Biogenic Synthesis of Copper Nanoparticles: A Systematic Review of Their Features and Main Applications

Nanotechnology is an innovative field of study that has made significant progress due to its potential versatility and wide range of applications, precisely because of the development of metal nanoparticles such as copper. Nanoparticles are bodies composed of a nanometric cluster of atoms (1–100 nm). Biogenic alternatives have replaced their chemical synthesis due to their environmental friendliness, dependability, sustainability, and low energy demand. This ecofriendly option has medical, pharmaceutical, food, and agricultural applications. When compared to their chemical counterparts, using biological agents, such as micro-organisms and plant extracts, as reducing and stabilizing agents has shown viability and acceptance. Therefore, it is a feasible alternative for rapid synthesis and scaling-up processes. Several research articles on the biogenic synthesis of copper nanoparticles have been published over the past decade. Still, none provided an organized, comprehensive overview of their properties and potential applications. Thus, this systematic review aims to assess research articles published over the past decade regarding the antioxidant, antitumor, antimicrobial, dye removal, and catalytic activities of biogenically synthesized copper nanoparticles using the scientific methodology of big data analytics. Plant extract and micro-organisms (bacteria and fungi) are addressed as biological agents. We intend to assist the scientific community in comprehending and locating helpful information for future research or application development.


Introduction
During the past two decades, extensive research has focused on synthesizing and utilizing nanoparticles, which play an essential role in multiple areas, including feed and pharmaceuticals, due to their unique properties [1]. Copper nanoparticles (CuNPs) have exhibited extraordinary properties due to their versatile activity, such as antitumor, antimicrobial, antioxidant, dye removal, and catalytic degradation. However, their traditional synthesis (chemical method) has issues, such as prolonged technical work and toxic raw materials, e.g., hydrazine, N-dimethylformamide, and sodium borohydride [2,3], which are potentially carcinogenic compounds [4]. In addition, the traditional chemical synthesis may present environmental risks, such as dispersion and exposure, to bioactive residues, with soil and water ecotoxicity.
According to Santhroshkumar et al. (2018) [1], green synthesis is most effective for reducing and stabilizing metal ions. Biological synthesis offers numerous benefits and

The Antioxidant Effect of CuNPs
The antioxidant properties of CuNPs synthesized from plant extracts have been demonstrated. Figure 2 reveals that a determining factor was the salt concentration, which, despite being variable, ranged from 1 to 100 mM in most cases ( Figure 2C). According to the research, the antioxidant capacity depends on the concentration due to the same behavior observed in metallic salts, such as copper acetate, copper chloride, and copper nitrate. However, copper sulfate exceeds these values [16].
Furthermore, the percentage of the antioxidant activity of 16 plants was screened by analyzing the antioxidant effects of CuNPs in each of the papers reviewed: this quantity is due to the complete available information. The antioxidant activity of Koelreuteria paniculata seed extract (14.54%) was lower than that of Solanum nigrum leaf extract (90%) (Figure 2A). Despite these values, the half maximal inhibitory concentration (IC50) of Tinospora cordifolia extracts was higher (566 µg/mL) than those of Solanum nigrum leaf extracts and Borreria hispida (60 µg/mL and 0.6 µg/mL, respectively) ( Figure 2B).

The Antioxidant Effect of CuNPs
The antioxidant properties of CuNPs synthesized from plant extracts have been demonstrated. Figure 2 reveals that a determining factor was the salt concentration, which, despite being variable, ranged from 1 to 100 mM in most cases ( Figure 2C). According to the research, the antioxidant capacity depends on the concentration due to the same behavior observed in metallic salts, such as copper acetate, copper chloride, and copper nitrate. However, copper sulfate exceeds these values [16].
Furthermore, the percentage of the antioxidant activity of 16 plants was screened by analyzing the antioxidant effects of CuNPs in each of the papers reviewed: this quantity is due to the complete available information. The antioxidant activity of Koelreuteria paniculata seed extract (14.54%) was lower than that of Solanum nigrum leaf extract (90%) (Figure 2A). Despite these values, the half maximal inhibitory concentration (IC 50 ) of Tinospora cordifolia extracts was higher (566 µg/mL) than those of Solanum nigrum leaf extracts and Borreria hispida (60 µg/mL and 0.6 µg/mL, respectively) ( Figure 2B). Table 1 presents an overview of different metallic salts and concentrations, with various plants and the component extract used in CuNPs. As a result, the part of the plant used for extract preparation, such as leaves, fruits, stems, roots, flowers, and seeds, is significant because of the relationship between the phytochemical characteristics and the total antioxidant capacity necessary for the reduction to synthesize CuNPs. Because phenolic components are considered the main contributors to plant extracts' total nonenzymatic antioxidant capacity, a decrease in phenolic compounds most likely resulted in reduced radical scavenging capacity [17]. In some cases, such as with tree bark, a direct relationship between antioxidant activity and total polyphenolic content was observed [18]. It is important to note that the phytochemicals present in the extract can be used as reducing and stabilizing agents in the overall synthesis [14].   Table 1 presents an overview of different metallic salts and concentrations, with various plants and the component extract used in CuNPs. As a result, the part of the plant used for extract preparation, such as leaves, fruits, stems, roots, flowers, and seeds, is significant because of the relationship between the phytochemical characteristics and the total antioxidant capacity necessary for the reduction to synthesize CuNPs. Because phenolic components are considered the main contributors to plant extracts' total non-enzymatic antioxidant capacity, a decrease in phenolic compounds most likely resulted in reduced radical scavenging capacity [17]. In some cases, such as with tree bark, a direct relationship between antioxidant activity and total polyphenolic content was observed [18]. It is important to note that the phytochemicals present in the extract can be used as reducing and stabilizing agents in the overall synthesis [14].
In contrast, the antioxidant effect underlies the inhibition of chain reactions, such as the breakdown of peroxides, the binding of transition metal ion catalysts, radical scavenging activity, and the inhibition of continued hydrogen abstraction [14]. The antioxidants present in plants can act as stabilizing agents during synthesis, preventing nanoparticle aggregation or clustering. These plant components with antioxidant activity can reduce oxidative stress during synthesis. Oxidative stress can lead to the formation of ROS, which can be detrimental to the synthesis and stability of nanoparticles [14]. In contrast, the antioxidant effect underlies the inhibition of chain reactions, such as the breakdown of peroxides, the binding of transition metal ion catalysts, radical scavenging activity, and the inhibition of continued hydrogen abstraction [14]. The antioxidants present in plants can act as stabilizing agents during synthesis, preventing nanoparticle aggregation or clustering. These plant components with antioxidant activity can reduce oxidative stress during synthesis. Oxidative stress can lead to the formation of ROS, which can be detrimental to the synthesis and stability of nanoparticles [14].

