Fabrication of Multifunctional Green-Synthesized Copper Oxide Nanoparticles Using Rumex vesicarius L. Leaves for Enhanced Photocatalytic and Biomedical Applications

Abstract

Recently, the use of plant extracts has emerged as an innovative approach for the production of various nanoparticles. Enhancing green methods for synthesizing copper oxide (CuO) nanoparticles (NPs) is a key focus in the field of nanotechnology. This study presents a novel and eco-friendly synthesis of CuO NPs using Rumex vesicarius L. leaf extracts, offering a cost-effective and efficient method. The synthesized CuO NPs were evaluated for their cytotoxic effects against human cervical carcinoma (HeLa) cells, as well as their photocatalytic and antimicrobial activities. The morphology, size, and structural properties of the CuO NPs were characterized using various analytical techniques. X-ray diffraction (XRD) analysis confirmed the pure crystalline structure of the CuO NPs with a size of 19 nm, while transmission electron microscopy (TEM) showed particle sizes ranging from 5 to 200 nm. The photocatalytic performance of the CuO NPs was assessed through the photodegradation of crystal violet (CV) and methylene blue (MB) dyes under UV light. The NPs exhibited excellent decolorization efficiency, effectively degrading dyes in aqueous solutions under irradiation. Furthermore, the green-synthesized CuO NPs displayed strong antibacterial and antifungal activities against a variety of human pathogens. They also demonstrated significant dose-dependent cytotoxicity against the HeLa cancer cell line, with an IC₅₀ value of 8 ± 0.54 μg/mL.

1. Introduction

Nanotechnology is an emerging field that integrates various disciplines of science, physics, chemistry, biology, materials science, and medicine. It primarily focuses on the nanonization of metals and their oxides to synthesize particles of varying dimensions chemical compositions, and dispersities [1,2]. Many researchers all over the world have begun to study and exploit the versatile applications offered by nanotechnology. Much effort has been put towards the synthesis and characterization of metal and metal oxide nanomaterials to generate novel materials that demonstrate electronic and optical properties which are different from those observed on the bulk scale. Nanomaterials have recently garnered immense attention for their potential biomedical applications including medicine, agriculture, food, cosmetics, paints, catalysis, and textiles [3,4].

Recently, metal oxide nanoparticles have been widely used in research, with researchers exploring their ability to alter the physical, optical, and electronic properties of compounds [5,6]. Among various types of transition metal nanoparticles, copper oxide nanoparticles (CuO NPs) are considered to be one of the most important metal oxide nanomaterials [7]. CuO is a native p-type semiconductor with a band gap in the range of 1.2–2.6 eV, which facilitates the absorbance of radiation in the visible region [8]. CuO NPs are viable candidates in several fields and have shown considerable utilization for their role in nanofluids, sensors, antimicrobial applications, catalysis, superconductors, energy storage systems, and as anticancer agents [9]. For example, the decomposition of synthetic dyes and organic effluents in wastewater using nanoparticles has been widely explored by researchers in the field of catalysis. Among many photocatalysts, CuO is an ideal photocatalytic system for the remediation of environmental contaminants because it is inexpensive, nontoxic, efficient, photostable, and abundant, and because it generates high photocatalytic performance under a solar spectrum [10]. In recent years, CuO NPs have been at the forefront owing to their antimicrobial and biocidal properties and are widely used for biomedical applications. CuO NPs have demonstrated significant microbial properties against microorganisms such as Bacillus, Staphylococcus aureus, Escherichia coli, and Pseudomonas bacteria [11,12], and a prominent fungicidal influence on Penicillium spp. [13]. CuO NPs have also been used for their antioxidant status and cytotoxic activity, for example in the antitumor activity that has been demonstrated preclinically in various cancer types, including hepatocarcinoma, lung carcinoma (A549), nasopharynx cancer, breast cancer, cervical carcinoma (HeLa), and pancreatic cancer [14,15,16].

