Echinacea purpurea Root, chemical structure, molecular formula, Reference Standards

Echinacea purpurea Root

»Echinacea purpureaRoot consists of the dried rhizome and roots of Echinacea purpurea(L.)Moench (Fam.Asteraceae).It is harvested in the fall after 3or more years of growth.It contains not less than 0.5percent of total phenols,calculated on the dried basis as the sum of caftaric acid (C13H12O9),chicoric acid (C22H18O12),chlorogenic acid (C16H18O9),and echinacoside (C35H46O20).It contains not less than 0.025percent of alkamides calculated as dodecatetraenoic acid isobutylamides (C16H25NO).

Packaging and storage— Store 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 á11ñ— USP Chlorogenic Acid RS.USP Powdered Echinacea purpurea Extract RS.USP2E,4E-Hexadienoic Acid Isobutylamide RS.

Botanic characteristics—

Macroscopic— The roots are cylindrical and irregularly branched.The outer surface is dark brown and longitudinally striated;fractures are short and tough.Transverse sections show a thin periderm and yellowish xylem with distinct rays.In older roots,the pith is spongy,with a brownish center surrounded by yellow.

Microscopic— Rhizomes and roots in transverse section show a thin outer bark separated from a wide xylem by a brown vascular cambium.The cork is composed of several rows of thin-walled cells containing brown pigment.Schizogenous resin canals are present in the cortex.The rhizome contains bast fibers and stone cells.The xylem,with distinct rays,contains tracheary elements composed of reticulated vessels and tracheids (about 80×30µm)with bordered pits and slanted end walls.Vessels and tracheids are surrounded by thick-walled parenchyma and fibers;fibers are elongated with narrow lumens and funnel-shaped ends (20to 40µm wide).Polygonal sclereids (about 50µm in diameter)are also present.Xylem fibers have minimal or no phytomelanin deposits (unlike Echinacea angustifoliaand Echinacea pallida).Amelanogenic layer is present between adjacent xylem parenchyma cell walls.The rhizome,with pith,is composed of pitted parenchyma cells containing inulin crystals.Starch is minimal to absent,and calcium oxalate crystals are absent.

Change to read:

Identification—

A:Thin-Layer Chromatographic Identification Test á201ñ—

PRESENCE OF CHICORIC ACID AND ABSENCE OF ECHINACOSIDE—

Test solution— Prepare as directed for Identificationtest Aunder Echinacea angustifolia,except to use Echinacea purpureaRoot instead of Echinacea angustifolia.

Standard solution 1— Proceed as directed for Identificationtest Aunder Echinacea angustifolia,except to use USP Powdered Echinacea purpureaExtract RSinstead of USP Powdered Echinacea angustifoliaExtract RS.

Standard solution 2,Developing solvent system,Spray reagent 1,and Spray reagent 2— Prepare as directed for Identificationtest Aunder Echinacea angustifolia.

Procedure— Proceed as directed in the chapter.Develop the chromatograms in Developing solvent systemuntil the solvent front has moved not less than 18cm,and dry the plate in a current of air.Spray the plate with Spray reagent 1followed by Spray reagent 2,and examine the plate under UVlight at 365nm:the chromatogram obtained from the Test solutionshows a yellowish-green zone at an RFvalue of 0.75due to chicoric acid and another yellowish-green zone at an RFvalue of 0.45due to caftaric acid,both zones corresponding in color and RFvalue to zones in the chromatogram obtained from Standard solution 1.The chromatogram obtained from the Test solutiondoes not show or shows only traces of a zone at an RFvalue of 0.1due to echinacoside (present in Echinacea angustifoliaand in Echinacea pallida)that corresponds to a yellowish spot in the chromatogram obtained from Standard solution 1,and does not show a zone that corresponds in color and RFvalue to the spot for

1,3-dicaffeoylquinic acid (cynarin)

USP28(present in Echinacea angustifolia)in the chromatogram obtained from Standard solution 2.Other colored zones of varying intensities may be observed in the chromatogram obtained from the Test solution.

B:Thin-Layer Chromatographic Identification Test á201ñ—

PRESENCE OF ISOBUTYLALKENYLAMIDES—

Test solution— Proceed as directed for Identificationtest Bunder Echinacea angustifolia,except to use the chloroform extract retained from Identificationtest Aunder Echinacea purpureaRoot instead of the chloroform extract retained from Identificationtest Aunder Echinacea angustifolia.

Standard solution 1— Dissolve an accurately weighed quantity of USPEchinacea purpureaExtract RSin methanol to obtain a solution having a known concentration of about

100

USP28mg per mL.

Standard solution 2,Developing solvent system,and Spray reagent— Proceed as directed for Identificationtest Bunder Echinacea angustifolia.

Procedure— Proceed as directed in the chapter.Develop the chromatograms in Developing solvent systemuntil the solvent front has moved not less than

USP28cm,and dry the plate in a current of air.Examine the plate under UVlight at 254nm:the chromatogram obtained from the Test solutionshows

USP28one main zone corresponding in RFvalue 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 1

and below this zone there are several other zones due to a,b,g,d-unsaturated isobutylamides.

