Evaluation of Antimicrobial, Cytotoxicity and Catalytic Activities of CuO-NPs Synthesized by Tanacetum Parthenium Extract

Development of ecient methods for treating microbial infections, cancer, and toxic organic dyes is a serious challenge in medical sciences. The purpose of this study is to synthesize CuO-NPs using T. parthenium extract and to evaluate its anticancer, antimicrobial, and catalytic activity. CuO-NPs were characterized by UV-Vis, XRD, FTIR, FESEM, and EDX. UV-Vis spectra exhibited surface plasmonic resonance at 298 nm of synthesized CuO-NPs. The synthesized CuO-NPs were pure, predominantly spherical with mean size of 16 nm. FTIR conrmed that CuO-NPs were reducted and stabilized with the biomolecules present in the T. parthenium extract. CuO-NPs indicated excellent degradation activity for the industrial dyes, i.e., MO (96.6% removal in 400s), Rh B (98.3% removal in 400s), MB (98.7% removal in 400s) and CR (99.6% removal in 180s). CuO-NPs showed excellent inhibition against selected microorganisms, especially E.coli and C. albicans. CuO-NPs have also shown good anticancer activity against A549, Hela, and MCF7 cancer cell lines (IC50 = 65.0, 57.4, and 71.8 µg/mL, respectively) while negligible cytotoxic effects were observed on L929 (IC50 = 226.1 µg/mL). The results proposed that synthesized CuO-NPs can be considered as a suitable candidate for biomedical, pharmaceutical, and environmental applications.


Introduction
Nanoparticles (NPs) have broad applications in pharmaceutics, biomedicine, cosmetics, space technology, electronics, catalytic, and environment 1 . NPs possess several exceptional and useful properties as compared with bulk materials with similar chemical composition. High yield strength, quantum size, high surface-to-volume ratio, rigidity, plasticity, hardness, and macro quantum tunneling effect are the most important properties of NPs 2 . Currently, CuO-NPs have attracted considerable interest due to their low cost and acceptable stability 3,4 . CuO-NPs are used as diagnostic tools and therapeutic agents against microbial infections, cancer and it is also applied as a clearing agent against synthetic organic dyes from water [5][6][7][8] . Physical and chemical methods can produce pure nanoparticles, but they are not e cient methods. Biosynthesis of NPs using green chemistry based methods is cost-effective, nontoxic, and eco-friendliness 9 . Several methods have been applied to produce green synthesized nanoparticles using bacteria 10 , plants 11 , yeast 12 , fungus 13 , and viruses 14 . Plant based nanoparticle synthesis has several advantages over other methods, including cost-effectiveness, fast production, simplicity, non-toxicity and biocompatibility 11 . The biosynthesis of CuO-NPs has been successfully attained using the extract of E. prostrate 15 , P. asiatica 16 and Rhuscoriaria L. 17 . However, the potential of plants aqueous extracts as natural materials for synthesizing metal nanoparticles is yet to be fully explored. The plant Tanacetum parthenium grows in moderate regions of Asia, Europe, North and South America. It belongs to the family of Asteraceae and which is known as a perennial plant 18 . In traditional medicine, this herb is used to treat thrombotic thrombocytopenic purpura, spasmodic pains, in ammation and microbial infections. These medicinal features have been related to the presence of several bioactive compounds such as sesquiterpenes, coumarins, avonoids, avones and tannin 18, 19 . In the present study, an environmentally compatible method for the preparation of CuO-NPs using T. parthenium extract as reducing and stabilizing agents has been described. The catalytic activity and clearing effects of synthesized CuO-NPs were also examined on organic dyes. The antimicrobial activity of CuO-NPs against E. coli, S. typhimurium, P. oryzae, F. thapsinum, C. albicans, and C. neoformans was tested using disc diffusion method. Using MTT assay, the inhibitory effect of CuO-NPs was also evaluated against Hela, MCF7, A549, L929 cell lines.

