Red Clover consists of the dried inflorescence of Trifolium pratense L. (Fam. Fabaceae). It contains NLT 0.5% of isoflavones, calculated on the dried basis as the sum of daidzein, genistein, formononetin, and biochanin A.
• 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: Transfer 1 g of the powdered plant material to a screw-capped centrifuge tube. Add 10 mL of a mixture of methanol and water (3:2), heat in a steam bath for 1015 min, cool, and filter.
Adsorbent: 0.25 mm Layer of chromatographic silica gel mixture
Application volume: 2030 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
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 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)
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, and the ratio of 5,7-dihydroxyisoflavones to 7-hydroxyisoflavones is between 0.1 and 10.
• 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.
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 about 2.5 g of ground plant material, accurately weighed, into a 120-mL flask with a stopper. Add exactly 100 mL of Solvent, close the flask, and shake on an orbital or wrist-action shaker for NLT 12 h.
Sample solution: Evaporate 50 mL of Sample stock solution to dryness under vacuum at 40. Add 15 mL of 2 N hydrochloric acid, and heat in a water bath for 30 min. Quantitatively transfer this solution, with the aid of 15 mL of alcohol, to a 50-mL volumetric flask, and dilute with Solvent to volume. Filter through a membrane having a 0.45-µm or finer pore size, discarding the first 4 mL of the filtrate.
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
Sample: Standard solution A
Chromatogram similarity: The chromatogram 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%
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 percentages of daidzein, genistein, formononetin, and biochanin A in the portion of Red Clover taken:
Result = (rU/rS) × CS × (V/W) × 1/F × D × 100
Acceptance criteria: Add the percentages of daidzein, genistein, formononetin, and biochanin A: NLT 0.5% of isoflavones on the dried basis
• Heavy Metals 231: NMT 10 µg/g
• Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561: Meets the requirements
• Microbial Enumeration Tests 2021: The total aerobic microbial count does not exceed 106 cfu/g, the total combined molds and yeast count does not exceed 104 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.
• Botanic Characteristics
Macroscopic: Red Clover inflorescences are ovoid with a rounded summit, mostly 1234 mm in length and width, usually on a very short stalk, shriveled, purplish, and more or less brown from drying, consisting of many papilionaceous flowers, crowded together and clothed at the base with broad, pointed, pale green ciliate stipules with darker veins. The flowers, which may or may not be accompanied by diminutive trifoliate leaves, are up to 15 mm in length and have the following: five green, hairy, subulate calyx teeth, one longer than the other four; petals united into a more or less campanulate tube, somewhat recurved, and colorless with pinkish purple veins; diadelphous stamens; slender style; a faintly aromatic, somewhat tea-like odor; and a sweet, then slightly bitter taste.
Microscopic: Epidermis of calyx composed of polygonal cells with faintly striated cuticle and occasional anomocytic stomata on the outer epidermis only; abundant, uniseriate, covering trichomes with two small, thin-walled basal cells and a thick-walled tapering end cell, up to 1 mm in length with a warty cuticle. Glandular trichomes are also present, particularly on the lower epidermis, each with a one- or two-celled stalk and a large, cylindrical head composed of several cells arranged in two rows. Epidermal cells of the corolla, papillose at the tip, are elongated with slightly wavy walls and a strongly striated cuticle; vascular strands of corolla and calyx are surrounded by a crystal sheath containing prismatic crystals of calcium oxalate. The following are also present: fibrous layer of anthers; subspherical pollen grains, 2048 µm in diameter with smooth exine, three distinct pores, and three furrows; upper epidermal cells of leaflets with sinuous and slightly beaded anticlinal walls; lower epidermis with sinuous to wavy walls; anomocytic stomata on both surfaces, but more frequent on the lower surface; abundant covering trichomes on both surfaces and on the margins; and fibrovascular strands surrounded by a crystal sheath containing prismatic crystals of calcium oxalate
• Loss on Drying 731: Dry 1 g 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, protected from moisture.
• Labeling: The label states the Latin binomial and, following the official name, the parts of the plant contained in the article.
• USP Reference Standards 11
USP Powdered Red Clover Extract RS
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USP35NF30 Page 1418Pharmacopeial Forum: Volume No. 30(2) Page 550