Date of Award:

5-2010

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biological Engineering

Committee Chair(s)

Charles D. Miller

Committee

Charles D. Miller

Committee

Ronald C. Sims

Committee

Marie K. Walsh

Committee

Soonjo Kwon

Abstract

An immediate demand exists for new tuberculosis (TB) antibiotics due to the ever-increasing spread of drug-resistant strains. The drug-development process goes through four phases, the first (Phase 0) of which is to demonstrate and investigate drug effectiveness and toxicity. This research investigated the effectiveness of the Hypericum perforatum herb (commonly St. John's wort (SJW)) in its growth inhibition of mycobacteria and its viability effect on human lung cells.

Organic-solvent SJW extracts were effective at inhibiting every nonpathogenic genetically sequenced Mycobacterium isolate currently available (six isolates) in preliminary studies. Quantitative studies of five Mycobacterium isolates showed an order of concentration sensitivity to the SJW methanol (MeOH) extract as (high to low) M. JLS, M. KMS, M. phlei (not sequenced), M. MCS, B. subtilis, M. smegmatis, and E. coli, with minimal bactericidal concentrations (MBCs) ranging from 0.33-2.66 mg extract/ml. The SJW compounds hyperforin (Hfn), hypericin (Hpn), and pseudohypericin (Phn) were quantified using a novel HPLC method that utilized common HPLC equipment. A crude MeOH extract solution of 133 mg extract/ml contained 2.26 mg Hfn/ml, 0.77 mg Hpn/ml, and 2.67 mg Phn/ml. Purified Hfn had a MBC of between 6-13 μg/ml for M. JLS in the absence of Tween 80. Tween 80 repressed Hfn (46 μg/ml) inhibition of M. JLS at ≥ 0.025% (v/v). Purified Hpn and Phn showed no inhibition of M. JLS at all assayed concentrations, which were ≤ 27 μg/ml and ≤ 25 μg/ml, respectively. Inhibitory results from the five quantitatively assayed Mycobacterium samples could be extrapolated to M. tuberculosis, as these isolates have as high as 72% genetic homology to the pathogen.

The crude MeOH extract and Hfn were lethal to the human carcinomic alveolar epithelial lung cell line A549 at 1.3 mg extract/ml (crude extract) and ≥ 11 μg/ml (Hfn), with a Hfn LD50 of 3-6 μg/ml (5.6-11.2 μmol/L). Because Hfn is antiproliferative to a list of other carcinomic cell lines in the same concentration range, the A549 cell line may be added to that list. The addition of M. JLS cells (5x106 cells/cm2) to penicillin-streptomycin-containing A549 culture (which killed the bacteria) did not affect A549 viability.

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Comments

This work made publicly available electronically on August 2, 2010.

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