Powdered Echinacea pallida
DEFINITION
Powdered Echinacea pallida is Echinacea pallida reduced to a powder or very fine powder.
IDENTIFICATION
• A. Presence of Echinacoside and Absence of Dicaffeoylquinic Acids (cynarin(e))
Standard solution A:
10 mg/mL of USP Powdered Echinacea pallida Extract RS in methanol
Standard solution B:
1 mg/mL of 1,3-dicaffeoylquinic acid in methanol
Sample solution:
Transfer 1 g of Powdered Echinacea pallida to a suitable extraction thimble. Transfer the thimble to a continuous extraction apparatus, and extract with 50 mL of chloroform for 1 h. Retain the chloroform extract for Identification test B. Continue the extraction with 50 mL of methanol, and concentrate to a small volume at 40
in vacuum. With the aid of methanol, transfer the extract to a 10-mL volumetric flask, and dilute with methanol to volume.
in vacuum. With the aid of methanol, transfer the extract to a 10-mL volumetric flask, and dilute with methanol to volume.
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture (TLC plates)
Application volume:
10 µL
Developing solvent system:
Ethyl acetate, formic acid, and water (17:2:1)
Spray reagent A:
10 mg/mL of diphenylborinic acid, ethanolamine ester in methanol
Spray reagent B:
50 mg/mL of polyethylene glycol 4000 in alcohol
Analysis
Samples:
Standard solution A, Standard solution B, and Sample solution
Develop the chromatograms until the solvent front has moved NLT 12 cm, and dry the plate in a current of air. Spray the plate with Spray reagent A followed by Spray reagent B, and then examine the plate under UV light at 365 nm.
Acceptance criteria:
The chromatogram obtained from the Sample solution shows a yellowish zone at an RF value of 0.14, characteristic of echinacoside (absent or traces in Echinacea purpurea), corresponding in color and RF value to that in the chromatogram of Standard solution A, and does not show a zone characteristic of 1,3-dicaffeoylquinic acid (present in Echinacea angustifolia) corresponding in color and RF value to that in the chromatogram of Standard solution B. Other colored zones of varying intensities may be observed in the chromatogram of the Sample solution.
• B. Presence of Ketoalkenynes
Standard solution A:
10 mg/mL of USP Powdered Echinacea pallida Extract RS in chloroform. Shake for 1 min, and centrifuge. Use the supernatant.
Standard solution B:
1 mg/mL of 
-sitosterol in methanol
-sitosterol in methanol
Sample solution:
Evaporate to dryness the chloroform extract retained from preparation of the Sample solution in Identification test A at 40 in vacuum. To the residue add 1 mL of alcohol, and pass through a nylon membrane filter having a 0.45-µm pore size.
in vacuum. To the residue add 1 mL of alcohol, and pass through a nylon membrane filter having a 0.45-µm pore size.
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture (TLC plates)
Application volume:
10 µL
Developing solvent system:
Toluene and ethyl acetate (7:3)
Spray reagent A:
1% solution of vanillin in alcohol
Spray reagent B:
10% solution of sulfuric acid in alcohol
Analysis
Samples:
Standard solution A, Standard solution B, and Sample solution
Develop the chromatograms until the solvent front has moved NLT 12 cm, and dry the plate in a current of air. Spray the plate with Spray reagent A followed by Spray reagent B, and heat the plate at 120 for 3 min.
for 3 min.
Acceptance criteria:
The chromatogram obtained from the Sample solution shows green, brown, and violet zones above the spot for -sitosterol (RF range 0.6–0.8). These zones (unlike those in Echinacea angustifolia and Echinacea purpurea) are characteristic of ketoalkenynes, and correspond in RF value to the zones in the chromatogram obtained from Standard solution A.
-sitosterol (RF range 0.6–0.8). These zones (unlike those in Echinacea angustifolia and Echinacea purpurea) are characteristic of ketoalkenynes, and correspond in RF value to the zones in the chromatogram obtained from Standard solution A.
• C.
The retention time of the major peak in the Sample solution corresponds to that of the echinacoside peak in the Standard solution A, as obtained in the test for Content of Total Phenols. The peak area of any peak detected at the locus of 1,3-dicaffeoylquinic acid is NMT 1% of the peak area for the echinacoside peak.
