Copper/copper oxide nanoparticles synthesis using Stachys lavandulifolia and its antibacterial activity

Abstract

Nanoparticles of copper/cuprous oxide (Cu/Cu₂ O) were successfully synthesised by a green chemistry route. The synthesis process was carried out using an extract of Stachys lavandulifolia as both reducing and capping agents with a facile procedure. The nanoparticles were characterised by different techniques including X‐ray diffraction, indicating that the synthesised sample comprised both copper and cuprous oxide entity. The nanoparticles had a mean size of 80 nm and represented an impressive bactericidal effect on Pseudomonas aeruginosa.

1 Introduction

In literature, components or ingredient with size range between 1–100 nm are called nanomaterials [1, 2]. Nanomaterials show novel biological and physicochemical properties in comparison with the bulk of counterparts [1, 2]. A large number of physicochemical and green approaches used to synthesise different nanostructures of carbon, noble metals, and transition metal oxides [3, 4, 5, 6, 7, 8, 9, 10].

The green approaches would be cheaper than chemical methods [11, 12, 13, 14, 15, 16, 17, 18, 19]. The physicochemical approaches can be hazardous to environment and human health. The green synthesis approaches are simple, cheap and saving energy consumptions, that can be used for synthesis of fixed nanoparticles with favourite size and shape, and without using toxic chemical agents [3, 10].

Herbal extracts used both as reducer and as stabiliser agents. The kind of bio‐resource, pH, temperature, time and metal ion concentration affect the green synthesis mechanism of nanoparticles. A research with functional extract can be useful for the development of green synthesis of nanoparticles.

There have been various researches on the synthesis and applications of nanostructures of copper and copper oxides in various fields. Some of the important applications of these materials include fabrication of sensors and biosensors [20, 21, 22], optical and electronic devices [23], energy storage devices [24], and antibacterial materials [25]. Copper and its oxides are relatively non‐toxic to mammals [26], low sensitivity for human tissue [27], and antimicrobial activity [25, 28].

Nowadays, the increasing pollutants have caused potentially harmful micrograms to grow extremely to dangerous levels making widespread use of antibiotics. On the other hand, indiscriminate use of antibiotics causes growing concern about antibacterial resistance. In addition, some antimicrobial agents are extremely irritant and toxic. Therefore, it is necessary to find new varieties of safe and cost‐effective antibacterial agents that would not have resistance [29, 30, 31]. In this regard, there are a few studies on the antibacterial activity of copper‐based nanomaterials [32, 33, 34, 35, 36], and it was found that they had bactericidal effects on various species and can be better than silver nanoparticles, for example, against Bacillus subtilis and Escherichia coli [32].

In the present study, a green synthesis of Cu/Cu₂ O nanoparticles was carried out using an extract of flowers of Stachys lavandulifolia for the first time.

Copper chloride was used as a precursor of copper ions, and the extract as the other reagent. After the pH adjustment of the extract of S. lavandulifolia flowers to alkali, the nanoparticles were synthesised. A set of structural characterisation methods such as transmission electron microscope (TEM), X‐ray diffraction (XRD), UV‐visible spectroscopy and Fourier transform infrared spectroscopy (FTIR) were applied for the characterisation of the nanoparticles. Finally, the antibacterial activity of the nanoparticles against Pseudomonas aeruginosa was evaluated by agar well diffusion method.

2 Materials and methods

2.1 Materials

Copper chloride (CuCl₂. 2H₂ O), hydrochloric acid and sodium hydroxide were purchased from Germany (Merck Co). The flowers of S. lavandulifolia (Fig. 1) were obtained from Bakhtiari Zagross Mountains of Iran.

Fig. 1.

Picture of S. lavandulifolia (purple flower), which was taken from, Zagross, Mountains of Iran

2.2 Preparation of the herbal extract

First, 5.0 g of S. lavandulifolia flowers were rinsed with deionised water (DW) and the sepals were transferred into an Erlenmeyer flask containing 100 ml hot DW and boiled for 20 min. Then, the supernatant phase was discarded and filtered using a filter paper. The filtered extract was used to synthesise the Cu/Cu₂ O nanoparticles.

2.3 Biosynthesis of Cu/Cu₂ O nanoparticles

50 ml copper chloride solution of 0.1 mol l⁻¹ was added drop by drop to 25 ml herbal extract with rapid stirring at 50°C. Then, the pH was adjusted to 10 by addition of 1 mol l⁻¹ NaOH solution. The obtained precipitate was separated by centrifugation, washed several times by water and ethanol, and dried at ambient temperature. The samples were kept at room temperature.

2.4 Characterisation of the Cu/Cu₂ O nanoparticles

UV‐vis spectra of the nanoparticles were recorded over a period of one month using a Analytik Jena spectrophotometer. TEM was performed by a LE9, LEO012‐AB to record microscopic images from the nanoparticles. XRD patterns were recorded using a Philips X'pert (the Netherlands) using CuKα. FTIR spectra were recorded using a Bruker, Tensor 27 (Germany).

Agar well diffusion method was applied for evolution of the antibacterial activity of the nanoparticles toward P. aeruginosa. The bacterial suspension was prepared as 0.5 McFarland, and cultured on Mueller Hinton agar medium. In each plate, three wells (5 mm of diameter) were prepared. In the wells, 100 μl of the tested samples, standard streptomycin (positive control) or the nanoparticles suspension were added. The plates were incubated at 37°C for 24 h, and the inhibition zones were measured. All measurements were performed in triplicate.

