Powdered Horse Chestnut Extract
Powdered Horse Chestnut Extract is prepared from Horse Chestnut by extraction with alcoholwater mixtures or methanolwater mixtures. The ratio of starting plant material to extract is between 5:1 and 8:1. It contains NLT 90.0% and NMT 110.0% of the labeled amount of triterpene glycosides, calculated on the dried basis as escin (C55H86O24). It may contain suitable added substances.
• A. Thin-Layer Chromatographic Identification Test
Standard solution: 5 mg/mL of USP Escin RS in methanol
Sample solution: To 10 mL of methanol add a quantity of Powdered Extract equivalent to 25 mg of the labeled amount of triterpene glycosides, and shake. Allow to stand for 15 min before use.
(See Chromatography 621, Thin-Layer Chromatography.)
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)
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.
• 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: Transfer a quantity of Powdered Extract, equivalent to 50 mg of the labeled content of triterpene glycosides, into a 50-mL flask. Add 20 mL of 0.1 N hydrochloric acid, and shake for 5 min. Filter into a 250-mL separatory funnel with the aid of two additional 5-mL portions of 0.1 N hydrochloric acid. 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 100-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.
Wavelength: 540 nm
Blank: Glacial acetic acid
Analysis: Transfer 1 mL each of Standard solutions A, B, and C, the Sample solution, and the 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 the labeled amount of triterpene glycosides in the portion of Powdered Extract taken:
Result = (C/CU) × 100
Acceptance criteria: 90.0%110.0% of the labeled amount of triterpene glycosides as escin (C55H86O24) on the dried basis
• Heavy Metals, Method II 231: NMT 20 µg/g
• Microbial Enumeration Tests 2021: The total aerobic microbial count does not exceed 104 cfu/g, the total combined molds and yeasts count does not exceed 102 cfu/g, and the count for enterobacteria does not exceed 103 cfu/g.
• Absence of Specified Microorganisms 2022: It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
• Loss on Drying 731: Dry 1 g at 105 for 2 h: it loses NMT 5.0% of its weight.
• Other Requirements: It meets the requirements in Botanical Extracts 565, General Pharmacopeial Requirements, for Packaging and Storage, Residual Solvents, and Pesticide Residues for powdered extracts.
• Packaging and Storage: Preserve in tight, light-resistant containers. Store in a cool place.
• Labeling: The label states the Latin binomial and, following the official name, the part of the plant from which the article was prepared. The label also indicates the content of triterpene glycosides, the extracting solvent or solvent mixture used for preparation, the ratio of the starting crude plant material to Powdered Extract, and the name and content of any added substance. It meets the requirements for labeling in Botanical Extracts 565.
• USP Reference Standards 11
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