1 Introduction

The multi-applications of metal oxide nanoparticles (MO NPs) in various fields including catalysis, energy, environment, agriculture, medicine and biological sciences, etc. gained consideration from scientific societies recently [1]. The size-dependent properties of MO NPs such as high surface area, enhanced solubility, and reactivity as well as optical, electronic, and magnetic properties, hold a pledge to their applications in biological sciences [2]. Moreover, the capacity of MO NPs to generate a high number of ROS (reactive oxygen species), with great selectivity and lower sensitivity to normal cells, urges their use in nanobiotechnology [3].

Among many biologically benign MO NPs that have been introduced into clinical fields, ZnO, and CuO nanoparticles have disclosed several benefits such as bioavailability, biocompatibility, and cost-effectiveness in biological applications [4,5,6]. Due to the semi-conductive nature and surface crystal defects of these NPs the electrons freely move from the valance band to conduction bands and hence could react with oxygen species to form free radicals which cause oxidative damage of in bacterial and cancerous cells. ZnO NPs is more appropriate in cancer therapy due to its inherent electrostatic nature and selective toxicity [7, 8]. ZnO nanoparticles have exhibited significant anticancer efficacies against a variety of cancer types, including lung cancer [9], breast cancer [10], ovarian cancer [11], and colorectal cancer [12]. The anticancer potency of ZnO NPs is mainly through zinc-mediated protein activity disequilibrium and oxidative stress [13, 14]. CuO NPs have shown enhanced anticancer efficacy against breast cancer [15], hepatocellular carcinoma [16], lung cancer [17], and cervical cancer [18]. The anticancer mechanism of CuO NPs is mainly through genotoxicity and apoptotic death in cancerous cells due to its ability to generate enhanced ROS [19]. The exceptional features of MgO NPs such as biocompatibility, recycling activity, low density, hygroscopic nature, and good functionality make it a promising anticancer remedy [20,21,22]. Moreover, nano-metal oxides of zinc, copper and magnesium have been broadly utilized in microbial infections [23,24,25]. However, the integration of ZnO, MgO, and CuO NPs together is preferred over monometallic counterparts due to their immensely improved synergistic effects including enriched surface area, multiple reactive sites, higher charge flow, and mass transfer for biological application [26,27,28].

For the synthesis of trimetallic oxide nanoparticles (TMO NPs), chemical, physical, and biological routes have all been used [29,30,31,32]. The fabrication of TMO NPs from biological entities has many benefits over other techniques. The bioreduction approach is effective, economical, and environmentally benign prohibiting the assembly of undesirable or toxic chemicals [33,34,35]. The green fabrication of MMO NPs has been implemented to accommodate diverse bio-systems, including algae, fungi, bacteria, and plant extracts. Moreover, using plant extracts has a low cost of handling, safety, and simple protocol to create TMO NPs at a large scale relative to bacteria and/or fungi-assisted fabrication [36,37,38,39]. Various secondary metabolites from the plant extracts, including phenolic compounds, flavonoids, alkaloids, terpenoids, etc., contain functional groups such as NH2, SH, COOH, C=O, and OH, contribute to the strong reduction and stabilization of NPs [40]. Moreover, medicinal plant mediated synthesized NPs could also exhibit enhanced biological activity due to the synergistic effects of phytochemicals [25, 41].

Cancer and infectious diseases have become a global burden that causes international health, economic, and communal crises [42, 43]. Due to their unpredictable spread, infectious diseases have deleterious impacts worldwide, including high mortality rates, enormous burdens, and impairment [43, 44]. However, several anticancer drugs have been reported recently, majority of them revealed different encounters in clinical use, like poor water solubility, limited membrane transport capacity, and swift clearance and degradation problems in the bloodstream during clinical use [45]. The path of physiological response to counter them as well as the cytotoxicity to a healthy body of conventional chemotherapy recently became the main focus of researchers [46]. Antibiotics are one of human medicine's most extensively utilized treatment techniques to fight pathogenic microorganisms [47]. However, changes in the ability of microorganisms to resist antibiotics, either by inactivating them or limiting their therapeutic efficacy, lead to microbial resistance, also known as multi-drug resistance [48].

In this study, Artemisia abyssinica leaf extract (AALE) was used as a greener reducing and stabilizing agent for the synthesis of ZMC NPs. Artemisia abyssinica belongs to the family Asteraceae indigenous medicinal plant regularly used for the treatment of different diseases traditional as well as modern medicine of Ethiopia [49]. AA was known to treat inflammation, cold, amenorrhea, headache, colic, anorexia, fever, and dysmenorrhea traditionally [50]. Moreover, the plant has also been reported as an antimalarial, antiparasitic, antirheumatic, anti-inflammation, antitumor, and antioxidant agent [51,52,53]. Essential oils, tannins, terpenoids, saponins, polyphenols, alkaloids, and flavonoids are the most common phytochemicals reported from the AALE [54, 55].

