Introduction

Skin pathogens usually associated with open wound ulcers, such as surgical infections and wounds, diabetic infections and foot ulcers, pressure ulcers, arterial ulcers, chronic ulcers, skin disorders and infections, traumatic wounds and venous ulcers, are the most common wound injuries that lead to human morbidity and mortality, including amputation and death (Nussbaum et al. 2018). Medicare cost estimates for the treatment of all wound types range from $28.1 billion to $96.8 billion, and the highest expenses are for surgical wounds, followed by diabetic foot ulcers (Nussbaum et al. 2018). Wounds infected by multidrug-resistant (MDR) and non-MDR skin pathogens are hardly treatable (Uckay et al. 2015; September et al. 2019; Wang et al. 2019). Broad-spectrum antibiotics, such as vancomycin, oxacillin, penicillin, cefoxitin and chloramphenicol, are ineffective to control the growth of MDR skin pathogens and are not preferred in colonized open wound ulcers (Hamzah et al. 2019). Besides, non-MDR Gram-positive Staphylococcus aureus is also one of the most common skin pathogens responsible for infectious wound ulcers (Wong et al. 2015). In past researchers found that, among 77 wound swab samples studied at Local Hospital, Malaysia, 82 isolates consist of Gram-negative (71.1%) and Gram-positive bacteria (27.7%) (Nur Hilda Hanina et al. 2015). Moreover, a recent study showed that roughly 23,000 deaths a year in the USA and more than 33,000 deaths in Europe are due to antibiotics failure in treating MDR strain-infected ulcers (Pacios et al. 2020).

Increasing antibiotic resistance has stimulated research on green-synthesized copper oxide (CuO) nanoparticles with different morphologies as a bactericidal agent to overcome open wound ulcers (Table 1). In this regard, many papers have reported the usage of different types of natural plant extract to synthesize desired properties of green-synthesized nanoparticles with excellent bactericidal properties (Akintelu et al. 2020; Thakur et al. 2019; Lediga et al. 2018; Mtambo et al. 2019). The rise of green-synthesized CuO in biomedical field can provide genuine support in facilitating the healing process of locally colonized open wound infections and repairing injured tissue. Currently, highly moisturized bactericidal dressings, including those that contain metals or metal oxide nanoparticles, are used locally to manage skin ulcer infection while accelerating wound healing (Janowska et al. 2019; Han and Ceilley, 2017; Djavid et al. 2020; Frykberg and Banks, 2015; Bogoslovskaya et al. 2022). These dressings help in accelerating the wound recovery process through the release of metal ions and the generation of reactive oxygen species (ROS) (Jadhav et al. 2017). In addition, calcination temperature predominantly affects the physicochemical (i.e., size and shape) as well as their bactericidal properties of metal and metal oxide nanoparticles (El Desouky et al. 2020; Yu et al. 2003; Saravanan and Sivasankar, 2016; Jiao et al. 2018; Azam et al. 2012).

Table 1 Physicochemical, bactericidal and cytocompatibility properties of green-synthesized copper oxide (CuO) nanoparticles
Full size table

Generally, low-temperature annealed green-synthesized nanoparticles were expected to have remarkable medicinal properties and exhibit steady and low ROS release which is safe and non-toxic against cell line (Umar et al. 2015; Azizi et al. 2016). Wider zone of inhibition against several skin pathogens and improved antioxidant activity was also captured for low-temperature annealed CuO nanoparticles than high-temperature calcined CuO nanoparticles (Hamid et al. 2021). Besides of that, the degree of crystallinity and clusters or boundaries of CuO grains size were further increased at extremely high annealing temperature between 600 and 900 °C (Luna et al. 2015; George et al. 2020). Larger grain size and crystallite size at high calcination temperature predominantly affect and weaken the antibacterial properties (Hamid et al. 2021). A similar phenomenon was also reported in previous research, where CuO nanoparticles crystallization was incomplete at very low temperature of 200 °C (Luna et al. 2015). As referred to Table 1, calcination temperature between 400 and 500 °C was most preferred in green synthesis of CuO nanoparticles. Furthermore, formation of pure Cu/Cu2O/CuO nanoparticles was seen during slight changes on full width at half maximum (FWHM) peak intensity of XRD at calcination temperature between 400 and 500 °C (Fuku et al. 2020).

