Amalgamation and Characterization of Copper Nanoparticles Alleviated With Stachytarpheta Cayennensis and Its Anti-cancer Activity in Both in Vitro and in Vivo Animal Model

Melanoma is an extremely malignant skin cancer with a probability of metastasis and accountable for the mainstream skin associated mortality. In the present study, we described the novel usage of Stachytarpheta cayennensis mediated copper nanoparticles and its anti-cancer activity in both in vitro and in vivo model of skin cancer. The synthesis of Cu-NPs was conrmed using UV-absorbance peak values ranging from 325-345 nm. The size of the nanoparticles was around 90nm, as deduced by the dynamic light scattering study.Furthermore, transmission electron microscopy (TEM) established that the morphology of the copper particles. Cytotoxicity of Cu-NPs of Stachytarpheta cayennensis illustrates the toxicity level of Stachytarpheta cayennensis. Also, the anti-cancer potential of Cu-NPs was evaluated in A375 cells. In experimental animals, body biochemical parameters like SOD, CAT, GSH were diminished in DMBA induced animals while Cu-NPs treatment raised the levels of the aforementioned enzymatic antioxidants compared to the control animals. Additionally, cytotoxicity assay, mitochondrial membrane potential (MMP), cell adhesion analysis, and the estimation of reactive oxygen species (ROS) in the presence of Cu-NPs was evaluated by standard protocols. The present study's outcomes conrm the defensive and valuable effects of copper loaded Stachytarpheta cayennensis against DMBA induced skin melanoma, animal model.


Introduction
Melanoma is the malignancy of melanocytes which exist as single cells inside the basal layer of cuticles.
Melanoma is an extremely malignant skin cancer with a probability of metastasis and accountable for the mainstream skin associated mortality. The 5-year existence of melanoma is about 10% (Miller et al., 2020). To date, melanoma is one of the world's critical medical challenges. The unsuccessful therapy is complemented with chemotherapy, which might lead to toxic and side effects on individuals. Also, few familiar drugs, such as metformin, were too used for melanoma cancer treatment in the clinic. In current years, the speedy propagation of nanotechnology and the nano-related treatment approach could offer a novel idea for treating melanoma cancer (Hong Feng et al. 2019). Nanoparticles (NPs) are progressively contributing to the opportunity of their usage in the expansion of new healing methods in the arena of cancer. Nanoscale particles are the emerging sources of useful medications due to their high exterior to capacity ratio and improved reactive surface area. The growing anticancer candidates nano-patterned from decorative metals like copper, silver, and gold are increasingly recognized (Li et al., 2020;Zottel et al. 2019). Since copper is inexpensive than gold and silver, the expansion of copper-related NP's as curative is preferred for developing therapeutics. The biomedical applications of copper nanoparticles were already proved earlier (Akintelu et al., 2020).
Though there are abundant ndings on the anticancer outcome of copper oxide nanoparticles, there is inadequate research available on the anticancer effectiveness of metallic copper nanoparticles ( are characteristically luxurious and necessitate harmful chemicals, while the green synthesis method is environmentally friendly and less costly. Plants form the most discovered collection of living organisms available for the green synthesis of nanoparticles (Silva et al. 2015). Herbal harvests can also play a central role in covering adaptable groups, avoiding the necessity for ligand interchange preceding to living or organic aspects. However, greener or biogenic approaches do not assure nanomaterials' production with enhanced or at undistinguishable possessions than those fashioned by outdated procedures (Metz et al. 2015).
Earlier reports suggest that wild owers, the unsolicited plants in the farming setups, can be employed for synthesizing nanoparticles (Francois et al. 2019). Stachytarpheta cayennensis is a ligneous weed with 0.5-1m height. This plant's leaves are used in traditional medication to improve digestion and as an antipyretic to cure chronic liver disorders, shafts, coughing and ache in bone, and high urine passage and excess sweating (Schapoval et al. 1998). It was already proved that the Stachytarpheta cayennensis has potent antimicrobial, antispasmodic, anti-in ammatory, and anti-ulcerogenic activities Panido et al., 2006). Stachytarpheta cayennensis extracts have proven useful as an antimicrobial, antispasmodic, and anti-diabetic agent. Phytochemical characterization exposed the occurrence of carbohydrates, avonoids, terpenoids, and saponins (Adebajo et al. 2007;) in the plant extract. In the present study, we describe the novel usage of Stachytarpheta cayennensis plant extract for copper nanoparticle preparation. Additionally, in-vitro antioxidant and anticancer trials show the e cient pharmacological usage of such nanoparticles.

