Antimicrobial activity of green synthesized tri-metallic oxide Ni/Cr/Cu nanoparticles

The tri-metallic oxide Ni/Cr/Cu nanoparticles (NPs) were synthesized using Coriander sativum extract as the reducing agent. The precursors namely CuSO4.5H2O, Ni(NO3)2·6H2O and Cr (NO3)3·9H2O were used for the green synthesis. Further, the prepared NPs were characterized using Ultraviolet-Visible (UV-Vis) spectroscopy and X-ray diffraction (XRD) studies. Its antimicrobial property against two fungal and two bacterial species was determined by measuring the respective zone of inhibition (ZOI) in well diffusion method. A dose dependent inhibition was observed in all the four species of pathogens including Escherichia coli, Staphylococcus aureus, Aspergillus flavus and Penicillium sp. This antimicrobial property of tri-metallic oxide NPs may be utilized in the field of medical research, pharmaceutical industries and environmental sciences. DOI:10.46481/jnsps.2021.237


Introduction
The common methods to synthesize of multi metallic NPs includes laser ablation, ultrasonication, sol-gel, hydrothermal, co-precipitation and green synthesis [1,2]. The physical and chemical methods involve higher cost and are not eco-friendly. On the other hand, use of green synthesis methods offer several advantages such as cost-effectiveness, simplicity, and environmentally friendliness. The green sources used for synthesizing NPs are micro-organisms and plants [3][4][5][6]. The choice of these sources as bioreducing agents should be delicately considered in the green synthesis approach [7,8]. On comparing, the choice of plants is less expensive than micro-organisms.
Coriander sativum is an important medical plant belonging to Umbelliferae family comprising several bio-active compounds such as flavonoid, phenol, terpenoid, tannin and glycosides with high level of in vitro radical scavenging activity [9,10]. These bio-active compounds were believed to play a major role in the synthesis of metal oxide NPs.
Due to the distinctive properties of bimetallic NPs, it has attracted the attention of research community [11,12]. For various applications, the most suitable methods were to fabricate multi-metallic NPs followed by core-shell and/or alloy forma-tion. Extensive research has been established on traditional single metallic methods. Exploration tri-metallic nanostructures have been initiated recently [13] since an elaborate study with respect to its application is still at its infancy.
The greatest obstacle for scientists trying to eradicate dangerous pathogens such as bacteria, moulds, yeast and viruses has been growing resistance to antibiotics. Multi-metallic NPs have been heavily studied against these dangerous pathogens as feasible therapeutic and diagnostic methods [14,15]. An effective approach to overcome these issues is to synergistically engineer these NPs with various formulations, such as the creation of multi-metallic composites, to strengthen current vulnerabilities.
The antimicrobial resistance has long been of concern to researchers and the use of metal oxide NPs has increased the interest in nanomaterial researchers. Hence in the present study, a green method to synthesize tri-metallic oxide NPs comprising Nickel (Ni), Copper (Cu), and Chromium (Cr) using the Coriander sativum extract will be developed. Subsequently, its antimicrobial efficacy against two bacterial species and two fungal species will be assessed. This investigation of antimicrobial activity of tri-metallic oxide NPs could be encouraging for a pharmaceutical application.

Materials required
Chemicals including CuSO 4 ·5H 2 O, Ni(NO 3 ) 2 ·6H 2 O, Cr (NO 3 ) 3 ·9H 2 O were purchased from SRL India. Double distilled water was used for the preparation of chemical reagents.. All antibacterial and antifungal media were purchased from Himedia. Laboratory grade solvents were used for our study. The bacterial and fungal species were obtained from MTCC.

Collection of plant
Coriander sativum leaves were used for our study. The collected leaves were washed with tap and double distilled water and finally dried. The leaves were crushed and grounded to fine powder. The extract was prepared using 5 g of fine powder in 100 mL distilled water for 15 min at 400W. It was filtered using Whatman no.1 filter paper and filtrate was used as a reducing agent for green synthesis of tri-metallic oxide NPs.

Green synthesis of tri-metallic oxide NPs
The method involves simultaneous reduction of precursor salts CuSO 4 ·5H 2 O, Ni(NO 3 ) 2 ·6H 2 O and Cr(NO 3 ) 3 ·9H 2 O with the C. sativum extract [16]. Typically, 0.01M of salt solution comprising all three metals was mixed with the leaf extract in 250 mL Erlenmeyer flask. The temperature of the reaction mixture was maintained 40 • C for 45 min in a water bath. After completion of the reaction, mixture was cooled to achieve room temperature. The mixture was centrifuged at 3000 rpm for 15 min and washed several time with distilled water. Finally the mixture was washed with ethanol to remove impurities. The sediment comprising the tri-metallic NPs were stored in vacuum oven at 45 • C to maintain its stability and integrity.

Characterization of NPs
The synthesized tri-metallic oxide NPs were characterized using UV-Vis spectroscopy and X-ray diffraction (XRD). The composition of the Ni/Cr/Cu NPs were investigated using a Empyrean X-ray diffractometer, operated at 45 kV, 40 mA, with CuKα1 radiation (wavelength λ=1.5406 Å) and copper filter. The X-ray diffractogram was recorded in the 2θ range from 10 • to 80 • at scanning steps of 0.026 • . An UV-Vis spectrum was measured between 190-800 nm using Jasco spectrophotometer to determine the characteristic peaks of the synthesized NPs.

