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3.3.5 Photocatalytic dye degradation

Once the CuO-NPs were synthesized using extracts from two different guava varieties, their photodegradation of MB and CV under the irradiation of UV light was evaluated. The spectral changes were observed for MB at various time intervals while being irradiated by the UV light (Fig. 5a, b). It was observed that CuO-NPs derived from Allahabad Safeda extract were more effective in degrading MB (82.31%) compared to CuO-NPs synthesized from the extracts of Hisar Safeda (72.69%), at the end of 150 min (Fig. 5c). The findings highlighted the superior photocatalytic activity of CuO-NPs derived from Allahabad Safeda extract under UV irradiation. To further assess the impact of the CuO-NPs, a controlled study without CuO-NPs was also performed under UV irradiation, where no obvious spectral changes were observed, indicating very limited dye degradation. In addition, the photodegradation efficiency was only 17% under irradiation of light at the end of 150 min. Furthermore, the photodegradation kinetics results showed that the degradation rate under CuO-NPs was relatively constant over time. The kinetics of the MB photocatalytic decolorization were fitted to the Langmuirnear–Hinshelwood model, suggesting that the use of CuO-NPs could be effective for the efficient degradation of dyes under the irradiation of UV light. The photodegradation kinetics parameters for dye removal are shown in Table 4.

Fig. 5
figure 5

MB photodegradation by CuO-NPs derived from a Allahabad Safeda and b Hisar Safeda extracts under UV irradiation, c MB photodegradation (%) by CuO-NPs; CV photodegradation by CuO-NPs derived from d Allahabad Safeda and e Hisar Safeda extracts under UV irradiation, f CV photodegradation (%) by CuO-NPs

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Table 4 UV photodegradation kinetics of MB and CV by CuO-NPs synthesized from Allahabad Safeda and Hisar Safeda extracts
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Similar to MB, CV photodegradation under UV light was examined (Fig. 5d, e). CuO-NPs derived from Allahabad Safeda extract showed a higher photodegradation of CV than from Hisar Safeda extract (Fig. 5g). Based on the UV photodegradation kinetics, a higher removal of CV was obtained by CuO-NPs from Allahabad Safeda extract (88.54%) than by CuO-NPs from Hisar Safeda extract (76.78%) at the end of experiments (Fig. 5f). Furthermore, a controlled study without CuO-NPs was also performed under UV irradiation, showing minimal spectral changes and little dye degradation. Specifically, the photodegradation efficiency was only 16% after 150 min of light irradiation. The results show that the degradation rate under CuO-NPs was relatively constant over time. The kinetics of the CV photocatalytic removal also followed the Langmuirnear–Hinshelwood model, as shown in Table 3.

The dye degradation potential of various metal oxide nanoparticles synthesized using diverse plant extracts has been reported. The findings of this study are in line with Muthukumar and Matheswaran [46] who demonstrated the potential of iron oxide nanoparticles derived from Amaranthus spinosus leaf extract, resulting in 75% and 69% decolorization of methyl orange and MB respectively. Faisal et al. [28] synthesized ZnO-NPs which showed 88% of MB degradation within 140 min. Chandraker et al. [7] utilized Ageratum houstonianum leaf extract to deriv  CuO-NPs and evaluated their degradation potential against azo dye and congo red dye. Moreover, Vishnu et al. [66] investigated the potential of a cobalt-doped cadmium ferrite nanoparticle for the degradation of MB resulting in 60% degradation after 2 h. In addition, Oliveira Gaber et al. [67], Sibhatu et al. [65], Selvam et al. and Kumar et al. [68] have also investigated the potential of different nanoparticles in the degradation of various dyes, showing effective dye degradation.

3.3.6 Mechanisms of dye degradation by CuO-NPs

The dye degradation process on the surface of CuO-NPs initiates with the absorption of a photon with energy equal to or greater than the band-gap energy of the CuO-NPs, as described by Rezaie et al. [69]. This photon absorption leads to the excitation of an electron (e−) from the valence band to the conduction band of CuO-NPs, resulting in the generation of a hole (h+) in the valence band. As a result of the generation of electrons and holes, reduction–oxidation reactions take place on the surface of CuO-NPs [70]. In the reduction reaction, the electrons in the conduction band can either reduce the dye or react with electron acceptors, such as O2 molecules adsorbed on Cu(II) surface or dissolved in water, which results in the reduction of O2 to superoxide radical anion (O2−·). In the oxidation reaction, the holes in the valence band can oxidize organic molecules, forming R+ species, or react with OH− ions or water, oxidizing them into hydroxyl radicals (OH·) [71]. These highly reactive species, including peroxide radicals, play a crucial role in the photo-decolorization of organic substrates such as dyes on CuO-NPs surfaces [72]. The resulting OH· radicals are an extremely potent oxidizing agent and can efficiently oxidize many azo dyes, ultimately converting them into mineral end-products. Consequently, the relevant reactions occurring at the CuO surface responsible for the decomposition of dyes can be expressed as follows, in alignment with the findings of Wardani et al. [73], Rezaie et al. [69], Faisal et al. [28], and Guleria et al. [39].

