1 St. John s Wort as an Antimycobacterial Agent, Ronald Sims, Marie Walsh, Soonjo Kwon, Charles Miller Utah State University
2 Introduction Mycobacterial diseases Leprosy Tuberculosis Extensively Drug-Resistant (XDR) strain St. John s Wort (SJW) Common herb usually used for depression Bactericidal against Gram+ bacteria M. tuberculosis St. John s Wort
3 The idea: Introduction Use mycobacterial surrogates to gauge SJW effect on pathogenic mycobacteria. Our surrogates: M. JLS M. KMS M. MCS M. Smegmatis M. Phlei
4 Research Outline Basic inhibitory testing Novel HPLC quantification method Minimal Bactericidal Concentration (MBC) studies Lung cell testing Conclusions
5 Basic Inhibitory Testing SJW + Gram(-) control Organic solvent + Mycobacteria + LB agar Inhibition of all mycobacteria Mycobacteria (Gram+)
6 Basic Inhibitory Testing Using different SJW extracts: Growth pattern at two weeks for streak plate of GNC SJW. =growth, X=inhibition
7 Basic Inhibitory Testing SJW dilutions in agar: MIC threshold using agar SJW dilution method for M. JLS, KMS, and MCS, with E. coli as a control
8 HPLC Quantification of SJW Compounds- SJW Solubility Testing Solubility in MeOH : 70% MeOH Solubility in Acetonitrile : 60% Acetonitrile 80% Solubility test for SJW in MeOH and ACN
9 HPLC Quantification of SJW Compounds- Novel Method 30-min, including washing and equilibrating Requires only two mobile phases Uses common photodiode array detection Uses C18 column (common type) HPLC machine used
10 HPLC Quantification of SJW Compounds- Chromatograms Standards (mixed) Crude Extract HPLC chromatograms for SJW. Standards on left. MeOH extract on right.
11 HPLC Quantification of SJW Compounds- Concentrations Standard curves for SJW active compounds In 5.76 mg SJW/ml solution ( 1X ) Pseudohypericin: 25 ug/ml Hyperforin: 52 ug/ml Hypericin: 27 ug/ml
12 Liquid culture with SJW MeOH extract Minimal Bactericidal Concentration Viable Cell-Count Method Cell dilutions Cell counting Cell spotting
13 Minimal Bactericidal Concentration MeOH Extract Results 5.76 mg SJW/ml 1.44 mg SJW/ml 2.88 mg SJW/ml 0.72 mg SJW/ml 2% MeOH Control Representative sample of M. KMS No MeOH/SJW Control
14 Minimal Bactericidal Concentration MeOH Extract Results Growth Curves Mycobacterium Gram negative Bactericidal Representative growth curves of M. KMS and E. coli
15 Minimal Bactericidal Concentration Purified SJW Compounds Results Hyperforin MIC/MBC assay. Awaiting further results. MIC/MBC assay of hyperforin against M. KMS. From left to right: 0, 0.1, 1.0, 5.0 µg/ml
16 Untreated M. KMS SEM Imaging After 1 hour After 48 hours MB-10-93 MB-16-19 MB-12-13 surface peeling SEM images of untreated M. KMS, 1 hour, and 48 hours after adding SJW extract
17 Effect on Human Lung Cells (A549) Toxicity testing Pseudohypericin Hyperforin Hypericin Mixture Crude MeOH extract Confluent human alveolar epithelial lung cells
18 Engineering Applications Use of SJW compounds in inhalants for XDR-TB Study of mass transfer in lung cell models Implementation of SJW compounds into topical treatments for mycobacterial skin infections Study of mass transfer in epidermal cell models Inhalant Topical treatment
19 Conclusions SJW inhibits and kills mycobacteria and other G+ bacteria MBC range: 2.88 11.52 mg SJW/ml culture Depends on strain A new HPLC quantification method Hyperforin has a bactericidal effect on G+ bacteria at 6.5 ug/ml
20 Acknowledgements USU Department Huntsman Environmental Research Center TC Shen for SEM images Renaguli Gayiti for A549 cell images
21 Questions
22 References SJW antidepressant, anticancer, antiviral, and anti-inflammatory activity Barnes J, Anderson L, Phillipson J (2001) St. John s Wort (Hypericum perforatum L.): a review of its chemistry, pharmacology and clinical properties. Journal of Pharmacy and Pharmacology 53: 583-600. Gaster B, Holroyd J (2000) St. John s Wort for depression. Archives of Internal Medicine 160: 152-156. Lawvere S, Mahoney M (2005) St. John s Wort. American Family Physician 72: 2249-2254. Agostinis P, Vantieghem A, Merlevede W, Witte P (2002) Hypericin in cancer treatment: more light on the way. The International Journal of Biochemistry and Cell Biology 34: 221-241. Jacobson J, Feinman L, Liebes L, Ostrow N, Koslowski V, Tobia A, Cabana B, Lee D, Spritzler J, Prince A (2001) Pharmacokinetics, safety, and antiviral effects of hypericin, a derivitive of St. John s Wort plant, in patients with chronic hepatitis C virus infection. Antimicrobial Agents and Chemotherapy 45: 517-524. Tedeschi E, Menegazzi M, Margotto D, Suzuki H, Forstermann U, Kleinert H (2003) Anti-inflammatory actions of St. John s Wort: inhibition of human inducible nitric-oxide synthase epression by down-regulating signal transducer and activator of transcription-1α (STAT 1α) activation. The Journal of Pharmacology and Experimental Therapeutics 307: 254-263. SJW antimicrobial activity Reichling J, Weseler A, Saller R (2001) A current review of the antimicrobial activity of Hypericum Perforatum L. Pharmacopsychiatry 34: S116-S118. Avato P, Raffo F, Guglielmi G, Vitali C, Rosato A (2004) Extracts from St. John s Wort and their antimicrobial activity. Phytotherapy Research 18: 230-232 SJW HPLC analysis Poutaraud A, Lobstein A, Girardin P, Weniger B (2001) Improved procedure for the quality control of Hypericum perforatum L. Phytochemical Analysis 12: 355-362. Reyes G, Koda R (2001) Development of a simple, rapid and reproducible HPLC assay for the simultaneous determination of hypericins and stabilized hyperforin Ganzera M, Zhao H, Khan I (2001) Hypericum perforatum chemical profiling and quantitative results of St. John s Wort products by an improved high-performance liquid chromatography method Figures: MeOH: http://www.methanolsales.co.uk/; M. tuberculosis: embryology.med.unsw.edu.au; Human lungs: www.glogster.com; SJW plant: www.daveingram.ca; SJW bottle: www.gnc.com; SJW pills: http://www.naturallygreen.co.uk; Nebulizer: http://www.salterlabs.com.com; Topical cream: http://img2.timeinc.net