Black Cohosh Tablets
DEFINITION
Black Cohosh Tablets contain Powdered Black Cohosh Extract or Black Cohosh Fluidextract. Tablets contain NLT 90.0% and NMT 110.0% of the labeled amount of Powdered Extract or Fluidextract, represented by the content of triterpene glycosides, calculated as 23-epi-26-deoxyactein (C37H56O10).
IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test 
201
201
Adsorbent:
Chromatographic silica gel mixture with an average particle size of 10–15 µm (TLC plates)
Sample solution:
10 mL of the Sample solution prepared for Identification test B. Evaporate to dryness, and redissolve in 1 mL of methanol.
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
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)
[Note—Store in a refrigerator. The reagent is colorless; discard if color appears. ]
Analysis
Samples:
Standard solution A, Standard solution B, and Sample solution
Develop until the solvent front has moved 15 cm, and dry the plate with the aid of a current of air. Examine the plate under UV light at a wavelength of 365 nm. Spray the plate with Spray reagent, heat at 100
for 5 min, and examine in daylight.
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. Examined under UV light, the Sample solution exhibits a blue fluorescent zone at the level of the zone due to isoferulic acid in Standard solution B, in the upper third of the plate. Examined after treatment with Spray reagent, 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 in Standard solution B.
• B. Thin-Layer Chromatographic Identification Test 201
201
Adsorbent:
Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)
Sample solution:
Transfer the equivalent of the labeled amount of Powdered Extract or Fluidextract, containing 25 mg of triterpene glycosides from a portion of powdered Tablets, to 25 mL of water; shake to disperse; and sonicate for 10 min. Add 75 mL of methanol, and sonicate for 10 min. Allow to stand for 15 min, and use the clear supernatant.
Standard solution A:
Methanol and Standard solution A prepared in Identification test A (3:1)
Standard solution B:
Methanol and Standard solution B prepared in Identification test A (4:1)
Application volume:
2 µL as an 8-mm band
Developing solvent system:
Toluene, ethyl formate, and formic acid (5:3:2)
Spray reagent:
Methanol, glacial acetic acid, sulfuric acid, and p-anisaldehyde (85:10:5:0.5)
[Note—Store in a refrigerator. The reagent is colorless; discard if color appears. ]
Analysis
Samples:
Standard solution A, Standard solution B, and Sample solution
Develop until the solvent front has moved 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.
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, two of which are red-violet zones at RF values of 0.5 and 0.4, similar in color and RF values to those due to actein and 23-epi-26-deoxyactein in Standard solution B.
• C.
The Sample solution exhibits peaks for cimiracemoside A, 26-deoxycimicifugoside, (26S) actein, 23-epi-26-deoxyactein, cimigenol–arabinoside, and cimigenol–xyloside at retention times corresponding to those compounds in the Standard solution, as obtained in the test for Content of Triterpene Glycosides. The ratio of the peak areas of cimigenol–arabinoside to cimigenol–xyloside is NLT 0.4 (distinction from Cimicifuga foetida).
STRENGTH
• Content of Triterpene Glycosides
Solution A:
Filtered and degassed 0.05% trifluoroacetic acid in water
Solution B:
Filtered and degassed acetonitrile
Mobile phase:
See the gradient table below.
| Time (min) |
Water (%) |
Solution A (%) |
Solution B (%) |
|---|---|---|---|
| 0 | 0 | 80 | 20 |
| 8 | 0 | 80 | 20 |
| 15 | 68 | 0 | 32 |
| 55 | 36 | 0 | 64 |
| 65 | 5 | 0 | 95 |
| 70 | 5 | 0 | 95 |
| 85 | 0 | 80 | 20 |
System suitability solution:
0.1 mg/mL each of USP Actein RS and USP 23-epi-26-Deoxyactein RS in methanol
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 having a 0.45-µm or finer porosity.
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 having a 0.45-µm or finer porosity.
Sample solution:
Weigh NLT 20 Tablets, and finely powder. Transfer a quantity of the powder, equivalent to 8 mg of triterpene glycosides, to a suitable polytef-capped centrifuge tube. Add 3 mL of water, shake to disperse, and sonicate for 10 min at 60. Add 3 mL of methanol, and sonicate for 10 min. Centrifuge, and transfer the clear supernatant to a 10-mL volumetric flask. Wash the residue twice with 1.5 mL of a mixture of methanol and water (1:1), and transfer the washings to the volumetric flask. Dilute with a mixture of methanol and water (1:1) to volume, and pass through a membrane filter having a 0.45-µm or finer porosity.