Therefore, the aims of this work were to synthesize green CuO nanoparticles with cytocompatibility properties using the aqueous extract of the giant milkweed medicinal plant and the effect of low calcinations temperature (400 and 500 °C) on the physicochemical and bactericidal properties of CuO was determined. The milky and evergreen flowering plant of giant milkweed is a genus from family of Asclepiadaceae, and the traditional use of this plant in treating open wound ulcers is well documented (Sharma et al. 2015). It is also known as “Erukku” and quite popular among Konar community of Tamil Nadu (Jayakumar et al. 2018). Additionally, the bioderived CuO was evaluated for bactericidal activities by employing skin-related pathogens using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill assay and Kirby-Bauer disc diffusion technique. At final stage, cytocompatibility assay was performed against green CuO on fibroblast cells lines model according to ISO 10993–5 (2009) guidelines.

Materials and methods

Materials and chemicals

Copper (II) nitrate trihydrate (99.5%) was purchased from Merck (Darmstadt, Germany). Dimethyl sulfoxide (DMSO) obtained from Sigma-Aldrich was used as diluents for samples preparation. Commercial CuO (< 10 µm) was used as commercial control purchased from Sigma-Aldrich. Luria–Bertani (LB) nutrient medium was purchased from Sigma-Aldrich (Darmstadt, Germany) and was used to culture non-MDR and MDR skin pathogens. Oxoid™ cefoxitin bactericidal susceptibility disk with concentration of 30 µg was introduced as an antibiotic positive control. The fibroblast cells lines model, L929 used in this work, was purchased from American Type Culture Collection (ATCC, USA). Fibroblast was maintained in Roswell Park Memorial Institute (RPMI) 1640 media (Gibco, Life technologies), and the media were supplemented with other nutrients. The alamarBlue™ cell viability reagent DAL1025 (Invitrogen, UK) was used for cell viability assay. 2,2-Diphenyl-1-picrylhydrazyl was purchased from Sigma-Aldrich for DPHH radical scavenging assay. The non-MDR and MDR pathogens of S. aureus 29213, E. coli 25922, K. pneumoniae 700603 and MRSA 38591 were obtained from American Type Culture Collection (ATCC).

Preparation of green-synthesized CuO nanoparticles

Copper (II) nitrate trihydrate was used as main precursor for green synthesis of CuO nanoparticles using giant milkweed plant. The fresh leaves of giant milkweed plant were collected from Perai Pulau Pinang, Malaysia. The species was authenticated by the expert of Unit Herbarium, Pusat Pengajian Sains Kajihayat USM Pulau Pinang (Herbarium No.: 11843). Green CuO nanoparticles were synthesized according to the following procedures (Sharma et al. 2015; Govindasamy et al. 2021a). Copper (II) nitrate trihydrate was dissolved in filtered giant milkweed leaves extracts at boiling temperature of 60–80 °C. The CuO was formed as green paste after the chemical reaction between leaves extracts and copper (II) nitrate trihydrate. The green paste (Fig. 1a) calcined at 400 and 500 °C was called as CuO-400C and CuO-500C, respectively. The final annealed powder gives the appearance of black and very stable monoclinic phase of CuO (Fig. 1b) (Zayyoun et al. 2016). It is proved that green synthesis at calcination temperature between 400 and 500 °C only produces chemically stable CuO without presence of orange Cu2O powder (Zayyoun et al. 2016).

Fig. 1
figure 1

Green synthesis of CuO nanoparticles; a Green paste before annealing and b final annealed black CuO powder

