Preparation , Characterization and Antibacterial Efficiency of Olive Leaves Extract and Chitosan-Silver Nanoparticles using Electrochemical Method

The present work involves the improvement of chitosan-olive leaves extract and silver nanoparticles (Cs-OLEAgNPs) using electrochemical approach followed by UV-irradiation reduction. The formation of silver nanoparticles (AgNPs) was characterized via UV–vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), EDX, thermal gravimetric analysis (TGA) and Swelling. The obtained nanoparticles average size was 36.19 nm. UV Spectroscopy show increase of peaks at 320 and 345 nm indicate the formation of a large cluster of AgNPs. Cs-OLE and Cs-OLE-AgNPs also demon started a relativity high antibacterial against both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacteria. AgNPs show broadspectrum antibacterial activity at lower concentration (0.56%) and may be a regarded as a good alternative therapeutic approach in the future.


Introduction
Chitosan is a linear polysaccharide comprises of β-(1→4)-2-acetamido-D-glucose and β-(1→4)-2-amino-Dglucose units obtain by alkaline deacetylation of chitin which is the second most abundant organic compound beside cellulose. Chitosan has three type of function group's free amino acid, hydroxyl andacetamide group, which influence the solubility of the polymer. Chitosan has proven to be non-toxic, biodegradable, biocompatible and has an antimicrobial characteristic, so it has variety application in the medicalfields such as antitumor, wound healing and hypercholesterolemia treatment [1][2][3].
Olive tree leaves consider agriculture waste, which contains flavonoid and large concentration of phenolic acid, of which oleuropein is most abundant [4]. Olive leaves have benefit in the treatment of conditions caused by virus, bacterium andprotozoan because it has antimicrobial activity, antioxidant properties, anti HIV properties, vasodilatation effect, hypoglycemic effect and anticancer properties [5][6][7].
The recent study has concentrated recently on analysis and improvement of nanoparticles (Ag, Au, Pt and Pd) owing to their uncommon properties and potential applications in various fields including magnetic, optical, catalytic, electrical, and sensing technologies compared to bulk metals [8].
Silver nanoparticles are one of the most universal antibacterial substances, which low concentration of it gives high antibacterial activity [9]. Applications have been found for silver nanoparticles in wide range fields from electronic, antimicrobial to diagnostics and therapeutics [10].
The inexperienced synthesis technique (the electrochemical method) delineate within the gift the gift study doesn't solely represents a cheap and comparatively fast technique for developing Cs-OLE-AgNPs, however, it's additionally provide varied benefits of biocompatibility and ecofriendliness for various biological applications as all the materials utilized in the work square measure renewable and environmentally benign.
The goals of the present study are to improve a film of (Cs-OLE) and (Cs-OLE-AgNPs) and investigate characterization, antibacterial and thermal difference between them. The major aim of this study focuses on the study the change of antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) happen owing to adding silver nanoparticles by the electrochemical method to chitosan-olive leaves extract.

Materials
Chitosan powder (degree of deacetylation of 93%) was obtained from Oxford laboratory reagent (Thani, India). Tween, glycerol, ethanol and acetic acid ( EL-Naser pharamatical chemical Egypt Co. Silver plate (20 × 40 × 5 ) mm with purity 99.99%, were provided by Sigma-Aldrich (St Louis, USA). The Ag sheet was used as the anode in the cell, polished by victimization fine abrasive, clean by dimethyl ketone, ethyl alcohol (90 %) and deionized water. Platinum sheet (20 × 40 × 2) mm obtained from Sigma, were used as the cathode. De-ionized water was utilized for all the samples preparation.

Preparation of olive leaves extract
The dried olive leaves powder using a mixer grinder and 200 g portions of finely olive leaves was mixed with 80% ethanol for 2 h at 60 °C then use Ultrasonic bath for 10min, The extracts were through filter paper (Waterman No. 1) and afterwards dried overnight in an electric oven at 50 °C in order to form crude. Finally, this crude was stored at 4 °C until further analysis.

Synthesis of chitosan and olive leave extract film
Cs solution prepared with 1% (w/w) Cs in 1% (w/w) acetic acid at room temperature (T= 30 o C) and After overnight stirring, the solution filters in order to remove any traces and impurities. Then, glycerol (glycerol/chitosan = 0.5, w/w) and Tween 80 at 0.5% (w/w) were blended and stirred for 30 min. Then, olive leaves extract was added to the chitosan solution (10g/l) with stirring overnight and use centrifuge (1500rps) to remove any insoluble particles. The solution poured into glass Petri dishes and put in the oven at 50 o C for 24 h.

