Nanotechnology is becoming an assurance for scientific advancement nowadays in areas like medicine, consumer products, energy, materials, and manufacturing. Copper nanoparticles (Cu NPs) were synthesized using Ardusi (Justicia adhatoda L) leaf extract via green synthetic pathway. The leaf of Justicia adhatoda L were known to have strong antimicrobial and anticancer properties arising due to the presence of various secondary metabolites, including, flavonoids, alkaloids, saponins, tannins, and phenolic compounds which serve as reducing, stabilizing, and capping agents for the Cu-Nanoparticles (NPs) synthesized. The biosynthesized A CuNPs were characterized based on Dynamic Light Scattering, Zeta- potential, Fourier transform infrared spectroscopy, X-ray diffraction spectroscopy, Scanning electron microscopy, and EDX. Justicia adhatoda L leaf extract mediated synthesis could produce A CuNPs with average hydrodynamic diameter size of 112.0 nm. The biosynthesized A CuNPs were further examined for antibacterial as well as antifungal activity with Gram-positive (S. aureus) and Gram-negative bacteria (E. coli and S. typhi) and with F. oxysporum f. sp. lycopersici respectively. The A CuNPs synthesized using Justicia adhatoda L leaf extract inhibited the growth of S. aureus, E. coli. and S. typhi. Whereas A CuNPs showed considerable inhibition against  F. oxysporum f. sp. lycopersici . The A CuNPs showed considerable inhibition against Human lung cancer (A-549) cell line in MTT assay. The newly synthesized  nanoparticles were found to be very effective antimicrobial and antifungal agents. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of Justicia adhatoda leaf extract revealed potent phytochemicals possessing antifungal, antibacterial, and anticancer properties.

Nanotechnology, Green synthesis, Phytochemicals, Antibacterial activity, Antifungal activity, Anticancer activity, Bioimaging.

The creation and application of materials that are at least one direction smaller than 100 nm (Njus et al., 2020) and that are produced utilizing top-down or bottom-up techniques (Chaloupka et al., 2010; Alanazi et al., 2018) is what is known as nanotechnology. Additionally, bottom-up approaches produce nanomaterials with better properties than top-down approaches because they promote the growth of nanocrystals at the atomic and molecular level, resulting in nanoparticles with enhanced properties and a larger surface area (Chaerun et al., 2022; Nwanya et al., 2019). Metallic nanoparticles (MNPs) have good optical, electrical, catalytic, magnetic, and biological activity (Jahan et al., 2021), Among the MNPs methods, there are several chemical-physical processes, such as vapor chemical deposition, laser ablation, gas evaporation, and sputtering (Da Costa et al. 2015). However, conventional physical-chemical methods involve a series of toxic and expensive reagents, limiting their applicability (Rajesh et al., 2018). 

Alternative synthesis techniques, like green or biosynthesis, which offer a reduction, nucleation, and stabilization process, must be used (Khanna et al., 2007). According to Suárez-Cerda et al. (2017), biosynthesis is defined by the use of biomolecules or extracts (functioning as biocatalysts) to reduce the metallic precursor and stabilize metallic nanoparticles while using fewer chemical reagents and leaving no environmentally hazardous residues behind. As a result, vitamins and plant, bacterial, algae, and fungal extracts are frequently employed as reduction agents (Wu et al., 2022; Kang et al., 2010; Ramyadevi et al., 2012; Latimer et al., 2014). Among the reducers, ascorbic acid (AA) has antioxidant qualities that support the immune system and promote wound healing in addition to its capacity to function as a reducing agent because of its oxidation (Gillespie et al., 2020; Li et al., 2020).

Even though they are costly, farmers all over the world now employ chemicals like fungicides, herbicides, and pesticides as the quickest means of controlling illnesses and pests. A number of issues have arisen from the misuse of these products, including detrimental effects on human health, the development of resistance to pests and diseases, issues with pollinating insects and domestic animals, and direct and indirect environmental effects when this material is incorporated into the soil and water (Chowdappa and Gowda 2013). The use of nanotechnology in plant disease prevention holds out a lot of promise for controlling various phytopathogens. Numerous metal-based nanoparticles (NPs) have gained popularity, such as silver (Ag), copper (Cu), zinc (Zn), titanium (Ti), magnesium (Mg), silica (Si), aluminum (Al), gold (Au).