Results And Discussion
Chemical characterization of CuO-NPs The UV-Vis absorption spectrum of T. parthenium extract (Fig. 1) demonstrates that peaks at 290 and 320 nm are allocated to the π→π* or n→π* transitions, which can be attributed to the presence of polyphenols compound. The change of solution color corroborated the synthesis of CuO-NPs using T. parthenium extract from pale yellow to caramel brown Fig. 1. Further veri cation was performed using UV-Vis spectroscopic analysis, and the maximum peak was viewed at 298 nm, emphasizing CuO production from copper sulfate (Fig. 1). According to the results, the main characteristics of resonance band of the sulfate Plasmon at 298 nm happened for copper nanoparticles. The result here is compatible with previous studies on the biosynthesis of CuO-NPs using R. tuberosa and P. guajava leaves extract 6,7 .
FTIR spectral was performed to identify the biomolecules from T. parthenium extract that might be responsible for the reduction, stability, and synthesis of CuO-NPs. The representative FTIR spectra of the aqueous extract and the biosynthesized CuO-NPs are showed in hispidum aqueous extract 20 .
The morphology and particle size of the synthesized CuO-NPs were evaluated by FESEM analysis. The FESEM results showed the globular gure with the range size nearly from 13 to 25 nm and average size around 16 nm, which corroborating the formation of CuO-NPs by extract of T. parthenium (Fig. 4a). Similar morphology was achieved for CuO-NPs when it was synthesized by R. Crispus extract and P.
hexapetalum leaf extract 27,28 . EDX was applied to identify the elements of the synthesized CuO-NPs by T. parthenium extract (Fig. 4b).
The signals corresponding to carbon (14.38%), oxygen (30.37%), copper (49.38%), phosphorus (3.88%), and sulfur (1.99%) identi ed in CuO-NPs EDX spectrum and the Cu signal intensity authenticated the formation of CuO-NPs. Carbon, phosphorus and sulfur signals are emanated from the biomolecules of T. parthenium extract detected on the nanoparticles plane. EDX result was in agreement with previously reported results on synthesized CuO-NPs 3, 25, 29 .
Catalytic activity evaluation for reduction of MO, RhB, MB and CR dyes Industrial dyes are released into the water and aqueous environments; hence they are considered as a major threat to the ecosystem, aquatic life, and creature's health. Methylene Orange (MO), Rhodamine B (RhB), Methylene Blue (MB), and Cango red (CR) lead to several health hazards such as breathing, vomiting, nausea, and diarrhea 7,30 . As a result, there is a great interest developing modern methodologies that can remove and degrade industrial dyes. These dyes are stable molecules, and their reduction by NaBH 4 in the absence of any suitable catalyst occurs at a prolonged rate. This prolonged rate may be due to the large redox potential difference between an electron donor (NaBH 4 ) and an electron acceptor (MO, RhB, MB, and CR) 31,32 . CuO-NPs serve as nanocatalyst, capable of accepting electrons from an electron donor and transfer them to the electron acceptors (industrial dyes). The catalytic reductions of MO, Rh B, MB, and CR with an extra amount of NaBH 4 were selected as model reactions to appraise the catalytic activity of CuO-NPs. As the results indicated, in the absence of CuO-NPs, the reduction reaction did not proceed. On the other hand, in the presence of CuO-NPs, catalytic reduction of the dyes happened. This