In-vitro Antioxidant and Cytotoxicity (sk-mel-3 cell) Activity of Green Synthesised Copper Nanoparticle using P. pellucida Plant Aqueous Extract

INTRODUCTION
There are numerous techniques for the biogenic development of metallic nanomaterials. Because of their fascinating physical characteristics and possible application in various fields (medicine, pharmacy, biotechnology, chemistry etc.), technical and scientific research on the synthesis and development of metallic nanoparticles has augmented. Principally tiny size and large volumes to surface area ratio. Special attention was paid to copper nanoparticles production within metallic nanoparticles due to its low price of the noble metal compared to Ag, Au and Pt and their varied research and industrial (1, 2). Synthesis of nanoparticles through a green approach, non-toxic and biodegradable chemicals have been used. Nanoparticles are synthesised from different plant sources (3,4).
Medicinal plant extractions for copper nanoparticles have generous benefits, such as catalysis, photocatalytic activity (5) and bactericidal (6), DNA binding and sensors (7), anticancer (8), free radical scavenging, etc. (9, 10).
The green synthesis of copper nanoparticles with medicinal plants extracts is biodegradable, profitable and stable (11). Biomolecules such as carbohydrates, phenols, tannins, flavonoids, and others in plant extract have been found to play a significant factor in nanocomposites reduction, aggregation and capping (12). Presently, the market for herbal supplements for health care is growing with each day.
Pellucida peperomia (shiny bush, silver bush) annual herb, and it belong to the Piperaceae family. The monsoon season grows to a height of 15-46 cm in humid soil, particularly underneath the trees (13). It is usually observed in southeast and southwest Nigeria and several tropical Asian and South American countries in the West African rainforest region. Pellucida peperomia used amazon region humans for the treatment of cardiac arrhythmia, diuretic, dementia disease. The leaves and stem aqueous blend is used to treat bleeding, fever, headache, stomach pain, injuries, and a cough suppressant recorded in the Ayurveda (14, 15, 16). The present study investigates in-vitro antioxidant and cytotoxicity of green synthesised copper nanoparticles using P. pellucida plant extract.

MATERIALS AND METHODS
Source of chemical and reagents
Cu (NO3)2·3H2O, acridine orange, ethidium bromide (AO/EB), PBS (1%), DMEM (Dulbecco’s Modified Eagle’s Medium), streptomycin, penicillin-G, L-glutamine, phosphate-buffered saline, MTT (3-(4,5 dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide), ethylene diamine tetraacetic acid, ethanol, ethidium bromide and acridine orange (EB/AO) and DMSO (dimethyl sulfoxide), Dehydrated methanol obtained from Sigma Aldrich Chemicals Pvt. Ltd (India).
Plant collection
P. pellucida was obtained from Patambi, Kerala, India, in August 2020. A plant taxonomist (Dr Dhanapal V, Professor and Principal of Sri Sasta College of Pharmacy) authenticated the plant (Fig. 1).

Preparation of plant extract
Plant leaves are cleaned with distilled water to eliminate any particulate matter. Plant leaves make a fine powder using mortar and pestle. The extract (aqueous) was prepared using a cold maceration process. 100 g powder of P. pellucida Linn was saturated in 1 litre of deionised water and held in a shaker at 30 ° C for 24 hrs for constant stirring at 100 rpm. It then dried out at normal temperature. The extract was then purified and processed at - 4 ° C for additional study (17, 18). 

Phytochemical screening
Preliminary phytochemical examination of the extract was carried out to classify active elements using basic methods (19). 

