Synthesis of Lawsonia inermis -encased silver – copper bimetallic nanoparticles with antioxidant, antibacterial, and cytotoxic activity

: The extract of the medicinal plant Lawsonia inermis , known as henna, was employed to synthesize silver – copper bimetallic nanoparticles (Ag – Cu NPs) in a unique, e ﬃ cient, and cost-e ﬀ ective method. The shape, size, and structural features of synthesized Ag – Cu NPs were determined by ultra – visible spectroscopy, Fourier transform infrared spectroscopy, X-ray di ﬀ ractometer, ﬁ eld emission scanning electron microscopy, transmission electron microscopy, and energy dispersive X-ray spectroscopy methods. The rod-shaped Ag – Cu nanoparticles, averaging 41.66 ± 17.18 nm in size, synthesized from L. inermis , exhibited potent antioxidant activity by inhibiting 2,2-diphenyl-1-picrylhydrazyl and 2,2 ′ -azinobis (3-ethylbenzothia-zoline-6-sulfonic acid) free radicals. They also displayed signi ﬁ cant antibacterial e ﬀ ects against Pseudomonas aeruginosa


Introduction
Nanotechnology is considered useful in fields of modern technology, such as health [1], pharmaceutical [2], and ecology [3].The cornerstone of nanoscience lies in the creation of innovative molecules or nanoparticles [NPs] through techniques centered around auto-assembly [4].Nanomaterials have demonstrated applications in the development of oral, transdermal, widespread, pulmonary, and other delivery techniques to facilitate drug transport, increase therapeutic bioavailability, and ensure medicine biocompatibility [5], including the utilization of NPs as catalysts [6].In particular, NPs produced by noble metals, including gold, silver, platinum, and zinc, are utilized in medicinal and consumer applications [7].However, NPs are regarded as chemical risks.In fact, they may combine with and absorb organic compounds [8].
Silver nanoparticles (AgNPs) have shown potentials in the pharmacy sector [9].AgNPs are used in the production of disinfection lotions and dressings for burns and wounds [10,11].Additionally, they could be used as visual components, such as fluorescent equipment and bioimaging devices [12].AgNPs have an extensive choice of applications in biomedicine because of their distinct properties, including antibacterial, antifungal [13,14], anti-inflammatory [15], anti-angiogenesis [16], antiviral agent [17], and anti-platelet effects, among others [18,19].
The advantages of plant-based sustainable nanoparticle synthesis over chemical methods have been repeatedly demonstrated by recent study [20].A viable and eco-friendly method for producing metal and metal oxide NPs, among other nanomaterials with a variety of uses, is called "green synthesis."Sustainable synthesis from plants yields non-toxic and biocompatible NPs, making it a safe and environmentally acceptable substitute for chemical processes, especially in the context of medical applications [21].Green fabrication of the metal NPs is produced by the utilization of chemical compounds of plant extracts [22].The inexpensive, less hazardous, and easily scalable singlestep green synthesis processes, on the other hand, are quick, sustainable, and environmentally friendly [23].Applications for metal NPs, including gold, silver, and platinum, are widely used and provided top priority in the bioimaging, photochemical, electrochemistry, and cosmetic sectors.Green synthesis approaches are used because they have the ability to minimize the toxicity of NPs [24].The monometallic NPs were shown to possess antibacterial abilities as well as anti-plasmodial, anticancer, pesticidal, and larvicidal effects [25,26].However, as compared to their monometallic counterparts, the combinatorial synthesis of various mixtures of bimetallic nanoparticles (BNPs) is recognized to have unique features.Investigation into the various BNPs might reveal special characteristics regarding their antagonistic or synergistic multifunctional action [27][28][29].The growth in the use of hybrid composites has accelerated because of their extensive wide range of applications, which include water purification, antibacterial characteristics, and photocatalysis [30,31].The plant components not only function as a strong reducing agent but also assist in the formation of steady NPs through their capping activity.The metal silver is now widely utilized as a protective coating for NPs in several areas of biosensors and antibacterial applications in medicine.It demonstrated notable characteristics, including non-toxicity, biodegradability, and biocompatibility [22].This article reports the synthesis of NPs containing L. inermis L. aqueous extract.This plant is worldwide known for its use in temporary tattoos and as a medicinal plant, under the names of the derived products henna or henné.The phytochemical studies revealed a complex chemical constitution of L. inermis, including phenolic, tartaric, citric acids, anthraquinones, stilbenoids, polyphenols, triterpenes, carotenoids, naphthalene, and other constituents, including hennosides, the glucosidic precursors of lawsone, which is the dying ingredient [32].The majority of these constituents, notably organic acids, function as strong reducers.
In the synthesis of AgNPs using phytochemicals, there are three phases.First, the precursor Ag + is reduced to Ag by constituents of the plant extract.Then, the produced Ag atoms develop NPs, and the newly manufactured AgNPs enlarge as a result of the surface reduction of the Ag + ion.Finally, the AgNP's size is controlled by electrostatic stabilization, and plant extract's negatively charged ionic species adhere to the surface of the AgNPs, triggering capping [33].
The study aimed to synthesize for the first time bimetallic (Ag-Cu) nanoparticles using L. inermis leaf extract.The resulting Ag-Cu NPs were characterized by various methods (ultra-visible spectroscopy [UV-Vis], Fourier transform infrared spectroscopy [FTIR], X-ray diffractometer [XRD], field emission scanning electron microscopy [FESEM], and transmission electron microscopy [TEM]), and the antioxidant, antibacterial, and antiproliferative activities were assessed.Furthermore, the biotoxicity was determined.In ecotoxicological studies that screen several metabolites for drug detection in plants, the A. nauplii assay is routinely used [34].Artemia-based assays are considered quick, convenient, and affordable.

