Collection and preparation of plant materials
Dried plant material identified as H. gordonii was kindly donated by Farm Vredelus in July 2014. Farm Vredelus is a commercial medicinal plant farm based in Mariental, Namibia. A mechanical blender was used to grind the plant material. Plant identification was done by Silke Rugheimer at the National Herbarium of Namibia. Voucher number M1 [H. gordonii (Masson) Sweet ex Decne].
Plant material (108.3 g) was macerated at room temperature in 1 L of ethanol for 48 h. The filtrate was then concentrated under reduced pressure using a rotary evaporator and half of the residue obtained was further extracted in ethyl acetate to exclude highly polar tannins which are regarded as non-specific enzyme inhibitors . The extracts obtained were dried in a fume hood and stored at room temperature until further use.
Qualitative phytochemical analysis was conducted using standard procedures previously described [17, 18]. The metabolites screened for were flavonoids, phenolics, alkaloids, terpenes, steroids, cardiac glycosides, tannins and quinones. The quantitative phytochemical analysis of H. gordonii extracts was also carried out to determine the total phenol and tannin contents, which are amongst the most popular natural antioxidants reported in plants [19, 20].
Test for flavonoids
Dilute ammonia solution (5 mL) was added to a portion of the crude extract followed by addition of concentrated H2SO4. A yellow coloration observed in each extract indicated the presence of flavonoids.
Test for phenolics
A few drops of 5 % ferric chloride were added to extracts dissolved in distilled water. A dark green colour indicated the presence of phenolic compounds.
Test for alkaloids
Extracts were mixed with 2 mL of Wagner’s reagent and a reddish brown coloured precipitate indicated the presence of alkaloids.
Test for terpenes
The extract (5 mL) was first mixed with 2 mL of chloroform and 3 mL of concentrated H2SO4 was slowly added to form a layer. A reddish brown coloration of the interface indicated the presence of terpenes.
Test for steroids
0.5 mL of crude extract was mixed with 2 mL of acetic anhydride. This was followed by the subsequent addition of 2 mL H2SO4. A colour change from violet to blue or green in samples indicates the presence of steroids.
Test for cardiac glycosides
Exactly 5 mL of extract was treated with 2 mL of glacial acetic acid containing one drop of ferric chloride solution. The mixture was layered with 1 mL of concentrated H2SO4. A brown ring at the interface is an indication of the presence of the cardiac glycoside constituent.
Test for tannins
Each extract (1 mL) was mixed with 1 mL of 0.008 M Potassium ferricyanide. 0.02 M Ferric chloride in 0.1 M HCl (1 mL) was added and observed for blue-black coloration.
Test for quinones
Dilute NaOH was added 1 mL of crude extract. The development of a blue green or red coloration indicates the presence of quinones.
Total phenolic content
The total phenolic content (TPC) of H. gordonii extracts was carried out following a method previously described , with modification. Extracts (0.5 g) macerated with 10 mL of 80 % ethanol were filtered and 2.5 mL of the filtrate was subsequently added to 0.25 mL of 2 M Folin–Ciocalteu reagent. The mixture was allowed to stand for 30 min and then 2 mL of 20 % sodium carbonate was added. The absorbance was measured at 650 nm using a SpectraMax M2 plate reader. A standard calibration curve (R2 = 0.944) was constructed using various concentrations of gallic acid (0.63, 1.25, 2.5, 5 and 10 mg/mL). TPC was expressed as milligrams (mg) of gallic acid equivalents per gram (g) of extract (mg GAE/g extract).
Total tannin content
The total tannin content (TTC) was conducted following a procedure previously described , with modification. Briefly, 100 mg of the sample was macerated with 5 mL of distilled water and filtered. The filtrate (1 mL) was transferred into test tubes and mixed with 2 mL of concentrated picric acid. Absorbance was measured at 530 nm using a SpectraMax M2 plate reader. TTC was determined from extrapolation of a standard calibration curve (R2 = 0.966) prepared using various concentrations of tannic acid (0.63, 1.25, 2.5, 5 and 10 mg/mL). TTC was expressed as mg tannic acid equivalents per g of extract (mg TAE/g extract).