Green synthesis of copper nanoparticle (CuNPs)
The aqueous plant extract was used to reduce Cu (NO3)2·3H2O. The Cu (NO3)2·3H2O was mixed with the aqueous plant extract and stirred continuously for at least one hr. The reaction process was repeatedly observed, and the colour change was noted. The reaction mixture was centrifuged at 12,000×g for 15 min, and the nanoparticle pellet was cleaned with distilled water (20) 

Characterisation of CuNPs
CuNPs were initially studied by ultraviolet spectroscopic analysis within a 200 – 800 nm range. FTIR was used to identify the structural features and selective phytochemical components. The results estimated 4000–400 cm−1 range. The powder form of the nanoparticles exposed to CuKα1X-Ray diffractometer radiation (λ = 1.5406 A°) at 40 kV and 30 mA with 2θ rang. CuNPs were placed on the sample holder and sputter-coated using gold, following that, the nanoparticles’ average particle size and shape were studied by using TESCAN MIRA3 LMH Schottky FE-SEM (Japan) (21, 22) 

Determination of antioxidant activity (CuNPs)
The ascorbic acid and CuNPs sample stock solutions prepared have a strength of 1.0 mg/ml. CuNPs and ascorbic acid concentrations of 10, 20, 40, 60, 80, 100 μg/mL, in methanol solution. CuNPs (0.5 ml), ascorbic acid transformed into 0.5ml of 0.5 mM DPPH in methanol solvent. After 30 min of storage at in the dark place at room temperature, the optical density was measured at 517 nm using Stat Fax 4200 Elisa reader (USA).  Each experiment was performed in duplicate. Inhibition (I%) calculated as follows: (23) 

I% antioxidant activity    =    
Abs control – Abs of antioxidant   X  100
            Abs control

Cell culture maintenance
Human skin cancer cells (SK-MEL-3) were obtained from the National Centre for Cell Sciences, Pune, India. Dulbecco’s Modified Eagle’s medium was used to preserve the cell line, complemented by 10% of Fetal Bovine Serum. Penicillin (100 U/mL) and streptomycin (100 μg/ml) was added to prevent microbial contamination. The cell culture medium was preserved in a pressurised environment with 5 per cent CO2 at 37°C.

Cytotoxicity activity
SK-MEL-3 cells were seeded in 96 well plates and incubated in a CO2 incubator for 24hr to facilitate the adhesion. Cells were treated with the control and CuNPs at various concentrations and incubated cell culture incubator after 24hr cell were washed with the cell culture media and added MTT dye (Incubated 4hr 37°C). After 4hr the cells were treated with formazan and observed cell viability using a multi-well plate reader (540 nm). In comparison to the control, the proportion of stable cells was calculated. CuNPs IC50 value was calculated, and the effective dose was analysed at a different period (24).
    

 IC50 of nanoparticles measured from the dose-responsive curve inhibit 50% cytotoxicity compared to control cells. Experiments were carried out three times (25). 

Measurement of apoptotic induction using acridine orange/ethidium bromide (AO/EB) dual staining method
Microscopic fluorescence analysis of apoptotic cell inhibition was carried out according to Liu et al. (26). SK-MEL-3 cells were seeded at 5 x 104 cells/well in a 96 six-well plate and incubated for 24 hours. After treatment with CuNPs for 24 hrs, the cells were detached, washed with cold PBS and then stained with a mixture of A.O. (100μg ml−1)/E.B. (100μg ml−1) ratio (1:1) at room temperature for 5 min. Treated cells were collected and rinsed three times with PBS. The plates were stained with acridine orange/ethidium bromide (AO/EB 1:1 ratio; 100 µg/mL) for 5 minutes and examined immediately under fluorescent microscope 40x magnification. A fluorescence microscope observed the stained cells at 40x magnifications.

Statistical analysis
Results descriptive as mean ± S.D. They restricted comparing statistical differences carried by (ANOVA) one-way analysis of variance accompanied by Duncan’s Multiple Range Test, using SPSS version 12.0 for windows. Values tested for significance if the p-value was less than 0.05.

RESULTS
Phytochemical analysis
All the findings of the phytochemical investigation are shown in Table 1. Aqueous extract gave promising outcomes for steroids, terpenoids confirmed by Salkowski and Liebermann-Burchard’s test in the present study. The presence of terpenoids, phenols and flavonoids were also confirmed in P. pellucida aqueous extract.

Evaluation of CuNPs
UV - Vis spectroscopic analysis
U.V. spectroscopy within the 200-800 nm range initially verified the development of CuNPs. A characteristic peak of 575 nm showed the absorption spectrum of green synthesised CuNPs.
FTIR spectroscopic analysis
A Fourier transform infrared spectroscopy was utilised as an affirmative method for the construction of nanoparticles (Fig. 2). This study offers an imprint of current molecules vibrational and rotational modes, thus helping to classify the functional and potential plant molecules involved in CuNPs nanoparticles reduction.