Reduction reaction:

$${{\text{e}}}^{-}({\text{conduction}} \, {\text{band}})+{{\text{O}}}_{2}\to {{{\text{O}}}_{2}}^{-\cdot }({\text{superoxide}} \, {\text{radical}} \, {\text{anion}})$$

Oxidation reaction:

$${{\text{h}}}^{+}({\text{valence}} \, {\text{band}})+{\text{Organic}} \, {\text{molecule}}\to {{\text{R}}}^{+}({\text{oxidized}} \, {\text{product}})$$
$${{\text{h}}}^{+}({\text{valence}} \, {\text{band}})+{{\text{OH}}}^{-}{\text{or}} \, {{\text{H}}}_{2}{\text{O}}\to {\text{OH}}\cdot ({\text{hydroxyl}} \, {\text{radical}})$$

These reactions collectively lead to the degradation and decolorization of organic dyes on CuO-NP surface through a series of reduction and oxidation processes.

3.3.7 Role of ROS in photocatalytic dye degradation

The scavenging activity results (Fig. 6) indicate the significant role of scavengers on the photodegradation of dyes (MB and CV) using CuO-NPs derived from Allahabad Safeda and Hisar Safeda extracts. The results revealed the critical role of superoxide and hydroxyl radicals in the photodegradation of both dyes. CuO-NPs from Allahabad Safeda extract presented degradation rates of 52.31% MB and 50.1% CV dyes when BQ was used as the scavenger. Similarly, in the presence of TBA scavenger, the degradation rate reached 51.23% and 48.23% for MB and CV dyes. In contrast, scavengers such as EDTA and silver nitrate, which scavenge holes and free electrons, had a minor impact on the degradation process (Fig. 6a, b). In the presence of EDTA and silver nitrate scavengers, MB degradation was 79.29% and 75.62% while CV degradation was 77.75% and 74.23%, respectively. On the other hand, using CuO-NPs derived from Hisar Safeda extract, the photodegradation of MB and CV dyes was significantly influenced by the presence of TBA and BQ scavengers (Fig. 6c, d). The utilization of TBA as a scavenger resulted in a degradation rate of 51.32% for MB and 50.61% for CV. Furthermore, in the presence of a BQ scavenger, the degradation rates reached 48.59% MB and 48.99% CV, highlighting the significant role of superoxide free radicals in the photodegradation process. In addition, scavengers like EDTA and silver nitrate had a limited impact on dye photodegradation (Fig. 6c). In the presence of EDTA as a scavenger, MB and CV photodegradation occurred at 68.21% and 66.32%, while for silver nitrate scavengers, the degradation rates were recorded at 64.32% of MB and 62.12% of CV.

Fig. 6
figure 6

Impact of scavengers on the photodegradation of dye by CuO-NPs derived from (a–b) Allahabad Safeda, and (c–d) Hisar Safeda extracts

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4 Conclusions

This research investigated the utilization of two varieties of Psidium guajava (Allahabad Safeda and Hisar Safeda) leaf extracts for the eco-friendly synthesis of CuO-NPs. The bioactive compounds in Allahabad Safeda and Hisar Safeda extracts played a critical role in shaping the nanoparticle formation process. The CuO-NPs synthesized using 3% Allahabad Safeda extract and 7% Hisar Safeda extract generated a particle size of 15.88 nm and 14.05 nm. The functional groups in CuO-NPs were identified through FTIR characterization. Notably, CuO-NPs derived from Allahabad Safeda extract displayed enhanced capabilities than those from Hisar Safeda extract, presenting remarkable antimicrobial activity against bacteria S. aureus, S. lactis, E. coli, and P. aeruginosa, with inhibition zones of 20.5 mm, 20.7 mm, 19.5 mm, and 19.7 mm, respectively. Additionally, these nanoparticles displayed significant DPPH scavenging activity (68.23%), indicative of their strong antioxidation properties. CuO-NPs derived from Allahabad Safeda achieved 71.45% inhibition of amylase activity at 100 µg/mL. Furthermore, CuO-NPs derived from Allahabad Safeda exhibited significant photodegradation of MB (82.31%) and CV (88.54%) in 150 min. In summary, the results demonstrated environmentally friendly CuO-NPs synthesis using Psidium guajava, which offered promising avenues for novel and sustainable approaches to tackle current global challenges in environmental protection.