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Category Antiarrhythmic; cardiotonic

        Digitalis Leaf is the dried leaf of Digitalis purpurea L. (Family Scrophulariaceae).  The leaves are rapidly dried, as soon as possible after collection, at a temperature not exceeding 60°.  It contains not less than 0.3 per cent of cardenolic glycosides, calculated as digitoxin C₄₁H₆₄O₁₃, on the dried basis.

Origin of plant Digitalis is native to Europe, the Mediterranean region, and the Canary Islands.

Constituents  Digitalis contains cardenolic glycosides, including digitoxin, gitoxin, purpurea glycoside A and purpurea glycoside B.  It also contains steroid saponin (e.g., desgalactotigonin, digitonine), anthraquinones and other four glycosides, namely acteoside, purpureaside A, calceolarioside B, and plaintainside D.  

Description  Odour, mild, characteristic; taste, bitter.
Macroscopical  Broken, brittle leaves, apex subacute, margin irregularly crenate, dentate or serrate, base decurrent; upper surface rugose and pubescent; lower surface densely pubescent with prominent lateral veins and raised veinlets.
Microscopical  Powdered drug of Digitalis shows fragments of upper epidermis, palisade parenchyma and fragments of lower epidermis.  Fragments of upper epidermis:  thick-walled, slightly sinuous-walled or pitted-walled cells covered with cuticle, and some with scars of covering trichomes.  Palisade parenchyma underlined the upper epidermis.  Fragments of lower epidermis:  in surface view, distinctly prominent cells and anomocytic stomata; multicellular uniseriate covering trichomes, usually 3- to 5-celled and often with one or more collapsed cells; unicellular and/or multicellular uniseriate glandular trichomes with stalks and unicellular or multicellular heads. 

Other relevant information  When given in large and quickly repeated doses, it may be toxic and even life-threatening. 

Packaging and storage  Digitalis Leaf shall be kept in well-closed containers, protected from light and moisture.

Identification
     A. To 1 g, in fine powder, add 20 mL of ethanol (50 per cent) and 10 mL of lead acetate TS, boil for 2 minutes, allow to cool, centrifuge, and extract the clear supernatant liquid with two 15-mL portions of chloroform, separating the layers, if necessary, by centrifuging.  Dry the combined chloroform extracts over anhydrous sodium sulfate and filter (solution A).  Reserve 15 mL of solution A for use in tests B and C, and evaporate 5 mL of the remainder to dryness on a water-bath.  To the residue add 2 mL of a 2 per cent w/v solution of 3,5-dinitrobenzoic acid in ethanol and 1 mL of
1 M sodium hydroxide:  a reddish violet colour is produced within 15 minutes.
     B. Evaporate 5 mL of solution A to dryness on a water-bath; to the residue add 3 mL of xanthydrol TS and heat for 3 minutes on a water-bath: a red colour is produced.
     C. Carry out the test as described in the “Thin-Layer Chromatography” (Appendix 3.1).
          Standard solution  Dissolve 5 mg of Purpureaglycoside A RS, 2 mg of Purpureaglycoside B RS, 5 mg of Digitoxin RS and 2 mg of Gitoxin RS in a mixture of equal volumes of chloroform and methanol and dilute to 10 mL with the same solvent.
          Test solution  Evaporate 10 mL of solution A to dryness on a water-bath and dissolve the residue in 1 mL of a mixture of equal volumes of chloroform and methanol.
          Adsorbent  Silica gel G.
          Mobile phase  Water, methanol, and ethyl acetate (7.5:10:75).
          Application  Apply 20 µL each of Standard solution and Test solution as (2-cm × 0.3-cm) bands.
          Development and drying  Allow the solvent front to ascend 10 cm above the line of application.
     Dry the developed plate at room temperature until the solvents have evaporated.
          Detection  Spray with a mixture of 2 volumes of a 1 per cent w/v solution of chloramine T and 8 volumes of a 25 per cent w/v solution of trichloroacetic acid in ethanol, then heat at 105° for 10 minutes and examine under ultraviolet light (366 nm).
          Results  When examined under ultraviolet light
(366 nm), the test solution shows a light blue fluorescent band due to purpureaglycoside B in the lower part, a brownish-yellow fluorescent band due to purpureaglycoside A just above it, a light blue fluorescent band due to gitoxin around the middle of the chromatogram and a brownish yellow fluorescent band due to digitoxin just above it, corresponding in colour and R_f to the bands shown by the standard solution.
Digitalis lanata Ehrh.  Leaves with few or no trichomes and with parallel venation or cells of the abaxial epidermis with beaded anticlinal walls and of cells of the adaxial epidermis with numerous stomata are found. 

Loss on drying  Not more than 6.0 per cent w/w after drying at 105° to constant weight (Appendix 4.15).

Foreign matter  Not more than 2.0 per cent w/w (Appendix 7.2).

Acid-insoluble ash  Not more than 5.0 per cent w/w (Appendix 7.6).

Total ash  Not more than 12.0 per cent w/w (Appendix 7.7).

Assay
     Standard preparation  Dissolve about 50 mg of Digitoxin RS, accurately weighed, in sufficient ethanol to produce 50.0 mL.  Dilute 5.0 mL of this solution to
50.0 mL with ethanol.  To 5.0 mL of the resulting solution add 25 mL of water and 3 mL of 4 M hydrochloric acid.  Heat on a water-bath under a reflux condenser for 1 hour and transfer to a separator, rinsing the flask with two 5-mL portions of water.  Extract with three 25-mL portions of chloroform.  Dry the combined chloroform extracts over anhydrous sodium sulfate and dilute to 100.0 mL with chloroform.  Evaporate 40.0 mL of the chloroform solution and dissolve the residue in 7 mL of ethanol (50 per cent).  Add 2 mL of dinitrobenzoic acid TS and 1 mL of 1 M sodium hydroxide.
     Assay preparation  Shake about 250 mg of Digitalis Leaf, in fine powder and accurately weighed, with 50 mL of water for 1 hour.  Add 5 mL of a 15 per cent w/v solution of lead acetate, and shake.  After a few minutes, add 7.5 mL of a 4 per cent w/v solution of disodium hydrogen phosphate dodecahydrate and filter.  To 50.0 mL of the filtrate add 5 mL of hydrochloric acid, heat on a water-bath under a reflux condenser for 1 hour and transfer to a separator, rinsing the flask with two 5-mL portions of water.  Extract with three 25-mL portions of chloroform.  Dry the combined chloroform extracts over anhydrous sodium sulfate and dilute to 100.0 mL with chloroform.  Evaporate 40.0 mL of the chloroform solution and dissolve the residue in 7 mL of ethanol (50 per cent).  Add 2 mL of dinitrobenzoic acid TS and 1 mL of 1 M sodium hydroxide.
     Procedure  Measure the absorbances of Standard preparation and Assay preparation at 540 nm several times during the first 12 minutes until the maximum is reached (Appendix 2.2), using a mixture of 7 mL of ethanol
(50 per cent), 2 mL of dinitrobenzoic acid TS and 1 mL of 1 M sodium hydroxide as the blank.
     Calculation  Calculate the content of C₄₁H₆₄O₁₃ in the portion of Digitalis Leaf taken, using the declared content of C₄₁H₆₄O₁₃ in Digitoxin RS.