Therefore, in this study we hereby report a novel, eco-benign synthesis of trimetallic ZMC NPs using biomolecules from AALE for the first time. For the characterizations of biosynthesized ZMC NPs the advanced techniques including UV–visible, XRD, FTIR, SEM, EDX, TEM/HRTEM and SAED were used. Furthermore, comprehensive studies of in-vitro cytotoxicity and antibacterial potentials as well as in-silico molecular docking analysis of biosynthesized ZMC NPs were also presented.

2 Materials and Methods

2.1 Chemicals and Media

Magnesium nitrate hexahydrate (Mg(NO3)2·6H2O, 99.8%), zinc nitrate tetrahydrate (Zn(NO3)2·4H2O, 99.9%), copper nitrate trihydrate, (Cu(NO3)2·3H2O, 99.8%), ethanol (C2H6O, purity ≥ 99.9%), and sodium hydroxide (NaOH, 97%), dimethyl sulfoxide (DMSO), ascorbic acid and Chloramphenicol, Sigma Aldrich, were purchased from Addis Ababa, Ethiopia. Mueller Hinton broth (MHB), Sabouraud Dextrose Agar (SDA), Potato Dextrose Agar (PDA), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), 3-(4,5-dimethylthiazol-2-yl)-2, were procured from institute of public health (IPH), Addis Ababa, Ethiopia. Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT), PenStrep, Trypsin, Spectramax I3X, CO2 incubator, and Doxorubicin (Invitrogen, USA) where obtained from, India.

2.2 Bacteria and Cancer Cell Lines

Bacteria strains, Escherichia coli (E. coli, ATCC 25922), Pseudomonas aeruginosa (P. aeruginosa, ATCC 27823), Streptococcus pneumoniae (S. pneumoniae, ATCC 11778), and Staphylococcus aureus (S. aureus ATCC 25923), were obtained from the Institute of Public Health, Addis Ababa, Ethiopia. Breast cancer (MCF-7) cells and peripheral blood mononuclear cells (PBMCs) (Invitrogen, USA) were acquired from National Center for Cell Sciences (NCCS), India.

2.3 Preparation of Artemisia abyssinica Leaf Extract (AALE)

The preparation of AALE was done according to our previous work with certain modifications [54]. The leaves part of the medicinal plant Artemisia abyssinica was collected from Tiyo Woreda, Arsi Zone, Tiyo Woreda, 7°45′55'' and 8°02′02''N latitude and 38°56′42'' to 39°18′31''E longitude, Oromia Region, Ethiopia, which is 175 km Southeast of Addis Ababa with an elevation ranges of 1850–4050 m [56]. The obtained leaves were washed with tap and distilled water (dH2O) repeatedly and dried for 15 days in the dark to remove moisture content. After grinding with a mechanical grinder, 10 g of powdered leaves were added with 100 mL of 50% ethanol (water and ethanol, 1:1 v/v) containing 250 mL conical flasks, and then the mixtures were shaken for 1 and 1/2 h at 120 rpm and 25 °C in a mechanical shaker and then heated for 50 min at 60 °C on a magnetic stirrer. After centrifuging the drift to produce a clear solution, it was chilled at room temperature (RT) overnight before the resulting solutions were filtered through Whatman filter paper. Finally, a clear brown color extract was obtained and stored at 4 °C for further study.

2.4 Bio-Preparation of ZMC NPs

Bio-preparation of ZMC NPs was carried out by mixing 10 mM of each salts [Cu(NO3)2·3H2O, Mg(NO3)2·6H2O, and Zn(NO3)2.4H2O] solution in a 1:1:1 ratio with AALE in a proportion of 3:1 (v/v). According to optimizations of our previous study [54], the solution was adjusted at pH 5, and heated for 1 h at 70 °C on a hot plate containing a magnetic stirrer. The light blue to deep brown color change has been observed for the spontaneous reduction and formation of MZC NPs. The reaction mixture was sonicated for 1 h at RT at 60 rpm to retain the particle dispersion. The resultant solution was then centrifuged at 6000 rpm for 30 min. The pellets were repeatedly cleaned with distilled water (dH2O) following ethanol to remove the impurities and then oven-dried for 12 h at 80 °C, annealed for 2 h at 400 °C; the fine deep brown powder was obtained and stored in protective sample holders at 4 °C for further analysis [57].

2.5 Characterizations

For the characterization of the biosynthesized ZMC NPs various techniques such as UV–visible (UV–vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDAX), transmission electron microscopy (TEM), and selected area electron diffraction (SAED) were used.

2.5.1 Absorption Edge Determination

For the estimation of absorption edge of the as biosynthesized ZMC NPs, UV–vis diffuse absorption was measured using SP65 spectrophotometer (Shimadzu UV–vis, SM-1600) at a scanning range of 200–800 nm.