Anticancer Activity of CuNPs
In recent decades, radiotherapy, chemotherapy, and surgery have been the options with which to treat cancer. However, these standard methods have cost and usage restrictions. Thus, a natural, cheap, non-toxic, and side-effect-free treatment and prevention option is urgently needed. The Food and Drug Administration of the United States (FDA) recognizes metallic nanoparticles (i.e., iron, gold, zinc, titanium, and silver) as safe therapeutic compounds. Thus, CuNPs (374 USD/lb) have attracted considerable interest from researchers because they are cheaper than gold (1973 USD/t oz), silver (24.17 USD/t oz), and platinum (997 USD/t oz) [54]. Therefore, approaches involving CuNPs will be highly cost-effective [16].
CuNPs are promising cancer diagnosis and evaluation agents. Due to their unique properties, including their high surface-to-volume ratio, diffusion, efficiency of synthesis, and optical properties, they are effective against many cancer cell lines. These crucial factors are essential for extending the drug's half-life and delivery application [55].
Depending on the source of CuNPs and the type of cell lines, CuNPs can act via various cytotoxic mechanisms, primarily ROS production, apoptosis, autophagy, and DNA damage. CuNPs interact actively with intracellular protein functional groups, nitrogen bases, and phosphate groups in DNA, causing cytotoxicity in tumor cell lines. Nanoparticles with anticancer properties are known for their potential ability to inhibit abnormally expressed signaling proteins, such as Akt and Ras, cytokine-based therapies, DNA-or protein-based vaccines against specific tumor markers, and tyrosine kinase inhibitors with a consistent antitumor effect [56].
In vitro research of various human cell lines, including neuronal cells, cardiac microvascular endothelial cells, kidney cells, liver cells, and lung epithelial cells, demonstrated that oxidative stress mediates the cytotoxicity of CuNPs. Thus, the excessive use and disposal of CuNPs increase their potential toxicity to the environment and human health. Therefore, the biocompatibility of synthesized CuNPs must be determined [57]. The high concentration of free radicals in normal cells causes numerous mutations in their DNA and RNA, accelerating the proliferation and growth of abnormal or cancerous cells.
Some reports have suggested that CuNPs could induce apoptosis in cancerous cells via ROS generation by modulating the uptake of P53 and Bax/Bcl-2. Previous research indicates that the mechanism of action involves the destruction of ROS generated during cancer cell proliferation and transported as radicals or free radicals [58]. Regarding the ROS species (·OH, ·O 2 , and H 2 O 2 ), they play a crucial role in the death of eukaryotic cells induced by biogenic CuNPs. The highly reactive ·OH is a significant oxidant that influences oxidative DNA damage, including single-and double-strand breaks, mutation discovery, and the production of oxidized nucleotides [59].
The anticancer efficacy of CuNPs seems to depend on their size, morphology, specific surface area, increase in oxygen vacancies, reactant molecule diffusion ability, and release of Cu 2+ . When particle size decreases, particle surface area increases dramatically. It increases the potential number of ROS groups on the particle surface, which could substantially impact adverse biological effects [60]. In addition, small NPs offer a larger surface area to produce ROS, such as hydrogen peroxide, superanion radicals, and hydroxyl radicals, in cancer cells [57]. The smaller size of CuNPs may result in widespread tissue distribution, deeper penetration within specific tissues, improved cellular uptake, and enhanced cytotoxicity against cancer cells [61]. Research shows particles smaller than 50 nm exhibit more activity in the different cancer cell lines [28]. NPs with dimensions less than 200 nm exhibit efficient extravasation into leaky tumor vasculature and accumulation in tumor tissues due to their increased permeability and retention effect [62]. In vivo studies on the biodistribution and toxicity of CuNPs have revealed that smaller particle sizes exhibit greater transvascular and interstitial transport. In tumors, 50 nm NPs have demonstrated significantly greater permeability than 125 nm NPs [63]. Therefore, an efficient drug carrier must be small enough to leave the bloodstream, enter the vessels, and reach the tumor site [64].