Full size image

Characterization of green-synthesized CuO nanoparticles

X-ray diffraction (XRD) patterns of CuO and powdered giant milkweed leaves were recorded with X-ray diffractometer (Bruker D8), and average crystallite sizes of CuO were calculated using Debye–Scherrer equation [1]:

$$d = \frac{K\lambda }{{\beta \cos \theta \prime }}$$
(1)

where λ is the X-ray wavelength of Cu Kα radiation (0.1541 nm), K = 0.9 is the shape factor, β is the FWHM of the respective diffraction peak, and θ is the Bragg diffraction angle. Scanning electron microscopy (SEM Fei Quanta FEG 650) and transmission electron microscopy (TEM FEI TECHNAI F20 G2) were used for the morphology and microstructure observation of the green CuO, respectively. Semi-quantitative analysis of nanoparticles was carried out by energy-dispersive X-ray spectroscopy (EDAX) which is equipped with SEM machine. The primary detection of CuO and natural compound of leaves extract such as carbonyl and phenol group were investigated via UV–Vis spectrophotometer (Varian) in the range of 200–700 nm. The CuO was prepared in a suspension form, by dispersing the powder in autoclaved deionized water for UV–Vis analysis. The functional groups involved in green synthesis and stabilization of CuO were examined using FTIR spectroscopy (Perkin Elmer). The FTIR spectra of the green samples were recorded in the range of 4000–400 cm−1 by the KBr pellet method.

Minimum inhibitory concentration/minimum bactericidal concentration

The MIC/MBC of CuO was determined by the broth dilution technique using 96-well plate as previous protocol (Govindasamy et al. 2021b). The absorbance reading for CuO was determined at appropriate wavelengths before and after 24-h incubation.

Time-kill assay

The bactericidal performance of 20 mg/mL of green CuO-500C sample against time was evaluated toward S. aureus, E. coli and MRSA according to the following time-kill assay method (Govindasamy et al. 2021b).

Kirby-Bauer disc diffusion assay

Bactericidal activity of green CuO and commercial CuO was evaluated against non-MDR and MDR pathogens using disk-diffusion susceptibility test (El-Kased et al. 2017). Subsequently, 2.5 mg/mL and 10 mg/mL sample’s solution was prepared for bactericidal studies.

Cytocompatibility assay ISO 10993–5 (2009)

The fibroblast cells lines model, L929, was cultured in RPMI 1640 media supplemented with 10% (v/v) fetal bovine serum (FBS), 12.5 g/mL HEPES, 1% (v/v) L-glutamine, sodium bicarbonate and 1% (v/v) PenStrep (Gibco, USA). Cells were maintained in T25 flasks and incubated at 37 °C in 5% CO2 humidified atmosphere and sub-cultured at 80–90% confluency prior experiments. 10% (v/v) of dimethyl sulfoxide (DMSO) was introduced as a negative control (strong cytotoxic material). Blank control (fibroblast cells lines model in culture medium without CuO) was set as 100% viability in the experiment.

Fibroblast cells lines model was used to measure the cytocompatibility of green CuO by calculating cell viability using the alamarBlue™ cell viability reagent DAL1025 (Invitrogen, UK) according to ISO 10993–5 (2009). The cytocompatibility assay was performed following the protocol recommendation in previous study (Harun et al. 2021). Briefly, fibroblast cells lines model was seeded at a density of 1 × 104 cells/well (100 µl/well) in 96-well plate and grown for 24 h in CO2 incubator at 37 °C. Green CuO was sterilized via autoclave machine at 121 °C prior to use. Then, different-concentrated green CuO (0, 2.5, 5, 10 and 20 mg/mL) was prepared in the culture media and suspension of green CuO nanoparticles was produced. Samples were kept for overnight for facilitating solubility and diffusion of bactericidal ions into media. After that, the suspension of green CuO nanoparticles added into the cells in serial dilution method and incubated for 24 h. The cytocompatibility test was performed in triplicate. After 24 h of incubation period, cell viability was determined using the (1:10) alamarBlue reagent. The cells were incubated for 20 h before measuring the absorbance of viability. The alamarBlue reagent stained treated fibroblast cells lines model (blank alamarBlue reagent media without cells and green CuO) was detected absorbance at wavelength 570 nm and 600 nm using a microplate reader (Bio-Tek Instruments, USA) (Harun et al. 2021). The absorbance reading from sedimentation of green CuO at bottom surface of well plate can be avoided by transferring color changed and increased fluorescence alamarBlue reagent media into new 96-well plate before reading the actual absorbance. The morphology of the cells was captured through an Olympus CKX41 optical light microscope, and images are taken with magnification of 20 X.