reduction effect is responsible for the degradation observed in the industrial dyes. It should be noted that the degradation process is continuously increased along with time. Speci c peaks for MO, RhB, MB, and CR disappeared thoroughly after 400, 400,400, and 190s, respectively, and the color became lucid, denoting the reaction's completion. Linear relationship between ln (C t /C 0 ) versus reaction time con rms that the reactions pursued rst-order kinetics. The apparent rate constants (k t ) were calculated from rstorder reaction kinetics using the slope of straight lines. The k t values of CuO-NPs for the reduction of MO, RhB, MB, and CR were 5.8 × 10 − 3 s − 1 , 9 × 10 − 3 s − 1 , 1.31 × 10 − 3 s − 1 and 2.5 × 10 − 3 s − 1 , respectively. The highest k t value of CuO-NPs was observed for the reduction of RhB (9 × 10 − 3 s − 1 ). The apparent rate constants and dye degradation time of CuO-NPs catalyzed reactions are identi ed to be comparable and, in some cases, even better than the reviewed catalysts in the articles for the reduction of MO, RhB, MB and CR 27,33,34 . The maximum degradation percentage of MO, RhB, MB, and CR was 96.6%, 98.3%, 98.7%, and 99.6%. The maximum and minimum degradation were observed in Congo red and Methylene Orange, respectively. Recycling and reusing heterogeneous catalysts are among the main subjects in the practical application of heterogeneous catalysts 35 . CuO-NPs heterogeneous catalyst can be retrieved after the completion of the reaction mixture by centrifugation. The UV-Vis absorption results demonstrated that the stability and turnover of the CuO-NPs catalyst in reducing and degrading MO, RhB, MB, and CR can remain unaltered after four consecutive cycles in catalytic reaction 27 . Antimicrobial Activity Of Synthesized Cuo-nps Signi cant antimicrobial activity was observed in all concentrations of CuO-NPs against examined bacteria and fungi strains ( Fig. 6 and Table 1). The size of inhibition zone increased with increasing CuO-NPs concentration; at 100 µg/ml concentration of CuO-NPs, the maximum inhibition was obtained for E. coli (22 mm) and S. typhimurium (21 mm). The maximum zone of inhibition (21mm) was observed in C. albicans followed by C. neoformans and F. thapsinum (20mm), and F. semitectum (19mm) with 130 µg/ml CuO-NPs ( Fig. 6 and Table 1). CuO-NPs showed potent antimicrobial activity against E. coli and F. semitectum when compared to the positive control. In comparison with bacteria, fungi had a smaller inhibition zone, probably because of the presence of chitin in their cell wall, which exhibits higher resistance to nanoparticle penetration into the inner layer of the cell wall. Similar results were observed when CuO-NPs synthesized using Cissus arnotiana extract 36 . The antimicrobial activity of CuO-NPs could be due to the smaller size and the larger surface-to-volume ratio of CuO-NPs allowing nanoparticles to expansively attach with the cell membrane and damage the genetic material, causing cell death 37 . The cell membrane damage caused by the electrostatic interaction between the phosphate groups in the cell membrane. Moreover, releasing the Cu 2+ ions disrupts cell membrane integrity leading to membrane leakage 34,37 . Furthermore, the production of reactive oxygen species (ROS) damages DNA, RNA, lipids, proteins, and the activity of certain periplasmic enzymes, restraining ATPase activities from reducing the ATP level 34 .