2.5.2 FTIR Analysis

Fourier transform-infrared spectroscopy (Perkin Elmer FT-IR, Spectrum 65) using KBr pellets in the range of 400–4000 cm−1 was used to study the responsible functional groups for the reduction and stability of biosynthesized ZMC NPs and their chemical bonding behavior.

2.5.3 XRD Study

The X-ray diffraction (XRD, Shimadzu, and XRD-7000) equipped with a Cu target for generating a Cu Kα radiation (wavelength 1.5406 Å) was used to study the actuality and crystallinity of as synthesized ZMC NPs. The measurements were made at room temperature and the accelerating voltage and the applied current were 40 kV and 30 mA, respectively. The instrument was operated under step scan type with step time and degree (2θ) of 1 s and 0.020°, respectively over 2θ range of 10° to 80°.

2.5.4 SEM/EDX Analysis

The morphology and composition of the biosynthesized ZMC NPs were characterized by scanning electron microscopy (SEM, JEOL-JSM 6500F, made in Japan); SEM coupled energy-dispersive X-ray spectroscopy (EDX).

2.5.5 TEM/HRTEM/SAED Analysis

The internal morphology, particle size, and interplanner distance of ZMC NPs were characterized by transmission electron microscope (TEM/HR-TEM/SAED, Tecnai F20 G2, Philips, and the Netherlands).

2.6 Cytotoxicity Test

The standard method was used to culture the MCF-7 and PBMC cells [58]. The cells were given injections of 10% (v/v) FBS, streptomycin (100 g/mL), and penicillin (100 IU/mL) until congestion was achieved while being cultured in DMEM with 5% CO2 at 37 °C. Following that the matured cells were isolated with cell separating solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS (Fetal Bovine Serum), then centrifuged and tested for viability. Furthermore, 5 × 104 cells /well were cultivated in a 96-well plate and nurtured for 24 h at 37 °C, under a 5% CO2 incubator.

The cytotoxicity of ZMC NPs and standard drug (Doxorubicin) against MCF-7 and PBMC cells were evaluated using MTT assay [59, 60]. The standard cells were trypsinized and adjusted with 5 × 104 cells/ml to count cells using respective media containing 10% FBS (v/v). The diluted cell suspension (5 × 104 cells/well) was poured into the 96-wells of the microliter plate in a quantity of 100 µL. The test solutions in the wells were rejected after incubation and 0.05 mg MTT was poured into the wells. The plates were kept for 4 h at 37 °C under a 5% CO2 atmosphere. The supernatants were removed from each plate and shaken gently and then 100 mL of DMSO was added to dissolve the formed formazan. The absorbance was recorded with a microplate reader at 570 nm. The growth inhibition percent (%) of the cells was computed using Eq. (1) and IC50 values were determined by using dose–response curves.

$$\mathrm{\%\, Inhibition}=\frac{\mathrm{OD \,of\, Control}- \mathrm{OD\, of \,sample}}{\mathrm{OD \,of\, Control}}\times 100$$
(1)

where, OD stands optical density.

2.7 Antibacterial Activity Test

The antibacterial efficacy of the bio-prepared ZMC NPs was evaluated using the agar disc diffusion method [61]. Mulberry Hinton Broth and Muller Hinton Agar were used as liquid and solid mediums, respectively. Using 0.5 McFarland standard the density of bacteria was adjusted and wiped on a plate covered with Muller Hinton agar from a suspension of fresh bacterial cultures such as E. coli, S. pneumoniae, S. aureus, and P. aeruginosa mixed to the liquid medium to obtain turbidity. The disks were soaked with 50 μL of (12.5, 25, 50, 100, and 200 μg/mL) ZMC NPs. Chloramphenicol (positive control) and DMSO (negative control) were used by impregnated standard discs (30 μg) as standard antibiotics. The cultures were incubated for 24 h at 37 °C, and then the zone of inhibition (ZOI) was recorded with a ruler (in mm) to ascertain the bacterial strains liability to the samples. Each trial was carried out in triplicate and reported in mean standard deviation using statistical analysis software (SPSS) (version 20).

2.8 Molecular Docking Study

The molecular interaction of ZMC NPs and the standard drugs (doxorubicin and chloramphenicol) were evaluated against the binding sites of estrogen receptor alpha (ERα; PDB: 5GS4), dihydrofolate reductase (PDB: 2w9h) (DHR), and DNA gyrase B (PDB: 6F86) using AutoDock 4.2.6 (MGL tools 1.5.7) programs. Estrogen receptor alpha, dihydrofolate reductase, and DNA gyrase B are the important protein targets of MCF-7, S. aureus, and E. coli respectively [62, 63]. The geometry of ZMC NPs and standard drugs were augmented using the B3LYP-GD3/6–311 +  + G(d,p)/LanL2DZ approach before the molecular docking investigation, and then translated to PDB files applying GaussView. The protein database was used to download the crystal structures of the targeted proteins. The structures of targeted proteins were then processed by adding polar hydrogen and cofactors, as well as eliminating the co-crystallized ligand and water molecules according to the AutoDock 4.2.6 (MGL tools 1.5.7) program. After cleaning the protein, only polar hydrogen and the Kollman charges were introduced. Nonpolar hydrogen atoms were merged and Gasteiger partial atomic charges were assigned to the compounds. The grid center coordinates were 65, 65, and 65 pointing in the respective x, y, and z directions with a grid point spacing of 0.375 Å. The grid box was centered at – 12.055, – 10.491, and 5.964 Å. Using 100 alternative conformations each standard drug and ZMC NPs were evaluated. Discovery Studio Visualizer was employed to visualize the binding interactions between ZMC NPs and standard drugs with the target receptors exhibiting the lowest binding free energies.