DPHH radical scavenging assay

The antioxidant activity of the giant milkweed leaf extract aqueous solution samples was determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma-Aldrich) assay as previously reported by Sachett et al. 2021. Absolute ethanol was labeled as negative control. DPHH control without treatment was introduced as well. Total five replicate sample solutions for each treatment were prepared using aluminum foil wrapped 1.5-mL microtubes and incubated at 25 °C for 24 h in the dark environment. Then, all the treated solutions were transferred into 96-well microplate and the absorbance (Abs) of the samples was read at 517 nm in a microplate spectrophotometer. The percentage of DPHH scavenging effect was measured using the Equation [2]:

$${\text{DPHH }}\left( \% \right) = \frac{{{\text{Abs}}\;{\text{control}}\;{\text{DPHH - (Abs}}\;{\text{sample - Abs}}\;{\text{negative}}\;{\text{control)}}}}{{{\text{Abs}}\;{\text{control}}\;{\text{DPHH}}}} \times 100$$
(2)

Statistical analysis

Statistical significance was determined by two-way ANOVA implemented in the GraphPad Prism. Results were considered statistically significant if P value is less than 0.05 with respect to control. Data are presented as mean values of three independent replicates (n = 3) and standard deviation (± SD).

Results and discussion

Characterization of the green-synthesized CuO nanoparticles

The morphology of the green CuO was studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and is shown in Fig. 2. The TEM average particle size of CuO nanoparticles was measured by ImageJ software analysis. The SEM and TEM images (Figs. 2a, c, e and f) show that the CuO-400C sample is a mixture of rod- and quasi-spherical-shaped nanoparticles and the CuO-500C sample is oval in shape with agglomerated nanosized morphology.

Fig. 2
figure 2

SEM and TEM analysis on green-synthesized CuO; a SEM micrograph of CuO-400C (5 µm), b EDAX spectrum of CuO-400C, c SEM micrograph of CuO-500C (5 µm), d EDAX spectrum of CuO-500C, e TEM micrograph of CuO-400C (100 nm) and f TEM micrograph of CuO-500C (100 nm)

Full size image

In this step, energy-dispersive spectroscopy (EDAX) analysis was used to evaluate the chemical composition and purity of the prepared samples. The EDAX analysis of the bioderived CuO confirms the presence of Cu and O, which are about 17.52 and 17.92 wt.%, respectively, in CuO-400C and 67.23 and 25.05 wt.%, respectively, in CuO-500C (Figs. 2b and d). The Cu and O atoms in the EDAX profile of CuO exhibit strong signals. High chemical weight composition in Cu ratio for CuO-500C sample was formed during green synthesis, and it might be correlated to the complete crystallization of monoclinic pure CuO at high annealing temperature of 500 °C (Luna et al. 2015). Formation of some additional peaks was natural bioderived elements from giant milkweed plant. The presence of the weak peaks of C, Ca, S, Cl, K and Mg atoms in the green CuO indicates the participation of the phytochemical compounds in giant milkweed leaf extract during the green synthesis (Bharathi et al. 2020; Fafal et al. 2017; Kannan et al. 2013; Majeed et al. 2021) (Table 2).

Table 2 SEM-EDAX chemical weight composition (%) results for different calcined CuO
Full size table

The diffraction peaks with Miller indices of CuO-400C and CuO-500C samples are presented in Fig. 3a. CuO-400C and CuO-500C have 12 main diffraction 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 correspond to the crystal faces (110), (− 111), (111), (202), (− 202), (020), (202), (− 113), (− 311), (220), (311) and (004), respectively. The green CuO nanoparticles have a monoclinic crystalline structure (ICDD number 01–089-5897) with the following lattice constant: a = 4.686486, b = 3.421156, c = 5.129263, α = 90°, β = 99.413°, γ = 90° along with d-spacing value of 2.52761 Å. Previously Ravele et al. (2022) stated that formation of monoclinic pure phase of CuO only obtained at calcination temperature above 350 °C. But, cubic phase of Cu2O or other Cu oxide mixture could appear when the nanoparticles produced at low calcination temperature below than 300 °C (Ravele et al. 2022). Basically, in this study, it confirms the green synthesis of pure CuO nanoparticles with monoclinic geometry. The X-ray diffraction peaks at 28.80°, 31.53°, 40.94°, 50.47°, 58.89°, 66.70°, 67.70° and 73.92° observed in the control powdered giant milkweed leaf sample (Fig. 2a) demonstrated that the medicinal plant is rich in bioderived constituents, mainly carbon (C) and calcium (Ca). These natural compounds could further accelerate the bactericidal activity of CuO synergistically (Mydin et al. 2018; Marquis et al. 2015; Dizaj et al. 2015). The crystallite size of CuO was estimated from the XRD pattern using Scherer’s equation at the highest FWHM peak (Table 3).

