Biosynthesis, characterisation and antimicrobial activity of zinc oxide and nickel doped zinc oxide nanoparticles using Euphorbia abyssinica bark extract

Abstract Biosynthesis of metallic oxide nanoparticles is being used and preferred over physical and chemical methods of synthesis since it is simple, inexpensive, environmentally friendly, and green. The aim of this study was to synthesise ZnO and nickel doped ZnO nanoparticles using Euphorbia abyssinica bark extract for antimicrobial activity studies via agar disk diffusion method against some selected microbes. The synthesised nanoparticles were characterised using X‐ray diffraction (XRD), ultraviolet–visible spectroscopy, and Fourier transform infrared spectroscopy. The study results revealed that the biosynthesised nanoparticles had good crystalline nature, with crystal sizes in the range of nanoparticles and structures of hexagonal wurtzite. Both undoped ZnO and nickel doped ZnO nanoparticles demonstrated antibacterial and antifungal activity against four bacterial strains and two fungal genus. Generally, nickel doped ZnO NPs were found to possess more antimicrobial activities than undoped ZnO NPs. Specially, 4% and 5% nickel doped ZnO NPs showed significantly enhanced activity against Enterococcus faecalis, Staphylococcus aureus, Aspergillus and Fusarium.


| INTRODUCTION
ZnO has high electron mobility, high thermal conductivity, good transparency and direct wide band gap of 3.37 eV. Moreover, ZnO is preferentially a stable hexagonal wurtzite, zinc blend and rock salt structure at room temperature and each oxygen ion surrounded by tetrahedral zinc ion [1]. ZnO has got remarkable research attention as a result of its innocent characteristics such as non-toxicity or bio-compatibility and stability as well as abundancy [2], and it also inhibits the action of pathogenic microbes when used in small concentration [3]. However, its wide band gap has limited the practical use of ZnO NPs as a photocatalyst and an antimicrobial agent. And doping of metals into ZnO NPs is receiving attention due to its improved properties and applications [4]. Fe [4], Mg [5], Ta [6] and Cu [7,8] doped ZnO NPs with enhanced antimicrobial activities have been reported. The inhibitory mechanism of ZnO NPs is by the generation of Reactive Oxygen Species (ROS), metal ion release and membrane damage [9][10][11][12][13], and increase in the antimicrobial activities of metal doped ZnO have been mechanistically suggested due to the enhanced generation of ROS as a result of doping [4][5][6][7][8]14]. However, a scholar suggests the requirement for further studies to exactly know the inhibitory mechanisms of the nanoparticles [9,11].
Different classical methods of nanoparticle synthesis such as coprecipitation, thermal decomposition, hydrothermal, solgel, and electrochemical are known. However, they involve the use of hazardous solvents and apply expensive reagents [15]. Consequently, researchers have developed the green method for the synthesis of nanoparticles [16], which is an eco-friendly method that uses minimum or no hazardous chemicals for the synthesis of nanomaterials thus receiving much attention [2,15]. Many nanomaterials have been synthesised using biomaterials extracted from plants which are more efficient, inexpensive, eco-friendly and of nontoxic approach [2]. Phytochemicals extracted from plants have been used for the synthesis of metal/metal oxide nanoparticles as reducing and stabilising agents [17], and Aloe vera [18], Calotropis procera [19], Trifolium pratense flower [3], and Citrus aurantifolia [20] extracts have been used for the synthesis of ZnO nanoparticles. Amaranthus spinosus leaf extract [4], Prunus cerasifera fruit extract [21] and Cannabis sativa leaf extract [14] have been used as reducing and stabilising agents for synthesis of Fe, Ag and Ag doped ZnO NPs respectively.
Traditionally, Euphorbia abyssinica plant parts have been used for curing fungal infection [22], cancer [23,24], abdominal pain [25], hemorrhoid and skin wound [26], and malaria [27]. Euphorbia abyssinica bark has been also used by people traditionally for curing diseases like gonorrhea and syphilis [28]. It has been reported that the Euphorbia abyssinica bark plant extract contains metabolites like terpenoids, flavonoids, tri-terpenoids and phenolic compounds [29]. However, to the best of our knowledge, there is no literature on the synthesis of ZnO and Ni doped ZnO nanoparticles using Euphorbia abyssinica plant extract. Thus, the main objective of this work was to synthesise ZnO and Ni doped ZnO nanoparticles by using Euphorbia abyssinica bark extract and to evaluate their antimicrobial activities.