Plant and Extract Composition
The test plants of Stachytarpheta cayennensis were obtained from Eziobodo communal, Owerri West, Nigeria (L.G.A of Imo state). The plant leaves were sliced into small pieces and cleaned under running tap water to remove sand particles. Leaves were then dehydrated in a room for a month and crushed into powder using a crushing machine. The crushed sample was then deposited in sealed decanters until used for the study (Iwu et al. 2018 The synthesized copper nanoparticles were characterized using a UV-visible spectrophotometer functioning at 1nm determination with an ophthalmic measurement of 10mm. Absorptions were determined after 24h of nanoparticle development recovered by centrifugation at 5000rpm within one hour. UV-Vis examination of the response combination was carried out for 300 seconds (Francois et al. 2019).

Dynamic light scattering
To normalize the particle magnitude or dimension of the synthesized copper nanoparticles, they were analyzed using dynamic light scattering (Shi et al., 2014). In this method, 0.01gm of polymer precipitation included 12mmol silver nitrate in 20ml puri ed solutions.

Transmission electron microscopy
The structure of the copper nanoparticles was resolved by transmission electron microscopy (JEOL JEM 1200, Czech Republic). From 1mg/ml suspension of copper nanoparticles, a drop was positioned on a 300-lattice carbon covered copper electron microscope grid and then systematically air-dried for 10hr. The 2.5 In vitro assays 2.5.1 MTT cytotoxicity assay Cell toxicity by Cu-NP was scrutinized using the typical MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, Sigma) assay (Mosmann 1983). The principle of MTT assay is that the mitochondrial dehydrogenase enzyme of viable cells breakdown the tetrazolium salts present in the membrane, which reacts with the MTT dye, resulting in the formation of purple formazan crystals, that is soluble in isopropanol or DMSO. The absorbance of these crystals can be measured at 580nm. The progressed inhibition of Cu-NP treated cells was articulated as the proportion inhibitory concentration (IC) compared to the unprocessed control cells. For the experiment, 4×10 4 cells per well were seeded in 24 well microtiter plates, mixed with MTT dye at a nal concentration of 0.5 mg/ml. The cells were incubated for 3-4hr in the dark condition at room temperature. The MTT solution was then discarded from the plated, supplemented with acidic isopropanol (Merck, USA), and incubated for further 10min to solubilize the formazan crystals. The absorbance was measured using a Shimadzu, UV-1900i instrument at 580nm.

Cell adhesion assay
Copper nanoparticles were prepared by succeeding amalgamation with trypsin. Then Stachytarpheta cayennensis were distributed to microtiter plates. Cu-NPs was disconnected at a different time, and wells were washed with phosphate buffer, and the resultant solution was eliminated excitedly in contrast to committed cells. Cells steadfast beneath the microtiter plate were stained with crystal violet dye and paraformaldehyde, further supplemented with the Cu-NPs plates and incubated for 20min. After that discoloration, excess crystal violet dye was cleaned by phosphate-buffered saline solution. Crystal violet binds to the intracellular proteins. The amount of crystal violet bound to the proteins was proportional to the number of cells in the dishes. The crystal violet dye was extracted using isopropanol, and the purple color was measured at 540nm in the reader (Ruan et al. 2012).

Estimation of intracellular ROS
The reactive oxygen species produced inside the cell was estimated by the method as reported earlier (Gunaseelan et al. 2017) using DCFDA (2, 7-dichloro uorescein diacetate, Thermo Fisher Scienti c, USA) dye. Cu-NPs at 5µg/mL and 10µg/mL was added to the microtiter plate. After one day of incubation, the cells were exposed to Cu-NPs for 4-5hr. Following this, the plates were washed three times by saline with phosphate as a buffering agent and incubated with DCFDA dye (20µM) for about 45mins at room temperature. The cells were then washed with 200µl of buffered saline phosphate, and the dye uptake was recorded in a multimode reader (Sigma, USA) at wavelengths of 540 and 490nm excitation/emission, respectively. The con rmation of the ROS generation was analyzed through a uorescence microscope (Sigma, USA).

