Coffee arabica-derived copper nanoparticles: A potent larvicidal agent against Aedes aegypti mosquitoes

The use of green biological chemistry has made it easy, cheap, and quick to create metallic nanoparticles such as copper, silver, gold, and selenium in recent years. The synthesis of these nanoparticles was done in a manner that was clean, non-toxic, and environment-friendly [1]. Synthesizing nanoparticles from plants is considered relatively faster, as they do not need specialized culture media or microorganism cultivation. In addition, plants contain primary and secondary compounds such as enzymes, proteins, flavonoids, and teraponids that act as reducing and capping agents [2, 3]. Today, nanoparticles are used in various fields of life, such as solar energy, water treatment, medicine, and agriculture, it may be considered a viable solution to challenges that pollute the environment [4, 5]. Biologically synthesized nanoparticles are toxic to harmful bacteria and insects due to their unique properties, including a large surface area that grants high catalytic capacity, facilitating the generation of reactive oxygen species (ROS). These ROS act as potent antimicrobial and insecticidal agents by causing damage to cellular structures and disrupting metabolic processes within target organisms [6, 7].
Many severe infections that cause fever, including dengue, Zika, and Chikungunya are transmitted mostly by mosquitoes [8]. Currently, mosquitoes contribute to the spread of many diseases in urban and rural areas as well. To deal with this problem, scientists recommend using nanotechnology as a solution for diseases transmitted by insects [9]. Green-synthesized metal nanoparticles show promise in controlling insect vectors, including mosquitoes. Copper, gold, palladium, silver, zinc, carbon, and silica nanoparticles have been identified as effective against mosquito populations [10]. 
Copper is one of the basic elements that a living organism needs physiologically and structurally [11]. Copper nanoparticles (CuNPs) are gaining popularity in various scientific fields due to their low toxicity, high stability, and unique properties. Researchers have also found that biologically synthesized CuNPs are safer, more environment-friendly, and more cost-effective than those produced using chemical or physical methods [12, 13].  A recent report suggests that CuNPs are safe, compatible with living organisms, and free of harmful substances [14]. At the same time, many scientific methods are available to synthesize CuNPs biologically from plant extracts such as dried leaves, seeds, and barks [15-18]. 
Coffea arabica, a plant belonging to the Rubiaceae family, possesses significant levels of phenolic compounds. These compounds exhibit powerful antioxidant properties and have the ability to hinder the infection caused by severe acute respiratory syndrome coronavirus type 2 [19]. Previous research has shown that an extract derived from C. arabica contains both chlorogenic acid and a small amount of caffeic acid. This extract has been shown to have antioxidant, anti-photoaging, and anti-inflammatory properties in both human skin fibroblasts and mice with no specific genetic background. It also effectively inhibits the production of ROS caused by ultraviolet (UV) radiation [20]. Another research investigation has provided further evidence of the remarkable properties of Coffea arabica extract. In a recent research investigation, the larvicidal activity of Coffea arabica extract was examined. The study found that the extract exhibited potent larvicidal effects against mosquito larvae, specifically those of Aedes aegypti, which is responsible for transmitting diseases such as dengue fever, Zika virus, and chikungunya [21]. Similarly, a research conducted by Kaitana et al. (2022) revealed that the use of Pangi leaf extract is successful in eradicating mosquito larvae and deterring insects [22]. Thus, this study aimed to investigate the potential of Coffee arabica extract synthesized CuNPs for biotoxicity testing against Aedes aegypti larvae and their pathological markers as compared to green and roasted Coffee arabica.

