Synthesis and Characterization of Copper Oxide Nanoparticles from Aqueous Extract of Cyanotis tuberosa (Roxb.) Schult. & Schult. F. and Analysis of Antioxidant, Antimicrobial and Anticancer Activity

Cyanotis tuberosa (Roxb.) Schult. & Schult.f family Commelinaceae is traditionally used as ethnomedicine for curing several health problems in India. In this study copper oxide CuONPs were synthesized using C. tuberosa tubers aqueous extract. The biosynthesized CuONPs were characterized using an Ultra violet–visible spectroscopy, Fourier-Transform Infra Red spectroscopy, X-RAY Diffraction Analysis, Scanning electron microscopy. The results obtained from SEM showed that the copper oxide nanoparticles were a face-centered cubic crystal structure. The characterization of the biosynthesized CuONPs through EDS also indicated that the reaction product was composed of highly pure CuO NPs. The invitro antimicrobial activity of different concentrations of green synthesized copper oxide nanoparticles of C. tuberosa showed that the highest inhibition zone was observed on Klebsiella pneumonia (15.25 mm). Furthermore, the antioxidant activity of green synthesized CuONPs of C. tuberosa tubers aqueous extract was also tested the highest percentage activity was exhibited at 100 μg/ml CuONPs (56.32). The human Original Research Article Ramakrishna et al.; JPRI, 33(53A): 138-153, 2021; Article no.JPRI.78552 139 cancer cell line was used for cytotoxicity analysis at 48 hrs of incubation period, a significant abatement in cell viability was observed against the treated cell lines. The study concludes that the C. tuberosa tubers aqueous extract can be successfully used for the synthesis of CuONPs that exhibit effective in vitro anti-bacterial, antioxidant and anticancer activity.


INTRODUCTION
Metal nanoparticles play a crucial role in various fields, most importantly in the fields of medicine and pharmacy. Since few decades metal nanoparticles made of Ag and Au have been used in the field of medicine [1,2,3]. The search for novel nanoparticles with potential biological properties is going on in many fields. Recent studies explored the importance of CuO nanoparticles and their potential in the fields such as gas sensors, catalyst, solar energy conversion and photovoltaic devices along with medical field [4,5,6]. Though CuO nanoparticles show excellent activity, their synthesis plays is a key role in their function. Several methods either physical or chemical are available for the synthesis of CuO nanoparticles such as sonochemical methods, thermal oxidation route, thermal decomposition, hydrothermal approach, precipitation methods and reverse micro emulsion methods [7,8,9,10,11,12]. All of these methods have their own drawbacks such as toxic chemicals usage, expensive to afford, hazardous materials and labor engagement. The toxicity produced through these methods contaminates the environment. Most reliable and ecofriendly method of synthesis of CuO nanoparticles is biological method where plant extracts can be employed to synthesize the nanoparticles. The nanoparticles synthesized through biological means have plenty of applications in medical and clinical fields. CuO nanoparticles have been synthesized using microorganisms such as bacteria, fungi, yeast and by using several types of leaf extracts such as neem leaf extract, tea leaf extract, coffee powder extract and aloe vera extracts [13,14,15] Recent reports have shown the synthesis of CuO nanoparticles using plant extracts and their applications in medical field. Copper nanoparticles synthesized from the aqueous extracts of black bean showed in-vitro anti cancer activity that can stimulate apoptosis and suppress the HeLa cells proliferation. Enhanced antibacterial activity against the microbes responsible for Urinary Tract Infections (UTI) by the copper nanoparticles prepared from cellulose gum was reported [16]. Numerous studies have been reported the antibacterial function of copper nanoparticles against different bacteria such as Enterococcus faecalis, Shigella flexneri, Salmonella typhimurium, Proteus vulgaris, Staphylococcus aureus, Klebsiella pneumonia [17], and Aeromonas hydrophila, Flavobacterium branchiophilum and Pseudomonas fluorescens, the most prominent fish pathogens [14]. Along with the copper nanoparticles other metal nanoparticles such as iron oxide and zinc oxide have been used in medical field for their antibacterial and anticancer activity [18,19]. The present study was aimed to study the biological functions of copper nanoparticles synthesized by the aqueous extracts of Cyanotis tuberosa.