Collection and preparation of leaf extract
L. inermis L. fresh leaves were collected in Idappadi (latitude: 11.5849°N, longitude: 77.8382°E), Salem, Tamil Nadu.Botanical voucher samples were deposited in the Periyar University, Natural Drug Research Laboratory (NDRL).The leaves were thoroughly cleaned under running water, allowed to dry at room temperature, and then cut into small pieces to remove any remaining debris.Dr. D. Natarajan, Associate Professor in the Department of Biotechnology at Periyar University in Salem, identified the plant.The fresh leaves were thoroughly washed with distilled water and then allowed to air dry in the shade at room temperature.The dried leaves were ground using an electric lab grinder into a well powder.The prepared fine powder (10 g) was mixed with distilled water (100 mL), and the combination was heated for 30 min at 80°C, while being continually stirred.Whatman filtration paper No. 1 was used to filter the obtained material.The extract was stored until necessary at 4°C [34].

Preliminary phytochemical analysis
The freshly obtained n-hexane, methanol, ethyl acetate, and aqueous extracts of L. inermis leaves were subjected to phytochemical analysis to identify the presence of several phytoingredients, including phenol, terpenoids, flavonoids, tannins, saponins, and alkaloids (Table 1) by the standard methodology reported by Balaei-Kahnamoei et al. [35].The results are in accordance with previous reports [32] and with different polarities of the constituents.

Biosynthesis of Ag-Cu NPs
Ag-Cu was used as a precursor material in the nanoparticle's synthesis.The bimetal nanoparticles were weighed (2 mM of silver nitrate [AgNO 3 ], Sigma Aldrich and copper acetate [0.2 M of CuCO 2 CH 3 , Sigma Aldrich]) and added to 1,000 mL of the plant extract.Then, the aqueous extract (10 g of plant leaf powder mixed with 100 mL of sterile distilled water) was added to the metal solution.Aqueous extract and metal solution were combined in a 1:1 ratio.The metal solution (Ag-Cu) was added along with 500 mL of L. inermis leaf extract and mixed using a magnetic stirrer for 1 h.The mixture was kept in a magnetic stirrer under vigorous agitation at 60°C for a further 3 h.The solution was allowed to stand overnight at room temperature.After this process, a yellowish-brown color was observed.Then, the mixture was centrifuged at 12,000 rpm for 15 min and the sediment was dissolved in distilled water.The pellet was stirred at 12,000 rpm for 20 min along with milli-Q-water.The pellet/sediment was transferred into the Petri plate.After the plate was dried using a hot air oven.Then, the plate was kept in a drier for 2 days.Finally, the dried nanopellets were stored in an airtight container and kept at 4°C until further use [36].

Characterization of Ag-Cu NPs
A UV-Vis spectrophotometer (Systronics, 2302, India) was used for the preliminary detection of Ag-Cu NPs.After agitating and maintaining the pH, the solution was scanned from 300 to 700 nm.Functional groups at distinct wavenumbers in L. inermis, as well as the resulting solution of Ag-Cu NPs, were evaluated using FTIR Bruker ATR Alpha-II (Bruker, India) in a range from 4,000 to 500 cm −1 [37].The phase and crystalline nature of the fabricated Ag-Cu NPs were examined using XRD.Cu K energy with a spectrum of 1.5 Å at 40 kV and 30 mA was used for the evaluation on an X-ray diffractometer (Bruker, Germany) with a scanning frequency of 0.02 cm −1 in the area of 2θ extending between 10°and 90° [35].The morphology, shape, and atomic constituents of metallic elements in fabricated Ag-Cu NPs were investigated using FESEM combined with energy dispersive X-ray spectroscopy (EDX).Using double-sided tape, the samples were secured to the tubular aluminum stub.The samples supported by stubs had a gold coating.A scanning electron microscope (EVO 18, ZEISS) with a 10,000× magnification was used to evaluate the surface morphology of the gold-coated samples [38].TEM analysis was used to categorize the NPs based on their size and form.Following the synthesis of Ag-Cu NPs, a small quantity of the sample was released onto a copper grid that had been covered with carbon.The excess solution was eliminated using tissue paper, and the grid's film was left to dehydrate under a mercury lamp for 6 min.The picture was then enlarged at various magnifications to observe Ag-Cu NPs.The instrument's software was used to examine the data.Subsequently, a JEOL JEM-2100 TEM (JEOL, Tokyo, Japan) was used to scan the grid at a voltage of 120 kV.The surface charge of the produced Ag-Cu NPs was estimated using zeta potential analysis, which served as a standard for the NPs' physical stability.After preparing the sample solution in distilled water and ultrasonically sonicating it twice or three times, it was centrifuged for 15 min at 5,000 rpm.The dispersed solution exhibited a viscosity of 0.875 mPa s.Subsequently, the conductivity was fine-tuned to 0.130 mS•cm −1 , and the electric voltage was set at 3.4 V for the nano-analyzer apparatus (Horiba Scientific Nanoparticle Analyzer SZ-100, Kyoto, Japan) [39].

Antibacterial activity
The antibacterial efficacies of the Ag-Cu NPs and the aqueous extract of L. inermis were examined using four strains, such as Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Bacillus subtilis (MTCC 1272), and Pseudomonas aeruginosa (MTCC 1035).They were periodically sub-cultured in the lab and kept in nutritional agar slants.For the production of the inoculums, a single colony was cultured overnight in nutrient broth, and the turbidity was subsequently adjusted to 0.5 McFarland standards.The experiment strains were swabbed onto Mueller Hinton agar (MHA) dishes and left to air dry for 10 min.Three distinct amounts (25, 50, and 75 µg•mL −1 ) were used.Chloramphenicol (5 µg•mL −1 ) served as the positive reference.After 24 h at 37°C, the area of inhibition on the plates was determined and tabulated.The results were obtained by calculating the zones of growth inhibition (mm), as described by Kiani et al. [40].