Cytotoxic Effect of CuNPs on Cancer Cell Lines
The cytotoxic effects and biocompatibility of CuNPs depend on their concentration and synthesis routes. The synthesis of CuNPs has been reported mainly by physical methods, like ball milling, chemical methods, such as the sol-gel method, and biological methods by different plants or animal extracts. Among these routes, the biological routes, often called 'green synthesis', have proven to be one of the most biocompatible and ecofriendly methods for CuNP synthesis due to using eco-compatible reagents for the synthesis process [65]. Both the synthesis route and the inherent nature of CuNPs are important considerations for their effectiveness. The choice of synthesis route affects their specific characteristics, while their inherent nature determines their functional properties and behavior in specific applications. Table 2 shows different plants that have been used as biological sources for the synthesis of CuNPs that have a cytotoxic effect on cancer cell lines.
CuNPs became of great interest due to their cytotoxicity for multiple types of cancer in a dose-dependent manner without affecting healthy cells, compared to chemically synthesized NPs, which cause cell death in both benign and cancerous cells.

(a) CuNPs against breast cancer
The effects of CuNPs derived from Prunus nepalensis on MCF7 were studied by analyzing the expression of oncogenes (Ras, Myc) and tumor suppressor genes (P14/P19, P53, P21, and Caspase 3). The results demonstrated that CuNPs increased the expression of the genes involved in apoptosis in a dose-dependent manner. Furthermore, CuNPs induced apoptosis in MCF-7 cells by downregulating oncogenes and upregulating tumor suppressor genes [56]. Additionally, XRD studies on CuNPs synthesized from G. Sylvestre leaf extract showed a smaller crystal size with a higher surface area, which increased their anticancer activity. The cytotoxicity results showed that green CuNPs were more effective against MCF-7 cells than chemically synthesized CuNPs [60].
The cytotoxicity of CuNPs derived from Echinophora platyloba was observed in Raji and MCF-7 cells. The interaction of CuNPs with circulating tumor DNA (ct-DNA) showed an unusual binding mode that combines the characteristics of groove binding and intercalation, suggesting that the cytotoxicity of CuNPs may result from their interaction with DNA [66].
Additionally, the treatment of MDA-MB-231 cells with CuNPs resulted in distinct morphological changes, such as shrinkage, detachment, membrane blebbing, and distortion [61].
On the other hand, it uses an endophytic bacterium. A low concentration of green synthesized CuNPs (2-28 nm) has antiproliferative effects on breast cancer (T47D) cell lines [67].
(b) CuNPs against cervical cancer CuNPs were synthesized by using the aqueous leaf extracts of Azadirachta indica, Hibiscus rosa-sinensis, Murraya koenigii, Moringa oleifera, and Tamarindus indica and were tested against cervical cancer cells, revealing changes such as caspase activation, plasma membrane blebbing, binucleation, cytoplasmic vacuolation, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal apoptotic bodies, and DNA fragmentation. The IC50 ranged between 20.32 to 26.73 µg/mL, with an average size of 12 nm [19]. Another study used black beans to produce NPs that caused cell death by activating apoptotic pathways initiated by intracellular ROS [68].
Similarly, CuNPs 40-45 nm in size were synthesized using Houttuynia cordata extract. The fluorescent staining analysis revealed the inhibition of cell proliferation and promotion of apoptotic cell death in HeLa cells targeting the PI3K/Akt signaling pathways at 5 and 7.5 µg/mL doses with an IC50 of 5 µg/mL [69].
(c) Copper/copper oxide NPs against lung cancer The cytotoxic effects of CuNPs were examined in A549 cells, where they induced apoptotic pathways and caused low cell viability with fragmented nuclei and loss of membrane integrity. Aqueous Ficus religiosa leaf extract was used to synthesize copper NPs, showing that, at higher concentrations (500 g/mL), cell viability was reduced by up to 6%. The presence of bioactive molecules in F. religiosa leaf extract may explain the improved cytotoxicity; in conclusion, copper oxide nanoparticles activate the apoptosis pathway via the generation of reactive oxygen species, and mitochondrial depolarization causes cell death via nuclear fragmentation [70].
Similarly, as previously stated, copper oxide NPs synthesized by the aqueous leaf extracts of A. indica, H. rosa-sinensis, M. koenigii, M. oleifera, and T. indica showed a cytotoxic effect on lung cancer cells. The induction of apoptosis was accompanied by membrane blebbing, cell shrinkage, caspase activation in the cytoplasm, and nuclear fragmentation [19].
(d) Copper/copper oxide NPs against ovarian cancer Green CuNPs synthesized from Camellia sinensis aqueous leaf extract showed high cell death and anti-human ovarian cancer properties against CAOV-3, SW-626, and SK-OV-3 cell lines. Even at high doses, the healthy cells were unharmed [53]. In another study, small CuNPs were obtained (6-15 nm) using Cressa leaf extract, inhibiting SKOV3 human ovarian carcinoma cell line growth with an IC 20 value of µg/mL [71].
(e) CuNPs against other cancer cells lines CuNPs synthesized from plant extracts showed promising results against other cancer cells. For instance, pumpkin seed extract was used to synthesize CuNPs against colorectal cancer cells (HCT-116) [73], in which the results showed significant apoptotic induction after treating the cells with 25 g/mL CuNP. CuNPs made from Allium noeanum extract against three endometrial cancer lines (Ishikawa, HEC-1-A, HEC-1-B, and KLE) showed that the percentage of cell viability decreases when increasing CuNP concentration [28]. In the case of melanoma (B16F10)-synthesized CuNPs from Quisqualis indica floral extract, the results indicate that CuNPs induced cytotoxicity via apoptosis involving LDH release, ROS generation, and GSH depletion in a dose-dependent manner [74].
In contrast, when CuNPs were synthesized using Vibrio sp. to research cytotoxicity activity in esophageal cancer cells (KYSE30), the viability of KYSE30 cells was significantly reduced over time [IC50 = 37.52 mg/L (24 h), IC50 = 18.26 mg/L (48 h), and IC50 = 13.96 mg/L (72 h)] [75]. In another study using the fresh biomass of the cyanobacteria Cylindrospermum stagnale, CuNPs were found to significantly increase the concentration in a time-dependent manner. At 24 h, increasing the particle concentration from 25 to 100 µg/mL increased cell viability inhibition from 45 to 66% [76].
As shown in Figure 3, the IC50 antitumor effect on the major types of cancer cell lines is demonstrated using biological sources, mainly plant extracts.  Additionally, in human bone marrow mesenchymal stem cells, studies on the effect of CuNPs have demonstrated a certain suppression of proliferation development due to the activation of apoptotic pathways [77]. Nonetheless, numerous other studies report a positive effect when applied to biomaterials, primarily due to osteogenic properties for the regeneration of bone tissue and cartilage, where an increase in porosity, mechanical strength, and cross-linking is observed in scaffolds. These results and those of a number of other authors are contradictory, indicating that the topic must be explored and studied further, as it appears that determining the appropriate forms and concentration of copper is essential for their safe and effective application [78][79][80].  Additionally, in human bone marrow mesenchymal stem cells, studies on the effect of CuNPs have demonstrated a certain suppression of proliferation development due to the activation of apoptotic pathways [77]. Nonetheless, numerous other studies report a positive effect when applied to biomaterials, primarily due to osteogenic properties for the regeneration of bone tissue and cartilage, where an increase in porosity, mechanical strength, and cross-linking is observed in scaffolds. These results and those of a number of other authors are contradictory, indicating that the topic must be explored and studied further, as it appears that determining the appropriate forms and concentration of copper is essential for their safe and effective application [78][79][80].

