Milk Thistle consists of the dried ripe fruit of Silybum marianum (L.) Gaertn. (Fam. Asteraceae), the pappus having been removed. It contains NLT 2.0% of silymarin, calculated as silybin (C25H22O10), on the dried basis.
• A. Thin-Layer Chromatographic Identification Test
Standard solution: 1.0 mg/mL of USP Silydianin RS in methanol
Sample solution: Use the Sample solution, prepared as directed in the test for Content of Silymarin.
Adsorbent: 0.25 mm layer of chromatographic silica gel, typically 20 cm long (TLC plates)
Application volume: 10 µL
Developing solvent system: Freshly prepared mixture of chloroform, acetone, and anhydrous formic acid (75: 16.5: 8.5)
Spray reagent A: 10-mg/mL solution of 2-aminoethyl diphenylborinate in methanol
Spray reagent B: 50-mg/mL solution of polyethylene glycol 4000 in alcohol
Samples: Standard solution and Sample solution
Develop the chromatograms until the solvent front has moved about three-fourths of the plate, and dry it for 30 min in a current of cold air. Spray the plate with Spray reagent A, allow to dry, and then spray with Spray reagent B. One h later, examine the plate under long-wavelength UV light.
Acceptance criteria: The chromatogram of the Sample solution exhibits an intense green-blue fluorescent zone at an RF value of about 0.5 (presence of silybin) and exhibits a gray-blue spot at an RF value of about 0.4, corresponding to a spot observed in the chromatogram of the Standard solution. The chromatogram of the Sample solution may exhibit other colored zones: an intense green-blue zone at an RF value of about 0.25 (presence of silychristin) and a red-orange zone at an RF value of about 0.3 (presence of taxifolin).
• B. HPLC Identification Test
Analysis: Proceed as directed for Content of Silymarin.
Acceptance criteria: The retention times of the peaks for silydianin, silychristin, silybin A, silybin B, isosilybin A, and isosilybin B in the chromatogram of the Sample solution correspond to those in the chromatogram of the Milk thistle standard solution.
• Content of Silymarin
Solution A: Methanol, phosphoric acid, and water (20: 0.5: 80)
Solution B: Methanol, phosphoric acid, and water (80: 0.5: 20)
Mobile phase: See Table 1.
Milk thistle standard solution: 0.7 mg/mL of USP Powdered Milk Thistle Extract RS in methanol. Sonicate for 20 min to dissolve. Pass through a membrane filter having a 0.45-µm or finer pore size. Dilute 1 in 5 with methanol to obtain a solution of 0.14 mg/mL of USP Powdered Milk Thistle Extract RS.
Silybin standard solutions: 0.20, 0.02, and 0.004 mg/mL of USP Silybin RS in methanol. Pass through a membrane filter having a 0.45-µm or finer pore size.
Sample stock solution: Transfer 10 g of finely powdered Milk Thistle to an extraction thimble, and cover with a small cotton ball. Transfer the thimble to a continuous-extraction apparatus fitted with a 250-mL round-bottom flask containing 150 mL of solvent hexane, and heat the flask on a heating mantle for 4 h. After the extraction, separate the round-bottom flask containing solvent hexane extract from the extraction apparatus, and discard the solvent hexane solution. Remove the adherent solvent hexane from the extraction thimble by drying, and transfer the thimble to an extraction apparatus suitable for hot extraction and fitted with a 250-mL round-bottom flask containing 100 mL of ethyl acetate. [NoteAdjust the volume of ethyl acetate, if necessary, to sustain a continuous extraction. ] Heat the flask on a heating mantle to allow the solvent to reflux gently. After 8 h, transfer the extract quantitatively into a 100-mL volumetric flask, and dilute with methanol to volume.
Sample solution: Dilute the Sample stock solution (1 in 25) with methanol.
Detector: UV 288 nm
Column: 4.6-mm × 15-cm; 5-µm packing L1
Column temperature: 40
Flow rate: 1 mL/min
Injection size: 10 µL
Sample: Milk thistle standard solution
[NoteFor the relative retention times, see Table 2. ]
Chromatogram similarity: The chromatogram from the Milk thistle standard solution is similar to the reference chromatogram provided with the lot of USP Powdered Milk Thislte Extract RS being used.