Fig. 3
figure 3

a XRD diffraction peaks of green-synthesized CuO [: C. gigantea leaves, CuO, filled diamond: additional peaks appeared after green synthesis]; b FTIR spectra of green CuO; and c UV–Vis absorbance spectra of green CuO

Full size image
Table 3 CuO nanoparticles size and shape
Full size table

Fourier transform infrared spectroscopy was performed to investigate the participation of phytochemical compounds in stabilizing and reducing green CuO (Fig. 3b). The sharp peak at 3438 cm−1 is assigned to O–H stretching polyphenols (flavonoids). In future, high-performance liquid chromatography (HPLC) method is recommended to further identify and quantify the flavonoids and phenolic compounds of giant milkweed leaves extract aqueous solution. This flavonoids compounds may act as a capping and reducing agent, thus providing greatest stability in the formation of green CuO during green synthesis (Hublikar et al. 2021a, b). The peaks between 1633 and 1765 cm−1 are assigned to the C = C (carbonyl group) and C = O stretching. The absorption peaks at 1385 cm−1 are assigned to the vibration mode of esters. The absorption peaks between 1110 and 1115 cm−1 belong to the C–O stretching of bioderived elements of giant milkweed leaf extract (Siddiqi and Husen, 2020; Bhavyasree and Xavier, 2020). The low absorption peak at 540 cm−1 is a characteristic peak of the CuO group (Das et al. 2018; Fouda et al. 2020). The intensity of broad absorbance peak of O–H stretching at 3438 cm−1 and small peaks of C = C and C = O stretching between 1633 and 1765 cm−1 were sharply decreased as calcination temperature increased to 500 °C, thus suggesting condensation of Cu(OH)2 into pure CuO phase with removal of water molecules (Hamid et al. 2021). The reduction of ionic copper into copper nanoparticles was successfully obtained under the effect of carbonyl and hydroxyl compounds in the giant milkweed leaves extract aqueous solution.

The UV–Vis spectrogram of giant milkweed extract and green CuO is shown in Fig. 3c. Giant milkweed extract as the control solution has two prominent absorbance peaks at 206 (carbonyl compounds) and 269 nm (phenolic compounds) in the UV spectrum (Mongkholrattanasit et al. 2011). The solution with CuO has a sharp distinct absorbance peak at 233 nm, which belongs to natural carbon (Son and Park, 2018). The produced nanoparticles can be identified by the appearance of a small band at around 307 nm in their respective spectra for the CuO-500C (Siddiqi and Husen, 2020). This similar trend was previously identified by Bharathi et al. (2019).

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

Skin-associated pathogens are a global public health threat, and the wound care and management of serious wounds incur huge economic costs. S. aureus, E. coli, K. pneumoniae and MRSA are the prevailing microbial pathogens that occur in patients with open colonized wounds. Gram-positive S. aureus is among the most common skin pathogens associated with open wound infections (Dunyach-Remy et al. 2016). So, this study mainly focuses on investigating the bactericidal performance of green CuO-400C and CuO-500C toward Gram-positive (S. aureus), Gram-negative (E. coli) and other MDR pathogens (K. pneumoniae and MRSA).

