Plant materials Aerial parts of U. urens were collected from the province of Bizerte in Noth-Africa during the spring 2010 which were the main plant material of the study.
Preparation of the plant extracts Aerial parts of U. urens were washed and dried in the open air at room temperature, frozen and grounded in liquid nitrogen. The resulting powder was used for the preparation of methanol (EMO) and acetone (ECO) extracts. The powder obtained was macerated in the appropriate solvent (like methanol and acetone) under stirring for 30 min at room temperature. The mixture was left at 4°C for 24 h to be subsequently filtered through a Whatman paper (No.4 Sigma Aldrich® ). The filtrate is collected and stored in dark at 4°C for next use.
Preparation of the aqueous extract In order to avoid any controversial contradiction between the effect of the solvent and the effect of the nettle itself, the antibacterial assay was conducted with the water extract (EAO) of nettle. To prepare the water extract plant powder was mixed with the boiled water and centrifuged at 4000 g for 20 min and finally filtered on filter paper. The filtrate was then stored at 4°C for further use.
Determination of total phenolic contents The assay for the determination of total phenolic content in U. urens was performed according to the method of Namjooyan et al. (2007). Amounts of 1.85 ml of distilled water, 0.125 ml of FolinCiocalteu reagent and 0.5 ml of a 20% sodium carbonate (Na2CO3 ) solution were added to 25 µl of liquid extract sample in a test tube, making a final volume of 2.5 ml. The solution was homogenized and left to stand for 30 min, and the absorbance was determined at 750 nm. The total phenols were calculated as milligrams of gallic acid equivalents per gram of dry matter (DM).
Determination of total carotenoid: Determination of total carotenoid was performed according to the method of Wellburn (1994) with few modifications. 50 mg of fresh plant material was ground in 10 ml of acetone (80%). The obtained extract was centrifuged and filtered. The reading of the absorbance is carried out at three different wavelengths of 470, 663 and 647 nm and the concentration is determined as follows: Carotenoid = 5 × DO470 + 2.846 × DO663 -14.87 × DO647 The result is expressed in mg per 100 g dry matter (DM).
Determination of total flavonoids The principle is based on the formation of the complex flavonoids-aluminum chloride (Djeridane et al., 2006; Chang et al., 2002). The color intensity is proportional to the concentration of the flavonoids. A volume of 250μL of the extract is diluted 5 times, supplemented with 75 μL of a solution of Na NO2 (5%), followed by a rest of 6 min before adding 150 μL of aluminum chloride (AlCl3 , 6H2O, 10%) after 5min of incubation, 500 μL of NaOH (1 M) are added. The final volume of the solution is adjusted to 2500 μL with distilled water. The absorbance of the mixture was read at 510 nm. The reference range is prepared with the catechin. The content of total flavonoids expressed as mg catechins per gram of dry matter (DM).
Determination of condensed tannins In the presence of concentrated sulfuric acid, condensed tannins depolymerize and transform in the presence of vanillin anthocyanidols red color whose intensity is measured spectrophotometrically at 500 nm. A volume of 3 ml of vanillin (4%) was added to 50 µl of the extract and 1500 µl of concentrated HCl. The resulting mixture was incubated for 15 min at room temperature and the absorbance is measured at 500 nm. The standard is like catechin flavonoids, condensed tannins content in mg catechin equivalents per gram of dry matter (mg CE / mg DM).
Determination of total anthocyanins Plant powder of 1 g was mixed with 12 M HCl (1%). The mixture is incubated at a temperature of 4°C for 48 h with continuous stirring followed by centrifugation and filtration of supernatant. The absorbance is read at two different wavelengths (530 nm and 675 nm). The concentration of anthocyanins was expressed in g per 100 g of dry matter (DM).
Total antioxidant capacity: Total antioxidant capacity of the extract was evaluated by the method of phosohomolybdenum described by Prieto et al. (1999). A volume of 1ml of a solution (0.6 M sulfuric acid, 28mM sodium phosphate and 4 mM ammonium molybdate) is added to 100 µL of the 10 times diluted extract. The mixture obtained is incubated at 95°C in the dark for 90 min. The absorbance reading was taken at 695 nm against a blank containing the solvent and the reagent. A standard range is prepared and the results are expressed in mg gallic acid equivalent per range of dry matter (DM).
Free radical scavenging activity The free radical scavenging activity of the nettle extract was measured by 1,1-diphenyl- 2picryl-hydrazil (DPPH) was evaluated using colorimetric method of Shimada et al. (1992) and improved by Islam et al (2013). At room temperature and in the presence of an antioxidant, the DPPH radical has an intense purple color. The transition to non-radical form DPPH (after saturation of its electronic layers) was accompanied by the disappearance of the violet color (Soler-Rivas et al., 2000). The decrease in the intensity of the color is monitored by spectrophotometry at 517 nm. The estimation of this activity was measured. The free radical scavenging activity of each sample is expressed as percent inhibition of DPPH using the following formula:
DPPH = [(A0-A)/A0] × 100 Where, A0 is absorbance at 517 nm DPPH without extract and A is sample absorbance.