Synthesis of CuONPs from C. tuberosa
The tubers collected were washed thrice with running tap water to remove the contaminants followed by Milli Q ultrapurified water. The material was dried up to 10 -15 days under shade conditions. The dried material was finally ground with blender for further use.

Characterization of CuONPs of C. tuberosa
The CuONPs synthesized with aqueous extract of C. tuberosa were characterized by using different spectroscopic and microscopic methods. Initial confirmation of nanoparticles was done by UV-VIS spectroscopy (Nano drop 8000 UV-Vis -spectrometer) to know which metals of the phytochemicals were actually involved in the reduction of nanoparticles by surface Plasmon resonance method. Stabilization of nanoparticles; Fourier-Transform Infra Red (FT-IR) spectra of synthesized SNPs were analyzed in the range of 4,000 to 500 cm-1 with an IR-AFFINITY-1, IR by ATR method. Zeta potential of synthesized nanoparticles was analyzed to know the average size and stability of particles (Nanoparticle analyzer, Horiba SZ 100, Japan). XRD (Shimadzu, XRD-6000) was used to analyze crystalline nature and calculate the average size of particles. Microscopic analysis with TEM (HF-3300, 300 kV TEM/ STEM, Hitachi) instrument reveals the size, shape, dispersed nature and agglomerated pattern of nanoparticles [22,23,24,25,26,27,28].

In-vitro Antioxidant Activity
Free radical scavenging activity of aqueous extract of C. tuberosa was determined by its ability to bleach the stable 2, 2diphenyl -1picryl hydrazyl (DPPH) radical. The stock solution of DPPH free radical was prepared by dissolving 4 mg of DPPH in 100 ml of methanol and stored at 20 °C. 2 ml of this stock solution was added to 1 ml of C. tuberosa aqueous extract and C. tuberosa CuONPs separately at different concentrations (25 -100 μg/ml). Ascorbic acid was used as a standard. The radical scavenging activity was calculated by using the formula

RSA = X100
Where RSA is Radical scavenging activity, Ac is the absorbance of the control, and As is the absorbance of the sample or standard [29].

Antimicrobial Studies
The antimicrobial activity of the CuONPs synthesized with aqueous extracts of C. tuberosa was analyzed against bacterial and fungal strains. The bacterial strains include two Gram positive bacterial strains namely Bacillus subtilis (MTCC-441), Staphylococcus aureus (MTCC-731) and two Gram negative bacterial strains namely Escherichia coli (MTCC-443), Klebsiella pneumoniae (MTCC-741). The fungal strains include Aspergillus niger (MTCC 281), and Candida albicans (MTCC-183). The antibacterial activity was determined by disc diffusion method [30]. The samples include rhizome extract as positive control, 5 mM copper sulphate as negative control, Streptomycin/Flucanozole (10 mg) as standard and CuONPs as test sample. The sterile discs of size 6 mm were prepared from Whatman No. 1 filter paper and these sterile discs were impregnated with 20 μl of each sample on separate discs with the help of micropipette and allowed to dry for one hour under aseptic conditions. Freshly prepared nutrient agar plates were spreaded with the microbial cultures and the discs impregnated with samples were placed on the plates and incubated at 37 °C for 24 to 48 h. After the incubation the zone of inhibition was measured.
The experiment was carried out in triplicates [31].

