Powdered Echinacea purpurea Extract

DEFINITION

Xi’an QingShuo Import And Export Trade Co.,Ltd.

Powdered Echinacea purpurea Extract is prepared from dried Echinacea purpurea Root, Echinacea purpurea Aerial Parts, or a mixture of them, by extraction with hydroalcoholic mixtures or other suitable solvents. The ratio of the starting crude plant material to Powdered Extract is between 2:1 and 8:1. It contains NLT 4.0% of total phenols, calculated as the sum of caftaric acid (C13H12O9), chicoric acid (C22H18O12), and chlorogenic acid (C16H18O9), on the dried basis. It contains NLT 0.025% of dodecatetraenoic acid isobutylamides (C16H25NO), calculated on the dried basis.

IDENTIFICATION

•  A. Presence of Isobutylalkenylamides

Standard solution A:  100 mg/mL of USP Powdered Echinacea purpurea Extract RS in methanol

Standard solution B:  1 mg/mL of -sitosterol in methanol

Sample solution:  Transfer about 1 g of Powdered Echinacea purpurea Extract to a suitable extraction thimble. Transfer the thimble to a continuous extraction apparatus, and extract with 50 mL of chloroform for 1 h. Evaporate the chloroform extract to dryness at 40 in vacuum. To the residue add 1 mL of alcohol, and pass through a nylon membrane filter of 0.45-μm pore size.

Adsorbent:  Chromatographic silica gel mixture with an average particle size of 10–15 μm (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, and then spray the plate with Spray reagent, and heat the plate at 100 for 5 min.

Acceptance criteria 

Under UV light at 254 nm: The chromatogram from the Sample solution shows one main zone corresponding in RF value to the zone due to dodeca-2E,4E,8Z,10E-tetraenoic acid isobutylamide and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide in the chromatogram of Standard solution A, and below this zone there are several other zones due to unsaturated isobutylamides.

After treatment with Spray reagent and heating: The zone due to dodeca-2E,4E,8Z,10E-tetraenoic acid isobutylamide and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide turns blue-black, and below this zone there are several other zones due to -unsaturated isobutylamides (not detectable in Echinacea pallida) that turn violet (unlike the corresponding zones in the chromatogram of Echinacea angustifolia that are mostly yellowish due to -unsaturated isobutylamides). A zone due to -sitosterol that corresponds in RF value to the principal spot in the chromatogram of Standard solution B is also observed.

•  B. The retention times of the peaks for chicoric and caftaric acids of the Sample solution correspond to those of Standard solution A, as obtained in the test for Content of Total Phenols. An echinacoside peak is not detectable or is very weak.

COMPOSITION

•  Content of Total Phenols

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. After dilution, 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 60 mg of Powdered Echinacea purpurea Extract to a round-bottom flask equipped with a condenser. Add 25 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.

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

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 of 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% for 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), and chlorogenic acid (C16H18O9) in the portion of Powdered Echinacea purpurea Extract taken:

Result = (rU/rS) × (CS/CU) × 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)
CU	=	= concentration of Echinacea purpurea in the Sample solution (mg/mL)
F	=	= response factor: chicoric acid, 0.695; caftaric acid, 0.881; and chlorogenic acid, 1.000

Calculate the percentage of total phenols in the portion of Powdered Echinacea purpurea Extract taken by adding the individual percentages calculated.

Acceptance criteria:  NLT 4.0% on the dried basis

•  Content of Dodecatetraenoic Acid Isobutylamides

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 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 500 mg of Powdered Echinacea purpurea Extract, accurately weighed, into a 100-mL volumetric flask. Add 80 mL of methanol, and sonicate for 30 min. Cool to room temperature, and dilute with methanol to volume. Pass through a membrane filter of 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 purpurea 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 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 Powdered Echinacea purpurea Extract taken:

Result = (rU/rS) × (CS/CU) × F × 100

rU	=	= sum of the peak responses of the relevant analytes from the Sample solution
rS	=	= peak response from Standard solution B
CS	=	= concentration of USP 2E,4E-Hexadienoic Acid Isobutylamide RS in Standard solution B (mg/mL)
CU	=	= concentration of Echinacea purpurea in the Sample solution (mg/mL)
F	=	= response factor to convert 2E,4E-hexadienoic acid isobutylamide into dodecatetraenoic acid isobutylamides, 1.353

Acceptance criteria:  NLT 0.025% on the dried basis

CONTAMINANTS

 NMT 20 ppm

 Meets the requirements

 The total bacterial count does not exceed 104 cfu/g. The total combined molds and yeasts count does not exceed 103 cfu/g.

 It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.

 Meets the requirements

SPECIFIC TESTS

Dry 1 g at 105 for 2 h: it loses NMT 5.0% of its weight.

ADDITIONAL REQUIREMENTS

•  Packaging and Storage: Preserve in tight, light-resistant containers, in a cool place.

•  Labeling: The label states the Latin binomial and, following the official name, the parts of the plant from which the article was prepared. If derived from root and aerial parts, indicate the corresponding percentages. Label it to indicate the content of total phenols and dodecatetraenoic isobutylamides. The label bears a statement indicating that Echinacea purpurea may cause rare allergic reactions, rashes, or aggravate asthma. It meets the requirements for Botanical Extracts 

 

USP Chlorogenic Acid RS 

USP Powdered Echinacea purpurea Extract RS

USP 2E,4E-Hexadienoic Acid Isobutylamide RS