Chemicals and tools 
Coffee arabica seeds were brought from the Bani Hammad area, Taiz, Yemen. Copper sulfate pentahydrate (CuSO₄·5H₂O) and gallic acid were prepared using the highest-purity materials sourced from Merck, Germany. Additionally, Folin Ciocalteu reagent, aluminum chloride, and Na₂CO₃ were obtained from WINLAB laboratory chemicals reagent, UK. Na acetate anhydrous, acetic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), NaOH, petroleum ether, sulfuric acid, phosphate buffer, as well as reagents such as Benedict's, Biuret's, Phenols, Dragendroff's, Braemer's, Shinoda's, Liebermann's, and Frothing's and methanol were procured from Loba Chemie Pvt Ltd, India. The research employed a spectrophotometer (UNICO 1100-CSO106027) and a centrifuge (800D) (50102002).

Statement on research ethics
The authors obtained ethical clearance from the Animal Ethics Committee at IPB University's Institute of Research and Community Development, Indonesia, with approval number 69-2017 for collecting animal samples in accordance with Indonesia's Regulation (PMK) No. 7 of 2016.

Rearing of target insects
Aedes aegypti mosquito eggs were collected from the colonies reared in laboratories of the Faculty of Medicine, Department of Parasitology, Hasanuddin University, Indonesia. The eggs hatched and turned into larvae under laboratory conditions at room temperature (28°C), humidity and natural lighting. The larvae were kept in non-chlorinated tap water and fed with aquarium fish food.

Plant extract and synthesis of CuNPs   
About 20 grams of green Coffee arabica powder and 20 grams of roasted Coffee arabica powder were individually weighed and then combined with 100 ml of distilled water. The mixture was then heated for 1 h at a temperature of 80°C using a magnetic stirrer, and subsequently filtered using ashless filter paper No. 11. 40 (mM) of copper sulfate (CuSO₄.5H₂O) was prepared and then combined with Coffee arabica extract in a ratio of (1:1). The formation of CuNPs was defined by color change from brown to light green. CuNPs were harvested in powder form after washing it three times with distilled water and centrifugation (HERMLE Z 366 K1) at 12,000 g for 15 minutes, they were put in the furnace to dry for 16 h at 80°C.

Characterization of phytochemicals of Coffee arabica 
Qualitative tests were conducted to determine the type of phytochemicals present in Arabic coffee extract according to international protocols [23]. Quantitative tests: the total phenolic content (TPC) of the plant extracts was estimated using the Folin Ciocalteu assay. The assay involved combining Folin Ciocalteu reagent and Na2CO3 with aliquots of the extract or a standard solution of gallic acid. The absorbance was measured at 760 nm after 40 minutes of incubation. A gallic acid curve was used to determine the total phenolic content [24]. The total flavonoid capacity (TFC) was determined using the aluminum chloride (AlCl3) colorimetric method. The extract or a standard quercetin solution was mixed with AlCl3, and the absorbance was measured at 430 nm after 40 minutes. A quercetin plot was used to determine the total flavonoid concentration [25]. 

Antioxidant potential of Coffee arabica
The antioxidant potential was calculated using the DPPH assay. Control, sample, and blank solutions were prepared, and their absorbance was measured at 517 nm after 30 minutes. The antioxidant activity was determined using the formula of radical scavenging activity (RSA) % = [(A control− A test)/A control] × 100%, and the IC₅₀ value was calculated.

Characterization of CuNPs
The spectrum of CuNPs was studied using a visible/UV spectrophotometer. Wavelengths range from 300 to 800 nm (UV-2600, Shimadzu Europa GmbH). To further characterize the optical properties of CuNPs synthesized from Coffee arabica were analyzed using the X-ray diffusion instrument (Shimadzu XRD-7000). Fourier-transform infrared spectroscopy (FTIR) using an infrared spectrometer (IR Prestige-21, Shimadzu Europa GmbH) was employed to identify the functional groups that contributed to the reduction and stability of CuNPs.