Antioxidant assay 2.7.1 DPPH scavenging assay
The DPPH scavenging procedure was used to assess the antioxidant properties of the Ag-Cu NP containing aqueous leaf extract of L. inermis [41].Briefly, 100 µL of different Ag-Cu NP doses, plant extract, and reference (ascorbic acid) (25-150 µg•mL −1 ) were combined with 100 µL of DPPH (0.1 mM in 80% ethanol) and kept for 30 min at 37°C in dark conditions.The intensity of the response solution was read at 517 nm against a blank (80% ethanol) using a spectrophotometer (Shimadzu1601 model, Japan).The % of DPPH scavenging free radicals was calculated using Eq. 1.

{( ) }
where A0 is the absorbance of the control, and A1 is the absorbance of the test.

ABTS assay
The ABTS radical ability of various amounts of aqueous extract and Ag-Cu NPs was measured using a proven methodology.ABTS (7 mM) and potassium persulfate (2.45 mM) were combined to produce the stock solution, which was then kept at RT in the dim for 16 h.Then, the stock ingredient was diluted with methanol to produce the working solution, which had a 734 nm absorbance.Then, 25-150 µg•mL −1 of different water extract and Ag-Cu NP concentrations were added to 180 µL of the ABTS test solution.Using a spectrophotometer (Shimadzu1601 model, Japan), the optical density (OD) at 734 nm was recorded after 30 min at ambient temperature.Ascorbic acid functioned as the standard.Formula 1 was used to estimate the % of free radicals that ABTS was able to scavenge [38].Percent inhibition of absorbance at 734 nm was calculated using Eq.2: where AB is the absorbance of ABTS radical + methanol and AA is the absorbance of ABTS radical + sample extract/ standard.Ascorbic acid was used as a standard substance.

Culturing of cancerous cells
The cytotoxicity effects of fabricated Ag-Cu NPs were evaluated using the breast cancer (BC) cells (MDA-MB-231).The cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a CO 2 atmosphere with 5% CO 2 .Trypan blue was used to determine the number of cells and the live cells in a hemocytometer.The cells were seeded at 1 × 10 4 in a 96-well plate and placed in the CO 2 incubator for 24 h at 37°C for the MTT evaluation.

Assessment of cytotoxic effects on cancer cells
The bimetallic synthesized Ag-Cu NPs were investigated in the MDA-MB-231 cells at different concentrations (25, 50, 75, and 100 µg•mL −1 ).The dilutions were obtained using DMSO.The cells received treatment and were then placed in a CO 2 incubator for 24 h.After a microscope observation of the cell morphology after 24 h, 20 µL of the MTT solution (5 mg•mL −1 ) was added, and the mixture was then maintained for 4 h at 37°C.About 100 µL of DMSO was added after incubation to help in thawing the formazan that had been produced.In order to measure the cell viability, the OD at 570 nm was calculated via a 96-well plate reader.

AO/EB dual staining
Ag-Cu NP-treated cells were exposed to acridine orange/ ethidium bromide (AO/EB) double labeling to look for structural signs of death.To stain 25 µL of treated and untreated cells (5 × 10 6 cells•mL −1 ), AO/EB dye combination (100 mg•mL −1 generated in PBS noticeably) was used.After that, fluorescence microscopy (Nikon Eclipse TS 100) was used to evaluate the samples [42].

Toxicity testing by A. nauplii
A. nauplii cysts were obtained at the Central Institute of Brackishwater and Aquaculture (CIBA, Chennai, Tamil Nadu).The synthetic saline water was produced by adding 35 g of NaCl in 1 L of purified water, and dehydrated cysts were then immersed in the solution.After 48 h of incubating at room temperature (28 ± 2.0°C) with active oxygenation and continuous light, the nauplii hatched within 48 h [34].
The toxicity of NPs in A. nauplii was evaluated using the previous procedures [43].The test was conducted on brine shrimp larvae (A.nauplii).For the production of a standard solution, 50 mg of NPs were placed in suspension in 1 mL of distilled water.The freshly prepared suspension with various amounts of NPs (2, 4, 6, 8, and 10 mg•L −1 ) was then arranged by serial dilution of the stock solutions of NPs in synthetic saline water (35 g•L −1 ) instantly before use.The 24-well microtiter plates were filled with 2 mL of a solution (different NP amounts).The 24-well plates were then filled with 10 nauplii each, and they were left to develop for 24 h at 37°C.After 24 h, the quantity of nauplii that survived in the respective well was recorded using a stereo microscope.For each concentration, the experiment was performed in triplicate.Only ten nauplii and prepared brine water were found in the negative control wells.The % of mortality was estimated by comparison of the amounts of survival in the test and control experiment help.The lethality was measured according to Abbott's formula: Test Control /Control 100

Statistical analysis
GraphPad Prism was used to perform a grouped analysis of variance (ANOVA) on the data (GraphPad Software, San Diego, California, USA).Between the treatment and control sets, a P < 0.05 was considered significant.The findings of experiments were stated as mean ± SD.

