Stinging Nettle
DEFINITION
Stinging Nettle consists of dried roots and rhizomes of Urtica dioica L. subsp. dioica (Fam. Urticaceae), and may contain Urtica urens L., known in commerce as dwarf nettle, as a minor component. It contains NLT 0.8% of total amino acids, NLT 0.05% of 
-sitosterol (C29H50O), and NLT 3 µg/g of scopoletin (C10H8O4), calculated on the dried basis.
-sitosterol (C29H50O), and NLT 3 µg/g of scopoletin (C10H8O4), calculated on the dried basis.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:
Extract 1 g of powder by refluxing with 10 mL of a solution containing toluene, ethyl acetate, and methanol (7:2:1) for 15 min, cool, and filter. Evaporate the filtrate to dryness under reduced pressure at less than 40
, and dissolve the residue in 2 mL of the mixture containing toluene, ethyl acetate, and methanol.
, and dissolve the residue in 2 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
Develop the plate and examine under UV light at 365 nm. Spray the plate with 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 in 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 in the Standard solution, weakly visible zones above and below -sitosterol, and a violet-red zone corresponding to -sitosterolglucoside.
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 with chloroform to 5 mL. 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 with chloroform to 5 mL. Dry 0.5 mL under reduced pressure, and add 1 mL of Derivatizing reagent.
Sample solution:
Transfer 50.0 g of finely powdered Stinging Nettle to a Soxhlet apparatus, add 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 Stinging Nettle taken:
-sitosterol in the portion of Stinging Nettle 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 Stinging Nettle taken to prepare the Sample solution (mg) |
Acceptance criteria:
NLT 0.05% on the dried basis
• 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:
Extract 160 mg of finely powdered Stinging Nettle in each mL of 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 detector; 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 Stinging Nettle taken:
Result = (rU/rS) × CS × (V/W) × D
| 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) |
| V | = | = volume of the Sample stock solution (mL) |
| W | = | = weight of Stinging Nettle taken to prepare the Sample solution (g) |
| D | = | = dilution factor to prepare the Sample solution from the Sample stock solution |
Acceptance criteria:
NLT 3 µg/g on the dried basis
• 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:
Finely powder Stinging Nettle, and transfer 1.0 g of it to 80 mL of water. Place in an ultrasonic bath for 25 min, and centrifuge. Transfer the supernatant to a 100-mL volumetric flask, dilute with water to volume, 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 Stinging Nettle taken:
Result = (AU/AS) × CS × (V/W) × F × 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 (µg/mL) |
| V | = | = volume of the Sample solution, 100 mL |
| W | = | = amount of Stinging Nettle taken to prepare the Sample solution (mg) |
| F | = | = unit conversion factor, 0.001 mg/µg |
Acceptance criteria:
NLT 0.8% on the dried basis
CONTAMINANTS
• Articles of Botanical Origin, Pesticide Residues 
561
:
Meets the requirements
561:
Meets the requirements
• Microbial Enumeration Tests 2021:
The total aerobic microbial count does not exceed 106 cfu/g, the total combined molds and yeasts count does not exceed 104 cfu/g, and the bile-tolerant Gram-negative bacteria count does not exceed 103 cfu/g.
2021:
The total aerobic microbial count does not exceed 106 cfu/g, the total combined molds and yeasts count does not exceed 104 cfu/g, and the bile-tolerant Gram-negative bacteria 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
• Botanic Characteristics
Macroscopic:
The rhizome is irregularly bent, about 3–10 mm thick, and light gray-brown on the outside; thin roots spring from the knotty bulges of a lengthwise furrow. A transverse cut of the rhizome shows it is fibrous, light yellowish white, and usually has a small medulla cave. The roots are often very long, usually 0.5–2 mm thick, light yellow-brown on the outside, and contain some deep longitudinal furrows; a transverse cut shows a pale and almost pure-white color.
Microscopic:
The transverse section of the rhizome and root shows the following characteristics. The rhizome has a narrow cork composed of brown, thin-walled cells, a few rows of tangentially elongated cortical parenchyma, and a pericyclic region with numerous fibers occurring singly or, more frequently, in small groups. Fibers are much elongated with very thick and lignified walls. Some cells of the pericycle and outer part of secondary phloem contain large globular compound crystals of calcium oxalate. The vascular cambial region is distinct and continuous with narrow radial groups of vascular tissue separated by wide medullary rays. The secondary phloem is mainly parenchymatous with groups of thin-walled sieve tissue. The xylem is dense and completely lignified, containing scattered vessels, isolated or in small groups, associated with moderately thickened xylem parenchyma cells and numerous thicker-walled xylem fibers with slit-shaped pits. Individual vessels have fairly large, closely arranged, bordered pits, while the adjacent parenchyma has simple or bordered pits. Medullary rays indicate alternating areas of lignified and unlignified cells, appearing as tangential bands between the vascular bundles, each composed of 5 or 6 layers of cells; the lignified cells have moderately thickened walls with simple pits. The pith is composed of rounded, unlignified parenchyma, collapsed in the central part to form a cavity. Mature roots show a thin cork, narrow phelloderm, and secondary phloem and xylem with alternating areas of lignified and unlignified parenchyma in the wide medullary rays, similar to that found in the rhizome.
• Articles of Botanical Origin, Total Ash 561:
NMT 10%
561:
NMT 10%
• Loss on Drying 731
Dry 1.0 g of Stinging Nettle, finely powdered, at 105 for 2 h: it loses NMT 12.0% of its weight.
731
Dry 1.0 g of Stinging Nettle, finely powdered, at 105 for 2 h: it loses NMT 12.0% of its weight.
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight containers, protected from light. Store at controlled room temperature.
• Labeling:
The label states the Latin binomial and, following the official name, the parts of the plant contained in the article.
• 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 1451
Pharmacopeial Forum: Volume No. 29(4) Page 1285