Biosynthesis of Silver Nanoparticles Using Eucalyptus Bicolor Bark and their Antimicrobial Activity

The current study examined the production of silver nanoparticles from silver nitrate(AgNO3) solution with the extract of Eucalyptus bicolor bark and the antimicrobial activity of the silver nanoparticles versus microorganisms. The silver nanoparticle was characterized by its color changes of extract, UV-Visible spectroscopy and SEM. The useful groups for alcoholic, extract were recognized by using Fourier transform infrared spectroscopy analysis (FTIR). In addition to we compared, between the biological effectiveness of alcoholic and nano extract of Eucalyptus bark against three types of pathogenic microbes: Staphylococcus. aureus, (Gram +ve), Pseudomonas, aeruginosa (Gram-ve), and yeast, Candida, albicans . Alcoholic extract offered the higher effect with Candida by inhibition zone of 14mm , S,aureus, of 13mm and pseudomonas of 10mm , While the nano extract has showed that the highest effect on growth of microorganism, Candida by inhibition zone of 20mm , staph of 19mm, and pseudomonas of 11mm .Furthermore,the combined effect of some antibiotics and alcoholic extract was studied against the same pathogenic microorganisms and the results showed that both :synergism and antagonism effects were seen, Carpenciline and the extract exhibited synergistic effect as evidenced by the increment of antimicrobial , activity with all bacteria(synergism) while Amoxicilline and the extract exhibited antagonistic effect against S.aureus (antagonism), and synergistic effect against P. aeruginosa


Introduction
Plants have been used in popular medicines for several thousand years and world health organization WHO expected that 80% of persons used popular medicines for their healthiness (1).Medical plants are thought to be a main source of chemical materials including beneficial effects (2).Eucalyptus be a part to the family mytaceae It is an active growing tree, aromatic evergreen tree and several natural substances having antagonistic activities against several microorganisms (2).The bark of the Eucalyptus plant was used in cure of ulcers, injuries, and other diseases (1). the ethanolic extracts from plants used for, antiviral, antimicrobial and antitumor agents (3).Nanotechnology increases much care among the students for the properties distribution, size, and morphology of nanomaterials (4).Biological means are used to production of nanoparticales using, an ecofriendly (green synthesis) method by using silver ion (5,6).Silvernanoparticles have showed to be greatest active as it has good anti-microbial effectiveness against Viruses, bacteria, and other micro-organisms (7).Some recommendations have been advanced about the effect of silver ion against bacteria and all opinion to the heavy metal effect on protiens,DNA,and respiratory enzyme that which leads to inactivation of the proteins and DNA (7).The objective of the present study was to produce silver nanopaticles from bark extract of Eucalyptus bicolor and exam the antimicrobial effect of the nanoparticles against bacteria and yeast, in order to find an alternative of antimicrobial agent by joining the antimicrobial properties of Eucalyptus bicolor bark extract with antibiotics in order to maximize the antimicrobial effect against bacteria and yeasts.

Materials and Methods
Sample of plant: E.bicolor bark were taken from; local, garden cleaned , dried for extract.

plant extract:
Fifty g of dried material was extracted by addition 250 ml of ethanol (70 %) using soxholet apparatus for two days.Next extract solution was filtered and the solvent was detached by rotary evaporator to get the crude extract.After that extract was saved in refrigerator at 4 °C for until use.

Microorganisms:
The examined microbial strains were obtained from microbiology laboratory Applied Science department University of Technology these involved Candida albicans pseudomonas aerugenosa and staph aureus.

Preparation of silver nanoparticles
Ten milliliter of E.bicolor bark extract was mixed with 90 mL of 0.01 mmol/mL aqueous The silver nanoparticles produced using E. bicolor bark extract was tested by agar well diffusion method against C. albicans (yeast), P. aeruginosa> (gram negative) and S. aureus> (gram positive).the pure cultures of the organism were subcultured on muller-hinton agar.
every strain was spreaded on the plates by spreader.wells were made on plates by gel puncture.50 µl of extract (10,20,40,60,80 and 80mg, concentration respectively) was transferred onto each well on all plates.disc diffusion method was used for evaluating the antimicrobial activity of antibiotic with ethanolic extract.antibiotic discs were applied to the inoculated MHA plates another collection of these antibiotic discs were soaked with ethanolic extract disc then located on the inoculated plates.plateswas incubation at 37 °c for overnight at the end the diameter of inhibition zone was calculated in millimeter and The tests were done in triplicate.

