Macroscopic— Horse chestnut seeds are dense and hard, subspherical to oval, slightly flattened, and from 2 to 4 cm in diameter. They have a dark brown seed coat from 1 to 1.5 mm thick, with a large, round, light brown spot (hilum). The seed coat is shiny, but only in fresh condition. The space under the coat is totally filled with the shiny, massive embryo and its large, pale yellow cotyledons lacking endosperm.

Microscopic— The epidermis of the testa in surface view has yellowish-brown cells of fairly uniform size, with the majority of cells rounded to polygonal, and a few that are square to obscurely triangular. The walls of these cells are considerably but rather unevenly thickened, and lack pits. In sectional view, the cells are columnar, approximately 3 to 4 times as high as they are wide, with the outer periclinal wall markedly thickened, uneven, and becoming thinner towards the base; beneath the epidermis there are a few layers of small collenchymatously thickened cells with small intercellular spaces; the greater part of the testa consists of larger, loosely-packed parenchymatous cells forming a spongy tissue; the walls are variably and unevenly thickened, with intracellular and large circular spaces well marked; the inner testa is a narrow zone, with ill-defined and thinner-walled cells. All the parenchymatous cells of the testa are darkly pigmented. The embryo has an outer layer of small colorless cells, almost square in sectional view, with outer and side walls thickened. In surface view, only the irregular and more or less polygonal lumens are discernible, giving a reticulate, pitted appearance. Cotyledons are moderately thickened and indistinctly pitted, having round to ovoid parenchymatous cells densely filled with starch. Starch granules, mainly simple, are present in two size ranges: from 15 to 30 µm and from 3 to 10 µm. The largest granules vary from circular, ovoid, and bluntly polygonal to pyriform, most of them with a well-marked cleft or stellate hilum, and lacking striations. The smaller starch granules are less variable, spherical to ovoid, with the hilum more often a point. Compound starch granules are very infrequently found.

Test solution— Transfer about 1 g of the powdered plant material to a screw-capped centrifuge tube, add 10 mL of a mixture of alcohol and water (7:3), and heat on a steam bath for 10 minutes. Centrifuge, and use the clear supernatant.

Standard solution— Dissolve an accurately weighed quantity of USP Escin RS in methanol to obtain a solution having a known concentration of about 5 mg per mL.

Developing solvent system— Use the upper phase of a mixture of 1-butanol, water, and glacial acetic acid (50:40:10).

Spray reagent— Prepare a mixture of methanol, glacial acetic acid, sulfuric acid, and p-anisaldehyde (85:10:5:0.5).

Procedure— Develop the chromatogram to a length of not less than 15 cm, and dry the plate in a current of air. Spray the plate with Spray reagent, heat the plate at 100 for 5 minutes, and examine the plate under daylight: the chromatogram obtained from the Test solution shows a blue-violet zone corresponding to escin, comparable in position and color to the main zone in the chromatogram obtained from the Standard solution. Above this zone, the chromatogram of the Test solution shows several narrow, brown to brownish-red zones that are less intense than the zone corresponding to escin.

Solvent 1— Prepare a mixture of methanol and water (65:35).

Solvent 2— Use the lower phase of a mixture of 30 mL of 0.1 N hydrochloric acid, 20 mL of 1-propanol, and 50 mL of chloroform.

Reagent— Dissolve 75 mg of ferric chloride in 50 mL of glacial acetic acid. Add 50 mL of sulfuric acid, while shaking and cooling. Prepare immediately before use.

Escin standard solutions— Dissolve an accurately weighed quantity of USP Escin RS in glacial acetic acid, shaking for 1 minute. Dilute quantitatively, and stepwise if necessary, to obtain solutions having known concentrations of about 0.6, 0.4, and 0.2 mg per mL.

Test solution— Accurately weigh 1.00 g of ground seeds, and place in a 250-mL round-bottom flask. Add exactly 100 mL of Solvent 1, and weigh the filled flask with a precision of ±0.1 g. Attach a condenser to the flask, reflux for 30 minutes, and allow to cool. Adjust to the initial weight by adding Solvent 1 as needed, mix, and filter. Transfer 30.0 mL of the filtrate to a round-bottom flask, and evaporate the solvents under vacuum. Dissolve the residue with 20 mL of 0.1 N hydrochloric acid, and quantitatively transfer with the aid of two additional 5-mL portions of 0.1 N hydrochloric acid to a 250-mL separation funnel. Add 20 mL of 1-propanol and 50 mL of chloroform, and shake vigorously for 2 minutes. Separate the chloroform layer, and add Solvent 2 to the upper phase remaining in the separation funnel. Shake vigorously for 2 minutes, and separate the chloroform layer. Combine the chloroform layers in a round-bottom flask, and evaporate to dryness under vacuum. Evaporate the remaining solvents with the aid of a current of air. Wash the residue with two 10-mL portions of ether, filter, wash the filter with 10 mL of ether, and discard the ether filtrates. After evaporation of the residual ether, add to the residue a 10-mL portion of glacial acetic acid, and pass through the previously used dried filter into a 50-mL volumetric flask. Repeat the addition of glacial acetic acid followed by filtration two additional times, combining the filtrates in the volumetric flask. Wash the round-bottom flask with small quantities of glacial acetic acid, and filter into the volumetric flask. Dilute with glacial acetic acid to volume.

Procedure— Transfer 1 mL each of the Escin standard solutions, the Test solution, and glacial acetic acid to separate test tubes with stoppers. Add 4.0 mL of Reagent to each tube, cap the tubes, and place them in a water bath at 60 for 25 minutes, shaking occasionally. Measure the absorbances at 540 nm of the reacted Test solution and the reacted Escin standard solutions, using glacial acetic acid as the blank. Plot the absorbances obtained from the reacted Escin standard solutions versus concentrations, in mg per mL, of USP Escin RS in the corresponding Escin standard solution. From the graphs so obtained, determine the concentration, C, in mg per mL, of triterpene glycosides as escin (C55H86O24) in the Test solution. Calculate the percentage of triterpene glycosides in the portion of Horse Chestnut taken by the formula: in which C is the concentration, in mg per mL, of triterpene glycosides in the Test solution as obtained above; and W is the weight, in g, of Horse Chestnut taken to prepare the Test solution.