Preparation of chitosan-olive leaves extract and silver nanoparticles
Cs-OLE-AgNPs were obtained using electrochemical oxidation /complexation method by using a constant potential equal 1.4 V for 2 h. The cell comprises of 5 cm-separated Ag (anode) and Pt (cathode) sheets. These two electrodes were immersed with in the electrolytic solution (Cs-OLE) via (Pt) platinum wires that sealed to a glass tube followed by UV Irradiation reduction ( λ max = 254 nm at 30 o C for 1h ) [11].

Instrumentation
The UV-Vis spectra of the Cs, Cs-OLE and Cs-OLE-AgNPs measured in the range of 200-600 nm utilizing ATI Unicom UV-Vis. Spectrophotometer. The measurements completed at room temperature using quartz cuvettes and the blank is 100 ml water and 1 ml acetic acid. The FTIR spectra of the Cs, OLE, Cs-OLE and Cs-OLE-AgNPs were measured in range (400-4000) cm -1 using a Mattson 5000 FTIR spectrometerat 303 Kwith resolution of 8 cm -1 . All sample place in the sample holder of the spectrometer. The X-ray diffraction patterns (XRD) of the Cs, Cs-OLE and Cs-OLE-AgNPs samples obtain utilize Philips PW 1390 X-ray diffractometer. The X-ray diffraction provides with a beam monochromatic and CuKα radiation at λ =1.5406 A°. The applied voltage was 40 KV and the current intensity was 40 mA. Sec. The (2θ) angle was scanned in the range of 4 o to 70 o , and the X-ray runs were applied at a scanning speed of 2θ=2 o /min. DSC analysis carries out using a NETZSCH STA 409C/CD instrument,samples place into an aluminum pan then scale with a crimped lid and heat from 20 o C to 800 o C at a rate of 10 o C/min with Him as carrier gas at a flow rate (50 ml/min). It was worked in the metallurgical institute (El-Tebbeen Helwan). Thermo gravimetric analyses achieved with rate of heating at 10 o C/min.

Swelling studies
The ensuing Cs-OLE and Cs-OLE-AgNPs complexes method caste in 10 cm Petri dishes and dry at temperature, then followed by drying at 50 o C for 1days. The samples (3x4) cm, then cut from the pure metal, Cs-OLE and Cs-OLE-AgNPs samples. The thickness of the samples was existed within vary of 250-300 μm employing micrometer. The swelling behavior was studied by sinking the weighted samples in deionized water at temperature for planned periods. The weighted samples were set as operate of your time once drying it exploitation paper to get rid of water drops. The swelling of the samples was expressed because the siharevainglorous (S%) as shown in the next equation.
Where W d is the weight of the dry film (g) and W s is the weight of the swelled film at time t.

Clinical isolates
Two identified clinical isolates particularly coccus aureus (S. aureus) and Escherichia coli (E. coli) provide from biological science department, faculty of Agriculture, Mansoura University. All microorganism clinical isolates maintain habitually on culture medium slants (Oxide) at 4 o C.

Preparation of bacterial inoculum
Twenty-fourhour nutrient broth cultures of take a look at microorganism to grow in associate orbital shaking brooder, centrifuge, wash doubly with PBS and so standardize to about 106 CFU ml victimisation broth medium.

Bacterial sensitivity test
Standard well agar diffusion methodology carries bent on find the activity of Cs-OLE and Cs-OLE-AgNPs against the clinical microorganism isolates in step with Cheesbrough (2000) [12]. For antibacterial drug activities of the compounds, wells build in plates contain culture medium seed with one hundred µl of twenty-four hour of every clinical isolate. The plates were left in icebox for two hour afterwards, incubated at 37 o C for twenty-four hour. The diameter of inhibition zones was measured and tabulated.

Result and Discussion
Fourier transform infrared spectroscopy (FTIR).
FT-IR spectroscopy use to confirm the crosslinking reaction between Cs-OLE and Cs-OLE-AgNPs. Figure1 , minor broadening and peak at 890 cm -1 suggested that there was good interaction between AgNPs and Cs-OLE [19].

