Ethiopia has very rich biodiversity in the world with 6, 500 species of higher plants. Most of the rural population of Ethiopia is dependent on traditional medicine who have little access to health services. In recent years numerous Ethiopian medicinal plants have been validated in a scientific empirical framework through phytochemical analysis and subsequent bioassays . The medicinal plants species have been applied to treat many diseases. Syzygium guineense (Willd.) DC is a medium-sized or tall evergreen tree with the height of 15-30 m. The ripe fruits are consumed by humans, birds, and some wild animals. The bark varies in subspecies and is greyish-white or silver mottled and smooth in young trees, turning rough, flaky, creamy, light grey, dark brown or black in older trees. Fruit of this plant is used as a remedy for dysentery, while a decoction of the bark is used as an anti-diarrhoeic. In traditional medicine, liquid from the pounded bark and roots, mixed with water, is used as a purgative. This plant has been identified for our present work to synthesise CuO NSs.
The research on the fabrication of plant mediated CuO NSs for biomedical, environmental and agricultural applications has gained significance in the recent years. The green CuO NSs have been used for photocatalytic, organic dye degradation, biomedical, pharmaceutical, cosmetic, energy and catalytic applications since many decades . These CuO nanostructures such as nanoparticles, nanocrystals, nanorods, nanotubes, nanosheets exhibit versatile properties and hence found to be more useful when combined as coatings in textiles for antifungal and antimicrobial applications. The nanoparticles of copper and its oxides have shown great promise as antibacterial agent. A very few medicinal plants such as Barleria prionitis , Dioscorea bulbifera , Caesalpinia bonducella , Hagenia abyssinica  have been used to synthesize silver, gold, copper and their oxides in the recent past for antimicrobial applications. But no significant work has been done especially with the application of medicinal plant extracts as reducing and capping agents for the synthesis of CuO NSs in Ethiopia for biomedical and antibacterial applications. Thus we present the eco-friendly green synthesis of CuO NSs using medicinal plant Syzygium guineense (SyG) leaf extract at low temperature to investigate the influence of phytochemicals capped CuO NSs on bacteria with evaluation of electrochemical properties.
MATERIALS AND METHODS
The chemical Cu (NO3)2·3H2O, ethanol, Dimethyl sulfoxide DMSO used in the experiments were of analytical grade (purchased from Merck chemical Industrial company).
Collection and authentication of plant materials
Syzygium guineense (Willd.) DC plant leaves were collected from the Arsi zone, Ethiopia. The identity of the plant Syzygium guineense (Willd.) DC. (Code EB006) was authenticated by a plant taxonomist at the National Herbarium, Department of Biology, Addis Ababa University Herbarium, Addis Ababa, Ethiopia.
Preparation of plant leaf extract
The young leaves of Syzygium guineense were picked and washed repeatedly with tap water followed by distilled water and then shade dried for 15 days to remove moisture contents from the leaves. The procedure to prepare the extract is followed as per our reported work  and hence not discussed here.
Green synthesis of SyG- CuO NSs
A standard 0.2M aqueous Cu (NO3)2·3H2O solution was prepared. The plant leaf extract (100 ml) was added to 400 ml of 0.2M Cu (NO3)2·3H2O solution (1:4) dropwise with constant stirring  at about 45 oC. Then the solution was centrifuged for 20 min at 8000 rpm after completion of incubation at room temperature for 24 hrs. The obtained SyG-CuO NSs were washed by deionized water and ethanol to remove any impurities. Thereafter, the NSs were dried ground and stored for further analysis .
The UV-visible absorbance and reflectance spectra of the samples were recorded in the range of 200–800 nm using Shimadzu’s UV-2600, UV-visible spectrophotometer. The thermal stability of the CuO was conducted with TGA/DTA instrument (DTG 60H Shimadzu Co. South Korea). Fourier transform-infrared spectroscopy (FT-IR) Spectrum was recorded using 65 FT-IR PerkinElmer. X-ray diffraction (XRD-Shimadzu x-ray diffractometer (PXRD-7000) analytical technique was used to reveal the crystalline nature of CuO NSs . Microscopic analysis has been conducted by scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX-EVO 18 model with low vacuum facility and ALTO 1000 Cryo attachment) and transmission electron microscope with high-resolution (JEOL JEM 2100 HRTEM) Gatan Digital Micrograph Software was used to evaluate d-spacing values of lattice fringes. Particle size was obtained by using imageJ application.
