SUPPLEMENTARY MATERIAL
Green Synthesis of Silver Nanoparticles using Eucalyptus leucoxylon extract and
Evaluating the Antioxidant Activities of Extract
Mehdi Rahimi-Nasarabadi1*, Seyed Ataollah Sadat Shandiz2, Seied Mahdi Pourmortazavi3,
Farhad Ahmadi4, Hossein Batooli5
1
Center of Nano Science, Imam Hossein University, Tehran, Iran
2
Department of biology, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran
3
Faculty of Material and Manufacturing Technologies, Malek Ashtar University of Technology, Tehran,
Iran
4
Department of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah University of Medical Sciences,
Kermanshah, Iran
5
Isfahan Research Center of Natural Sources and Agriculture, Kashan Station, Kashan, Iran
Abstract:
This study was designed to examine in vitro antioxidant activity of essential oil and methanol
extracts of Eucalyptus leocoxylon. Furthermore the polar fraction of extract was used as a
reducing agent for green synthesis of silver nanoparticles (Ag NPs). Antioxidant activities of the
samples were determined by three different test systems namely DPPH and β-carotene/linoleic
acid and reducing power. The aqueous extract of the spices was used as reducing agent for
synthesis of Ag NP. The structure and composition of the prepared Ag NPs were characterized
by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron
microscopy (TEM) and UV-Vis spectroscopy. Biosynthesized Ag NPs was almost spherical in
shape with average diameter of about ~ 50 nm were synthesized within 120 min reaction time at
room temperature.
Keywords: Eucalyptus leocoxylon, Antioxidant activity, Silver nanoparticles, Green synthesis
*Corresponding
author:
Tel.:+98
2177104930;
rahiminasrabadi@gmail.com(M. Rahimi-Nasrabadi)
1
fax:
+98
2177104930.
E-mail:
Experimental (materials and methods)
Materials
Plant material
The leaves of E. leocoxylon were collected in Sep. 2009 from Kashan (Isfahan province,
Iran). The leaves were dried in the shade (at room temperature). A voucher specimen of the plant
was deposited at the Herbarium of Kashan botanical garden (with voucher specimen number of
KBG 1500).
Chemicals
Linoleic acid, 2,6-di-tert-butyl-4-methylphenol (butylated hydroxyl toluene, BHT), 2,2Diphenyl-1-picrylhydrazyl (DPPH, 95%), gallic acid and β-carotene,
were procured from
Sigma–Aldrich Chemie (Steinheim, Germany). Analytical grade methanol, ethanol, and dimethyl
sulphoxide (DMSO), HPLC grade chloroform, standard Folin–Ciocalteu’s phenol reagent,
anhydrous sodium sulphate, ferric chloride, sodium carbonate, potassium ferricyanide, Phosphate
buffer solution (PBS), silver nitrate (AgNO3) and Tween 40 were obtained from Merck
(Darmstadt, Germany).
Preparation of the methanol extract
Thirty grams of the dried and powdered plant materials were extracted with methanol by using
Soxhlet apparatus at 60 ˚C for 12 h. The extract was filtered and concentrated under vacuum at
40 ˚C by using a rotary evaporator (Heidolph, Laborota 4000, Schwabach, Germany), yielding a
waxy material (2.5 g, 8.3% w/w). This extract was suspended in water and extracted with
chloroform (4 × 100 ml) to obtain 1.4 g (4.7%) polar and 1.0 (3.3%) nonpolar extracts. The
extract was stored in the dark at 4 ˚C until used within a maximum period of one week. The polar
subfraction of methanol extract dissolved in distilled water has been used for synthesis of Ag
nanoparticle.
Synthesis of silver nanoparticles
2
Aqueous solution (0.01 M) of silver nitrate (AgNO3) (Merck) and polar subfraction of methanol
extract (5.0 mg/mL) was prepared and used for the synthesis of silver nanoparticles. 5.0 mL of
ammonia solution was added to 50 ml of AgNO3 solution and then 4 ml of polar subfraction of
methanol extract (5.0 mg/mL) solution was added to the above mixture. The reaction was carried
out at room temperature. The reaction flask was covered with aluminium foil. The experiments
were performed in room temperature at 27 °C and stirring rate of electrolyte solution was 500 rpm.
Addition of E. leocoxylon extract to aqueous solutions of AgNO3 changed the solution from
colorless to yellowish and finally to dark brown. The reduction process of Ag+ ions was
monitored at fixed intervals based on surface plasmon resonance by UV visible spectrometer at
430 nm. Shape and size of the produced nanoparticles was determined using scanning electron
microscope
Characterization of silver nanoparticles
All samples were characterized using scanning electron microscopy (SEM) by a Philips XL30
series instrument using a gold film for loading the dried particles on the instrument. Gold films
were prepared by a sputter coater model SCD005 made by BAL-TEC (Switzerland).
