SUPPLEMENTARY MATERIAL
Evaluation of the antioxidant and antibacterial properties of ethanol
extracts from berries, leaves, and stems of Hedera pastuchovii Woron.
ex Grossh.
Robabeh Baharfar1,*, Zahra Rahmani1, Mojtaba Mohseni2, Razieh Azimi1
1
Department of Organic Chemistry, Faculty of Chemistry, University of Mazandaran,
Babolsar, Iran
2
Department of Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran
*
Corresponding author, Fax: +98 1125342350; E-mail: baharfar@umz.ac.ir
Abstract
This study was designed to examine the total phenolic and flavonoid contents,
radical scavenging and antibacterial activity of the ethanolic extracts from leaves,
berries, and stems of Hedera pastuchovii. The berry extract which contained the
highest phenolic and flavonoid compounds, showed an appreciable DPPH•
scavenging ability in comparison with leaf and stem extracts. The various extracts
exhibited moderate to good activity against both Gram-negative and Gram-positive
bacteria and the effectiveness of leaf extract was higher for all tested bacteria.
Keywords: Hedera pastuchovii; phenolic content; antioxidant; antibacterial
3. Experimental
3.1. Chemicals
Folin-Ciocalteau, gallic acid, quercetin, ascorbic acid, butylated hydroxytoluene
(BHT) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich
Chemical Company. All other reagents and organic solvents were obtained from
commercial suppliers and used without further purification.
3.2. Plant material
Hedera pastuchovii Woron. ex Grossh. was collected from Bandpay region of
Mazandaran, north of Iran, in Nov. 2013. The plant was identified and authenticated by
Dr. A. Naqinezhad at Department of Biology, University of Mazandran, Babolsar, Iran.
A specimen is deposited in the Herbarium of this Institution, under the voucher number
5201.
3.3. Preparation of ethanol extracts
Approximately 50 g of the powdered sample was placed in a Soxhlet extractor
and extracted with ethanol for 8 h. The solvent was recovered by distillation in vaccuo,
and the residue was stored at -10 °C for subsequent experiments.
3.4. Total phenolic contents (TPC)
The TPC of the extracts were determined spectrophotometrically by FolinCiocalteu reagent (Meda et al., 2005) and calculated in terms of mg gallic acid
equivalent (GAE) / g dry extract, with reference to standard curve (y = 0.0054x +
0.0628, R2 = 0.987). Accordingly, 0.5 mL of extract (0.2 mg mL-1, in methanol) was
mixed with 2.5 mL of 0.2 N Folin-Ciocalteau reagent at room temperature for 5 min
and 2.0 mL of 75 g L-1 sodium carbonate was then added. The absorbance of reaction
mixtures was measured at 760 nm using UV-vis spectrometer (Braic 2100 Double
Beam) after 30 min of incubation at room temperature. Total phenol values were
presented as means of triplicate analysis.
3.5. Total flavonoid contents (TFC)
Total flavonoid content was estimated using aluminum chloride method (Meda
et al., 2005). Accordingly, 0.5 mL of extracts (0.2 mg mL-1) was separately mixed with
1.5 mL of methanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1.0 M potassium
acetate and 2.8 mL of distilled water and left at room temperature for 30 min. The
absorbance of the reaction mixture was measured at 415 nm with a double beam
spectrophotometer. TFC were calculated as mg quercetin equivalent (QE) / g dry
extract with reference to standard curve (y = 0.0063x, r2 = 0.999), and presented as the
means of triplicate analysis.
3.6. DPPH radical scavenging
The stable 1,1-diphenyl-2-picryl hydrazyl radical (DPPH•) was used for
determination of free radical-scavenging activity of the extracts (Blois, 1985). 2.0 mL
of different concentration of extracts (50-200 µg mL-1, in methanol) were added to 2.0
mL of methanol solution of DPPH (0.1 mM). The tubes were shaken properly and
incubated for 30 min in the dark. The changes in the absorbance of the samples were
measured at 517 nm using a spectrophotometer. Ascorbic acid and BHT were used as
standard controls for comparison. IC50 values denote the concentration of sample,
which is required to scavenge 50% of DPPH free radicals.
3.7. Antibacterial activity
The antibacterial activity of the extracts was assayed onto LB medium
contained: bactoTM tryptone, 10.0 g L-1; yeast extract, 5.0 g L-1; NaCl, 5.0 g L-1;
glucose, 1.0 g L-1; and agar 12.0 g L-1 (Mohseni et al., 2014). The medium was
dispersed into universal bottles and sterilised at 121 ◦C for 15 min. The extracts were
dissolved into dimethyl sulfoxide (DMSO) and added into LB medium to give a final
concentration of 1-300 g mL-1 as required. The antibacterial activity of the extracts
was also compared with known antibiotic tetracycline and DMSO as a negative control
at the same concentration (Lakouraj et al., 2013). Minimum inhibitory concentration
(MIC) of the extracts was assayed using a standard method against some bacteria
including Escherichia coli PTCC 1330, Pseudomonas aeruginosa PTCC 1074,
Staphylococcus aureus ATCC 35923 and Bacillus subtilis PTCC 1023. Late
exponential phase of the bacteria was prepared by inoculating 1% (v/v) of the cultures
into the fresh LB medium and incubating on an orbital shaker at 37 ◦C and 100 rpm
overnight. Before using the cultures, they were standardised with a final cell density of
approximately 108 cfu mL-1. A 1% (v/v) inoculums of each culture was inoculated into
the LB medium containing different concentration of the extracts and incubated on the
orbital shaker at 37 ◦C and 100 rpm. The extracts sensitivity of the strains was assayed
for positive or negative growth after 24-48 hours.
3.8. Statistical analysis
Experimental results were mean ± SD of three parallel measurements and
analyzed by SPSS 18. Differences between means were determined using ANOVA and
Tukey multiple comparisons and least significant difference (LSD). p values ≤ 0.05
were regarded significant.
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