2.9 Statistical Data Analysis

The experimental results were computed using the one-way analysis of variance (ANOVA) function of the statistical package for social science (SPSS) version 20 and presented as mean ± standard deviation for triplicate experiments. ImageJ (imajej153-win java8 imagej.exe), Gatan Microscopy Suite software (GMS 64bit) version 2.x, and Origin software (Originpro 9.0 64bit) were also utilized for data processing.

3 Results and Discussion

3.1 One-Pot Bio-Preparation of ZMC NPs

Trimetallic ZMC NPs were synthesized by one-pot green preparation method using ethanollic (1:1, v/v) AALE. The addition of AALE to the mixed salts solution after 1 h of reaction at 70 °C on a hot plate resulted in color change from reddish-brown to a black-brown. Initially, the formation of ZMC NPs was confirmed qualitatively by the steady color change from reddish-brown to a black-brown (Fig. 1). Furthermore, the UV–visible spectroscopy was used to test the formation of ZMC NPs as presented in Fig. 2. The significant surface Plasmon resonance (SPR) peak at 367 nm confirmed the bioreduction of salts [Cu(NO3)2·3H2O, Mg(NO3)2·6H2O, and Zn(NO3)2·4H2O] solution to ZnO–MgO–CuO (ZMC) NPs [16, 64].

Fig. 1
figure 1

The schemes of reaction mechanism for biopreparation of ZMC NPs

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Fig. 2
figure 2

UV–visible spectra of AALE, and biosynthesized ZMC NPs

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Figure 1 illustrates the mechanism of interactions between precursor salts and phytochemicals from the extract of A. abyssinica during the synthesis of MZC NPs, whereas Fig. 2 presents the UV-visible spectra. The main secondary metabolites in A. abyssinica extract are phenolic compounds such as polyphenols, alkaloids, and flavonoids used as reducing and stabilizing ligands [54]. Polyphenols are potential agents that could participate as reducing and capping ligands among the phytochemicals mentioned, that are also illustrated in the FTIR study (Fig. 3). However, polyphenols most commonly contain phenolic acids, stilbenes, and lignans [65]. Among the phenolic acids accounts for the majority of polyphenols in most reported works of literature and also are those with the most active sites than the others [66]. In light of this, a proposed reaction scheme for forming ZMC NPs is presented as follows.

Fig. 3
figure 3

FTIR spectra of AALE, and biosynthesized ZMC NPs

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3.2 UV–visible Study

UV–visible (UV–vis) spectroscopy is an essential technique used to determine NPs physico-chemical properties, electronic structure, and optical activities. 5 mM of AALE and ZMC NPs aqueous solutions were prepared and their respective absorption peaks were measured using UV–visible spectroscopy. AALE revealed various absorption peaks at 254, 360, and 402 nm. This could be due to various active biomolecules present in AALE, which are responsible for the reduction and stability of synthesized nanoparticles [67]. The biosynthesized ZMC NPs revealed the SPR band at 367 nm. The interaction between the light wave and the ZMC NPs free electrons may be what causes the SPR absorption [68, 69]. According to recent research, spherical NPs only display one SPR band, whereas anisotropic particles evince two or more SPR bands [70]. Hence the single SPR peak in the UV–vis spectrum of ZMC NPs illustrates the formation of iso-morphological particles [69]. The symmetrical shape of SPR bands also suggests that the biosynthesized ZMC NPs have well-dispersed and uniform sizes [54, 71]. Since non-uniform particles will have a broad absorption peak with a split Plasmon band [72]. Moreover, the higher intensity in the UV–vis spectra of ZMC NPs could also use to carry out the quantitative analysis of yields of nanoparticles [73]. Hence absorbance of the peak has direct relation to the concentration of the absorbing species in the medium. Therefore, the UV–vis spectra of ZMC NPs (Fig. 2) demonstrated the formation of well dispersed, iso-morphological particles with high yield.

