Degree Type

Dissertation

Date of Award

2008

Degree Name

Doctor of Philosophy

Department

Theses & dissertations (Interdisciplinary)

Major

Genetics

First Advisor

Diane F. Birt

Second Advisor

Marian Kohut

Third Advisor

Jeff Essner

Abstract

Hypericum perforatum (Hp) is commonly known for its anti-viral, anti-depressant, and anti-proliferative properties, but traditionally Hp was also used to treat inflammation. Studies on the anti-inflammatory activity of Hp are far less advanced than studies on other bioactivities. In fact until recently, there was still debate as to which constituents present in Hp may be responsible for the anti-inflammatory properties.;In this study, the anti-inflammatory activity and cytotoxicity of different Hp extractions and accessions and constituents present within Hp extracts were characterized in RAW 264.7 mouse macrophage cells. In contrast to the light-dependent anti-viral activity of Hp, the anti-inflammatory activity observed with all Hp extracts was light-independent. Extracts made from Hp Elixir(TM) plant material using Soxhlet ethanol or chloroform for extraction displayed superior activity to extracts made from other accessions. When pure constituents were tested, the flavonoids quercetin, quercitrin, isoquercitrin, and hyperoside, the bi-flavonoid amentoflavone, the phloroglucinol hyperforin, and light-activated naphthodianthrone pseudohypericin displayed significant reduction in lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) production, albeit at concentrations generally higher than the amount present in the Hp extracts. Constituents that were present in the Hp extracts at concentrations close to those that inhibited the production of PGE2 were pseudohypericin and hyperforin, suggesting that they are the primary anti-inflammatory constituents along with the flavonoids, and perhaps the interactions of these constituents and other unidentified compounds are important for the anti-inflammatory activity of the Hp extracts.;To further delineate the constituents that may be important for anti-inflammatory activity, an ethanol extract of Hp was fractionated with the guidance of an anti-inflammatory bioassay, LPS-induced PGE2 production, through 4 rounds of fractionation. Four constituents were identified as putative bioactive constituents for the reduction in PGE2. When combined together at concentrations detected in the third round Hp fraction to make a 4 component system, these constituents (0.2 muM chlorogenic acid, 0.08 muM amentoflavone, 0.07 muM quercetin, and 0.03 muM pseudohypericin) explained the majority of the activity of the fraction when activated by light, but only partially explained the activity of this Hp fraction in dark conditions. One of the constituents, light-activated pseudohypericin, was necessary, but not sufficient to explain the reduction in LPS-induced PGE2 of the 4 component system. The Hp fraction and the 4 component system inhibited cyclooxygenase (COX-2) and cytosolic phospholipase A2 (cPLA2), two enzymes in the PGE2-mediated inflammatory response, in a similar way.;To further explore the potential of the four putative bioactive constituents combined into a 4 component system, we used microarray gene expression analysis to identify key gene targets of the 4 component system and the Hp fraction, a third round subfraction from an Hp ethanol extract. Twelve genes were implicated in the activity of the 4 component system +LPS and the fraction +LPS and 8 of the 12 were part of either the janus kinase and signal transducer and activator of transcription (JAK-STAT) or eicosanoid biosynthesis pathways. Additionally, these two pathways, which are important in inflammation, could be linked via the mitogen-activated protein kinase (MAPK) pathways. The 4 component system explained some of the activity of the fraction through inflammatory pathways, however; the fraction also affected genes that the 4 component system did not affect. Thus, other known or unknown compounds in the fraction may be responsible for activities that were identified in the microarray analysis, such as effects on cell cycle.

DOI

https://doi.org/10.31274/rtd-180813-16977

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Kimberly Dawn Petry Hammer

Language

en

Proquest ID

AAI3337385

OCLC Number

313368495

ISBN

9780549924005

File Format

application/pdf

File Size

210 pages

Share

COinS