Powdered Milk Thistle
DEFINITION
Powdered Milk Thistle is Milk Thistle reduced to a fine or very fine powder. It contains NLT 2.0% of silymarin, calculated as silybin (C25H22O10), on the dried basis.
IDENTIFICATION
• 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.
Chromatographic system
Adsorbent:
0.25-mm layer of chormatographic silica gel, typically 20 cm long
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
Analysis
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 in the test 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.
COMPOSITION
• 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.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 0 | 0 |
| 5 | 85 | 15 |
| 20 | 55 | 45 |
| 40 | 55 | 45 |
| 41 | 85 | 15 |
| 55 | 85 | 15 |
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 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. [Note—Adjust 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.
Chromatographic system
Mode:
LC
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
System suitability
Sample:
Milk thistle standard solution
[Note—For the relative retention times, see Table 2. ]
Table 2
| Relative Retention Time |
|
|---|---|
| Silychristin | 0.68 |
| Silydianin | 0.73 |
| Silybin A | 1.00 |
| Silybin B | 1.05 |
| Dehydrosilybin | 1.09 |
| Isosilybin A | 1.15 |
| Isosilybin B | 1.19 |
Suitability requirements
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:
0.8–2.0
Relative standard deviation:
NMT 2.0% for the sum of peak responses due to silybin A and silybin B
Analysis
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 Powdered Milk Thistle taken:
Result = C × (V/W) × D × 100
| C | = | = concentration of the relevant component in the Sample solution as interpolated from the calibration graph (mg/mL) |
| V | = | = volume of the Sample stock solution (mL) |
| W | = | = weight of the portion of Powdered Milk Thistle taken (mg) |
| D | = | = dilution factor to prepare the Sample solution from Sample stock solution, 25 |
Calculate the content of silymarin, as a percentage, in the portion of Powdered Milk Thistle taken by adding the individual percentages.
Acceptance criteria:
NLT 2.0% of silymarin, calculated as silybin (C25H22O10), on the dried basis
CONTAMINANTS
• Heavy Metals 
231
:
NMT 20 µg/g
231:
NMT 20 µg/g
• Articles of Botanical Origin, Pesticide Residues 561:
Meets the requirements
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.
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.
2022:
It meets the requirements of the tests for the absence of Salmonella species and Escherichia coli and for absence of Staphylococcus aureus.
SPECIFIC TESTS
• Botanic Characteristics:
The powder is characterized by fragments of colorless cells, up to approximately 75 µm in length and 8 µm in width, from the palisade layer of the epidermis of the fruit wall, with their adherent pigment layer (they assume a red color in chloral hydrate preparations), and by gray pieces, viewed from above, with the slitlike lumen produced by the pronounced wall thickening or the nodes on the cell wall formed by the thickened ridges; fragments of the pigment layer viewed from above, with red coloration diffusing out of them in chloral hydrate preparations, pigment cells alternating with colorless parenchymal cells; conspicuously stippled colorless cells through which pigment cells are visible when viewed from above; cigar-shaped or monoclinic calcium oxalate prisms, lying free or in groups of cells; numerous fragments of the lemon yellow palisade cells of the seed coat, up to roughly 150 µm in length, having a very narrow lumen and conspicuously stippled when viewed from above; pale yellowish fragments from the net cell layer together with portions of the embryo consisting of thin-walled cells with small glands and lipophilic substances.
• Articles of Botanical Origin, Total Ash 561:
NMT 8.0%, determined on 1.0 g of Powdered Milk Thistle
561:
NMT 8.0%, determined on 1.0 g of Powdered Milk Thistle
• Loss on Drying 731:
Dry 1.0 g of Powdered Milk Thistle at 105 for 2 h: it loses NMT 8.0% of its weight.
731:
Dry 1.0 g of Powdered Milk Thistle at 105 for 2 h: it loses NMT 8.0% of its weight.
ADDITIONAL REQUIREMENTS
• 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 source from which the article was derived.
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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 1386
Pharmacopeial Forum: Volume No. 28(2) Page 417