3 Results and discussion

The reduction of copper ions to Cu/Cu₂ O nanoparticles could be confirmed by the naked eye through the color change of the S. lavandulifolia flower extract from light brown to dark brown, after pH adjustment of reaction mixture to 10 and maintaining at 50°C, as shown in Fig. 2. UV‐vis spectra of the extract and the synthesised nanoparticles are also shown in Fig. 3. While the extract sample did not show any specific absorption in a wavelength range of 375–675 nm, a strong absorption peak was observed after synthesis of the nanoparticles at about 590 nm (Fig. 3). The results confirmed the formation of the nanoparticles.

Fig. 2.

Change in the color of S. lavandulifolia flowers extract from bright brown (middle sample) to dark brown (right sample) after pH adjusting to 10 with NaOH at 50°C

Fig. 3.

UV‐visible spectra of the extract of S. lavandulifolia flowers, and the synthesised of the Cu/Cu₂ O nanoparticles using the extract

Fig. 4 shows TEM images of the Cu/Cu₂ O nanoparticles with two different magnifications. The nanoparticles had a uniform structure with a near‐spherical morphology. The mean diameter of the nanoparticles was 80 ± 8 nm (n  = 20). Because there was no difference in the structure of the nanoparticles in the high‐magnified TEM image, it can be deduced that the nanoparticles are a composite/blend of copper and cuprous oxide.

Fig. 4.

TEM images of the bio‐synthesised Cu/Cu₂ O nanoparticles at different magnifications

An XRD pattern of the nanoparticles is presented in Fig. 5. The pattern included some diffraction peaks appeared at 2θ values of 29, 36, 42, 43, 50, 61 and 74° assigning to (110), (111), (200), (111), (200), (220), and (220) planes of the nanoparticles. From the diffraction peaks in the pattern, those appearing at 2θ values of 29, 36, 42, and 61° are related to Cu₂ O species which correspond to JCPDS 05‐0667. On the other hand, the peaks appearing at 2θ values of 43, 50, and 74° are related to copper which correspond to JCPDS 04‐0836. Therefore, the entity of the nanoparticles is a mixture of Cu and Cu₂ O.

Fig. 5.

XRD pattern of the Cu/Cu₂ O nanoparticles

A FTIR spectrum of S. lavandulifolia extract is shown in Fig. 6 which included different peaks. A band at 1067 cm⁻¹ is assigned to the ‐C‐O‐C stretching vibration, C‐N bond or amine group [37]. A band at 1634 corresponds to N‐C = O, C = O or C‐O groups. The band at 3434 is related to the O‐H [37]. The results indicate that the extract contained some polyhidroxy compounds, maybe sucrose polyester and terpenoids, flavonoids or alkaloids. These compounds acted as ligands to control the size of the Cu/Cu₂ O nanoparticles.

Fig. 6.

FTIR spectrum for the extract of the flowers of S. lavandulifolia

3.1 Antibacterial activity of the Cu/Cu₂ O nanoparticles

The S. lavandulifolia extract did not show any antibacterial effect while Cu/Cu₂ O nanoparticles showed a significant bactericidal effect toward P. areoginoza. A sample image of the results obtained is shown in Fig. 7, and they are quantitatively summarised in Table 1.

Fig. 7.

Agar plate showing the well diffusion tests

(a) Standard streptomycin, (b) Cu/Cu₂ O nanoparticles, (c) S. lavandulifolia flower extract

Table 1.

Antibacterial activities of the S. lavandulifolia extract, the synthesised Cu/Cu₂ O nanoparticles and streptomycin (as a control) using agar well diffusion tests

Bacterial strain	Zone of inhibition, mm
	Cu/Cu2 O nanoparticles	S. lavandulifolia extract	Streptomycin
P. aeruginosa	12	1	14

The results and a comparison with streptomycin revealed that the nanoparticles had a comparable bactericidal activity toward the strain. As for the green synthesis of copper‐based materials, Behera and Giri [38] reported a green synthesis procedure for cuprous oxide nanoparticles using the extract of arka leaves (a perennial shrub), using copper sulphate hydrazine hydrate as precursors. Vaseem et al. [39] synthesised copper nanoparticles in an alkaline medium. Yan et al. [40] reported synthesis of cuprous oxide nanoparticles by a polyol process. The values of the standard reduction potentials of cupric/copper and cupric/cuprous and cuprous/cupric systems are 0.34, 0.15, and 0.52 V (versus standard hydrogen potential). Therefore, there are limited natural compounds that can reduce cupric ions, and there should be relatively strong reducing agent(s) in the herbal extract to employ for the green synthesis of copper‐based nanostructure. Regarding the mechanism of the antibacterial activity of copper‐based nanomaterials, release of the cupric ions [33], and attachment to the bacterium surface [34] have been reported. The isoelectric point (IEP) of cuprous oxide has been reported to be 11.8 < IEP < 11.5; [41] therefore, the nanoparticles bear a net positive surface charge. On the other hand, bacteria possess a negative surface charge due to bearing an excess number of carboxylic (and other) groups [42]. Therefore, electrostatic attractions are the first force causing the nanoparticles’ attachment to the surface of Pseudomonas areoginoza. The resultant effect will then be membrane alteration causing death. Simultaneously, the release of copper ions can be an alternative to bacteria killing.

4 Conclusion

Here, Cu/Cu₂ O nanoparticles were synthesised using S. lavandulifolia flower extract without using any additional reagents at room temperature and pressure. The results indicated a high potential of S. lavandulifolia for the synthesis of Cu/Cu₂ O nanoparticles with a size of <100 nm. The S. lavandulifolia has the ability to synthesise the stable Cu/Cu₂ O nanoparticles without need to any additional surfactants, polymers or chemical reagents at room temperature and pressure. This is an attempt to further develop the green synthesis approach of nanoparticles.