AANNTTIBIBAACCTTEERRIAIALL AACCTTIVIVITITYY OOFF HHEERRBBAALL EEXXTTRRAACCTTSS UUSSEEDD ININ NNAATTIVIVEE AAMMEERRICICAANN TTRRAADDITITIOIONNAALL MMEEDDICICININEE Jonathan Tolentino and Claudia Briones Biology Department, Skyline College, San Bruno CA Results Abstract Table 1. Native American Uses of Plants Tested Table 2. Zones of inhibition against S. aureus and E. coli by plant extract. Data in mm. Due to increasing numbers of bacteria strains resistant to antibiotics, there • None of the aqueous extracts inhibit the test bacteria. Methanolic and Against S. aureus Against E. coli is a need for effective alternative sources of antimicrobial agents. Native acetone extracts of T. officinale leaves and the acetone extract of C. Passiflora incarnata American folklore recognizes various common plants for medicinal Dandelion MeOH EtOH Ace H2O Dandelion MeOH EtOH Ace H2O pycnocephalus leaves inhibit both S. aureus and E. coli with zones of inhibition at 11 mm. Ethanolic extracts of R. californicus flowers and purposes, but their efficacy has not been tested. Several Native American flower 0 0 0 0 flower 0 0 0 0 plants were hypothesized to have antibacterial properties. Passiflora Passionflower acetone extracts of D. sativus flowers inhibit E. coli (Table 2). Cherokee incarnata, Taraxacum officinale, Dipsacus sativus, Carduus leaf 11 0 11 0 leaf 11 0 11 0 • The MIC of the methanolic and acetone extracts of T. officinale leaves Wounds pycnocephalus, Sonchus oleraceus, and Ranunculus californicus were stem 0 0 0 0 stem 0 0 0 0 against both bacteria is 0.25 g/mL. The MIC of C. pycnocephalus leaf evaluated for their antibacterial activity against gram-positive Ref: 3 in acetone against S. aureus is 0.25 g/mL, and 0.5 g/mL against E. coli Plumeless Plumeless Staphylococcus aureus and gram-negative Escherichia coli bacteria. (Figure 1). thistle MeOH EtOH Ace H2O thistle MeOH EtOH Ace H2O • The MBC of the acetone extract of D. sativus flower against S. aureus flower 0 0 0 0 flower 0 0 0 0 is 4.50 g/mL. MBC of the ethanolic extract of R. californicus flower Aim leaf 0 0 11 0 leaf 0 0 11 0 against E. coli is 15 mg/mL. Evaluate antimicrobial activity and identify active compounds in plants Taraxacum officinale stem 0 0 0 0 stem 0 0 0 0 Paper chromatographic segments of D. sativus flower extract and T. traditionally used to treat infections • Indian California officinale leaf extract produced zones of inhibition against S. aureus Common Dandelion teasel MeOH EtOH Ace H O buttercup MeOH EtOH Ace H O (Figure 2). Bella Coola 2 2 Background Stomachache flower 0 0 11 0 flower 0 13 0 0 • One HPLC fraction from D. sativus flower acetone extract (Figure 3) and another from T. officinale acetone leaf inhibited S. aureus. No other Ref: 6 • Indigenous cultures throughout the world use herbal medicine to cure leaf 0 0 0 0 leaf 0 0 0 0 fractions showed inhibition from the two plants. illness. stem 0 0 0 0 stem 0 0 0 0 • Many invasive plants are regarded as pests, but certain species are • NMR data from acetone extracts of D. sativus flower and T. officinale valued in Native American folklore for healing properties (2). leaf indicate one or more compounds in each extract. A second HPLC • Such a source of alternative medicine can provide an abundant supply analysis of the compounds determined that they are fairly pure, but are that is readily available, and potentially contribute to managing the Dipsacus sativus still unidentified. health of the ecosystem. • Traditional uses of the medicinal plants we tested are shown in Table 1. Indian Teasel Iroquois Discussion & Conclusions Acne Materials & Methods Ref: 4 • The T. officinale leaf methanolic and acetone extracts of inhibited both gram-positive and gram-negative bacteria (MIC=0.25 g/mL). Plant extracts: • The C. pycnocephalus leaf-acetone extract inhibited gram-positive S. 1. Plants (Table 1) were gathered and separated by flowers, stems, and aureus (MIC=0.25 g/mL) and gram-negative E. coli (MIC= 0.5 g/mL). leaves. 