| Plant sample collection and preparation
The bark of Euphorbia abyssinica (Adaammi in Afaan Oromo) was collected from Kiremu District, East Wollega, Oromia, Ethiopia. The collected Euphorbia abyssinica bark was washed with tap water thoroughly to remove debris and other contaminants, then dried in the sun shade and powdered using mortal and pistil. The maceration method of extraction was employed. 60 g of Euphorbia abyssinica bark was added to distilled water in a 300 ml beaker, and kept for 24 h for extraction. Then, the extract was filtered and stored for further experimental analysis.

| Preparation of ZnO and Ni doped ZnO nanoparticles
The synthesis of ZnO nanoparticles was made by mixing bark extract (50 ml) and zinc nitrate hexahydrate (2 g) in a 75 ml beaker. The mixture was stirred well for 30 min using a magnetic stirrer. Then, the mixture was kept for 12 h for paste formation and this paste was collected in ceramic crucibles.
The aforementioned ZnO NPs synthesis procedure was adopted for the synthesis of nickel doped ZnO NPs, and 1%-5% mole of nickel nitrate hexahydrate was added to the adjusted amount of zinc nitrate hexahydrate. The mass of zinc nitrate hexahydrate, the volume of extract and the calcination temperatures used were obtained by optimisation. All the products were calcinated for 2 h in a muffle furnace at 500°C and stored for characterisation. At the time of calcinations, the colour of the brown black samples changed to white in both ZnO and Ni doped ZnO samples.

| Characterisation of ZnO and Ni doped ZnO nanoparticles
The synthesised ZnO and Ni doped ZnO nanoparticles were characterised by ultraviolet-visible spectroscopy (UV-Vis) absorption spectra in the wavelength range of 230-800 nm. The existence of functional groups left after calcination as capping and stabilisation agents were characterised using Fourier transform infrared spectroscopy (FTIR) spectra. The structural parameters of ZnO and nickel doped ZnO nanoparticles have been calculated from the X-ray diffraction (XRD) pattern using Scherer equations [30].

| Antimicrobial activity
Antimicrobial studies were conducted at the Jimma University in the Biology Department Laboratory and done using the agar disc diffusion method [30]. The biological screening effects of the synthesised nanoparticles were tested against the bacterial strains such as Enterococcus faecalis, Staphylococcus aureus, Bacillus subtilis and Escherichia coli. And fungal activities were also tested against Aspergillus and Fusarium genuses. Culture containing test tubes with approximately equal concentration or density of 0.5 McFarland standards [31] was used for inoculation of media. And freshly grown liquid culture of test bacteria and fungi solution having similar turbidity with 0.5 McFarland were seeded over Muller-Hinton Agar (MHA) and Potato Dextrose agar respectively. DMSO was used as negative and the antimicrobial activity of nanoparticles was demonstrated by the diameter of the zone of inhibition developed around the sample.

| Phytochemical screening
The result of phytochemical screening showed that Euphorbia abyssinica plant bark extract possesses flavonoids, phenols, saponoids, steroids and tannin metabolites (Table 1). This finding agrees with the previous phytochemical screening results of Euphorbia species [32]. The existence of these secondary metabolites enables the Euphorbia abyssinica plant extract to be used as a reducing and stabilising agent during the synthesis of ZnO and Ni doped ZnO nanoparticles.

| UV-visible spectral analysis
The amount of Zn(NO 3 ) 2 ·6H 2 O and the volume of the extract were optimised using the UV-visible spectra of ZnO nanoparticles synthesised from these two substances in different ratio. Based on the appearance of the peak and maximum wavelength, the optimum amount of Zn (NO 3 ) 2 ·6H 2 O and the volume of the extract used in the synthesis of ZnO were found to be 2 g and 50 ml respectively. For Ni doped ZnO nanoparticle synthesis, the amount of Ni(NO 3 ) 2 ·6H 2 O which was doped is indicated in Table 2. The UV-visible spectra of all the synthesised nanoparticles are indicated in Figure 1. The UV-visible spectra of the extract showed λ max at 288 nm and that of zinc nitrate salt at 242 nm. The two peaks disappeared in the synthesised NPs and new peaks were observed that suggest the synthesis of ZnO and nickel doped ZnO NPs. The absorption peaks were observed in the wavelength range of 350-380 nm [33], and indicated the synthesis of ZnO and nickel doped ZnO nanoparticles. It is also strongly believed that the phytochemicals like phenols, phenolic, steroids and tannins are working as reducing agents and are responsible for conversion of metal salts into ZnO nanoparticles [17,34].