The Antibacterial Effect of CuNPs
The antimicrobial effect of nanoparticles on bacteria is due to the effect caused at the cell wall level, primarily composed of the polymers of peptidoglycans, sugars, and amino acids, which, due to their porosity, facilitate the passage of nanoparticles [104]. This effect is also highly dependent on the type of bacteria: Gram-negative cell walls comprise a single layer of peptidoglycan, whereas Gram-positive cell walls comprise multiple layers [26]. Gram-negative bacteria are less resistant due to greater negative surface charge [105].
Gram-positive micro-organisms have walls composed of amines and carboxyl groups, which, when combined with aryls in the presence of copper, result in an amination reaction, and increased membrane permeability [106,107].
The inhibitory or bactericidal effect of CuNPs results precisely from the inhibition of cell membrane enzymes caused by the attraction between NPs and the membrane, thereby promoting the oxidation of NPs that are electrostatically attracted to membrane-based plasma reductases [108]. These ions enter through the lipid layers and move towards the cytosol, causing the production of oxygen species as O 2 , which leads to the formation of H 2 O 2 , which is responsible for the oxidation of proteins and lipids [109,110].
Particularly when nanoparticles are small and spherical, their size and shape could have potential as inhibitors [112]. Nanoparticles enhance antimicrobial activity and membrane performance by increasing the surface area [113]. After interacting with compounds, such as sulfur and phosphorus, the nanoparticles appear to have introduced reactive hydroxyl radicals capable of causing irreversible damage, oxidizing the proteins, and causing damage at the RNA and DNA levels, thereby altering and destroying the helical structure [114].
The phytochemical composition of the source (such as phenolic compounds and other antioxidants) that allows for the synthesis and stability of the nanoparticles will significantly impact these characteristics and effects [115]. CuNPs combined with plant extracts can further enhance the antimicrobial effect due to terpenoids (present in essential oils), phenolic compounds, tannins, flavonoids, and alkaloids that can cause ion transport disruptions and alter the activity of ion transport [116,117].
The destruction of proton efflux bombs results in the release of toxic metal ions, which affects the permeability of pathogen membranes and the respiratory system [118].
According to research by Selvan et al., 2018 [118], CuNPs have an anti-larvicidal effect on the Anopheles subpartus due to their accumulation in the alimentary and respiratory channels, which causes a rupture in the layers of tissues.
Alternatively, when compared to bacteria, fungi can be less sensitive to CuNPs due to the nature of their cell walls. Fungi have cell walls composed of polysaccharides, such as chitin (N-acetylglucosamine) and lipids, which provide stiffness and resistance to nanoparticles [119]. Table 3 includes columns for salt concentration, shape, and biological source, allowing for a comprehensive analysis of the antibacterial effect in different micro-organisms.

