Black Cohosh consists of the dried rhizome and roots of Actaea racemosa L. [Cimicifuga racemosa (L.) Nutt.] (Ranunculaceae). It is harvested in the summer. It contains NLT 0.4% of triterpene glycosides, calculated as 23-epi-26-deoxyactein1 (C37H56O10) on the dried basis.
• A. Thin-Layer Chromatographic Identification Test
Standard solution A: 100 mg/mL of USP Powdered Black Cohosh Extract RS in methanol
Standard solution B: 1 mg/mL each of USP Actein RS, USP 23-epi-26-Deoxyactein RS, and isoferulic acid in methanol
Sample solution: Transfer 5 g of powdered Black Cohosh 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.
Adsorbent: Chromatographic silica gel mixture with an average particle size of 1015 µm (TLC plates)
Application volume: 10 µL
Developing solvent system: Use the upper phase of a mixture of butyl alcohol, glacial acetic acid, and water (5:1:4).
Spray reagent: Methanol, glacial acetic acid, sulfuric acid, and p-anisaldehyde (85:10:5:0.5). [NoteStore in a refrigerator. The reagent is colorless; discard if color appears. ]
Samples: Standard solution A, Standard solution B, and Sample solution
Develop the chromatograms until the solvent front has moved about 15 cm, and dry the plate with the aid of a current of air.
Acceptance criteria: Examine the plate under UV light at 365 nm. The chromatogram of the Sample solution exhibits main zones similar in position and color to the main zones of Standard solution A. In the upper third of the plate, the Sample solution exhibits a blue fluorescent zone at the level of the zone due to isoferulic acid of Standard solution B. Spray the plate with Spray reagent, heat at 100 for 5 min, and examine in daylight. The Sample solution exhibits main zones similar in position and color to the main zones of Standard solution A. Standard solution B exhibits red-violet zones due to actein and 23-epi-26-deoxyactein. The Sample solution exhibits several greenish-brown spots in the lower third of the plate and several violet zones above; two of these violet zones occur at RF values similar to those due to actein and 23-epi-26-deoxyactein of Standard solution B.
• B. Thin-Layer Chromatographic Identification Test
Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)
Standard solution A: 0.5 mL of Standard solution A prepared in Identification test A, diluted with methanol to 2 mL
Standard solution B: 1.0 mL of Standard solution B prepared in Identification test A, diluted with methanol to 5 mL
Sample solution: Transfer 0.5 g of powdered Black Cohosh to a screw-capped tube, add 5 mL of methanol, sonicate for 10 min, and filter into a 10-mL volumetric flask. Wash the residue on the filter paper four times, using 1 mL of methanol for each washing; add the washings to the volumetric flask; and dilute with methanol to volume.
Application volume: 2 µL as an 8-mm band
Developing solvent system: Toluene, ethyl formate, and formic acid (5:3:2)
Spray reagent: Proceed as directed for Identification test A.
Samples: Standard solution A, Standard solution B, and Sample solution
Develop the chromatograms until the solvent front has moved about two-thirds of the length of the plate, and dry the plate with the aid of a current of air. Spray the plate with Spray reagent, heat at 100 for 5 min, and examine in daylight.
Acceptance criteria: The Sample solution exhibits main zones similar in position and color to the main zones of Standard solution A. Standard solution B exhibits red-violet zones due to actein and 23-epi-26-deoxyactein at RF values of about 0.5 and 0.4, respectively. The Sample solution exhibits zones similar in color and RF values to those due to actein and 23-epi-26-deoxyactein of Standard solution B.
• C. The Sample solution exhibits peaks for cimiracemoside A, 26-deoxycimicifugoside, (26S)-actein, 23-epi-26-deoxyactein, cimigenolarabinoside, and cimigenolxyloside at retention times corresponding to these compounds in the Standard solution, as obtained in the test for Content of Triterpene Glycosides. The ratio of the peak areas of cimigenolarabinoside to cimigenolxyloside is NLT 0.4 (distinction from Cimicifuga foetida).
• Content of Triterpene Glycosides
Standard solution: Dissolve a quantity of USP Powdered Black Cohosh Extract RS in methanol with shaking for 1 min, and dilute with methanol to obtain a solution having a known concentration of 30 mg/mL. Pass through a membrane filter of 0.45-µm or finer pore size.
23-epi-26-Deoxyactein standard solutions: Dissolve USP 23-epi-26-Deoxyactein RS in methanol with shaking for 1 min. Dilute quantitatively, and stepwise if necessary, to obtain solutions having known concentrations of 500, 100, 50, 25, and 12.5 µg/mL. Pass through a membrane filter of 0.45-µm or finer pore size.
System suitability solution: 0.1 mg/mL each of USP Actein RS and USP 23-epi-26-Deoxyactein RS in methanol
Sample solution: Accurately weigh about 750 mg of ground plant material, and place in a 20-mL polytef-capped centrifuge tube. Pipet 15 mL of methanol, sonicate for 30 min, centrifuge, and transfer the supernatant to an evaporation flask. Repeat the extraction twice. Evaporate the combined extracts under vacuum at 4550. Dissolve the residue in methanol, and quantitatively transfer to a 10-mL volumetric flask. Dilute with methanol to volume, and pass through a membrane filter of 0.45-µm or finer pore size.
Solution A: 0.05% trifluoroacetic acid in water
Solution B: Acetonitrile
Mobile phase: See Table 1.
