Electrochemical properties of biogenic silver nanoparticles synthesized using Hagenia abyssinica (Brace) JF. Gmel. medicinal plant leaf extract

The biogenic/green silver nanoparticles (g-Ag NPs) were synthesised by using the extract of indigenous medicinal plant of Ethiopia, Hagenia abyssinica (Brace) JF. Gmel. leaf extract for the first time, to investigate the synergistic effect of biomolecules towards the enhancement of electrochemical properties of NPs. The synthesized g-Ag NPs were characterized by UV-visible, UV-DRS, FT-IR, XRD, SEM, EDXA, TEM, HRTEM and SAED techniques. The maximum absorbance, λmax was found to be 461 nm for g-Ag NPs due to surface plasmon resonance. The energy gap, Eg of NPs, was found to be 2.31 eV. FTIR spectrum confirmed the presence of bioactive compounds responsible for possible capping and stabilisation of g-Ag NPs. The XRD analysis revealed that the g-Ag NPs are highly crystalline exhibiting sharp peaks for (111), (200), (220) and (311) planes in the diffraction pattern. SEM and TEM micrographs showed differently shaped Ag particles in addition to spherical shape. The average particle size of NPs was found to be 24.08 nm using imageJ analysis. EDX analysis confirmed the presence of Ag in the g-Ag NPs. In addition, the SAED pattern of g-Ag NPs presented concentric patterns for 4 major planes of crystalline silver. The d-spacing values of 0.2428 nm, 0.2126 nm, 0.1483 nm and 0.1263 nm corresponds to d111Ag, d200Ag, d220Ag and d311Ag lattice fringes respectively. The cyclic voltammetry (CV) results suggest that g-Ag NPs possess better electrochemical properties due to its lower charge transfer resistance value of 17 Ω. EIS studies too revealed better stability of g-Ag NPs as electrode materials.


Introduction
The green synthesis of metallic nanoparticles has been proposed as a cost-effective and environmentally friendly alternative to chemical and physical methods. In recent years, Ag nanoparticles (Ag NPs) have attracted much attention of researchers due to their wide applications in biotechnology, biomedicine and other industries. Because of chemical stability, good conductivity, catalytic nature, nano silver finds applications as an antimicrobial agent, in textiles, home water purification systems, medical devices, cosmetics, electronics, and household appliances [1]. In addition, due to its conducting nature, its electrochemical properties are worthy to investigate. Silver is also used in wound dressings, topical creams, antiseptic sprays and fabrics [2].
Many plants parts or whole plants have been used for the green synthesis of Ag NPs due to the presence of large number of bioactive compounds in plants. The extracts of plants and various parts of plants, Aloe fleurentiniorum plant [3], Coffea arabica seed [4], Jatropa Gossypifolia and Jatropa Glandulifera leaf [5], M. balbisiana (banana), A. indica (neem) and O. tenuiflorum (black tulsi) leaves [6], Alysicarpus monilifer leaves [7], Atrocarpus altilis leaves [8], Pimpinella anisum seeds [9] Aloe Vera plant [10], Azadirachta indica [11], olive Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
In addition, Algae Parachlorella kessleri (syn. Chlorella kessleri) was used to investigate the effect of culture age [19] and long-term stability of green Ag NPs [20]. At last the bacteria coral (Favites sp) was also used to synthesize Ag NPs for combating against urinary tract infections [21].
The silver NPs have been synthesized by using extracts of various plants found all over the globe. But no research has been conducted as far as green synthesis of Ag NPs is concerned using extracts of medicinal plants of Ethiopia. Therefore, the present research work was proposed to explore and provide an alternate green material for the synthesis of green Ag NPs (g-Ag NPs) using extracts of medicinal plant of Ethiopia. More than 95% of traditional medical preparations in Ethiopia are of plant origin [22]. A medicinal plant species of Ethiopia identified for the biogenic synthesis of g-Ag NPs in aqueous media is Hagenia abyssinica (Brace) JF. Gmel.
Hagenia abyssinica (Brace) JF. Gmel, a member of the Rosaceae family, is species of flowering plant native to the high-elevation Afromontane regions of central and eastern Africa from Sudan and Ethiopia. It is known in English as African redwood, East African rosewood and in Amharic as kosso. Hagenia abyssinica is a slender tree up to 20 m tall, with a short trunk and thick branches. The roots are cooked with meat and the soup drunk for general illness and malaria, while the dried and pounded female inflorescence is used as an anthelmintic (especially for tapeworm). Bark may be pounded, added to cold water and the liquid drunk as a remedy for diarrhea and stomach-ache. Generally, this is a strong medicine that must not be taken in large quantities; it is sometimes taken as an abortifacient [23].
The aim of this research work was to synthesize g-Ag NPs mediated by Hagenia abyssinica (Brace) JF. Gmel, medicinal plant leaf extract. The integration of bioactive compounds of this medicinal plant with Ag NPs is believed to be extremely beneficial for varieties of ailments. This green synthesis method is devised to eliminate the use of toxic chemicals which play roles of reducing agents and capping agents during chemical synthesis. The biogenically synthesized g-Ag NPs were characterized using UV-visible, UV-DRS, FT-IR, XRD, SEM, EDXA, TEM, HRTEM and SAED techniques. The electrochemical properties of g-Ag NPs have also been evaluated using cyclic voltammetry and EIS studies. The present research work gives comprehensive report on synthesis, characterization and electrochemical activities of g-Ag NPs which in turn will have significant impact on electrochemical sensor applications.

