Extract analysis

Screening tests for flavonoids, alkaloids, tannins, saponins, cardiac glycosides and sterols were done based on the standard methods as follows:

Flavonoids of the extracts are often detected by Cyanidin test. One gram of extract was dissolved in methanol (50%), HCl (37%) powder of magnesium and amyl alcohol (50%). Flavonoids appeared as orange to red zone. Extract of the Chamomile flowers was used as a standard of flavonoids (positive control) (Sharifzadeh et al. 2006).

For the detection of alkaloids, three drops of Dragendroff reagents were added to the fraction that is separated by using HCl, NaCl, methanol and chloroform from 1 g of extract. Alkaloids appeared as brown, blue or whitish zone. A thin-layer chromatogram (TLC) spot test with Dragendroff, Wagner and Mayer’s reagents was performed for checking the results (Sharifzadeh et al. 2006).

Plant extract (about 1.0 g) was stirred with sterile-distilled water (10 ml) and filtered (using Whatman number 1 filter paper). A blue colouration resulting from the addition of two drops of 10% FeCl₃ reagent to the filtrate indicated the presence of tannins (pseudo tannins) (Abioye et al. 2013).

Also, according to the standard protocol, tannins were detected by adding NaCl (10%) and gelatin (1%) to the dissolved fraction of 1 g of extract in 20 ml of boiled water. The amount of precipitation showed the presence of tannins. As a standard positive control, the extract of Quercus infectoria fruits was used for tannins evaluation (Sharifzadeh et al. 2006).

For saponins determination, the height of the foam produced after shaking the extract (1 g) in distilled water (10 ml) is one of the standard ways to determine the amount of saponins. Liquiritiae radix root extract was used as a standard for saponins (Sharifzadeh et al. 2006).

The extract (about 0.5 g) was dissolved in glacial acetic acid (2 mL) containing 1 drop of 1% FeCl₃. This was underlaid with concentrated H₂SO₄. A brown ring at the interface indicated the presence of a deoxy sugar, a characteristic of cardiac glycosides. A violet ring may form just above the brown ring and gradually spreads through this layer (Abioye et al. 2013).

Both Salkowski test and Liebermann–Burchard test were performed.

2 ml of chloroform and 2 ml of concentrated H₂SO₄ was added to 2 ml of plant extract, and shaken well. The chloroform layer appeared red and the acid layer greenish yellow fluorescent. This confirms the presence of sterols.

2 ml of methanolic plant extract was mixed with chloroform. About 1–2 ml acetic anhydride and two drops of concentrated H₂SO₄ from the side of the test tube was added in the mixture. First red, then blue and finally green colour indicates the presence of sterols (Rimjhim et al. 2014).

Preliminary Phytochemical screening showed that Sterols, Flavonoids, pseudo tannins (FeCl₃ test), Saponins and Cardiac glycosides are present in the extract, while Tannins and Alkaloids are absent.

Total phenols were determined using Folin–Ciocalteu reagent as described by (Velioglu et al. 1998) with slight modifications. The extract (200 μL) was mixed with 1.5 ml of Folin–Ciocalteu reagent (previously diluted 10 times with distilled water) and allowed to stand at room temperature for 5 min. 1.5 ml sodium bicarbonate solution (60 g/L) was added to the mixture and after incubation for 90 min at room temperature, the absorbance level was measured at 750 nm using a UV–Visible spectrophotometer. Total phenolics were quantified by calibration curve obtained from measuring the absorbance of the known concentrations of Gallic acid standard solutions (25–150 μg/mL in 80% methanol). The results were calculated as Gallic acid equivalent (GAE) per 250 μg dry extract (Velioglu et al. 1998; Jia et al. 1999; Shams Ardekani et al. 2011).

Total flavonoids content was measured by the aluminium chloride colorimetric assay. An aliquot (1 mL) of extracts or standard solution of catechin (50, 100, 150, 200, 250 and 300 mg/L) was added to 10 ml volumetric flask containing 4 mL of double-distilled water. Then 0.3 mL 5% NaNO₂ was added to the flask and, after 5 min, 0.3 ml AlCl₃ (10%) was also added. At the sixth minute, 2 ml NaOH (1 M) was added and the total volume was made up to 10 ml with double-distilled water. The solution was mixed completely and the absorbance level was measured versus prepared reagent blank at 510 nm. Total flavonoids’s content was expressed as catechin equivalent (CE) per 500 μg dry extract (Table 3) (Velioglu et al. 1998; Jia et al. 1999; Shams Ardekani et al. 2011).

Total phenol content of the extract was 40.66 ± 2.5 μg GAE/250 μg extract, while total flavonoids content of the extract was 100.9 ± 8.7 μg CE/500 μg extract, whereas GAE is Gallic acid equivalent, and CE is Catechin equivalent.