The Catalytic Effect of CuNPs
The catalytic effect depends on chemical production, elimination, and industrial catalytic process efficiency, selectivity, and yield. Selectivity reduces waste and impurities, making products safer and greener [170]. Copper oxide nanoparticles are reactive metal oxide semiconductors. Their high surface area facilitates catalytic, antimicrobial, and antifouling effects [171]. They are relevant in two areas: a reduction in components, characterized by the reduction or elimination of dyes and colorants, and the synthesis and catalysis of elements. Table 4 summarizes the screened data.
When a dye solution containing nanoparticles is exposed to sunlight, a free electron and a hole are generated from the nanoparticles; the electron interacts with oxygen to form superoxide free radicals, whereas the hole interacts with water to form hydroxyl ions. The dye is decolored by the formed superoxide free radicals and hydroxyl ions [172].
Each reaction or product has its pathway in component synthesis and catalysis. Quantum effects and a large surface-to-volume ratio give metal nanoparticles fascinating UVvisible, catalytic, and antibacterial properties [173]. The catalytic activity might be affected by nanoparticle size [42], shape, and exposed crystal planes [32]. CuNPs with a high surface area are popular due to their stability, cost, toxicity, manufacturability, and potential as catalysts [174].

Reducing and Capping Agents in the Synthesis of CuNPs
The reducing agent is an important component of nanoparticle synthesis. All research is included due to the ecofriendly approach, where the preference of agents derived from micro-organisms or plant species is critical. Thus, this review covers some of the most important nanoparticle-synthesizing plants. Most of the papers in this section use agricultural waste like calli [98], rhizomes [175,176], fruit hulls [177], aerial parts [178], beans [179], leaves, peels, flowers, juice, and peels [180]. There are also some byproducts, such as gum [181], and some species from another kingdom, such as the algae Cystoseira trinodis [42] and the bacteria Escherichia sp. SINT7 [182]. We can also highlight the application of plants like Plukenetia volubilis [183] and Moringa oleifera [184], which are widely cultivated in Peru and can be used for CuNP synthesis.

Factors That Affect the Synthesis and Catalysis of CuNPs
(a) Particle size: Most of the papers reviewed have synthesized nanoparticles with a maximum size of 100 nm, noting that decreasing particle size will increase catalytic activity [185] and that the solvent concentration affects the particles' size and shape [186]. Furthermore, it has been discovered that a larger extract volume is required to produce nanoparticles with a narrower size range [155]; (b) Temperature: The temperature of the reaction is an essential factor to consider when synthesizing nanoparticles. Some of the papers reviewed emphasized the importance of a high temperature for synthesis; (c) pH: pH is essential in nanoparticle synthesis, ranging from 9 [155,187] to 10 [188] and to 12 [172,189].  In the process of the degradation and reduction of both dyes and organic compounds using CuNPs, nanoparticle and sample' concentration, pH, time, and light are the parameters that must be considered in these processes. The best yields can be obtained using a higher nanoparticle concentration, a lower sample concentration [200], and a smaller nanoparticle size [185]. Alkaline pH improves the degradation or reduction process, and time in many studies is directly proportional to the light factor and the use of reducing agents.

Mechanism of Compound Reduction Using CuNPs as a Catalyst
The reduction process consists of the following steps. The initial step involved the adsorption of reactants to the surface of nanoparticles. The adsorption of BH4-onto synthesized CuNPs transfers surface hydrogen to the nanoparticles. The second step is desorption, which produces a product on the nanoparticles' surface. Immediately after the final product (amino compounds) desorption, the metal surface is made available for the catalytic cycle [219]. The pH of the solution primarily determines the removal of heavy metals from contaminated water [223]. CuNPs are an excellent alternative to adsorption for removing Cr (VI) toxicity from water [200]. Organic compounds require a reducing agent, such as NaBH4 or CuNPs, for a faster reduction [199]. One study found that using NaBH4 as a reducing agent under alkaline conditions reduced 97% of para-nitrophenol in 14 min [218]. (e) Salt concentration frequency: The frequency of salt concentration was also screened through all the papers reviewed for catalytic application. The four main metallic salts synthesizing CuNPs were copper sulfate, copper acetate, copper nitrate, and copper chloride ( Figure 4C). Copper sulfate was the most used salt, followed by copper chloride, copper nitrate, and copper acetate. The most frequently used concentration for copper sulfate was 0.01 M, copper acetate was 0.001 M, copper chloride was 0.003 M, and copper nitrate was 0.1 M. The concentration range for the most used salts, copper sulfate, and copper chloride ranged from 1 mM to 10 mM ( Figure 4D).

