Echinacea purpurea Aerial Parts
DEFINITION
Echinacea purpurea Aerial Parts consists of the aerial parts of Echinacea purpurea (L.) Moench (Fam. Asteraceae). It is harvested during the flowering stage. It contains NLT 1.0% of the sum of caftaric acid (C13H12O9) and chicoric acid (C22H18O12), and NLT 0.01% of dodecatetraenoic acid isobutylamides (C16H25NO), calculated on the dried basis.
IDENTIFICATION
• A. Presence of Chicoric Acid and Absence of Echinacoside
Standard solution:
10 mg/mL of USP Powdered Echinacea purpurea Extract RS in methanol
Sample solution:
Add 5 mL of diluted alcohol (7:3) to 0.5 g of the powdered plant material, and shake for 1 min. Centrifuge, and use the supernatant.
Adsorbent:
Chromatographic silica gel mixture with an average particle size of 10–15 µm (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 and Sample solution
Develop the chromatograms until the solvent front has moved NLT 18 cm, and dry the plate in a stream 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 from the Sample solution shows a yellowish-green zone at an RF value of 0.75 due to chicoric acid and another yellowish-green zone at an RF value of 0.45 due to caftaric acid, both zones corresponding in color and RF value to zones in the chromatogram from the Standard solution. The chromatogram from the Sample solution does not show or shows only traces of a zone at an RF value of 0.1 due to echinacoside (present in Echinacea angustifolia and in Echinacea pallida). Other colored zones of varying intensities may be observed in the chromatogram from the Sample solution.
• B.
The retention times for the relevant peaks of the Sample solution, mainly due to caftaric acid and chicoric acid, correspond to those of Standard solution A, as obtained in the test for Content of Chicoric Acid and Caftaric Acid. A peak for echinacoside is not detected or is very weak (difference with E. pallida and E. angustifolia).
• C.
The retention times for the relevant peaks of the Sample solution, mainly due to dodecatetraenoic isobutyl amides, correspond to those of Standard solution A, as obtained in the test for Content of Dodecatetraenoic Isobutylamides.
COMPOSITION
• Content of Chicoric Acid and Caftaric Acid
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 |
| 17.5 | 60 | 40 |
| 18 | 90 | 10 |
| 30 | 90 | 10 |
Solvent:
Alcohol and water (7:3)
Standard solution A:
5 mg/mL of USP Powdered Echinacea purpurea Extract RS in Solvent. Dissolve by shaking for 1 min, dilute to volume, and filter through a filter of 0.45-µm or finer pore size.
Standard solution B:
40 µg/mL of USP Chlorogenic Acid RS in Solvent. Dissolve by shaking for 1 min, dilute to volume, and pass through a filter of 0.45-µm or finer pore size.
Sample solution:
Transfer about 125 mg, accurately weighed, of finely powdered Echinacea purpurea Aerial Parts (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 of 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 from Standard solution A is similar to the Reference Chromatogram for total phenols provided with USP Powdered Echinacea purpurea Extract RS.
Relative standard deviation:
NMT 2.0% 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 percentages of caftaric acid (C13H12O9) and chicoric acid (C22H18O12) in the portion of Echinacea purpurea Aerial Parts taken:
Result = (rU/rS) × CS × (V/W) × F × 100
| rU | = | = peak area of the relevant analyte from the Sample solution |
| rS | = | = peak area of 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 Echinacea purpurea Aerial Parts taken to prepare the Sample solution (mg) |
| F | = | = response factor: chicoric acid, 0.695; caftaric acid, 0.881; and chlorogenic acid, 1.000 |
Calculate the percentage of the sum of chicoric acid and caftaric acid in the portion of Echinacea purpurea Aerial Parts taken by adding the individual percentages calculated.
Acceptance criteria:
NLT 1.0% on the dried basis
• Content of Dodecatetraenoic Acid Isobutylamides
Mobile phase:
Acetonitrile and water (55:45)
Standard solution A:
5 mg/mL of USP Powdered Echinacea purpurea Extract RS in methanol. Dissolve using sonication and shaking for 10 min. After dilution, pass through a membrane filter of 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 finely powdered Echinacea purpurea Aerial Parts (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 of 0.45-µm or finer pore size.
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 from Standard solution A is similar to the Reference Chromatogram for alkamides provided with USP Powdered Echinacea purpurea Extract RS.
Resolution:
NLT 1.0 between dodecatetraenoic acid isobutylamide peaks, Standard solution A
Tailing factor:
NMT 2.0 for 2E,4E-hexadienoic acid isobutylamide, 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 of the two isomers of dodecatetraenoic acid isobutylamides 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.
