Ulcerative skin infections arising from the colonisation and development of Gram-positive bacteria, Gram-negative bacteria, and multidrug-resistant bacteria are significant health-care problems that seriously affect human skin. A prospective quantitative study reported that the prevalence rates of skin pressure ulcers (PUs) are 15.5% in Kuala Lumpur, Malaysia (2013)1, 33% in Palestine (2017)2, and 16% in Bandung, Indonesia (2017)3. Skin infection has been found in 60 (74.0%) of the collected samples from PUs of hospitalised patients, and these PUs primarily comprise Enterobacteriaceae strains (49.0%), such as Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Enterobacter spp., and Proteus spp.; followed by Staphylococcus aureus (S. aureus) (28.0%) and nonfermenting GNB (23.0%), mostly Pseudomonas aeruginosa (P. aeruginosa), Acinetobacter spp., and methicillin-resistant S. aureus (MRSA)4,5,6,7. PUs are open infected wounds that develop on the skin as result of pressure on one spot of the body for too long or from friction on the skin. Some studies have found that new inorganic oxide antimicrobial agents synthesised from natural plants can be remarkable alternatives for infectious skin treatments of PUs because they are rich in numerous varieties of metal oxides that release ions and in reactive oxygen species (ROS), such as hydroxyl radical (·OH) and superoxide (·O2−) which cause increased cell permeability, rupture, and death in microorganisms8,9.

The incorporation of inorganic metal and metal oxides in sponges10, hydrogels11,12, and bandages13,14 has become a research hotspot because of these materials’ advantages as antimicrobial agents for treating locally infected skin ulcers. Mixed inorganic metal and metal oxides are effective disinfectants because of their relatively nontoxicity, chemical stability, and efficient antibacterial activity (Table 1). The use of binary antimicrobial agents (e.g., CuO, ZnO, and Ag–ZnO) has been highlighted over single antimicrobial agents given the stronger synergic effect of the former in eliminating bacterial colonies at low concentrations10,25,39, more pronounced wound-healing ability10, lower cytotoxicity10, better biocompatibility25, and improved cell viability which indicates safe human application 25. The combined use of binary antimicrobial agents could reduce the cytotoxicity but not the antimicrobial effect10,25. Furthermore, several studies have shown that the incorporation of antimicrobial agents such as CuO40, CuSO441, ZnO42, ZnO-SiO243, and Re-ZnO44 into biopolymers can effectively combat Gram-positive and Gram-negative bacteria in a concentration-dependent manner. However, binary ZnO/CuO nanocomposites prepared from Calotropis gigantea (C. gigantea) leaves in the current work were found to exert a strong antimicrobial effect on multi-drug resistant (MDR) pathogens such as P. aeruginosa and MRSA compared with other previously reported antimicrobial binary inorganic oxides nanocomposites (Table 2). It can effectively work against MDR pathogens at a very low minimum bactericidal concentration (MBC) of about 0.3125 mg/mL.

Table 1 Antimicrobial properties of different mixed metal/metal oxides towards various microorganisms.
Table 2 MIC and MBC concentration of binary antimicrobial agent.

Accordingly, the present study focused on the preparation of green synthesised binary ZnO-CuO nanocomposites using C. gigantea leaf extract. The microbial activity of these nanocomposites was investigated by culturing with skin ulcer pathogens such as E. coli, K. pneumoniae, S. aureus, P. aeruginosa, and MRSA. Furthermore, the effects of different compositions on ZnO-CuO nanocomposites were explored with respect to their prospective antimicrobial application.

