Transcript B. pilularis

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
Methods
Over the past 60 years, overuse of antibiotics has selected antibioticresistant 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.
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.
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 2. Disk
diffusion assay
of leaf extracts
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.
References
45
41.73 41.73
40
Baccharis extract (mg/mL)
Abstract
35
MIC
30
MBC
25
20.85 20.85
20
15
10.42
10
5
0
0.33
Acetone
70% Ethanol
95% Methanol
Figure 3: MICs and MBCs of leaf extracts against S. aureus.
1. 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.
2. 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.
3. Bocek, B. R. “Ethnobotany of Costanoan Indians, California, Based on
Collections by John P. Harrington.” Economic Botany 38(2):240-255,
1984.
4. Guo, Y. et al. “Clerodane diterpenoids and flavonoids with NGFpotentiating activity from the aerial parts of Baccharis
guadichaidiana.” Chemical and Pharmaceutical Bulletin 55(10):15321534, 2007.
5. 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.
6. University of Michigan. Native American Ethnobotany Database.
<http://herb.umd.umich.edu/>
Results
•
•
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 mutans in 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).
•
•
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.
Table 1. Minimal bactericidal concentrations against S.aureus
Extract
Acetonic leaf
Ethanolic leaf
Methanolic leaf
Acetonic bark
Ethanolic bark
Methanolic bark
MIC (mg/mL)
0.33
20.85
41.73
83.36
41.68
41.68
MBC (mg/mL)
10.43
20.85
41.73
166.77
83.38
83.38
Acknowledgements
Figure 4: Active Compound Isolation. Pieces of the paper
chromatogram (Rf values 0.83 and 0.94) of the methanolic extract
inhibited S. aureus.
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