Anticancer Activity
CuONPs of C. tuberosa tubers aqueous extract was subjected to MTT 3-(4, 5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide for colorimetric assay used for the determination of cell proliferation and cytotoxicity, based on reduction of the yellow colored water soluble tetrazolium dye (MTT) to formazan crystals. Mitochondrial lactate dehydrogenase produced by live cells reduces MTT to insoluble formazan crystals, which upon dissolution into an appropriate solvent exhibits purple color, the intensity of which is proportional to the number of viable cells and can be measured spectrophotometrically at 570 nm [32,33]. MDA MB 453 (human breast cancer cell line) cell line is procured from National Centre for Cell Sciences (NCCS), Pune, India. The Dulbecco's Modified Eagle's Medium with high glucose is used to growing up 2 × 104 cells per well in 96well plates and incubated in 5% CO2 atmosphere at 370C for 24 h supplemented with 2 mM/L glutamine, 10% Foetal Bovine Serum (FBS) with 10 g/ml of Ciprofloxacin [34]. Afterwards medium was expelled and treated with different concentrations (12.5, 25, 50, 100 and 200 μl/ml) CuONPs of C. tuberosa incubated for 24 hrs. Further, remove the spent media and add 100 μl of MTT reagent with the 0.5 mg/ml concentration and incubate the plate for 2.5 hrs for the reaction. Later, remove MTT reagent completely and add 100 μl of 100% Dimethyl sulfoxide (DMSO) to solubilize the formazone crystals completely and measure the absorbance at 570 nm using 96 well Plate reader. The 0.1% of DMSO used to dissolve the nanoparticles and set as negative control and 15 μM Camptothecin treated cell lines were set as positive control. The initial experiment was maintained for 0 to 24 hrs of timeline period with 12 hrs of time gap period to check probability of cell toxicity. It provides specific time course period to allow functional cell mortality to understand the experiment in a flexible and adaptable way. According to the results, significant cytotoxicity was observed at 24 hrs at 370 C incubation period. The percentage of cell viability was calculated by the following formula [35].

UV Spectroscopy of CuONPs of C. tuberosa
The brown colored fresh aqueous tuber extract of C. tuberosa has an ability to convert copper oxide solution into the ionic form. In this reaction, the tuber extract reacts with copper oxide solution and changes to green colour. The SPR spectrum peak of biosynthesized C. tuberosa CuONPs was obtained at 270 nm (Fig. 2).

FTIR Spectrum of C. tuberosa CuONPs
The

Particle Size and Zeta Potential Analysis of CuONPs
The particle size of CuONPs prepared from tuber extracts of C. tuberosa were analyzed by Dynamic Light Scattering experiment. The particle size distribution curve clearly showed that the particles have an average size of 14.5 nm (Fig. 4). Zeta potential analysis gives clear picture about the surface of nanoparticles and predicts the long-term stability of the nanoparticles formed. In the present study, the CT-Cu copper oxide nanoparticles of C. tuberosa have showed Zeta potential of -20.2 mV (Fig. 5). The negative value indicates that the surface of nanoparticles is charged negatively. The negatively charged particles in the medium experience repulsion and hence exhibit stability. It is well known fact that the charge and size of the nanoparticles play important roles in the biological activity of prepared NPs.

X-RAY Diffraction Analysis of CuONPs of C. tuberosa
The

High-Resolution Transmission Electron Microscopy (HR-TEM) Analysis
HR-TEM was used to analyze the morphology and size of the Copper oxide nanoparticles. The selected area of electron diffraction (SAED) shows clear diffused concentric rings (Fig. 7A), which are due to crystalline and polycrystalline spots. These results indicate that the synthesized nanoparticles were crystalline in nature. 20 nm scales bar TEM image (Fig. 7B) signified the synthesized nanoparticles with a size range from 1.52 to 3.31 nm. The 50 nm scale bar image (Fig. 7C) showed the particle size as 1.42 to 2.89 nm. The 100 nm scale bar image (Fig. 7D) showed the particles are spherical in shape.

EDX Spectrum of CuONPs of C. tuberosa
The experimental results of energy-dispersive spectrum of the synthesized CuONPs illustrate the presence of Cu as the ingredient element (Fig. 8). The elemental analysis of copper nanoparticles of C. tuberosa through EDX microanalysis revealed the presence of 49.38% weight percentage of copper metal along with 0.00% of carbon, 23.85% of sodium, 23.42% of oxygen, 0.92% of sulfur, 0.67% of chlorine, 1.04% of potassium and 0.72% of magnesium.
The above results clearly show the abundance (49.38%) of copper in the preparation ( Table 2).

