Jonathan Tolentino and Claudia Briones Biology Department, Skyline College, San Bruno CA

1. ANTIBACTERIAL ACTIVITY OF HERBAL EXTRACTS USED IN NATIVE AMERICAN TRADITIONAL MEDICINE ANTIBACTERIAL ACTIVITY OF HERBAL EXTRACTS USED IN NATIVE AMERICAN TRADITIONAL MEDICINE Jonathan Tolentino and Claudia Briones Biology Department, Skyline College, San Bruno CA • Results • None of the aqueous extracts inhibit the test bacteria. Methanolic and acetone extracts of T. officinale leaves and the acetone extract of C. pycnocephalus leaves inhibit both S. aureus and E. coli with zones of inhibition at 11 mm. Ethanolic extracts of R. californicus flowers and acetone extracts of D. sativus flowers inhibit E. coli (Table 2). • The MIC of the methanolic and acetone extracts of T. officinale leaves against both bacteria is 0.25 g/mL. The MIC of C. pycnocephalus leaf in acetone against S. aureus is 0.25 g/mL, and 0.5 g/mL against E. coli (Figure 1). • TheMBC of the acetone extract of D. sativus flower against S. aureus is 4.50 g/mL. MBC of the ethanolic extract of R. californicus flower against E. coli is 15 mg/mL. • Paper chromatographic segments of D. sativus flower extract and T. officinale leaf extract produced zones of inhibition against S. aureus (Figure 2). • One HPLC fraction from D. sativus flower acetone extract (Figure3) and another from T. officinale acetone leaf inhibited S. aureus. No other fractions showed inhibition from the two plants. • NMR data from acetone extracts of D. sativus flower and T. officinale leaf indicate one or more compounds in each extract. A second HPLC analysis of the compounds determined that they are fairly pure, but are still unidentified. Abstract Due to increasing numbers of bacteria strains resistant to antibiotics, there is a need for effective alternative sources of antimicrobial agents. Native American folklore recognizes various common plants for medicinal purposes, but their efficacy has not been tested. Several Native American plants were hypothesized to have antibacterial properties. Passiflora incarnata, Taraxacum officinale, Dipsacus sativus, Carduus pycnocephalus, Sonchus oleraceus, and Ranunculus californicus were evaluated for their antibacterial activity against gram-positive Staphylococcus aureus and gram-negative Escherichia coli bacteria. Aim Evaluate antimicrobial activity and identify active compoundsin plants traditionally used to treat infections • Background • Indigenous cultures throughout the world use herbal medicine to cure illness. • Many invasive plants are regarded as pests, but certain species are valued in Native American folklore for healing properties (2). • Such a source of alternative medicine can provide an abundant supply that is readily available, and potentially contribute to managing the health of the ecosystem. • Traditional uses of the medicinal plants we tested are shown in Table 1. Figure 1.Inhibitory concentrations of plant extracts against (a) S. aureus and (b) E. coli. • Discussion & Conclusions • The T. officinale leaf methanolic and acetone extracts of inhibited both gram-positive and gram-negative bacteria (MIC=0.25 g/mL). • The C. pycnocephalus leaf-acetone extract inhibited gram-positive S. aureus (MIC=0.25 g/mL) and gram-negative E. coli (MIC= 0.5 g/mL). • The D. sativus flower-acetone extract was bactericidal against gram-positive bacteria (MBC=4.38 g/mL). • The R. californicus flower-ethanolic extract was bactericidal against gram-negative bacteria (MBC=26 mg/mL). • Results confirm that these plants have antimicrobial properties. Exploring folkloric herbal plants may provide rewarding natural resources for medicine. Continued testing should determine whether these plants can be processed into potential drugs to control certain infectious diseases. Materials & Methods Plant extracts: 1. Plants (Table 1) were gathered and separated by flowers, stems, and leaves. 2. A food processor & mortar and pestle were used to grind plant parts to be mixed with solvents. 3. Methanolic, ethanolic, acetone, and aqueous extracts were made in concentrations at a minimum of 1 mL/g. Disk diffusion assay: Filter paper disks immersed in an extract or solvent (control) were applied to nutrient agar plates inoculated with the test bacteria: Staphylococcus aureus or Escherichia coli and incubated for 24 hr at 35°C. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC): 1. Microdilutions of plant extracts (500 mg/mL to 15 mg/mL) were made in nutrient broth, inoculated with the test bacteria: S. aureus or E. coli, and incubated for 24 hr at 35°C 2. Dilutions showing no growth were subcultured in nutrient broth to determine the MBC. Paper Chromatography Paper chromatography was performed on the extracts, which determined Rf values to help identify active compounds. Isopropanol was used as solvent, and sections of strips were assayed against test bacteria to locate antibacterial activity. High performance Liquid Chromatography HPLC separated the crude extract samples into fractions, which were concentrated using high pressure vacuum. Fractions were assayed by the disk-diffusion method. Nuclear Magnetic Resonance Spectrometry NMR data were gathered from active fractions to help determine chemical structure of the active compound. Mass Spectrometry Mass spectrometry was performed to determine elemental composition of active samples. a. Inhibition of S. aureus. b. Inhibition of E. coli. Figure 2. Paper chromatograms were cut to use in a disk diffusion assay. Rf =0.75 of the acetone extract of D.sativus flower inhibited growth of S. aureus (at arrow). Rf =0.05 of the acetone extract of dandelion leaf inhibited growth of S. aureus (not shown). Literature Cited 1. Barrett, S. A. and E. W. Gifford. 1933. Miwok Material Culture. Bulletin of the Public Museum of the City of Milwaukee 2(4):11. 2. Cowan, M. M. 1999. “Plant Products as Antimicrobial Agents.” Clinical Microbiology Review 12(4): 564–582. 3. Hamel, P. B. and M. U. Chiltoskey. 1975. Cherokee Plants and Their Uses—A 400 Year History. Sylva, N.C. Herald Publishing Co. 4. Herrick, J. W. 1977. Iroquois Medical Botany. State University of New York, Albany, Ph.D. Thesis. 5. Smith, H. H. 1932. Ethnobotany of the Ojibwe Indians. Bulletin of the Public Museum of Milwaukee 4:327-525. 6. Smith, H. I. 1929. Materia Medica of the Bella Coola and Neighboring Tribes of British Columbia. National Museum of Canada Bulletin 56:47-68. 7. Speck, F. G. 1941. “A List of Plant Curatives Obtained From the Houma Indians of Louisiana.” Primitive Man 14:49-75. Figure 3.One HPLC fraction from D. sativus flower acetone extract inhibited S. aureus. No other fractions produced inhibition. • Acknowledgements • Dr. Christine Case, Professor of Biology, Skyline College. • Tiffany Reardon, Assistant Director, California MESA. • Patricia Carter, Biology Technician, Skyline College. • Marc Anderson, Professor of Chemistry, San Francisco State University • Ulla Andersen, Chemistry Mass Spectrometry Facility, UC Berkeley • Funded by NIH/SFSU Bridges to the Baccalaureate.

2. Figure 6. Paper chromatograms were cut to use in a disk diffusion assay. (a) Rf =0.05 of the acetone extract of dandelion leaf inhibited growth of S. aureus. (a) Rf =0.75 of the acetone extract of Indian teasel flower inhibited growth of S. aureus. Dandelion leaf fraction A vs. S. aureus Indian teasel flower fraction AVs. S. aureus NMR data from acetone extracts of D. sativus flower and T. officinale leaf indicate one or more compounds in each extract. A second HPLC analysis of the compounds determined that they are fairly pure, but are still unidentified. Figure 2.Bactericidal concentrations of plant extracts.