Horse Chestnut
DEFINITION
Horse Chestnut consists of the dried seeds of Aesculus hippocastanum L. (Fam. Hippocastanaceae). It is harvested in the fall. It contains NLT 3.0% of triterpene glycosides, calculated on the dried basis as escin (C55H86O24).
IDENTIFICATION
•  A. Thin-Layer Chromatographic Identification Test
Standard solution:  5 mg/mL of USP Escin RS in methanol
Sample solution:  Transfer 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 min. Centrifuge, and use the clear supernatant.
Chromatographic system 
Adsorbent:  0.25-mm layer of chromatographic silica gel
Application volume:  10 µL
Developing solvent system:  Use the upper phase of a mixture of 1-butanol, glacial acetic acid, and water (5:1:4).
Spray reagent:  Methanol, glacial acetic acid, sulfuric acid, and p-anisaldehyde (85: 10: 5: 0.5)
Analysis 
Samples:  Standard solution and Sample solution
Develop the chromatograms to a length of NLT 15 cm, and dry the plate in a current of air. Spray the plate with Spray reagent, heat the plate at 100 for 5 min, and examine the plate under daylight.
Acceptance criteria:  The chromatogram from the Sample solution shows a blue-violet zone corresponding to escin, comparable in position and color to the main zone in the chromatogram from the Standard solution. Above this zone, the chromatogram of the Sample solution shows several narrow, brown to brownish-red zones that are less intense than the zone corresponding to escin.
COMPOSITION
•  Content of Triterpene Glycosides
Solvent A:  Methanol and water (13:7)
Solvent B:  Use the lower phase of a mixture of chloroform, 0.1 N hydrochloric acid, and 1-propanol (5:3:2).
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.
Standard solution A:  0.2 mg/mL of USP Escin RS in glacial acetic acid, shaking for 1 min
Standard solution B:  0.4 mg/mL of USP Escin RS in glacial acetic acid, shaking for 1 min
Standard solution C:  0.6 mg/mL of USP Escin RS in glacial acetic acid, shaking for 1 min
Sample solution:  Weigh 1 g of ground seeds, and place in a 250-mL round-bottom flask. Add exactly 100 mL of Solvent A, and weigh the filled flask with a precision of ±0.1 g. Attach a condenser to the flask, reflux for 30 min, and allow to cool. Adjust to the initial weight by adding Solvent A as needed, 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 min. Separate the chloroform layer, and add Solvent B to the upper phase remaining in the separation funnel. Shake vigorously for 2 min, 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.
Instrumental conditions 
Wavelength:  540 nm
Blank:  Glacial acetic acid
Analysis:  Transfer 1 mL each of Standard solutions A, B, and C, Sample solution, and Blank 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 min, shaking occasionally. Measure the absorbances of the reacted Sample solution and the reacted Standard solutions A, B and C, and correct for the Blank. Plot the absorbances of the reacted Standard solutions A, B, and C versus concentrations, in mg/mL of USP Escin RS in the corresponding Standard solution. From the graphs determine the concentration, C, in mg/mL, of triterpene glycosides as escin (C55H86O24) in the Sample solution.
Calculate the percentage of triterpene glycosides as escin in the portion of Horse Chestnut taken:
Result = (C/W) × (50/3)
C== concentration of triterpene glycosides in the Sample solution as obtained above (mg/mL)
W== weight of Horse Chestnut taken to prepare the Sample solution (g)
Acceptance criteria:  NLT 3.0% of triterpene glycosides, calculated as escin (C55H86O24), on the dried basis
CONTAMINANTS
•  Heavy Metals, Method III 231: NMT 20 µg/g
•  Microbial Enumeration Tests 2021: The total aerobic microbial count does not exceed 106 cfu/g, the total combined molds and yeast count does not exceed 104 cfu/g, and the enterobacterial count is NMT 103 cfu/g.
•  Absence of Specified Microorganisms 2022: It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
SPECIFIC TESTS
•  Botanic Characteristics
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 the sectional view, the cells are columnar, approximately 3–4 times as high as they are wide, with the outer periclinal wall markedly thickened, uneven, and becoming thinner toward 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 layer of the 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 the 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 found very infrequently.
•  Extractable Matter
Analysis:  Proceed as directed for Articles of Botanical Origin 561, Alcohol-Soluble Extractives, Method 2, except use a mixture of methanol and water (8:2) instead of alcohol.
Acceptance criteria:  NLT 18.0%
•  Loss on Drying 731: Dry a sample at 105 for 2 h: it loses NMT 10.0% of its weight.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in a well-closed, light-resistant container, protected from moisture.
•  Labeling: The label states the Latin binomial and, following the official name, the part of the plant contained in the article.
•  USP Reference Standards 11
USP Escin RS Click to View Structure
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