The preliminary screening MIC and MBC study concentrates on determining bactericidal activity of CuO-400C and CuO-500C against skin pathogens such as S. aureus, E. coli, K. pneumoniae and MRSA. The MIC and MBC results against non-MDR and MDR strains are shown in Table 4 and Fig. 4. It was clearly noticed that a significant decrease in S. aureus colony count from 4.3 log10 to 2.72 log10 at concentration of 10 mg/mL for CuO-500C was seen (Figure S1; Supplementary material). But for CuO-400C sample did not show any bactericidal activity at dosage of 10 mg/mL (Figure S1; Supplementary material); thus, it considered less effective in killing toward S. aureus at low concentration. However, the MBC of CuO-400C and CuO-500C for S. aureus is 20 mg/mL. The results showed that CuO-400C, which was produced at low annealing temperature, is less effective in killing microbes at low dosage compared with CuO-500C because of its reduced specific surface area (SSA) (Damm et al. 2008). In general, CuO nanoparticles exhibited shape-dependent bactericidal activity toward various bacterial strains (Laha et al. 2014; Tavakoli et al. 2019). The strong bactericidal effect of CuO-500C might be attributed to the highest SSA of uniform oval-shaped pure monoclinic CuO produced at high calcination temperature (Lee et al. 2021).

Table 4 MIC and MBC of green CuO against various skin pathogen associated with open wound infections
Full size table
Fig. 4
figure 4

Evolution of MIC and MBC of green CuO-400C and CuO-500C toward S. aureus, E. coli, K. pneumoniae and MRSA; a MIC results and b MBC results. Statistical significance was calculated using analysis of two-way ANOVA (GraphPad Prism). ****P < 0.0001

Full size image

In addition, the bactericidal performance of CuO against non-MDR Gram-negative and MDR pathogens was further investigated. Based on MIC result shows that the CuO-500C was most potent bactericidal agent in tackling E. coli, K. pneumoniae and MRSA at lowest concentration level ranging from 0.3125 to 5 mg/mL, while CuO-400C bactericidal activity ability was depending on the higher concentration level (1.25–10 mg/mL). This study recommends that the killing mechanisms of CuO toward different species of pathogens should be further investigated. We concluded that CuO-500C, which has uniform oval shape, is a more potential bactericidal agent than CuO-400C. The rate of the bacterial killing activity was further improved for green-synthesized CuO-500C sample, and it might be due to closer interaction established between the small nanosized and high surface area to volume ratio of the bioderived oval-shaped CuO and bacterial outer cell membrane (Mahamuni et al. 2019; Dulta et al. 2022). The strong bactericidal effectiveness of green CuO-500C is most likely due to the pronounced copper ion release (Cu2+) (Govindasamy et al. 2021c) and oxidative stress caused by ROS (Fouda et al. 2020). Meanwhile, mixture of large-sized rod-shaped CuO-400C sample with length of 58.46 ± 4.00 nm expected to have smaller surface-to-volume ratio, resulting in fewer Cu2+ ions release than uniform oval-shaped CuO-500C sample with diameter of 25.48 ± 1.433 nm (Table 3) (Naqvi et al. 2019). Bacterial metabolism process might be disrupted when Cu2+ ions effectively interact and bind the DNA molecules, leading to helical structure disorder (Dulta et al. 2022).

Time-kill assay

The time-dependent bactericidal activity of the optimized green CuO-500C was determined against non-MDR and MDR skin pathogens. In this investigation, we have only studied the time-kill bactericidal performance of CuO-500C sample against selected skin pathogens based on the results obtained from the preliminary study of MIC and MBC (Table 4) and cytocompatibility study (Fig. 7). The non-toxic behavior and lowest MIC value of CuO-500C sample create a great extent for utilization of this green-synthesized nanoparticles as strong bactericidal agent in future biomedical field. The outcomes of the experiment are shown in Fig. 5 and Fig. 6. A ≤ 2.5 log10 reduction in S. aureus colonies proportional with time was observed in those treated with the green CuO-500C. A reduction in S. aureus viable count from 4.3 log10 to 2.3 log10 after 6 h (Fig. 5c) and complete killing at 12 h (Fig. 5d) was recorded. The average log reduction of the E. coli colonies at 3.3 log10 cfu/mL was seen after 6 h of incubation and bactericidal effect was attained at 12 h (Figs. 5h and i). However, MRSA growth inhibition was more apparent at 12 h of incubation, with complete inactivation at 24 h (Figs. 5n and o). The killing activity of CuO-500C was strain-dependent where it required prolonged period (up to 24 h) to attain bactericidal effect against pathogenic MDR MRSA strain (Kannan et al. 2021). The untreated S. aureus, E. coli and MRSA skin pathogen revealed no decrease in colony counts after 24 h of incubation.

