Obvious peak at the wavelength range of 1000–1200 nm was found for CuNCs at the periodicity of 20 min for 80 min, which showed the growth of NCs during this time (Fig. 1e). XRD spectrum revealed the availability of carbon, iron sulfate hydrate (FeSO4.xH2O), and tenorite (CuO) with copper (Fig. 1f). There were several functional groups indicated by FTIR for CuNCs, CuNCs-Am, CuNCs-Pe, and CuNCs-Te (Fig. 1g). C = C stretch and the C–H stretch were indicated for the bonds related to the phytochemicals such as carbohydrates, proteins, phenolic compounds, and alkaloids in the leaf extract of Adhatoda vasica Nees for green synthesis of CuO/carbon NCs [25]. The FT-IR spectra of CuFeS2 nanocrystals illustrated peaks at 2346 cm− 1 related to stretching of C-S bonded to nanocrystal, significant peaks at 2918 cm− 1 for stretching vibrations of C-H assigned responded to the capping ligand of 1-dodecathiol as well as 1446 and 1554 cm− 1 for stretching vibrations of –CH2 and C-C, respectively [26]. Hydroxyl group was a common bond for creating the nano-complex of amoxicillin and ampicillin with AgNPs. Furthermore, formulation of ciprofloxacin-ZnONPs was based on interaction of carboxylic acid and aryl amine of antibiotic with ZnONPs [27].
3.1. TEM micrographs and DLS analysis
As presented in Figs. 2a and b, Carbon/FeSO4/Cu/CuO NCs had zeta potential, Z-average, and PDI of -5.09 mV, 460.2 d.nm, and 0.293 (mono dispersed particles), respectively. TEM images illustrated 44.58 ± 6.78 nm with various morphologies including triangular, rod, and hexagonal shapes (Figs. 2c and d). According to zeta potential result, aggregation stability is expected for these negative charged colloidal NCs. In a green synthesis way, bimetallic ZnO–CuO NCs fabricated by aqueous leaves extract of Calotropis gigantean plant species with three weight percentages of 75 wt%, 50 wt%, and 25 wt% of metallic salt (zinc nitrate hexahydrate) showed porous agglomerated morphology, irregular rod-shaped NPs, and honeycomb shape at agglomerated morphology, respectively [28]. Mixture of 0.01 M copper sulphate (CuSO4 .5H2O) solution and 1g of the aqueous leaves extract of A. vasica was used to synthesize nanoflakes of CuO/carbon having a mean diameter of 7–11nm [25]. The presence of natural compounds such as polyphenols, flavonoids, tannins, alkaloids, saponins, and reducing carbohydrates may be responsible for the reduction and stabilization of metal or metal oxides NCs, as it was confirmed for green synthesized CuO nanosheets (a mean size of 20 nm) by aqueous arial parts of Rhazya stricta plant species [29].
3.2. Antibacterial activity
Higher antibacterial activity was found for CuNCs/Te toward E. coli, P. aeruginosa, and S. aureus as IZDs of 25.31, 23.07, and 27.21 mm, respectively (Table 2). E. coli as more sensitive bacteria revealed 27.37, 23.35, and 23.23 mm of IZD values upon stress of CuNCs, CuNCs/Am, CuNCs/Pe, sequentially. Resistance of Gram-negative bacteria of P. aeruginosa towards CuNCs and CuNCs/Am was indicated as IZDs of 11.77 and 14.24 mm, wherein Gram-positive bacteria, S. aureus, had the lowest sensitivity to CuNCs/Pe as value of 1.03 mm. MIC and MBC assays confirmed disc diffusion test as lower bactericidal effects by values of 100, 100, and 250 ppm toward E. coli, S. aureus, and P. aeruginosa, respectively (Fig. 3B-C). Biogenic CuO/carbon NCs fabricated by the aqueous leaves extract of A. vasica showed zones of inhibition 11, 12, 14, and 11 mm against P. aeruginosa, Klebsiella pneumoniae, S. aureus, respectively [25]. Compared to bacterial sources for synthesis Cu or CuO NPs, Streptomyces capillispiralis Ca-1 strain isolated from Convolvulus arvensis as the endophytic actinomycete (Gram-positive mycelial bacteria) was exploited to prepare CuNPs with spherical shape and diameter of 3.6–59 nm and antifungal effect against Pythium spp., Aspergillus niger, and Alternaria spp., by the inhibition of 58.05%, 63.81, and 57.14, respectively at amount 20 mM of NPs [30]. In compared to ciprofloxacin antibiotic with zone of inhibition values of 21.3 and 21.3 mm, biosynthesized CuO NPs by Actinomycete sp. VITBN4 exhibited 14.3 and 15.6mm against S. aureus and Bacillus cereus, respectively at 10µg/mL concentration more than cell free supernatant of actinomycetes. The mean size, zeta potential, and shape of these NPs were 61.7 nm, − 31.1 mV, and spherical with aggregated form, respectively [31]. Optimized synthesis of CuNPs with face cubic center (fcc) crystal structure and average size of 56–73 nm was prepared by supernatant of culture medium of Halomonas elongate IBRC-M 10214, wherein IZDs for this NP were 10 and 8.7 mm toward E. coli and S. aureus [32].
