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2.2. Extract preparation

The extracts of the powered samples were prepared using maceration technique (Harborne,
1973). The sample was initially defatted using water and extract was obtained from this

defatted marc. The extract thus obtained was stored in centrifuge tubes for further study.
2.3. Phytochemical analysis

The plant extracts obtained using solvent extraction technique were then subjected to thin
layer chromatography technique, 80% ethanol (8:2 ethanol: distilled water) was used as

mobile phase. The different observations thus obtained were recorded. The standard

procedures were used for the qualitative estimation of different bioactive constituents of the
extracts. The tests performed were – tests for carbohydrates, flavonoid, glycosides and

alkaloids were done according to Trease and Evans(1978); tests for terpinoids and saponins

was done according to Saibu, (2017); the presence of saponins was further confirmed using
haemolysis test to eliminate false positives (Sofowora, 1993) and tests for steroids were done

according to Libermann (1885). The quantitative phytochemical estimations to determine the
total phenolic content (TPC) and total flavonoid content (TFC) were also done according to

Singleton et. al, (1999).
2.4. In-vitro Antioxidant activity

2.4.1. Reducing power activity

The reducing power activity is based on the samples ability to donate an electron (Singleton
et. al. 1999). This method determines the ability of a compound to donate an electron and

3+
produce Fe 2+ by reduction of Fe . The antioxidants present in the sample facilitate this
reduction process and higher the no. of antioxidants higher reading will be observed at

700nm (Yildirim et. al., 2000). We plotted a graph of readings of our sample against ascorbic
acid as a standard and obtained the value for our sample.

2.4.2. DPPH radical scavenging assay

This is a preliminary test often used to determine the antioxidant property of a sample
(Ferreira et. al. 2007). The antioxidant materials have an electron donating nature and thus

when an antioxidant material interacts with the DPPH assay it scavenges it which can be seen

as a colour change of the solution.
DPPH gives a strong absorbance at 515nm (de Anos et. al. 2000). The concentration of

DPPH in the reaction medium was calculated from the calibration curve plotted against
ascorbic acid concentraction versus percent of DPPH scavenged. The percentage of DPPH

scavenged was calculated using following formula :
%DPPH = A0 - As ÷ A0 × 100

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