Materials and methods

Preparation of leaf extract and binary inorganic oxides

Whole C. gigantea plant was collected from Perai Pulau Pinang, Malaysia and identified by an expert from the Unit Herbarium, Pusat Pengajian Sains Kajihayat USM Pulau Pinang (Herbarium No.: 11843). C. gigantea leaves were extracted using deionised water and boiled using hot plate47,48. Then, the filtered leaf extracts were taken and boiled with a stirrer–heater. Binary ZnO–CuO nanocomposites were prepared by adding copper (II) nitrate trihydrate and zinc nitrate hexahydrate into the extract solutions simultaneously and then boiled until they were reduced to pastes. These pastes were calcined in an air-heated furnace47,48. Notably, the mixing composition of copper (II) nitrate trihydrate and zinc nitrate hexahydrate was varied with constant rotation speed and calcination temperatures (Table 3). The samples prepared at weight percentages of 25 wt%, 50 wt%, and 75 wt% of zinc nitrate hexahydrate were denoted as B1Z3C, B1Z1C, and B3Z1C, respectively. Commercial B3Z1C sample was prepared by mixing ZnO (< 100 nm; Aldrich) and CuO (< 10 µm; Sigma–Aldrich) with an agate mortar (Table 3).

Table 3 Composition of binary ZnO-CuO nanocomposites samples.

Physicochemical characterisation

The crystal phases of BZC nanocomposites were studied by X-ray diffraction (XRD; Bruker D8 powder diffractometer) operated in reflection mode with a Cu Kα radiation (40 kV, 30 mA) diffracted beam monochromator. The step scan mode with a step size of 0.030° within the range of 10° to 90° was used. Scanning electron microscopy (SEM; Fei Quanta FEG 650) was used for morphology and microstructure observations of BZC nanocomposites. The purity of BZC was identified by energy-dispersive X-ray (EDAX) spectroscopy which was equipped with SEM. Detailed morphology of B3Z1C nanocomposites was further confirmed by transmission electron microscopy (TEM; FEI TECHNAI F20 G2).The characteristic optical properties of BZC nanocomposites were studied using a UV–Vis spectrophotometer (Varian) at room temperature within the range of 200–900 nm. FTIR spectroscopy (Perkin Elmer) was recorded within the range of 4000–400 cm−1 through the KBr pellet method to observe the functional groups involved in the natural-plant green synthesis and stabilization of B3Z1C nanocomposites.

Minimum inhibitory concentration (MIC)/MBC determination and tolerance level

Antibacterial activity of BZC nanocomposites against S. aureus 29213, E. coli 25922, P. aeruginosa 27853, K. pneumoniae 700603, and MRSA 38591 were assessed using broth-dilution method on 96-well plates as described by Harun et al46. Absorbance was read at 980 nm wavelength46. High wavelength was selected because of BZC nanoparticle deposition. The bactericidal and bacteriostatic capacity of the samples was determined by the tolerance level46.

Time-kill assay

The antibacterial activity of BZC nanocomposites against time was performed using time-kill assay as illustrated in a previous protocol46. S. aureus bacterial suspension adjusted to 0.5 McFarland standard turbidity was used and diluted with sample solution to a final concentration of 2.5 mg/mL.

Kirby–Bauer disc-diffusion test

The antibacterial activity of BZC nanocomposites against S. aureus was further evaluated using Kirby–Bauer disc-diffusion test49. BZC nanocomposite solutions (2.5 and 10 mg/mL) were prepared and used further for antibacterial studies. About 20 µL of BZC nanocomposite solution, negative control (10% DMSO + distilled water), and C. gigantea leaf extract were loaded into 6 mm sterile filter papers, and the solution was allowed to be diffused within 15–30 min. Then, all discs were properly placed on agar which was already previously spread with bacterial culture. A standard antibiotic comprising 10 µg of Oxoid streptomycin antimicrobial susceptibility discs served as a positive control. After 24 h of incubation at 37 °C, the different levels of zone of inhibition were measured.