Detector: Evaporative light-scattering
[NoteThe detector is set up according to the manufacturer's instruction in order to achieve a signal-to-noise ratio of NLT 10 for the 12.5-µg/mL 23-epi-26-Deoxyactein standard solution. ]
Column: 4.6-mm × 25-cm; 5-µm packing L1
Column temperature: 35
Flow rate: 1.6 mL/min
Injection size: 20 µL
Samples: System suitability solution, Standard solution, and 100-µg/mL 23-epi-26-Deoxyactein standard solution
Chromatogram similarity: The chromatogram of the Standard solution is similar to the Reference Chromatogram provided with the lot of USP Powdered Black Cohosh Extract RS being used.
Resolution: NLT 1.0 between the (26S)-actein and the 23-epi-26-deoxyactein peaks, System suitability solution
Tailing factor: NMT 2.0 for the 23-epi-26-deoxyactein peak, 100-µg/mL 23-epi-26-Deoxyactein standard solution
Relative standard deviation: NMT 2.0% of the logarithm of the area response of the 23-epi-26-deoxyactein peak in repeated injections, 100-µg/mL 23-epi-26-Deoxyactein standard solution
Samples: System suitability solution, Standard solution, 23-epi-26-Deoxyactein standard solutions, and Sample solution
Using the chromatogram of the Standard solution and the Reference Chromatogram provided with the lot of USP Powdered Black Cohosh Extract RS, identify the retention times of the peaks corresponding to the triterpene glycosides. The approximate relative retention times of the triterpene glycosides are provided in Table 2.
Plot the logarithms of the peak area responses versus the logarithms of the concentrations, in µg/mL, of the 23-epi-26-Deoxyactein standard solutions, and determine the regression line using a least-squares analysis. The correlation coefficient for the regression line is NLT 0.995. From the graphs so obtained, determine the concentration, C, in µg/mL, of the relevant analyte in the Sample solution. Separately calculate the percentages of cimicifugoside H-1, cimiracemoside A, (26R)-actein, 26-deoxycimicifugoside, (26S)-actein, 23-epi-26-deoxyactein, acetyl-shengmanolxyloside, cimigenolarabinoside, cimigenolxyloside (cimicifugoside), 26-deoxyactein, 25-acetyl-cimigenolarabinoside, (24S)-25-acetyl-cimigenolxyloside, 25-O-methyl-cimigenolarabinoside, and 25-O-methyl-cimigenolxyloside as 23-epi-26-deoxyactein (C37H56O10) in the portion of Black Cohosh taken:
Result = (V × C)/(F × W) × 100
Calculate the percentage of triterpene glycosides in the portion of Black Cohosh taken by adding all of the percentages calculated for the individual analytes.
Acceptance criteria: NLT 0.4% on the dried basis
• Heavy Metals 231: NMT 10 ppm
• Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561: Meets the requirements
• Microbial Enumeration Tests 2021: It meets the requirements of the tests for absence of Salmonella species and Escherichia coli. The total aerobic microbial count does not exceed 105 cfu/g; the total combined molds and yeasts count does not exceed 103 cfu/g; and the bile-tolerant Gram-negative bacteria count does not exceed 103 cfu/g.
• Botanic Characteristics
Macroscopic: The Black Cohosh rhizome is dark brown, longitudinally grooved, rough, strongly knotty, and somewhat curled and irregular. It is 15 cm long and up to 2.5 cm thick. The upper surface is covered with numerous round scars of the earlier stalks; laterally, it is clearly curled, and the lower surface is covered with thin, longitudinally grooved, dark brown, easily breakable roots. The fracture is horny and fibrous. The transverse cut shows a thin outer bark surrounding a ring of numerous pale, narrow wedges of vascular tissue alternating with dark medullary rays and a large central pith. Black Cohosh roots are dark brown, between 1 and 3 mm in diameter, brittle, nearly cylindrical or obtusely quadrangular, and longitudinally wrinkled. The fracture is short. The transverse cut shows a distinct cambium line separating a wide outer bark from a central region composed of three to six wedges of lignified xylem tissue united by their apices and separated by broad nonlignified medullary rays.
Microscopic: In a surface view, suberous epidermal cells are tabular with moderately thickened walls. The parenchymatous cortex is filled with starch. Xylem wedges are lignified and composed of numerous small vessels with bordered pits or reticulately thickened walls, thin-walled fibers, and xylem parenchyma. The parenchyma of the pith is unlignified. Medullary rays are filled with starch granules, which are spherical or polygonal and are mostly simple or two to three compounded but can be up to six compounded. Individual starch granules are between 3 and 15 µm in diameter, each with a somewhat central slit-shaped hilum.
• Articles of Botanical Origin, Foreign Organic Matter 561: NMT 2.0% of foreign organic matter, and NMT 5.0% of stem bases
• Articles of Botanical Origin, Alcohol-Soluble Extractives, Method 2 561: NLT 8.0%, using a mixture of alcohol and water (1:1) instead of alcohol
• Loss on Drying 731: Dry at 105 for 2 h: it loses NMT 12.0% of its weight.
• Articles of Botanical Origin, Total Ash 561: NMT 10.0%
• Packaging and Storage: Preserve in a well-closed, light-resistant container. Protect from moisture, and store at room temperature.
• Labeling: The label states the Latin binomial and, following the official name, the parts of the plant contained in the article. Dosage forms prepared with this article should bear the following statement: Discontinue use and consult a healthcare practitioner if you have a liver disorder or develop symptoms of liver trouble, such as abdominal pain, dark urine, or jaundice.
• USP Reference Standards 11
USP Powdered Black Cohosh Extract RS
USP 23-epi-26-Deoxyactein RS
1 23-epi-26-Deoxyactein is sometimes referred to as 27-deoxyactein.
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USP35NF30 Page 1204Pharmacopeial Forum: Volume No. 33(5) Page 954