Phytochemical analysis of L. inermis
The antibacterial activity of plants is mostly attributed to secondary metabolites, including tannins and phenolic compounds.Phytochemical analyses were carried out to determine the metabolites contained in the various L. inermis extracts.Table 1 shows that the aqueous, methanol, and acetone extracts all included cardioglycosides, terpenoids, carbohydrates, phenols, and quinones.All four of these extracts lacked significant amounts of proteins.The methanol extract also contained tannins, cardioglycosides, carbohydrates, phenols, and quinones, but not alkaloids or terpenoids, at least not in concentrations that could be detected.These metabolites dissolve in the solvent based on their polarity.

UV-Vis spectroscopic examination
Color changes were noticed by pictorial analysis of the L. inermis leaf extract of the synthesized Ag-Cu NP solution.The L. inermis leaf extract alone does not result in any changed color.When the plant extract was added to the Ag-Cu solution, the color changed to light brown (in an hour), and 24 h later, it changed to light green (Figure 1).Ag-Cu NPs produced from the L. inermis leaf extract were absorbed in the UV spectrum at different wavelengths between 200 and 600 nm.The peak of Cu NPs is 295 nm, the highest wavelength of AgNPs is 415 nm, and the peak of Ag-Cu NPs is 455 nm (Figure 2).

FTIR analysis of Ag-Cu NPs
The synthesized Ag-Cu NPs were subjected to FTIR spectroscopy, which demonstrated the presence of multiple functional groups at various wavelengths in the range of   2 and Figure 3.In summary, the FTIR analysis demonstrates that L. inermis contains proteins, amino acids, alcohols, and metabolites that include carboxylic acids.These substances may have served as stabilizing and reducing mediators while Ag-Cu NPs were being formed.

X-ray diffraction analysis
The XRD pattern study proved the crystalline aspects of the Ag-Cu NPs (Figure 4).The most intense peak was observed at 32.12°, 38°, 44°, and 77.30°.This confirmed the position of the Ag-Cu nanocrystals as the peak positions at 2θ (43.21°,

FESEM and EDX analysis
The FE-SEM micrograph provided further information about the morphological characteristics of the particles synthesized.The average size of acquired Ag-Cu NPs is 41.66 nm.The scanning electron micrograph of the NPs at 123× magnification is shown in Figure 5a-e.The EDX findings confirmed that the Ag-Cu NPs are the source of the metals Ag and Cu, with a 51.7% atomic proportion.Other maxima in the EDX were seen in addition to Ag, C, Cu, and O, each having an atomic proportion of 47.6%, 23.4%, 4.1%, and 25.0%, respectively.The oxygen single was produced from the absorbed parts of the leaf extract and the device's coating.In contrast to Ag, Cu, and indicated the production of pure Ag-Cu NPs, all other elements, with the exception of carbon, have a very low atomic amount (Figure 5F).The zeta potential was used to examine the stability of the nanoparticle in the aqueous solution and its surface charge potential.The zeta potential of the produced Ag-Cu NPs revealed good stability with a strong peak at −21.0 mV (Figure 5g).

Antibacterial activity
The three different concentrations of L. inermis aqueous extract and Ag-Cu NPs (25, 50, and 75 µg•mL −1 ) were tested for antibacterial activity against selected pathogens using the agar well diffusion method.The antibacterial activity of leaf extract from L. inermis was compared to a positive reference (chloramphenicol, 5 µg•mL −1 ).The inhibitory zones are presented in Figures 6 and 7 and Tables 3 and 4  effective against human pathogens, which leads to the capability of the Ag-Cu NPs to inhibit bacterial growth.The growth of inhibition was validated for each of the pathogens and recorded.

Antioxidant activity using DPPH and ABTS assay
DPPH and ABTS evaluations were used to determine the radical potential of the aqueous extract of L. inermis and Ag-Cu NPs.Using ascorbic acid as a reference, the analyses demonstrated that the tested sample increased the scavenging ability of DPPH (Figure 8a) and ABTS radicals (Figure 8b) in a concentration-dependent pattern.The Ag-Cu NPs had a better free radical inhibition rate with 90.5 and 85.24 μg•mL −1 as associated with the extract with 70 and 68 µg•mL −1 using DPPH and ABTS, respectively.The capacity of the nanomaterials to quench neutral and cationic radicals was evaluated, and the result showed that Ag-Cu NPs were effective against both constant unbiassed radicals produced by DPPH and free cationic radicals generated via ABTS.

Evaluations of cytotoxicity on BC cells
To investigate the cytotoxic effects of the environmentally produced Ag-Cu NPs, MDA-MB-231 cancer cells were treated with varying concentrations of NPs for 24 h.The MTT assay results demonstrate that cell viability gradually decreases when Ag-Cu NP concentration increases in MDA-MB-231, which were determined to be 37.40 µg•mL −1 , respectively.The cell appearance was examined using an inverted microscope at the IC 50 concentration of Ag-Cu NPs.Based on the findings of the cytotoxic activity investigation, the researchers can conclude that the biosynthesized Ag-Cu NPs are 100 µg•mL −1 hazardous to MDA-MB-231 cells, as depicted in Figure 9.The study's findings indicate that the cytotoxicity of phyto-synthesized Ag-Cu NPs increased with nanoparticle concentration.

Morphological sign of cancer cells
The AO/EB double staining method was used to observe the apoptosis-related structural changes induced by both Ag-Cu NPs.The stained cells were classified as follows: alive (light green) (Figure 9c), primary apoptotic (bright green color with packed chromatin), late apoptotic (orange color), or completely unviable (fluorescence of red color) (Figure 9d).Cells exposed to Ag-Cu NPs displayed cytotoxic bodies, membrane blebbing, and contracted nuclei.The nuclear framework of the control cells, however, was undamaged.