3.3 FTIR Study

FTIR spectroscopy is a helpful tool for identifying functional groups of active phytoconstituents that serve as reductants and stabilizers in the bio-fabrication of ZMC NPs. FTIR spectra of the bio-fabricated ZMC NPs and AALE are presented in Fig. 3. The absorption band positions (Table 1) at 3436, 2914, 2072, 1392, 1268, 1076, and 512 cm−1 represent the FTIR spectra of the AALE. The bending and stretching vibrational frequencies of phenolic –OH is presumed to be involved inthe broadband at 3436 cm−1 [74]. The narrow peak at 2914 cm−1 is also ascribed and sharp peak at 2072 cm−1 are due to the bending and stretching frequency of C-H from vinyl groups C≡C from terminal alkyl groups respectively [54, 74]. The prominent peak at 1392 cm−1 could be accountable for the stretching vibration of C=O of polyphenols. The intense peaks at 1268, and 1076 cm−1 were due to stretching vibrations of aromatic C-O and N–H from phenolic groups respectively [75, 76]. The broad peak at 512 cm−1 could be connected to the esters C–O or C–O–C stretching [75].

Table 1 Assignment of characteristic absorption peaks from FTIR spectra of AALE and ZMC NPs
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Bio-prepared ZMC NPs nanoparticles have shown FTIR spectra absorption band positions of 3354–3206, 2914, 2072, 1381, 1105, 833, 614, and 524 cm−1 (Fig. 3 and Table 1). The broad peak at 3354–3206 cm−1 is attributed to the stretching frequency of phenolic O–H from extract, which is used as a reducing and capping ligand. The broad peak at 2914 cm−1 was due to the stretching vibration of methylene from vinyl groups. The demanding peak at 2072 cm−1 was due to the stretching vibration of C≡C from the terminal alkyl group [77]. The intense peak at 1381 was due to the C=O vibrational frequency from phenolic compounds during the production of ZMC NPs. The prominent peaks at 1105, 833, and 614 cm−1 correspond to the stretching vibrations of the Cu–O, Zn–O, and Mg-O bonds [64, 77, 78]. The broad peak at 588 cm−1 could be assigned due to the stretching of C–O–C. In general, FT-IR spectra of synthesized materials revealed that NPs are coated with active phytoconstituents, particularly with the O–H, C=O, and C–N residues of phenolic compounds and alkaloids. O–H, C=O, and C-N residues can bond with metal by coating their surface and minimizing agglomeration, which are crucial for stabilization [75].

3.4 XRD Study

The X-ray diffractometer (XRD) pattern of the bio-fabricated ZMC NPs is illustrated in Fig. 4. The prominent peaks at 27.75°, 31.73°, 34.36°, 36.12°, 38.74°, 42.98°, 47.58°, 57.10°, 61.82°, 73.06° and 78.54° associated with the Miller indices (hkl) values of (110), (100), (002), (002), (111), (200), (102), (110), (220), (311), and (222) respectively. The typical diffraction peaks at (100), (002), (102), and (110) planes with 2θ values 31.73°, 34.36°, 47.58°, and 57.10° are associated with the crystalline structure of zinc oxide NPs with the (ICSD card No. 00–036-1451, Zincite-P63mc) [79]. The characteristic diffraction peaks at (002), (111), (220), and (311) planes with peak positions of 36.12°, 38.74°, 61.82°, and 73.06° are associated with the CuO NPs with the (ICSD card No. No.00–048-1548, Tenorite-C2/c) [80]. The prominent diffraction peaks at (110), (200), and (222) planes with diffraction positions of 18.57°, 42.98°, and 78.54° are also associated with the MgO NPs with the (ICSD card No. 00–045-0946, Periclase-Fm-3 m) [81]. These XRD spectra results revealed the successful synthesis of highly crystalline ZMC NPs. The sharper to broader diffraction peaks are observed, and the displayed (101), (100), (002), (002), (111), (200), (102), (110), (220), (311), and (222) planes are indicating the formation of the hexagonal structures lattice with monoclinic symmetry [82].

Fig. 4
figure 4

XRD pattern of ZMC NPs

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The mean crystallite size of the biosynthesized ZMC NPs was computed from the full width at half-maximum (FWHM) of the diffraction peaks at the (101), (100), (002), (200), and (311) planes. The Scherrer’s equation was applied to approximate the crystallite size of the synthesized LDHs from the significant diffraction peaks utilizing the equation. Using the Scherrer’s Eq. (2), the average crystalline size was calculated to be 14.67 nm.

$$\mathrm{D}=\frac{0.9\uplambda }{\mathrm{\beta cos\theta }};$$
(2)

where, ‘D’ is the average crystal size, ‘λ’ is the wavelength of the X-ray radiation (Cu Kα = 0.15418 nm), ‘0.9’ stands the Scherrer’s constant ‘K’, and ‘β’ is FWHM.

3.5 SEM–EDX Analysis

The surface topology, structure, and composition of the bio-fabricated ZMC NPs were revealed using SEM–EDX data. The SEM micrographs of ZMC NPs at different magnifications were presented in Fig. 5a, b. The micrographs indicate that the bio-prepared ZMC NPs were found to be flower-like aggregates with spherical morphology. Furthermore, the particles are dispersed across the surface more uniformly with a low level of agglomeration/clustered forms, and that may be allied with the presence of phytochemicals from the extract of Artemisia abyssinica.

