Antibacterial mechanism of biogenic copper nanoparticles synthesized using Heliconia psittacorum leaf extract

2.1 Materials

Fresh leaves of Heliconia psittacorum plants (shown in Figure 1) were collected from a local farm and authenticated before preparing the leaf extract. Analytical-grade copper sulfate was purchased from Merck India Ltd (Mumbai, India). The culture medium (nutrient agar) required for the antibacterial study was procured from Himedia (Mumbai, India). Deionized (DI) water was used for performing all experiments. Glassware was cleaned with DI water and dried in the air.

Figure 1:

Heliconia psittacorum plants.

2.2 Methods

2.2.1 Biosynthesis of copper nanoparticles

Around 100 g of Heliconia psittacorum leaves was cleaned, chopped, and then ground with 50 ml DI water inside a grinder for 10 min. The supernatant was filtered out, and the obtained soup was further used as the standard aqueous leaf extract of H. psittacorum. A 20 mm stock solution of copper sulfate was prepared by dissolving 0.49 g of CuSO₄ in 100 ml of DI water. The biological synthesis of copper nanoparticles was carried out by adding 100 ml of leaf extract of H. psittacorum to 100 ml stock solution of CuSO₄ (conc. 20 mm), keeping the resulting concentration of the reacting solution as 10 mm. The solution was incubated at ambient temperature (25°C) for the next 24 h. The color of the reacting solution began to change from sky blue to reddish brown after almost 6 h of observation (refer to Figure 2), indicating generation of copper nanoparticles inside the medium. The solution color darkened with the time of incubation and finally turned into dark red after 24 h. The bioreduction of copper sulfate to Cu nanoparticles was examined periodically with an ultraviolet-visible (UV-Vis) spectrometer. The biosynthesized nanoparticles were separated out from the reacting solution by centrifugation at 5590 g for 20 min. The formed precipitate was redispersed in DI water (small amount) and again centrifuged at 894 g for nearly 10 min for better removal of extract residues. Then, the pellet deposited at the bottom of centrifugation tubes after repeated centrifugation was carefully collected and dried overnight in a desiccator to make the dried powder of biosynthesized copper nanoparticles for further experiments.

Figure 2:

Color change of the reacting solution.

3.1 Preparation of biogenic Cu nanoparticles

Heliconia psittacorum or parrot’s flower is a perennial herbaceous plant extensively found in South Asia and South America. The green leaves and stem of this plant contain strong bioactive molecules that can reduce copper cations in the reacting medium [17]. When leaf extract of H. psittacorum was added dropwise to the light blue solution of CuSO₄, the color of the mixture remained light blue initially. After observation for 6 h at ambient temperature, expectedly the color of the reacting solution began to change from light blue to reddish brown, as shown in Figure 2. This may be owing to the reduction of Cu²⁺ by biomolecules present in H. psittacorum leaf extract and further stabilization of the colloidal particles in the solution [18]. Gradual change in the solution color was noticed over a period of 24 h as the color intensified with time and turned into dark red, denoting the saturation point of nanoparticle production [19]. The reacting solution was scanned at regular intervals (every 3 h) under a UV-Vis spectrophotometer to investigate the production of Cu nanoparticles and further kinetic study of this green synthetic route. The absorbance spectra (shown in Figure 3A) feature maximum absorbance near 550 nm wavelength, confirming the synthesis of copper nanoparticles and spectral recordings at different time intervals provide an insight to the rate of nanoparticle formation in the reacting solution. Figure 3B shows the variance of peak absorbance with time, where it is clear that initially maximum absorbance increases with time almost linearly because of higher colloidal particle production during exposure to bioactive molecules [20]. However, after 12 h of incubation, the formation rate began to saturate and finally saturated after 24 h, as shown in Figure 3B.

Figure 3:

(A) UV-Vis absorption spectra of Cu nanoparticles recorded at regular intervals. (B) Variation of peak absorbance with incubation time.

3.2 Structural analysis using XRD and HRTEM

The XRD curve of biogenic copper nanoparticles (refer to Figure 4) shows three distinct peaks at 2θ=43.34°, 50.48°, and 74.26° that may be attributed to the (111), (200), and (220) crystal planes of metallic copper as per JCPDS card file no.-04-0836 [21]. Thus, the XRD result verified that the green synthesized Cu nanoparticles have a crystalline nature with fcc (face centered cubic) structure.

Figure 4:

XRD pattern of biogenic copper nanoparticles.

The HRTEM images of copper nanoparticles show the shape, size, and surface morphology of the particles as depicted in Figure 5. It may be observed from the images that the leaf extract-derived copper nanoparticles have a near spherical shape and average diameter between 8 and 12 nm. The fringes of the lattice structure suggest high crystallinity, and spacing between the planes was measured to be around 0.21 nm, which may correspond to the (111) crystal planes of copper.

Figure 5:

HRTEM images of green copper nanoparticles.

