Cascara Sagrada— Usually in flattened or transversely curved pieces, occasionally in quills of variable length and from 1 to 5 mm in thickness. The outer surface is brown, purplish brown, or brownish red, longitudinally ridged, with or without grayish or whitish lichen patches, sometimes with numerous lenticels and occasionally with moss attached. The inner surface is longitudinally striate, light yellow, weak reddish brown, or moderate yellowish brown. The fracture is short with projections of phloem fiber bundles in the inner bark.

Histology— It shows a yellowish brown, purple, or reddish brown cork of up to 10 or more rows of small cells; stone cells in yellowish, tangentially elongated groups of 20 to 50 cells in the cortex, pericycle, and outer phloem regions; phloem rays 1 to 4 cells wide, 15 to 25 cells deep, frequently diagonal or curved, forming converging groups; phloem fibers in small bundles, more or less surrounded by crystal fibers and located between the phloem rays; parenchyma with brown walls and containing starch grains and calcium oxalate crystals.

Powdered Cascara Sagrada— Moderate yellowish brown to dusky yellowish orange. It shows numerous broken phloem fiber bundles with accompanying crystal fibers containing monoclinic prisms of calcium oxalate; stone cells more or less adherent, in small groups with thick, finely lamellated and porous walls; fragments of reddish brown to yellow cork; masses of parenchyma and phloem ray cells colored reddish brown to orange upon the addition of a solution of an alkali; starch grains spheroidal, up to 8 µm in diameter; calcium oxalate in monoclinic prisms or rosette aggregates from 6 to 20 µm in diameter, occasionally up to 45 µm in diameter.

A: Add 100 mg of powdered Cascara Sagrada to 10 mL of hot water, shake the mixture occasionally until it is cold, filter, dilute the filtrate with water to 10 mL, and add 10 mL of 6 N ammonium hydroxide: an orange color is produced.

B: It becomes red to reddish brown in color when treated with 6 N ammonium hydroxide.

C: Macerate 100 mg of powdered Cascara Sagrada with 1 mL of alcohol, add 10 mL of water, boil the mixture, then cool, filter, and shake the filtrate with 10 mL of ether: a greenish yellow ether solution separates. Shake 3 mL of this ether solution with 3 mL of 6 N ammonium hydroxide, and dilute the separated ammonia solution with 20 mL of water: a distinct orange-pink color remains.

Ferric chloride solution— Dissolve 100 g of ferric chloride in water to make 100 mL.

Assay solution— Add about 1 g of Cascara Sagrada, accurately weighed, to about 70 mL of boiling water, boil for several minutes, with stirring, allow to cool, and transfer with the aid of water to a 100-mL volumetric flask. Dilute with water to volume, mix, and filter through suitable filter paper.

Assay preparation— Pipet 10 mL of Assay solution into a separatory funnel containing 5 mL of water and 2 drops of 1 N hydrochloric acid. Extract with 40 mL of methylene chloride, and transfer the lower layer to a second separatory funnel. Add 10 mL of water to the second separatory funnel, and shake. Allow to separate, discard the lower layer, and transfer the water layer to the first separatory funnel. Extract the combined water layers with 40 mL of methylene chloride, and transfer the lower layer to the second separatory funnel. Add 10 mL of water to the second separatory funnel, and shake. Allow to separate, discard the lower layer, and transfer the water layer to the first separatory funnel. Extract the combined aqueous phase with 30 mL of clear, freshly prepared water-saturated ethyl acetate, and transfer the water layer to another separatory funnel. Repeat the extraction with two additional 30-mL portions of the freshly prepared water-saturated ethyl acetate. Add 5 mL of water to the combined ethyl acetate extracts, shake, allow the phases to separate, discard the ethyl acetate extracts, and add 30 mL of the freshly prepared water-saturated ethyl acetate to the water wash. Shake, allow the phases to separate, and discard the ethyl acetate phase. Transfer the combined aqueous phases, with the aid of water, to a 50-mL volumetric flask. Dilute with water to volume, and mix.

Procedure— Pipet 15 mL of Assay preparation into a flask containing 2 mL of Ferric chloride solution and 12 mL of hydrochloric acid. Attach a condenser arranged for refluxing, and heat for 3 hours by keeping the flask immersed in boiling water or continuously exposed to steam heat. Cool, wash down the condenser, and transfer to a separatory funnel with the aid of 4 mL of 1 N sodium hydroxide and five 6-mL portions of water. Extract with 20 mL of methylene chloride, and transfer the lower layer to another separatory funnel. Repeat the extraction with three additional 20-mL portions of methylene chloride, wash the combined methylene chloride extracts with two 10-mL portions of water, shaking each time for 2 minutes, and discard the water washings. Transfer the washed methylene chloride extract to a 100-mL volumetric flask, dilute with methylene chloride to volume, and mix. Evaporate a 20.0-mL portion carefully on a water bath to dryness, and dissolve the residue in 10.0 mL of a 1 in 200 solution of magnesium acetate in methanol. Determine the absorbance, against methanol as a reference, in 1-cm cells at the wavelength of maximum absorbance at about 515 nm. Calculate the quantity, in mg, of cascarosides in the portion of Cascara Sagrada taken by the formula: in which AU is the absorbance of the solution from the Assay preparation.

Ferric chloride solution and Assay solution—Prepare as directed in the Assay for cascarosides.

Assay preparation— Pipet 10 mL of Assay solution into a separatory funnel containing 5 mL of water and 2 drops of 1 N hydrochloric acid. Extract with 40 mL of methylene chloride, and transfer the lower layer to a second separatory funnel. Add 10 mL of water to the second separatory funnel, and shake. Allow to separate, discard the lower layer, and transfer the water layer to the first separatory funnel. Extract the combined water layers with 40 mL of methylene chloride, and transfer the lower layer to the second separatory funnel. Add 10 mL of water to the second separatory funnel, and shake. Allow to separate, and discard the lower layer. Transfer the combined water layers, with the aid of water, to a 50-mL volumetric flask, dilute with water to volume, and mix.

Procedure— Proceed as directed for Procedure in the Assay for cascarosides, except to evaporate a 15.0-mL portion of the methylene chloride solution instead of 20.0 mL. Calculate the quantity, in mg, of total hydroxyanthracene derivatives in the portion of Cascara Sagrada taken by the formula: in which AU is the absorbance of the solution from the Assay preparation.