Toxicity to A. nauplii
The primary assessment of the environmental toxicity of chemicals and other substances can be obtained through ecotoxicological investigations using microcrustaceans such as brine shrimp.Based on the present study, mortality rates have been determined for exposure to different Ag-Cu NP concentrations for 24 and 48 h.The lethality assays using     5). A. nauplii mortality was noticed in all doses, as shown in Figure 10, and was more pronounced at the concentration of 10 mg•L −1 .
The LC 50 and LC 90 values of Ag-Cu NPs synthesized against A. nauplii larvae treatments were better (3.58, 5.83 mg•L −1 for 24 h and 1.51, 2.59 mg•L −1 for 48 h).The aquatic behavior of A. nauplii larvae exposed to Ag-Cu NPs at the highest concentration (10 mg•L −1 ) was significantly changed, and the following alterations were noticed: disruption and change of behavior, which predominantly resulted in reduced swimming ability, feeding (Figure 10c).Furthermore, body damages, such as thorax swelling, appendages loss, and antennal disfigurement (treated), were observed.In control tests, all nanoparticle doses had no lethal impact on A. nauplii after 24 h of exposure (Figure 10d).Overall, the results indicate that the mortality rate increased with fabricated nanomaterials and exposure duration.

Discussion
In recent decades, the biosynthesis of AgNPs from phytoextracts has gained prominence owing to the potent antibacterial effect of zerovalent silver and the simple reduction of silver(I) ions [44].The development of an active, environmentally friendly, and biocompatible process for synthesizing metal NPs is an attractive and well-defined research area.The aqueous extract of L. inermis contains phytochemicals that lead to the production of Ag-Cu bimetallic NPs.The color of the solution changed from light brown to dark yellow when L. inermis extract was added to the combination of Ag and Cu solution, providing the first indication of NP production.Figure 12 depicts the green synthesis of Ag-Cu bimetallic NPs.The reduction of Ag + and Cu 2+ occurs in a one-pot procedure, employing phytochemicals found in L. inermis leaf extract as reducing and stabilizing/capping molecules.As a result, during the onepot reduction of two metal ions, Ag ions were first reduced by reducing botanical compounds, such as hennosides, coumarins, and isoplumbagin from an aqueous extract of leaves from L. inermis, and then Cu 2+ ions were reduced on the surface of Ag to correspond with their potential deposition.The color change from brown to dark yellow confirms the production of Ag-Cu NPs.Similarly, Al-Haddad et al.
[45] also reported on the production of Ag-Cu NPs using Phoenix dactylifera, which exhibits UV spectrum maximum at 295 nm.The UV spectrum of Ag-Cu NPs fabricated using L. inermis leaf extract was noted at 395 nm, previously reported by Al-Haddad et al. [45].Synthesized biogenic silver nanoparticles' surface plasmon resonance (SPR) noticed a peak with a 420 nm center that was connected to the absorbance of AgNPs [46].The fabrication of Ag-Cu NPs using L. inermis leaf extract was analyzed by XRD.The XRD evaluations of synthesized Ag-Cu NPs show five strongest peak values at (32.12°), (38°), (44°), (64.41°), and (77.30°), which corresponds to the IC.Sagar et al. [47] found that the green-produced AgNPs from Saussurea obvallata had peak values in the XRD data of 38.49°, 44.62°, 64.85°, and 77.68°; these values corresponded to the crystalline planes of the FCC framework of AgNPs ( 111), (220), and (311), respectively.The crystallinity and transparency of the manufactured NPs were confirmed by the sharp peaks and removal of  The FTIR analysis of nanomaterials showed the relation between the metal and bioactive substances present in the fabricated Ag-Cu NPs.The existence of NPs was proved by the presence of crucial functional groups, such as alcohol, alkanes, amides, aliphatic, methyl, and halides.The development of the peaks is assigned to interactions between C]O (C-O) groups and NPs, indicating that C]O and C-O groups are the anchoring ligands of the NPs with a strong affinity for coordination [49].The existence of functional groups, viz.3,234 cm −1 , which can be assigned to phenolic functions, indicates the presence of O-H bonds.When Eltaweil et al. [50] studied the C. ambrosioides synthesized Ag@biochar, a spectral band for the O−H stretching vibration was detected at 3,274 cm −1 , which they attributed to the presence of phytoconstituents containing OH functional groups, such as flavonoids, tannins, with alkaloids.Similarly, Velidandi et al. [51] stated that the green fabrications of novel Ag-Cu and Ag-Zn bimetallic NPs from Annona muricata showed the presence of functional groups aromatics and carboxylic acids.Ag-Cu NPs attached molecules appear to have both bound and free nitro groups, as shown by the peaks at 1,724 cm −1 , which are carboxylic acids from phenols.For the fabrication of copper nanoparticles (Cu NPs), the presence of coumarins was determined in produced NPs and the aqueous extracts of Hippophae rhamnoides L. Comparable outcomes were also reported in the literature [52,53].All those functional groups were involved in the potential reduction of Ag-Cu and the steadying of the produced Ag-Cu NPs.Similar findings demonstrated that the protein found in Balanites aegyptiaca functions as an agent of reduction and stabilizing agent for the CuO NPs and indicated that common functional groups of biologic molecules (such as phenols, carbonyls, and aliphatic amines) from the stem bark extract may potentially be engaged in the bio-conversion of copper ions to CuO NPs [54].EDX of the synthesized product provides distinct elemental signals of Ag-Cu.In the present study, the element signal includes C and O. Similarly, Gopinath et al. [55] suggested that the other elements signals containing C and O are assured to the external towards Ag-Cu NPs.The EDX spectrum evidenced the signal peaks of Cu at 0.9 keV.The result was correlated with the findings observed in a strong peak of Ag at 3.4 keV peak and showed the purity of the sample.It is noticeable that metallic Ag-Cu NPs are usually shown by the absorption at 3.4 keV.FESEM analysis helps to attain the size and shapes of Ag-Cu NPs.The size of the fabricated NPs has been observed with different ranges of 41.66 nm.Sallam et al. [56] have previously published results that were similar.Porous-shaped silver/ zinc nanoparticles made from Persicaria salicifolia biomass have been produced by Hosny et al. [31].Overall, the slight aggregated form was observed in fabricated Ag-Cu NPs due to the occurrence of phytochemicals, such as phenol, polysaccharides, and other reducing sugars.
Green-synthesized Ag-Cu NPs were found to be active against targeted clinically important pathogens.The diameter of the inhibition zones is fully dependent on the species of bacteria and plant NPs.Ag-Cu NPs have potential to attach to the outer membrane of bacteria due to the occurrence of hydroxyl groups [57].The current study noticed the green fabricated Ag-Cu NPs demonstrated strong antibacterial activity against P. aeruginosa [58].In the work of Rathi et al. [59], bio-mediated synthesis of Ag/CuO and Ag/TiO 2 from Caesalpinia pulcherrima demonstrated excellent antibacterial potential by inhibiting E. coli and S. aureus.Likewise, Gopinath et al. [55] investigated the sustainable production of Ag, Au, and Ag-Au BNPs utilizing leaf extract from Gloriosa superba, which showed potent antibacterial properties against pathogens that affect humans.The precise process underlying metal nanoparticle antimicrobial action is still undetermined [60].However, the interaction between the negatively electric bacterial membrane and the positively electric AgNPs could be connected with their ability to generate electrostatic interactions.The relationship disturbs the structure of the bacterial membrane, which results in cell destruction and a bactericidal effect.Reactive oxygen species are produced more frequently by the binding of AgNPs with the cell membrane and mesosomes, which disrupts mesosomal function and probably kills the cell.Furthermore, the antibacterial action of Ag Cu NPs has interacted with bacterial inter-cellular cell communication and its disruption of cellular metabolisms.Second, change may result from bacterium cell viability being lost.When related to Ag and Cu NPs, it has a strong synergistic potency.Likewise, Merugu et al. [61] reported the antibacterial activity against targeted pathogens by Toddy palm synthesized bimetallic Ag/Cu NPs.According to Hosny et al. [31], the Ag/ZnO@Biohar nanocomposite-derived NPs showed bactericidal function.This is believed to be the result of two processes: first, the small particles bind to protein thiol groups, triggering deactivation; second, they communicate with microbial DNA, compressing the DNA and preventing DNA replication, which leads to death.
