Echinacea pallida
DEFINITION
Echinacea pallida consists of the dried rhizome and roots of Echinacea pallida (Nutt.) Nutt. (Fam. Asteraceae). It is harvested in the fall after 3 or more years of growth. It contains NLT 0.5% of total phenols, calculated on the dried basis as the sum of caftaric acid (C13H12O9), chicoric acid (C22H18O12), chlorogenic acid (C16H18O9), and echinacoside (C35H46O20).
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:  Weigh and finely pulverize about 10 g of Echinacea pallida, and transfer about 1 g of powder, 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.
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 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
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 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:  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.
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.
•  C. The retention time of the major peak in the Sample solution corresponds to that of the echinacoside peak in 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 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. Pass through a membrane filter having a 0.45-µm or finer pore size.
Sample solution:  Transfer 125 mg of finely 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 obtained 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 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== volume of the Sample solution (mL)
W== weight of powdered Echinacea pallida used 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
SPECIFIC TESTS
•  Botanic Characteristics
Macroscopic:  The outer surface of the rhizome is pale to yellowish-brown, crowned with the remains of the aerial stem, and sometimes shows surface annulations up to 15 mm in diameter. The roots are pale to yellowish-brown, cylindrical or slightly tapering, sometimes spirally twisted, longitudinally wrinkled and deeply furrowed, up to 4–10 mm in diameter, and pass imperceptibly into rhizome. The short fracture, when dry, becomes tough and pliable on exposure to air.
Microscopic:  The rhizomes and roots in transverse section show a thin outer bark separated from a wide xylem by a distinct cambial line. The cork is composed of several rows of thin-walled cells containing yellowish-brown pigment. The rhizome has a small circular pith, occasional small groups of thick-walled, lignified fibers in the pericycle, and a parenchymatous cortex. The phloem and xylem are composed of narrow strands of vascular tissue separated by wide, nonlignified medullary rays. Xylem vessels are lignified, 25–75 µm in diameter, usually with reticulate thickening but occasionally with spiral or annular thickening. Sclereids occur singly or in small groups, varying considerably in size and shape from rounded to rectangular to elongated and fiber-like, are up to 300 µm long and 20–40 µm wide, with intercellular spaces forming schizogenous oleoresin canals that are 80–150 µm in diameter and contain a dense black deposit present both inside and outside of the central cylinder (unlike Echinacea angustifolia, where the canals are present only outside of the central cylinder). Spherocrystalline masses of inulin occur throughout the parenchymatous tissues. Lignified fibers, present in the periphery of the cortex, are 100–300 µm long and occur singly with phytomelanin often absent (unlike Echinacea angustifolia, where the fibers occur scattered in groups, are 300–800 µm long, and are usually surrounded by phytomelanin).
•  Loss on Drying 731: Dry a sample at 105 for 2 h: it loses NMT 10.0% of its weight.
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 parts of the plant contained in the article.
•  USP Reference Standards 11
USP Chlorogenic Acid RS Click to View Structure
USP Powdered Echinacea pallida Extract RS
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