Antimicrobial activity
The antimicrobial activity of tri-metallic NPs was evaluated against two bacterial and two fungal species by well diffusion method [17,18]. 1 mL of the selected microbial species was inoculated over the entire agar surface. A well with 5 mm diameter was bored aseptically, and 50µL of the NPs solution at desired concentration was introduced into the well. The inoculated plates were incubated under suitable conditions depending upon the test microorganism. The details of the medium, species and incubation conditions are provided below.

Antibacterial activity
Mueller Hinton Agar plates were prepared and inoculated with standardized bacterial strains comprising E. coli and S. aureus in individual plate. Five different wells were bored on the agar plates where, four different concentrations of the NPs were added in four separate wells and control (amoxicillin) was added in the other well. The plates were incubated (37 • C/24 hours) and observed for zone of inhibition (ZOI).

Antifungal activity
Sabouraud Dextrose Agar plates were prepared and inoculated with standardized fungal strains of A. flavus and Penicillium sp. Five different wells were bore on the agar plates where, four different concentrations of the NPs were added in four individual wells and control (fluconazole) was added in the other well. The plates were then incubated at 25 • C for 72 hours and observed for zone of inhibition (ZOI).

Statistical analysis
All results of antimicrobial activity are expressed only numerically as mean ± standard deviation (SD) without any analysis.

Synthesis and characterization of tri-metallic NPs
Initially, the extract and the precursor solution looked tanned brown colour. After the reduction reaction, the solution turned dark brown in colour. After centrifugation and drying, a dark brown powder was collected. The UV-Vis absorption of the C. sativum extract was compared with the collected tri-metallic oxide NPs. The tri-metallic oxide NPs showed three characteristic peaks at 261 nm, 426 nm and 564 nm (Figure 1) that correspond to tri-metallic oxide Ni/Cr/Cu NPs reported earlier 145  [16]. Similar results were obtained in monometallic NPs synthesized using individual metallic precursors [19][20][21].
The Ni, Cu, and Cr NPs tend to show characteristic XRD peaks at 40-50 • and 50-60 • [19][20][21] individually. A combination of peaks was observed in XRD ( Figure 2) and new peak at 20 • characteristic to that of graphene nanomaterials was also observed. This may be due to the reduction reaction of phytochemical that is present in the leaf extract of C. sativum. Using Scherrer's equation, the crystallite size of the tri-metallic oxide NPs was calculated as 17.72 nm from the XRD. In the present study, the cumulative peaks of all the three metal were exhibited in both UV-Vis and XRD. This shows that tri-metallic oxide NPs comprising Ni, Cr and Cu were synthesized.

Antibacterial activity
The antibacterial activity of the tri-metallic NPs was determined against E. coli and Staph. aureus by measuring the ZOI after incubation (37 • C/ 24 h) ( Table 1). The observed results are presented in figure 3. It can be seen that the tri-metallic NPs exhibited dose-dependent antibacterial activity. That is, the zone of inhibition increased proportionally with the concentration of NPs. Among the tested species, the tri-metallic NPs showed better antibacterial activity against E. coli than Staph. aureus. There are reports that discussed the antibacterial activity of these metals. In a study by O. Dlugosz et. al., cop- per showed improved antibacterial activity against E. coli and Staph. aureus with respect to increasing size. But its effect reduced when it is combined with silver. On the other hand, it exhibited inhibition against broader spectrum of microbes. In another study, the bimetallic NPs made of gold and silver showed that the size and the antimicrobial property are inversely proportional to each other [15]. There is only one study which discussed similar antibacterial property of similar tri-metallic NPs wherein all the three metals showed synergistic antimicrobial effect [16]. Hence as per the earlier studies and the present results, it can be understood that the antibacterial property of the metallic NPs was due to the size and the charge of the active surface area.

Antifungal activity
The antifungal activity of the tri-metallic NPs was determined against A. flavus and Penicillium sp. by a similar method to that of antibacterial activity. The measured ZOI and its respective plates are provided in Table 1 and figure 4. It was observed that the organism showed resistance towards the trimetallic NPs at 125 µg/mL concentration. But as the nanoparticle's concentration increased, the diameter of the zone of inhibition also increased. Thus, the dose and inhibition were directly proportional to each other. In earlier studies, the bimetallic NPs showed good activity towards fungal species Candida sp. and B. cinerea [22,23] due to their external charge and size. The tri-metallic NPs showed better antifungal activity against Penicillium sp. than A. flavus that can be attributed towards even 146 Table 1. Zone of inhibition (mm) measured for each microbes across the concentration gradient. All the values are given as mean value.

Conclusion
Tri-metallic NPs comprising Ni, Cr and Cu were synthesized using the leaf extract of Coriander sativum. Characteristic peaks were observed in both UV-Vis spectroscopy and XRD diffractogram. The main objective of this research is to investigate the recognition and targeting capability of the tri-metallic oxide NPs and use it to deliver the antimicrobial drugs for resistant species. As per the results, these NPs showed good antimicrobial property against E. coli, Staph. aureus, Penicillium sp. and A. flavus. Further investigation is required to determine other parameters like minimum inhibitory concentration, critical dose and lethal dose.