DPPH is one of the most significant techniques to study free radical properties that have been described in recent years [40].The antioxidant abilities of the manufactured Ag/Cu NPs were assessed using the DPPH assay.DPPH radicals are purple in organic solvents, but yellow when showing to reducing agents or hydrogen donors.At a frequency of 517 nm, such a shift can be detected.Chemicals, as well as organic molecules with healing activities, can counteract such radicals, and a reduction in color concentration at 517 nm indicates a higher neutralizing action.The NPs' potential to scavenge both radicals improved in a concentration-dependent pattern.At the higher doses (150 µg•mL −1 ), the measured DPPH free radical potentiality of the phyto-fabricated Ag-Cu by L. inermis leaf extract was found to be 68.84 ± 0.56%, whereas for the ascorbic standard (reference) was found to be 97.45 ± 0.45% at the same concentration.Similarly, Nguyen et al. [62] reported on the phytosynthesis of CuO, ZnO, and CuO/ZnO nanomaterials by leaf extract from Annona glabra.These NPs demonstrated higher antioxidant capacity by neutralizing DPPH radicals.The inhibitory rate for Ag-Cu NPs derived from L. inermis leaf extract resulted at the doses of 150 µg•mL −1 ; however, it was 90.23 ± 0.65% compared to 65.34 ± 0.65% for water extract in the ABTS scavenging activity.Similarly, ascorbic acid had an inhibition of 99.5 ± 90.45% at the same concentration.The outcomes also indicated that aqueous extract synthesized NPs had better radical scavenging potential in the DPPH test than they had in the ABTS assay.These studies showed that this interaction demonstrates the NPs' reducing capacity and that the antioxidant capabilities of the Ag-Cu NPs produced by the phytoextract have been maintained, maybe due to the covering of the Ag-Cu NPs [63].Chitturi et al. [64] observed 71.56 ± 0.55% inhibition in the DPPH radical scavenging assay at 100 µg•mL −1 by bimetallic Ag-Cu synthesized using palmyra fruit.Furthermore, Sharma et al. [65] demonstrated that the phyto-derived production of Pt and Au/Pt NPs using Syzygium aromaticum extract had powerful anti-potential by reducing DPPH (IC 50 -27.2µg•mL −1 ) and ABTS (IC 50 -14 µg•mL −1 ).The high antioxidative capacity of L. inermis biofabricated via Ag-Cu NPs may be due to the presence of phenolic chemicals on their surface.Globally, BC is the most prevalent disease that affects women.With an expected 2.3 million new cases, or 11.7% of all tumor patients worldwide in 2020, it has presently exceeded lung cancer as the most common cause of cancer rate [66].Radiation, chemotherapy, hormone treatment, and surgery (mastectomy and lumpectomy) are all right now employed to treat BC [66].The absence of specificity in these treatments represents a serious drawback.Cancer nanomedicine, which is a multidisciplinary field that focuses on developing and therapeutic uses of materials and technology at the nanoscale (usually up to 100 nm), has made significant advances in the development of cancer medicines over the past several decades [67].
Nanoparticle-based targeted delivery may provide biocompatibility, multifunctional encapsulation of active ingredients, decreased degradation during the bloodstream, inactive or active targeting, efficient delivery, and fewer adverse effects.The medicinal benefits of biosynthesized NPs beyond those produced chemically are higher.The Ag-Cu NPs synthesized in this study displayed a higher inhibitory effect on BC cells, especially at the dosages of 100 µg•mL −1 .Pechyen et al. [68] stated the green fabrication of Au NPs exhibited cytotoxicity properties against human breast tumor cells at maximum doses of 400 µg•mL −1 .Likewise, AuNPs produced from Musa paradisiaca revealed Synthesis of Lawsonia inermis-encased Ag-Cu NPs  15 cytotoxic efficacy against human lung tumor (A549) cells at maximum doses of 100 mg•mL −1 [69].Similar to this, synthetic AuNPs from Annona muricata showed cytotoxic impacts on Hep2 liver cancer cells while being non-toxic to Vero cells [70].On the other hand, the AgNPs manufactured from mushrooms were active in killing MDA-MB-231 cells at a low concentration (6 mg•mL −1 ).The anticancer strategy of NPs comprises intracellular enzyme and protein interactions, excessive ROS formation, and failing of mitochondrial membrane potential [71].The presence of oxidative stress nanomaterials to be harmful to tumor cells, and DNA injury and cell alteration can affect the growth of cancer cells [72].
Artemia salina is recognized as one of the utmost valuable experimental microcrustaceans suitable for aquatic eco-toxicity screening [73].Artemia sp. are a highly appropriate, labor-intensive-minimum living food source for aquatic cultivation due to its commonly used as a beneficial and energetic food to the larvae of many marine faunas [74].The biotoxicity of phyto-fabricated Ag-Cu NPs from L. inermis was estimated on non-target microcrustacean A. nauplii.The outcomes showed minimal toxicity to A. nauplii (two time periods of 24 and 48 h), with LC 50 and LC 90 values ranging from 3.58, 5.83, and 1.51, 2.59 mg•L −1 .In the control group, no notable Ag-Cu NPs were observed.The overall accumulation amounts of Ag-Cu NPs increased significantly with cumulative concentration and time exposure, confirming a time-and dosedependent accumulation that showed a deposition of NPs in higher concentration.Similarly, Velidandi et al. [51] reported that the green fabricated platinum-palladium bimetallic NPs evaluated against Artemia nauplii at 100 μg•mL −1 demonstrated 100% mortality.Additionally, Dinesh et al. [75] studied the Phyllanthus emblica fabricated of PdNPs toxicity assessment against non-target organism A. nauplii with least LC 50 value is 1.25 μg•mL −1 .Madhav et al. [76] investigated the synthesized CuO NP was accumulated in the gut of A. salina and showed significant toxicity also, associated changes in biochemical markers such as catalase, reduced glutathione, and glutathione-S-transferase were evident.Many studies have evidenced a close relationship between oxidative stress and all aspects of cancer [77,78], including carcinogenesis and the tumor-bearing state, asking for necessary treatment and prevention [79].The oxidative stress can arise from endogenous origins (at the cellular level where mitochondria are involved) [80,81], as well as exogenous origins (e.g., bacterial attack) [82].This study evidences that technology, as always, affords new solutions to the profit of nature's resources, also in the case of a very old medicinal plant like L. inermis [83,84].When oxidative stress exceeds the capacity of the oxidationreduction system of the body, the multiform activity of medicinal plant extracts, renewed by nanotechnology, can be the source of novel solutions against old challenges (Figure 11).