Method of antimicrobial evaluation
All the antibacterial tests were conducted at Oromia Public Health Research, Capacity Building & Quality Assurance Laboratory, Adama, Ethiopia. The in-vitro antibacterial activity of SyG-CuO NSs was evaluated using Agar disc-diffusion method against selected one Gram positive bacteria (Staphylococcus aureus) and three Gram negative pathogenic bacteria (Escherichia coli, Pseudomonas aeruginosa and Enterobacter aerogenes). A standardized inoculum of the bacteria was swabbed onto the surface of Mueller-Hinton Agar (MHA) plate. The actively growing bacterial cultures were inoculated/spread onto the MHA plate (turbidity was adjusted with TSB to match 0.5 McFarland standard) The nanoparticles extract was prepared with four different concentrations in Dimethyl Sulfoxide. Four concentrations (6.25, 12.5, 25 and 50 µg/µl) of the synthesized nanoparticles were added to the respectively labeled wells.
The antibiotic discs of 6 mm diameter were applied to agar surface using forceps with gentle pressure and then impregnated with the dissolved extract. Chloramphenicol disc was used as a positive control while DMSO was taken as negative control. The plates were incubated at 35 ±2°C in an ambient air incubator for 18-24 hours. The antimicrobial activity was evaluated in terms of zone of inhibition, measured to the nearest millimeters (mm) using a ruler and recorded .
Cyclic voltammetric tests were conducted by using CHI608E potentiostat. The potential range utilized during these studies is ranging between −0.6 V and 0.6 V. EIS studies were carried out in the frequency range of 1 Hz to 1 MHz at AC amplitude of 5 mV .
RESULTS AND DISCUSSION
Synthesis and Characterization of SyG-CuO NSs
The SyG-CuO NSs were synthesized by using copper nitrate as a precursor and Syzygium guineense plant leaf extract as a reducing and capping agent. The biomolecules present in the extract were alkaloids, phenolic compounds, tannins, saponins, anthraquinone glycosides and cardiac glycosides. The details of the phytochemical screening are as given in Table 1. The reactive hydrogen atom released during tautomeric transformation of polyphenols from enol form to keto form would reduce copper ions to copper nanoparticles. In addition, the enzymes of leaf extract also assist copper ions to form an enzyme substrate complex resulting into formation of protein capped copper NSs . It is also understood that polyphenols bind copper ions and reduce them and cap them to form nanostructures. These polyphenolic ligands are believed to influence the morphology of formed particle as reported by the earlier researcher .
The as synthesised SyG-CuO NSs were characterized using UV-visible, UV-DRS, FT-IR, XRD, SEM, EDXA, TEM, HRTEM and SAED techniques. The UV-visible absorbance spectrum recorded for instantaneously synthesised SyG-CuO NSs exhibited λmax of 423 as shown in Fig. 1a, just after 10 min of mixing plant extract with copper nitrate solution. This absorption band is basically due to surface plasmon resonance of SyG-CuO NSs. The absorbance spectrum recorded after 40 minutes of forming homogeneous mixture, exhibited 2 maxima, one λmax at 423 nm and the other at λmax of 451 nm (Fig. 1b). The splitting of absorbance band into two, clearly confirm the formation of more than one type of nanoparticles.
The presence of two maxima is the clear indication of a mixture of copper and its oxides NSs. Initially Cu-NSs formed resulted in a single band with λmax of 423 nm. But at later stage, Cu is found to react slowly and undergo surface oxidation to CuO. This is responsible for the presence of 2 maxima in the spectrum after 40 minutes of beginning of synthesis process of NSs. A similar result was observed during the analysis of synthesized CuO NSs using the Zingiber and Allium sp.  and Psidium guajava leaves extract . The surface plasmon absorbance presents a set of different λmax values for NSs synthesised using different plant extracts which is possibly influenced by morphological features of the NSs.
Similarly, the UV-visible diffused reflectance spectrum was recorded (Fig. 1c). The band gap energy of SyG-CuO NSs obtained by Tauc plot using the data obtained in reflectance spectra utilizing Kubelka-Munk function is as shown in Fig. 1d. The band gap energy, Eg of SyG-CuO NSs was found to be 1.93 eV.
