Pygeum Extract
DEFINITION
Pygeum Extract is prepared from pulverized Pygeum, using suitable solvents. It contains NLT 90% and NMT 110% of the labeled amount of docosyl ferulate and NLT 90% and NMT 110% of the labeled amount of total sterols as 
-sitosterol, calculated on the dried basis.
-sitosterol, calculated on the dried basis.IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test
Standard solution A:
15 mg/mL of USP Pygeum Extract RS in chloroform
Standard solution B:
2 mg/mL of USP -Sitosterol RS in chloroform
-Sitosterol RS in chloroform
Sample solution:
Dissolve 150 mg of Extract in 10 mL of chloroform.
Chromatographic system
Adsorbent:
0.25-mm layer of chromatographic silica gel
Application volume:
10 µL
Developing solvent system:
Methylene chloride in a saturated chamber
Spray reagent:
Sulfuric acid and water (1:1)
Analysis
Samples:
Standard solution A, Standard solution B, and Sample solution
Develop the chromatograms to a length of NLT 15 cm, and dry the plate in a current of air. Spray the plate with Spray reagent, and heat the plate at 100
for 10 min. Examine the plate under white light.
for 10 min. Examine the plate under white light.
Acceptance criteria:
The chromatogram from the Sample solution shows one red-violet zone turning to grayish-brown near the origin that corresponds in color and RF value to that of Standard solution A, and one red-violet zone turning to grayish-brown at an RF of 0.08 corresponding in color and RF value to that in the chromatogram of Standard solution B; above these spots a grayish-brown zone may be present, corresponding in color and RF value to that of Standard solution A; and other colored zones of varying intensities may be observed in the Sample solution.
• B. HPLC Identification Test
Analysis:
Proceed as directed in the test for Content of Docosyl Ferulate.
Acceptance criteria:
The chromatogram of the Sample solution presents a peak for docosyl ferulate that corresponds in retention time to the principal peak in the chromatogram of the Standard solution.
COMPOSITION
• Content of Sterols
Derivatizing solution:
Bis(trimethylsilyl)acetamide and trimethylchlorosilane (9:1)
Internal standard solution:
2 mg/mL of 5
-cholestane in chloroform
-cholestane in chloroform
System suitability stock solution:
2 mg/mL each of campesterol, stigmasterol, and USP -Sitosterol RS
-Sitosterol RS
System suitability solution:
Mix 2.0 mL of the System suitability stock solution and 2.0 mL of the Internal standard solution, and dilute with chloroform to 10 mL. Evaporate 500 µL of this solution to dryness using a stream of nitrogen. Dissolve the residue in 80 µL of Derivatizing solution and 20 µL of pyridine. Allow to stand for NLT 10 min at room temperature.
Standard stock solution:
2.0 mg/mL of USP -Sitosterol RS in chloroform
-Sitosterol RS in chloroform
Standard solution:
Mix 2.0 mL of the Standard stock solution and 2.0 mL of the Internal standard solution, and dilute with chloroform to 10 mL. Evaporate 500 µL of this solution to dryness using a stream of nitrogen. Dissolve the residue in 80 µL of Derivatizing solution and 20 µL of pyridine. Allow to stand for NLT 10 min at room temperature.
Sample solution:
Transfer 100 mg of Extract into a 100-mL round-bottom flask. Add 2.0 mL of Internal standard solution and 20 mL of diluted hydrochloric acid. Attach a condenser, and reflux in a bath at 100 for 30 min. Cool the solution to room temperature, and adjust by the addition of about 5 mL of 10 N sodium hydroxide to a pH of 8. Extract twice using 50 mL of ether each time, wash the collected organic phases with 50 mL of water, and evaporate the organic phase to dryness under vacuum. Dissolve the residue with 4 mL of chloroform, and transfer to a cartridge containing 500 mg of packing L81 that has been conditioned with a 2-column volume of n-hexane . Collect the eluate. Elute twice with a 1-column volume of a mixture of chloroform and isopropanol (2:1). Combine the eluates, and evaporate to dryness. Dissolve the residue in 10 mL of chloroform. Evaporate 500 µL of this solution to dryness under a stream of nitrogen. Dissolve the residue with 80 µL of Derivatizing solution and 20 µL of pyridine. Allow to stand for NLT 10 min at room temperature.
for 30 min. Cool the solution to room temperature, and adjust by the addition of about 5 mL of 10 N sodium hydroxide to a pH of 8. Extract twice using 50 mL of ether each time, wash the collected organic phases with 50 mL of water, and evaporate the organic phase to dryness under vacuum. Dissolve the residue with 4 mL of chloroform, and transfer to a cartridge containing 500 mg of packing L81 that has been conditioned with a 2-column volume of n-hexane . Collect the eluate. Elute twice with a 1-column volume of a mixture of chloroform and isopropanol (2:1). Combine the eluates, and evaporate to dryness. Dissolve the residue in 10 mL of chloroform. Evaporate 500 µL of this solution to dryness under a stream of nitrogen. Dissolve the residue with 80 µL of Derivatizing solution and 20 µL of pyridine. Allow to stand for NLT 10 min at room temperature.
Chromatographic system
Mode:
GC
Detector:
Flame ionization
Column:
0.32-mm × 30-m capillary; G27 phase coating of 0.25-µm thickness
Temperature:
Injector:
285
Detector:
285
Column:
See Table 1.