Descriptions of Silver Nanoparticles
Produced nanoparticles were check by measuring the UV-visible spectroscopy, UV-Vis spectral analysis was determined by using (TechcompUV2300) spectrophotometer.The morphology of the nanoparticles was investigation by Scanning Electron Microscope (SEM).

Results and discussion
UV-visible spectrum analysis: The E.bicolor bark extract was yellowish in colour , after addition of silver nitrate solution and showing to sunlight for 1 h turned reddish brown (Figure 1) .Creation of nano silver was established using UV-vis spectral analysis and displayed silver surface plasmon resonance band at 410 nm (Figure 2) .

Fourier Transform Infrared Spectroscopy (FTIR).
Fourier transform infrared spectroscopy is often used to characterize the beneficial groups.
The FTIR exhibited the presence of bands at 3427.62 cm-1 as a result of phenolic OH.The  Table (1) showed the antimicrobial effect of both ethanolic and nano extract was more marked against bacteria G+ve than bacteria G-ve and nano extract exhibited good inhibition activity against C. albicans, that's may be because the differences of the cell wall structure between Gram positive and negative bacteria (11,12) .The mechanism of the antimicrobial effect of nanosilver remains unclear,but Some studies suggest that the great affinity of Ag+ to phosphorus and sulfur and affects on microorganisms (13).Because of the bacterial cell membrane containing abundance of sulfur-proteins Ag+ can react with sulfur-containing amino acids inside the cell membrane and affects bacterial cell viability (14,15).Ag + can interact with phosphorus moieties in DNA and increases DNA mutation (15).as well as, nanosilver interacts with NADH dehydrogenase resulting in the separation of respiration from ATP synthesis (15).

Antimicrobial activity of antibiotics:
Eucalyptus bicolor ethanolic extract had antimicrobial activity on the bacteria and yeast when combined with different antibiotics.Disc diffusion method was used for evaluating the antimicrobial activity.Table (2) shows the inhibition zones diameter of antibiotics with and without Eucalyptus bicolor extract against bacteria (G ve+ ,G ve-)and yeast .The mixture of antibiotics with Eucalyptus bicolor extract had synergism and antagonism effect against microorganisim.Carpencilin with Eucalyptus bicolor extract exhibited an increase in its antimicrobial activity : S.aureus from (7mm) to (15mm), P.aerugenosa from (nil) to (12).
Amoxicillin when joined with Eucalyptus bicolor extract showed decrease in its antimicrobial against S.aureus from ( 15) to ( 14), but it exhibited an increase against P.aerugenosa from (nil) to ( 10) Fig (6).2) exhibited antimicrobial effect of antibiotics was more marked against gram-positive bacteria than gram-negative that's may be because the differences of the cell wall structure between Gram positive and negative bacteria (11,12).Mixture of Eucalyptus bicolor extract with antibiotics exhibited synergistic affect, in most tests, table (2,3) The mechanism of the synergistic effect unclear, but, studies suggest that Plant antimicrobials not have a strong antimicrobial activity alone, but when they were taken with antibiotics they increase the effect of that antibiotics (16,17).Eucalyptus bicolor extract has the phenols, flavonoids, tannins and alkaloids.Alkaloids have ability association with bacterial DNA leading to destroy it, while phenol effect on microbial enzyme leading to missing their function (1,2).An antagonistic effect was seen in some of the tests, that's may be because of several compounds that inhibited the antimicrobial activity of the effective compounds as well as some compounds interfered with crude extract that may have affected on the act of one another, therefore purification and separation, of the crude extract will increase in bioactivity than the crude extract (18,19,20).

Figure 1 :
Figure 1: synthesis of silver nanoparticles using E.bicolor bark extract

Vol: 14 Figure 6 :
Figure 6: Combined effect of antibiotics and ethanolic extract of Eucalyptus bicolor selective pathogens.against some E: extract AM: Amoxicillin, CAR: Carpencilin

Table 1 :
Antimicrobial activity of the ethanolic and nano bark extract of E.bicolor Concentration mg

Table 2 :
Antimicrobial activity of antibiotics and combination of ethanolic extract of Eucalyptu

Table 3 :
Percentage of synergistic/antagonistic effect of combination of Eucalyptus bicolor ethanolic