Materials and methods
All the chemicals AgNO 3 , ethanol, Dimethyl sulfoxide (DMSO) used in the experiments were of analytical grade (purchased from Merck chemical Industrial company) and used without any further purification.

Collection and authentication of plant materials
Hagenia abyssinica (Brace) JF. Gmel., plant leaves were collected from Ethiopian Institute of Agricultural Research, Wondo Genet, Oromia Regional State, Southern Ethiopia after conducting the field surveys.

Preparation of plant leaf extract
The leaves of Hagenia abyssinica plant were surface cleaned and washed repeatedly with tap water followed by distilled water to remove dust particles and then allowed to dry under shadow for 15 days to remove moisture contents from the leaves. The dried leaves were ground using grinding machine followed by packing in a brown bottle. The extraction was carried out by taking 20 g of powdered leaves of Hagenia abyssinica in a 500 ml of conical flask containing 400 ml of deionized water. The flask was later covered with aluminum foil, to prevent the effect of light. After that the mixture was shaken using mechanical shaker for 30 min and allowed to warm at 50°C for 1 h on magnetic stirrer, then it was allowed to cool down to room temperature overnight. The prepared solution was filtered through whatman No.1 filter paper to get clear solution. The filtrate was stored at 4°C for future experiments. The mixture has been incubated at room temperature for 24 h. The color change was checked periodically after 30 min of duration. The formation of reddish brown color visually indicates the formation of silver nanoparticles and then the solution was centrifuged for 15 min at 10000 rpm. The obtained g-Ag NPs (figure 1) were washed by deionized water and ethanol to remove any impurities. Thereafter, the NPs were allowed to dry and ground so as to be used for further analysis.

Preparation of carbon paste electrode
For the preparation of carbon paste electrode, the prepared sample (g-Ag NPs), graphite powder (<20 μm, 98% purity) and silicon oil (at 20°C weight is 0.98-1.0 g per ml and 370-500 mPas viscosity) with a mass ratio of 15:70:15 were systematically mixed in an agate mortar for about 30 min. The obtained paste was then filled into a Teflon cavity tube (surface area-0.3 mm) fabricated by our group. The surface of the packed carbon paste was then smoothened by pressing gently on a weighing paper [24].

Characterization
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. Fourier transform-infrared spectroscopy (FT-IR) Spectrum (65 FT-IR PerkinElmer) was recorded using KBr pellets in the range of 400-4000 cm −1 [25]. X-ray diffraction (XRD-Shimadzu x-ray diffractometer (PXRD-7000) analytical technique was used to reveal the crystalline nature of g-Ag NPs. The 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) were used for understanding morphological and structural features of g-Ag NPs. Gatan Digital Micrograph Software was used to evaluate d-spacing values of lattice fringes. Particle size was computed by using imageJ application.
Cyclic voltammetric tests were performed on a CHI608E potentiostat, using a three electrode system, comprising of carbon paste electrode, platinum wire and Ag/AgCl as working, counter and reference electrodes respectively in 6M KOH. 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.