NorA protein as NorA multidrug efflux pump in S. aureus was inhibited by interference of ciprofloxacin modified ZnONPs in function of this protein [27]. The interruptions of bacterial envelopes by NCs and break-down of bacterial enzymes are main antibacterial mechanisms for metallic NPs or NCs [3]. Surprisingly, at the genome level, both CuONPs and CuO in micro size can impact on the plasmid pUC19 as converting its supercoiled form to open circular one. However, in contrast to micro CuO, there was not any supercoiled form of plasmid on gel electrophoresis for CuONPs [33]. Figures 3 and 4 illustrate breaking of envelopes of S. aureus (thick peptidoglycan and cell membrane) and E. coli (outer membrane, thin peptidoglycan, and inner membrane) upon effect of CuNCs/Te, respectively. Synergistic effect was observed for the combination of 30 µg/mL of AgNPs biosynthesized via cyanobacteria of Phormidium sp. with chloramephnicol (0.5%) against methicillin-resistant S. aureus (MRSA) as IZDs of 28 mm compared to 20 and 15 mm for AgNPs and chloramphenicol, respectively [34]. In a similar investigation, AgNPs by a mean size of 29.8 nm were combined with several antibiotics including tetracycline, ampicillin, kanamycin, enoxacin, neomycin, and penicillin antibiotics. At the concentrations of 16, 8, 2, and 0.5 µM, significant bacterial inactivation of Salmonella typhimurium DT104 was observed as value of ~ 100% for AgNPs-tetracycline nanocompsites other AgNO3, AgNPs, and nanocomposites [35]. As presented in Fig. 4e, three main pathways including antibacterial effects of CuNCs/Te and each CuNCs and tetracycline alone on bacteria may be considered, which most probable case can be antibacterial activity of CuNCs/Te.
Table 2. IZDs results of E. coli, S. aureus and P. aeruginosa under effect of CuNPs, CuNPs/Am, CuNPs/Pe, and CuNPs/Te.
Bacteria
|
Antibacterial agents with IZD (mm)±SD
|
E. coli
|
CuNCs
|
CuNCs/Am
|
CuNCs/Pe
|
CuNCs/Te
|
27.37±0.85
|
23.35±0.70
|
23.23±0.92
|
25.31±1.14
|
P. aeruginosa
|
11.77±1.53
|
14.24±1.25
|
9.52±0.51
|
23.07±1.36
|
S. aureus
|
14.18±1.20
|
26.92±0.48
|
1.03±1.05
|
27.21±1.05
|
3.3. Molecular docking results
Table 3 presents type of amino acids interacting with each enterotoxin (A and B) by their binding affinity values. In the respect to enterotoxin A, highest and lowest affinity were respectively observed for daphnauranol C and (E)-Nerolidol by -6.2 and − 4 Kcal/mol. However, the difference of affinity between other compounds was not significant. Similarly, HDOCK confirmed this result as more negative docking score of -107.46 for daphnauranol C with RMSD of 23.77 Å. In the case of daphnauranol C, HIS50, LEU48, PHE47, ASP70, ARG214, TYR108, LEU68, ALA97, TYR92, and GLN95 interacted with enterotoxin A. Docking poses illustrated at Fig. 5, wherein tyrosine, asparagine, and histidine were common interacted amino acids with enterotoxin A. Best affinity was found for nootkatin with value of -6.7 Kcal/mol towards enterotoxin B, which (E)-Nerolidol showed lowest one by -4.6 Kcal/mol. Amino acids of PRO1006, LYS1007, PRO1008, ASP1009, GLU1010, LEU1011, THR1184, TYR1186, TYR1233, and THR1235 were responded to docking of ligand of nootkatin with receptor of enterotoxin B at hydrogen bond and steric interaction (Table 3 and Fig. 5). In addition, HDOCK showed similar affinity for nootkatin with docking score of -111.67 and ligand RSMD of 51.17 Å (Table 4). According to HDOCK result, docking interactions of each compound additionally are presented in Figs. 6. Betulin and 28-Norolean-12-en-3-one as triterpene metabolites showed free energy values of − 147.39 and − 83.97 Kcal/mol toward staphylococcal enterotoxin A, respectively [37]. In another study, for the natural metabolite of catechin (flavan-3-ol), there was binding affinity between the hydroxyl group at position 3 of the galloyl group and the active sites staphylococcal enterotoxin A [38]. Autodock scores were − 48.4, − 41.4, − 42.2, and − 35.5 kJ/mol for (-)-epigallocatechin-3-gallate, (-)-epigallocatechin, kaempferol-3-glucoside, and kaempferol, respectively [39]. Additionally, as antibacterial mechanism, these terpenoides can hinder growth of bacteria by the disruption of the cellular membrane integrity [40].
