Transcript Scientific Poster

ANTIBACTERIAL ACTIVITY OF HERBAL EXTRACTS
USED IN NATIVE AMERICAN TRADITIONAL MEDICINE
Jonathan Tolentino and Claudia Briones
Biology Department, Skyline College, San Bruno CA
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.
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.
Against S. aureus
Passiflora incarnata
Passionflower
Cherokee
Wounds
Ref: 3
Dandelion
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.
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.
Taraxacum officinale
Common Dandelion
Bella Coola
Stomachache
Ref: 6
Dipsacus sativus
Indian Teasel
Iroquois
Acne
Ref: 4
C. pycnocephalus
EtOH
Ace
H2O
flower
0
0
0
0
leaf
11
0
11
stem
0
0
0
Sonchus oleraceus
Common Sowthistle
Houma
Anti-diarrheal
Ref: 7
R. californicus
California Buttercup
Miwok
Food Supplement
Ref: 1
Dandelion
MeOH
EtOH
Ace
H2O
flower
0
0
0
0
0
leaf
11
0
11
0
0
stem
0
0
0
0
MeOH
EtOH
Ace
H2O
Plumeless
thistle
MeOH
EtOH
Ace
H2O
flower
0
0
0
0
flower
0
0
0
0
leaf
0
0
11
0
leaf
0
0
11
0
stem
0
0
0
0
stem
0
0
0
0
MeOH
EtOH
Ace
Indian
teasel
California
buttercup
MeOH
EtOH
Ace
H2O
flower
0
0
11
0
flower
0
13
0
0
leaf
0
0
0
0
leaf
0
0
0
0
stem
0
0
0
0
stem
0
0
0
0
D. sativus
flower in
Acetone
R. californicus
flower in EOH
C. pycnoc.
leaf in
Acetone
C. pycnoc.
leaf in
Acetone
T. officinale
leaf in
Acetone
T. officinale
leaf in
Acetone
T. officinale
leaf in MeOH
T. officinale
leaf in MeOH
0.0
Plumeless Thistle
Ojibwa
Bowel tonic
Ref: 5
Against E. coli
MeOH
Plumeless
thistle
Aim
Evaluate antimicrobial activity and identify active compounds in plants
traditionally used to treat infections
Results
0.5
1.0
1.5
2.0
2.5
H2O
• 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).
• The MBC 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 (Figure 3)
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.
Discussion & Conclusions
0.0
0.2
0.4
0.6
MIC (g/ml)
a. Inhibition of S. aureus.
• 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).
b. Inhibition of E. coli.
Figure 1. Inhibitory concentrations of plant extracts against (a) S. aureus and (b) 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).
Figure 3. One HPLC
fraction from D. sativus
flower acetone extract
inhibited S. aureus. No
other fractions produced
inhibition.
• 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 grampositive 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.
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.
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.
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 A Vs. S. aureus
5.0
4.5
4.0
MBC (g/ml)
3.5
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.
3.0
2.5
2.0
1.5
1.0
0.5
0.0
D. sativus flower in
acetone
R. californicus
flower in EOH
Figure 2. Bactericidal concentrations of
plant extracts.