Powdered Black Cohosh Extract
DEFINITION
Powdered Black Cohosh Extract is prepared from Black Cohosh by extraction with hydroalcoholic mixtures or other suitable solvents. It contains NLT 90.0% and NMT 110.0% of the labeled amount of triterpene glycosides, calculated as 23-epi-26-deoxyactein (C37H56O10) on the dried basis.
IDENTIFICATION
• 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:
Shake a quantity of Powdered Extract, equivalent to 25 mg of triterpene glycosides, in 10 mL of methanol. Allow to stand for 15 min before use.
Adsorbent:
Chromatographic silica gel mixture with an average particle size of 10–15 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). [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 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 in the chromatogram of Standard solution A. In the upper third of the plate, the chromatogram of the Sample solution exhibits a blue fluorescent zone at the level of the zone due to isoferulic acid in the chromatogram of Standard solution B. Spray the plate with Spray reagent, heat at 100
for 5 min, and examine in daylight. The chromatogram of the Sample solution exhibits main zones similar in position and color to the main zones in the chromatogram of Standard solution A. The chromatogram of Standard solution B exhibits red-violet zones due to actein and 23-epi-26-deoxyactein. The chromatogram of 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 the chromatogram of Standard solution B.
for 5 min, and examine in daylight. The chromatogram of the Sample solution exhibits main zones similar in position and color to the main zones in the chromatogram of Standard solution A. The chromatogram of Standard solution B exhibits red-violet zones due to actein and 23-epi-26-deoxyactein. The chromatogram of 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 the chromatogram of Standard solution B.
• B. Thin-Layer Chromatographic Identification Test
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:
Dilute 1 mL of the Sample solution prepared in Identification test A with methanol to 10 mL.
Adsorbent:
Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)
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.
Analysis
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.
for 5 min, and examine in daylight.
Acceptance criteria:
The chromatogram of the Sample solution exhibits main zones similar in position and color to the main zones in the chromatogram of Standard solution A. The chromatogram of 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 chromatogram of the Sample solution exhibits zones similar in color and RF values to those due to actein and 23-epi-26-deoxyactein in the chromatogram of Standard solution B.
• C.
The chromatogram of 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 chromatogram of 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).
COMPOSITION
• 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:
Transfer a quantity of Powdered Extract, equivalent to 7.5 mg of triterpene glycosides, to a 10-mL volumetric flask, add 7 mL of methanol, and sonicate for 30 min. Dilute with methanol to volume. Centrifuge, or pass through a 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.
Table 1
| 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 |
Chromatographic system
Mode:
LC
Detector:
Evaporative light-scattering
[Note—The 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, Standard solution, and 100-µg/mL 23-epi-26-Deoxyactein standard solution
Suitability requirements
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
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 Table 2.
Table 2
| 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. Separately calculate the percentages of cimicifugoside H-1, cimiracemoside A, (26R)-actein, 26-deoxycimicifugoside, (26S)-actein, 23-epi-26-deoxyactein, acetyl-shengmanol–xyloside, cimigenol–arabinoside, cimigenol–xyloside (cimicifugoside), 26-deoxyactein, 25-acetyl-cimigenol–arabinoside, (24S)-25-acetyl-cimigenol–xyloside, 25-O-methyl-cimigenol–arabinoside, and 25-O-methyl-cimigenol–xyloside as 23-epi-26-deoxyactein (C37H56O10) in the portion of Extract taken:
Result = (V × C)/(F × W) × 100
| V | = | = volume of the Sample solution (mL) |
| C | = | = concentration of the relevant analyte in the Sample solution (µg/mL) |
| F | = | = factor to convert mg to µg, 1000 µg/mg |
| W | = | = weight of the Powdered Extract taken to prepare the Sample solution (mg) |
Calculate the percentage of the labeled amount of triterpene glycosides in the portion of Extract taken by adding all of the percentages calculated for individual analytes.
Acceptance criteria:
90.0%–110.0% on the dried basis
CONTAMINANTS
• Heavy Metals 
231
:
NMT 10 ppm
231:
NMT 10 ppm
• Microbial Enumeration Tests 2021:
It meets the requirements of the tests for absence of Salmonella species and Escherichia coli. 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:
It meets the requirements of the tests for absence of Salmonella species and Escherichia coli. The total bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 103 cfu/g.
• Other Requirements:
It meets the requirements under Botanical Extracts 565, Pesticide Residues.
565, Pesticide Residues.
SPECIFIC TESTS
• Loss on Drying 731:
NMT 5.0%
731:
NMT 5.0%
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight, light-resistant containers, and store in a cool place.
• Labeling:
It meets the requirements under Botanical Extracts 565. Label it to indicate the content of triterpene glycosides, in percentage, calculated as 23-epi-26-deoxyactein. 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.”
565. Label it to indicate the content of triterpene glycosides, in percentage, calculated as 23-epi-26-deoxyactein. 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
11
USP Actein RS 
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USP Powdered Black Cohosh Extract RS
USP 23-epi-26-Deoxyactein RS
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 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1210
Pharmacopeial Forum: Volume No. 33(5) Page 960