2.1 Solvents, Reagent and Materials
Glycerine (VWR Chemicals BDH, USA), Ferric chloride (BDH Laboratory Supplies, England), HCl (AvonChem, U.K), Na2CO3 (Wardle Chemicals, Calveley).
2.2 Sampling and Sample Preparation
The Mentha spicata leaves and the neem seeds were collected from the School of Biological Sciences herbarium, University of Cape Coast, Ghana. The Mentha spicata leaves and the neem seeds were air dried for four weeks at laboratory conditions (26.4 ± 2°C) to reduce the moisture content and was milled into fine powder to increase the surface area for extraction. The armyworms were collected from an unsprayed maize field at Kwaprow, a suburb in Cape Coast, Central Region, Ghana, and kept in a perforated container at laboratory conditions (26.4 ± 2°C). The army worms were fed with maize leaves collected for 12-30 days. About 50 4th instar larvae were collected and reared until a sufficient population of second-generation larvae was obtained for the study.
2.3 Preparation of reagents - Mayer’s reagent
The Mayer’s reagent was prepared by dissolving 1.36 g of mercuric chloride and 5 g of potassium iodide in 100 mL of distilled water. This reagent would be used in the detection of alkaloids.
2.4 Extraction of Samples using Selective Solvents
A glass syrup bottle was loaded with 5 g of the blended Mentha spicata leaves powder and 100 mL of the aqueous solvent was poured over it to cover the samples. This was put under cold maceration for three days with intermittent agitations to ensure thorough diffusion of solvent into the plant tissues and to enhance solubilization of extracts. The residue was separated from the filtrate by suction filtration and the filtrate was stored in glass syrup bottle at laboratory conditions (26.4 ± 2°C) and kept away from sunlight until usage. This was done for all three selected aqueous solvents namely, distilled water, glycerine and glycerine plus water (in the ratio of 6:4). These solvents were chosen because their low cost, easy accessibility and their safety to the environment. The neem seeds powder was also taken through the same procedure using water as solvent.
2.5 Phytochemical Screening of Extracts
2.5.1 Detection of phenolics
The detection of phenolics was done using a method described by Tiwari et al., 2011. 0.5 mL of the extract was measured into separate test tubes and 1 mL of 1% ferric chloride solution was added into each test tube. The formation of a bluish-black colour indicated the presence of phenolic compounds. The chemical structures of common plant derived phenolics, namely, Rutin, Caffeic acid and Resveratrol are shown in Figure 1.
2.5.2 Detection of alkaloids
The detection of alkaloids was done using the Mayer’s test (Potassium Mercuric Iodine solution) as described by Ramamurthy and Sathiyadevi . Extracts were dissolved individually in 5 mL dilute HCl and filtered. To 1 mL of the filtrate, 0.5 mL of Mayer’s reagent was added. The formation of a yellow cream precipitate indicated the presence of alkaloids. The chemical structures of common plant derived alkaloids, namely, Caffeine and Berberine are shown in Figure 2.
2.5.3 Detection of tannins
The detection of tannins was done using the method described by Kumar et al.,  with slight modifications. 0.5 mL of the extract was measured into separate test tubes and mixed with 2.5 mL of water. The mixture was heated for 10 minutes and filtered. 1 mL of 1% ferric chloride solution was then added into the filtrate in each test tube. The formation of a green colour indicated the presence of tannins. The chemical structures of common plant derived tannis, namely, Gallic acid, Daidzein, Genistein and Glycitein are shown in Figure 3.
2.5.4 Detection of saponins
The detection of saponins was done using the foam test as described by Ekwueme et al., . 5 mL of the extracts was added to 5 mL of distilled water and shaken thoroughly. The persistence of foam for ten minutes indicated the presence of saponins. The chemical structures of common plant derived saponins, namely, Dammarenediol and Cucubitadienol are shown in Figure 4.
2.6 Quantitative Analysis of Extract
For each parameter, three repetitions were carried out to determine the concentration of the phyto constituents.
2.6.1 Total Tannins Content (TTC)
The quantitative tannin content in the extracts was estimated by Folin-phenol method a described by Tamilselvi et al, . 0.1 mL of the sample/extract was added to 7.5 mL of distilled water and 0.5 mL of Folin-phenol reagent was added. After that 1.0 mL of 35 % Na2CO3 solution was added, and the mixture was diluted to 10mL with distilled water. The mixture was shaken well and kept at room temperature for 30 minutes. A set of reference standard solutions of gallic acid (20, 40, 60, 80 and 100) μg/mL were prepared in the same manner as described earlier from a stock solution of 100 μg/mL (i.e. 2 mg/20 mL) and water was used as the blank. Absorbance for test and standard solutions were measured at a wavelength of 725 nm with an UV/VIS spectrophotometer (UVD-3200, Labomed Inc., Los Angeles, California, USA). The tannin content was determined from the gallic acid standard curve.
2.6.2 Total phenolic content
The phenolic content detected using a method described by Singleton and Rossi . 1.5 mL of the extracts was put into separate test tube and 0.2 mL of 0.1% Folin Ciocalteu reagent was added. The test tubes were incubated at room temperature for 4 mins. 0.5 mL of 20% sodium carbonate was added and kept in the dark for 30 mins. The absorbance was read at a wavelength of 650 nm. Gallic acid of concentration: (20, 40, 60, 80 and 100) ug / mL were used as standard for constructing calibration curve and water was used as the blank. The phenolic content was determined from the gallic acid standard curve.
2.6.3 Test for Antifeedancy
Maize leaves of approximately 14 cm x 2 cm were cut out. The leaves were dipped into the various extracts. This was done for glycerine, water, glycerine (60%) plus water (40%) extracts and for Azadirachta indica (neem seed) extracts which served as a positive control. A negative control was also set using ordinary distilled water. The armyworms were exposed to the leaves in plastic containers with perforated lids. Per each treatment, one armyworm pre-starved for 2 hours was used to prevent cannibalism. The antifeedant activity was monitored for a period of 24 hours of exposure, i.e., first 4 hours continuum and left overnight until after 24 hours. After 24 hours, the antifeedant activity of the extracts was accessed based on the rate of feeding by the armyworms. Percentage (%) antifeedancy was observed and estimated using the formula: