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 verification 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 extract6, 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 Fig. 2. The absorption bands at 3430.44 and 3429.25 cm− 1 are due to O-H stretching of phenols and alcohols20, 21. The bands at 2926.0 and 2924.14 cm− 1 originated from C-H stretching vibrations of methyl groups of the lipids3. The absorption bands at 1625.5 and 1624.5cm− 1 have corresponded to the amide I in proteins and C = C in aromatic compounds20. The bands at 1408.1 and 1386.3 cm− 1 were probably related to COO- in the amino acid residue of protein and CH3 stretch of fatty acids3, 22. The bands at 1256.5 and 1260.1 cm− 1 could be due to the C-O group stretching vibration of carbohydrates and amide III for protein23. The bands at 1069.4 and 1057.6 cm− 1 might be related to the C-O stretching band of oligosaccharide residue3. The bands at 616.9 and 611.1 cm− 1 are probably attributable to the alkyl halide24. The first and second numbers belong to the aqueous extract and CuO-NPs, respectively. Comparing the result obtained from aqueous extract and CuO-NPs showed that the peaks observed in the spectrum of the aqueous extract are also present in the spectra of synthesized CuO-NPs with lower severities and a slight variation. After synthesizing CuO-NPs, the absorption peaks at 3,430, 2926, 1625, 1408, and 1,069 cm− 1 observed in aqueous extract get thinner and changed to low-frequency regions. FTIR analysis results were in agreement with the FTIR spectrum pattern of the B. tomentosa leaves extract, which proposed phytochemical compounds such as proteins and polyphenol contributed to the synthesis of CuO-NPs25.
Crystalline feature confirmation of CuO-NPs was characterized by XRD analysis (Fig. 3). XRD patterns of CuO-NPs synthesized by green method demonstrate notable peaks at 2θ of 32.65˚, 38.7˚, 48.8˚, 53.4˚, 58.35˚, 61.65˚, 66.35˚ and 75.45˚ which were allocated to the (110), (111), (202), (020), (202), (113), (311) and (004) planes. The observed diffraction data were comparable with JCPDS No. 45-093720. The present results are in good agreement with the previous report on the synthesis of CuO-NPs using A. hispidum aqueous extract20.
The morphology and particle size of the synthesized CuO-NPs were evaluated by FESEM analysis. The FESEM results showed the globular figure 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 extract27, 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%) identified 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-NPs3, 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 diarrhea7, 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 NaBH4 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 (NaBH4) 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 NaBH4 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. Specific 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 (Ct/C0) versus reaction time confirms that the reactions pursued first-order kinetics. The apparent rate constants (kt) were calculated from first-order reaction kinetics using the slope of straight lines. The kt 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 kt 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 identified to be comparable and, in some cases, even better than the reviewed catalysts in the articles for the reduction of MO, RhB, MB and CR27, 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 catalysts35. 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 reaction27.
Significant 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 extract36. 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 death37. The cell membrane damage caused by the electrostatic interaction between the phosphate groups in the cell membrane. Moreover, releasing the Cu2+ ions disrupts cell membrane integrity leading to membrane leakage34, 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 level34.
In vitro cytotoxic activity of CuO-NPs indicated high level cytotoxicity effect against all of the cancer cell lines compared to L929 normal cells at various concentrations (25–130 µg/mL) (Fig. 7). Cell death recorded at 130 µg/mL concentration of CuO-NPs was almost 12.1%, 8.8%, and 19.9% for A549, Hela, and MCF7 cell lines, while in the case of L929 cell lines, the cell death was only 73% at the same concentration. Both cancer cell lines and normal cell line demonstrated a reducing percentage of cell viability with augmenting concentration of CuO-NPs (Fig. 7).
It can be concluded that increased number and aggregation of CuO-NPs within the cell lines result in enhanced oxidative stress, causing cell death28. IC50 values of CuO-NPs were discovered to be 65.0 µg/mL, 57.4 µg/mL, 71.8 µg/mL and 226.1 µg/mL for A549, Hela, MCF7 and L929 cell lines, respectively. Compared to the normal cell lines, lower IC50 value of CuO-NPs in the cancer cell lines can be related to the high reproduction rate, abnormal metabolism, and high uptake of CuO-NPs in cancer cell lines38. High cytotoxic effect of CuO-NPs is probably due to their size, shape, and large surface-to-volume ratio which enable the nanoparticles to readily enter the cells39. CuO-NPs not only can interact with mitochondria but also can interrupt the cellular electron transition chain, causing to elevated level of ROS which in turn causes
DNA damage, activates apoptosis and signaling pathway of MAPK, and consequently cancer cell lines death40. Several studies reported increased level of- ROS formation in cancer cell lines during treatment with nanoparticles41, 42.