The mode of synthesis plays a key role in the field of nanotechnology. There have been several techniques reported for the fabrication of CuO NPs, which are categorized as physical, biological, and chemical methods [17]. Recently, biosynthesis has emerged as an important mode in the preparation of metal oxide nanoparticles by excluding the use of toxic chemicals produced by chemical reactions and avoiding the use of organic solvents. Current trends in research indicate that plant-derived metal nanoparticles are safe, reliable, and eco-friendly compared with the physical or chemical systems. Plant-mediated nanoparticle synthesis is also inexpensive and therefore economically viable for large-scale production [18]. Several studies on the green synthesis of CuO NPs using plant extracts have been published, including Musa acuminata [19], Aglaia elaeagnoidea flower [20], Aloe vera [21], Saraca indica leaves [22], Piper betle [23], and Carica papaya [24].

Rumex vesicarius L. (R. vesicarius), commonly known as bladder dock (Arabic: Humeidh), is an annual plant belonging to the family Polygonaceae. The word Rumex takes its origin from the Latin word for dart, suggesting the shape of the leaves [25]. Previous literature reports numerous ethnobotanical and ethnopharmacological studies referring to the occurrence and traditional uses of Rumex species [26]. R. vesicarius is widely utilized as a medicinal and culinary herb. It is widely found throughout Saudi Arabia [27]. R. vesicarius was identified as a plant that showed a significant level of antiangiogenic activity [28], and it is used in traditional medicine as an antiflatulent, tonic, digestion enhancer, laxative, antiemetic, analgesic, and an antiangiogenic agent, and for the treatment of bronchitis, spleen disorders, asthma, and some hepatic diseases prevalent in Egypt, India, and Saudi Arabia [29].

With this premise, the present study focused on synthesizing CuO nanoparticles using green chemistry, with Rumex vesicarius extract serving a stabilizing and shaping role in the process. The synthesized CuO NPs were characterized with advanced techniques, including X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), and photoluminescence (PL) spectroscopy. The major objectives of the present study are to investigate the diverse applications of the prepared CuO NPs. These include evaluating their photocatalytic efficiency in degrading pollutant dyes, specifically crystal violet (CV) and methylene blue (MB), assessing their antimicrobial activity against both Gram-negative and Gram-positive bacteria as well as three fungal strains, and investigating their cytotoxic effects on the HeLa human cervical cancer cell line. To our knowledge, this is the first report on the phytomediated synthesis of CuO NPs using an aqueous R. vesicarius extract [30].

2. Results and Discussion

2.1. Analysis of the Optical Properties of Synthesized CuO NPs

The optical absorption properties of the synthesized CuO NPs were determined using UV–Vis and photoluminescence (PL) spectroscopies. The UV–Vis spectra of the resulting CuO NPs exhibited absorption peaks in the range of 200–600 nm. The absorption spectra of the synthesized CuO NPs showed two peaks (Figure 1a), which is in line with previous reports [30,31]. The sharp absorption peak at 279 nm in Figure 1a corresponds to the surface plasmon resonance (SPR) of CuO NPs, which is attributed to the oscillation of surface conduction electrons excited by the incident electromagnetic radiation [32]. The weak and broad peak observed at 377 nm was attributed to the SPR band of some metallic Cu colloids from nonoxidized Cu NPs [33]. This is within the framework of Mie’s theory that postulates that the SPR frequency or wavelength depends on the sizes and shapes of the nanoparticles; spherical nanoparticles have a single SPR band [34,35].

The photoluminescence (PL) spectrum of synthesized CuO NPs is shown in Figure 1b, where the excitation wavelength was 279 nm. The PL spectra of the synthesized CuO NPs displayed a sharp emission peak in the blue region at 450 nm, which is attributed to singly ionized Cu vacancies [36]. Muthuvel et al. reported that in green-synthesized CuO NPs oxygen vacancies and defects will bind to a photoinduced electron to easily form excitons, leading to a decrease in the PL intensity. Overall, the enhanced PL intensity and the highly crystalline nature of the green-synthesized CuO NPs are desirable properties for use in catalysis [37].

The band gap of the green-synthesized CuO NPs was determined from the absorption data using the Tauc plot method, as written in Equation (1):

$$\left(\alpha h\nu \right)={A\left({h\nu -E}_g\right)}^n$$

(1)

where $\alpha$ is the absorption coefficient, h is Planck’s constant, ν is the frequency, A is a constant related to the material and the matrix element of the transition, $E_g$ is the band gap, and n is a coefficient that depends on the nature of the transition, where n = 1/2 for a direct transition [38]. For indirect band gap materials, the energy gap is determined by plotting (Ahν)^1/2 versus hν and finding the intercept on the hν axis. The best linear relationship is obtained by plotting against photon energy (hν), indicating that the absorption edge is due to a direct allowed transition in the CuO NPs. From the Tauc plot, the indirect band gaps were determined to be approximately 3.52 and 2.93 eV, as shown in Figure 1c. These results are in moderate consensus with the data reported by Agam et al. and Talluri et al. [39,40].