Conclusions
Plant-based materials often exhibit advantageous properties in terms of both pest control [85,86] and medicinal applications [87].Our method involves an environmentally friendly and cost-effective approach to synthesizing BNPs (Ag-Cu) using phytofabrication derived from L. inermis leaves, all accomplished in a single step.The leaves' secondary metabolites, comprising phenolics, flavonoids, and tannins, act as surface-active ingredients, ensuring stability in the BNPs.Flavonoids and phenols play a role in reducing metal ions to form Ag-Cu NPs.Initial qualitative phytochemical analysis of L. inermis leaf extract revealed abundant phenols, tannins, and flavonoids.The production of nanocrystals was confirmed through spectroscopic and surface morphological methods.UV-Vis spectrophotometry showcased an SPR band at λ max 455 nm of Ag-Cu NPs.SEM images exhibited various shapes of NPs, including elongated, truncated, capsule, cubical shapes, and some aggregates.TEM and SEM images showcased spherical NPs within a size range of 20-43 nm.Moreover, the synthesized L. inermis Ag-Cu NPs displayed notable antibacterial activity, exhibiting significant growth inhibition zones against pathogenic bacteria such as P. aeruginosa (22 mm) and E. coli (19 mm) at the highest concentration.They also demonstrated remarkable antioxidant activity as DPPH radical scavengers, with the highest percentage of inhibition recorded at a concentration of 100 µg•mL −1 Ag-Cu NPs.However, acute exposure of A. nauplii to varying amounts of synthesized Ag-Cu NPs mediated by L. inermis resulted in significant behavioral changes, notable deaths, or oxidative stress within 24 h of contact.Prolonged exposure to NPs for 48 h induced oxidative stress but had minimal mortality effects.
Overall, this study underscores the potential of L. inermis-derived Ag-Cu NPs in various applications, emphasizing the need for balanced consideration of their beneficial properties alongside a thorough evaluation of their environmental and biological impacts for responsible and sustainable utilization.Further investigations into their safety profiles and long-term effects will contribute to their safe and effective implementation in diverse fields.
there is more carboxylic acid with O-H stretches involved.On the other hand, compounds containing alkanes are allocated the C-H stretching vibration of one band at 2,914 cm −1 .There is aldehyde with C-H stretches present, as indicated by the peak at 2,847.92 cm −1 .The dense band at 1,010 cm −1 is ascribed to the -C-O of ether found in coumarins, while the signatures at 1,724.04 cm −1 are caused by the distinctive peaks of coumarins (biomolecules responsible for reduction).The spectrum of absorption at 1,595 cm −1 is evidence of the stretching vibration of amines (N-H) in proteins.The value observed at 1,352.79 cm −1 denoted nitro with N]O stretches.The smaller concentration signal at 627.68 cm −1 provides chloride with C-Cl stretching.The corresponding assignments to functional groups are reported in Table

