2.1. Chemicals and Instrumental
Aluminum chloride, Folin-Ciocalteu reagent, quercetin, sodium acetate, sodium bicarbonate, butylated hydroxyltoluene (BHT), and the other solvents were prepared from Merck (Germany). Gallic acid and 1,1-diphenyl-2-picrylhedrazyl (DPPH) were purchased from Sigma-Aldrich (US). For colorimetric methods and DPPH assay, UV-visible spectrophotometer (CECIL, CE 7800, UK) was used. MHA (Muller-Hinton agar), MHB (Muller-Hinton broth) and BHIA (brain-heart infusion agar) were purchased from Merck (Germany) and all the antibiotics and antifungal agents were provided from Sigma-Aldrich (US).
2.2. Plant Material
The aerial parts of Nepeta binaludensis Jamzad were collected from Zoshk Village region in Binalood Mountains (3000 m height), in Khorasan Province, Iran on June 2014. Voucher specimen (No.48860) was deposited at the Herbarium of the Research Institute of Forests and Rangelands (TARI), Tehran, Iran.
2.3. Preparation of the Extracts
To prepare the extract, 10 g of dried powdered aerial parts of N. binaludensis was mixed with 100 mL of the following solvents separately: methanol, 50% hydro-methanol, deionized water, and ethyl acetate. The mixture was stirred occasionally in one week and then, was passed through a filter paper. The filtrate was concentrated under reduced pressure and used for assays.
2.4. Total Phenolic Contents
Total phenolic contents of the extracts were determined by the Folin-Ciocalteu method (7). Briefly, 0.75 mL of Folin-Ciocalteu reagent (10-fold diluted by deionized water) was added to 100 μL of the extracts (methanol, 50% hydro-methanol, and distilled water) (20 mg/mL) at room temperature. After 5 minutes, 0.75 mL of sodium bicarbonate (60 g/L) was added and mixed thoroughly.
The absorbance rate of each extract was measured by UV-Vis spectrophotometer after 90 minutes.
A calibration curve was plotted for the standard solutions of gallic acid (0 - 100 ppm) and total phenolic contents of the extracts were evaluated in terms of gallic acid equivalent (mg/L).
The results were expressed as mean ± standard deviation (SD) for three independent experiments.
2.5. Total Flavonoid Contents
Aluminum chloride colorimetric method was used to estimate total flavonoid contents of the extracts (8).
Briefly, 0.5 mL of each extract (methanol, 50% hydro-methanol, and deionized water) (20 mg/mL) was dissolved in methanol (1.5 mL) and then, 10% aluminum chloride (0.1 mL), deionized water (2.8 mL), and 1.0 M sodium acetate (0.1 mL) were added. The solutions were incubated for 30 minutes at room temperature. Then, the absorbance of the solutions was measured at 415 nm by UV-Visible spectrophotometer. The same procedure was used for the standard solutions of quercetin (0 - 100 ppm) and a calibration curve was plotted. The flavonoid contents of the extracts were expressed in terms of quercetin equivalent (mg/L). The results were expressed as mean ± SD for three independent experiments.
2.6. Antioxidant Activity
Radical scavenging activity of the extracts and the standard (BHT) was measured from the bleaching of the purple-colored methanolic solution of DPPH (9). For this purpose, a solution of DPPH in methanol (40 μg/mL) was freshly prepared. Then, 2.5 µL of the solution was added to 10μL of each extract. Finally, the absorbance of the test tubes was measured by the spectrophotometer at 517 nm after 30 minutes incubation in dark. To calculate the percentage of radical scavenging activity, following equation was employed:
Where As is the absorbance of sample and Ac is the absorbance of control (containing all reagents except the test sample).
The average of two independent experiments was expressed as the final result.
The IC50 value (extract concentration providing 50% inhibition) was calculated by the graph plotting inhibition percentage against the extracts concentration.
2.7. Antimicrobial Activity
Crude extracts of N. binaludensis was used to evaluate antimicrobial activity based on the macrodilution method. In this method, the ability of microorganisms to produce visible growth in the tubes containing serial dilutions of the antimicrobial agents is tested. For this purpose, 50% hydro-methanol (117 mg/mL) and ethyl acetate (64 mg/mL) extracts of the plant were used. The microorganisms evaluated in the study were Streptococcus pyogenes (ATCC 8668), Staphylococcus aureus (ATCC 25923), Salmonella paratyphi B (ATCC 1231), Escherichia coli (ATCC 11229), and a fungus Candida albicans (ATCC 10231) prepared from research center of biotechnology and industrial center of fungi and bacteria collections, Iran. The microorganisms were maintained in viable state via incubation on MHA and overnight incubation at 37°C. Broth inoculums were standardized to 0.5 McFarland by mean of dilution with sterile MHB and measuring the optical density at 620 nm wavelength. Absorbance readings were set to 0.09 - 0.13 (~ 1.5 × 108 CFU/mL).
The standard antibiotics, including tetracycline, ampicillin, kanamycin (100 mg/mL), and an antifungal agent as nystatin (2.5 mg/mL), were used as positive controls. To determine the possible inhibitory activity of solvents on the tested bacteria, 50% hydro-methanol and ethyl acetate were used simultaneously as positive controls.
A twofold serial dilution of each extract was prepared in 12 tubes containing 1 mL Muller-Hinton broth; then, 0.1 mL of bacterial suspension with 0.5 McFarland turbidity was added to all the tubes, except the tube No. 12 (as the control). Then, the tubes were incubated at 37°C for 24 hours. To determine the MBC (minimum bacterial concentration, the lowest concentration of an antibacterial agent required to kill a particular bacterium), 25 μL of each dilution was incubated at 37°C in BHIA for 24 hours. The highest dilution (least concentration) that inhibited colony formation on this solid medium was considered as MBC (10).
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