2.2. Dynamic Light Scattering (DLS) Measurements

DLS is an advanced tool used to analyze the size and distribution of synthesized nanoparticles, and it is one of the most commonly used techniques to determine the size of nanoparticles. The Brownian motion of nanoparticles disperses the light at varying intensities. By analyzing these dispersed light intensities, DLS can be used to determine the average size of the nanoparticles in solution. The size distribution ranged from about 90 to 200 nm (Figure 2), and the polydispersity index indicated narrowly sized particles with agglomeration and diversity in size.

2.3. TEM and HR-TEM Analysis of Synthesized CuO NPs

The particle size and morphology of the synthesized nanoparticles were determined using TEM. The TEM micrographs (Figure 3a) show that the synthesized CuO NPs are roughly spherical and irregular in shape, with variable sizes and some degree of agglomeration, corresponding with the UV–Vis analysis. Further analysis using high-resolution TEM (HR-TEM) showed the lattice fringes of the nanoparticles (Figure 3b), indicating the crystalline nature of the synthesized nanoparticles.

2.4. Structural Analysis of Synthesized CuO NPs

The synthesized CuO NPs were characterized using a variety of techniques. The XRD pattern of the CuO NPs, prepared using copper acetate, is presented in Figure 4. XRD measurements of the synthesized CuO NPs were obtained by coating glass substrates with the NPs in the form of a powder. The spectrometer was a Bruker D8 ADVANCE X-ray diffractometer operating at 40 kV and 40 mA with Cu K<alpha> radiation at a wavelength of 1.5418 Å. The XRD analysis indicated major diffraction peaks at 2θ values of 34°, 38°, 48°, 58°, 61°, 68°, and 74°. The peaks at 38°, 58°, and 74° correspond to the crystal planes (111), (200), and (220), respectively, of the cubic phase of CuO NPs. The other peaks are assumed to be the reflection lines of monoclinic CuO NPs. The pattern (COD 9012954) is consistent with the planes [41,42]. The crystallite size (L) of synthesized CuO NPs was found to be 19.8 nm, calculated from XRD data using Scherrer’s equation:

$$A={\displaystyle \frac{K\lambda }{\beta \mathrm{c}\mathrm{o}\mathrm{s}\left(\theta \right)}}$$

where A is the crystallite size (in nanometers), K is the Scherrer constant, λ is the wavelength of the X-rays, β is the full width at half maximum (FWHM) of the diffraction peak in radians, and θ is the Bragg angle (the angle corresponding to the peak position).

2.5. EDX Analysis of Synthesized CuO NPs

EDX and elementary mapping were used to determine the purity and elemental composition of the NPs. The constituent elements of the as prepared CuO NPs were determined by EDX analysis carried out at 8 keV and are shown in Figure 5. The measurements revealed the presence of copper (Cu) and oxygen (O) elements in the CuO NPs and indicated that the nanoparticles were nearly stoichiometric. There were no other elemental impurities found in the EDX spectra, confirming the formation of pure CuO NPs. The elemental analysis of the sample showed that the prepared sample was copper oxide, which is in good agreement with the XRD analysis. In the EDX spectra, a peak was observed at 8 keV, which was attributed to the absorption of copper oxide nanocrystallites arising from SPR [43]. The optical absorption band for the NPs was in the range of 1 to 9 keV, which is typical for the absorption of CuO NPs [44].