The larvicide bioassay
The parasitic activity was evaluated using the WHO protocol test [26]. Aedes aegypti larvae in the fourth instar were used to assess the effectiveness of CuNPs and Coffee arabica in controlling the parasites. The biotest was conducted in transparent 250 ml cups and repeated three times to ensure the reliability of the statistical outcomes. Each cup contained 20 fresh larvae exposed to different concentrations of CuNPs (2, 12, 25, 50, and 100 ppm), as well as roasted and green Coffee arabica extract (100, 200, 300, 400, and 500 ppm). The results were recorded after 24 h, and the half-lethal concentration (LC50) was determined using probability analysis. The LC50 and LC90 values were calculated using SPSS software, with a significant threshold set at p ≤ 0.05.

Morphological study 
The dead larvae of Aedes aegypti treated with CuNPs of Coffee arabica were carefully examined under a microscope (Nikon Microsystems, Japan) to study morphological changes. The outer body parts, including the siphon, setae, eyes, antennae, and anal gills, as well as changes to the abdomen, thorax, and head were carefully monitored.  Any visible deviation or discoloration in the body of the treated larvae was recorded and compared to the control group. 

Histopathological study
Aedes aegypti larvae were treated with CuNPs of Arabic coffee and the control were fixed with 10 percent buffer formalin for 24 h, then dehydrated, cleared, and sectioned (5 to 7 microns) and stained with hematoxylin-eosin according to a previous study [27].

Ecotoxicology study
The study investigated the effects of green CuNPs on Artemia salina. Brine shrimp eggs hatched and the nauplii were exposed to different concentrations of CuNPs (100, 200, 300, 400 and 500 ppm). After 24 h, the survival rate was calculated using the formula: Mortality Rate = (Number of Deaths) / (Number of Lives) x 100.

Statistical analysis
The statistics software for social sciences SPSS version 25 was used to conduct statistical analysis. Means plus standard deviation were used to represent the data. The single-way analysis of variance (ANOVA) was used to divide variations in the variables into more than two groups, and the variations between the groups were determined.

Production and characterization of CuNPs
CuNPs were produced, leading to a visible shift in color from brown to green. Following this, the nanoparticles underwent characterization using advanced methods including UV spectroscopy, FTIR, and X-ray diffraction. More information about the characterization can be found in our previous research [28]. The average size of CuNPs was measured to be around 16.3 nm phytochemical screening.

Phytochemical profile of Coffee arabica
Table 1 shows the phytochemical content of both green and roasted coffee beans. The roasted coffee beans contained alkaloids, flavonoids, steroids, tannins, phenols, and saponins as phytochemicals. The green coffee samples had similar phytochemicals, except saponins. 

Total phenolic content and total flavonoid capacity of Coffee arabica
The TPC and TFC of green and roasted coffee are displayed in Figure 1. The TPC and TFC of green coffee were 68.78 ± 0.868 and 21.95 ± 0.331, respectively which are generally higher than those of roasted coffee (65.12 ±0.853 and 19.80±0.259). A statistically significant difference was observed (p < 0.000). 

Effect of Coffee arabica-mediated CuNPs on antioxidant potentials
Based on the data presented in Figure 2, it is evident that both roasted and green Coffee arabica extracts exhibited potent free radical scavenging activity. This was supported by high values of DPPH RSA% and low IC₅₀ values, indicating their effectiveness in neutralizing free radicals. Green Coffee arabica extract generally exhibits higher scavenging activity and lower IC₅₀ values (3.37±1.2) compared to roasted Coffee arabica extract (3.21±1.0) at the same concentration. These findings suggest that both extracts have the potential to act as antioxidants. 