The XRD analysis was executed to explore the in-depth details of crystal structure of VeA-CuO NPs. The XRD spectrum of SyG-CuO NSs (Fig. 2) demonstrates a total of 11 prominent peaks  .
The other peaks at 2θ values of 32.46o, 35.52o, 38.73o, 48.81o, 53.42o, 58.01o, 61.53o, 66.09o 67.98o, 72.48o and 75.13o correlates to 110, 002, 111, 20-2. 020, 202, 11-3, 31-1, 113 and 400 planes of CuO (fcc) (ICSD card No. 00-048-1548, Tenorite-C2/c) ) . This confirms the formation of crystalline CuO from the plant extract via green synthetic route . The main draw back in the green synthesis of copper nanoparticles is their rapid oxidation in air to yield Cu2O and CuO . It is believed that Cu and Cu2O formed at the initial stages of biosynthesis would have oxidized on exposure to air for a longer period of time. However, trace amounts of Cu and Cu2O were detected during selective area electron diffraction (SAED) experimentation which has been clearly discussed under SAED analysis section.
The FTIR spectra of SyG plant extract and SyG-CuO NSs are shown in Fig. 3. FTIR spectra confirmed the presence of biomolecules in the extract and NSs. The broad peaks appeared in the region between 3391 and 2926 cm-1 corresponds to –OH stretching vibration and sp3 C-H stretching vibrations, respectively. The peak at 1623 cm-1 corresponds to C=O stretching of carbonyl groups. The sharp peak around 1450 cm−1 shows the presence of −COO group of carboxylic acid. It is also believed that the amine and carboxylate group present in the SyG plant leaf extract responsible for the binding of proteins with the surface of CuO and thereby leading to the stabilization of the biosynthesized nanoparticles. The intense peaks shown at 3391 cm-1, 1278 cm-1 and 1230 cm-1 corresponds to phenolic –OH stretching and bending vibrations.
The peaks at 1450 cm−1, 1623 cm-1 and 2926 cm−1 corresponds to −COO group of carboxylic acid, C=O stretching of ketonic groups and alkane C−H stretching mode respectively. A less intense peak at around 750 cm-1 confirms the presence of glycosidic linkage. The presence of prominent peaks at 3391 cm-1, 2926 cm-1, 1623 cm−1, 1450 cm−1 and 665 cm-1 in the FTIR spectra of both plant extract and NSs, clearly indicate the presence of bioactive molecules around the NSs. These phytochemical have played a significant role in the nucleation and growth of SyG-CuO NSs and hence found to be at the surface of nanostructures .
The bending vibrations of Cu–O–H bonds resulted in a small peak at 750 cm−1 which can be attributed to the presence of the Cu–O bond. The last peak at 665 cm-1 corresponds to bending modes of vibrations of C–H bond. FTIR analysis results confirmed the presence of various phytochemicals of the leaf extract such as phenolics, tannins, glycosides and proteins which played the roles of reducing agent and stabilizing agent during the synthesis of SyG-CuO NSs.
Fig. 4 presents the TGA-DTA curves for the thermal transformation of SyG-CuO NPs on heating from 0 to 800 °C under oxygen. The TGA curve revealed the three-step decomposition of CuO NSs, with the weight loss occurring in the temperature ranges of 30−160 oC, 160−270 oC and 270−480 °C. About 16 % weight loss was observed in the first temperature region due to usual water evaporation from the NSs. Similarly, 2nd and 3rd weigh losses were recorded to an extent of 17 % and 48 % corresponding to the decomposition of capped biomolecules on the NSs. The presence of different types of capped biomolecules are believed to be responsible for two stages of decomposition during TGA studies.
Figure 4 also shows a small endothermic DTA peak at the region of 91 °C, which corresponds to the first mass loss in the TGA curve. The second weight loss occurring around 160−270 °C in TGA is associated with a prominent DTA exothermic peak at 252 °C. The third major weight loss of 48 % at 405 oC (DTA exothermic peak) corresponds to complete decomposition of bioorganic phytoconstituents present around CuO NPs.
The morphological features of synthesized SyG-CuO NSs as depicted by SEM micrographs are shown in Fig. 5a and 5b. The SEM images also presented various types of nanoparticles in terms of their shape and size.