Mechanism of Compound Reduction Using CuNPs as a Catalyst
The reduction process consists of the following steps. The initial step involved the adsorption of reactants to the surface of nanoparticles. The adsorption of BH4-onto synthesized CuNPs transfers surface hydrogen to the nanoparticles. The second step is desorption, which produces a product on the nanoparticles' surface. Immediately after the final product (amino compounds) desorption, the metal surface is made available for the catalytic cycle [219]. The pH of the solution primarily determines the removal of heavy metals from contaminated water [223]. CuNPs are an excellent alternative to adsorption for removing Cr (VI) toxicity from water [200]. Organic compounds require a reducing agent, such as NaBH4 or CuNPs, for a faster reduction [199]. One study found that using NaBH4 as a reducing agent under alkaline conditions reduced 97% of para-nitrophenol in 14 min [218].
Arsenic (V) was reduced by 98% in 50 min at pH 8 [194]; it was concluded that if no reducing agents were used, the treatment time would be longer. Lead is another heavy metal with a significant reduction percentage, with a value of 89% at pH 6. Mahmoud's study discovered relatively low levels of cadmium and nickel, which could be attributed to their use of neutral pH rather than alkaline conditions. CuNPs are an excellent nanoabsorbent for purifying heavy metal-contaminated water, and their regeneration and reuse should be researched further [223].
Case I. Reduction of aromatic compounds: There are four steps in this reaction: hydrogen absorption, aromatic nitro compound absorption to metal surfaces, electron transfer from BH4-to aromatic nitro compounds, and aromatic amino compound desorption. Both azobenzene intermediates and hydroxyl amine reduction pathways most likely accomplish nitroarene reduction. The NPs (on their active surface) are reduced by the hydrogen liberated during sodium borohydride decomposition [229].
Case IV. Reduction of 4-nitrophenol: Another example of the catalytic properties of nanoparticles is the reduction of 4-NP to 4-aminophenol (4-AP). The result demonstrates that the natrolite zeolite/Cu NPs are required to reduce 4-NP to 4-AP [233].
Case IV. Homocoupling reactions: The base-free homocoupling reaction was also studied for various boronic acids. The reaction conditions are compatible with aryl functional groups such as aldehyde, ether, methyl, and nitro. Surprisingly, the bromo and chloro groups do not change. Other boronic acid surrogates, such as phenylboronic acid neopentylglycol ester, were also found to be suitable homocoupling reaction substrates [234].
Case V. N-arylation of compounds: It should be noted that the reaction of indole with iodobenzene was considered as a model reaction to determine the best reaction conditions for the catalytic N-arylation process [170].
Case VI. Aza-Michael reaction: The Aza-Michael reaction is ideal for creating the carbon-nitrogen (C-N) bond and other types of molecular bonds [189].
Case VII. Heterocoupling reactions: In organic synthesis, the Ullmann C−C homocoupling, C−N, and C−O hetero coupling reactions are required to prepare the bi-aryl, di-aryl amine, and di-aryl ether structures [235,236]. It should be noted that the C-S coupling reaction is also being investigated for thioether synthesis in the absence of a ligand [230].
Case VIII. Biginelli reaction: The catalytic efficacy of Cu/CuO/Cu 2 O nanoparticles in the Biginelli reaction to synthesize the biologically active compound 3,4-dihydropyrimidinone (DHPM). DHPM was prepared by loading various catalysts for the condensation of benzaldehyde with urea and ethylacetoacetate [131].
Case IX. Cyanation of aldehydes: This reaction occurs in the presence of non-toxic K4Fe(CN)6 as the cyanide source. The results confirmed the high stability and absence of impurities in reducing Cu II ions to CuO [210].
Case XI. Oxidation of oils: As an oxidant, H 2 O 2 can convert model oils (DBT in n-heptane) into the corresponding sulfone or sulfoxide. Peracetic acid and a CuNP oxide intermediate, hydroox-ocuprate, are formed by the nucleophilic attack of hydrogen peroxide on acetic acid and CuNPs, respectively [214].
Case XIII. Synthesis of pyrimidines: Another application of NPs as catalysts is the synthesis of pyrano [2,3-d] pyrimidines. Aromatic aldehydes, methylene compounds, barbituric acid, and Cu 2 O NPs were combined with solvents [53].
Case XIV. Ipso-hydroxylation of arylboronic acids: A typical reaction involves the ipso-hydroxylation of aryl and hetero-aryl boronic acids by phenyl-boronic acid, CuNPs, and H 2 O 2 . The reaction products are typically extracted with diethyl ether after the reaction [238]. Figure 5 is an example of the degradation mechanism of toxic organic dyes and a compilation of previously studied articles. In this instance, three dyes were considered: methylene blue, rhodamine, and congo red [216]. In most of the articles, the three dyes had similar concentrations of 10 mg/L plus 10 mg of CuNPs. This mixture was stirred constantly for 30 min ( Figure 5A). After, the solution was exposed to UV light and stirred continuously for 60 min ( Figure 5B). Following these procedures, the sample was analyzed using a UV-visible spectrophotometer ( Figure 5C), determining the percentage of dye degradation. The synthesis of methylene blue was also analyzed ( Figure 5D). CuNPs degrade most dyes more efficiently than silver nanoparticles, particularly crystal violet [177]. Several studies have examined the significance of stirring the dye solution with the CuNPs in the dark for a few minutes to achieve the equilibrium of the adsorption and desorption of the dye with the nanoparticle surface before exposing them to sunlight or UV light, which degrades them rapidly [187].

