References

1. Antimicrobial Activity of Baccharis pilularis (Asteraceae), A Traditional Native American Herbal Medicine Bardo Castro, Pamela Rios, and Christine Case Biology Department, Skyline College, San Bruno CA Abstract Over the past 60 years, overuse of antibiotics has selected antibiotic-resistant strains of many bacterial pathogens. The emergence of these resistant pathogens demands new antibacterial compounds to deal with this crisis. The plants used for centuries by traditional healers are logical places to search for new antibacterial agents due to their proven ability to treat infections. Baccharis pilulars, coyote brush, is one such plant. It has been used to treat skin wounds and gastrointestinal ailments in Latin American and Native American traditional medicine. We have demonstrated that ethanolic, methanolic, and acetonic extracts of B. pilularis leaves and bark kill Staphylococcus aureus. We determined the minimal inhibitory concentrations of the extracts: methanolic extract (10.4 mg/mL), ethanolic extract (20.9 mg/mL), acetonic extract (0.33 mg/mL). The purpose of this study is to isolate and characterize the antibacterial compound in the extracts. We are testing the antibacterial activity of compounds separated by chromatography. These findings may lead to development of plant-based, affordable antimicrobials to which bacteria are not resistant. • Methods • Baccharis pilularis (Figure 1) leaves were collected from off-road, natural sites. The plants had not been exposed to herbicides or highway emissions. • Extract Preparation • Fresh B. pilularis leaves and bark were ground in distilled water, 70% methanol, 80% ethanol, or 95% acetone to a final concentration of 250 mg/mL. • Extracts were prepared by constant stirring for 48-72 hr at 23°C. • Extracts were filtered using cheese cloth and supernatant was collected. • Agar Diffusion Assay • Nutrient agar plates were aseptically inoculated with Escherichia coli (ATCC 11775), Staphylococcus aureus (ATCC 27659), Saccharomyces cervesiae (ATCC 9763), and Aspergillus niger (ATCC 16404). • Sterile 10-mm filter disks were saturated with extracts and placed on inoculated plates. • The solvents alone were used as controls. • Plates were incubated at 35°C for 24 hr. • Minimal inhibitory concentration (MIC) • Serial dilutions of methanolic, ethanolic, and acetonic extracts (0.16–333 mg/mL) were prepared in nutrient broth in cell well plates. • Each dilution was inoculated with 100 µL of S. aureus and incubated at 35°C for 24 hr. • Minimal bactericidal concentration (MBC) • 100 µL from the MIC wells showing no growth was transferred to nutrient broth and incubated at 35°C for 24 hr. • Paper Chromatography • Methanolic and ethanolic extracts were separated by paper chromatography in petroleum ether and the acetonic extract was separated in 95% isopropanol. • Cut pieces from the chromatogram were used in a disk diffusion assay against S. aureus. • Column Chromatography • Extracts were separated through Sephadex G-25 beads. • The same solvents used in the extracts were used as elution buffer. Three separated aliquots were used in a disk-diffusion assay against S. aureus. • Discussion & Conclusion • The antistaphylococcal compound of both bark and leaf is not soluble in water however it is soluble in methanol, ethanol, and acetone. • The antistaphylococcal compound is more abundant in the leaves. • The MIC for the acetonic leaf extracts was lowest (0.33 mg/mL). • The MBC for the acetonic leaf extracts was lowest (20.84 mg/mL). • The active compound for the leaf extracts were separated by both paper chromatography and column chromatography. • It was not possible to separate the bark extracts through paper chromatography. This is most likely because of the low concentration of antibacterial agent in the bark. Figure 2. Disk diffusion assay of leaf extracts against S. aureus. References Andrade, N. S. et al. “Lack of clastogenic/genotoxic effect of Baccharis dracunculifolia extract on Swiss mouse peripheral blood cells.” Genetics and Molecular Research 7(4):1414-1421, 2008. Betoni , J. E. et al. “Synergism between plant extract and antimicrobial drugs used on Staphylococcus aureus diseases.” Memórias do Instituto Oswaldo Cruz 101(4):387-390, 2006. Bocek, B. R. “Ethnobotany of Costanoan Indians, California, Based on Collections by John P. Harrington.” Economic Botany 38(2):240-255, 1984. Guo, Y. et al. “Clerodane diterpenoids and flavonoids with NGF-potentiating activity from the aerial parts of Baccharis guadichaidiana.” Chemical and Pharmaceutical Bulletin 55(10):1532-1534, 2007. Pimenta da Silva, D. et al. “Comparative evolution of in-vitro effects of Brazilian green propolis and Baccharis dracunculifolia extracts on cariogenic factors of Streptococcus mutants.”Biological and Pharmaceutical Bulletin 27(11):1834-1839, 2004. University of Michigan. Native American Ethnobotany Database. <http://herb.umd.umich.edu/> Hypothesis The purpose of this study is to investigate the antibacterial activity of B. pilularis (Figure1). Figure1:Baccharis pilularis, coyote brush.This Asteraceae plant is the indicator species for the coastal scrub community in northern California. Figure 3: MICs and MBCs of leaf extracts against S. aureus. • Results • B. pilularis leaf and bark extracts inhibit gram-positive S. aureus bacteria (Figure 2). The leaf extracts are bactericidal (Figure 3). • B. pilularis leaf does not inhibit gram-negative E. coli, the yeast, or the mold. • Methanolic, ethanolic, and acetonic extracts inhibited S.aureus (Table 1). • The antistaphylococcal compound was isolated by paper chromatography (Figure 4) and by column chromatography. • Future Work • The active compound may provide an alternative treatment for antibiotic-resistant S. aureus. • The active compound needs to be purified and characterized. • Background • There is a growing need for new antimicrobials to combat bacterial infections. • Plants in the Baccharis genus are widely used in the Americas for their medicinal properties. • B. dracunculifolia is used as an antipyretic and a stomach remedy in Brazil. This plant has cytotoxic activity against leukemia cells (1). • B. trimer inhibits a variety of bacteria (2) • B.dracunculifolia inhibits Streptococcus mutansin vitro (5). • In Paraguay, B. guaduchaudiana is commonly used as a folk remedy for the treatment of gastrointestinal disease (4). • Native Americans used a decoction of B. douglasii to wash and disinfect wounds (3). • The Costanoan people, indigenous to the San Francisco Bay Area, used a B. pilularis infusion as a general remedy (6). Acknowledgements Pat Carter, Biology Lab Technician, Skyline College Ruth Arce, Biology Stockroom Assistant, Skyline College Stephen Fredricks, MESA Director, Skyline College Tiffany Reardon, Assistant Director, California MESA Colleagues from the Skyline SACNAS Chapter Figure 4: Active Compound Isolation. Pieces of the paper chromatogram (Rf values 0.83 and 0.94) of the methanolic extract inhibited S. aureus.