Antibacterial Activity of Biosynthesized CuONPs from Aqueous Extract of C. tuberosa
The green synthesized copper oxide nanoparticles of C. tuberosa were assessed for antimicrobial activities against two gram positive (K. pneumonia and B. subtilis) and two gram negative bacterial strains (E. coli and S. aureus). The aqueous extract of C. tuberosa and CuSO 4 .5H 2 O solutions were used as controls. The antibiotic streptomycin was used as positive control for antibacterial activity. The antibacterial activity was measured by measuring the zone of inhibition formed in the agar plate. The zone of inhibition was observed in the order of K. pneumonia (15.25 Table 3). The above result clearly showed that the synthesized CuONPs have potent antibacterial activity against microbial pathogens compared to the aqueous extract of C. tuberosa. The results obtained with biosynthesized CuONPs are on par with the standard drug streptomycin.    6.75 ± 0.25 *** 6 ± 0 *** 12.75 ± 0.25 *** 16.5 ± 0.29 S. aureus 6 ± 0 *** 7 ± 0 *** 11.75 ± 0.25 *** 17.5 ± 0.29 All the data are expressed as mean ±S EM: **p<0.01,* p<0.05 as compared to Control group, n=4: (One -way ANOVA followed by Dunnett's test)

Antifungal Activity of Biosynthesized CuONPs from Aqueous Extract of C. tuberosa
The green synthesized copper oxide nanoparticles of C. tuberosa were assessed for antifungal activities against two fungal strains namely C. albicans and A. niger. The aqueous extract of C. tuberosa and CuSO 4 .5H 2 O were also tested separately for antifungal activity. The antibiotic fluconazole was used as a positive control for antifungal activity. The antifungal activity was measured by measuring the zone of inhibition on plate. Among the two fungal strains the highest inhibition zone was observed on C. albicans (9. Table 4). The above result clearly showed that the synthesized CuONPs have potent antifungal activity against microbial pathogens. The results obtained with C. tuberosa CuONPs have nearest values with that of the standard drug fluconazole.

Antioxidant Activity of C. tuberosa Tubers Aqueous Extract
The antioxidant activity of aqueous tuber extract and CuONPs synthesized from tuber extracts of C. tuberosa were analyzed by DPPH radical scavenging assay. Ascorbic acid was used as a positive control for the assay.

Anticancer Activity of CuONPs of C. tuberosa Tuber Extract
The CuONPs synthesized from C. tuberosa were tested for their anticancer activity by using MDA MB 453 (human breast cancer) cell line by MTT assay. The assay is a sensitive colorimetric method for the determination of number of viable cells in the cytotoxicity assays. In the present study, DMSO was used as negative control whereas Camptothecin was used as a positive control for anticancer activity or cytotoxicity. The cell lines were treated with the samples and incubated for 48 hours to see the result. During the treatment of cell lines, CuONPs were used in the concentration range of 12.5 µg/ml to 200 µg/ml. After incubation, a significant abatement in cell viability was observed in the CuONPs treated cell lines which are comparable to the positive control Camptothecin. The result clearly shows a decrease in proliferation rate with an increase in the concentration of CuONPs. DMSO, which was used as negative control exhibited 100% healthy proliferated cells (Fig. 14 & Table 6). The concentration that exhibits 50% reduction in the growth of tumor cells in culture is generally termed as half maximal inhibitory concentration (IC 50

CONCLUSION
The present study was succeeded in synthesizing the copper oxide nanoparticles by using tubers aqueous extract of C. tuberosa. The plant has plenty full of several natural bioactive compounds that are capable of reducing copper ions and convert them to nano particles. The CuONPs that are synthesized were characterized in a sequential manner by employing various analytical techniques. The CuONPs thus characterized were tested for their antibacterial, antioxidant and anticancer properties. The present study clearly showed the antibacterial activity along with antioxidant and anticancer activity.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.