2.6. Cytotoxicity Assessments

The antiproliferative activity of the synthesized CuO NPs was evaluated by an in vitro assay with the HeLa cell line. The MTT assay was employed to assess the anticancer activity of the CuO NPs at different concentrations (5, 10, 20, 30, 50, and 100 µg mL⁻¹) against the HeLa cells. The results of the MTT assay indicated that the synthesized CuO NPs significantly decreased the viability of a cervical adenocarcinoma cell line (HeLa) in a dose-dependent manner compared to the R. vesicarius leaf extract. The inhibitory concentration, IC₅₀ (cytotoxicity level of 50% dead/alive cells), was observed to be >10 μg mL⁻¹ for the CuO NPs and >100 μg mL⁻¹ for the R. vesicarius extract. At this concentration, the CuO NPs showed a remarkable inhibition of growth of the HeLa cells by 50%. These results indicate that CuO NPs have a potent cytotoxic effect as compared to R. vesicarius leaf extract against the HeLa cell line (Figure 6).

The cytotoxicity results show significant agreement with previous studies. However, CuO NPs synthesized from R. vesicarius showed more profound cytotoxicity compared to the findings reported in other studies that used green-synthesized CuO NPs. Vinardell Rehana et al. reported the synthesis of CuO NPs using a green approach with different plant extracts and compared their cytotoxic efficacy against the HeLa cell line and other cell lines [45,46]. Nagajyothi et al. reported that CuO NPs efficiently reduced the proliferation of HeLa cells via apoptosis [47]. Synthesized CuO NPs in the present study showed higher cytotoxic potential against all cell lines in a dose-dependent manner with IC₅₀ > 10 μg mL⁻¹ than these reported by Vinardell and Nagajyothi. Previous studies also reported that the nanoparticles showed no toxicity on normal human dermal fibroblast cells [45,47]. Some studies reported that cancer cells treated with CuO NPs displayed an altered morphology of the mitochondria, where they exhibited condensed clump structures. Condensed nuclei and the clumped structure of the cancer cell mitochondria indicate apoptosis, i.e., reactive oxygen species (ROS)-mediated DNA and mitochondrial damage leading to apoptosis [15,48]. Others reports confirm that CuO NPs may cause disarray in the cellular integrity of cancer cells by damaging their DNA and other vital molecules required for successful survival and progression. The CuO NPs were found to induce cytotoxicity in a human carcinoma cell line in a dose-dependent manner, which is likely mediated through ROS generation and oxidative stress [49]. Overall, the findings suggest that the release of copper ions contributes to CuO NP-induced vascular endothelial cell death [50].

2.7. Photodegradation Results

The photocatalytic activity of the CuO NPs is demonstrated in Figure 7a,b. The synthesized CuO NPs showed a DE for the CV dye of 92.126% after 36 h under UV irradiation (Figure 7a) and 99.8435% for the MB dye after 12 h (Figure 7b). For the CV dye, after 4 h of UV irradiation a weak indication of Cu⁺ ion generation was detected by a change in the degradation percentage, and after 32 h a complete disappearance of color was observed. For the MB dye, after 4 h of UV irradiation a strong indication of Cu⁺ ion generation was detected by a change in the degradation percentage, and after 12 h a complete disappearance of color was observed. In the presence of CuO NPs, the primary absorption peak of the dyes decreased gradually with an increase of UV exposure time. This demonstrates the photocatalytic degradation of the CV and MB dyes in the presence of CuO NPs. Previous reports showed that the crystallographic structure, morphology, and size of the particles affects the photocatalytic activity of metallic nanoparticles [51]. Seerangaraj et al. [35] reported that CuO NPs synthesized using Ruellia tuberosa were effective as coating agents over cotton fabrics and in the photocatalytic degradation of CV dye under direct sunlight. Similarly, Roy et al. reported the photodegradation of methylene blue and Congo red after 72 h using CuO NPs synthesized with Impatiens balsamina leaf extract [52]. Navid Rabiee et al. also demonstrated the degradation of MB dye by CuO NPs biosynthesized from Achillea millefolium leaf extract [53].

According to Lahmar and Vivek [54,55], the possible photocatalytic degradation mechanism for MB and CV dyes using CuO NPs is shown in Figure 8 and below in Equations (2)–(11):

CuO + hv → (h⁺) + (e⁻)

(2)

e⁻ + O₂ → O₂^•−

(3)

H₂O + O₂^•− → OOH^• + OH⁻

(4)

2OOH^• → O₂ + H₂O₂

(5)

H₂O₂ + e⁻ → HO^• + OH⁻

(6)

H₂O₂ + O₂^•− → HO^• + OH⁻ + O²⁻

(7)

H₂O + h⁺ → OH^• + H⁺

(8)