Mechanism of Degradation of Toxic Organic Dyes by Biogenically Synthesized CuNPs
As mentioned above, CuNPs generate electron-hole pairs via photon absorption, which is the basis for explaining the preceding technique. Electrons created in the valence band migrate to the hole in the conduction band. These valence band holes combine with hydroxyl ions to produce hydroxyl radicals (•OH). Superoxide radicals form when conduction band electrons react with dissolved oxygen. Superoxide radicals react with water to increase the concentration of •OH [239]. Due to their high oxidizing potential, •OH radicals degrade organic pollutants effectively. Bonds are broken, rings are opened, and oxidation occurs in degradation. Due to the substitution of •OH, polyhydroxylated products are easily removed from the benzene ring under radical attack [240] and break down into less hazardous fragments, such as NO 3 , SO 4 2− , CO 2 , and H 2 O [227]. Based on the presented data, it is strongly recommended to prioritize and intensify further research on the effect of plant extracts on the synthesis of CuNPs. Synthesis utilizing plant extracts is a sustainable and environmentally friendly method with promising applications. However, additional research is required to fully explore the potential of plant extracts since specific data on the proper effect or synergic interaction are explicitly reported. Plant extracts are a rich source of different bioactive compounds, and their synergistic interactions with CuNPs can enhance the activities under study. In addition, by examining the effect of plant extracts, specific antibacterial or antitumor properties can be identified, allowing for targeted applications against antibiotic-resistant strains and cancer cell lines. More research in this area will unlock the potential of plant extract-mediated synthesis, resulting in the development of effective, sustainable, and economically viable solutions for addressing bacterial infections and cancer cell line resistance. Based on the presented data, it is strongly recommended to prioritize and intensify further research on the effect of plant extracts on the synthesis of CuNPs. Synthesis utilizing plant extracts is a sustainable and environmentally friendly method with promising applications. However, additional research is required to fully explore the potential of plant extracts since specific data on the proper effect or synergic interaction are explicitly reported. Plant extracts are a rich source of different bioactive compounds, and their synergistic interactions with CuNPs can enhance the activities under study. In addition, by examining the effect of plant extracts, specific antibacterial or antitumor properties can be identified, allowing for targeted applications against antibiotic-resistant strains and cancer cell lines. More research in this area will unlock the potential of plant extract-mediated synthesis, resulting in the development of effective, sustainable, and economically viable solutions for addressing bacterial infections and cancer cell line resistance.

Search Strategy
We systematically reviewed research articles published in English, excluding non-English publications, case reports, books, letters, and patents. We analyzed the research on CuNP biogenic or green synthesis and their primary applications.
We formulate the problem based on the premise that there is no systematic review of the principal applications of biogenically synthesized CuNPs.
A bibliographic search was conducted from 2012 to date, using the PubMed, Web of

Search Strategy
We systematically reviewed research articles published in English, excluding non-English publications, case reports, books, letters, and patents. We analyzed the research on CuNP biogenic or green synthesis and their primary applications.
We formulate the problem based on the premise that there is no systematic review of the principal applications of biogenically synthesized CuNPs.
A bibliographic search was conducted from 2012 to date, using the PubMed, Web of Science (WOS) Core Collection, and SCOPUS databases (September 2014 to January 2023). The results were deduplicated and uploaded to EndNote (Clarivate Analytics) and Rayyan Software. The preferred reporting items for systematic reviews and metaanalyses (PRISMA) recommendations were followed. The protocol for this systematic review was registered on the International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY) (INPLASY202350109) and is available in full at inplasy.com (https://inplasy.com/inplasy-2023--5-0109/, accessed on 15 June 2023). The systematic review has been elaborated according to the PRISMA 2020 checklist (Table S5).

Search Criteria
We carried a screening set for the biogenic or green synthesis of CuNPs in the databases mentioned above, using the keywords in the title and abstract: (nanoparticle*) AND (biogenic) OR (green AND synthesis) AND (copper) OR (cu) OR (cu2o) OR (cuo).

