1. Foam test: To a small quantity of diosgenin add 5 ml water, shake gently and set aside. There is formation of a persistent foam confirming the presence of a saponin. Prepare 10 ml of solution of diosgenin in chloroform and perform the following tests:
2. Liebermann–Burchard test: To 1 ml, add few drops of acetic anhydride followed by concentrated sulphuric acid along the sides of the test tube. A violet blue ring is formed at the junction of the two layers confirming the presence of a steroid.
3. Salkowski test: To 1 ml, add a few drops of concentrated sulphuric acid along the sides of the test tube. A yellow-coloured ring turning to red is observed at the junction of the two layers. This test is indicative of the presence of a steroid.
4. Antimony trichloride test: To 1 ml, add a saturated solution of antimony trichloride in chloroform containing 20% acetic anhydride. Formation of a pink colour on heating indicates presence of steroid and triterpenoids.
5. TLC test: Spot 20 μl of the prepared solution of diosgenin along with a similarly prepared solution of a standard sample of diosgenin on a precoated silica gel plate. Develop in a solvent system of toluene: ethyl acetate (7:3). Activate it by spraying with anisaldehyde sulphuric acid reagent. A dark green spot (Rf – 0.37) corresponding to diosgenin is seen in both standard and test tracks.

Estimation

Diosgenin content in plant material may be determined by gravimetric, spectrophotometric, GLC, IR, HPTLC and HPLC methods.

A. Method I (HPTLC Densitometry)

1. Reflux 1 g of dried powdered sample of dioscorea with 25 ml 2.5 N HCl for 4 h. Cool, filter and wash the residue with water until free from acid.
2. Dry the residue in an oven at NLT 80°C and extract with petroleum ether (60° to 80°C) in a Soxhlet for 4 h.
3. Evaporate the petroleum ether to dryness and dissolve the residue in 5 ml chloroform and make up the volume to 10 ml with the same solvent. This is the test sample.
4. Prepare standard solutions of diosgenin (2–25 µg/ml) using a reference standard sample.
5. Apply 5 µl each of the standard and test solutions on a precoated silica gel G 60 plate. Develop in a solvent system of toluene: ethyl acetate (7:3) to a distance of 8 cm.
6. Air dry the plate and spray it with Liebermann-Burchard reagent and heat at 120°C until the spot corresponding to diosgenin turns black.
7. Cool and scan the different tracks in a densitometer in reflectance mode at 600 nm. Note the areas corresponding to diosgenin in tracks developed from standard and test samples. Integrate the peak height and peak areas and calculate the percentage of diosgenin in the test sample from the calibration curve (peak area versus concentration) of the standard.

B. Method II (UV Spectrophotometry)

1. To 1 g of the powdered drug add 10 ml of ethanol (70 % v/v). Heat under reflux on a water bath for 30 min. Allow to cool and filter.
2. Rinse the filter. Combine the filtrate and the rinsing solution in a 10 ml volumetric flask, and dilute to 10 ml with ethanol (70 % v/v).
3. Transfer 5 ml of this solution into a 100 ml volumetric flask, and dilute with methanol. Mix well and take 1 ml of this solution into a small porcelain dish and evaporate to dryness. Dissolve the residue in 10 ml of sulfuric acid R. Leave in contact for 1 h. This is the test sample.
4. Standard sample: Dissolve 2.5 mg of diosgenin R in methanol R in a 200 ml standard flask and dilute with the same solvent. Mix well and transfer 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ml solutions into individual labeled (S1–S10) porcelain dishes and evaporate to dryness. Dissolve the residues in 10 ml of sulphuric acid R each. Leave in contact for 1 h.
5. Measure the absorbance values of the test and standard solutions at 410 nm against sulphuric acid R. Calculate the percentage content of diosgenin from the standard curve of diosgenin.