UV Spectroscopy
UV spectroscopy for Cs shows two peaks at 213 and 243 nm which happen occur attributed to π-system of benzene ring [20]. By adding OLE to Cs the peak at 243 nm shifted to 251 nm and other peaks appear at 275, 320,345 nm refer to OLE. In figure.3 λ max shift from 243 to 320 nm and energy gap decrease from 5.10 to 3.87eV, so chitosan and olive leave extract have lower energy difference, more conjugated system and more stable than chitosan. All of this indicates the presence of good interaction between chitosan and extract. In figure.3(c) by using electrochemical method to add AgNPs to Cs-OLE solution, there's increase in absorption of peaks at 320 and 345nm refer to formation of large cluster of silver nanoparticles and this may occur as a result of chitosan and olive leaves extract solution acidity (PH=4.1), so this may be lead to strong reducing power [21,22]. In figure. 4 there is an increase in all peaks absorption after 1 h of irradiation which suggests that expose the sample to UV irradiation leads to enhancement of interaction between chitosan, olive leave extract and silver nanoparticle (Cs-OLE-AgNPs).
The band gap can be estimated easily by this formula [23].

X-ray diffraction (XRD)
XRD in figure 5 (a)  where D is the crystallite size, n is a constant (=0.9, when the particles are spherical), λ is the wavelength, β is the line width (obtained after correction for the instrumental broadening) and θ is the diffraction angle [25].

Differential scanning calorimetry (DSC)
DSC in the figure.6 (a) shows that first peak at 125 o C may be ascribed to loss of water while the second peak at 285 o C related to decomposition of amine. For (b) and (c) show that water holding capacity of Cs-OLE is higher than Cs-OLE-AgNPs, this means there's good interaction happen between Cs-OLE and AgNPs. At Cs-OLE sample the peak appears at 45 o C is due to water evaporation and the glass transition temperature T g equal 106 o C. Due to increase crystallinity degree of Cs-OLE-AgNPs the glass transition temperature T g cannot be indicated [26].

Thermal Gravimetric Analysis (TGA)
Thermal gravity (TG) is the branch of thermal analysis which investigates the mass change of sample as a function of temperature in scanning mode. The thermal decomposition curves provide as the inflection points depict the peak maximum to determine the degradation characteristics (Table 3). Simultaneously, percentage mass loss during thermal decomposition investigates (Table 4) [27].  [27,28]. Add AgNPs to Cs-OLE lead to shift of first, third, fourth stages and disappear of the second stage as a result of decreasing water holding capacity. The high of weight loss indicates that the bio-organic material crystallizes with AgNPs [29]. The thermal decomposition of the sample contained silver is faster than sample without silver at 800 o C the residues mass equal 29.181%.

Antibacterial assay
Silver nanoparticles AgNPs have high antibacterial activity. The antibacterial activity of different samples demonstrates that both gram -positive and gram-negative bacteria were inhibited by a various solution. The results of the antibacterial activity are depicting in (table 5 and figure 8). The mechanism of antibacterial of solutions may be occur as result of interaction with lipophilic components of the bacterial membrane, which may cause varies in H + and K + permeability, and lastly, destroy the main functions and lead to death of the cell.
In addition, the antibacterial activity against S.aureus in the solution incorporated with 10 g/L OLE was remarkable higher than that incorporated with chitosan or extract alone, suggesting that there is a synergistic action.
Adding silver nanoparticle to chitosan and olive leaves extract solution by Electrochemical, the ability of chitosan, olive leaves extract and silver nanoparticles to inhibit growth greater than chitosan and olive leaves extract.

Swelling
The swelling behavior of chitosan, chitosan and olive leave extract and chitosan, olive leave and silver nanoparticle are obtained as shown in figure 9 by adding olive leave to chitosan then silver nanoparticle by the electrochemical method to sample the swelling ratio decrease this may be happen due to crosslinking with chitosan [11]. This result agrees with water capacity from DSC result.

Conclusion
FTIR, UV Spectroscopy and XRD prove that there's good interaction between Chitosan-Olive leaves extract and silver nanoparticles. Expose Cs-OLE-AgNPs to UV irradiation for 1 h enhance the interaction between the components. Large clusters of AgNPs synthesis using Cs-OLE solution with average size 36.1 nm by an electrochemical method for 2 h. Cs-OLE and Cs-OLE-AgNPs also demon started a relativity high antibacterial activity against E-coli and S-aureus bacteria. Low concentration of AgNPs (0.56%) of sample increase inhibition zone and this is a small amount.