USP28Spray the plate with Spray reagent,and then heat the plate at 100

for 5minutes.Examine the plate under long-wavelength UVlight: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 a,b,g,d-unsaturated isobutylamides (not detectable in Echinacea pallida)that turn violet (unlike the corresponding zones in the chromatogram of Echinacea angustifoliathat are mostly yellowish due to a,b-unsaturated isobutylamides).

Azone due to b-sitosterol that corresponds in RFvalue to the principal spot in the chromatogram of Standard solution 2is also observed.

USP28

C: The retention times for the relevant peaks in the chromatogram of the Test solution,mainly due to caftaric acid and chicoric acid,correspond to those in the chromatogram of Standard solution 1,as obtained in the test for Content of total phenols.An echinacoside peak is not detectable or is very weak.

Content of total phenols—

Solvent,Solution A,Solution B,Mobile phase,and Standard solution 2— Proceed as directed in the test for Content of total phenolsunder Echinacea angustifolia.

Standard solution 1— Dissolve an accurately weighed quantity of USP Powdered Echinacea purpureaExtract RSin Solvent,shaking for 1minute,and dilute with Solventto obtain a solution having a known concentration of about 5mg per mL.Pass through a membrane filter having a 0.45-µm or finer porosity.

Test solution— Proceed as directed for Content of phenolsunder Echinacea angustifolia,except to use finely powdered Echinacea purpureaRoot instead of Echinacea angustifolia.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 330-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L1.The column temperature is maintained at 35

.The flow rate is about 1.5mLper minute.The chromatograph is programmed as follows:

Time (minutes)	Solution A (%)	Solution B (%)	Elution
0–13	90®78	10®22	linear gradient
13–14	78®60	22®40	linear gradient
14–17.5	60	40	isocratic
17.5–18	60®90	40®10	linear gradient
18–30	90	10	equilibration

Chromatograph Standard solution 1,and record the peak responses as directed for Procedure:the chromatogram obtained is similar to the Reference Chromatogram for total phenols provided with USP Powdered Echinacea purpureaExtract RS.Chromatograph Standard solution 2,and record the responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2%.

Procedure— Proceed as directed in the test for Content of total phenolsunder Echinacea angustifolia.Separately calculate the percentage of caftaric acid (C13H12O9),chicoric acid (C22H18O12),chlorogenic acid (C16H18O9),and echinacoside (C35H46O20)in the portion of Echinacea purpureaRoot taken by the formula:

2500F(C/W)(ri/rS),

in which Fis the response factor and is equal to 0.695for chicoric acid,0.881for caftaric acid,1.000for chlorogenic acid,and 2.220for echinacoside;Cis the concentration,in mg per mL,of USP Chlorogenic Acid RSin Standard solution 2;Wis the weight,in mg,of Echinacea purpureaRoot taken;and riand rSare the peak responses for the relevant analyte obtained from the Test solutionand Standard solution 2,respectively.Calculate the percentage of total phenols in the portion of Echinacea purpureaRoot taken by adding the individual percentages calculated.

Content of alkamides—

Mobile phase and Standard solution 2— Proceed as directed in the test for Content of dodecatetraenoic acid isobutylamidesunder Echinacea angustifolia.

Standard solution 1— Proceed as directed for Content of dodecatetraenoic acid isobutylamidesunder Echinacea angustifolia,except to use USP Powdered Echinacea purpureaExtract RSinstead of USP Powdered Echinacea angustifoliaExtract RS.

Test solution— Proceed as directed for Content of dodecatetraenoic acid isobutylamidesunder Echinacea angustifolia,except to use Echinacea purpureaRoot instead of Echinacea angustifolia.

Chromatographic system (see Chromatography á621ñ)— Proceed as directed for Content of dodecatetraenoic acid isobutylamidesunder Echinacea angustifolia,except to use the Reference Chromatogram for alkamides provided with USP Powdered Echinacea purpureaExtract RSinstead of the Reference Chromatogram provided with USP Powdered Echinacea angustifoliaExtract RS.

Procedure— Proceed as directed in the test for Content of dodecatetraenoic acid isobutylamidesunder Echinacea angustifolia.Identify the peaks of the ten major alkamides in the chromatogram obtained from the Test solutionby comparison with the chromatogram obtained from Standard solution 1.Calculate the percentage of alkamides in the portion of Echinacea purpureaRoot taken by the formula:

10(1.353)(C/W)(ri/rS),

in which 1.353is the response factor for 2E,4E-hexadienoic acid isobutylamide;Cis the concentration,in mg per mL,of USP2E,4E-Hexadienoic Acid Isobutylamide RSin Standard solution 2;Wis the weight,in g,of Echinacea purpureaRoot taken;riis the sum of the peak responses of the relevant analytes obtained from the Test solution;and rSis the peak response obtained from Standard solution 2.

Other requirements— It meets the requirements of the tests for Loss on drying,Foreign organic matter,Total ash,Acid-insoluble ash,Pesticide residues,and Heavy metalsunder Echinacea angustifolia.

Auxiliary Information— Staff Liaison:Gabriel I.Giancaspro,Ph.D.,Senior Scientist and Latin American Specialist

Expert Committee:(DSB)Dietary Supplements:Botanicals

USP28–NF23Page 2081

Pharmacopeial Forum:Volume No.30(2)Page 561

Phone Number:1-301-816-8343