Powdered Stinging Nettle Extract
DEFINITION
Powdered Stinging Nettle Extract is prepared from comminuted Stinging Nettle with 60% alcohol or other suitable solvents. It contains NLT 5.0% of total amino acids, NLT 0.1% of 
-sitosterol (C29H50O), and NLT 30 µg/g of scopoletin (C10H8O4). The ratio of the starting crude plant material to Powdered Extract is 10:1.
-sitosterol (C29H50O), and NLT 30 µg/g of scopoletin (C10H8O4). The ratio of the starting crude plant material to Powdered Extract is 10:1.IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test
Standard solution:
0.05 mg/mL of USP Scopoletin RS and 0.5 mg/mL of USP -Sitosterol RS in methanol
-Sitosterol RS in methanol
Sample solution:
Dissolve 0.6 g of Powdered Extract, in a mixture of toluene, ethyl acetate, and methanol (7:2:1). Filter, and dry under reduced pressure at a temperature below 40
. Dissolve the residue in 2.0 mL of the mixture containing toluene, ethyl acetate, and methanol.
. Dissolve the residue in 2.0 mL of the mixture containing toluene, ethyl acetate, and methanol.
Chromatographic system
Adsorbent:
0.50-mm layer of chromatographic silica gel mixture
Application volume:
20 µL for the Sample solution; 10 µL for the Standard solution
Developing solvent system:
Diethyl ether and methanol (9:1)
Spray reagent:
85% phosphoric acid, 10% vanillin in 96% ethanol, and water (4.5: 1: 4.5)
Analysis
Samples:
Standard solution and Sample solution
Proceed as directed in the chapter. Examine the plates under UV light at 365 nm. Spray the plate with 10 mL of Spray reagent, heat between 100 and 105 for 10 min, and examine under daylight.
and 105 for 10 min, and examine under daylight.
Acceptance criteria:
The chromatogram of the Sample solution exhibits a violet-red zone corresponding to -sitosterol at the same RF value as -sitosterol from the Standard solution, weakly visible zones above and below -sitosterol, and a violet-red zone corresponding to -sitosterolglucoside.
-sitosterol at the same RF value as -sitosterol from the Standard solution, weakly visible zones above and below -sitosterol, and a violet-red zone corresponding to -sitosterolglucoside.
• B. Gas Chromatography Identification Test
Analysis:
Proceed as directed in the test for Content of -Sitosterol.
-Sitosterol.
Acceptance criteria:
The retention time of -sitosterol of the Sample solution corresponds to that of the Standard solution.
-sitosterol of the Sample solution corresponds to that of the Standard solution.
• C. HPLC Identification Test
Analysis:
Proceed as directed in the test for Content of Scopoletin.
Acceptance criteria:
The retention time of scopoletin of the Sample solution corresponds to that of the Standard solution.
COMPOSITION
• Content of -Sitosterol
-Sitosterol
Derivatizing reagent:
A solution containing equal volumes (1:1:1) of BSTFA [N,O-bis(trimethylsilyl)trifluoroacetamide], anhydrous pyridine, and a mixture of BSA [N,O-(trimethylsilyl)acetamide], TMSI (N-trimethylsilylimidazole), and TMCS (trimethylchlorosilane) (3:3:2)
Internal standard solution:
10 mg/mL of cholesterol in chloroform
Standard solution:
Dissolve 50.0 mg of USP -Sitosterol RS in 2 mL of chloroform, add 1.0 mL of Internal standard solution, and dilute to 5 mL with chloroform. Dry 0.5 mL under reduced pressure, and add 1 mL of Derivatizing reagent.
-Sitosterol RS in 2 mL of chloroform, add 1.0 mL of Internal standard solution, and dilute to 5 mL with chloroform. Dry 0.5 mL under reduced pressure, and add 1 mL of Derivatizing reagent.
Sample solution:
Transfer 20.0 g of Powdered Extract to a Soxhlet apparatus, treat with chloroform, and extract for 6 h. The volume of chloroform used is at least twice the volume of the thimble with an appropriately sized flask. Dry the solvent under reduced pressure, add 1.0 mL of Internal standard solution, and dilute with chloroform to 10 mL. Transfer 0.5 mL of this solution to a 10-mL round-bottom flask, dry the solvent under reduced pressure, and add 0.5 mL of Derivatizing reagent.
Chromatographic system
Mode:
GC
Detector:
Flame ionization
Column:
0.20-mm × 25-m fused-silica capillary; 0.35-µm film of phase G2 coating
Temperature
Injector:
325
Detector:
325
Column:
300, for 60 min
, for 60 min
Carrier gas:
Helium
Flow rate:
0.5 mL/min
Injection size:
1 µL
System suitability
Sample:
Standard solution
Suitability requirements
Tailing factor:
NMT 2.0 for each sterol peak
Relative standard deviation:
NMT 5.0% determined from each sterol peak
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of -sitosterol in the portion of Powdered Extract taken:
-sitosterol in the portion of Powdered Extract taken: Result = (RU/RS) × (WS/WU) × 100
| RU | = | = peak response ratio of -sitosterol to the internal standard from the Sample solution |
| RS | = | = peak response ratio of -sitosterol to the internal standard from the Standard solution |
| WS | = | = weight of USP -Sitosterol RS used to prepare the Standard solution (mg) |
| WU | = | = weight of Powdered Extract in the Sample solution (mg) |
Acceptance criteria:
NLT 0.1%
• Content of Scopoletin
Solution A:
Water
Solution B:
Methanol
Mobile phase:
See Table 1.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 75 | 25 |
| 2 | 60 | 40 |
| 8 | 60 | 40 |
| 10 | 0 | 100 |
| 15 | 0 | 100 |
| 20 | 75 | 25 |
| 30 | 75 | 25 |
Standard solution:
0.02 µg/mL of USP Scopoletin RS in methanol
Sample stock solution:
8 mg/mL of Powdered Extract in methanol. Place in an ultrasonic bath for 25 min, and centrifuge.