Effect of Coffee arabica-mediated CuNPs on mortality of mosquito larvae
Table 2 provides data on the effects of green and roasted C. arabica on A. aegypti larvae after 24 h. It included information on mortality, concentrations in ppm, standard deviation, total and mean mortality, as well as LC₅₀ and LC₉₀ values. The LC₅₀ and LC₉₀ concentrations for green C. arabica were 124.5 ppm and 456.5 ppm, respectively, while for roasted C. arabica, they were 73.5 ppm and 371.5 ppm, respectively. In addition, CuNPs concentrations ranging from 2 to 100 ppm caused mortality rates of 20% to 96.3% in exposed larvae, with corresponding LC₅₀ and LC₉₀ concentrations of 5.796 ppm and 36.595 ppm after 24 h. The chi-square value (df - 3) for green, roasted C. arabica and CuNPs were 6.482, 4.610 and 1.573, respectively. The results demonstrate a correlation between mosquito mortality and LC₅₀. 

Effect of Coffee arabica-mediated CuNPs on morphology of Aedes aegypti 
The findings of morphological analysis revealed the presence of damaged areas in the mid-gut region, characterized by pronounced pigmentation and darkening (Figure 3). Furthermore, the analysis identifies ruptured cuticles and deformed bodies, with a noticeable reduction in body size, particularly in the head and siphon.

Effect of Coffee arabica-mediated CuNPs on histopathological alterations
In the current study, histological investigation was conducted as represented in Figure 4. The results revealed a significant level of tissue degradation in fourth instar Aedes aegypti larvae treated with CuNPs. Notably, the head region experienced complete tissue breakdown, while the stomach pouches in the larva's thorax region exhibited only minor changes. The cells in the treated larvae appeared similar to those in the control larvae, with intact nuclei, except for the presence of splits in the protective layer surrounding the gut and localized destruction of gut lining cells. The larva's digestive tract showed various indications of damage, including lesions. Additionally, the microvilli layer on the surface of mid-gut epithelial cells was noticeably thinner compared to the intact and tightly packed brush border observed in the gut epithelial cells of healthy larvae. 

Effect of Coffee arabica-mediated CuNPs on ecotoxicological parameters
The study investigated the impact of varying concentrations of CuNPs on the survival of Artemia salina larvae. Results in Table 3 showed that higher concentrations of CuNPs led to increased larval mortality rates. The LC₅₀ and LC₉₀ values were determined to be 344.3 ppm and 1073.2 ppm, respectively. The findings suggest a significant relationship between CuNPs concentration and larval mortality. 