Mitochondrial membrane potential
This method is interesting to inspect the transmembrane e cacy, which employs the probe 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (Srilatha Sakamuru et al. 2016 ). All mice were divided into four groups of 6 mice (n = 6 each). The Group I mice were considered control and administered normal diet, and acetone alone was used during experimentation. Group II mice were treated with 25µg of DMBA in 100µL acetone through the dorsal region injection. Groups III mice were treated with DMBA and CuNPs at 10mg/kg b.w in 1% DMSO. Group IV mice were administered with CuNPs (10mg/kg b.w) alone. Oral administration of CuNps (10mg/kg bw) was carried out thrice a week, early from the week of commencement DMBA treatment till 25 weeks. At the end of the 25 weeks, biochemical and molecular level studies were carried out, and skin tissue was dichotomized out control and investigational animals.

Assessment of antioxidant levels in the serum of experimental animals
The level of antioxidants in the serum of control and experimental animals were analyzed by standard methods. Primarily, a blank trial containing 2mL reagent and 1mL distilled water were utilized. The reaction was initiated by adding up of 10% trichloroacetic acid and thiobarbituric acid, TBA (0.375%) to 0.025 normality HCl, and mixed gently till TBA entirely lique ed (Rahmat et al. 2017). The serum of 0.5mL was mixed with 0.5mL sterile water in a 1.5mL centrifuge tube. The reaction mixture was added to each sample, incubated at 37°C in a water bath for 10 min, and centrifuged at 2000×g for 20 minutes. The optical density values were recorded at 540nm, and the supernatant was transferred to new tubes. Likewise, the antioxidative molecules were estimated by the malondialdehyde standard index. Superoxide dismutase enzyme action was performed according to the previously described method ).
The catalase enzyme action was studied by spectrophotometry, followed by the hydrogen peroxide deprivation speed per minute, according to the standard method described earlier (Goth 1991). Other additional biochemical assays like GSH, and GPx were determined by the standard protocol (Rahmat et al. 2017).

Assessment for pro-in ammatory markers and interleukin levels
The level of pro-in ammatory markers, i.e., IL-6, IL-1β, and TNF-α in the serum of both control and experimental animals, were analyzed by enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fischer Scienti c, USA). All the experimental procedures were followed according to the user manual instructions.

RT-PCR analysis
The total RNA was extracted from the skin tissues of both control and Cu-NP-treated animals with the help of Trizol RNA extracting kit (Santacruz Biotech, USA) by the manufacturer's protocols. The extracted RNA from the experimental animals was utilized to construct the cDNA using the a commercial RT-PCR assay kit (Santacruz Biotech, USA). The primers used for NF-κB forward: 5′-GTGGTGCCTCACTGCTAACT-3′ and reverse: 5′-GGATGCACTTCAGCTTCTGT-3′; COX-2 forward: 5′-ACACACTCTATCACTGGCACC-3′ and reverse: 5′-TTCAGGGAGAAGCGTTTGC-3′; iNOS forward: 5′-CAGCTGGGCTGTACAAACCTT-3′ and reverse: 5′-CATTGGAAGTGAAGCGGTTCG-3′. The reaction was sustained with initial denaturation for the 30s at 95°C, subsequently 40 PCR cycles with 5s of denaturation at 95°C, annealing for 30s at 60°C and extension for 15s at 95°C. The whole test was executed in triplicate for precise measurements, and the obtained results were expressed as relative mRNA expression (fold change).

Histopathology
For histopathological investigations, the formalin embedded skin tissue segments from both control and experimental animals were xed in para n, subdivided, and then located onto miniature in nitesimal slides followed by standard histopathological measures. The skin tissue sections were stained with hematoxylin and eosin (H&E staining) and imaged using light microscopy (Attalla and El-Kott 2015).

Statistical analysis
The experimentations were carried out in triplicates, and the results were represented as mean ± standard deviation (SD). Values with p < 0.05 were considered statistically signi cant.