Fig. 5
figure 5

SEM images with a low, b high resolution and c EDX spectrum of ZMC NPs

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Figure 5c demonstrates the EDX spectrum of bio-prepared ZMC NPs. The result indicates the presence of zinc, copper, magnesium, and oxygen at high percentages and carbon and nitrogen at minimum extents. The weight percentages (%) of the zinc, copper, magnesium, and oxygen were 22.46, 20.56, 13.87, and 26.50%, respectively. The compositions of these elements clearly indicate the successful synthesis of pure ZMC NPs. However, 13.30 and 3.31% of carbon and nitrogen imply some impurity level due to the consisting active biomolecules binding with the surface of ZMC NPs. The intense peaks in (Fig. 6c) around 0.5, 1.2, 1.4, 8.2, and 8.7 keV energies also confirm the crystalline nature of bio-prepared ZMC NPs.

Fig. 6
figure 6

TEM images of ZMC NPs a at 50 nm, b Histogram of particle size distribution, c SAED pattern, d HRTEM at 2 nm, e profile of IFFT of CuO, f profile of IFFT of ZnO, and g profile of IFFT of MgO

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3.6 TEM-HRTEM-SAED Analysis

The morphology, particle size, and crystallinity of bio-fabricated ZMC NPs were further studied by TEM-HRTEM-SAED images (Fig. 6a–f). The bio-prepared ZMC NPs have well-defined spherical morphology with sizes ranging from 4 to 26 nm and an average particle size of 15.13 nm as presented in Fig. 6a, b. The presence of small particles with the size of 4 nm demonstrates the effectiveness of biomolecules from the extract of Artemisia abyssinica as capping and stabilizing agents. The nine major circular concentric circles on the SAED pattern (Fig. 6c) correspond to (110), (100), (002), (002), (111), (200), (102), (110), (220) and (311) crystal planes of ZMC NPs. Colored concentric circles also correlated the nine most noticeable patterns found in the XRD measurements in (Fig. 4). The average inter-planer spacing (IPS) values of 0.276, 0.261, and 0.211 nm (Fig. 6d), which was computed from profile IFFT in (Fig. 6e–g) well associated with the (002), (100) and (110) planes of CuO, ZnO, and MgO NPs respectively. The stacking faults on the HRTEM (IFFT) image and the presence of bright circular spots in the SAED pattern confirms the preparations of polycrystalline ZMC NPs [83].

3.7 Biological Efficacy

The in-vitro cytotoxicity and antibacterial efficacy of bio-prepared ZMC NPs was scrutinized against MCF-7 cell lines and pathogenic bacterial strains, respectively. The in-silico molecular docking efficiency of biosynthesized ZMC NPs also estimated against amino acids of estrogen receptor (ERα), S. aureus, and E. coli, respectively.

3.7.1 Cytotoxicity Test

The in-vitro cytotoxicity of bio-prepared trimetallic ZMC NPs and doxorubicin (the standard drug) have been assessed against PBMCs and MCF-7 cell lines. Evaluating the cytotoxicity on normal human cell lines was thought to be the initial step in determining the safety of the fabricated NPs [84]. The cytotoxicity results of the bio-prepared ZMC NPs and doxorubicin against MCF-7 and PBMC are presented in Table 2 and Figs. 7 and 8. The obtained results corroborated that the % inhibition of ZMC NPs against PBMC cell lines was 55.36 ± 0.11. Doxorubicin has shown a % inhibition value of 61.86 ± 0.02 against PBMC cell lines. ZMC NPs have shown remarkably lower cytotoxicity against PBMC cell lines than the standard drug. The IC50 of ZMC NPs and Doxorubicin were 227.69, and 175.56 mg/mL, respectively. The obtained high value of IC50, which is ≥ 90 μg/mL, confirmed that ZMC NPs are non-cytotoxic to the PBMC cell lines [85]. This may be due to the synthesis of NPs from biological precursors and more physiologically functioning (magnesium, zinc, and copper) metal ions, making them biocompatible and less cytotoxic to normal cells.

Table 2 Inhibition percent (%) of ZMC NPs and standard drug against PBMC, and MCF7 Cell lines on MTT assay
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The bio-prepared ZMC NPs and doxorubicin were also evaluated against MCF-7 cell lines. The results in Table 2 and Figs. 7 and 8 have shown a decline in cell viability in a concentration-dependent manner. The highest inhibition percent of ZMC NPs and doxorubicin were 94.37 ± 0.14 and 86.62 ± 0.04 at 250 mg/mL, respectively. ZMC NPs and doxorubicin have also revealed the respective IC50 values of 24.83 mg/mL and 31.19 mg/mL, respectively. The results reveal that the bio-fabricated ZMC NPs have enhanced cytotoxicity activity more than the standard drug. The result could be due to the synergism of different metals, improved surface-to-volume ratio, and richer electrochemical characteristics [86]. Consequently, bio-prepared ZMC NPs can be a good candidate for developing and designing therapeutic agents mainly for breast cancer ailments.