CuNPs are formed through a synthesis process. Copper ions from the copper sulfate pentahydrate solution react with reducing agents in the Coffee arabica extract. The reduction process converts the ions into metallic copper atoms, which form the core of the nanoparticles. The core is surrounded by a shell composed of organic molecules from the extract, which stabilize the nanoparticles and control their size and dispersibility. The core-shell structure provides stability and unique properties to the CuNPs [28].
The anti-inflammatory, antioxidant, and potential health benefits of coffee are attributed to its components. Tannins, phenols, and flavonoids act as antioxidants, while caffeine promotes wakefulness. Saponins and terpenoids have anti-parasitic properties. With its abundant phytochemical content, coffee is a widely consumed beverage that may offer health advantages. This study revealed the presence of certain phytochemicals in Coffee arabica extract, consistent with findings from other research [29, 30].
Green coffee exhibits higher levels of total phenolic content and total flavonoid capacity compared to roasted coffee, which can be attributed to the breakdown of phenolic components during the roasting process. Both green and roasted coffee contain TPC and TFC. Although the concentrations may be slightly higher in green coffee, a statistically significant difference was observed (p < 0.000). However, it is worth noting that many individuals prefer the robust flavor profile of roasted coffee. Several researchers have conducted similar studies on TPC and TFC in green and roasted Arabica coffee, and their findings correspond relatively closely with our own [31, 32]. According to Sualeh et al. (2020), the average chlorogenic acid content was statistically significant and the caffeine content was not significant [33].
Antioxidants are important for protecting cells from damage caused by free radicals, which are associated with various chronic diseases. Therefore, the antioxidant properties of roasted and green Coffee arabica extracts may contribute to the potential health benefits attributed to coffee consumption. Similar to the findings of this investigation, a study by Jung et al. found that the DPPH radical scavenging activity decreased with increasing roasting time [32]. Additionally, Cho et al. have found that light roasting increases the DPPH radical scavenging activity of coffee extracts. On the other hand, as roasting temperatures increased, the DPPH radical scavenging activity reduced [34]. Consistent with Herawati et al., once the first crack is completed, the DPPH IC50 of coffee brewed from Robusta coffee beans increases as the roasting temperature rises [35]. Additionally, coffee has antioxidant properties in addition to its well-known toxicological effects, which makes it a good natural dietary supplement for beehives to increase their resistance to pesticides and other environmental stresses [36]. 
The present study observed that roasted coffee exhibited greater efficacy in terms of larvicidal effects when compared to green coffee. This finding aligns with previous research conducted in the field [37-39]. Thanasoponkul et al. (2023) investigated the impact of using 50 g/L of used coffee grounds and reported complete mortality of larvae within a span of 48 h. Similarly, Sharawi (2023) determined an effective concentration of Coffee arabica extract for this purpose. Furthermore, Tangtrakulwanich et al. (2022) conducted a study that demonstrated the inhibitory effects of lightly roasted used coffee grounds on larval survival. Mortality in mosquito larvae is attributed to alkaloid compounds, flavonoids, and tannins that hinder the insect's nervous system. The bitterness of coffee grounds can also cause irritation and disrupt digestion, leading to larval demise. Caffeine, found in coffee and other beverages, has been extensively studied for its toxicological effects on various organisms [39]. 
CuNPs have been found to have a more significant impact on mosquito larvae compared to coffee extract alone. To the best of our knowledge no available data on the larvicidal activity of CuNPs synthesized from Coffee arabica plants. Rajagopal et al. (2021b) demonstrated the larvicidal effects of CuNPs derived from Wrightia tinctoria extract on Aedes aegypti larvae, with an LC50 value of 0.0321 mg/ml. Similar outcomes were observed with Tridax procumbens, Artocarpus heterophyllus, and Metarhizium robertsii leaf extracts [40, 41, 42]. CuO/SiO₂ nanoparticles synthesized from gum arabic extract showed larvicidal activity with an LC₅₀ value of 4.17 mg/l. Cu/Ni and Cu/Co nanoparticles derived from palmyra palm fruit extract exhibited LC50 values of 18.50 and 16.076 ppm against Culex quinquefasciatus larvae, respectively [43-46]. CuNPs synthesized from Lantana camara leaf extracts were effective against Anopheles multicolor larvae, with an LC50 value of 12.6 ppm [47]. Aegle marmelos leaf extract-derived CuNPs demonstrated increased larvicidal effects against Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus, with LC₅₀ values of 508.31, 538.25, and 582.73 ppm, respectively [48]. CuNPs derived from Achillea fragrantissima and Nigella sativa extracts exhibited larvicidal effects against Culex pipiens mosquitoes, with an LC₅₀ value of 0.98 ppm [49]. The accumulation of CuNPs in the alimentary canal and their toxic effects on mosquito larvae contribute to their impact. Morphological changes, such as body shrinkage, darkening, pigmentation, and neck prolongation, have been observed in treated larvae as shown in Figure 3. Similar damage and abnormalities have been reported in other studies [50-52]. 
Corresponding to histopathology, the findings match the observations of other researchers who have suggested the impact of nanoparticles on mosquito tissue. According to Sandhu et al. [27, 53], exposing fourth instar larvae of Aedes aegypti to a ZnS-based hybrid nano emulsion resulted in the breakdown of various tissues and organs located in different regions. For instance, Mahmoud et al. [54] investigated the larvicidal potential of Piper nigrum extracts and observed significant histopathological alterations. The larva exhibited elongated cells with large vacuoles in the lining of the digestive tract. The protective layer surrounding the gut was torn, and the supporting tissue layer for the epithelial cells became detached. The surface projections of the epithelial cells appeared disorganized, and damage was observed in the muscular layer of the abdomen. Similarly, Raguvaran et al. [55] described the detrimental effects of silver nanoparticles on brush border cells and epithelial cells compared to untreated control larvae. The interaction between CuNPs and protein molecules in cells can hinder physiological enzymatic reactions in organisms, potentially leading to toxic reactions in insects. 
In relation to ecotoxicological research, Vivekanandhan et al. (2021) observed decreased toxicity of CuNPs towards Artemia salina, with LC50 and LC90 values ranging from 293.9 to 980.2 μg/mL [42]. Another study by Kumaresn et al. (2015) found that environmentally feasible levels of CuO NP suspensions were not acutely toxic to Artemia salina larvae, but prolonged exposure resulted in significant toxicity [56]. The aggregation of CuO NPs into larger particles in seawater reduced their toxicity, and the effects varied with nanoparticle concentration. Cimen et al. (2020) found that the accumulation and elimination rates of copper and copper oxide nanoparticles in Artemia salina varied depending on the concentration and application time, with higher concentrations leading to increased mortality rates [57]. Khan et al. (2020) evaluated the toxicity of silver nanoparticles synthesized from Pinus wallichiana stem extract on Artemia salina. They observed mortality rates of 53%, 36%, and 30% at concentrations of 1000, 100, and 10 μg/mL, respectively, and a LD50 value of 717.8 for silver nanoparticles (AgNPs) on brine shrimp [58]. 
Kamaraj et al. (2023) found that the utilization of Cassia auriculata (CA) extract for synthesizing AgNPs resulted in low toxicity towards Artemia nauplii, with a mortality rate of 28.14% [59]. Pecoraro et al. (2021) investigated the toxicity of different metal nanoparticles (AuNPs, AgNPs, TiO2 NPs) on Artemia salina, focusing on their effects on cyst hatching and larval mortality. They observed that TiO2 NPs were non-toxic to both larvae and cysts, while AuNPs had toxic effects on hatching but did not impact larval development. AgNPs caused larval mortality and inhibited cyst hatching [60]. Ragavendran et al. (2023) conducted a recent study on the synthesis of zinc oxide nanoparticles (ZnO NPs) using Ficus racemosa leaf extract. The study found that these ZnO NPs exhibited low toxicity towards Artemia salina, suggesting their potential as environmentally friendly and versatile products [61]. Overall, these studies provide valuable insights into the safety and potential benefits of nanoparticles synthesized using plant extracts, paving the way for further exploration and utilization of this method in nanotechnology and related fields.