Calculate the percentage of dodecatetraenoic acid isobutylamides in the portion of Echinacea purpurea Aerial Parts taken:
Result = (rU/rS) × CS × (V/W) × F × 100
| rU | = | = sum of the peak areas of the relevant analytes from the Sample solution |
| rS | = | = peak area of 2E,4E-hexadienoic acid isobutylamide from Standard solution B |
| CS | = | = concentration of USP 2E,4E-Hexadienoic Acid Isobutylamide RS in Standard solution B (mg/mL) |
| V | = | = final volume of the Sample solution (mL) |
| W | = | = weight of Echinacea purpurea Aerial Parts taken to prepare the Sample solution (mg) |
| F | = | = response factor to convert 2E,4E-hexadienoic acid isobutylamide into dodecatetraenoic acid isobutylamides, 1.353 |
Acceptance criteria:
NLT 0.01% of dodecatetraenoic acid isobutylamides on the dried basis
CONTAMINANTS
• Heavy Metals, Method III 
231
:
NMT 10 ppm
231:
NMT 10 ppm
• Articles of Botanical Origin, General Method for Pesticides Residues Analysis 561:
Meet the requirements
561:
Meet the requirements
• Microbial Enumeration Tests 2021:
The total aerobic microbial count does not exceed 105 cfu/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the enterobacterial count does not exceed 103 cfu/g.
2021:
The total aerobic microbial count does not exceed 105 cfu/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the enterobacterial count does not exceed 103 cfu/g.
• Microbiological Procedures for 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 herb is an erect, coarse, rough-hairy perennial, usually up to 90 cm tall, rarely up to 180 cm. The leaves are alternate and simple; the lowermost leaves are slender, long, and petioled, ovate to broadly lanceolate, mostly penta-nerved, acute or acuminated at the apex, abruptly narrowed or rarely cordate at the base, usually sharply dentate, and 7–20 cm long and 2.5–7.5 cm wide; the petioles are mostly winged at the summit. The upper leaves are narrower, often almost entirely sessile, lanceolate or ovate lanceolate, and usually with 3 veins.
The flower heads are radiate, up to 15 cm across, solitary or few, and long-peduncled, with 12–20 rays, purple, crimson, or rarely pale; the bristle disks are often orange, 3.5–7.5 cm long; the involucre is depressed-hemispheric; the bracts are lanceolate, spreading or appressed, imbricated in 2–4 series, and hairy on the outer surface with ciliate margins; the receptacle is conical, the scales of the receptacle stiff, spinescent, and conspicuously longer than the disc flowers; the chaff is carinate and cuspidate; the achenes are 3–4 mm in length, tetrasided, obypyramidal, and thick; the pappus has a short, dentate crown.
Microscopic
Leaf:
The leaf has a thickness of 200–350 µm, with an epidermis 9–13 µm thick, largely without chloroplasts; the stomata are 28–35 µm, abundant on the ventral surface and fewer on the dorsal surface; the mesophyll is clearly divided into palisade parenchyma and sponge parenchyma. The palisade parenchyma is one layer thick, with elongated cells 50–65 µm in length, oriented at right angles to the leaf surface, containing numerous chloroplasts. The sponge parenchyma is 150–250 µm thick, with cells of irregular shape, and has multiple cell layers, few chloroplasts, and large intercellular spaces. The phloem bundles of the lateral veins within the sponge parenchyma are bound by a one-layer sheath of small parenchymous cells, with vascular elements of the midrib surrounded by large-celled parenchyma. The uniseriate trichomes are few in the ventral surface, numerous on the dorsal surface, typically tricelled, occasionally tetra- or pentacelled, 250–500 µm in length, each arising from an epidermal cell; the epidermal cell walls appear with moderate thickening; the vessels are various, scalariform, with variable reticulated width.
Petiole:
The parenchyma appear without chloroplasts, in several layers adjacent to a layer of collenchyma; 5–7 phloem bundles of small- to medium-sized vessels are weakly lignified and embedded in the parenchyma in the form of an arc; the wing ribs of the upper surface of the slightly hollowed petiole are marginal.
Inflorescence:
The epidermal cells of the ray florets are square, 50 µm, with a transparent, beaded cell wall; various elements of the Asteraceous exhibit inflorescence; numerous multicellular jointed trichomes of the involucral bracts are 500–800 µm in length; tangential sections of the paleae with numerous fiber bundles are 10–15 µm in diameter and 100–150 µm in length; cell walls are thin. The epidermis of ray florets is reddish to violet; the epidermal cells from the end of the corolla form rounded papillae; a stigma of papillary cells is present; Asteraceous pollen grains are 20–30 µm and spherical with a warty exine.
Calcium oxalate is negative; crystals of inulin and starch granules are rare.
• Loss on Drying 731:
Dry 1 g of the powdered plant material: it loses NMT 12% of its weight.
731:
Dry 1 g of the powdered plant material: it loses NMT 12% of its weight.
• Articles of Botanical Origin, Total Ash 561:
NMT 10.0%, determined on 3 g
561:
NMT 10.0%, determined on 3 g
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Store in tight, light-resistant containers 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 Chlorogenic Acid RS 
') + '&img=../../images/v35300/c11rs1412.gif',600,500);)
USP 2E,4E-Hexadienoic Acid Isobutylamide RS
USP Powdered Echinacea purpurea 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 |
| 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 1275
Pharmacopeial Forum: Volume No. 30(2) Page 557