OH⁻ + h⁺ → OH^•

(9)

Dye + h⁺ → Dye⁺

(10)

Dye + (O₂^•−, OOH⁻ or OH^•) → degradation products + CO₂ + H₂O

(11)

The photochemical reaction of UV light with CuO NPs leads to the formation of holes (h⁺) and electrons (e⁻) (Equation (2)). UV light excites an electron from the valence band (VB) to the conduction band (CB) when the energy of the light is equal to or greater than the band gap of the CuO nanoparticles. The movement of the excited electron from the VB to the CB produces a hole (h⁺) in the VB and an electron (e⁻) in the CB. The photogenerated electron in the CB acts as a reducing agent in the reaction with an oxygen molecule (O₂) to generate the superoxide anion (O₂^•−) (Equation (3)), hydroperoxide radical (Equation (4)), and hydrogen peroxide (Equation (5)). In the VB, the photogenerated hole acts as an oxidizing agent to form hydroxyl radicals (OH^•) from water. In addition, the number of hydroxyl radicals can be increased by the degradation of hydrogen peroxide or hydroperoxide radicals (Equations (6) and (7)). The resulting ROS, including hydroxyl radicals (OH^•−) (Equation (9)) and superoxide anions (O₂^•−) (Equation (3)), generated by the oxidation and reduction processes are responsible for degrading the CV or MB dyes to water and carbon dioxide under UV light irradiation [56]. The efficient degradation of the organic dyes in aqueous solution by the CuO NPs synthesized using R. vesicarius leaf extract suggests a wide range of additional photocatalytic applications, such as water remediation and pollution control.

A comparison of the photocatalytic activity of copper oxide synthesized in this study is made with copper oxide prepared using various plant extracts, as well as with other metal oxides, such as titanium and zinc, synthesized through different methods and commercially available as shown in

Table 1. Performance of the synthesized CuO NPs compared with published data on CuO prepared using plant extracts and other metal oxides (ZnO and TiO₂ nanoparticles) under UV and sunlight irradiation.

Decolorization of Dye (%)	Irradiation Time (min)	Source of Light	Band Gap (eV)	Particles Size (nm)/Morphology	Nano-Material	Source of Plants
84%/MB dye	150	Solar	2.23	11/agglomerated shape	CuO NPs	Amaranthus dubius leaf [57]
84.23%/methyl orange dye	120	Lamp	3.1	58.7/spherical shape	CuO NPs	Tribulus terrestris seed [58]
97.35%/AR88 dye	80	UV	2.59	36/irregular morphology shape	CuO NPs	Arundinaria gigantea (giant cane) [59]
72%/methyl orange dye	240	Sunlight	-	27.99/less agglomeration	CuO NPs	Lemon peel [60]
96%/MB and 99%/methyl orange dyes	540	Sunlight	3.57 (330 nm)	32/rectangular shape	CuO NPs	Aegle marmelos leaf [61]
86%/RB21 dye	60	UV	2.04	25/spherical shape	CuO NPs	Tragacanth gum [62]
76%/MB dye	240	Sunlight	-	30–40/agglomerated NPs	CuO NPs	Elaeagnus indica leaf [63]
91%/aniline blue dye	1000	Sunlight	3.56	20–80/spherical shapes	CuO NPs	Santa Maria feverfew [64]
82.31%/MB dye and 88.54%/CV dye	150	UV	-	15.88 nm/irregular surface shape	CuO NPs	Allahabad Safeda [65]
88.37%/MB dye	90	UV	2.97	55.73/fakes and irregular spherical shape	ZnO NPs	Trigonella foenum-graecum aqueous seed [66]
97%/rhodamine B dye	160	UV	3.6	35/nanorod needle shape	ZnO NPs	Cymbopogon proximus [67]
75.8%/acid red-88 (AR-88) dye	120	UV	2.79	30/irregular shape	ZnO NPs	Aloe vera latex [68]
79%/MB dye	30	UV	3.38 (366 nm)	582.35 ± 52.40 nm/flower-shape	ZnO NPs	Stevia rebaudiana leaves [69]
88%/red-141 azo dye	120	UV	3.36	22.13/spherical shape	ZnO NPs	Chemical method [70]
93.1%/CV dye, 90.6%/MB dye, 76.7%/methyl orange, 72.4%/alizarin red	240	UV	3.58	36–81/spherical shape	TiO2 NPs	Ludwigia octovalvis [71]
56%/MB	40	UV	3.47	15–28/spherical morphology	TiO2 NPs	Nervilia aragoana leaf [72]
97%/MB, 99%/methyl orange	90	Sunlight	3.2	22/spherical shape	TiO2 NPs	Wrightia tinctoria [73]
47% and 32%/MB	240	UV	-	32.3/spherical shape	TiO2 NPs	Commercial P25 [74]
92% CV dye, 99% MB dye	36 h, and 12 h	UV	3.52, 2.93	19.8/roughly spherical and irregular in shape	CuO NPs	Rumex vesicarius L. [current study]