Depiction of UV-Visible Spectroscopy
The reduction of copper loaded Stachytarpheta cayennensis extract was con rmed by UV-Vis Spectrophotometer. The UV visible spectra of Cu-NP's have been displayed in Fig. 1. The UV-VIS absorption of Cu-NP's was the highest around 370nm, which is equivalent to that of copper nanoparticles. Consequent to development, the color was temporarily biased of the excitation state of surface plasmon situation in the Cu-NPs. The decrease of copper was examined through UV-VIS Spectrophotometer. Absorption curves of Cu-NP's have a range around 300nm, and enlargement of peak speci es that the vital essentials are disconnected. The steadiness also ranges the surface plasmon captivation, which relies on the volume of the metal nanoparticles and the dielectric constant (Melvin et al. 2009). Stachytarpheta cayennensis and copper acetate changed the shade of the Cu-NPs. The highest absorption at 370nm was tentative, which denotes the entire corrosion of copper ions. The UV-Vis accessory range of Cu-NPs is recognized with a penetrating optical density at 370nm, which designates a projected consistent arrangement of the nanoparticles.

Dynamic light scattering analysis
Dynamic light scattering (DLS) is a technique used to measure the hydrodynamic size of the synthesized nanoparticles. Figure 2 shows Cu-NP's particle proportions in the nanosheets. It was established through subsequent tentative statistics that copper loaded Stachytarpheta cayennensis extract had a size of 100nm. The highest percentage of copper loaded Stachytarpheta existing in the solution was of 52.50nm.

Representation by Fourier Transform Infrared
Spectroscopy for NP's FTIR examination is an excellent and approved technique to differentiate the biological compounds that remained accountable for breaking down metal ions into Cu-NPs in the presence of Stachytarpheta cayennensis. (Fig. 3). The phyto-component in the extracts was responsible for the con guration of the variability of nanoparticles. The FTIR array of Stachytarpheta cayennensis demonstrated recurrent combined peaks from 3266cm − 1 to 660cm − 1 . Figure

Cu-NPs absorption examined by transmission electron microscope
We assessed the morphology and the size range of nanoparticles through transmission electron microscopy in the current study. TEM images of synthesized Cu-NP's are illustrated in Fig. 4. The TEM images of Cu-NPs revealed that they possessed irregular and partially oval-shaped structures with an average size ranging from 20-100nm.

Cytotoxicity outcomes of Stachytarpheta cayennensis Cu-NPs
The cytotoxicity effect of Cu-NPs in A375 cells was assessed by MTT assay. Figure 5 discloses the cell probability assay for normal A375 cells, which were primarily examined in a concentration-dependent manner (1, 2.5, 5, 7.5, and 10µg/ml), and the viability was normalized between 5 to 10µg/ml. Therefore, A375 cells were exposed to Stachytarpheta cayennensis Cu-NPs for about 24hr. Accordingly, the MTT outcomes documented the concentration needed for Cu-NP's cytotoxicity, as displayed in Fig. 5. The amount of mitochondrial damage investigated after 24hr exposure to two different concentrations of Cu-NP's, i.e., 5, 7.5 mg/ml, was 95% and 85%.
3.6 Cell adhesion evaluation using Stachytarpheta cayennensis copper nanoparticles Figure 6 demonstrates the examination of adhesive cells in control, Stachytarpheta cayennensis copper nanoparticles treated, and other experimental groups. The cells were evaluated for 24hr to get improved adherent consequences. Both control, Stachytarpheta cayennensis copper nanoparticles (5&7.5µg/ml) showed reasonable adherent cells compared to other groups. While in gestation time, intermission of 24hr control and Stachytarpheta cayennensis clusters exempli es attached cells. Eventually, our fallouts display the optimistic outcome of Stachytarpheta cayennensis in adhesion assay.

Stachytarpheta cayennensis on intracellular ROS production
The A375 cells treated with copper nanoparticles (5&10µg/ml) for 6hr demonstrated a signi cant decrease in ROS production. This was deceptive that DCF light emission considering the quantity and quality in Fig. 7(A). Group I control doesn't exhibit much reactive oxygen species development. Group II depicts mild ROS production where it was treated with the copper nanoparticles of Stachytarpheta cayennensis (5µg/ml). At the same time, Group III illustrates elevated ROS formation where it was treated with the copper nanoparticles of Stachytarpheta cayennensis at (7.5µg/ml).

