Powdered Milk Thistle Extract
DEFINITION
Powdered Milk Thistle Extract is prepared from Milk Thistle fruits or seeds by fat removal and subsequent extraction with suitable solvents. It contains NLT 90.0% and NMT 110.0% of the labeled amount of silymarin, calculated as silybin (C25H22O10), on the dried basis, consisting of NLT 20.0% and NMT 45.0% for the sum of silydianin and silychristin, NLT 40.0% and NMT 65.0% for the sum of silybin A and silybin B, and NLT 10.0% and NMT 20.0% for the sum of isosilybin A and isosilybin B.
IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test
Standard solution:
1.0 mg/mL of USP Silydianin RS in methanol
Sample solution:
10 mg/mL of Powdered Extract in methanol, and allow to stand for 15 min before use.
Chromatographic system
Adsorbent:
0.25-mm layer of chromatographic 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.
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 solution:
Dissolve a sample of Extract in methanol to obtain a solution equivalent to 0.4 mg/mL of silymarin. Sonicate for 20 min, cool to 20
, and pass through a filter of 0.45-µm or finer pore size.
, and pass through a filter of 0.45-µm or finer pore size.
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 |
| Tsosilybin 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 isolybin 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 silybin (C25H22O10) in the portion of Powdered Extract taken:
Result = C × (V/W) × 100
| C | = | = concentration of the relevant component in the Sample solution as interpolated from the calibration graph (mg/mL) |
| V | = | = volume of the Sample solution (mL) |
| W | = | = weight of the portion of Powdered Extract taken (mg) |
Calculate the percentage of the labeled amount of silymarin in the portion of Powdered Extract taken:
Result = SS/L × 100
| SS | = | = sum of the percentages of the each relevant component of silymarin |
| L | = | = labeled amount of silymarin, as a percentage, in the Powdered Extract |
Acceptance criteria:
90.0%–110.0% of the labeled amount of silymarin, calculated as silybin (C25H22O10), on the dried basis, consisting of: 20.0%–45.0% for the sum of silydianin and silychristin; 40.0%–65.0% for the sum of silybin A and silybin B; and 10.0%–20.0% for the sum of isosilybin A and isosilybin B
CONTAMINANTS
• Heavy Metals, Method II 
231
:
NMT 20 µg/g
231:
NMT 20 µg/g
• Microbial Enumeration Tests 2021:
The total bacterial count does not exceed 104/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the enterobacterial count does not exceed 103 cfu/g.
2021:
The total bacterial count does not exceed 104/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the enterobacterial count does not exceed 103 cfu/g.
• Absence of Specified Microorganisms 2022:
It meets the requirements of the tests for the absence of Salmonella species and Escherichia coli.
2022:
It meets the requirements of the tests for the absence of Salmonella species and Escherichia coli.
• Other Requirements:
It meets the requirements in Botanical Extracts 565 for the tests in sections Pesticide Residues and Residual Solvents.
565 for the tests in sections Pesticide Residues and Residual Solvents.
SPECIFIC TESTS
• Loss on Drying 731:
Dry a sample at 105 for 2 h: it loses NMT 5.0% of its weight.
731:
Dry a sample at 105 for 2 h: it loses NMT 5.0% of its weight.
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. It meets the requirements for Botanical Extracts 565, Labeling.
565, Labeling.
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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 1387
Pharmacopeial Forum: Volume No. 28(2) Page 417