Fig. 5
figure 5

Time-kill assay showing bactericidal activity exhibited by 20 mg/mL of CuO-500C against different skin pathogens for 0.5-h (30 min), 3-h, 6-h, 12-h and 24-h treatment periods; a CuO-500C at 0.5 h (30 min) against S. aureus, b CuO-500C at 3 h against S. aureus, c CuO-500C at 6 h against S. aureus, d CuO-500C at 12 h against S. aureus, e CuO-500C at 24 h against S. aureus, f CuO-500C at 0.5 h (30 min) against E. coli, g CuO-500C at 3 h against E. coli, h CuO-500C at 6 h against E. coli, (i) CuO-500C at 12 h against E. coli, j CuO-500C at 24 h against E. coli, k CuO-500C at 0.5 h (30 min) against MRSA, l CuO-500C at 3 h against MRSA, m CuO-500C at 6 h against MRSA, n CuO-500C at 12 h against MRSA and o CuO-500C at 24 h against MRSA

Full size image
Fig. 6
figure 6

Optical density (OD) measurement at 570 nm measured after 24-h incubation with CuO-500C. These data represent mean (± SD) of three replicates. Statistical significance was calculated using analysis of two-way ANOVA (GraphPad Prism). ****P < 0.0001

Full size image

The growth rate of the skin pathogens was further assessed via optical density measurement through microplate reader (Bio-Tek Instruments, USA) at 570 nm after 24-h incubation with CuO-500C, and the result is presented in Fig. 6. As it is seen from Fig. 6, the cloudy bacterial media (control strain) turned into transparent and clear blue color solution with presence of CuO, indicating an absence of bacterial colonies and decomposition of CuO. Moreover, the absorbance reading had been dropped significantly for CuO-500C treated samples. Overall the results of the time-kill analysis showed that a remarkable reduction in non-MDR and MDR colonies was achieved between 12 and 24 h of exposure. The bactericidal action of the green CuO might be from the release of free metal oxide ions and the generation of ROS (•O2−, •OH−, Cu2+ and Ca2+) which lead to severe rupture of cell membranes of bacteria (Jadhav et al. 2017; Yang et al. 2018b, a). Further investigation on metal ion release (Cu2+) and generation of ROS is recommended.

Kirby–Bauer disc diffusion test

The bactericidal activities of green CuO and commercial CuO at two different concentrations (2.5 and 10 mg/mL), as well as giant milkweed leaf extract, were examined on S. aureus, E. coli, K. pneumoniae and MRSA. The diameter ZOI reveals that CuO-500C was effective toward skin pathogen as the concentration increased from 2.5 mg/mL to 10 mg/mL. The MIC and MBC results agree with the finding that the bactericidal effectiveness of CuO is concentration-dependent (Table 5 and Figure S2; Supplementary material). The results of the Kirby–Bauer disc diffusion showed that the bactericidal activity of 10 mg/mL green CuO-500C was greater than those of other samples. CuO-500C at concentration 10 mg/mL against skin pathogens produced respective inhibition zones between 6.50 ± 0.00 and 7.33 ± 0.33 mm. Comparatively, commercial CuO had smaller zone of inhibition which ranges from 6 to 6.25 mm at 10 mg/mL. The increased bactericidal activity might be attributed to the increase in free metal oxide ions and ROS along with bioderived elements (C and Ca) at higher dosage.

Table 5 Measurement of the diameter zone of inhibition of CuO against skin pathogen. “NA” be a sign of no bactericidal activity observed for this sample
Full size table

Cytocompatibility study

Cytocompatibility of green CuO has been studied on the fibroblast cells lines model (Fig. 7 and Figure S3; Supplementary material). The fibroblast cells lines were able to survive and proliferate healthily above 90% after 24-h incubation with addition of 2.5–10 mg/mL of CuO-500C and 2.5–5 mg/mL of CuO-400C. Most of the cell lines were highly deteriorated and appeared in dull, shrink, rounded shape with reduction in cells density when high concentrated of CuO-500C (20 mg/mL) and CuO-400C (10–20 mg/mL) was introduced. Conversely, healthy live cells appeared in extended proliferation with elongated filopodia as referred to control cell line without an addition of CuO (blank control). Comparatively, 10% (v/v) DMSO had a strong cytotoxic effect on fibroblast cells lines. The sedimentation of agglomerated CuO appeared to be black, as indicated by yellow circle in the image.