Outcome of Stachytarpheta cayennensis on mitochondrial membrane e cacy
Programmed cell death triggered by the modi cation of mitochondrial membrane potential was assessed by JC-1 staining dye (Fig. 7B). The control cells release increased light emission of green color, representing differentiated mitochondria membrane e cacy in Fig. 7B. Simultaneously, Stachytarpheta cayennensis copper nanoparticles (5µg/ml) established a noteworthy adjustment of ΔΨM, which consistently abridged green color emission displays. Copper nanoparticles of Stachytarpheta cayennensis at about 7.5µg/ml were recognized notable in alteration of ΔΨM, which reliably exposed green color uorescence.

Ethidium bromide and Acridine orange staining
The e cacy of Cu-NPs consequences in a different concentration-dependent manner in several viable cells well improved in late apoptotic and necrotic cells (Fig. 7C). The EB/AO staining assay is appropriate for copper nanoparticles in concurrence with the cell layer worsening viably. Control assemblage compared to untreated cells does not demonstrate any color variation. Group II treated with Cu-NP's of Stachytarpheta cayennensis (5µg/ml) showed a normalized number of viable cells. Simultaneously group III Stachytarpheta cayennensis Cu-NP's (7.5µg/ml) exhibited viable A375 cells with an increase in the number of early apoptotic cells.

Effect of copper nanoparticles of Stachytarpheta cayennensis on biochemical parameters
The results from Fig. 8 depict that SOD, CAT, GPx, and GSH activities were signi cantly diminished in DMBA only treated group II wherein TBARS was production augmented. At the same time group III mice treated with DMBA along with Cu-NP's of Stachytarpheta cayennensis (10 mg/kg bw) restored these enzymatic antioxidants levels. This was reversed with TBARS levels in which group III animals showed reduced TBARS. However, group IV animals exhibit no noteworthy decrease or increase in SOD, GSH, CAT, and GPx levels, similar to the control group I animals. Subsequently, TBARS also exhibited similar results.
3.11 Effect of copper nanoparticles on pro-in ammatory cytokines level Figure 9 demonstrates that the levels of in ammatory markers were well improved by copper loaded Stachytarpheta cayennensis. It represents the effect of DMBA and copper nanoparticles of Stachytarpheta cayennensis (10 mg/kg bw) on pro-in ammatory cytokines IL-6, IL-1β, and TNF-α, levels.
A signi cant augmentation in the planes of pro-in ammatory cytokines, TNF-α, IL-6, IL-1β altitudes was evident in the DMBA alone administered group (Group II). Whereas in group III, treatment of copper nanoparticles of Stachytarpheta and DMBA promisingly (P < 0.05) restored the levels of these in ammatory cytokines, which were comparable to the control group and the drug alone treated group IV animals.
3.12 Effect of Cu-NPs of Stachytarpheta on transcription signaling molecules of skin segments of control and experimental mice. Figure 10 illustrates the levels of transcription signaling molecules, which were also ampli ed by copper loaded Stachytarpheta cayennensis. This signi es the effect of DMBA provoked and copper nanoparticles of Stachytarpheta cayennensis (10 mg/kg bw) on transcription molecules like NF-κB, COX-2, and iNOS, levels. An increase in the levels of COX-2, iNOS, and NF-κB planes was empirical in the DMBA only managed group (Group II). While in group III treatment with copper nanoparticles of Stachytarpheta and DMBA (p < 0.05) reinstated the levels of these signaling molecules comparable to the control group and group IV mice.
3.13 In uence of copper loaded Stachytarpheta cayennensis on the lung histology of tissues Figure 11 displays the histological inspection of the skin melanoma segment of control and experimental animals. Group I control mice exhibited standard architecture and slightly unaffected centers. Skin cancer holding or DMBA induced animals (group II) showed a tumoral structure with an asymmetrical architecture and bulky cells with dark nuclei and several minuscule hyper-refractile assemblies. Copper loaded Stachytarpheta cayennensis (10 mg/kg bw) along with DMBA induced group III animals unveiled recovered architecture, demonstrating the non-toxic feature of Stachytarpheta cayennensis. Pre-treatment with Stachytarpheta cayennensis alone (10 mg/kg bw) condensed the structural impairment in group IV animals, therefore, shielding the near usual architecture.

