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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

1Department of Organic Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran

2Department of Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran

*Corresponding author, Fax: +98 1125342350; E-mail:

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

1. Experimental
2. 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 Folin-Ciocalteu 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.

References

Blois, M. S. (1985). Antioxidant determinations by the use of a stable free radical. Nature, 181, 1199-1200.

Meda, A., Lamien, C. E., Romito, M., Millogo, J., & Nacoulma, O. G. (2005). Determination of the total phenolic, flavonoid and praline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chemistry, 91, 571-577.

Lakouraj, M. M., Rahpaima, G., & Mohseni, M. (2013). Synthesis, characterization, and biological activities of organosoluble and thermally stable xanthone-based polyamides. Journal of Materials Science, 48, 2520-2529.

Mohseni, M., Khosravi, F., Mohajerani, M., & Chaichi, M. J. (2014). Bioremediation activity of Pb (II) resistance Citrobacter sp. MKH2 isolated from heavy metal contaminated sites in Iran. Journal of Sciences, Islamic Republic of Iran, 25, 105-110.