Tomatoes (Solanum lycopersicum L), are a nutritious vegetable crop that are widely grown. Packed with vitamins, antioxidants, and lycopene, it helps support a healthy diet. However, tomato production is hampered by plant diseases like fusarium wilt and early blight. Because of their antimicrobial qualities, nanoparticles offer a possible treatment for these illnesses. Therefore, new directions for disease management and sustainable agriculture are opened by the combination of nanotechnology and plant-mediated synthesis. The potential of nanoparticles, particularly copper nanoparticles, to transform plant protection and agrochemical delivery is demonstrated by their many applications. As a result, the green synthesis of nanoparticles not only offers an environmentally friendly substitute but also improves the stability and efficacy of the nanoparticles, enabling a variety of uses such as disease management and crop protection.

A. Chemicals

Copper sulphate pentahydrate, Ascorbic acid and MTT (3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium 98 bromide) and other chemicals were procured from Sigma Aldrich, Navrangpura, Ahmedabad, Gujarat 380009. 

B. Green synthesis of Ardusi- Copper nanoparticles (A-CuNPs)

A-CuNPs were prepared by adding 4 ml of of Ardusi plant extract along with 20 ml of 0.1 M Ascorbic acid to 20 ml CuSO4 (7 mM) solution. The mixture pH (9.0) was kept on stirring for 10 minutes at 50℃ on Magnetic stirrer for reduction of Cu+ ions followed by 15 hrs of incubation at 60℃. The synthesis of nanoparticles was indicated with the colour change of the solution. The procedure was adopted from the literature Cai et al. (2020) along with some modifications.

C. Characterization of A-CuNPs

Particle Size Analyzer, Malvern, ZS 90 was used to examine DLS based particle size and zeta potential. Fourier transform infrared  spectroscopy (FTIR) was performed by using a Bruker, Invenio R. instrument. Scanning Electron Microscopy (SEM) and EDX analysis were carried out by using Scanning Electron Microscop, Carl Zeiss, EVO 18 equipped with AMETEK EDAX detector at 200 kV to examine scanning electron microscopic images to know the morphology and elemental composition of A-CuNPs.

D. Antimicrobial properties of A-CuNPS

Antifungal properties of A-CuNPs was evaluated by poisoned food technique against F. oxysporum f. sp. lycopersici. Bavistin was used as a positive control (Pariona et al., 2019).

Antibacterial properties of A-CuNPs was examined by agar-well diffusion method against both gram positive (S. aureus)  and gram negative bacterias (E. coli, S. typhi). Ampicillin was used as a positive control. (Dayana et al., 2021).

E. Cell imaging and Cytotoxicity of A-CuNPs

Yeast (Saccharomyces cerevisiae) cells were used to investigate the bioimaging ability of A-CuNPs. The Saccharomyces cerevisiae cells (100 µl) were treated with CuNPs (50 µl) and kept in incubation for 10 minutes in dark. Further, Yeast cells treated with CuNPs were heat-fixed on slides to make smears for further visualization of cells under Fluorescence microscope (Borse et al., 2022).

The A549 human lung carcinoma cells and NRK E Rat kidney cells were procured from Sardar Patel University, Gujarat, and the National Center for Cell Science, Maharashtra. A549 human lung carcinoma cells (1 × 105) were seeded and cultured in a 25 mm T Flask containing Dulbecco’s Modified Eagle Medium (DMEM), 10% Fasting Blood Sugar (FBS), 1% antibiotic solution, and incubated at 37°C under 5% CO2. Every third day, cells were trypsinized using Trypsin Phosphate Versene Glucose (TPVG) solution. NRK E Rat kidney cells (2.5 × 105) were grown overnight in a 96-well plate at 37°C. The cells were exposed to nanoparticle concentrations ranging from 20 µg/mL to 100 µg/mL for 24 hours before being incubated for 4 hours with MTT reagent at 37°C (Lokina et al., 2014; Thakore et al., 2019).

A. Green Synthesis of A-CuNPs

In this present experiment, CuNPs were prepared by mixing 20 ml of plant extracts of Ardusi, with 20 ml CuSO4 (7 mM, 9mM and 10mM) solutions, respectively, along with addition of 2 ml of 0.5M of NaOH with continuous stirring followed by heating the mixture at 50ºC for 10 min on magnetic stirrer for reduction of Cu+ ions. The synthesisation of nanoparticles was indicated with the colour change from Blue to transparent greenish brown at the end of the incubation (Cai et al., 2020) (Image 1).