Discussion
For nanocrystals, the improved overlay of the electron and the wave functions in the space and narrowed structure augments the excited binding a nity, thus re ecting the excitonic peak probability, even at room temperature (Francois et al. 2019). In the present study, the UV absorption spectrum offers a qualitative suggestion of the size dispersal. That is, a sharp absorbance peak in the absorption spectrum in the case of small nanocrystals (Fig. 1) is undoubtedly an indication of the thin size dispersal of the nanoparticles of Stachytarpheta. This indication is veri ed in the ultraviolet-visible range using the surface plasmon resonance of copper nanoparticles. Current results were correlated with the preceding works (Mulvaney 1996). The FTIR spectra array of Stachytarpheta cayennensis established persistent shared peaks extending from 3266cm − 1 to 660cm − 1 . Figure 3 displays the expressive FTIR range attained from Stachytarpheta cayennensis. The occurrence originates from 3266cm − 1 , which portrays the OH ranging signal, which shows amino acids and sugar molecules. The existence of 2909cm − 1 peaks signify the hydrogen and carbon layer predominantly from a lipid molecule.  (Azizi et al., 2017). Consequently, the present study was designed to perceive whether Cu-NP generates ROS in A375 cells. In this case, the minimal ROS production in which cells were induced by the copper nanoparticles of Stachytarpheta cayennensis (5µg/ml). Moreover, group III mice show raised ROS production, which was induced by the copper nanoparticles of Stachytarpheta cayennensis at 7.5µg/ml. The electric charge distribution of the mitochondrial membrane was strongly connected to cell death. To examine whether Cu-NP's depolarized mitochondria of A375 cells, in the present study was executed. For this, A375 cells were treated with two different doses of Cu-NP (5µg/ml & 7.5µg/ml). The outcome showed that with increasing Cu-NP concentration, there was an increase in the mitochondrial membrane depolarization. The ultrastructural classi cation of Cu-NPs was conducted using TEM.
Though the Cu-NP's structure is pro cient from TEM (200nm and 20 nm) it is slightly renowned than measurement considered using DLS (90 nm). This alteration on accretion was assigned to the diverse range of methods that provide irregular consequences in substance on the procedures that employ NP's (Sharma et al. 2009). Preceding studies determined the copper nanoparticles and copper concentrations distinguishingly disturbed the enzymatic antioxidants like CAT, SOD, and planes of TBARS in the mice serum (Rahmat et al. 2017). In the present study SOD, CAT, GPx, and GSH actions were downregulated in DMBA only treated group II. While group III mice, which were treated with DMBA as well as CuO NP's of Stachytarpheta cayennensis (10 mg/kg bw) reinstated these antioxidants levels. This was overturned in group III animals that displayed lowered levels of TBARS. Though, group IV animals revealed no notable decrease or upsurge in SOD, GSH, CAT, and GPx, which was comparable to control animals.
Previous work illustrates that melatonin could effectively mediate antitumor effect in melanoma cells by the diminished production of COX-2 and iNOS by regulating the nuclear translocation of NF-kB and retracting their binding a nity on COX-2 (Canhui et al. 2014). Likewise, in the current study, the in ammatory cytokines such as IL-6, IL-1β, and TNF-α and transcriptional signaling molecules like NF-kB, iNOS, and COX-2 were augmented in DMBA only treated group II animals. Treatment with copper nanoparticles of Stachytarpheta cayennensis (5µg/ml and 10µg/ml) showed downregulated expression of the aforementioned signaling molecules in a concentration-dependent manner wherein 10µg/ml of Cu-NPs exhibited lowered expressions. Thus, copper loaded Stachytarpheta cayennensis management restored these markers' levels comparable to normal, suggesting its anticancer effect over skin melanoma cancer.

Conclusion
The current study of Cu-NP's synthesis was carried out using the plant extract of Stachytarpheta cayennensis followed by characterization using UV-Visible spectroscopy, dynamic light scattering, and FTIR. Further, we assessed the anti-melanoma e ciency of biosynthesized copper nanoparticles from Stachytarpheta cayennensis. The consequences propose that Cu-NPs induced cytotoxicity through a ROS generation and membrane potential. Cell viability assays and adhesion assay was carried out in a concentration-dependent manner. Pro-in ammatory marker and cytokine levels were also estimated to con rm the e cacy of Stachytarpheta cayennensis. Molecular mechanisms of cellular transcription molecules were done by RT-PCR studies. Ultimately we propose that biosynthesized copper nanoparticles from Stachytarpheta cayennensis might be an excellent choice to treat skin melanoma cancer.   Transmission electron microscopy analysis of synthesized Cu-NPs