Synthesis of g-Ag NPs
The g-Ag NPs were synthesized by using 1:4 ratio of plant leaf extract as a reducing and capping agent and silver nitrate as a precursor. The as-synthesized g-Ag NPs (figure 1) were later subjected to various characterization methods.
The presence of tannins, phenolic compounds and different glycosides was confirmed during the screening of phytoconstituents of Hagenia abyssinica (Brace) JF. Gmel. leaf extract. The details are as given in table 1. It is  possibly believed that the bioactive compounds such as polyphenols act as ligand and bind to silver ions and reduce them and cap them to form nanoparticles. These ligands also act as particle size controllers as reported by the earlier researcher [26]. Primary components of Hagenia abyssinica extract are tannins, phenols, anthraquinone glycosides and cardiac glycosides. The antioxidant properties of polyphenolic compounds are primarily due to their high inclination towards chelating the metals. Phenolic compounds contain hydroxyl and carboxylic groups which have very high tendency to bind metal ions. Metal ions in solution interact with polyphenolic compound and helps in the nucleation and formation of Ag NPs. Formation of nanoparticles is believed to be due to the combinational effect of bioactive compounds of plant extract [27].

UV-visible spectral analysis
The UV-visible absorbance spectrum recorded for g-Ag NPs exhibited λ max of 461 as shown in figure 2(a). This absorption band is basically due to surface plasmon resonance of g-Ag NPs. A similar result was reported while synthesizing Ag NPs using the Persea americana seed extract [28]. Similarly, the UV-visible diffused reflectance spectrum was recorded ( figure 2(b)). The band gap energy of g-Ag NPs was evaluated using Tauc plot as shown in figure 2(c) by using the data obtained in reflectance spectra utilizing Kubelka-Munk function. The band gap energy, E g of g-Ag NPs was found to be 2.31 eV.

FT-IR spectral analysis
The  The peaks represented by 1350 cm −1 shows C-N stretching of amide. The medium peak at 1120 cm −1 corresponds to C-O stretching of phenolic compounds. The C-O-C and C-N stretching appears at 1050 cm −1 . The last peak at 501 cm −1 corresponds to bending modes of vibrations of -C-H bond. FTIR analysis results confirmed the presence of various phytochemicals of Hagenia abyssinica (Brace) JF. Gmel. leaf extract such as phenolics, tannins and proteins involved in the synthesis of g-Ag NPs [30]. In addition to this, the FTIR spectrum of g-Ag NPs shows peaks corresponding to the broad band centered at 768 cm −1 which is possibly due to the interaction of Ag with protein molecules of extract.

Morphological and compositional analysis by SEM-EDAX
The FESEM micrographs depicted morphological features of synthesized g-Ag NPs as shown in figures 4(a) and (b). The SEM images also demonstrated the non-homogeneity of the particles in terms of their shape and size. All the possible spherical and irregular shapes such as truncated hexagonal, cylindrical, triangular, prismatic shapes of Ag NPs with varying particle sizes were found in the micrograph [31].
The average particle size of Ag NPs was found to be in the range of 8-42 nm. The chemical compositions of the NPs were studied by EDAX analysis. Figure 4(c) shows the EDAX spectrum obtained for the g-Ag NPs. The peaks corresponding to elemental Ag, C and O were clearly identified and additional peak for Cl was present, which demonstrated the purity of the synthesized NPs and this is in consistent with the XRD studies.
It is also possibly believed that the presence of C and O is basically from the capped bioactive compounds. The reduction of silver ions to Ag NPs is facilitated by the biomolecules of plant extract containing surface hydroxyl groups [32].

