Comparison of 1H-NMR fingerprints of Echinacea purpurea extracts with stimulation of myelopoiesis in rat to identify active constituents

We have previously described stimulation of myelopoiesis by Echinacea herbal supplement (Spring Valley Natural Whole Herb, Idea Sphere Inc., American Fork, UT; E. purpurea aerial part) (Ramasahayam et al., 2011). A 70% increase in femur myeloid progenitors, i.e., CFU-GMs, upon culture in methylcellulose with csf2, IL-3 and SCF (Cameo assay, Hemogenix, CS, CO) occurs with oral administration of a 75% ethanol extract for 7 days at 50--100 mg/kg/d to female Sprague-Dawley rats. Comparable CFU-GM activity was obtained with ethanol extract of plant source material (EtCP) of the supplement (Ray Jaglowski, Twinlab Corp., Grand Rapids, MI). To assess whether the activity was a general property of this species, we tested aerial parts of another E. purpurea sample (ULM) harvested in summer 2012 from the campus of the University of Louisiana-Monroe (Long. /Lat., 32.536728°,-92.068448°) for myelostimulatory activity. Treatment with ULM ethanolic extract (EtULM; 25-200 mg/kg/d) did not show a significant increase in CFU-GMs. Since Echinacea phytoconstituent class, N-alkylamides, and endophyte lipopolysaccharide (LPS) and lipoprotein have been implicated as mediators of its immunological effects, we analyzed EtCP and EtULM for these constituents. N-alkylamides were assessed upon concentration in n-hexane washes HxW of EtCP and EtULM by 1H NMR (400 MHz, 20 mg/700 microl MeOH-d4) and HPTLC. 1H NMR spectral peaks with shifts equivalent to those of dodeca-2,4,8,10-tetraenoic acid isobutylamide (PhytoLab, Vestenbergsgreuth, Germany) were evident in HxW of EtULM, but absent from HxW of EtCP. HPTLC with apolar development confirmed that this constituent, as well as beta-sitosterol and other lipophilic constituents, were stripped from ethanolic extracts by n-hexane. Removal of n-hexane soluble constituents did not unmask myelostimulatory activity of EtULM and HxW of EtCP and of EtULM were inactive. LPS assayed with the Limulus Amebocyte Lysate assay (Assoc. of Cape Cod) was 117, 16.8, 83, and 3.2 EU/g for EtCP, HxEtCP, EtULM, and HxEtULM, respectively. EtCP, HxEtCP, EtULM, and HxEtULM extracts enhanced NF-kB activation, an indicator of lipoprotein content, to levels 4.2%, 6.3%, 6.0%, and 17.7% of LPS standard, respectively. Neither LPS nor components that activate monocytes correlated with myelostimulatory activity. In conclusion, these studies demonstrated that LPS, other components that activate monocytes, or lipophilic constituents, including N-alkylamides did not mediate or inhibit the myelostimulatory activity of E. purpurea.