XRD Analysis
The structure of the nanoparticles is indicated by the XRD analysis of the synthesised CuNPs. At 2θ values of 35.45°, 44.32°, and 65.25° degrees, diffraction peaks were observed (Fig. 3).

FE-SEM analysis
The SEM study was carried out using the Schottky FE-SEM (Japan) TESCAN MIRA3 LMH model. FE-SEM analysis was used to classify the structure and scale of CuNPs. Microscopy of CuNPs has shown that they have a nano-range particle size (500nm) spherical and homogeneous in distribution under the FE-SEM Microscope (Fig. 4).

EDAX analysis
The elemental analysis of the CuNPs from the EDAX spectrum of the FE-SEM image is shown in Fig-5. The percentage of molecular mass and atomic value of Cu respectively 69.7%. The EDAX spectrum is consistent with the presence of copper in the nanoparticle.

Antioxidant activity
The antioxidant properties of CuNPs using DPPH were evaluated and compared to ascorbic acid. The ratio of free radical inhibition of CuNPs was observed at different concentrations. The free radical inhibition visually detected colour transformation from purple to yellow suggests that DPPH reduced exhibiting better scavenging action (Fig. 6).

Cytotoxic activity (MTT assay)
Cytotoxic activity against SK-MEL-3 cancer cells with different concentrations ranging from 5μg/mL, 10μg/mL, 15μg/mL, 20μg/mL, 25μg/mL and 30μg/mL tested P. Pellucida mediated CuNPs. The In-vitro cytotoxicity evaluation was evaluated after 48hrs. Fig 7 and 8 shows the altered structure of SK-MEL-3 cells after dose-dependent treatment with CuNPs. Compared with control cell viability, CuNPs (16μg) significantly reduced the proliferation of SK-MEL-3 cells.

DISCUSSION
In the present investigation, CuNPs were bio-synthesised from the P. pellucida plant extract. Microscopical observation of CuNPs have shown that they have a particle size in the nano range; they are irregular in distribution. The dimensions, shape of the CuNPs were observed under FE-SEM. Cu particles grow slowly, form small structures.
Several investigations described biogenic preparation copper nanoparticles as environmentally approachable effective antioxidant and antitumor agents (27). The nanoparticle has a wide surface nature, permeability and biodegradability (28). Green synthesised CuNPs studied antioxidant activity using the DPPH method and showed antioxidant activity (standard ascorbic acid). Further analysis of copper nanoparticles studied cytotoxicity against SK-MEL-3 cell lines and showing good cytotoxic activity (IC50 value 16μg/mL). 
CONCLUSION
The convenient and environmentally friendly method to prepare CuNPs using reducing Cu (NO3)2·3H2O with P. pellucida aqueous extract was established. P. pellucida aqueous extract acting as a better reducing and covering agent. Green synthesised CuNPs characterised using EDAX, U.V, FT-IR, FE-SEM and XRD techniques. The development of CuNPs shows the deviations in the colour of the solution. U.V. spectroscopy showed the 200-800 nm range initially verified the story of CuNPs. A characteristic peak of 575 nm showed the absorption spectrum of CuNPs. The crystalline nature of the synthesised nanoparticles identified by XRD analysis has diffraction values at 35.45°, 44.32°, and 65.25° degrees. FE-SEM analysis conforms to the nanoparticle size and shape. The CuNPs investigated antioxidant and cytotoxicity. The experimental results concluded that biogenic synthesised nanoparticles had significant antioxidant (DPPH) and cytotoxicity activity (SK-MEL-3 cell).
 
ACKNOWLEDGEMENT
The authors acknowledge the Sanjo College of Pharmaceutical Studies and Centre for Biotechnology and Phyto-Pharmacognosy Research (CBPPR), Coimbatore, to provide necessary facilities to carry out the antioxidant cytotoxicity studies.

CONFLICT OF INTEREST 
Nil

FUNDING SUPPORT 
Nil