Image 1. Green Synthesization of A-CuNPs.

B. Physiological Characterization of CuNPS

(i) DLS with zeta potential. The hydrodynamic diameter distribution of A-CuNPs was measured by DLS. DLS measures the fluctuations in the intensity of light scattered by particles undergoing Brownian motion. The DLS analysis of A-CuNPs, showed that 100 % particles were under 22.12 nm size (average size 112.0 nm) (Fig. 1). The DLS measurements give hydrodynamic diameter and includes thickness of capping layer present on the surface of nanoparticles reported by Nagajyothi  et al. (2014).  

Zeta potential is a measure of the surface charge of nanoparticles in solution, which influences their stability. The Zeta potential of A- CuNPs, was found to be –27.9 mV (Fig. 2), which shows that repulsive forces between synthesised particles were less and thus synthesized nanoparticles exhibit high stability, similar result was reported by Ramesh et al. (2015).

Fig. 1. DLS based size distribution of A-CuNPs.

Fig. 2. Zeta potential distribution of A-CuNPs.

(ii) Fourier Transform Infrared (FTIR) Spectroscopy Analysis. The FTIR analysis was used to understand the reducing and stabilizing action of Ardusi leaf extract.  The FTIR spectra of Ardusi plant extract showed peaks (Fig. 3) at around 3732.1 and 3702.9  cm–1 corresponds to O–H stretching vibrations, whereas 2358.7 cm–1 accredited to the symmetric and asymmetric C–H stretching vibration of flavonoids/ phenolic, respectively. The peaks at 1623.1 cm–1 corresponds to the CONH amide group whereas 1459.1 cm–1 corresponds to -OH bend in aromatic rings. The FTIR spectra of A-CuNPs (Fig. 4) showed peaks at 3737.6 corresponds to O–H stretching vibrations, whereas 2325.5 accredited to the symmetric and asymmetric C–H stretching vibration of flavonoids/ phenolic, respectively. The peaks at 1647.5 cm–1 corresponds to the CONH amide group whereas 1538.9 cm–1 corresponds to -OH bend in aromatic rings.  Similarly, the absorbance peaks at 1087.1 confirms for C–O–C and secondary – OH of the aromatic phenolic group.  Various phytochemical constituents like alkaloids, amino acids, flavonoids, saponins, steroids, glycosides, carbohydrates, tannins, and phenolic compounds in the ardusi leaf extract were involved in the formation and stabilization of nanoparticles. There was a change in the intensity and a small shift is observed in the spectra of nanoparticles compared to leaf extract. This is due to the interaction of phytochemicals with metal surface (Singh et al., 2012).

Fig. 3. FTIR Spectra of Ardusi Plant Extract: (A) and B) Accredited to O-H stretching vibrations, (C) Accredited to symmetric and asymmetric C–H stretching vibration of flavonoids/phenolics, (D) Accredited to the CONH amide group, (E) Accredited to -OH bend in aromatic rings.

Fig. 4. FTIR Spectra of A-CuNPs: (A) Accredited to O–H stretching vibrations, (B) Accredited to the symmetric and asymmetric C–H stretching vibration of flavonoids/ phenolics, (C) Accredited to the CONH amide group, (D) Accredited -OH bend in aromatic rings, (E) Accredited to C–O–C and secondary -OH of the aromatic phenolic group.

(iii) X-Ray Diffraction (XRD) Analysis. The crystalline nature and lattice parameters of A-CuNPs were examined by XRD Analysis. XRD works on the principle of constructive interference of X-Rays scattered by atoms within a crystalline material. By analysing this diffraction pattern, we can determine the crystal structure of nanoparticles. 

The XRD pattern of A-CuNPs is given in (Fig. 5). The XRD peaks at 2Ø angles of 34.24, 37.027, 51.51 and 64.94 were indexed to lattice planes (002), (111), (151) and (152) which can be assigned to Face Centered Cubic nature of A- CuNPs [JCPDS (048-1548)].  Similarly, Hegde and Kadre (2023) reported face cantered cubic structure of copper nanoparticles.

Fig. 5. XRD Pattern of A-CuNPs.

