Green Synthesis of Copper Oxide Nanoparticles Using Coix lacryma jobi Leaves Extract and Screening of its Potential Anticancer Activities

Background: Copper oxide nanoparticles(CuO NPs) have been powerful evidence in several in vitro studies such as cytotoxicity and antimicrobial compared with other metal oxide. Here, we have synthesized green CuO NPs using Coix lacryma jobi leaves extracts. Place and Duration of Study: Department of Chemistry Manipur University, Manipur, India and Regional Institute of Medical Sciences, Imphal, India between February 2019 to March 2021. Methodology: Green CuO NPs nanoparticles were synthesized from Copper chloride dihydrate (CuCl2.2H2O) using Coix lacryma jobi leaves extract, and the synthesized green CuO NPs were characterized using Field Emission Scanning Electron Microscopy (FESEM) Energy Dispersive Spectroscopy, IR Spectroscopy, UV-Visible Spectroscopy, Powder X-Ray diffraction Spectroscopy and HR-TEM where FESEM-EDS determined the purity of CuO NPs. Results: No other impurities present were observed in EDS, and the PXRD spectra show the crystallite size of CuO NPs with respect to the (002) plane is found to be 25.2 nm, and the Original Research Article Devi et al.; JPRI, 33(52A): 128-139, 2021; Article no.JPRI.77688 129 presence of CuO NPs at adsorption spectrum with a distinct peak at 282 nm was determined by UV-Visible spectroscopy and the homogenous morphology and crystalline nature of the CuO NPs were determined from TEM micrograph and SAED pattern. In applications, the substantial anticancer activity of green CuO NPs (synthesized using Coix lacryma jobi leaves extract) was proved on human cervical and lung cancer cell lines with IC50 values of 31.88 μg/ml and 15.61 μg/ml, respectively

The non-toxic form of nanoparticles have been used in cancer therapy [39,40]. Considering the biological activities of Coix lacryma jobi, which acts as antioxidant [41], cytotoxic activities [42] and its leaves extracts at a different medium that inhibits various cancer cells such as Hela, HepG2, and SGC-7901 [43,44], hepatoprotective and anti-inflammatory activity [45] antitumor activities in human cervical cells [46] antibacterial and anthelmintic activities [47] were observed. However, the preparation of green CuO NPs using Coix lacryma jobi leaves has not yet been reported to the best of our knowledge.
In this work, we have synthesized green CuO NPs using leaves extract of Coix lacryma jobi as the reducing agent from CuCl 2 .2H 2 O using an eco-friendly solvent such as ethanol and distilled water. Substantial anticancer activity of green CuO NPs on human cervical and lung cancer cell line was carried out. And the present study explores the green synthesis of CuO NPs, their characterization, and investigation of in vitro cytotoxic activities.

Prepration of the Extract
Fresh leaves of Coix lacryma jobi were collected locally from Imphal West, Manipur, India. The leaves were repeatedly washed with distilled water and air-dried for one day, and the leaves were cut into small pieces. About 30 g of Coix lacryma jobi leaves were soaked in 100 ml of doubled distilled water in a conical flask and reflux at 100 o C for 30 min. And then cooled at room temperature and filtered with whatman No. 1 filter paper to remove the plant residues, and then the extract was stored at room temperature for future use. The reaction mixture's color was changed to dark brown. The solution was kept at room temperature overnight, and then the black powder CuO NPs was collected by annealing at 300 o C for 1 hour after centrifuging at 8000 rpm for 10 min followed by continuous washed with distilled water and absolute ethanol.

Characterization Technique
The purity of the CuO NPs was analyzed using Energy Dispersive X-ray analysis and FESEM (Zeiss Sigma model). IR-spectra were recorded using Perkin Elmer FT-IR spectrum 400 in the range of 4000-400 cm -1 using activated KBr pellets. The UV-Visible spectra were recorded using Shimadzu 2450 Spectrophotometer, and the diffraction pattern was recorded using PANalytical (X'Pert Pro) X-ray diffractometer with Cu Kα (1.54060 Å) radiation. To access the morphology and the size of CuO NPs was found using JOEL JEM-F200 HR-TEM.

Cell Lines and Culture Condition
Human cervical (HeLa) and lung (A549) cancer cell lines were obtained from the National Centre for Cell Science (NCCS), Pune, India. The cells were cultured in RPMI 1640 (Gibco, USA) media with 10% heat-inactivated fetal bovine serum (Gibco, USA) and 1% PenStrep (Gibco, USA). Cell lines were incubated in a humidified atmosphere with 5% of CO 2 at 37 °C. The cell lines were maintained at 37 °C in 5% CO 2, and media was changed frequently.

Cell Viability Assay
For evaluation of potential cytotoxic effects of synthesized CuO NPs leave extracts of Coix lacryma jobi, we used two human cell lines: HeLa and A549. Upon reaching confluency, they were trypsinized, and the concentration of viable cells was determined using the Trypan blue assay. Cells were cultured at a density of 1×10 4 cells per well in 200 μl RPMI (without phenol red), 10% FBS, and incubated with 5% CO 2 at 37ºC in a 96-well tissue culture plate. The final concentration of DMSO in all the experiments did not exceed 0.5% (v/v). Untreated cells served as a control and were assumed to be 100% viable. In the present toxicity analysis, various concentrations (3.12, 6.25, 12.5, 25, 50, 100, and 200 µg/ml) of CuO NPs were tested for 24 hr in triplicates. According to the manufacturer's protocol, the cytotoxic effect of synthesized CuO NPs leaves extracts was evaluated using the CellTiter 96® Non-Radioactive Cell Proliferation Assay (Promega, USA). These assays are based on the cellular conversion of a tetrazolium salt into a formazan product which the plate reader detects. After 24 hr incubation, a premixed optimized Dye Solution was added to each well, and the cells were further incubated at 37 ºC for 4 hr. During 4 hour incubation, living cells convert the tetrazolium component of the Dye Solution into a formazan product. The plates were shaken, and the optical density was measured at 570 nm using a microplate reader (Thermo Scientific Multiskan Spectrum, Thermo Fisher Scientific, Inc., Waltham, MA). The IC50 values of CuO nanoparticles on various cell lines were determined using Graph Pad Prism version 8.0.1 (GraphPad, San Diego, CA).

FESEM-EDX (Field Emission Scanning
Electron Microscopy-Energy Dispersive X-Ray Spectroscopy) Analysis FESEM determined the phase purity of CuO NPs with EDX analysis, and it is clear that no other impurity peaks were observed with the EDX taken at different spectrums. FESEM with EDX images of green CuO NPs is reported in Fig. 1.

Infra-Red Spectra Study
Infra-Red spectra of green CuO NPs are shown in Fig.2

UV-Visible( Ultra Violet) Spectra Study
UV-visible spectra of green synthesized CuO NPs using Coix lacryma jobi leaves Extract shown in Fig.3. A broad peak from 300-340 nm was observed in the absorption spectrum for the leaves extract and the absorption spectrum with a distinct peak centred near 282 nm (λ max ) indicates the presence of CuO NPs, which was confirmed from the maximum Surface Plasmon absorption band with a maximum at 250-350 nm [48,49].

XRD(X-Ray Diffraction) Analysis
The X-ray diffraction displayed in Fig.4

HR-TEM( High Resolution Transmission Electron Microscopy) Analysis
The HR-TEM micrograph is shown in Fig. 5a. Suggests the formation of CuO NPs with good lattice fringes. The spacing in this region is 2.3 Å which corresponds to (111 planes) ( space group C2/c, ICSB: 16025). Fig. 5b.shows the SAED pattern of the green synthesized CuO NPs. It confirms the crystalline nature of the prepared sample [51,52]. Cytotoxicity of CuO NPs against HeLa cell was measured. IC50 value of CuO NPs was found to be 31.88 g/ml, but less effective than Doxorubicin (13.21 g/ml) of 2.41 times which is used as standard. (Fig. 7) Further cytotoxicity of CuO NPs against A549 cell line with IC50 value of Doxorubicin (8.24 g/ml) was found, the corresponding the IC50 value of CuO NPs was 15.61g/ml (Fig. 8). Our results showed that the synthesized copper oxide nanoparticles have proved significant cytotoxicity effect on both Hela and A549 cells with IC50 value of 31.88 g/ml and 15.61g/ml respectively Fig 6 (e). The percentage of cell viability was declined further when the concentration of the CuO NPs was gradually increased to a maximum of 200 µg/ml on the cancer cell line tested. The reduced cell viability of HeLa and A549 cells observed in this study suggest anticancer effects of CuO NPs. Further studies are required to understand the process of cell death by apoptosis or necrosis pathway.

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.