TEM, HRTEM and SAED analysis
To get the further deep insight on the morphology, size and crystalline nature of the g-Ag NPs, TEM, HRTEM and SAED analysis was employed.
The high resolution transmission electron microscopic (HRTEM) image of as-synthesized g-Ag NPs ( figure 5) shows that the synthesized silver nanoparticles are mostly spherical [33] but not agglomerated.
The presence of smaller NPs as small as 8 nm confirm the efficient role of bioactive components of Hagenia abyssinica (Brace) JF. Gmel, plant extract as capping agents and stabilizing agents. In addition, the variation in size of g-Ag NPs is probably due to the presence of polyphenolic compounds (from Hagenia abyssinica (Brace) JF. Gmel, leaf extract) which have strong attractive forces between and holds the particles together.
The TEM micrographs which exhibited very finely grained g-Ag NPs with spherical, cylindrical, prismatic, hexagonal, triangular, as well as near spherical shapes are presented in figures 5(a) to (d).
All these near spherical particles with varying sizes from 8.51 nm to 42.1 nm with an average particle size of 24.08 nm as determined by imageJ application are as shown in figures 6(a) and (b). The Ag NPs appeared to have no direct contact with each other even within the small aggregates, indicating efficient stabilization of the nanoparticles by a capping agents of the plant leaf extract [34]. The SAED pattern of g-Ag NPs (figure 6(c)) contained four spots each corresponding to specific crystal planes. One of such planes is presented with d-spacing value of 0.2427 nm as shown in figure 5(d).
HRTEM morphology of g-Ag NPs with magnified lattice fringes, IFFT patterns and Profile of IFFT with d-spacing value for a specified plane  Figure 8 shows the CV curve of g-Ag NPs at different scan rates. The presence of redox peaks in their cathodic and anodic scan is an indication of significant share of pseudo capacitance in the electrochemical process. The reversibility of the redox reaction is measured by the difference (ΔE a,c ) in the anodic (E pa ) peak potential and cathodic (E pc ) peak potential [35]. The anodic and cathodic peaks are correlated to oxidation potential and reduction potential E pa and E pc respectively. Oxidation potential (E pa ), reduction potential (E pc ) and the difference between E pa and E a,c (ΔE a,c ) for g-Ag NPs electrode is given in table 3. It was found that the difference in the peak potentials ΔE a,c, is as low as 0.38 V.

Electrochemical activity
In order to understand the electrode stability of the prepared oxides, a series of CV scans (of 25 cycles), at a scanning rate of 50 mVs −1 , for g-Ag NPs sample was carried out (figure 9). During the test, the locations of anodic and cathodic peaks of electrode did not show any significant deviation with the growing cycles, revealing good electrode stability [36]. Figure 10 shows the Nyquist plot of the g-Ag NPs electrode. Based on the frequency, Nyquist plot comprises of two regions, (i) a high-frequency region representing charge transfer at the electrode/electrolyte interface and     denoted by a semicircle and (ii) a low-frequency region representing the capacitance of electrode and denoted by a straight line. The charge transfer resistance R ct is a direct measure of the diameter of the semicircle arc on the real axis. It can be observed that the impedance curve of g-Ag NPs is inclined towards Y axis; this suggests a low capacitance and minimum charge-transfer resistance of prepared electrode. The resistance of the nonmaterial can be obtained using the Nyquist plot. The semicircular portion at a higher frequency is equal to the electron transfer resistance (R ct ) at the contact interface of the electrode and electrolyte solution [37]. As seen in figure 9, the semicircular diameter of g-Ag NPs is small. The value of R ct is equal to the diameter of the semicircle. The R ct value of g-Ag NPs is found to be 17 Ω. It indicates that material has considerably good conductivity and thus can be better alternative as electrode material.

Conclusion
The green silver nanoparticles (g-Ag NPs) were successfully synthesised by using medicinal plant Hagenia abyssinica (Brace) JF. Gmel. leaf extract. The presence of phytoconstituents such as polyphenols of tannins played roles of reducing and capping agents during the formation of g-Ag NPs. The UV-visible absorbance and reflectance spectra showing λ max of 461 nm and E g of 2.31 eV, respectively confirmed the formation of g-Ag NPs. FTIR spectra supported the presence of capping agents on the surface of g-Ag NPs. The crystalline nature and composition of g-Ag NPs were confirmed by XRD pattern and EDX spectrum respectively. The SEM and TEM micrographs provided enough evidence towards the nano-morphology of g-Ag NPs with all possible shapes including spherical, triangular, hexagonal and cylindrical shapes with particle size varying between 8 nm to 42 nm with average particle size of 24.08 nm. HRTEM micrographs and SAED patterns confirmed the presence of Ag. The EIS and CV studies confirmed better electrochemical behavior of g-Ag NPs with good conductivity and stability to suit as electrode materials. This green synthetic route proves to be an efficient alternative method to synthesize silver nanoparticle using medicinal plants.