(iv) Scanning Electron Microscopy (SEM) Analysis of A-CuNPs. A-CuNPs, were characterized by Scanning Electron Microscopy (SEM) to determine the surface morphology synthesized CuNPs. SEM (Image 2) of the synthesized CuNPs are spherical in shape with agglomeration. The agglomeration of CuNPs is due to the presence of biomolecules involved during green reactions on the surface of nanoparticles. The biomolecules on the surface of nanoparticles will attract with the adjacent other biomolecules and its result with the highly agglomerative nature of green treated CuNPs nanoparticles reported by  Rajeswari et al. (2014).

Image 2. SEM Image of A-CuNPs.

(v) Energy dispersive X-ray (EDX) analysis. The elemental analysis of the synthesized CuNPs were carried out by energy dispersive X-ray analysis (EDX). It is based on the basic principle that each element has a unique atomic structure, giving a unique set of peaks in its electromagnetic emission spectrum. The vertical axis of the spectra represents the number of X-ray counts and the horizontal axis represents energy in keV. In the EDX spectra of A-CuNPs, signals of copper are found at 1.0 keV (Fig. 6).  The spectra showed the presence of C, Cu, and O elements in the sample and it confirms the formation of CuNPs. The obtained results were compared with the studies carried out by Cai et al. (2020).

Fig. 6. EDX spectra of A-CuNPs.

C. LC-MS Analysis of Aqueous Extract of Ardusi Plant 

The phytochemical compounds present in the aqueous extracts of Ardusi, was identified by LC-MS analysis and chromatogram was showed in Fig. 7. The active principles with their retention time (RT), molecular formula (MF), mass to charge ratio of ions (m/z) and Database score (DB) in the extracts were presented (Table 1)

Totally 19 compounds were identified from the aqueous extract of Ardusi are presented in Table 1. The aqueous extract of Ardusi plant revealed presenece of some important phytochemicals such as Indole (DB 77.08 %), Eflornithine (DB 92.65 %), Monobenzone (DB 97.12 %), Pyocyanin (DB 97.84 %), Cadralazine (DB 9.12 %), Rinderine (DB 82.68 %) and Pangamic acid (DB 83.84 %). Among these detected phytochemicals, Indole has been reported as a significant anticarcinogenic agent against various human cancer cell lines (Yao et al., 2022). Eflornithine was found to be active against some leukemias and solid tumors, such as breast, colon, cervical, small-cell lung cancer and melanoma (Yang et al., 2023). Monobenzone has been stated as a potent anticancer agent against gastric cancer cell lines (Ma et al., 2021). Pyocyanin has been reported as a potent antibacterial, antifungal, and anticancer agent (Mudliar and Prasad 2024).

Table 1: Identified phytochemicals in aqueous extract of Ardusi (LC-MS).

Fig. 7. Chromatogram of aqueous extract of Ardusi plant.

D. Antifungal Properties

In vitro antifungal activity of A-CuNPs was studied against F. oxysporum f. sp. lycopersici using poison food technique (Image 3).  Bavistin was used as positive control. A-CuNPs at a concentration of 500 ppm gave maximum inhibition against F. oxysporum f. sp. lycopersici by 75.6% as compared to the positive control (Bavistin). Whereas minimum inhibition of 33.6% was observed at concentration of 100 ppm. The action of A-CuNPs as an antifungal agent was better than that shown by Bavistin with 12.6% mycelial inhibition at 500 ppm concentration (Table 2). Cu-NPs forms unusual bulges on the surface of the mycelium. Higher concentrations caused stronger morphological alterations of the mycelium; the mycelium lose their smoothness and is strongly deforms, additionally, many fractures are generated, a rough and peeled mycelium surface, it indicates considerable damage of the cell wall, which promotes the outflow of intracellular components and shrinkage of hyphae leads to killing of fungi. Similarly, a comparative analysis of the antifungal activity of various concentrations of CuNPs, was done against F. Oxysporum and A. alternata (Pariona et al., 2019). Duvvi et al. (2019) also reported antifungal properties of CuNPs against phytopathogenic fungi like Rhizopus artocarpi, Penicillium citrinum, Fusarium roseae, Alternaria alternata, Fusarium oryzae, and Cladosporium cladosporoides.

