Powdered Echinacea angustifolia
DEFINITION
Powdered Echinacea angustifolia is Echinacea angustifolia reduced to a powder or very fine powder.
IDENTIFICATION
• A. Presence of Echinacoside and Dicaffeoylquinic Acids (cynarin(e))
Standard solution A:
20 mg/mL of USP Powdered Echinacea angustifolia Extract RS in methanol
Standard solution B:
1 mg/mL of 1,3-dicaffeoylquinic acid in methanol
Sample solution:
Transfer about 1 g of Powdered Echinacea angustifolia 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, typically 20 cm long (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 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 only traces present in Echinacea purpurea) that corresponds in color and RF value to that in the chromatogram of Standard solution A, and another zone characteristic of 1,3-dicaffeoylquinic acid (absent in Echinacea pallida and Echinacea purpurea) 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 Isobutylalkenylamides
Standard solution A:
Transfer a quantity of USP Powdered Echinacea angustifolia Extract RS to a centrifuge tube, and add chloroform to obtain a solution of about 100 mg/mL. Shake by hand to disperse, sonicate for 5 min, and centrifuge. Use the supernatant.
Standard solution B:
1 mg/mL of 
-sitosterol in methanol
-sitosterol in methanol
Sample solution:
Evaporate the chloroform extract retained from preparation of the Sample solution in Identification test A to dryness at 40 in vacuum. To the residue add 1 mL of alcohol, and pass through a nylon membrane filter having a pore size of 0.45 µm.
in vacuum. To the residue add 1 mL of alcohol, and pass through a nylon membrane filter having a pore size of 0.45 µm.
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture, typically 20 cm long (TLC plates)
Application volume:
10 µL
Developing solvent system:
Hexane and ethyl acetate (2:1)
Spray reagent:
Prepare a mixture of glacial acetic acid, sulfuric acid, and p-anisaldehyde (10:5:0.5) in an ice bath.
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. Examine the plate under UV light at 254 nm. Spray the plate with Spray reagent, and then heat the plate at 100 for 5 min.
for 5 min.
Acceptance criteria
Under UV light: The chromatogram obtained from the Sample solution shows one main zone at an RF value of about 0.25 due to 2E,4E,8Z,10E-dodecatetraenoic acid isobutylamide and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (absent in E. pallida) that corresponds in RF value to that in the chromatogram of Standard solution A.
After treatment with Spray reagent and heating: The chromatogram obtained from the Sample solution shows a zone due to -sitosterol that corresponds in RF value to the principal spot in the chromatogram of Standard solution B. Below this spot, there is a zone due to dodeca-2E,4E,8Z,10E-tetraenoic acid isobutylamide and to dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide that corresponds in RF value to that in the chromatogram of Standard solution A; and below this spot, there are several yellowish zones due to 
,-unsaturated isobutylamides (absent in Echinacea pallida and mostly violet in Echinacea purpurea due to the presence of ,,
,
-unsaturated isobutylamides) that are not visible or are very weak when viewed under UV light at 254 nm.
-sitosterol that corresponds in RF value to the principal spot in the chromatogram of Standard solution B. Below this spot, there is a zone due to dodeca-2E,4E,8Z,10E-tetraenoic acid isobutylamide and to dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide that corresponds in RF value to that in the chromatogram of Standard solution A; and below this spot, there are several yellowish zones due to ,-unsaturated isobutylamides (absent in Echinacea pallida and mostly violet in Echinacea purpurea due to the presence of ,,, -unsaturated isobutylamides) that are not visible or are very weak when viewed under UV light at 254 nm.
• C.
In the test for Content of Total Phenols the retention time of the major peak of the Sample solution corresponds to that of the echinacoside peak of Standard solution A, and a peak is detected at the retention time for 1,3-dicaffeoylquinic acid from the Sample solution corresponding to that of Standard solution A.
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 angustifolia Extract RS in Solvent, shaking and heating in a water bath. Dilute with Solvent to obtain a solution having a known concentration of 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 about 125 mg of Powdered Echinacea angustifolia (capable of passing through a 40-mesh sieve), accurately weighed, 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
Chromatogram similarity:
The chromatogram obtained from Standard solution A is similar to the Reference Chromatogram for total phenols provided with the USP Powdered Echinacea angustifolia Extract RS.