Resolution: NLT 1.0 between silybin A and silybin B
Tailing factor: 0.82.0
Relative standard deviation: NMT 2.0% for the sum of peak responses due to silybin A and silybin B
Samples: Milk thistle standard solution, each of the Silybin standard solutions, and Sample solution
Identify the peaks due to silychristin, silydianin, silybin A, silybin B, isosilybin A, and isosilybin B by comparison with the chromatogram of the Milk thistle standard solution, and measure the peak areas of the relevant peaks. Plot the areas of the sum of silybin A and silybin B peaks versus the concentration of USP Silybin RS in the Silybin standard solutions, and obtain a regression line for calibration.
Separately calculate the percentage of each relevant component of silymarin as silibin (C25H22O10) in the portion of Milk Thistle taken:
Result = C × (V/W) × D × 100
Calculate the content of silymarin, as a percentage, in the portion of Milk Thistle taken by adding the individual percentages.
Acceptance criteria: NLT 2.0% of silymarin, calculated as silybin (C25H22O10), on the dried basis
• Heavy Metals 231: NMT 10 µg/g
• Articles of Botanical Origin, Pesticide Residues 561: Meets the requirements
• Microbial Enumeration Tests 2021: The total bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 102 cfu/g.
• Absence of Specified Microorganisms 2022: It meets the requirements of the tests for the absence of Salmonella species and Escherichia coli and for absence of Staphylococcus aureus.
• Botanic Characteristics
Macroscopic: The fruits (achenes) are elongated ovoid, slightly crooked, somewhat flattened, roughly 67 mm in length, up to 3 mm in width, and 1.5 mm in thickness, and have a projecting cartilaginous, glossy, yellowish edge on the upper surface and a grooved hilum at the base. The fruit coat is glossy brown-black or mat gray-brown with dark or pale gray streaks; it encloses the straight embryo having two thick, flattened cotyledons that contain fatty oil and aleurone granules.
Microscopic: The fruit wall epidermis consists of almost colorless palisade cells arranged at right angles to the surface. They have greatly thickened outer walls, into which the lumen continues for some distance in the form of a slit. Viewed from above under high magnification, the cells show only a slit-shaped lumen. They have thickened ridges that appear as nodular thickenings of the cell wall when viewed from above. The subepidermal layer of the fruit wall is made up of unlignified thin-walled parenchymal cells and constitutes a pigment layer. Colorless cells and groups of cells alternate with pigment cells, the latter varying in number; this gives the fruit wall its mottled appearance. Next comes the fruit wall tissue, about 8 cell layers thick, with stippled parenchymal cells elongated in the longitudinal axis of the fruit. The cells of the innermost layer of the fruit wall may be collapsed; they contain large cigar-shaped or monoclinic calcium oxalate prisms. The seed coat epidermis is formed from large yellow palisade cells. The cells have a narrow lumen, somewhat expanded at each end of the cell, and the cell walls show conspicuous lamination. The subepidermal cells of the seed coat consist of peculiar stippled cells; their lignified cell membranes have prominent, close-set ridges or thickenings (net cells). Next to them is a single layer of cells having tough, somewhat swollen walls and lipophilic contents (endosperm residue). The embryo consists of thin-walled cells that, in addition to small glands, contain clumps of crystals and fat droplets.
• Articles of Botanical Origin, Total Ash 561: NMT 8.0%, determined on 1.0 g of finely powdered Milk Thistle
• Loss on Drying 731: Dry 1.0 g of finely powdered Milk Thistle at 105 for 2 h: it loses NMT 8.0% of its weight.
• Packaging and Storage: Preserve in well-closed containers, protected from light and moisture.
• Labeling: The label states the Latin binomial and, following the official name, the part of the plant contained in the article.
• USP Reference Standards 11
USP Powdered Milk Thistle Extract RS
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USP35NF30 Page 1384Pharmacopeial Forum: Volume No. 28(2) Page 414