Results and discussion

Surface morphology of binary ZnO–CuO nanocomposites

The SEM images of BZC nanocomposites are shown in Fig. 1. B1Z1C had a porous nature (Fig. 1c) with few irregular rod-shaped particles (inset in Fig. 1c). Meanwhile, B1Z3C (Fig. 1a) and B3Z1C (Fig. 1e) had porous honeycomb structures with agglomerated morphology (inset in Fig. 1a,e). The large porous honeycomb structures further increased the available surface area for antimicrobial activity26. These uniform pores were produced during green synthesis owing to the escape of gases at high temperatures26. The EDAX profile of the green synthesised B3Z1C nanocomposites confirmed the presence of Zn, Cu, and O, which were about 49.97 wt%, 20.34 wt%, and 21.32 wt%, respectively. Some weak signals for C, Mg, S, Cl, K, Na, and Ca atoms were found for all BZC nanocomposites (Fig. 1b,d,f). Similar results have been reported for green nanoparticles derived from Artemisia haussknechtii leaf extract50, aqueous Artemisia haussknechtii flower extract9, Protoparmeliopsis muralis lichen51, Ochradenus baccatus leaves52, and Jatropha curcas L. leaf53. The presence of elements such as C, Mg, S, Cl, K, Na, and Ca in small amounts indicated the participation of plant phytochemical groups in reducing and capping the green synthesised BZC nanocomposites9,50,51,52,53. Meanwhile, the TEM image of B3Z1C nanocomposites revealed irregular oval and quasi-spherical shape with an average length of 8.126 nm and diameter of 7.515 nm in size (Fig. 1g). These structures could increase the available surface area for reaction. The magnified TEM image of the B3Z1C nanocomposites along with the lattice fringes with an interfringe distance of 0.248 and 0.254 nm belonged to ZnO and CuO, respectively (Fig. 1h).

Figure 1
figure 1figure 1

Morphology of BZC nanocomposites; (a) SEM image of B1Z3C (10.00 µm), (b) EDAX of B1Z3C, (c) SEM image of B1Z1C (10.00 µm), (d) EDAX of B1Z1C, (e) SEM image of B3Z1C (10.00 µm), (f) EDAX of B3Z1C, (g) TEM image of B3Z1C (10 nm) and (h) Magnified TEM image of B3Z1C nanocomposites along with lattice fringes (2 nm).

Crystal analysis of binary ZnO–CuO nanocomposites

Prominent diffractive peaks on the differential ratio of binary ZnO–CuO nanocomposites were indexed by comparing the green ZnO and CuO diffraction angle 2θ with ICDD ZnO 01-089-0510 and ICDD CuO 01-089-5897, as presented in Fig. 2. Green CuO was observed to have 12 characteristic peaks at 32.32°, 35.50°, 38.71°, 45.01°, 48.37°, 53.29°, 58.15°, 61.09°, 65.56°, 67.90°, 72.16°, and 75.13°, which corresponded to the crystal surfaces (110), (− 111), (111), (202), (− 202), (020), (202), (− 113), (− 311), (220), (311), and (004), respectively. It had the following lattice parameters: a = 4.686486, b = 3.421156, c = 5.129263, α = 90°, β = 99.413°, γ = 90°, and d-spacing of 2.52761 Å with a monoclinic crystalline structure. Green ZnO was observed to have 12 characteristic peaks at 31.87°, 34.57°, 36.37°, 47.62°, 56.68°, 62.92°, 66.43°, 68.02°, 72.28°, 76.87°, 81.04°, and 89.44°, which corresponded to the crystal surfaces (100), (002), (101), (102), (110), (103), (200), (201), (004), (202), (104) and (203), respectively. It had the following lattice parameters: a = 3.252352, b = 3.252352, c = 5.209155, α = 90°, β = 90°, γ = 120°, and d-spacing of 2.47193 Å with a hexagonal wurtzite crystalline structure.