3.3 FTIR analysis: role of capping agent

Another prospect of green synthesis is the importance of bioactive organic molecules (found in the extract) that contribute to capping and stability of biogenic nanoparticles. Figure 6 demonstrates the infrared spectra (in mode of absorbance) of the leaf extract and green-synthesized copper nanoparticles. The infrared spectrum of the leaf extract consists of eight notable absorbance peaks throughout the entire range of wavenumber, i.e. 500–4000 cm⁻¹. Two distinct peaks at 1618 and 3096 cm⁻¹ may be attributed to the stretching vibration of C=O and O-H bonds present in amides and aromatic organic compounds, respectively [22]. Bands at 977 and 1419 cm⁻¹ may refer to bending of C-H bonds present in alkenes and alkanes, respectively [23]. The band noticed at 1097 cm⁻¹ may indicate the stretching vibration of C-N bonds present in amines, and a band at 1147 cm⁻¹ points to the stretching of the C-O bonds found in phenolic compounds [24]. Two other bands at 619 and 777 cm⁻¹ may be attributed to the stretching vibrations arising from haloalkanes [25]. As observed in Figure 6, the FTIR spectrum of green-synthesized copper nanoparticles appears less intensified and broadened. In addition, the absence of impurity peaks in the recorded spectrum denotes the purity of the bioengineered nanoparticles, though small peaks of C-O and C-N bonds are shifted, suggesting the capping of copper nanoparticles by amine groups [26]. From the FTIR study, it may be concluded that the amine groups along with the aromatic compounds (like phenol, etc.) probably capped and stabilized the copper nanoparticles during production in the reacting medium [27].

Figure 6:

FTIR spectra of (A) Heliconia psittacorum leaf extract and (B) biosynthesized copper nanoparticles.

3.4 Analysis of antibacterial activity

The antibacterial efficacy of the bioengineered copper nanoparticles was determined toward a few pathogenic bacteria – Staphylococcus aureus, Pseudomonas putida, and Escherichia coli. Staphylococcus aureus is a Gram-positive bacteria, whereas P. putida and E. coli are Gram-negative types. As seen from Figure 7, the largest inhibition zone was noticed against S. aureus irrespective of the concentration of Cu nanoparticles added. Pseudomonas putida and E. coli showed comparatively lower levels of inhibition. Quantitative analysis of the inhibitory effect of the copper nanoparticles toward the tested pathogenic strains has been demonstrated in Figure 8, where a sharp rise in the inhibitory effect may be observed depending on higher concentrations of biosynthesized copper nanoparticles. The Heliconia psittacorum extract (taken as S3) showed zero inhibition, suggesting no role in the antibacterial mechanism.

Figure 7:

Zone of inhibition observed after 24 h.

Figure 8:

Quantitative antibacterial study of biosynthesized copper nanoparticles.

The zone of inhibition observed here against Staphylococcus aureus is greater than that of different leaf extract-mediated silver nanoparticles as reported by earlier studies. For example, Abdel-Aziz et al. studied the antibacterial efficacy of plant-mediated Ag nanoparticles toward S. aureus and observed the zone of inhibition around 12.63 mm [28]. Roy et al. reported that the inhibition zones caused by parsley leaf extract-mediated Ag nanoparticles toward S. aureus and Escherichia coli were 12.50 and 14.15 mm, respectively [29]. Recently, Gupta et al. showed that the green silver nanoparticles curbed the bacterial growth of S. aureus as well as E. coli efficiently, and the inhibition zones were found to be nearly 12 and 14 mm, respectively [30]. The inhibition zones obtained in this study were around 13.01, 11.92, and 12.23 mm for S. aureus, P. putida, and E. coli, respectively. It is clear from these reports that copper nanoparticles show more activity in preventing the growth of Gram-positive S. aureus, whereas Ag nanoparticles are more efficient to curb the growth of Gram-negative strains like E. coli and P. putida.

The probable reason for the higher activity of Cu nanoparticles toward Gram-positive Staphylococcus aureus may be the cell wall components of Gram-positive bacteria that contain different amines and carboxyl group compounds. Chemically, amines can couple with aryls in the presence of copper, leading to copper-catalyzed amination, as shown below [31]. Here, the aromatic compounds, like phenol, involved in the capping of colloidal particles may act as aryls and couple with amines, resulting into the alteration of composition and permeability of bacterial cell walls [32].

C6H5OH+RO-NH2 →CuRO-NH-C6H5+H2O

Accordingly, carboxyl group compounds may decompose in the presence of copper [33].

CH3CH2COOH→CuCH3-CH3+CO2

This catalytic reactivity of copper toward carboxyl groups and amines found in the cell wall of Gram-positive bacteria is possibly responsible for the better antibacterial efficacy of copper nanoparticles against Gram-positive Staphylococcus aureus than Gram-negative strains [34].

Although true mechanism for the antibacterial action of copper nanoparticles is not fully understood, there are few theories to realize the action of metallic nanoparticles on bacterial cells. Earlier studies claim that the metal nanoparticles like silver and copper have the capability to get attached on the microbial cell walls due to electrostatic attraction and further penetrate it [35]. “Pits” formation on cell membrane degrades the selective permeability of the membrane, causing leakage of cytoplasmic fluid. As a result, cellular transport is interrupted and the cells expire gradually [36]. Another reason for cell death may be that the metal ions released from nanoparticles during interaction with bacterial cells affect the cellular respiratory chain by inhibiting respiratory enzymes, leading to reactive oxygen species (ROS) generation. Then, ROS imparts oxidative stress to the cells, causing significant cellular damage [37]. In addition, metal nanoparticles may react with soft bases like phosphorous and sulfur present in the cellular DNA, preventing its replication and protein binding. Consequently, bacterial cells stop all functions and eventually die [38, 39].