Unground Alexandrian senna leaf— Inequilaterally lanceolate or lance-ovate leaflets, frequently broken; from 1.5 cm to 3.5 cm in length and from 5 mm to 10 mm in width, unequal at the base, with very short, stout petiolules. The leaflets are acutely cuspidate, entire, brittle, and subcoriaceous, with short and somewhat appressed hairs, few on the upper surface, more numerous on the lower surface, where they occur spreading on the midrib, especially on its lower part. The color is weak yellow to light grayish-green to pale olive. The odor is characteristic.

Unground Tinnevelly senna leaf— Usually unbroken leaflets, from 2 cm to 5 cm in length and from 6 mm to 15 mm in width; acute at the apex; and slightly hairy. The color of the leaves is weak yellow to pale olive.

Histology— Senna leaf shows polygonal epidermal cells with straight walls and frequently containing mucilage; numerous, broadly elliptical stomata mostly from 20 to 35 µm in length, usually bordered by two neighbor-cells with their long axes parallel to that of the stoma, and rarely, though more frequently in Alexandrian senna leaf, a third epidermal cell at the end of the stoma. The hairs are nonglandular, one-celled, conical, often curved, with thick papillose walls, from 100 to 350 µm in length. Palisade cells in a single layer underlie both surfaces except in the midrib region where they occur only beneath the upper epidermis. A meristele occurs in the midrib composed of several radially arranged fibrovascular bundles, the latter separated by narrow vascular rays and supported above and below by arcs of lignified pericyclic fibers. Calcium oxalate occurs in rosette aggregates in the spongy parenchyma and in six- to eight-sided prisms in the crystal fibers, which lie on the outer surface of each group of pericyclic fibers.

Powdered senna leaf— Dusky greenish-yellow to light olive-brown, displaying fragments of veins bearing lignified vessels, tracheids, and crystal fibers, isolated hairs, masses of palisade and spongy parenchyma, fragments of epidermis with stomata, free calcium oxalate rosette aggregates, and prisms from 10 to 20 µm in length. In powdered Alexandrian senna leaf, the hairs are more numerous than in powdered Tinnevelly senna leaf.

Ferric chloride solution— Dissolve 10.5 g of ferric chloride in 100 mL of water.

Methanolic magnesium acetate solution— Dissolve 5.0 g of magnesium acetate in 1 L of methanol.

Sodium bicarbonate solution— Dissolve 5.0 g of sodium bicarbonate in 1 L of water.

Standard preparation— Dissolve accurately weighed quantities of USP Sennosides RS in Sodium bicarbonate solution to obtain a solution having a known concentration of about 0.13 mg per mL.

Assay preparation— Weigh and pulverize about 10 g of Senna Leaf. Transfer about 0.15 g, accurately weighed, to a 100-mL round-bottom flask, add 30 mL of water, mix, weigh, attach a condenser, and reflux in a water bath for 15 minutes. Cool to room temperature, weigh, and adjust to the original weight with water. Centrifuge, and transfer 20.0 mL of the supernatant to a 150-mL separatory funnel. Add 0.1 mL of diluted hydrochloric acid, and shake with three quantities, each of 15 mL, of chloroform. Allow to separate, and discard the chloroform layer after each addition. Add about 0.1 g of sodium bicarbonate, shake for 3 minutes, and centrifuge. Use the supernatant as the Assay preparation.

Procedure— Transfer 10.0 mL each of the Standard preparation and the Assay preparation to separate 100-mL round-bottom flasks equipped with condensers, add 20 mL of Ferric chloride solution, and mix. Reflux in a water bath for 20 minutes. Add 1 mL of hydrochloric acid, and reflux for an additional 20 minutes, with frequent shaking, to dissolve the precipitates. Cool to room temperature, transfer the mixtures to separate 100-mL separatory funnels, and shake with three quantities, each of 25 mL, of ether previously used to rinse the flasks. Combine the ether extracts, mix, and wash with two quantities, each of 15 mL, of water. Tranfer the ether layers to separate 100-mL volumetric flasks, dilute with ether to volume, and mix. Evaporate 10.0 mL of the ether extracts to dryness, and dissolve the residue in 10.0 mL of Methanolic magnesium acetate solution. Determine the absorbance of the resulting solution from the Standard preparation and the Assay preparation at 515 nm, with a suitable spectrophotometer fitted with matched quartz cells, using methanol as the blank. Calculate the percentage of sennosides in the Senna Leaf by the formula: in which AU and AS are the absorbances of the solutions from the Assay preparation and the Standard preparation, respectively; CS is the concentration, in mg per mL, of sennosides in the Standard preparation; and W is the weight, in mg, of powdered Senna Leaf used.