Powdered Red Clover Extract
DEFINITION
Powdered Red Clover Extract is prepared from Red Clover by extraction with hydroalcoholic mixtures or other suitable solvents. The ratio of plant material to extract is 3:1–25:1. It contains NLT 90.0% and NMT 110.0% of the labeled amount of isoflavones, calculated on the dried basis as the sum of daidzein, genistein, formononetin, and biochanin A. It may contain suitable added substances.
IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test
Standard solution:
Transfer 100 mg of USP Powdered Red Clover Extract RS to a screw-capped centrifuge tube. Add 1 mL of a mixture of alcohol and water (7:3), and heat in a steam bath for 10 min. Centrifuge, and use the clear supernatant.
Sample solution:
Shake a quantity of Powdered Extract, equivalent to 25 mg of the labeled amount of isoflavones, in 20 mL of methanol. Allow to stand for 15 min before use.
Chromatographic system
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture.
Application volume:
20–30 µL, in bands 2 cm long
Developing solvent system:
Ethyl acetate, formic acid, glacial acetic acid, and water (100:11:11:27)
Spray reagent A:
10 mg/mL of 2-aminoethyl diphenylborinate in methanol
Spray reagent B:
50 mg/mL of polyethylene glycol 4000 in alcohol
Analysis
Samples:
Standard solution and Sample solution
Develop the chromatograms to a length of NLT 18 cm, and dry the plate in a current of air. Spray the plate with Spray reagent A followed by Spray reagent B, and examine the plate under UV light at 365 nm.
Acceptance criteria:
The Sample solution chromatogram exhibits a blue zone, a yellowish-green zone, and a yellowish-orange zone at RF values of about 0.70, 0.55, and 0.50, respectively, corresponding in color and RF to zones in the chromatogram of the Standard solution. Other colored zones of varying intensities may be observed in the chromatogram from the Sample solution.
• B. HPLC Identification Test
Analysis:
Proceed as directed in the test for Content of Isoflavones.
Using the values obtained in the test for Content of Isoflavones, calculate the ratio of 5,7-dihydroxyisoflavones to 7-hydroxyisoflavones:
Result = (B + G)/(D + F)
| B | = | = percentage of biochanin A |
| G | = | = percentage of genistein |
| D | = | = percentage of daidzein |
| F | = | = percentage of formononetin |
Acceptance criteria:
The chromatogram of the Sample solution exhibits peaks for daidzein, genistein, formononetin, and biochanin A at retention times that correspond to those in the chromatogram of Standard solution A. The ratio of 5,7-dihydroxyisoflavones to 7-hydroxyisoflavones is between 0.1 and 10.0.
COMPOSITION
• Content of Isoflavones
Solution A:
Acetonitrile and water (1:3) containing 0.05% trifluoroacetic acid
Solution B:
Acetonitrile containing 0.05% trifluoroacetic acid
Mobile phase:
See Table 1.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 100 | 0 |
| 2 | 100 | 0 |
| 2.5 | 87 | 13 |
| 7.5 | 80 | 20 |
| 7.8 | 73 | 27 |
| 8.0 | 55 | 45 |
| 11.0 | 50 | 50 |
| 13.0 | 40 | 60 |
| 15.0 | 26 | 74 |
| 16.0 | 0 | 100 |
| 18.1 | 100 | 0 |
| 23.0 | 100 | 0 |
Solvent:
Alcohol and water (1:1)
Standard stock solution A:
Transfer a quantity of USP Powdered Red Clover Extract RS, equivalent to 30 mg of the labeled content of isoflavones, to a 250-mL volumetric flask. Add 15 mL of dehydrated alcohol, sonicate until dissolved, and dilute with Solvent to volume.
Standard solution A:
Evaporate 50 mL of Standard stock solution A to dryness under vacuum. Add 15 mL of 2 N hydrochloric acid, and heat in a water bath for 30 min. Quantitatively transfer the resulting solution, with the aid of 15 mL of alcohol, to a 50-mL volumetric flask, and dilute with Solvent to volume. Centrifuge, or filter through a membrane having a 0.45-µm or finer pore size.
Standard solution B:
0.1 mg/mL of USP Formononetin RS in a mixture of n-propanol and water (1:1). Sonicate, and filter through a membrane having a 0.45-µm or finer pore size.
Sample stock solution:
Transfer a quantity of Powdered Extract, equivalent to 30 mg of the labeled content of isoflavones, to a 250-mL volumetric flask. Add 15 mL of dehydrated alcohol, sonicate until dissolved, and dilute with Solvent to volume.
Sample solution:
Evaporate 50.0 mL of Sample stock solution to dryness under vacuum. Add 15 mL of 2 N hydrochloric acid, and heat in a water bath for 30 min. Quantitatively transfer the resulting solution with the aid of 15 mL of alcohol to a 50-mL volumetric flask, and dilute with Solvent to volume. Centrifuge, or pass through a filter of 0.45-µm or finer pore size.
Chromatographic system
Mode:
LC
Detector:
UV 254 nm
Column:
4.6-mm × 25-cm; end-capped 5-µm packing L1
Column temperature:
45
Flow rate:
1 mL/min
Injection size:
10 µL
System suitability
Sample:
Standard solution A
Suitability requirements
Chromatogram similarity:
The chromatogram obtained from Standard solution A is similar to the reference chromatogram provided with the lot of USP Powdered Red Clover Extract RS being used.
Tailing factor:
NMT 2.0 for the formononetin peak
Relative standard deviation:
NMT 2.0%
Analysis
Samples:
Standard solution A, Standard solution B, and Sample solution
Identify the peaks corresponding to daidzein, genistein, formononetin, and biochanin A in the Sample solution chromatogram by comparison with the chromatogram obtained from Standard solution A and the reference chromatogram. Measure the areas of the analyte peaks.
Separately calculate the percentage of each isoflavone in the portion of Powdered Extract taken:
Result = (rU/rS) × (CS/CU) × 1/F × 100
| rU | = | = peak response for each relevant isoflavone from Sample solution |
| rS | = | = peak response for formononetin from Standard solution B |
| CS | = | = concentration of USP Formononetin RS in Standard solution B (mg/mL) |
| CU | = | = nominal concentration of isoflavones in the Sample solution (mg/mL) |
| F | = | = conversion factor for each analyte: daidzein, 0.97; genistein,1.13; formononetin, 1.00; biochanin A, 1.05 |
Acceptance criteria:
90.0%–110.0% of the labeled amount of isoflavones as the sum of daidzein, genistein, formononetin, and biochanin A, calculated on the dried basis
CONTAMINANTS
• Heavy Metals, Method II 
231
:
NMT 10 µg/g
231:
NMT 10 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 103 cfu/g, and the enterobacterial count is NMT 103 cfu/g.
2021:
The total aerobic microbial count does not exceed 104 cfu/g, the total combined molds and yeasts count does not exceed 103 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.
2022:
It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
SPECIFIC TESTS
• Loss on Drying 731:
Dry 1 g at 105 for 2 h: it loses NMT 5.0% of its weight.
731:
Dry 1 g at 105 for 2 h: it loses NMT 5.0% of its weight.
• Other Requirements:
It meets the requirements for Packaging and Storage, Residual Solvents, and Pesticide Residues in Botanical Extracts 565.
565.
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight, light-resistant containers, 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 isoflavones, the extracting solvent or solvent mixture used for preparation, and the ratio of the starting crude plant material to Powdered Extract. It meets the requirements for labeling in Botanical Extracts 565.
565.
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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 1421
Pharmacopeial Forum: Volume No. 30(2) Page 552