Determining the particle size distribution of the sample suspended in distilled water was
performed by dynamic light scattering (DLS) using Malvern instrument (England). UV–Vis
absorption spectra of the samples dispersed in distilled water in the range 200–700nm, were
recorded using a Perkin–Elemer Lambda 35 UV–Vis spectrophotometer. The IR spectra were
acquired by an FT-IR spectrophotometer (Perkin–Elmer Spectrum 100) using KBr pellet
technique. X-ray powder diffraction (XRD) analysis was carried out using a Rigaku D/max 2500
V diffractometer equipped with a graphite monochromator and a Cu target.TEM image was
obtained using a transmission electron microscope (Ziess- EM900). Prior to the measurement,
the sample was treated with coating on the Cu-carbon coated grid.
Antioxidant properties
Scavenging capacity on DPPH radical
3
The free radical scavenging activities of extracts were measured by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as described by Gholivand (Gholivand, Rahimi-Nasrabadi, Batooli 
Ebrahimabadi, 2010). BHT and ascorbic acid were used as positive standards in this test.
β-Carotene linoleic acid assay
The antioxidant activity was evaluated according to the method described by Gholivand
(Gholivand, Rahimi-Nasrabadi, Batooli  Samimi, 2011). Briefly, 1.5 mL of β-carotene solution
(1 mg/ mL in chloroform), 3 mL of linoleic acid solution (10 mg/mL in chloroform), and 1.0 mL
of Tween 40 solution (300 mg/mL in chloroform) were pipetted into a 250 mL flask. The
chloroform was removed by rotary vacuum evaporator, and 150 mL deionized water was added
to the residue and the mixture was shaken to form an emulsion. 350μl of test sample in methanol
(2 mg/ml) was mixed with 2.5 mL of this reagent, and the emulsion system was incubated for up
to 48 h at room temperature. The same procedure was repeated with the synthetic antioxidant,
BHT as positive control, and a blank containing only 350 μl of methanol. After this incubation
period, absorbance of the mixtures was measured at 490 nm. Antioxidative capacities of the oil
and the extract were compared with those of BHT and blank (RAA = Asample/ABHT).
Reducing power
Different concentrations of methanolic extract and oil of plant in methanol (1.0 ml) were mixed
with 2.5 ml of phosphate buffer (200 mM, pH 6.6) and 2.5 ml of 1% potassium ferricyanide, and
the mixture was then incubated at 50˚C for 20 min. Then, 2.5ml of trichloroacetic acid (10%)
was added to the mixture to stop the reaction, the mixture was then centrifuged at 3000 rpm for
10 min. 2.5 ml from upper layer was mixed with 2.5 ml of de-ionized water and 0.5 ml of 0.1%
ferric chloride, and the absorbance was measured at 670 nm against a blank. The assays were
carried out in triplicate and the results were expressed as mean values. Increased absorbance
values indicate a higher reducing power.
Determination of total phenolic contents
4
Total phenolic contents of the extract and the oil were determined using the Folin–Ciocalteu
reagent according to the method of Singleton and Rossi (Singleton  Rossi, 1965) using gallic
acid as standard, with some modifications. The oil or the extract solution (0.1 ml) containing
1000 μg of the oil and/or extract was mixed with 46 ml of distilled water in a volumetric flask
and 1 ml Folin–Ciocalteu reagent were added, and the flask was thoroughly shaken. The mixture
was allowed to react for 3 min and 3 ml aqueous solution of 2% Na2CO3 was added. At the end
of incubation of 2 hours at room temperature, absorbance of each mixture was measured at 760
nm. The same procedure was also applied to the standard solutions of gallic acid and a standard
curve was obtained. Total phenol contents were expressed as µg gallic acid equivalents per mg
of the extract or the oil. All tests were carried out in triplicate and gallic acid equivalent values
were reported as means ± SD of triplicates.
5
References
Gholivand, M.B., Rahimi-Nasrabadi, M., Batooli, H., Ebrahimabadi, A. H. (2010). Chemical
composition and antioxidant activities of the essential oil and methanol extracts of
Psammogetoncanescens.Food and Chemical Toxicology 48, 24–28.
Gholivand, M.B., Rahimi-Nasrabadi, M., Batooli, H., Samimi, M. (2011). Chemical composition
and
antioxidant
activity
of
the
essential
oil
and
various
extracts
of
HaplophyllumrobustumBge. Nat. Prod. Res.26, 883-891.
Singleton, V.L., Rossi, J.A., (1965). Colorimetry of total phenolics with phosphomolybdic–
phosphotungstic acid reagents. Am J Enol Vitic, 16, 144-158.
6
Fig .S1. Reducing power for different concentrations of essential oil and methanol extract of E. leocoxylon
compared to ascorbic acid, (Spectrophotometric detection of the Fe+3–Fe+2 transformations). EO: essential
oil; PS: polar subfraction of methanol extract; NS: nonpolar subfraction of methanol extract; AA: ascorbic
acid.
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Fig.S2. UV-Vis absorption spectra of the Ag NPs recorded after 10, 20, 30 and 60 min.
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Fig.S3. XRD pattern for synthesized Ag NPs by green method via green synthesis method
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Fig.S4. SEM image of Silver nanoparticles prepared via green synthesis method
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