Figure 2
figure 2

XRD diffraction peaks of BZC nanocomposites prepared at different composition. (a) C. gigantea leaves powder, (b) Green ZnO, (c) Green CuO, (d) B1Z3C, (e) B1Z1C and (f) B3Z1C [open circle: C. gigantea leaves, filled balck circle: ZnO, filled black rhombus: CuO, open red rhombus: additional peaks after green synthesis].

Meanwhile, six characteristic peaks of ZnO for sample B3Z1C were identified at 31.72°, 34.45°, 36.25°, 47.35°, 56.41°, and 62.71° and deemed to correspond to the (100), (002), (101), (102), (110), and (103) crystal surfaces, respectively. Two other characteristic peaks of CuO at 38.62° and 67.78° were found and deemed to correspond to the (111) and (220) crystal surfaces, respectively. For sample B1Z3C, the peaks at 31.72°, 34.45°, 36.25°, 47.35°, 56.41°, 62.71°, and 68.05° belonged to the (100), (002), (101), (102), (110), (103), and (201) indices of ZnO nanoparticles, respectively. The diffractive peaks of CuO detected at 35.68°, 38.62°, 58.33°, 61.27°, and 65.80° corresponded to the (–111), (111), (202), (–113), and (–311) crystal surfaces, respectively. All 2θ values of ZnO and CuO for BZC nanocomposites slightly shifted, indicating that some modifications of ZnO with CuO occurred and a strong crosslinking framework structure of Zn–O–Cu atoms formed. Moreover, the binary mixing of CuO and ZnO resulted in decreased crystallinity of BZC nanocomposites. The peak intensity drastically increased with increased amount of ZnO or CuO in the BZC nanocomposites (Fig. 2), thereby indicating the variation in composition (25 wt%, 50 wt%, and 75 wt% of ZnO) during green synthesis. A few additional peaks were observed at 23.65°, 25.69°, 27.73°, 29.47°, and 40.78° (Fig. 2). This finding was possibly due to the presence of the phytochemical element of C. gigantea leaves as a capping and reducing agent47. The XRD patterns of powdered C. gigantea leaves successfully revealed trace natural elements such as calcium and carbon (Fig. 2). C. gigantea natural plant is rich in calcium and carbon elements. Calcium was observed to have six characteristic peaks at 28.80°, 50.47°, 58.89°, 66.70°, 67.70°, and 73.92°. The additional peaks detected at 31.53° and 40.94° were attributed to the natural graphene-like carbon present in the BZC nanocomposites54 as carbon is the main phytochemical element in the leaves of the C. gigantea medicinal plant55.

The main novelty of this study was the detection of pythochemical elements such natural calcium56 and carbon54,57 in leaf extract, which could further boost the antimicrobial activity of BZC nanocomposites. Calcium and carbon elements have never been reported before in the studies of Sharma et al., Gawade et al., and C R Rajith Kumar et al. performed on the same C. gigantea medicinal plant24,47,48.

FT-IR analysis of binary ZnO–CuO nanocomposites

The FTIR spectra of B3Z1C nanocomposites and C. gigantea leaves are shown in Fig. 3. The presence of capping and stabilization agents such as flavonoids, polyphenolics, and terpenoids can be confirmed from this analysis. The weak absorption band at 447 cm–1 was characteristic of the ZnO functional group58,59. However, the CuO functional group was not visible owing to its low composition in the B3Z1C nanocomposite binary system. The spectra further showed a very intense band at 3438 cm−1 associated with the O–H stretching polyphenols (flavonoids) present in the plant extract. The characteristic peaks at 1633 and 1765 cm−1 can be attributed to C=C (carbonyl group) and C=O stretching, respectively. The absorption band between 1110 and 1115 cm−1 could be attributed to C–O stretching owing to the biomolecules of C. gigantea leaves. The broad absorption band at 1385 cm−1 was observed owing to the O–C–O stretching modes of vibration of esters. The absorption band observed at 680 cm−1 belonged to primary amines, indicating proteins. Therefore, the presence of phenolic and carbonyl compounds of C. gigantea leaves played vital roles in the stabilisation of green B3Z1C nanocomposite formation and antimicrobial activity15.