The average grain size of CuO NSs was found to be in the range between 5 and 50 nm . The presence of mixed type of NSs is possibly due to the amount of capping agents around the particles . EDAX analysis revealed the elemental composition of the SyG-CuO NSs as depicted in Fig. 5c. The peaks corresponding to elemental Cu, C and O were clearly identified demonstrating the purity of the synthesized NSs and this is consistent with the XRD studies. No additional impurity peaks were observed.
The presence of elements, C and O can be attributed to capped bioactive compounds. In addition, it is also apprehended that copper present at the surface had been converted into CuO. In addition, it is also expected that copper, on exposure to air, is oxidized to yield very small amounts of CuO and Cu2O. The reduction of copper ions to Cu NSs is facilitated by the biomolecules of plant extract containing surface hydroxyl groups. In order to explore deep insights on the morphology, size and crystalline nature of the SyG-CuO NSs, TEM, HRTEM and SAED micrographs and patterns were utilized. The HRTEM images of as-synthesized SyG-CuO NSs (Fig. 6) shows that the synthesized NSs are nearly spherical but exhibited varieties of shapes. The presence of small particle with size 4.6 nm confirm efficiency of bioactive components of plant extract as capping and stabilizing agents. The nearly spherical particles with varying sizes from 8.1 nm to 42.1 nm with an average particle size of 24.08 nm as determined by imageJ application are as shown in Fig. 6a and 6b.
The six spots corresponding to specific crystal planes were observed on SAED pattern of SyG-CuO NSs (Fig. 6c). One of such planes is presented with d-spacing of 0.2423 nm as shown in Fig. 5d.
The Fig. 7a, 7b and 7c presents the HRTEM micrographs with SyG-CuO NSs with magnified lattice fringes, IFFT patterns and profile of IFFT with d-spacing value for a specified plane (Fig. 6d) respectively. The dhkl value of 0.2423 nm for a specific set of crystal planes at the surface of Cu NSs was deduced by using Gatan Digital Micrograph Software application.
The d-spacing values for all the spots depicted in the SAED pattern of SyG-CuO NSs (Fig. 6c) are presented in Table 2. Each spot on the SAED pattern corresponds to specific set of lattice planes.
The presence of 4 major peaks corresponding to (111), (200), (220) and (311) planes of fcc structure of pure CuO in the XRD pattern of NSs was presented earlier as Fig. 2. The d-spacing values of the derived diffraction planes from spot 1 to spot 4 corresponds to d-111CuO = 0.2854 nm, d111Cu2O = 0.2432 nm, d200Cu = 0.2271 nm and d111Cu = 0.2040 nm respectively. The absence of Cu and Cu2O peaks in the XRD pattern can be possibly due to their presence in very small quantities.
This SAED-HRTEM analysis is in good agreement with the previously arrived XRD results for the SyG-CuO NSs. The d-spacing value of lattice fringes at the surface of the SyG-CuO NSs was found to be 0.2395 nm, which is similar to the dhkl value of 0.24 nm for (111) plane of fcc structured Cu2O obtained in the previous reported work . This confirms that the copper atoms at the surface of NSs reacted with air to form their respective oxides, Cu2O and CuO.
The SyG-CuO NSs exhibited broad range of antibacterial activities against all tested pathogens; S. aureus, E. coli, P. aeruginosa, and E. aerogenes. The present work evaluated synergistic influence of biomolecules with NSs against 4 pathogens. The zone of inhibitions for Chloramphenicol, DMSO and NSs with four concentrations (6.25, 12.5, 25 and 50 µg/µl) are as shown in Fig. 8.
SyG-CuO NSs were found to show better antimicrobial activity against gram positive bacteria than gram negative bacteria which is believed to be due to the structural differences in the cell walls of bacteria. The antimicrobial activity of NSs can be partially attributed to the presence of bioactive compounds on the surface of NSs as capping and stabilizing agents. In this regard, the activity was pronounceable against S. aureus. The highest zone of inhibition (mm) recorded with 50 µg/µl of SyG-CuO NSs against S. aureus, E. coli and E. aerogenes bacteria was 12 mm and the lowest zone of inhibition (mm) recorded against P. aeruginosa bacteria was 10 mm (Table 3).