This study explored the larvicidal activity of Coffee arabica-synthesized CuNPs against Aedes aegypti mosquito larvae. The CuNPs were synthesized using Coffee arabica extract and characterized using advanced techniques. The extract contained bioactive compounds with antioxidant properties. Green coffee had a higher total phenolic content and total flavonoid capacity compared to roasted coffee. Green coffee extract displayed stronger antioxidant activity. CuNPs showed concentration-dependent mortality, with lower LC50 values compared to coffee extracts alone. Histological analysis revealed tissue degradation in treated larvae, particularly in the head region. CuNPs also had a detrimental effect on Artemia salina larvae, with higher concentrations resulting in increased mortality. The study suggests that Coffee arabica-synthesized CuNPs have strong larvicidal activity against Aedes aegypti mosquitoes, presenting potential for eco-friendly mosquito control strategies.

The authors express their gratitude to the Hasanuddin University Mathematics and Natural Science Faculty (FMIPA) for their support in conducting this study, and also extend special thanks to the Medicine Science Faculty at Taiz University, Yemen, for providing laboratory facilities.

YA, AA, IW, PT, and HK collaborated as a team to execute the research plan, conduct practical research, collect data, and prepare the manuscript. The finalized manuscript has been reviewed and approved by each author.

There is no conflict of interest among the authors.