Figure 3
figure 3

FTIR spectra (a) C. gigantea leaves and (b) B3Z1C nanocomposites.

UV–Vis spectroscopy analysis of binary ZnO–CuO nanocomposites

The UV–Vis diffuse reflectance spectra of C. gigantea extract and B3Z1C nanocomposites are shown in Fig. 4. The appearance of a small broad peak at approximately 317 nm indicated the formation of irregular oval and quasi-spherical B3Z1C nanocomposites. Absorption peaks at 206 nm could be attributed to various chromophores, including the C=C bond of various compounds, the C=O bond of carbonyl compounds, and the benzene ring, whereas the absorption peak at 269 nm may be related to the various aromatic compounds, such as phenolics60. A sharp distinct peak was found at 233 nm owing to the formation of natural graphene-like carbon which played an important role in antimicrobial efficacy against MDR strains61.

Figure 4
figure 4

UV–Vis diffuse reflectance spectra (a) C. gigantea leaves and (b) B3Z1C nanocomposites.

Antimicrobial properties of binary ZnO–CuO nanocomposites

About 37% of patients with skin-ulcer disease are infected with Gram-positive S. aureus pathogen62. The antimicrobial characterisation BZC nanocomposites with different ratios is presented in Fig. S1 and Table 4. The MICs of B1Z3C, B1Z1C, and B3Z1C were 5, 2.5, and 0.625 mg/mL for S. aureus, respectively. Similar to the MIC values, B1Z3C and B1Z1C had MBCs of 20 mg/mL, and the counterpart for B3Z1C was 2.5 mg/mL for S. aureus. B3Z1C exerted a higher bactericidal effect against the S. aureus strain at the lowest MIC/MBC values (0.625 mg/mL/2.5 mg/mL). Antimicrobial activity was further enhanced by increasing the amount of ZnO nanoparticles in the binary compound (ZnO-CuO). This finding can be explained by the fact that the binary B3Z1C nanocomposites were highly diffusible and able to generate more Zn2+ ions19. Moreover, Cu2+ ions bound the cell wall of host cells through surface proteins and entered the cell19. Subsequently, the change in cell metabolism led to the microbe’s cell death19. Commercial B3Z1C was also prepared and tested against S. aureus for comparison. Results showed that commercial B3Z1C was a bacteriostatic agent because the MBC/MIC ratio was ≥ 1646 (Table 4 and Fig. S1). However, the green B3Z1C was labelled as a strong bactericidal agent because the tolerance ratio was ≤ 4.

Table 4 MIC and MBC of BZC nanocomposites against S. aureus.

Further antimicrobial analysis of B3Z1C nanocomposites was conducted on selected skin-ulcer pathogens, and results are shown in Table 5. These pathogens are commonly associated with skin-ulcer disease4,5,6,7. Also, the inhibitory activities of binary antimicrobial agents on bacterial colonies highly depend on the antimicrobial efficacy of dual-ionic systems and types of microbial pathogens, such as non-MDR Gram-positive bacteria (S. aureus), Gram-negative bacteria (E. coli) and MDR bacteria (P. aeruginosa, K. pneumoniae, and MRSA). The MIC amounts for B3Z1C were 0.625, 0.15625, 0.625, and 0.15625 mg/mL for E. coli, P. aeruginosa, K. pneumoniae, and MRSA, respectively. MBC values with 2.5, 0.3125, 1.25, and 0.3125 mg/mL were also observed for this green binary inorganic oxide sample. Table 5 indicates that for all tested microbes, the tolerance levels for B3Z1C were less than 4, indicating that the sample was a strong bactericidal agent. Binary B3Z1C has strong antimicrobial activity against Gram-negative bacteria (E. coli). Table 5 is the evidence for this finding. Clearly, B3Z1C showed very promising results against all tested MDR microbes such as P. aeruginosa, K. pneumoniae, and MRSA. This outcome may be due to the B3Z1C nanoparticles’ larger surface-to-volume ratio and the cell-membrane penetration of the bacteria by its ions. Some studies have reported that the antimicrobial effectiveness of green synthesised inorganic oxide nanoparticles depends on high particle dosage and small nanoparticle size, which could explain the higher antimicrobial activities of B3Z1C. The antimicrobial activity of B3Z1C was due to the electrostatic interaction between positively charged zinc and copper ions (Zn2+ and Cu2+) and negatively charged microbial cell membranes21. The antimicrobial activity of B3Z1C nanocomposites relied on the generation of ROS as well17,19. Moreover, free ions from natural organic carbon and calcium derived from C. gigantea leaf extract played an important role in exerting the synergic effect that killed MDR microbes at very low concentrations54,56.