Table 1
| Initial Temperature ( ) |
Temperature Ramp ( /min) |
Final Temperature ( ) |
Hold Time at Final Temperature (min) |
|---|---|---|---|
| 250 | 0 | 250 | 5 |
| 250 | 5 | 320 | 0 |
Carrier gas:
Helium. [Note—The carrier gas flow rate should be adjusted to obtain a retention time of about 19 min for -sitosterol. ]
-sitosterol. ]
Makeup gas:
Helium
Injection size:
2 µL
Injection type:
Split injection system
Split ratio:
1:50
System suitability
Sample:
System suitability solution
[Note—The relative retention times for 5-cholestane, campesterol, stigmasterol, and -sitosterol are about 0.66, 0.94, 0.96, and 1.00, respectively. ]
-cholestane, campesterol, stigmasterol, and -sitosterol are about 0.66, 0.94, 0.96, and 1.00, respectively. ]
Suitability requirements
Resolution:
NLT 2 between campesterol and stigmasterol
Column efficiency:
NLT 150,000 theoretical plates for the 5-cholestane peak
-cholestane peak
Tailing factor:
NMT 2.0 for each relevant peak
Analysis
Samples:
Standard solution and Sample solution
Identify the signals corresponding to the relevant analytes by comparison with the chromatograms obtained with the System suitability solution.
Separately calculate the individual percentages of campesterol, stigmasterol, and -sitosterol, as -sitosterol, respectively, in the portion of Pygeum Extract taken:
-sitosterol, as -sitosterol, respectively, in the portion of Pygeum Extract taken:
Ci = (RU/RS) × CS × (V/W) × 100
| RU | = | = ratio of the appropriate sterol peak to the internal standard from the Sample solution |
| RS | = | = ratio of the -sitosterol peak to the 5-cholestane internal standard from the Standard solution |
| CS | = | = concentration of -sitosterol in the Standard stock solution (mg/mL) |
| V | = | = volume of the Standard stock solution taken to prepare the Standard solution (mL) |
| W | = | = weight of Pygeum Extract taken to prepare the Sample solution (mg) |
Calculate the percentage of the labeled amount of total sterols as -sitosterol:
-sitosterol: Result = (SCi /L) × 100
| Ci | = | = individual percentage of each sterol as calculated above |
| L | = | = labeled amount of total sterols in the Pygeum Extract taken |
Acceptance criteria:
90%–110% of the labeled amount of total sterols as -sitosterol on the dried basis
-sitosterol on the dried basis
• Content of Docosyl Ferulate
Solution A:
Methanol and water (95:5)
Solution B:
Acetonitrile
Mobile phase:
Solution A and Solution B (17:3)
Standard solution:
Dissolve a quantity of USP Docosyl Ferulate RS in chloroform, and dilute with acetonitrile to obtain a concentration of 0.02 mg/mL. Pass through a filter of 0.45-µm or finer pore size.
Sample solution:
To 250 mg of Extract add 5 mL of chloroform, and dilute with acetonitrile to 25 mL. Pass through a filter of 0.45-µm or finer pore size, discarding the first 4 mL of filtrate.
Chromatographic system
Mode:
LC
Detector:
UV 323 nm
Column:
4-mm × 25-cm; packing L7
Column temperature:
25
Flow rate:
1 mL/min
Injection size:
20 µL
System suitability
Sample:
Standard solution
Suitability requirements
Column efficiency:
NLT 1700 theoretical plates for the docosyl ferulate peak
Tailing factor:
NMT 2.0 for docosyl ferulate
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of docosyl ferulate (P) in the portion of Extract taken:
P = (rU/rS) × CS × (V/W) × 100
| rU | = | = peak area for docosyl ferulate from the Sample solution |
| rS | = | = peak area for docosyl ferulate from the Standard solution |
| CS | = | = concentration of USP Docosyl Ferulate RS in the Standard solution (mg/mL) |
| V | = | = volume of the Sample solution (mL) |
| W | = | = weight of Extract taken to prepare the Sample solution (mg) |
Calculate the percentage of the labeled amount of docosyl ferulate:
Result = (P/L) × 100
| P | = | = percent of docosyl ferulate in the portion of Extract taken |
| L | = | = labeled amount of docosyl ferulate in the Pygeum Extract (%) |
Acceptance criteria:
90%–110% of the labeled amount of docosyl ferulate on the dried basis
CONTAMINANTS
• Heavy Metals, Method II 
231
:
20 µg/g
231:
20 µg/g
• Articles of Botanical Origin, Test for Aflatoxins 561:
NMT 4 µg/kg of total aflatoxins B1, B2, G1, and G2; NMT 2 µg/kg of aflatoxin B1
561:
NMT 4 µg/kg of total aflatoxins B1, B2, G1, and G2; NMT 2 µg/kg of aflatoxin B1
• Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561:
Meets the requirement
561:
Meets the requirement
• Botanical Extracts, Preparations 565:
Meets the requirements in General Pharmacopeial Requirements, Residual Solvents
565:
Meets the requirements in General Pharmacopeial Requirements, Residual Solvents
• Microbial Enumeration Tests 2021:
The total aerobic microbial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 103 cfu/g.
2021:
The total aerobic microbial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 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 1.0 g of Extract for 3 h at 110: it loses NMT 10% of its weight.
731:
Dry 1.0 g of Extract for 3 h at 110: it loses NMT 10% of its weight.
• Articles of Botanical Origin, Total Ash 561:
NMT 0.5%
561:
NMT 0.5%
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Store in tight containers, protected from light.
• Labeling:
The label states the Latin binomial and, following the official name, the part of the plant from which the article was prepared. Label the content as a percentage of total sterols as -sitosterol and the content as a percentage of docosyl ferulate. It also meets the requirements in Botanical Extracts 565, Labeling.
-sitosterol and the content as a percentage of docosyl ferulate. It also meets the requirements in Botanical Extracts 565, Labeling.
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A suitable cartridge is Chromabond NH2, manufactured by Macheray Nagel, or equivalent.
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 1415
Pharmacopeial Forum: Volume No. 30(3) Page 956