Fig. 7
figure 7

Cytotoxicity of ZMC NPs against MCF-7 cell lines: a untreated, b treated with 7.81 μg/mL, c 62.5 μg/mL, and d 250 μg/mL

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Fig. 8
figure 8

Cytotoxicity of ZMC NPs and Doxorubicin against PBMC and MCF-7 cell lines respectively

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3.7.2 Antibacterial Activity

The antibacterial efficacy of bio-prepared ZMC NPs against selected pathogenic bacterial strains is presented in Table 3 and Fig. 9. Bio-prepared ZMC NPs have shown substantial antibacterial efficacy against S. aureus (+), S. pneumoniae (+), P. aeruginosa (−), and E. coli (−). At 200 μg/mL ZMC NPs have shown the highest ZOI (35 ± 0.03) with respective MIC of 5 μg/mL for S. aureus strains which slightly preceded the inhibition zone (31 ± 0.11) of chloramphenicol. Comparably, the lowest zone of inhibition was observed (31 ± 0.11with MIC 10 μg/mL) against E. coli with a similar concentration (200 μg/mL). The antibacterial efficacies of ZMC NPs against all tested strains are higher than the standard drug (chloramphenicol). Moreover, the bio-prepared trimetallic ZMC NPs also revealed enhanced antibacterial efficacy than its monometallic counterpart, reported in our previous work [54]. This could be due to the synergistic effects of three metals as well as the modification of physicochemical features including size, surface area, shape, and charge distribution of trimetallic ZMC NPs [87, 88] Therefore, the as-synthesized ZMC NPs can be designed and developed as a drug for the remedies of multidrug-resistant bacterial infections.

Table 3 The ZOI (mean ± SD, in mm) of ZMC NPs and chloramphenicol against S. pneumoniae, P. aeruginosa, E. coli, and S. aureus
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Fig. 9
figure 9

Antibacterial performance of ZMC NPs against selected bacterial strains

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3.7.3 Molecular Docking Study

One of the most significant aspects in the design and development of drugs is the evaluation of interaction efficiency and the binding affinity of molecules with particular targets. Using AutoDock 4.2.6 (MGL tools 1.5.7) and a molecular docking database, interactions between bio-prepared ZMC NPs and doxorubicin (standard drug) were examined against the estrogen receptor alpha (ERα; PDB: 5GS4) [62, 89]. Estrogen receptor (ERα) is the primary clinical biomarker employed to subtype cancers of the breast. The estrogen receptor α (ERα) plays an essential role in the progression and development of hormonal-dependent type breast cancer [90]. The molecular docking analysis of ZMC NPs in (Table 4 and Fig. 10a, b) has revealed that two active sites of ERα, Arg363, and Lys449 amino acids which took part in hydrogen bonding. Four amino acid residues, namely Ala322, Glu323, Trp360, and Trp393 involved in van der Waals interactions. Glu353, His356, Met357, and Arg394 are the four amino acid residues participating in π-Sigma/π-Alkyl interaction. On the other hand, doxorubicin in (Table 4 and Fig. 10c, d) has revealed a total of eleven interactions: four hydrogen bonding (Glu353, Arg394, Trp393, Glu323), four π-Sigma/π-Alkyl (Met357, Trp360, Ile386, and His356) interaction and three van der Waals (Gly390, Leu387, and Lys449) interactions.

Table 4 Molecular docking scores and the corresponding prominent residual amino acid interactions of ZMC NPs and standard drugs against estrogen receptor alpha, S. aureus and E. coli
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Fig. 10
figure 10

The binding interactions of ZMC NPs a 3D, b 2D and doxorubicin c 3D, d 2D against estrogen receptor alpha (ERα; PDB: 5GS4)

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The binding affinities and inhibition constants of ZMC NPs and doxorubicin were found to be – 9.85 and 1.75, – 7.54 kcal/mol, and 2.99 µM, respectively. The results revealed that both ZMC NPs and the standard drug had shown promising interactions with active sites of amino acid residues of estrogen receptors (ERα). However, the binding affinity of ZMC NPs (– 9.85) kcal/mol) was remarkably higher than the standard drug which is – 7.54 kcal/mol. These demonstrate that the biosynthesized ZMC NPs will have stronger interactions with estrogen receptor alpha (ERα; PDB: 5GS4) than the standard drug. These strong binding with amino acids of estrogen receptors (ERα) is important to prevent the proliferation rates of breast cancer cells [63]. Moreover, the molecular docking interactions are also correlated with the experimental results presented in Table 2, confirming the enhanced cytotoxicity efficacy of ZMC NPs against MCF-7 cell lines. Therefore, bio-prepared ZMC NPs can be promising candidates for breast cancer ailments.