Table 5 MIC and MBC of B3Z1C nanocomposites against different microbes.

Results of time-kill assay were presented in terms of the changes in log10 CFU/mL of viable S. aureus colonies, as shown in Fig. S2. The green synthesised B3Z1C nanocomposites were found to have significant bactericidal activity. Figure 5 presents the time-kill curve graph for the strain. Generally, bacterial growth includes a log or exponential phase in which bacterial-cell doubling occur and their biomass increases from day 1 to day 263,64. A reduction in viable count from 4.3 log10 to 3.4 log10 was observed after 6 h of incubation for S. aureus. By 12 h, only 1.3 log10 of bacterial colonies were found. At 24 h, the bacteria were completely killed. Thus, Gram-positive S. aureus bacteria were effectively controlled by the synergistic combination of 75 wt% of ZnO and 25 wt% of CuO nanoparticles in the presence of natural graphene-like carbon, calcium, and phytochemical constituents such as cardiac glycosides, tannins, saponins, terpenes, flavonoids, and phenolics in C. gigantea leaf extract54,56,65,66,67,68.

Figure 5
figure 5

Time-kill curves against S. aureus strains using 2.5 mg/mL of green B3Z1C sample for 0.5 h (30 min), 3 h, 6 h, 12 h and 24 h treatment periods. These data represent mean (± SD) of three replicates.

Furthermore, Kirby–Bauer disc-diffusion method was used to evaluate the antimicrobial activity of BZC nanocomposites against Gram-positive S. aureus. The cultures exposed to negative control sample did not show any inhibition zones around the filters, indicating that they did not have any antibacterial properties. However, B3Z1C exhibited a wider zone of inhibition (ZOI) than other BZC samples possibly because of the nanoparticle size and the fast diffusion of metal ions into agar medium (Fig. S3 and Table 6). The antimicrobial activity of all green BZC samples further improved with increased concentration. C. gigantea extract also exhibited a slight ZOI toward S. aureus which could be attributed to bioactive compounds such as carbonyl and phenolic groups. The antibiotic streptomycin serving as a positive control exhibited a larger ZOI, as shown in Fig. S3 and Table 6.

Table 6 Kirby–Bauer disc diffusion ZOI (mm) of BZC nanocomposites against S. aureus.


Binary B3Z1C nanocomposites prepared at compositions of 75 wt% of ZnO and 25 wt% CuO demonstrated significant antimicrobial property against non-MDR and MDR pathogens with tolerance ratio of ≤ 4 and ≤ 2, respectively. Besides, promising antimicrobial effect of B3Z1C sample towards non-MDR bacteria (S. aureus) were seen from disc diffusion assay and time kill analysis. The mechanisms underlying the biocidal activity of B3Z1C nanocomposites may involve the presence of natural carbon, free ions (i.e., Cu2+, Zn2+ and Ca2+), and ROS. Further In vitro and In vivo toxicity studies are needed to understand B3Z1C efficiency in treating PU infections.