.

2.8. Evaluation of the Antimicrobial Activity of Synthesized CuO NPs

The antimicrobial activity of CuO NPs synthesized using R. vesicarius leaf extract was assessed using a standard well-diffusion method with Mueller–Hinton agar media for various strains of microbes. The Gram-positive bacterial strains, Gram-negative Bacillus cereus (B. cereus) and Staphylococcus aureus (S. aureus), and the Gram-negative bacterial species Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) were used as model bacterial species, while Helminthosporium, Alternaria alternata (A. alternata), and Fusarium oxysporum (F. oxysporum) were the model fungal species used to investigate the antimicrobial activity of the green-synthesized CuO NPs. The antimicrobial activities of the synthesized CuO NPs are shown in Figure 9.

The in vitro antibacterial activity of the CuO NPs against E. coli, P. aeruginosa, S. aureus, and B. cereus were assessed by determining their zones of inhibition. The synthesized CuO NPs at a concentration of 10 µg mL⁻¹ showed the highest antibacterial performance against S. aureus, B. cereus, and P. aeruginosa, demonstrating inhibition zones of 20.0 ± 0.03, 16.3 ± 0.3, and 15.3 ± 0.3 mm, respectively. The least antibacterial activity was observed against E. coli with an inhibition zone of 5 ± 0.0 mm. No inhibitory ability of the R. vesicarius extract was observed against any strains of bacteria. These results clearly show that Gram-positive bacteria were more sensitive to CuO NPs than Gram-negative bacteria. This is in consensus with previous studies that showed the inhibition zone of synthesized CuO NPs with Ailanthus altissima leaf extract against S. aureus and E. coli [75], with Adhatoda vasica Nees extract against P. aeruginosa [76], and with Eryngium caucasicum extract against B. cereus [77].

The findings of the antibacterial performance analysis revealed that the CuO NPs were highly effective in inhibiting the growth of bacterial species. The plausible explanation for this marked performance of antibacterial activity could be the direct interaction of the NPs with the cell membranes of the bacteria. The CuO NPs generate ROS, and their interaction with the bacterial cell membrane could facilitate the penetration of individual NPs into the cell. The inhibition of bacterial growth is also possibly due to dysfunction of the cell membrane caused by the NPs that results in a malfunction of the cell enzymes affecting cellular integrity. The NPs could also destroy bacterial membranes with ROS (superoxide and hydroxyl radicals) due to the lipid peroxidation causing oxidative damage to the bacterial cell. Another probable explanation for the potent anti-microbial activity of the CuO NPs is the presence of amines and carboxyl groups on the cell surface of bacterial species which enhance the affinity of Cu²⁺ ions for the cells [1].

The antifungal activity of the CuO NPs prepared with R. vesicarius extract was evaluated against three types of fungi: Fusarium oxysporum, Aspergillus flavus, and Alternaria alternata. The results demonstrated that the synthesized CuO NPs inhibited the growth of F. oxysporum with an inhibition zone of 30 ± 0.0 mm, slightly higher than the aqueous extract (28.5 mm). For A. flavus, the inhibition zone was also 30 ± 0.0 mm, but no inhibition was observed for the aqueous extract. Finally, the inhibition zone of A. alternata was 20.5 ± 0.03 mm and 6 mm for the CuO NPs and aqueous extract, respectively (Figure 9). The results indicated that the CuO NPs were more efficacious than the aqueous extract against all the fungal strains evaluated. Similar studies have been reported on the antifungal activity of CuO NPs synthesized from Celastrus paniculatus Willd. leaf extract [78], Pseudomonas fluorescens extract [79], and Brassica oleracea var. italica extract [7]. The possible mechanisms of the activity of CuO NPs are based on induced changes in the structure and function of the fungi. Furthermore, the NPs can affect the DNA, hindering replication and protein synthesis, which eventually leads to death of the fungi [80].

