Preparation of plant extracts
Fresh R. arvensis (L.) was collected in May 2011 from F. R. Bannu (32°56′ 33°16′ North latitudes and 70°22′ 70°52′ longitudes), located on the East of Bannu District, Khyber Pakhtunkhwa, Pakistan. Taxonomic identification of the plant was done by taxonomist Department of Plant Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan and Department of Botany, Government Post Graduate College, Bannu, Pakistan. The voucher specimen (AR-57) was deposited in the herbarium. The plant was rinsed with distilled water and shade dried. The extracts were prepared by soaking 30 g of ground plant powder in 300 mL of various solvents i.e. chloroform, chloroform:methanol (1:1), methanol, methanol:acetone (1:1), acetone, methanol:water (1:1) and water. They were placed in a shaking incubator (1575-2, Shel Lab., USA) at 150 rpm for 24 h at room temperature (28 ± 2°C) and sonicated for 5 min after 12 h. It was filtered with Whatmann No. 41 filter paper and concentrated in rotary evaporator (BUHI Rotavapor R-20, Switzerland) at 40°C. Fully dried extracts were packed in seal-pack containers and stored at −20°C for further experiments.
Chemicals
Aluminum chloride, ammonium molybdate, ascorbic acid (Vitamin-C), caffeic acid, catechin, dibasic sodium phosphate, 1, 1-diphenyl-2-picrylhydrazyl (DPPH), ferric chloride, Folin–Ciocalteu reagent, gallic acid, hydrogen chloride, hydrogen peroxide (H2O2), kaempferol, monobasic sodium phosphate, myrecitin, nitric acid, potassium acetate, potassium ferricyanide, quercetin, rutin sodium carbonate, sodium phosphate, trichloroacetic acid, chloroform, methanol, acetone, and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich chemical co.
DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging assay
The free radical scavenging potential of different extracts were determined according to the procedure of Kulisic with some modifications [16]. An aliquot of 50 µL of sample solution of various concentrations (25–400 μg/mL) were mixed with 950 µL of methanolic solution of DPPH (3.4 mg/100 mL). The reaction mixture was incubated at 37°C for 1 h in the dark. The free radical scavenging potential of the extracts were expressed as the disappearance of the initial purple color. The absorbance of the reaction mixture was recorded at 517 nm using UV–Visible spectrophotometer (Agilent 8453, Germany). Ascorbic acid was used as the positive control. DPPH scavenging capacity was calculated by using the following formula:
$${\text{Scavenging}}\;{\text{activity}}\;(\% ) = \left( {\frac{{{\text{Absorbance}}^{\text{control}} - {\text{Absorbance}}^{\text{sample}} }}{{{\text{Absorbance}}^{\text{control}} }}} \right)\; \times \;100$$
Hydrogen peroxide scavenging assay
The ability of the extract to scavenge hydrogen peroxide (H2O2) was determined according to the method of Ruch et al. [17]. Aliquot of 0.1 mL of extracts (25–400 μg/mL) was transferred into the eppendorf tubes and their volume was made up to 0.4 mL with 50 mM phosphate buffer (pH 7.4) followed by the addition of 0.6 mL of H2O2 solution (2 mM). The reaction mixture was vortexed and after 10 min of reaction time, its absorbance was measured at 230 nm. Ascorbic acid was used as the positive control. The ability of the extracts to scavenge the H2O2 was calculated using the following equation:
$${\text{H}}_{2} {\text{O}}_{2} \;{\text{scavenging}}\;{\text{activity}}\;{\text{percentage}} = [(A_{0} - A_{1} )/A_{0} ] \, \times \, 100$$
where: A0 = Absorbance of control, A1 = Absorbance of sample.
