2.2. Preparation of extracts

After each harvest, the aerial part of the plant (stem and leaves for preflowering and stem, leaves and flowers for flowering and postflowering) were rinsed with distilled water and blotted dry with a paper towel. Thereafter, they were oven-dried at 40 °C for 72 h until fixed weight was achieved. The dried leaves were pulverized with an industrial electric blender (Hamilton Beach, HBF500s series, Canada). To prepare crude extract of the sample, 200 g of the ground sample were agigtated constantly for 72 h using a shaker (Gallenkamp incubator orbital shaker) in 1.5 L of distilled water, acetone and methanol. The solution was thereafter filtered using Whatman No. 1 filter paper, a Buchner funnel and a vacumm pump. The aqueous extract (APRE, AFLW and APST) was freezed at −40 °C with a shell freezer and freeze-dried (Vir Tis Co, Vir Tis benchtop K, Gardiner, NY). The solvents acetone (AcPRE, AcFLW an AcPST) and methanol (MPRE, MFLW and MPST) were vaporized using a rotary evaporator (Strike-202 Steroglass, Italy), at their respective boiling points. The extaction solvents were chosen based on their extractive abilities and solubility. Water was chosen as one of the solvents of extraction because it is the medium used for cooking and preparing decoctions of medicinal plants in folklore medicine. Methanol was used because of its ability to extract polar and non-polar substances. Also, because of the lower boiling point of methanol (64.7 °C) when compared to ethanol (78.4 °C), lower temperature is required to evaporate the solvent from the methanol extract in the rotary evaporator; hence the extract is less damaged. Acetone was chosen because of it polar and non-polar characteristics. The crude extracts were stored at 4 °C in a refrigerator (Polyscience AD15R-40-A12E, USA) until needed. Percentage yield of solvent was determined and recorded. All dry extract samples were reconstituted in methanol (1mg per ml) to prepare the stock solution needed for various antioxidant assays. The mixture was then sonicated (Branson 2510, 220–230V, 50–60Hz, 130W and 0.6A; Branson Ultrasonics Corporation, DANBURY, CT06813, USA) for 40 min at 27 °C. The mixture was vortexed thoroughly and dissolved samples were used for all analyses in the study.