The total cardenolide content of D. lanata is 1%, about two to three times that found in D. purpurea. The presence of acetyl group on the terminal digitoxose renders the lanatosides in general easier to isolate from the plant material and makes crystallization easier. D. lanata glycosides are based on 5 aglycones, digitoxigenin, gitoxigenin, gitaloxigenin, digoxigenin and diginatigenin. While the primary glycosides with the tetrasaccharide unit of 2 digitoxose, 1 acetyl digitoxose and 1 glucose are called lanatosides, lanatosides A and C constitute 50% to 70% of the major components in the fresh leaf. These are based on aglycones digitoxigenin and digoxigenin respectively and lanatosides B, D and E are minor components derived from gitoxigenin, diginatigenin and gitaloxigenin respectively. Drying of the leaf is also accompanied by partial hydrolysis leading to the loss of both the terminal glucose and the acetyl group.

1. 100 g of finely ground, dried powdered leaves of D. lanata are packed in a Soxhlet apparatus and extracted exhaustively with 80% ethanol.
2. The alcoholic extract is then shaken with petroleum ether (5 × 75 ml) for the removal of plant pigments.
3. The aqueous alcoholic layer is then evaporated to about one-fifth its original volume, when a whitish or slightly coloured crystalline material separates out.
4. Separate it by filtration and wash with ice cold ethanol. To the mother liquor from which the crystals are separated, add equal volume of 80% alcohol and extract with acetone. Discard the acetone layer and further concentrate the aqueous layer for the separation of crystalline material. Repeat the recrystallization till no further crystals are formed.
5. Separate the crystals by filtration and wash with acetone.
6. Purify by repeated recrystallization from methanol.

Identification

1. Keller–Kiliani test for digitoxose: Dissolve the drug in 3 ml glacial acetic acid containing 2 drops of 5% ferric chloride solution. Carefully transfer this solution to the surface of 2 ml of sulphuric acid, a reddish-brown layer forms at the junction of the two liquids and the upper layer slowly becomes bluish-green, darkening on standing. This test confirms the presence of a deoxy sugar.
2. Legal’s test: To a few milligrams of the drug dissolved in a few drops of pyridine add a drop of 2%w/v solution of sodium nitroprusside. A pink or a deep red colour is formed confirming the presence of a 5-membered lactone ring.
3. Kedde’s test: To 1 ml of the solution of the drug in 70% alcohol, add 2 drops of 2% 3, 5–dinitro benzoic acid in 90% alcohol. Make it alkaline with a few drops of 20% sodium hydroxide solution. A purple colour is produced indicating the presence of an unsaturated lactone.
4. Baljet’s test: To 1 ml of a solution of the drug in 70% alcohol, add a few drops of alkaline sodium picrate reagent. Formation of an orange-red colour indicates the presence of a lactone.
5. Raymond’s test: Dissolve a small quantity of the drug in 1 ml 50% ethanol and add 0.1 ml of Raymond’s reagent (1% w/v solution of m-dinitrobenzene in ethanol) and 2 to 3 drops of 20% sodium hydroxide solution. Appearance of a violet colour slowly changing to blue is indicative of the presence of methylene group (at C-21 position on the lactone ring).
6. Tollen’s test: Dissolve a small quantity of the drug in a few drops of pyridine. To this add 1ml Tollen’s reagent (0.1 N silver nitrate solution is treated with dilute ammonia solution till the initially formed white precipitate dissolves on further addition of ammonia) and heat gently if required. Appearance of a silver mirror indicates the presence of a monosaccharide.
7. Xanthydrol test: To a solution of the drug in 70% alcohol, add 0.5 ml of xanthydrol reagent (0.125% w/v solution of xanthydrol in glacial acetic acid containing 1% hydrochloric acid). Formation of red colour indicates the presence of an unsaturated lactone.
8. Antimony trichloride test: To a solution of the drug in 70% alcohol add a solution of antimony trichloride and trichloroacetic acid and heat the mixture. A blue or violet colour is formed.