Macroscopic— Mature chaste tree fruits are spherical to ovoid, 2 to 4 mm in diameter, very hard, usually with a short pedicil. The fruit is reddish brown to black, slightly rough, and covered with glandular hairs. There are four grooves perpendicular to one another, and a slight depression on the apex, more evident on large fruits. The internal appearance of the fruit is yellowish. The internal structure of the fruit includes four compartments, each containing an oblong seed with a high fat content. A group of up to six spongy, light tan, immature fruits may also accompany mature fruits. The fruit is often covered by a tubular, greenish-gray, fine tomentous calyx, which is persistent and has five teeth.

Microscopic— The exocarp is brown and narrow, consisting of parenchymatous cells with thin walls and partially lignified cells with many pitted thickenings on the inside. In surface view, the exocarp shows an epidermis of polygonal cells with irregular thickenings and glandular hairs, each with a short single-celled stalk and a four-celled head containing essential oil. The outer mesocarp consists of several layers of brown, isodiametric parenchyma cells. The inner mesocarp consists of finely pitted sclerenchymatous cells, some with moderately thickened walls, others consisting of isodiametric stone cells with small lumen. The endocarp consists of a layer of small brown sclereid cells. The seeds are small, having large cotyledons surrounded by thin-walled, large parenchymatous cells that have ribbed thickenings. The nutritive tissue and the cells of the germ contain aleuron grains and oil globules. Starch is absent. The outer epidermis of calyx is composed of polygonal cells, covered by abundant unicellular or multicellular curved trichomes. The inner epidermis of calyx is glabrous and composed of rectangular, elongated cells with slightly wavy walls.

A: Thin-Layer Chromatographic Identification Test 201—

B: The chromatogram of the Test solution in the test for Content of casticin shows a peak at the retention time corresponding to the casticin peak in the chromatogram of the Standard solution.

Solution A— Use filtered and degassed methanol.

Solution B— Use a filtered and degassed solution of 5.88 g of phosphoric acid in 1000 mL of water.

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve an accurately weighed quantity of USP Casticin RS in methanol, with sonication. Dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 0.05 mg per mL. Filter through a cellulose membrane having a 0.45-µm or finer porosity.

Test solution— Accurately weigh approximately 1000 mg of ground plant material, and place in a suitable container with a stopper. Extract twice with 40 mL of methanol, using a hand homogenizer at 19,000 rpm for 2 minutes. Filter each supernatant, and transfer to a 250-mL round-bottom flask. Rinse the residue with methanol, and filter the resulting solution into the flask. Evaporate the combined extract to dryness. Dissolve the residue in methanol, quantitatively transfer to a 20-mL volumetric flask, and dilute with methanol to volume. Filter through a cellulose membrane having a 0.45-µm or finer porosity.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 348-nm detector and a 3.1-mm × 12.5-cm column that contains 5-µm packing L1. The column temperature is maintained at 25. The flow rate is about 1.0 mL per minute. The chromatograph is programmed as follows. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the tailing factor for casticin is not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas of the analyte peaks. Identify the retention time of the peak corresponding to casticin in the Test solution by comparison with the chromatogram of the Standard solution. Calculate the percentage of casticin in the portion of Chaste Tree taken by the formula: in which C is the concentration, in mg per mL, of USP Casticin RS in the Standard solution; W is the weight, in mg, of the Chaste Tree taken to prepare the Test solution; and rU and rS are the peak responses of casticin obtained from the Test solution and the Standard solution, respectively.

Solvent: a mixture of water and methanol (95:5).

Solution A— Use filtered and degassed acetonitrile.

Solution B— Use a filtered and degassed solution of 5.88 g of phosphoric acid in 1000 mL of water.

Standard solution— Dissolve an accurately weighed quantity of USP Agnuside RS in Solvent, with sonication. Dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 0.125 mg per mL. Filter through a cellulose membrane having a 0.45-µm or finer porosity.

Test solution— Accurately weigh approximately 1000 mg of ground plant material, and place in a suitable container with a stopper. Extract twice with 40 mL of methanol, using a hand homogenizer at 19,000 rpm for 2 minutes. Centrifuge, and transfer each supernatant to a 250-mL round-bottom flask. Rinse the residue with methanol, and filter the resulting solution into the flask. Evaporate the combined extract to dryness, and dissolve the residue in 2 mL of Solvent. Quantitatively transfer the solution to a solid-phase extraction cartridge packed with neutral aluminum oxide previously conditioned with 5 mL of Solvent. Connect the cartridge to a vacuum pressure not exceeding 300 mbar, and collect the eluate. Rinse the round-bottom flask with 2 mL of Solvent, and pass this solution through the cartridge, apply the vacuum, and collect the eluate. Rinse the cartridge with 4 mL of Solvent, and collect the eluate. Combine the eluates from the cartridge, transfer to a 10-mL volumetric flask, and dilute with Solvent to volume.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 258-nm detector and a 3.1-mm × 12.5-cm column that contains 5-µm packing L1. The column temperature is maintained at 25. The flow rate is about 1.3 mL per minute. The chromatograph is programmed as follows. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the tailing factor for agnuside is not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas for the analyte peaks. Identify the retention time of the peak corresponding to agnuside in the Test solution by comparison with the chromatogram obtained from the Standard solution. Calculate the percentage of agnuside in the portion of Chaste Tree taken by the formula: in which C is the concentration, in mg per mL, of USP Agnuside RS in the Standard solution; W is the weight, in mg, of the Chaste Tree taken to prepare the Test solution; and rU and rS are the peak responses of agnuside obtained from the Test solution and the Standard solution, respectively.