POSTER PRESENTATION ABSTRACTS
Please note: All posters will be displayed in
Salons 10-12.
FRIDAY, 10:15-11:00 AM
Cech NB, Hovater BA, Farcas A, Tutor K, Wenner CA.
Cytochrome P450 metabolized fresh root extracts from
Echinacea purpurea induce TNF expression by differentiated monocytes
in vitro.
University of North Carolina Greensboro, Department
of Chemistry and Biochemistry nadja_cech@uncg.edu
PURPOSE: In this study, the hypothesis was tested
that Echinacea purpurea extracts and metabolized constituents
thereof induce cytokine expression by human immune cells in vitro.
METHODS: Extracts were prepared from fresh roots
of E. purpurea using various ethanol/water mixtures as extraction
solvents. Diluted extracts were tested for endotoxin contamination using
the limulus amebocyte lysate assay. Liquid chromatography/mass spectrometry
(LC-MS) was employed to determine quantity and identity of a number
of alkylamides and caffeic acid derivatives present in the extracts.
The extracts and alkylamide isolates were subjected to in vitro
metabolism by human liver microsomes, and the resulting metabolized
products were again analyzed with LC-MS to characterize the structures
of the metabolites. U937 monocytic cells were treated with PMA to initiate
differentiation, and 48 hr later, washed and treated with both metabolized
and un-metabolized extracts, individual alkylamides, and appropriate
controls. After 8 hr incubation, supernatants were analyzed using ELISA
techniques to determine the concentration of TNF expressed by the cells.
RESULTS: All extracts tested negative for endotoxin
contamination. The E. purpurea root extract prepared with 95%
ethanol solvent caused an approximately 2-fold increase in TNF expression
by differentiated U937 cells. The TNF-inducing effect increased with
increasing ethanol content in the extraction solvent, with the strongest
enhancement induced by the 95% ethanol extract. Alkylamide and caffeic
acid derivative content also increased with increasing ethanol content
in the extraction solvent. However, individual alkylamides and caffeic
acid derivatives, when tested in the same concentration range present
in the extracts, did not cause a significant increase in TNF production.
A number of hydroxylated or epoxidated products of alkylamides were
formed by incubation of the Echinacea extracts and isolated alkylamides
with liver microsomes. The metabolized Echinacea extracts also
had an inductive effect on TNF production by differentiated monocytes,
but the TNF inducing activity of the extracts did not appear to increase
as a result of liver metabolism.
CONCLUSIONS: Fresh root extracts of E. purpurea,
with and without metabolism by cytochrome P450 enzymes, induce TNF expression
by differentiated monocytes in vitro. Alkylamides, caffeic acid derivatives,
and metabolites of alkylamides are all present in these extracts. These
isolated constituents do not appear to induce TNF induction by themselves.
However, it is possible that the activity of the complex extracts results
from synergistic interactions among these constituents. The role of
synergistic interactions between alkylamides, caffeic acid derivatives,
and metabolites thereof in the immunomodulatory activity of Echinacea
purpurea extracts is the subject of continuing investigations.
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