3. Materials and Methods

3.1. Preparation of Rumex vesicarius L. Extract

Fresh leaves of Rumex vesicarius L. (known as Humeidh) were collected from the Al Muzahimiyah area and sun dried for a week. The dried leaves of R. vesicarius were washed with tap water several times before a final wash with deionized water, and again allowed to dry. Next, 100 mL of boiled deionized water was added to 10 g of the dried and cleaned R. vesicarius leaves. The mixture was gently stirred at a slow speed and allowed to soak overnight at room temperature with a cover to prevent evaporation or contamination. The liquid extract was then isolated by filtration with a grade 1 Whatman paper and stored at 4 °C.

3.2. Preparation of Copper Oxide Nanoparticles

Two grams of copper acetate monohydrate (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in 50 mL of deionized water and stirred for 10 min at room temperature to prepare a copper acetate solution. Then, 100 mL of R. vesicarius extract (prepared as described earlier) was added to the copper acetate solution. The mixture was stirred for 30 min at room temperature, during which the initial light blue color changed to light green. The stirring continued for an additional 3 h at room temperature, after which the temperature was raised to 60 °C. Next, a specific amount of ammonia solution was added dropwise to the mixture with continuous magnetic stirring to adjust the pH to 11. This led to the formation of a brown precipitate, indicating the preliminary formation of CuO nanoparticles (CuO NPs). Once the addition was complete, the precipitate was isolated by centrifugation at 10,000 rpm for 10 min to separate the liquid from the solid. The resulting pellet was transferred to a covered ceramic vessel and dried in an oven at 80 °C for 12 h. Finally, the dried material was calcined at 400 °C for 4 h in a muffle furnace (CWF 1300, Carbolite, Hope Valley, UK) to produce the CuO NPs [30].

3.3. Characterization of the Copper Oxide Nanoparticles

The synthesized CuO NPs were characterized using a variety of techniques. The morphology of the copper oxide nanoparticles was characterized using TEM with a JEM-2100 (JEOL, Peabody, MA, USA). The optical absorption spectrum of the CuO NPs was recorded using a UV–Vis spectrometer (UV-1800, Shimadzu, Muttenz, Switzerland). The photoluminescence (PL) spectra were measured for the CuO NPs dispersed in double-distilled water using a Perkin–Elmer photoluminescence spectrophotometer equipped with a xenon lamp as the excitation source. All experiments were conducted at room temperature. Energy dispersive X-ray (EDX) spectroscopy was conducted (JSM-2100F, JEOL, USA), and a SEM (JOEL JSM-7600F, USA) was used to identify and map the elements in the CuO NPs samples. For the microscope measurements, an aqueous solution of CuO NPs was sonicated, and a drop was placed on carbon-coated Cu grids and allowed to dry.

3.4. Anticancer Activities of the Copper Oxide Nanoparticles

Trypan blue dye was used as a metabolic indicator for assessing cell viability. A methyl thiazol tetrazolium (MTT) assay was used to determine the cytotoxicity of the synthesized CuO NPs for the HeLa cervical carcinoma cell line (obtained from American Type Culture Collection, Manassas, VA, USA). HeLa cells were cultured at the optimal growth conditions of 5% CO₂ in air and 37 °C and passaged regularly until use. The total number of cells used in the experiments was determined with a 0.4% trypan blue exclusion test using a cell counter. HeLa cells were seeded in a 96-well plate at a density of 2 × 10⁵ cells/well in 100 µL of the optimized medium (Dulbecco’s Modified Eagles Medium (DMEM)). After 24 h, the cells were fed with fresh medium and treated with one of six concentrations of CuO NPs (5, 10, 20, 30, 50, and 100 µg mL⁻¹) and R. vesicarius leaf extract as a positive control. The treated cells were incubated and allowed to grow further for 24 h. After the incubation time, 20 μL of the MTT solution (Cell Titer 96^® Aqueous One Solution Reagent, Catalog Number G3581, Promega Corporation, Madison, WI, USA) was added to each well and further incubated for 4 h. The absorbance was then read at 540 nm using an automatic microplate reader (Molecular Devices–SPECTRA max–PLUS384, San Jose, CA, USA). Each experiment was performed in four replicates.