Phosphomolybdenum assay
For the conduction of the phosphomolybdenum assay, the method of Prieto et al. was followed [18]. An aliquot of 0.1 mL of sample solution of different concentrations (25–400 μg/mL) treated with 1 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were incubated at 95°C in a water bath for 90 min. The samples were cooled to room temperature and their absorbance was recorded at 765 nm. Ascorbic acid was used as the positive control. Antioxidant capacity was estimated by using following equation:
$${\text{Antioxidant}}\;{\text{activity }}\% \; = \;[({\text{Absorbance control }} - {\text{ Absorbance sample}})/{\text{Absorbance control}}] \, \times \, 100.$$
Reducing power assay
The reducing power was determined according to the Oyaizu et al. method with some modifications [19]. Aliquot of 0.2 mL of various concentrations of the extracts (25–400 μg/mL) were mixed separately with 0.5 mL of phosphate buffer (0.2 M, pH 6.6) and 0.5 mL of 1% potassium ferricyanide. The mixture was incubated in a water bath at 50°C for 20 min. After cooling at room temperature, 0.5 mL of 10% trichloroacetic acid was added to it followed by centrifugation at 3,000 rpm for 10 min. Supernatant (0.5 mL) was collected and mixed with 0.5 mL of distilled water. Ferric chloride (0.1 mL of 0.1%) was added to it and the mixture was left at room temperature for 10 min. The absorbance was measured at 700 nm. Ascorbic acid was used as positive control.
Determination of total flavonoid content
The total flavonoid content was determined by the aluminum chloride colorimetric method as described by Chang et al. with some modifications [20]. Aliquot of 0.5 mL of various extracts (1 mg/mL) were mixed with 1.5 mL of methanol, followed by the addition of 0.1 mL of 10% aluminum chloride, 0.1 mL of potassium acetate (1 M) and 2.8 mL of distilled water. The reaction mixture was kept at room temperature for 30 min. Absorbance of the reaction mixture was recorded at 415 nm. The calibration curve (0–8 µg/mL) was plotted using rutin as a standard. The total flavonoids were expressed as mg of rutin equivalent/gram dry weight.
Determination of total phenolic content
The amount of total phenolic content was determined according to the Velioglu method using the Folin–Ciocalteu reagent [21]. Aliquot of 0.1 mL of various extracts (4 mg/mL) was mixed with 0.75 mL of Folin–Ciocalteu reagent (10-fold diluted with dH2O). The mixture was kept at room temperature for 5 min and 0.75 mL of 6% sodium carbonate was added. After 90 min of reaction, its absorbance was recorded at 725 nm. The standard calibration (0–25 μg/mL) curve was plotted using gallic acid. The total phenolics were expressed as mg gallic acid equivalent/gram dry weight. Negative control was prepared by adding 0.1 mL of DMSO instead of extract.
High performance liquid chromatography analysis
For the analysis of flavonoids and phenolics, stock solutions of caffeic acid, catechin, kaempferol, myricetin, rutin, quercetin and gallic acid were prepared in methanol (1 mg/mL). Solutions were filtered by 0.2 µm Sartolon Polyamide membrane filter (Sartorius). The calibration curve was raised by 10, 20, 50, 100, 150 and 200 µg/mL. The crude extracts of R. arvensis were prepared at concentration of 10 mg/mL in methanol. The extracts were dissolved in methanol with the aid of sonication and were filtered through 0.2 µm Sartolon Polyamide membrane filter (Sartorius). All the samples were prepared fresh and used for analysis immediately.
The analysis was carried out by using Agilent Chem. station Rev.B.02-01-SR1(260) software and Agilent 1200 series binary gradient pump coupled with a diode array detector (DAD; Agilent technologies, Germany) having Discovery-C18 analytical column (4.6 × 250 mm, 5 µm particle size, Supelco, USA). Method followed was as described by Zu et al. with slight modification according to the system suitability [22]. Briefly, mobile phase-A was methanol:acetonitrile:water:aectic acid (10:5:85:1) and mobile phase B was methanol:acetonitrile:acetic acid (60:40:1). A gradient of time 0–20 min for 0–50% B, 20–25 min 50–100% B and then isocratic 100% B till 30 min was used. Flow rate was 1 mL/min and injection volume was 20 µL. Rutin and gallic acid were analyzed at 257 nm, catechin at 279 nm, caffeic acid at 325 nm and quercetin, myricetin, kampferol was analyzed at 368 nm. Each time the column was preconditioned for 10 min before the next analysis.
Statistical analysis
Results were expressed as mean ± standard deviation of three replicates. CoStat statistical program 6.400® (2008©, USA) was used for statistical analysis. Analysis of variance (ANOVA) was performed through Bartlett’s Test. Latin square design (LSD) was applied to testify the significance of concentrations and extracts.