Study Selection, Data Extraction, and Quality Assessment
Four authors selected the studies and reviewed the titles and abstracts of all published articles using the Rayyan software, based on the selected criteria and keywords. To compile research articles on biogenic synthesis, the patents, clinical trials, reviews, duplicates, and in vivo test-related studies and articles were excluded.
CMLJ, ACL, TEU, JCDC, and LDGM carried out the review process and management by extracting the authors' and content's raw data in accordance with a standard procedure.
After the initial database data collection, we screened and categorized the major applications using four screening sets. The first set contained antitumor terms, the second set contained antioxidant terms, the third set contained antibacterial terms, and the fourth set contained catalytic effect and dye removal terms.
The quality and risk of bias were evaluated based on the Cochrane Handbook for Systematic Interventions recommendations (http://www.handbook.cochrane.org, accessed on 12 December 2023). Independently, the authors evaluated the possibility of bias during the review process. At any stage of the reviewing process, disagreements among the authors were discussed and resolved.
The keywords for inclusion in the Rayyan Software were 1.
For the antitumoral analysis, the following keywords in the title and abstract were used in the research fields: Green synthesis, Biogenic, Cancer, Anticarcinogenic, Antitumoral, and Cytotoxic; they were also used as keywords to collect data that might be under one of these terms; 2.
For the antioxidant analysis, the following keywords in the title and abstract were used in the research fields: Green synthesis, Biogenic, Antioxidant, and Oxidative, and these were also used to collect data that might fall under one of these terms; 3.
For the antibacterial analysis, the following keywords in the title and abstract were used in the research fields: Nanoparticles, Copper, Antibacterial, Green synthesis, antimicrobial, and biogenic, which were also used as keywords to collect data that might fall under one of these terms; 4.
For the catalytic effect and dye removal analysis, the following keywords in the title and abstract were used in the research fields: Green synthesis, Catalytic, Photocatalytic, and Biogenic, which were also used as keywords to collect data that might fall under one of these terms.
A MeaSurement Tool to Assess Systematic Reviews (AMSTAR) was used to assess and evaluate the validity, quality, and reliability of the systematic review, as the content of each article was evaluated by following a set of criteria. The 11-item eligibility instrument was used to determine these scores, with each item in each article receiving a score of 1 or 0: Can't Answer (CA) and Not Applicable (NA). The lowest score was 5, and the highest score was 8. (Table 5). The quality of research is proportional to the score: a score between 8 and 11: good quality; a score between 4 and 7: moderate quality; a score between 0 and 3: lower quality [38]. Was the status of publication (i.e., grey literature) used as an inclusion criterion? 5 Was a list of studies (included and excluded) provided? 6 Were the characteristics of the included studies provided? 7 Was the scientific quality of the included studies assessed and documented? 8 Was the scientific quality of the included studies used appropriately in formulating the conclusions? 9 Were the methods used to combine the findings of studies appropriate? 10 Was the likelihood of publication bias assessed? 11 Were potential conflicts of interest included?

Data Analysis
During the systematic review, we collected qualitative data. Using the free VOSviewer server software (Version 1.6.15; https://www.vosviewer.com, accessed on 23 January 2023), we compiled, analyzed, and plotted the publication datasets, observing the trends and the most studied fields and applications on the biogenic synthesis of CuNPs. We established a threshold of 30 keyword co-occurrences. These co-occurrences were words that appeared in the analysis more than 29 times. The strategy process is summarized in Figure 6. It was unnecessary to conduct a meta-analysis because the current study did not reveal any discrepant findings for CuO. between 8 and 11: good quality; a score between 4 and 7: moderate quality; a score between 0 and 3: lower quality [38]. Was the scientific quality of the included studies used appropriately in formulating the conclusions? 9 Were the methods used to combine the findings of studies appropriate? 10 Was the likelihood of publication bias assessed? 11 Were potential conflicts of interest included?

Data Analysis
During the systematic review, we collected qualitative data. Using the free VOSviewer server software (Version 1.6.15; https://www.vosviewer.com, accessed on 23 January 2023), we compiled, analyzed, and plotted the publication datasets, observing the trends and the most studied fields and applications on the biogenic synthesis of CuNPs. We established a threshold of 30 keyword co-occurrences. These co-occurrences were words that appeared in the analysis more than 29 times. The strategy process is summarized in Figure 6. It was unnecessary to conduct a meta-analysis because the current study did not reveal any discrepant findings for CuO.

Conclusions
The biogenic synthesis of CuNPs is a promising method for producing functional and stable nanoparticles with numerous potential applications. Biogenic synthesis has several advantages over conventional chemical synthesis methods, including low toxicity, cost-effectiveness, and mild reaction conditions. In addition, these nanoparticles have antioxidant, antibacterial, and antitumor properties, making them useful for healthcare and medical applications.
Biologically synthesized CuNPs have demonstrated catalytic potential in a variety of chemical reactions due to their large surface area and unique surface properties. As effective catalysts, they can enhance reaction rates and selectivity, and their production can support environmentally friendly and sustainable chemical industry processes. Due to their antioxidant and antitumor properties, the biogenic synthesis of CuNPs has shown great promise in medicine as antimicrobial agents and for cancer treatment and prevention.
The potential applications of biogenic CuNPs, which include antibacterial, antitumor, antioxidant, and catalytic properties, make them an exciting study area. However, there are still challenges that need to be overcome for the further advancement and utilization of CuNPs, such as optimizing the synthesis parameters, tailoring the properties for specific applications, ensuring biodegradability, achieving scalability, understanding the mechanisms of action, addressing safety concerns, and integrating CuNPs into emerging technologies. By overcoming these challenges through research, the full potential of CuNPs will be unlocked and expand their applications in diverse fields.
The integration of CuNPs with emerging technologies, such as nanomedicine and nanoelectronics, can create new diagnostics, therapeutics, and material applications. Collaboration between researchers, regulatory bodies, and industry stakeholders will be essential in establishing safety guidelines, standardizing protocols, and translating the potential of biogenic CuNPs into real-world applications, which will ultimately benefit sectors such as healthcare, catalysis, energy, and environmental remediation.