Effects of chemically characterized fractions from aerial parts of Echinacea purpurea and E. angustifolia on myelopoiesis in rats

Abstract

Echinacea species are used for beneficial effects on immune function, and various prevalent phytochemicals have immunomodulatory effects. Using a commercial E. purpurea (L.) Moench product, we have evaluated the myelopoietic effect on bone marrow of rats treated with various extracts and correlated this with their chemical class composition. Granulocyte/macrophage-colony forming cells (GM-CFCs) from femurs of female Sprague-Dawley rats were assessed at 24 h after 7 daily oral treatments. A 75% ethanolic extract at 50 mg dried weight (derived from 227 mg aerial parts) per kg body weight increased GM-CFCs by 70% but at 100 mg/kg was without effect. Ethanolic extracts from aerial parts of E. angustifolia DC. var. angustifolia and E. purpurea from the USDA North Central Regional Plant Introduction Station increased GM-CFCs by 3- and 2-fold, respectively, at 200 mg/kg (~1400 mg/kg plant material). Extract from another USDA E. angustifolia was inactive. Proton and APT NMR, MS, and TLC indicated alkylamides and caffeic-acid derivatives (CADs) present in ethanolic extracts of both the commercial and USDA-derived material. Cichoric and caftaric acids were prominent in both E. purpurea ethanolic extracts but absent in E. angustifolia. Aqueous extract of the commercial material exhibited polysaccharide and CAD signatures and was without effect on GM-CFCs. A methanol-CHCl3 fraction of commercial source, also inactive, was almost exclusively 1:4 nonanoic: decanoic acids, which were also abundant in commercial ethanolic extract but absent from USDA material. In conclusion, we have demonstrated an ethanolextractable myelostimulatory activity in Echinacea aerial parts that, when obtained from commercial herbal supplements, may be antagonized by medium-chain fatty acids presumably derived from a non-plant additive.

Fig. 1

APT spectrum of 75% ethanolic extract of aerial parts of a commercial E. purpurea in DMSO-d₆ at 100 MHz. The region circled in the top spectra is expanded below. The region within the square corresponds to amide carbon. DMSO signal is evident at 39.5 ppm.

Fig. 2

¹H NMR spectra (400 MHz, in DMSO-d₆) of 75% ethanol extracts of aerial parts of a commercial E. purpurea (A), two accessions of E. angustifolia (B and D) and one accession of E. purpurea (C) obtained from the USDA North Central Regional Plant Introduction Station. DMSO signals are evident at 2.50 ppm. Insert in C shows expanded δ = 6.0– 7.30 ppm region to illustrate spin-spin coupling.

Fig. 3

APT spectra of 75% ethanolic extracts (100 Hz in DMSO-d₆) of aerial parts of one accession of E. purpurea (A) and two accessions of E. angustifolia (B and C) obtained from the USDA North Central Regional Plant Introduction Station. DMSO signals are evident at 39.5 ppm.

Fig. 4

HPTLC of 75% ethanolic extracts developed with a polar mobile phase. Extracts from USDA E. angustifolia accessions PI 649029 and PI 649026, E. purpurea PI 649040, and 2 lots of commercial E. purpurea were chromatographed. Standards are cichoric acid, caftaric acid, and echinaco-side. Chromatograms were developed with EtOAc-acetone-formic acid-water (15:9:1:1). Origin and mobile phase front are indicated by an arrow and arrowhead, respectively. Diphenylborinic acid aminoethylester spray reagent and UV light, λ₃₆₆ nm, were used for visualization.

Fig. 5

HPTLC of 75 % ethanolic extracts developed with an apolar mobile phase. Extracts of E. angustifolia 649029 and 649026, E. purpurea 649040 (lanes 1,2, and 3, resp.), and 2 lots of commercial E. purpurea were chromatographed (lanes 4, 5). MeOH-CH₃Cl wash is in lane 6. Chromatograms were developed with toluene-EtOAc-cyclohexane-formic acid (24:6:3:0.9). β-sitosterol (band E, R_f 0.52) and C₉-C₁₀ fatty acids (band H) are indicated by arrows and arrowheads, respectively. p-Anisaldehyde-sulfuric acid was used for visualization.

Fig. 6

Effects of 75% ethanolic extract of commercial E. purpurea and cultivars of E. angustifolia 649029 and 649026 and E. purpurea 649040 on femur GM-CFCs (A) and total bone marrow cells (B) of rats treated with 50, 100, and 200 mg/kg/d. Rats received 7 daily doses of extract, and bone marrow cells were processed 24 h after the last dose. Means ± SEM for n = 4 rats are shown. Means that differed from vehicle control (0 mg/kg) by a statistically significant amount are indicated by * p < 0.05 and ** p < 0.01.