3.5. Photodegradation of Dyes by Copper Oxide Nanoparticles

The photocatalytic activity of the synthesized CuO NPs was demonstrated in the degradation of pollutant dyes, specifically crystal violet (CV) and methylene blue (MB) dyes, under UV lamp irradiation. A 1 mg L⁻¹ solution of the synthesized CuO NPs (i.e., 0.03 mg CuO NPs) was added to 30 mL of either CV or MB dye solution (dye solutions were 0.1 wt.% aqueous solutions) in 50 mL glass beakers. The synthesized CuO NP/dye solutions were stirred and irradiated at a set distance from a UV lamp (365 nm wavelength: 0.7 AMPS). The range of wavelengths applied was from 190–950 nm. Optical absorption spectra were collected after different light exposure durations using a UV–Vis spectrophotometer. The rate of degradation was monitored by recording the absorption intensity of the dye at the maximum wavelength as it decreased. The degradation efficiency (DE) was calculated according to the following equation:

$$\left(\%DE\right)=\left[{\displaystyle \frac{A_0-A}{A_0}} \right]\times 100$$

(12)

where $\left(\%DE\right)$ is the degradation efficiency, $A_0$ is the initial absorption intensity, and $A$ is the absorption intensity after the photodegradation of the dyes.

3.6. Antibacterial Activities of the Copper Oxide Nanoparticles

The CuO NPs were tested for antimicrobial activity against various bacterial and fungal strains. Minimum inhibitory concentration (MIC) of the CuO NPs against each bacterial pathogen was determined by the micro-dilution broth method. To match the McFarland (turbidity) standard, all microbial dilutions were standardized, showing a bacterial density of 1.5 × 10⁸ CFU/mL. The antibacterial activity of the synthesized CuO NPs was evaluated using the agar well-diffusion method. Four bacterial strains, Gram-negative Bacillus cereus (B. cereus) and Staphylococcus aureus (S. aureus), and the Gram-negative bacterial species Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), along with three fungal strains Helminthosporium, Alternaria alternata (A. alternata), and Fusarium oxysporum (F. oxysporum) were tested. The bacteria were grown in blood agar at 37 °C for 18 h, collected and suspended in 0.85% NaCl, and adjusted to 0.5 McFarland (10⁸ CFU/mL) turbidity. The fungal and bacterial suspensions were smeared on Muller–Hinton agar (MHA) plates. 10 µg mL⁻¹ of the synthesized CuO NPs and R. vesicarius leaf extract was loaded in the agar, and the plates were incubated at 37 °C for 18–24 h for bacteria and at 28 °C for 48–72 h for fungi. After incubation, clear zones of inhibition appeared around the wells, and the diameter of each inhibition zone was measured and recorded.

3.7. Statistical Analysis

Statistical significance among groups was analyzed by one-way analysis of variance (ANOVA) using SPSS version 17. Group comparisons were made using Tukey’s post hoc test. Values of p ≤ 0.05 were considered statistically significant.

4. Conclusions

In summary, the key findings of this study highlight a simple, cost-effective, and rapid method for the green synthesis of CuO nanoparticles (NPs) using copper acetate and the aqueous extract of R. vesicarius at room temperature. Various standard techniques, including UV–Vis, XRD, EDS, PL spectroscopy, as well as TEM and SEM microscopy, confirmed the successful formation of CuO NPs. The synthesized nanoparticles were irregular in shape and formed a network due to agglomeration, with particle sizes ranging from 5 to 200 nm.

Cytotoxicity studies revealed the anti-proliferative effects of CuO NPs against the HeLa cancer cell line, with an IC50 value of 8 µg/L, while the R. vesicarius extract itself showed no cytotoxic effects, even at an IC50 of 50 µg/L. Additionally, the CuO NPs exhibited significant antimicrobial activity, as demonstrated by inhibition zones against various fungal and bacterial strains, indicating their therapeutic potential for treating infectious diseases. Overall, the multifunctionality of the synthesized CuO NPs suggests their promising potential for a wide range of future applications.