Table 3
Interacting amino acids and binding affinities (Kcal/mol) for six natural metabolites toward enterotoxin toxin A and B.
Ligands | Enterotoxin A | Interacting residues for enterotoxin A | Enterotoxin B | Interacting residues for enterotoxin B |
(Z)-α-Bisabolene epoxide | -5.2 | GLN19, GLY20, TYR64, GLY93, TYR 94, GLN95, CYS96, ASN102, and THR104 | -5.6 | PHE1044, LEU1045, ARG1065, GLU1067, TYR1089, GLN1092, TYR1094, SER1096, LYS1097, and SER1211 |
(E)-Nerolidol | -4 | LYS10, ILE7, GLU2, LEU183, SER1, ASN128, ASP227, VAL185, ASN195, SER193, HIS187, and HIS225 | -4.6 | TYR2091, CYS2093, TYR2094, PHE2095, SER2096, ASP2108, and LYS2111 |
α-Cyperone | -5.3 | ASN33, GLY93, TYR94, ASN25, SER206, and TYR205 | -6.6 | PRO1006, LYS1007, PRO1008, ASP1009, GLU1010, LEU1011, THR1184, TYR1186, TYR1233, and THR1235 |
Daphnauranol C | -6.2 | HIS50, LEU48, PHE47, ASP70, ARG214, TYR108, LEU68, ALA97, TYR92, and GLN95 | -6.4 | LYS1025, ASP1029, VAL1169, LYS1170, LYS1173, TYR1175, GLU1176, LYS2170, |
Nootkatin | -5 | HIS50, ARG214, ARG211, TYR108, ASP70, LEU48, PHE47, ASN207, ALA97, and TYR92 | -6.7 | PRO1006, LYS1007, PRO1008, ASP1009, GLU1010, LEU1011, THR1184, TYR1186, TYR1233, and THR1235 |
Nootkatone | -5.6 | THR38, LYS37, PHE57, THR59, PHE58, ALA36, LYS35, TYR91 | -5.9 | PHE2044, LEU2045, ARG2065, GLU2067, TYR2094, SER2096, and LYS2097 |
Table 4
Results of HDOCK indicating docking score and ligand RMSD in the best mode of docking.
Ligands | Enterotoxin A | Enterotoxin B |
Docking score | Ligand RMSD (Å) | Docking score | Ligand RMSD (Å) |
(Z)-α-Bisabolene epoxide | -88.30 | 51.24 | -101.46 | 51.31 |
(E)-Nerolidol | -91.86 | 27.62 | -92.54 | 50.99 |
α-Cyperone | -88.98 | 26.36 | -99.64 | 51.03 |
Daphnauranol C | -107.46 | 23.77 | -101.88 | 49.72 |
Nootkatin | -99.03 | 26.45 | -111.67 | 51.17 |
Nootkatone | -89.49 | 25.57 | -98.01 | 51.16 |
3.4. Interaction of albumin and hemoglobin with CuNCs
Interaction of green prepared CuNCs with two main blood proteins including albumin and hemoglobin was evaluated by FTIR, AFM, and SEM techniques. Size and concentration of proteins can impact on roughness and self-assembly of proteins around the metal NPs or NCs [41], wherein larger size of protein may result in a higher aggregation and roughness. In this regard, size of human serum albumin and hemoglobin are 8 − 13 nm and 5-6.9 nm, respectively [42, 43], which CuNC/Te-A showed roughness of 38.12 ± 19.96 nm compared to CuNC/Te-H having 20.74 ± 5.33 nm (Figs. 7a-c). Electrophoretic mobility of these proteins can be changed by binding of metal ions to the metal binding sites, which can induce a protein aggregation. As reported in a previous study, a total molar ratio of 8 metal ions of cobalt to one human serum albumin can promoted aggregation of these proteins [23]. As presented in Fig. 8, SEM images showed rod shape of CuNC/Te-A (diameter = 109.26 ± 40.30 nm and length = 1055.64 ± 271.62 nm) in contrast to globular shape of CuNC/Te-H by average size of 321.75 ± 194.28 nm. Spectra of FTIR exhibited main peaks at 3442, 2919, and 1095 cm− 1 for functional groups of -O-H, -C-H and -C-O stretch, separately (Fig. 7d). There was higher intensity of transmittance for CuNC-Te-A relative to CuNC-Te-H showing a major role of -O-H group in creating of complex of CuNC-Te-A.