Trypsin inhibitor was assayed by determining residual trypsin activity using Kakade et al. (1974) method with slight modification. Briefly, samples (1.0 g) were extracted with 0.01 N sodium hydroxide for 3 hrs in shaker and the supernatant was collected after centrifugation. To the known aliquots, 2 ml of trypsin solution was added and kept in a temperature bath at 37°C for 10 min. 5 ml of Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA) pre-warmed to 37°C was added. The reaction was terminated exactly 10 min later by adding 30% of acetic acid. A blank and control were also run simultaneously. The absorbance was read at 410 nm in UV-spectrophotometer. Decrease of 0.019 of ∆A indicates the presence of 1µg of trypsin inhibitor in the sample.
Phytic acid
The estimation of phytic acid was carried out by Davis and Reid (1979) method after extracting the samples with 0.5 N nitric acid for 3 h. To the known volume of extract, 1ml ferric ammonium sulphate was added and placed in a boiling water bath for 20 min followed by 5 ml of amyl alcohol. The test tubes were shaken well, and then centrifuged at 3000 rpm for 10 min. Finally the colour intensity was read at 465 nm in UV-spectrophotometer against amyl alcohol blank exactly after 15 min of addition of ammonium thiocyanate.
Tannin
Vanillin-HCl assay (Price et al. 1978) was used to determine the quantity of tannin content after extracted with absolute methanol for 20 min. The content was centrifuged at 3000 g and the supernatant was used for analysis. To the 1 ml of extracted aliquots, 5 ml of vanillin-HCl reagent was added and kept in a water bath for 20 min. The intensity of colour developed was read at 500 nm in UV-spectrophotometer against 4% hydrochloric acid as a blank. Catechin as equivalent to tannin was used as a standard with different concentration.
Saponin
Saponin was extracted from the acetone extracted residual matter for 3 h using methanol in Soxhlet extraction unit (AOAC 1997). To 1 ml of methanolic extract, water and organic solvent (chloroform and methanol) was added at the ration of 1:2 and allowed to separate the layers after mixing thoroughly. The upper aqueous layer (1 ml) was kept at 110°C in hot air oven till complete evaporation of solvent. To which 0.1 ml and 0.4 ml of vannilin reagent and perchloric acid were added, respectively and kept at 70°C for 10 min. The intensity of colour developed was read at 540 nm in UV-spectrophotometer after adding 2.5 ml acetic acid. Diosgenin was used as a standard at different concentration to calculate the saponin content.
Glucosinolate
Glucosinolate was estimated by McGhee et al. (1965) method. Briefly, 10 g of samples were extracted with hot distilled water for 5 min and the contents were filtered using Buchner funnel. The residue was repeatedly washed with hot water and made up to known volume. To 25 ml of extract, 10 ml of silver nitrate, 25 ml of ethanol were added and kept in a boiling water bath for 45 min. After cooling to room temperature, it was titrated against 0.01 N potassium thiocyanate in the presence of 6 N nitric acid and ferric ammonium sulphate till the pale salmon colour was obtained.
Guar gum
The estimation of gum content in guar meal was carried out by Das et al. (1977) method with slight modifications. Briefly, guar meal was soaked with distilled water at the proportionate of 1:10 for overnight. The soaked material was filtered using Whatmann filter paper and the residue was rinsed for 3 to 5 times with distilled water. All the filtrates were pooled together in a container. The gum content was precipitated by the addition of 50 to 100 ml of isopropanol and the process was repeated for 5 to 10 times. Meanwhile the precipitated gum was collected completely in a pre-weighed petridish and the exact quantity of gum content was measured after drying the excess of moisture.