Conclusions
We have successfully biosynthesized CuO-NPs through eco-friendly, low cost, and simple method using aqueous extract of T. parthenium as the reducing and stabilizing agent. UV-vis spectra and FT-IR results showed that polyphenols, proteins, polysaccharides, and lipids could be useful for bio-reduction, capping, and stabilizing the particles. The crystalline structure and spherical shape of particles with a size range from 13-25 nm were designated by XRD and FTIR analysis-. CuO-NPs showed suitable catalytic activities in degradation process of the Methylene Blue, Methylene Orange, Rhodamine B, and Congo red by NaBH 4 . CuO-NPs revealed vigorous antimicrobial and cytotoxic activities against -pathogenic microbial strains (two bacterial and four fungal strains) and A549, Hela, and MCF7 cancer cell lines. This antimicrobial and anticancer activity could be related to the smaller size and the larger surface area of CuO-NPs. Moreover, electrostatic interactions, increased ROS generation, DNA damage and apoptosis induction might be the other effects of CuO-NPs. Our results suggested that CuO-NPs synthesis using aqueous extract of T. parthenium can be considered as a suitable candidate for biomedical, pharmaceutical, and environmental applications.

Material And Method
Preparation of T. parthenium extract Fresh leaves and owers of T. parthenium were collected from Kepin, Mazandaran, Iran. The leaves and owers were washed to eliminate the dust particles. Washed leaves and owers were then dried and chopped using a mixer. 20g of prepared leaf and ower powder were added to 150 mL warm deionized water and the mixture was maintained in magnetic stirrer for 30 min at 80ºC. The mixture was then centrifuged at 6500 rpm, and the supernatant was ltered by Whatman No.1 lter paper.

Synthesis Of Cuo-nps
In order to prepare CuO-NPs, 35 ml of prepared extract was added to a stirred CuSO 4 solution (100 ml, 0.03 M). The mixture was stirred on a magnetic stirrer for 2h at 70ºC and then the caramel brown precipitate was centrifuged at 6500 rpm for 30 min. The resulting precipitate was washed with DI water and then dried at 60˚C in the oven.

Characterization Of Cuo-nps
Different analytical techniques were used to con rm the biosynthesized CuO-NPs. UV-Vis Spectroscopy was used with a different wavelength between 200-650nm for con rming the production CuO-NPs mediated by T. parthenium extract. Identi cation of chemical composition of CuO-NPs was done by FT-IR Spectroscopy with a wide range of wavenumbers of 400 cm − 1 to 4000 cm − 1 . X-ray Diffractometer (XRD) was conducted to identify the crystalline structure of the CuO-NPs in the 2θ domain of 20-80º. The structural characterization of CuO-NPs was determined using FESEM and EDX.

Catalytic Reduction Study Of Dyes
200 µg of CuO-NPs catalyst and 3ml of each dye solution (Congo red and Methylene orange 0.08 mM; Methylene blue 0.03 mM and Rhodamine B 0.05M) were mixed and then 0.5 ml of NaBH4 aqueous solution (6 × 10 − 3 M) was added to this mixture. The reduction process was monitored using UV-vis spectroscopy and the absorbance was recorded every 25 seconds intervals. The rate constant could be appraised by pseudo-rst-order kinetics for dyes reduction. The following equation was applied to determine the rate constant (k t ) of CuO-NPs: k t = -ln (C t /C 0 ). Where k is the rate constant at the given time and t is the reaction time. C 0 and C t are the concentrations of dyes at initial and at time t respectively.
The degradation percentage was computed from the formula, R (%) = (A 0 − A t /A 0 ) × 100 Where R (%) is the degradation percentage, A 0 and A t are the absorbance of a dye at times t = 0 and t = t, respectively. For the recyclability test, the recovered catalyst was washed with distilled water, dried and used in the previous condition.
Antimicrobial Properties Of Cuo-nps Disc di usion method was used to investigate the growth inhibition activity of CuO-NPs against some microbes. The bacterial and fungal strains used for the study were Escherichia coli, Salmonella typhimurium, Fusarium semitectum, Fusarium thapsinum, Candida albicans, and Cryptococcus neoformans. Mueller-Hinton Agar and Sabouraud dextrose agar plates were inoculated with a microbe suspension for bacterial and fungal strains. Various concentrations of CuO-NPs were loaded on sterile paper discs (6 mm in diameter). The bacteria and the fungi plates were incubated at 37°C for 24h and 28°C for 48h, respectively.

Cytotoxic Activity
In vitro inhibitory activities of CuO-NPs obtained by T. parthenium extract on MCF7, Hela, A549, and normal broblast L29 cells were examined using MTT assay. The cell lines were cultivated on the RPMI-1640 media containing fetal bovine serum 1% (10%), glutamine (1%) Streptomycin and penicillin (100 U/ml) under CO2 5% for 24h at 37°C. The medium was sent out and re lled with fresh medium along with various concentrations of CuO-NPs (25-130 µg/mL) and incubated for 24h at 37°C. Then tetrazolium MTT solution was added to each well and incubated for a further four h. The MTT solution was then discarded, and the crystals formed were solved by adding DMSO. The optical density of the solutions was read at 570 nm by a microplate reader. The IC50 values and cell viability percentage for each cell line were computed by the following formula.