Sample solution:
Sample stock solution diluted with methanol 1 in 20
Chromatographic system
Mode:
LC
Detector:
Fluorescence; set at an excitation wavelength of 366 nm and an emission wavelength of 420 nm
Column:
4.6-mm × 25-cm; packing L1
Flow rate:
1 mL/min
Injection size:
10 µL
System suitability
Sample:
Standard solution
Suitability requirements
Capacity factor (k¢):
NLT 5 determined from the scopoletin peak
Tailing factor:
NMT 2.0 for the scopoletin peak
Relative standard deviation:
NMT 5.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak responses for scopoletin.
Calculate the content of scopoletin (C10H8O4), in µg/g, in the portion of Powdered Extract taken:
Result = (rU/rS) × (CS/CU)
| rU | = | = peak area of scopoletin from the Sample solution |
| rS | = | = peak area of scopoletin from the Standard solution |
| CS | = | = concentration of USP Scopoletin RS in the Standard solution (µg/mL) |
| CU | = | = concentration of Powdered Extract in the Sample solution (g/mL) |
Acceptance criteria:
NLT 30 µg/g
• Content of Total Amino Acids
Buffer:
Mix 5.40 g of anhydrous sodium acetate, 0.3 mL of glacial acetic acid, and water to a final volume of 100 mL. Adjust to pH 5.5.
Reagent solution:
Solution containing 1.00 g of ninhydrin, 1.50 g of hydrindantin, and 37.5 mL of propylene glycol. Adjust with Buffer to 50.0 mL. [Note—Prepare the Reagent solution daily. ]
Standard solution:
20 µg/mL each of USP Glutamic Acid RS and USP Aspartic Acid RS in water
Sample solution:
Dissolve 50 mg of Powdered Extract in 80 mL of water, shake for 10 min, dilute with water to 100 mL, and filter.
Blank:
Water
Instrumental conditions
Mode:
Visible
Analytical wavelength:
570 nm
Cell:
1 cm
Analysis
Samples:
Standard solution, Sample solution, and Blank
Transfer 5.0 mL of the Standard solution, Sample solution, and Blank to separate 50-mL volumetric flasks. Add 5.0 mL of Reagent solution to each flask. Heat in a boiling water bath for 30 min, cool, and adjust with a mixture of ethanol and water (1:1) to volume.
Calculate the percentage of total amino acids in the portion of Powdered Extract taken:
Result = (AU/AS) × (CS/CU) × 100
| AU | = | = absorbance of the Sample solution |
| AS | = | = absorbance of the Standard solution |
| CS | = | = sum of the concentration of USP Glutamic Acid RS and USP Aspartic Acid RS in the Standard solution (mg/mL) |
| CU | = | = concentration of Powdered Extract in the Sample solution (mg/mL) |
Acceptance criteria:
NLT 5.0%
CONTAMINANTS
• Articles of Botanical Origin, Pesticide Residues 
561
:
Meets the requirements
561:
Meets the requirements
• Alcohol Determination, Method II 611:
NMT 1.0%, if present
611:
NMT 1.0%, if present
• Microbial Enumeration Tests 2021:
The total aerobic microbial count does not exceed 103 cfu/g, and the total combined molds and yeasts count does not exceed 102 cfu/g.
2021:
The total aerobic microbial count does not exceed 103 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 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 Powdered Extract at 105 for 2 h: it loses NMT 8.0% of its weight.
731:
Dry 1.0 g of Powdered Extract at 105 for 2 h: it loses NMT 8.0% of its weight.
• Articles of Botanical Origin, Total Ash 561:
NMT 20.0%
561:
NMT 20.0%
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight containers, protected from light. Store at controlled room temperature.
• Labeling:
The label states the official name of the article, the Latin binomial, and, following the official name, the part of the plant from which the article was prepared. Label it to indicate the content of total amino acids, -sitosterol, scopoletin, the extracting solvent used for preparation, and the ratio of the starting crude plant material to Powdered Extract.
-sitosterol, scopoletin, the extracting solvent used for preparation, and the ratio of the starting crude plant material to Powdered Extract.
• USP Reference Standards 11
11
USP Aspartic Acid RS 
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USP Glutamic Acid RS
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USP Scopoletin RS
USP -Sitosterol RS
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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 1454
Pharmacopeial Forum: Volume No. 29(4) Page 1289