Fig. 7
figure 7

Effect of different concentrated green CuO-400C and CuO-500C on cytocompatibility of fibroblast cells lines (n = 3). Statistical significance was calculated using analysis of two-way ANOVA (GraphPad Prism). ****P < 0.0001

Full size image

Our cell viability experiment demonstrated that the green-synthesized CuO-500C and CuO-400C were shown concentration-dependent cytocompatibility effect toward fibroblast cells lines where at high doses the cell line was severely affected and induced abnormal morphologies. It might be attributed to the higher release of free radicals at high dosage level (Ayoubi et al. 2017). It is believed that overproduced hydroxyl radicals in short duration of time have reactive and hazardous free radicals which causes disturbance to wound healing-related cells and fibroblast (Fu et al. 2014). However, the natural carbon and calcium-wrapped CuO-500C at concentration of 2.5–10 mg/mL might prolong the release time of free radicals in steady manner for long-term bactericidal application and create stable environment that might provide cytocompatibility advantage (Yang et al. 2018b, a). Successful control on steady and slow release of free radicals such as •O2− and •OH− from this green-synthesized nanoparticle might present with cytocompatibility properties toward human cells and can accelerate wound healing properties (Sung et al. 2020).

Generally, shape and size of nanoparticles play an important role in effecting cell viability (Yin et al. 2012; Sirelkhatim et al. 2016). The findings noticeably revealed the difference of size and shape between CuO-500C and CuO-400C in responding to cell cytocompatibility. Smaller size (< 10 nm) and dominant quasi-spherical-like structures CuO-400C displayed strong cytotoxic effects toward fibroblast cells lines at concentration of 10 mg/mL (Yin et al. 2012; Sirelkhatim et al. 2016), while uniform oval-shaped CuO-500C (~ 20 nm) offer better cell proliferation and maintain live cells up to 90% in wound healing. We believed that smaller size dominant quasi-spherical-like structures CuO-400C might further accelerate uncontrollable amounts of ROS release at elevated level and promote inhibition of cell growth (Yin et al. 2012; Sirelkhatim et al. 2016). However, high-concentrated green CuO can be safely introduced to the cells by embedding them into biopolymer like chitosan for future biomedical application (Gopal et al. 2014). Collectively, these outcomes exhibited that CuO-500C would be suitable candidates for wound healing application at low dosage level (2.5–10 mg/mL). Finally, further investigation on the long-term (48–72 h) cytocompatibility effect of CuO-500C on fibroblast cells lines is recommended.

DPHH assay

The storage-dependent antioxidant assay for giant milkweed leaf extract solutions was evaluated according to the in vitro protocol illustrated by Sachett et al. 2021. As shown in Table 6, giant milkweed leaf extract aqueous solution which stored at –4 °C for approximately 1 year and 2 years, presented the free radical scavenging activity of 52.85 and 35.05%, respectively. This observation revealed that prolongation of storage time could decrease the bioactive elements from giant milkweed leaf extract aqueous solution (Xu et al. 2018). Hence, the storage condition plays a vital role in determining inhibition of the DPPH radical.

Table 6 Storage-dependent DPHH scavenging (%) for giant milkweed leaf extract solutions
Full size table

Conclusions

This study revealed the potential of bioderived natural constituents decorated on plant-mediated copper oxide (CuO-500C) nanoparticles from giant milkweed as strong bactericidal agent against non-MDR and MDR wound-associated pathogens. This bactericidal agent worked effectively in a concentration-dependent manner and demonstrated strong killing effect against S. aureus, E. coli, K. pneumoniae and MRSA. The outcome of the cytocompatibility study on fibroblast cells lines model proved that the low-concentrated CuO-500C (2.5 – 10 mg/mL) had cell viability of approximately above 90% at 24-h treatment. A further understanding on the killing mechanism of CuO-500C toward skin pathogens, especially on metal oxide ion and ROS release profile, is needed. In addition, the cell viability study for longer period (48 – 72 h) should be carried out in future to understand the long-term cytocompatibility effect of green CuO-500C toward fibroblast cells lines model.