Figure 1 :
Figure 1: Fabrications of bimetallic Ag-Cu NPs from L. inermis.(a) Aqueous leaf extract of L. inermis.(b) 2 mM silver nitrate (AgNO 3 ), (c) 0.2 M copper acetate, and (d) color shift of the extract from light brown to light green after development at room temperature (synthesized Ag-Cu NPs).

Figure 2 :
Figure 2: UV spectrum of the Ag, Cu, and Ag-Cu NPs fabricated from the L. inermis.

54 .
10°, and 75.10°) were indexed to the normal planes of Ag-Cu nanocrystals.These planes showed that copper metal face-centered cubic (FCC) in its structure.These high-pitched Bragg peak strengths have been confirmed due to the stabilization of the Ag-Cu NPs.The XRD pattern of fabricated Ag-Cu NPs shows five strongest peak values at (32.12°), (38°), (44°), (64.41°), and (77.30°) which is corresponding to the intense count (IC).

Figure 3 :
Figure 3: FTIR spectra of the biosynthesis of Ag-Cu NPs fabricated from L. inermis aqueous extract.

Figure 4 :
Figure 4: XRD analysis of the biosynthesis of Ag-Cu NPs fabricated from the L. inermis.
. At 75 µg•mL −1 concentration, the maximum concentration of leaf extract showing the highest growth inhibition was observed, such as E. coli (19 mm), B. subtilis (23 mm), P. aeruginosa (26 mm), and S. aureus (16 mm).Strong antibacterial properties were demonstrated by synthesized Ag-Cu NPs against the targeted human pathogens.The results revealed that P. aeruginosa and S. aureus had the maximum inhibition zones, measuring 25 mm, followed by E. coli, which had the second-largest zones, measuring 22 mm, and B. subtilis, which had the lowest zones, measuring 20 mm.Overall, these results demonstrated that the presence of strong bioactive components may affect the antibacterial properties of plant substances.The antimicrobial activities of produced Ag-Cu NPs covered with bioactive plant compounds may effectively control new bacterial strains, including multidrug-resistant strains, based on the results of this experiment.According to the results, the antibacterial activity of nanomaterials depends mostly on their size and concentration, and it is more evident against Gram negative than Gram positive pathogens.Ag-Cu NPs are strongly

Figure 5 :
Figure 5: Electron microscopy study: (a) FESEM analysis, (b-d) TEM analysis of Ag-Cu NPs, (e) particle size distribution, (f) EDX spectra of Ag-Cu NPs, and (g) zeta potential analysis of synthesized Ag-Cu NPs from leaf extract of L. inermis.

Figure 6 :
Figure 6: Antibacterial activity of L. inermis aqueous extract tested against human clinical pathogens at different concentrations.

Figure 7 :
Figure 7: Antibacterial activity of synthesized Ag-Cu NPs tested against human clinical pathogens.

Figure 8 :
Figure 8: Scavenging activity of various amounts of the aqueous leaf extract of L. inermis and Ag-Cu NPs using (a) DPPH assay and (b) ABTS assay.

Figure 9 :
Figure 9: Morphological sign of apoptosis by AO/EB dual staining.(a) Untreated cells (control); (b) cells exposed to synthesized Ag-Cu NPs of L. inermis show unchanged morphology; (c) untreated cells (control) appear to be systematically green with a normal framework; and (d) cells treated (100 µg•mL −1 ) with Ag-Cu NPs displays apoptotic bodies and disorganized nuclei (400× magnification).(e) The percentage of living cells among MDA-MB-231 human breast cancer cells, as well as apoptotic and necrotic cells, after 24 h of treatment with synthesized Ag-Cu NPs from L. inermis.

Figure 10 :
Figure 10: Visible abnormalities in Artemia nauplii exposed to various Ag-Cu NP amounts.(a) A. nauplii in the control was not subjected to any NP effect.(b) Treatment of 2 mg•L −1 of NPS did not alter morphological features.NPs (6 mg•L −1 ) exposed nauplii had altered eyeball form (c), lost eye pigment, and had fading eyes, whereas the typical eye is rounded and black (control).(d) A. nauplii was occasionally observed using a stereomicroscope (10 mg•L −1 ) (Labomed at 40×).Note: The yellow arrow indicates no accumulation of NPs, and the red arrow indicates the accumulation of NPs in A. nauplii's intestine.

Figure 12 :
Figure 12: Schematic illustration of the possible mechanism of the green synthesis process of Ag-Cu BNPs.

Table 1 :
Qualitative phytochemical analysis of different extracts of

Table 2 :
FTIR analysis of Ag-Cu NPs synthesized using L. inermis

Table 3 :
Antibacterial activity in L. inermis plant extract against clinical pathogens

Table 4 :
Antibacterial activity of Ag-Cu NPs tested against human pathogens

Table 5 :
Non-target toxicity assay of Artemia nauplii lethality using Ag-Cu NPs at 24 and 48 h exposure