In order to propose the binding mechanism of the bio-prepared nanoparticles and the standard drug (chloramphenicol) with bacterial cells, their interaction efficiency and binding affinity were evaluated against S. aureus dihydrofolate reductase and E. coli DNA gyrase B. Dihydrofolate reductase and DNA gyrase B are the most prominent enzymes for the survival of the respective bacterial species [91]. They are ubiquitously expressed in bacterial species and crucial for cell growth and proliferation. The molecules' interactions with these enzymes are supposed to hinder the DNA replications of this species [92]. Therefore, the good interactions with the amino acid residues of these enzymes could be the antibacterial drug targets of synthesized materials.

The results of molecular docking analysis of ZMC NPs and chloramphenicol (standard drug) against S. aureus have been presented in Table 4, Fig. 11a–d. ZMC NPs has demonstrated binding interaction with amino acids of S. aureus through hydrogen bond (Ser49), π-Sigma/π-Alkyl (Leu28, Val31, Leu54, and Phe92), and van der Waals (Leu20, Ile50, Arg57, and Asp27) interactions with a binding affinity – 12.31 kcal/mol and an inhibition constant (IC) of 9.5 × 10–4 µM. Chloramphenicol has revealed the interactions through hydrogen bond (Trp121, Gln95, and Trp46), and van der Waals (Ile14, Gly93, Gly94, and Ser49) interactions with a respective binding affinity and an IC of – 7.16 kcal/mol and 29.02 µM. respectively. In general, the bioprepared ZMC NPs have strong binding interactions with the prominent bacterial proteins, and that is also associated with the experimental results.

Fig. 11
figure 11

The binding interactions of ZMC NPs a 3D, b 2D, and doxorubicin c 3D, d 2D against S. aureus dihydrofolate reductase (PDB: 2w9h)

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Table 4 and Fig. 12a–d presented the molecular docking assessment of ZMC NPs and chloramphenicol against E. coli DNA gyrase B. ZMC NPs have shown the binding interactions through hydrogen bond (Arg76), π-Sigma/π-Alkyl (Asp49, Ile94, Thr165, and Val167), and van der Waals (Asn46, Glu50, Asp73, and Ile78) interactions with a binding affinity and IC of – 6.04 kcal/mol and an of 37.42 µM, respectively. Chloramphenicol has revealed the interactions through hydrogen bond (Asp73, Asn46, and Glu50), π-Sigma/π-Alkyl (Val43), and van der Waals (Ile78, Arg76, and Trp165) interactions with a binding affinity and an IC of – 6.19 kcal/mol and 5.67 µM, respectively. These results are feasibly complemented with in-vitro activities; hence, the bio-prepared ZMC NPs can be further studied and possibly exploited as antibacterial agents.

Fig. 12
figure 12

The binding interactions of ZMC NPs a 3D, b 2D and doxorubicin c 3D, d 2D against E. coli DNA gyrase B (PDB: 6F86)

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4 Conclusions

This study encompasses a one-pot, cost-effective, and environmentally benign synthesis of novel trimetallic ZMC NPs using the medicinal plant A. abyssinica. The SPR peak of the biosynthesized ZMC NPs at 375 nm UV spectrum affirmed the preparations of iso-morphological and spherical nanoparticles. The FTIR and EDX data ascertained the existence of characteristic phytochemicals from AALE accountable for the fabrication of ZMC NPs. The bio-fabricated ZMC NPs have been analyzed by using XRD, FESEM/EDX, TEM/HRTEM/SAED, and the results have revealed a pure, spherical shape with average crystalline and particle sizes of 14.67 and 15.13 nm, respectively. The biological functionalities of bio-fabricated ZMC NPs has been evaluated and revealed enhanced in-vitro cytotoxicity and antibacterial efficacies. Results revealed that the bio-prepared ZMC NPs exhibited high cytotoxicity (94.37 ± 0.14) against MCF-7 cell lines and low cytotoxicity (52.82 ± 0.11) against normal PBMC cell lines by MTT assay. The antibacterial efficacy of ZMC NPs have evaluated against S. pneumoniae, S. aureus, P. aeruginosa, and E. coli strains and has revealed high zones of inhibition such as 35 ± 0.03, 34 ± 0.04, 33 ± 0.14, and 31 ± 0.11 mm, respectively. Furthermore, ZMC NPS also have shown strong interactions with amino acids of estrogen receptor (ERα), S. aureus, and E. coli with binding energies of – 9.85, – 12.31, and – 6.04 kcal/mol, respectively. Consequently, the biosynthesized ZMC NPs have a remarkable cytotoxicity and antibacterial efficacy and can be designed as a therapeutic agent for these ailments. However, further in-vivo biological efficacy studies against MCF-7 cell lines and the selected bacterial strains are recommended.