Pure Cu and its oxides were proved to exhibit excellent antimicrobial activity. The wide zone of inhibitions (Fig. 8) of SyG-CuO NSs against pathogens confirm their superior potentiality to inhibit and cause death of pathogens. It was found that the bacterial resistance decreased with increase in the concentration of the nanoparticles for all the bacteria except E. aerogenes. The antimicrobial results obtained using SyG-CuO NSs, were found to be better when compared with earlier results presented by many researchers (Table 4).
The highest zone of inhibition (mm) recorded with CuO NSs against bacteria was 12 mm which is comparatively better than those of zone of CuO NSs synthesised by various researchers using various plant extracts as mentioned in the Table 4. Thus it can be concluded that the cumulative effect of CuO NSs coupled with bioactive compounds such as tannins, saponins and glycosides of Syzygium guineense leaf extract proved to be detrimental for bacteria .
Even though many antimicrobial mechanisms were proposed by earlier researchers, the action of SyG-CuO NSs on the bacteria is yet to be explored fully. Nanoparticles were found to cause the death of bacteria by attacking its cell wall, inhibiting RNA synthesis and preventing DNA replication. It is assumed that the positive copper oxide in the NSs get adsorbed on to the cell wall of bacteria and interacts with the negatively charged species. This results in disruption of the cell wall and damage occurred by entering into the cell through the generation of reactive oxygen species (ROS) by the effect of visible/UV light radiation. It is also believed that the synergistic effect of SyG-CuO NSs with bioactive compounds of extract would have played significant influence to inhibit the activity of pathogenic bacteria as suggested by the recent researcher.
Most studies have shown that Gram-negative bacterial growth is more affected by NSs than Gram-positive bacteria which can be attributed to thin cell walls that are easily penetrated, while Gram-positive bacteria have thicker cell wall . In addition, electrochemical potential across the cell membrane decreases up on interaction with the released Cu metal ion by SyG-CuO NSs affecting integrity of the membrane . The maximum antibacterial activity is recorded with Gram-negative bacteria than the Gram-positive bacteria due to their differences in their cell structure .
In order to evaluate the electrode stability of the prepared oxides, a series of CV scans (of 25 cycles), at a scanning rate of 50 mVs-1, for SyG-CuO NSs sample was carried out (Fig. 9). During the test, the locations of anodic and cathodic peaks of electrode did not show any significant deviation with the growing cycles, which confirms good electrode stability  .
The charge transfer resistance Rct is a direct measure of the diameter of the semicircle arc on the real axis. It can be observed that the impedance curve of SyG-CuO NSs is inclined towards Y axis; this suggests a low capacitance and minimum charge-transfer resistance of prepared electrode .
The resistance of the nanomaterial can be obtained using the Nyquist plot. The semicircular portion at a higher frequency is equal to the electron transfer resistance (Rct) at the contact interface of the electrode and electrolyte solution . As seen in Fig. 9, the semicircular diameter of SyG-CuO NSs is small. The value of Rct is equal to the diameter of the semicircle. The Rct value of SyG-CuO NSs is found to be 49 Ω which confirms that the nanomaterial has considerably good conductivity and thus can be better alternative as electrode material.
The application of medicinal plant, Syzygium guineense leaf extract towards the green synthesis of copper and its oxides (SyG-CuO) nanostructures was successful. The UV-visible spectra, XRD pattern and FTIR spectra confirmed the formation crystalline SyG-CuO NSs in the presence of biomolecules such as phenolics, tannins, glycosides and proteins of the plant extract. Thermal stability of CuO NSs was confirmed by TGA-DTA analysis. TEM-HRTEM-SAED analysis confirmed the presence of Cu and its oxides. The synergistic influence of bioactive compounds and SyG-CuO NSs proved to be highly effective antibacterial agent against pathogens, Gram positive bacteria S. aureus, and Gram negative bacteria, E. coli, P. aeruginosa, and E. aerogenes. The EIS and CV studies confirmed better electrochemical behavior of SyG-CuO NSs with superior electrode property. The green copper oxide NSs exhibited better future perspective for antimicrobial and electrochemical applications.
The authors are grateful and thankful to Adama Science and Technology University for financial support and laboratory facility to conduct this research work which was funded from the project (ANSD/04/0453/11-2018) approved by Research and Technology Transfer Office, sanctioned by Adama Science and Technology University, Ethiopia.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.