. Add 3 mL of methanol, and sonicate for 10 min. Centrifuge, and transfer the clear supernatant to a 10-mL volumetric flask. Wash the residue twice with 1.5 mL of a mixture of methanol and water (1:1), and transfer the washings to the volumetric flask. Dilute with a mixture of methanol and water (1:1) to volume, and pass through a membrane filter having a 0.45-µm or finer porosity.
Chromatographic system
Mode:
LC
Detector:
Evaporative light-scattering
[Note—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
System suitability
Samples:
System suitability solution and 100 µg/mL of 23-epi-26-Deoxyactein standard solution
Suitability requirements
Chromatographic profile:
The chromatogram of the Standard solution is similar to the Reference Chromatogram provided with the lot of USP Powdered Black Cohosh Extract RS.
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% for the logarithm of the area responses for replicate injections, 100 µg/mL 23-epi-26-Deoxyactein standard solution
Analysis
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 the following table.
| Name | Relative Retention Time |
|---|---|
| Cimicifugoside H-1 | 0.61 |
| Cimiracemoside A | 0.78 |
| (26R)-Actein | 0.94 |
| 26-Deoxycimicifugoside | 0.96 |
| (26S)-Actein | 0.98 |
| 23-epi-26-Deoxyactein | 1.00 |
| Acetyl-shengmanol–xyloside | 1.03 |
| Cimigenol–arabinoside | 1.08 |
| Cimigenol–xyloside (cimicifugoside) | 1.13 |
| 26-Deoxyactein | 1.22 |
| 25-Acetyl-cimigenol–arabinoside | 1.60 |
| (24S)-25-Acetyl-cimigenol–xyloside | 1.64 |
| 25-O-Methyl-cimigenol–arabinoside | 1.90 |
| 25-O-Methyl-cimigenol–xyloside | 1.93 |
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.
Calculate the quantity, in mg, of triterpene glycosides in the portion of Tablets taken:
Result = CT/100
| CT | = | = sum of the concentrations C, in µg/mL, of all the relevant triterpene glycosides, calculated as 23-epi-26-deoxyactein |
Calculate the percentage of the labeled amount of Extract in the portion of Tablets taken:
Result = (AWT/W) × (100/LE) × (100/L) × CTT
| AWT | = | = average weight of Tablets |
| W | = | = weight of sample |
| LE | = | = labeled content, as percentage, of triterpenes in the Extract used to prepare the Tablets |
| L | = | = labeled amount of Extract per Tablet |
| CTT | = | = content, in mg, of triterpenes in the sample |
Acceptance criteria:
90.0%–110.0% of the labeled amount of Powdered Extract or Fluidextract, represented by the content of triterpene glycosides
PERFORMANCE TESTS
• Disintegration and Dissolution of Dietary Supplements 2040:
Meet the requirements for Disintegration
2040:
Meet the requirements for Disintegration
• Weight Variation of Dietary Supplements 2091:
Meet the requirements
2091:
Meet the requirements
CONTAMINANTS
• Microbial Enumeration Tests—Nutritional and Dietary Supplements 2021:
The total bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 103 cfu/g.
2021:
The total bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 103 cfu/g.
• Microbial Procedures for Absence of Specified Microorganisms—Nutritional and Dietary Supplements 2022:
Tablets meet the requirements of the tests for absence of Salmonella species and Escherichia coli.
2022:
Tablets meet the requirements of the tests for absence of Salmonella species and Escherichia coli.
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight, light-resistant containers, and store at room temperature.
• Labeling:
The label states the Latin binomial and, following the official name, the article from which the Tablets were prepared. The label also indicates the amount, in mg/Tablet, of Powdered Extract or Fluidextract; the solvents used to prepare the Powdered Extract or Fluidextract; and the ratio of starting crude plant material to Powdered Extract or Fluidextract. Label it to indicate the content, in percentage, of triterpene glycosides as 23-epi-26-deoxyactein in the Powdered Extract or Fluidextract used to prepare the Tablets.
The label bears 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
11
USP Actein RS 
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USP Powdered Black Cohosh Extract RS
USP 23-epi-26-Deoxyactein RS
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Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Maged H. Sharaf, Ph.D.
Principal Scientific Liaison 1-301-816-8318 |
(DS2010) Monographs - Dietary Supplements |
| 2021 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| 2022 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1212
Pharmacopeial Forum: Volume No. 36(1) Page 150