Resolution:
NLT 1.0 between the 1,3-dicaffeoylquinic acid isomer and echinacoside peaks, Standard solution A
Capacity factor (k¢):
NLT 3.0, 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), dicaffeoylquinic acids (C25H24O12), and echinacoside (C35H46O20) in the portion of Powdered Echinacea angustifolia 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 | = | = volume of the Sample solution (mL) |
| W | = | = weight of Powdered Echinacea angustifolia used to prepare the Sample solution (mg) |
| F | = | = response factor: chicoric acid, 0.695; dicaffeoylquinic acids, 0.729; caftaric acid, 0.881; chlorogenic acid, 1.000; and echinacoside, 2.220 |
Calculate the percentage of total phenols in the portion of Powdered Echinacea angustifolia taken by adding the individual percentages calculated.
Acceptance criteria:
NLT 0.5% of total phenols on the dried basis
• Content of Dodecatetraenoic Acid Isobutylamides
Standard solution A:
Dissolve, with sonication, USP Powdered Echinacea angustifolia Extract RS in methanol, shaking for 10 min, and dilute with methanol to obtain a solution having a concentration of 5 mg/mL. Pass through a membrane filter having a 0.45-µm or finer pore size.
Standard solution B:
10 µg/mL of USP 2E,4E-Hexadienoic Acid Isobutylamide RS in methanol
Sample solution:
Transfer about 2.5 g of Powdered Echinacea angustifolia (capable of passing through a 40-mesh sieve), accurately weighed, into a round-bottom flask. Add 80 mL of methanol, and reflux for 30 min. Cool to room temperature, and filter into a 100-mL volumetric flask, using small portions of methanol to rinse the flask and the filter. Dilute with methanol to volume. Pass through a membrane filter having a 0.45-µm or finer pore size.
Mobile phase:
Acetonitrile and water (55:45)
Chromatographic system
Mode:
LC
Detector:
UV 254 nm
Column:
4.6-mm × 25-cm; 5-µm packing L1
Column temperature:
30
Flow rate:
1.5 mL/min
Injection size:
25 µL
System suitability
Samples:
Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity:
The chromatogram obtained from Standard solution A is similar to the Reference Chromatogram for alkamides provided with USP Powdered Echinacea angustifolia Extract RS.
Resolution:
NLT 1.0 between dodecatetraenoic acid isobutylamide peaks, Standard solution A
Tailing factor:
NMT 2.0 for the 2E,4E-hexadienoic acid isobutylamide peak, Standard solution B
Relative standard deviation:
NMT 2.5% for the 2E,4E-hexadienoic acid isobutylamide peak in repeated injections, Standard solution B
Analysis
Samples:
Standard solution A, Standard solution B, and Sample solution
Identify the peaks due to 2E,4E,8Z,10E-dodecatetraenoic acid isobutylamide and 2E,4E,8Z,10Z-dodecatetraenoic acid isobutylamide in the chromatogram from the Sample solution by comparison with the chromatogram from Standard solution A. Measure the areas for the relevant peaks.
Calculate the percentage of dodecatetraenoic acid isobutylamides in the portion of Powdered Echinacea angustifolia taken:
Result = (rU/rS) × CS × (V/W) × F × 100
| rU | = | = sum of the peak responses of the relevant analytes from the Sample solution |
| rS | = | = peak response of 2E,4E-hexadienoic acid isobutylamide from the Standard solution B |
| CS | = | = concentration of USP 2E,4E-Hexadienoic Acid Isobutylamide RS in Standard solution B (mg/mL) |
| V | = | = volume of the Sample solution (mL) |
| W | = | = weight of Powdered Echinacea angustifolia used to prepare the Sample solution (mg) |
| F | = | = response factor for 2E,4E-hexadienoic acid isobutylamide, 1.353 |
Acceptance criteria:
NLT 0.075% of dodecatetraenoic acid isobutylamides (C16H25NO) on the dried basis
CONTAMINANTS
• Heavy Metals, Method III 
231
:
NMT 10 ppm
231:
NMT 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 angustifolia is a brown powder with a slight aromatic odor and a sweet taste that quickly becomes bitter, leaving a tingling sensation on the tongue. Under a microscope, the following characteristics are observed: thin-walled polygonal cork cells with red-brown contents; lignified reticulate vessels; abundant stone cells of various shapes; fragments of oleoresin canals with reddish-brown contents; and abundant thin-walled parenchyma with spherocrystalline masses of inulin.
• 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.
• Articles of Botanical Origin, Total Ash 561:
NMT 7.0%
561:
NMT 7.0%
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in well-closed, 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.
• USP Reference Standards 11
11
USP Chlorogenic Acid RS 
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USP 2E,4E-Hexadienoic Acid Isobutylamide RS
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USP Powdered Echinacea angustifolia Extract RS
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 1267
Pharmacopeial Forum: Volume No. 26(6) Page 1583