| Fourier transform infrared spectroscopy spectral analysis
Fourier transform infrared spectroscopy analysis confirms the presence of different organic and inorganic functional groups. The metal oxide absorption peaks range less than 1000 cm −1 [35]. FTIR result clearly indicated that the extract contains functional groups of alcohol, carboxylic acid, ether, phenols, tannins and ketone which may act as capping or stabilising agents for the formation nanoparticles [36]. The FTIR spectra ( Figure 2) and the stretching of major functional groups presented in Table 3

| X-ray diffraction analysis
X-ray diffraction study result confirmed that the synthesised ZnO and Ni doped ZnO NPs were with hexagonal wurtzite phase ( Figure 3 and  [37]. The XRD data were recorded by using Cu Kα radiation (λ = 1.5406 Å).

Synthesised nanoparticles Grain size (nm) Wavelength (nm) Lattice parameters (Å) u (KJ m −3 ) c/a Bandgap (eV) V (Å 3 )
Undoped The average crystallite size (D) of ZnO and Ni doped ZnO NPs were calculated from the most intense peak (101) using the Debye Scherer formula (D = kλ/β 1/2 cosθ) [32,38], where k denotes the Scherer constant k = 0.90 [39], λ = 0.15406 nm is the wavelength of the incident Cu Kα radiation; β represents the full-width at half-maximum (FWHM) of the respective peak. The diffraction peak of each sample was strong, sharp and narrow that indicates the good crystalline nature of biosynthesised ZnO and Ni doped ZnO NPs [40]. The size of the particles (Table 4) was found to be in the range of the nanoparticle material size of 1 to 100 nm [32,41,42]. Lattice constants a, b, and c, inter planar angle and unit cell volumes were calculated by using Bragg's equation, λ = 2dsinθ and Lattice geometry equation [36]. The lattice parameters were calculated by using sinθ = λ/2a (h 2 + k 2 + l 2 ) 1/2 , a = λ/ (3) 1/2 sinθhkl, c = λ/sinθ, and d hkl = 1/((4/3) 1/2 (h 2 + k 2 + hk)/ a 2 + l 2 /c 2 ). Based on these formulas, the grain size or d-spacing and hkl values were determined (Tables 5 and 6). When compared with the synthesised ZnO nanoparticles, there were no extra peak impurities observed in the synthesised Ni doped ZnO nanoparticles [43], and hence no significant change was observed in the hexagonal wurtzite structure of ZnO nanoparticles as a result of Ni doping. However, the diffraction angle at intensity (101) reflection of Ni doped ZnO NPs was slightly shifted relative to the diffraction angle 36.37°of ZnO nanoparticles which may indicate the doping of Ni into ZnO lattice and assures Ni is doped into ZnO nanoparticles [4,41]. The bond length (L) was calculated by using L = ((a 2 /3 + (1/2 − u) 2 C 2 ) 1/2 , and the positional parameter (u) in the Wurtzite structure related to c/a ratio was determined by u = a 2 /3c 2 + 0.25 equation. Positional parameter is the measure of the amount by which each atom is displaced with respect to 'c' axis. The increasing amount of dopant (Ni) from 1% to 5% resulted in the increase of particle size from 8.11 to 14.90 nm and decrease in the band gab from 3.53 to 3.46 eV, and the result was found to be similar with what was reported [4].

| Antimicrobial activity
The antimicrobial activities of ZnO  the zone of inhibition is indicated in Figure 4. The antimicrobial activity study results of undoped ZnO, 1% Ni doped ZnO and 2% Ni doped NPs were found to be similar (Table 7) which is probably due to their close similarities in particle sizes (Tables 4 and 5

| CONCLUSION
ZnO and Ni doped ZnO NPs have been successfully synthesised by using Euphorbia abyssinica bark extract, and confirmed that this plant contains reducing and stabilisation agents, and could be used as potential agents for the biosynthesis of ZnO and Ni doped ZnO NPs. The characterisation results confirmed the synthesis of hexagonal wurtzite structure ZnO and Ni doped ZnO nanoparticles. As the amount of dopant (Ni) increases from 1% to 5%, the band gap was decreased and the particle size increased. Generally, 4% and 5% Ni doped ZnO NPs showed significantly enhanced antimicrobial activity when compared with the antimicrobial activity of the synthesised undoped ZnO NPs. Thus, the band gab and antimicrobial activity of ZnO NPs can be adjusted by using nickel as a dopant. -31