COMPOSITION
• Content of Total Phenols
Solution A:
Phosphoric acid (0.1 in 100) in water
Solution B:
Acetonitrile
Mobile phase:
See Table 1.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 90 | 10 |
| 13 | 78 | 22 |
| 14 | 60 | 40 |
| 17 | 60 | 40 |
| 17.5 | 90 | 10 |
| 22 | 90 | 10 |
Solvent:
Alcohol and water (7:3)
Standard solution A:
Dissolve USP Powdered Echinacea pallida Extract RS in Solvent, by shaking and heating in a water bath. Dilute with Solvent to obtain a solution having a known concentration of about 1 mg/mL. Pass through a membrane filter having a 0.45-µm or finer pore size.
Standard solution B:
40 µg/mL of USP Chlorogenic Acid RS in Solvent
Sample solution:
Transfer 125 mg of Powdered Echinacea pallida (capable of passing through a 40-mesh sieve), to a round-bottom flask equipped with a condenser. Add 25.0 mL of Solvent, and heat under reflux, while shaking by mechanical means, for 15 min. Centrifuge, or pass through a membrane filter having a 0.45-µm or finer pore size.
Chromatographic system
Mode:
LC
Detector:
UV 330 nm
Column:
4.6-mm × 25-cm; 5-µm packing L1
Column temperature:
35
Flow rate:
1.5 mL/min
Injection size:
5 µL
System suitability
Samples:
Standard solution A and Standard solution B
Suitability requirements
Chromatographic similarity:
The chromatogram of Standard solution A is similar to the Reference Chromatogram for total phenols provided with USP Powdered Echinacea pallida Extract RS.
Capacity factor (k¢):
NLT 3.0 for the chlorogenic acid peak, Standard solution B
Tailing factor:
NMT 2.0 for the chlorogenic acid peak, Standard solution B
Relative standard deviation:
NMT 2.5% for the chlorogenic acid peak in repeated injections, Standard solution B
Analysis
Samples:
Standard solution A, Standard solution B, and Sample solution
Identify the relevant analytes in the chromatogram obtained from the Sample solution by comparison with the chromatogram obtained from Standard solution A. Measure the areas for the relevant peaks.
Separately calculate the percentage of caftaric acid (C13H12O9), chicoric acid (C22H18O12), chlorogenic acid (C16H18O9), and echinacoside (C35H46O20) in the portion of Powdered Echinacea pallida taken:
Result = (rU/rS) × CS × (V/W) × F × 100
| rU | = | = peak response for the relevant analyte from the Sample solution |
| rS | = | = peak response for chlorogenic acid from Standard solution B |
| CS | = | = concentration of USP Chlorogenic Acid RS in Standard solution B (mg/mL) |
| V | = | = final volume of the Sample solution (mL) |
| W | = | = weight of Powdered Echinacea pallida taken to prepare the Sample solution (mg) |
| F | = | = response factor: chicoric acid, 0.695; caftaric acid, 0.881; chlorogenic acid, 1.000; and echinacoside, 2.220 |
Calculate the percentage of total phenols in the portion of Echinacea pallida taken by adding the individual percentages calculated.
Acceptance criteria:
NLT 0.5% of total phenols on the dried basis
CONTAMINANTS
• Heavy Metals, Method III 
231
:
10 ppm
231:
10 ppm
• Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561:
Meets the requirements
561:
Meets the requirements
SPECIFIC TESTS
• Botanic Characteristics:
Powdered Echinacea pallida is a brown powder with a faint aromatic odor and a slightly acrid, persistent taste. It turns yellow when mounted in sodium hydroxide solution. Under a microscope, the following characteristics are observed: groups of secretory canals with brown contents, surrounded by parenchymatous cells containing cluster crystals of calcium oxalate; and parenchymatous cells with small starch granules; thick-walled lignified fibers and fragments of reticulate and pitted vessels.
• Articles of Botanical Origin, Volatile Oil Determination 561:
1.0–2.0 mL/100 g
561:
1.0–2.0 mL/100 g
• Articles of Botanical Origin, Total Ash 561:
NMT 7.0%
561:
NMT 7.0%
• Loss on Drying 731:
Dry a sample at 105 for 2 h: it loses NMT 10.0% of its weight.
731:
Dry a sample at 105 for 2 h: it loses NMT 10.0% of its weight.
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight, light-resistant containers.
• Labeling:
The label states the Latin binomial and, following the official name, the part of the plant 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 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1273
Pharmacopeial Forum: Volume No. 26(6) Page 1588