Degree Type

Dissertation

Date of Award

2009

Degree Name

Doctor of Philosophy

Department

Food Science and Human Nutrition

First Advisor

Diane F. Birt

Abstract

Preparations of Echinacea as a dietary supplement are most commonly used to treat or lessen the severity of symptoms associated with the common cold or other upper respiratory tract infections, although the use Echinacea as a medicinal herb dates back hundreds of years when Native American peoples used it to treat various ailments ranging from snake bite antidotes to venereal diseases to rheumatism. With the popularity of this botanical growing throughout the United States, and the world, studies have been ongoing to understand how Echinacea and its constituents modulate the immune system.

The purpose of this study was to identify Echinacea species, fractions, and constituents responsible for the anti-inflammatory properties associated with Echinacea , as well as establish a mechanistic basis for these properties. The model system chosen to carry out these studies was the RAW264.7 mouse macrophage cell line which is a strongly established model used to mimic the inflammatory response when induced with lipopolysaccharide (LPS). Inhibition of the production of the pro-inflammatory lipid mediator, prostaglandin E2 (PGE2), along with parallel cytotoxicity studies were used to identify Echinacea species with the greatest anti-inflammatory potential. From the screening of Soxhlet ethanol root extractions (prepared in Dr. Patricia A. Murphy's laboratory) from six of the nine Echinacea species, it was determined that Echinacea angustifolia , Echinacea pallida , Echinacea simulata , and Echinacea sanguinea were the strongest inhibitors of LPS induced PGE2 production, showing no cytotoxic effects. Common alkylamides of Echinacea were also chemically synthesized in Dr. George A. Kraus's laboratory and examined for their ability to inhibit PGE2 production, identifying 13 alkylamides capable of significant inhibition of the lipid mediator at 50 μM, five alkylamides capable of significant inhibition at 25 μM, and only one alkylamide, Bauer alkylamide 14, capable of significant inhibition at 10 μM. Again, these constituents of Echinacea did not show cytotoxic effects at concentrations at or below 50 μM. Alkylamides present in the Echinacea species extracts were present at concentrations much lower than those screened in the PGE2 assay, implying that although alkylamides are anti-inflammatory as synthetic constituents, other constituents present in the plant, either previously identified or not, or interactions among these constituents are important for the anti-inflammatory properties of Echinacea preparations.

In order to unravel the complex mixture of Echinacea constituents to identify key contributors to the anti-inflammatory activity, bioactivity guided semi-preparative reverse phased HPLC was used to fractionate four species of Echinacea in Dr. Patricia A. Murphy's laboratory. From the fractionation of an Echinacea pallida extract, it was determined that Bauer ketones 23 and 24 were important for the identified PGE2 inhibitory capabilities of a ketone rich first round fraction, necessitating further study of this group of compounds for their anti-inflammatory potential. Following the inhibition of PGE2 production through three rounds of fractionation with an Echinacea angustifolia extract led to the identification of Bauer alkylamide 11 and Bauer ketone 23 at concentrations present in their respective third round fractions capable of partially explaining the PGE2 inhibition observed prior with their corresponding fraction. Synthetic Bauer alkylamide 11 was also capable of significant inhibition of nitric oxide production. The knowledge that Bauer alkylamide 11 and Bauer ketone 23 were key contributors to the anti-inflammatory properties of Echinacea at endogenous concentrations led to the hypothesis that through the enrichment of a first round alkylamide rich fraction of E. angustifolia with synthetic Bauer alkylamide 11 and Bauer ketone 23 an enhanced anti-inflammatory potential could be achievable. By enriching the fraction with synthetic Bauer alkylamide 11 and Bauer ketone 23 to concentrations determined to be important from the third round fractions, a greater inhibition of PGE2 production was identified than that observed with the fraction alone.

In order to identify key gene targets for the Echinacea angustifolia fraction, enriched fraction, the combination of Bauer alkylamide 11 and Bauer ketone 23, and these constituents individually, microarray and time course qRT-PCR studies were conducted. These studies indicated from the microarray study that the selected Echinacea treatments to LPS induced RAW264.7 cells did not alter gene expression after eight hours, with only eight differentially expressed (DE) genes being identified with false discovery rates (FDR) ranging from 50% to 75% in the microarray. Although, 3,257 DE genes were identified between the media + DMSO and the media + DMSO + LPS controls with a FDR of 0.0001%, establishing the expected LPS effect. The qRT-PCR data showed a decrease in TNF-α gene expression after treatment with all samples and an increase in iNOS expression after treatment with enriched fraction. Although PGE2 production had been decreased by these treatments, COX-2 mRNA levels were not significantly different between treatments compared the media + DMSO + LPS at any time point analyzed (0.5 hr, 1 hr, 2hr, 4 hr, 8 hr, and 24 hr). COX-2 protein levels were increased after an 8 hour treatment of E. angustifolia fraction 3, and Bauer ketone 23. Furthermore the activity of COX-2 was inhibited by all treatments. Combined these studies suggest that Echinacea extracts, fractions and certain classes of constituents have anti-inflammatory potential through the direct modulation of a key enzyme in the eicosanoid pathway and that the noted inhibition of NO production and TNF-α gene expression may be due to the effect these treatments have on divergent signal transduction pathways.

Copyright Owner

Carlie Ann Lalone

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

210 pages

Included in

Nutrition Commons

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