One gram each of pulverised samples of the plant was mixed with 20 ml of petroleum ether. This was poured out into a test tube and then left to evaporate to dryness. Later, 0.2 ml of chloroform–acetic anhydride (1:1, v/v) was added to the residue and 2 ml of TCA-chloroform (1:1, v/v) was added to the solution obtained. Vitamin A standard (retinol) was prepared using the same procedure for the samples. Final concentration of 1 mg/ml was made for both standard and plant extracts and serial dilution of the concentration was made given concentration levels 1, 0.8, 0.6, 0.4 and 0.2 mg/ml. The absorbance of the resulting solution and vitamin A standard was measured at 620 nm. The concentration of vitamin A in the sample was deduced from the standard curve. The vitamin A content is measured (in milligrams) as retinol activity equivalence/100 g (RAE/100 g DW).

Vitamin C content of the samples was evaluated by pouring 20 ml of 0.4% oxalic acid into each of the samples (1 g each). The mixture was filtered and indophenol reagent was reacted with the filtrate in the ratio of 9 to 1 (filtrate: indophenol). Vitamin C standard (ascorbic acid) was prepared using the same procedure for the samples. The final concentration of 1 mg/ml was made for both standard and samples. Serial dilution of the concentration was later done giving concentration levels 1, 0.8, 0.6, 0.4 and 0.2 mg/ml. The absorbance of the resulting sample solution and standard was read at 520 nm. Vitamin C content in milligram ascorbic acid/100 g DW of the each of the samples was extrapolated from the standard curve.

Vitamin E content was determined by mixing 1 g each of the samples with 20 ml ethanol. The mixture was filtered and to 1 ml of the filtrate, 1 ml each of 0.5% α-α-dipyridine and 0.2% ferric chloride in ethanol was added. The solution was made up to 5 ml with distilled water. Vitamin E standard (tocopherol) solution was also prepared using the same procedure as samples. The final concentration of 1 mg/ml was made for both standard and samples. Serial dilution of the concentration was later done giving concentration levels 1, 0.8, 0.6, 0.4 and 0.2 mg/ml. The absorbance of both samples solution and Vitamin E standard solution was read. Vitamin E content in milligrams alpha tocopherol/100 g DW was deduced from the standard curve.

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