Table 2: In vitro antifungal activity of A-CuNPs against F. oxysporum f. sp. Lycopersici

Image 3. In vitro antifungal activity of A-CuNPs against F. oxysporum f. sp. Lycopersici (A) Control, (B) 500 ppm Bavistin, (C) 100 ppm A-CuNPs, (D) 300 ppm A-CuNPs, (E) 500 ppm A-CuNPs.

E. Antibacterial Properties

The antibacterial activity of A-CuNPS was evaluated against both Gram (+ve) and Gram (-ve) bacteria. Clear zones of inhibition were observed around the well, which showed their inhibitory effect on bacterial growth (Image 4). 

In-vitro antibacterial activity was carried out by agar well diffusion method. A-CuNPs sample solutions (150 µg/ml to 250 µg/ml) were used for antibacterial activity against E. coli, S.typhi, S. aureus. The positive control (20 µg/ml) was antibiotic ampicillin. A-CuNPs had the biggest zones of inhibition against Salmonella typhi (22 mm), followed by E. coli (21 mm), and Staphylococcus aureus (19 mm) at 250 µg/ml concentration (Table 3).

These results show that this green synthesis of A-CuNPs have higher tendency to show better antibacterial activities against these different bacterial strains. The current study clearly demonstrates that CuNPs are solely responsible for antibacterial activity against Gram (+ve) and Gram (-ve) bacteria, and the results are quite promising when compared to previous findings (Chatterjee et al., 2024; Mehta et al., 2022). Mohindru and Garg (2017) also reported antibacterial activity of Geraniol based Copper Nanoparticles.

Table 3: In vitro antibacterial activity of A-CuNPs against E. coli, S. typhi, S. aureus.

Image 4. In vitro antibacterial activity of A-CuNPs against E. coli, S. typhi, S. aureus.

F. Cell Imaging 

Yeast (Saccharomyces cerevisiae) cells were used to investigate the bioimaging ability of CuNPs. To this, fluorescence microscopy was utilized to visualize Saccharomyces cerevisiae cells using CuNPs as imaging probe by applying excitation wavelength at 488 nm (Image 5). It can be observed CuNPs were easily internalized yeast cells, leading to exhibit blue as well as green color fluorescence. These findings demonstrate A-CuNPs has strong capacity to emit distinct bluish green emission, cross cell membranes, and enter intracellular spaces, indicating A-CuNPs could be used as cell imaging probes. Similar results were reported by Borse et al. (2022).

Image 5. Cell imaging of Yeast (Saccharomyces cerevisiae) cells using A-CuNPs.

Cytotoxicity of CuNPs. The MTT assay was used to assess the cytotoxicity of A-CuNPs, B-CuNPs, and N-CuNPs on the NRK 52 rat kidney cell line, with exposures to various concentrations (7.5, 15.25, 31.25, 62.5 µg/mL) of each type. At higher concentrations, cell viability declined slightly. At the respective concentrations, the cell viability for A-CuNPs were 97.60%, 95.40%, 94.25%, and 91.75% (Fig. 8). A-CuNPs exhibited nontoxic and biocompatible properties, making them suitable for use as fluorescent probes in cellular imaging (Borse et al., 2022).

The anticancer property of green synthesized A-CuNPs was scrutinized using Human lung cancer (A-549) cell line by MTT assay. Different concentrations of (7.5,15.25,31.25,62.5 µg/mL) A-CuNPs was treated with A-549 cell line, the IC50 value of synthesized A-CuNPs at a concentration of 62.5 µg/mL caused 41.75% of inhibition of cell proliferation in cell lines (Fig. 9). This is due to number of A-CuNPs will accumulate inside of the cells; it creates stress that leads to cell death. These results obviously proved the effectiveness of A-CuNPs against cancer cells. The cell death was increased by increasing concentrations of A-CuNPs. Similar kinds of results were also previously reported by Lokina et al. (2014); Thakore et al. (2015).

Fig. 8. In-vitro cytotoxicity of A-CuNPs on NRK 52 E rat kidney cell line.

Fig. 9. In-vitro cytotoxicity of A-CuNPs on A-549 lung cancer cell line.

Kunj Bhatt, Sanjay Jha, Vaibhav Mehta, Hitesh Mehta, Vasudev Thakkar and Anjali Thakkkar (2025). Biosynthesis of Cu Nanoparticles using Leaf Extract of Justicia adhatoda L for their Potent Antimicrobial and Anticancer Activity. Biological Forum, 17(3): 12-20.