2. A food processor & mortar and pestle were used to grind plant parts to • The D. sativus flower-acetone extract was bactericidal against gram- be mixed with solvents. C. pycnocephalus positive bacteria (MBC=4.38 g/mL). 3. Methanolic, ethanolic, acetone, and aqueous extracts were made in • The R. californicus flower-ethanolic extract was bactericidal against concentrations at a minimum of 1 mL/g. Plumeless Thistle gram-negative bacteria (MBC=26 mg/mL). Disk diffusion assay: Ojibwa a. Inhibition of S. aureus. b. Inhibition of E. coli. • Results confirm that these plants have antimicrobial properties. Filter paper disks immersed in an extract or solvent (control) were Bowel tonic Figure 1. Inhibitory concentrations of plant extracts against (a) S. aureus and (b) E. coli. Exploring folkloric herbal plants may provide rewarding natural applied to nutrient agar plates inoculated with the test bacteria: Ref: 5 resources for medicine. Continued testing should determine whether Staphylococcus aureus or Escherichia coli and incubated for 24 hr at these plants can be processed into potential drugs to control certain 35°C. Figure 2. Paper chromatograms infectious diseases. Minimal Inhibitory Concentration (MIC) and were cut to use in a disk Minimal Bactericidal Concentration (MBC): diffusion assay. Rf =0.75 of the 1. Microdilutions of plant extracts (500 mg/mL to 15 mg/mL) were made acetone extract of D. sativus Literature Cited Sonchus oleraceus in nutrient broth, inoculated with the test bacteria: S. aureus or E. coli, flower inhibited growth of S. 1. Barrett, S. A. and E. W. Gifford. 1933. Miwok Material Culture. Bulletin of the Public and incubated for 24 hr at 35°C aureus (at arrow). Rf =0.05 of Museum of the City of Milwaukee 2(4):11. 2. Dilutions showing no growth were subcultured in nutrient broth to Common Sowthistle the acetone extract of dandelion 2. Cowan, M. M. 1999. “Plant Products as Antimicrobial Agents.” Clinical Microbiology Review 12(4): 564–582. determine the MBC. Houma leaf inhibited growth of S. 3. Hamel, P. B. and M. U. Chiltoskey. 1975. Cherokee Plants and Their Uses—A 400 Year aureus (not shown). Paper Chromatography Anti-diarrheal History. Sylva, N.C. Herald Publishing Co. 4. Herrick, J. W. 1977. Iroquois Medical Botany. State University of New York, Albany, Ph.D. Paper chromatography was performed on the extracts, which Ref: 7 Thesis. determined Rf values to help identify active compounds. Isopropanol 5. Smith, H. H. 1932. Ethnobotany of the Ojibwe Indians. Bulletin of the Public Museum of was used as solvent, and sections of strips were assayed against test Milwaukee 4:327-525. 6. Smith, H. I. 1929. Materia Medica of the Bella Coola and Neighboring Tribes of British bacteria to locate antibacterial activity. Columbia. National Museum of Canada Bulletin 56:47-68. Figure 3. One HPLC High performance Liquid Chromatography 7. Speck, F. G. 1941. “A List of Plant Curatives Obtained From the Houma Indians of fraction from D. sativus Louisiana.” Primitive Man 14:49-75. HPLC separated the crude extract samples into fractions, which were R. californicus flower acetone extract concentrated using high pressure vacuum. Fractions were assayed by inhibited S. aureus. No the disk-diffusion method. California Buttercup other fractions produced Acknowledgements Nuclear Magnetic Resonance Spectrometry Miwok inhibition. NMR data were gathered from active fractions to help determine • Dr. Christine Case, Professor of Biology, Skyline College. chemical structure of the active compound. Food Supplement • Tiffany Reardon, Assistant Director, California MESA. Ref: 1 • Patricia Carter, Biology Technician, Skyline College. Mass Spectrometry • Marc Anderson, Professor of Chemistry, San Francisco State University Mass spectrometry was performed to determine elemental • Ulla Andersen, Chemistry Mass Spectrometry Facility, UC Berkeley composition of active samples. • Funded by NIH/SFSU Bridges to the Baccalaureate.