Transcript Mohammed Abbas & Martin Cordero

Evaluating the Antibacterial
Properties of Ceanothus thyrsiflorus
Mohammed Abbas & Martin Cordero
Biology Department, Skyline College, San Bruno CA
Aim
To evaluate the antimicrobial properties of Ceanothus thyrsiflorus.
Background
There are many other types of antibiotics, each with unique mechanisms
that vary in their approach. However, due to the rapid reproduction of
bacteria and overuse and misuse of antibiotics, antibiotic resistant
pathogens have emerged as a major health concern (Mistcher). New
antimicrobics must be found.
Plants have historically been used as remedies for a variety of diseases.
Ceanothus spp. have been used by Native Americans to treat wounds and
gastrointestinal illnesses (Moerman, Hedges).
C. coeruleus flower extracts inhibit S. aureus (Salazar-Aranda),and C.
americanus seed extracts have been proven to be effective free radical
scavengers (Borchardt).
Results
1.8
Extract Preparation
•C. thyrsiflorus (Figure 1) was collected from Skyline College.
•Leaves were ground in 95% methanol (500 mg/mL) with a mortar and
pestle and centrifuged at 78 g for two minutes.
•The supernatant was tested for antibacterial activity.
• The methanolic Ceanothus leaf extract (500 mg/mL) did not inhibit
any gram-negative bacteria. Of the three gram-positive bacteria being
tested, only S. aureus was inhibited by the extract (Figures 2 & 3).
1.6
Well-diffusion Assay
• 4-mm holes were aseptically punched into nutrient agar plates using a
sterile cork borer.
• The plates were inoculated with:
• Escherichia coli (ATCC 11775)
• Salmonella enterica (ATCC 14028)
• Shigella sonnei (ATCC 9290)
• Staphylococcus aureus (ATCC 27659)
• Streptococcus pyogenes (ATCC 12228)
• Enterococcus faecalis (ATCC 19433).
• 20 µL methanolic Ceanothus leaf extract (500 mg/mL) was placed
into each well; 20 µL 95% methanol was pipetted into the control
well.
• The cultures were incubated at 35°C for 24 hours.
Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal
Concentration (MBC) Determination
• Serial dilutions of methanolic Ceanothus leaf extracts were made in a
well cell plate.
• Each well was inoculated with 200 µL S. aureus.
• The plate was incubated for 24 hours at 35°C.
• After incubation, the plates were observed for bacterial growth.
• Wells with no growth were subcultured on a nutrient agar plate to
determine the MBC.
Lysozyme Activity
•A 1:10 dilution of leaf extract (500 mg/mL) was made in lysozyme buffer
and inoculated with Micrococcus luteus in a spectrophotometer tube.
•The absorbance was recorded at 30-second intervals for five minutes.
•This was repeated replacing leaf extract with egg-white lysozyme
(control).
Gel Filtration
•A 500 mg/mL extract was filtered through a Sephadex (G-25) column.
•25-drop aliquots were collected across 20 test tubes. 200 µL of each
collection were used in a well-diffusion assay against S. aureus.
•The extracts were also centrifuged at 313 g, 704 g, and 1252 g and
gel filtrated in separate trials.
•Each collected filtered extract was tested for inhibition as well.
• The MIC was 125 mg/mL, and the MBC was 250 mg/mL. The extract
had no lysozyme activity (Figure 4). The active compound was not
separated by gel filtration.
• Gel filtration: No zones of inhibition were obtained.
1.2
1.0
Ceanothus
0.8
Egg white
lysozyme
0.6
0.4
0.2
0.0
30
230
430
630
830
1030
Time, seconds
Figure 4. No lysozyme activity was found in the methanolic
Ceanothus extract.
Discussion & Conclusion
Figure 2. The methanolic Ceanothus extract is 30%
as effective as penicillin against S. aureus.
C. thyrsiflorus leaf extract was tested against three gram-positive bacteria
and three gram-negative bacteria. The methanolic leaf extract inhibited S.
aureus but did not inhibit S. pyogenes or E. faecalis. C. thyrsiflorus is a
possible source of an antistaphylococcal agent, however further research
is required for confirmation. Paper chromatography with an appropriate
solvent or high performance liquid chromatography may isolate the active
compound.
16
14
Literature Cited
12
Borchardt, J. R., et al. 2009. ”Antioxidant and Antimicrobial Activity of
Seed from Plants of the Mississippi River Basin.” Journal of
Medicinal Plants Research 3(10): 707-718.
Hedges, Ken. 1986. Santa Ysabel Ethnobotany. San Diego Museum of
Man Ethnic Technology Notes, page 15.
Mitscher, Lester A. 2008. “Coevolution: Mankind and Microbes.“ The
American Chemical Society and American Society of Pharmacognosy
71(3): 497-509.
Moerman, D. (ed). “Native American Ethnobotany.” University of
Michigan <http://herb.umd.umich.edu/>
Salazar-Aranda, R., et al. 2009. “Antimicrobial and Antioxidant Activities
of Plants from Northeast of Mexico.“ Evidence-based Complementary
and Alternative Medicine (epub doi 10.1093/).
10
8
6
4
2
0
Ceanothus
S. aureus
Methanol
Penicillin (10 µg)
E. coli
Figure 3. Methanolic Ceanothus extract (500 mg/mL)
inhibited gram-positive S. aureus. Error bars: 1 S. D.
Figure 1.
Ceanothus
thyrsiflorus, a
plant native to
the Pacific
Coast, is in the
Rhamnaceae.
1.4
Absorbance, 540 nm
Antibiotics have been widely used to treat bacterial infections since the
1940’s. However their over-use has caused selection of antibiotic resistant
pathogens. Indigenous North American people traditionally used
Ceanothus thyrsiflorus to treat urinary, intestinal, and respiratory
infections. Our purpose was to determine whether C. thyrsiflorus has
antibacterial activity. Methanolic leaf extracts (500 mg/mL) were
prepared. Well-diffusion assays were performed to test the extract against
three gram-positive (Staphylococcus aureus, Streptococcus pyogenes,
Enterococcus faecalis) bacteria and three gram-negative (Escherichia coli,
Salmonella enterica, Shigella sonnei). The methanolic Ceanothus extract
inhibited S. aureus, with a 3-mm zone of inhibition. C. thyrsiflorus did not
inhibit the gram-negative bacteria. Serial dilutions were used to determine
the minimal inhibitory concentration (125 mg/mL) and the minimal
bactericidal concentration (250 mg/mL). There is no lysozyme activity in
the extract. The antibacterial fraction was unable to be isolated by
Sephadex gel filtration. Further investigation of the active compound is
warranted for confirmation.
Materials & Methods
Averagezoneof inhibition, mm
Abstract
Acknowledgements
First and foremost, we would like to thank our mentor Dr. Christine Case
for donating her time, energy, and intellect to ensure a first-class learning
environment, providing us and our peers with invaluable laboratory
experience and biological knowledge. Patricia Carter and Ruth Arce, our
lab technicians, for their help and countless hours spent in lab readily
available to us, and Stephen Fredricks for handling the logistics between
Skyline College and SACNAS. Lastly, we thank SACNAS for giving
Skyline College the opportunity to participate in a national scientific
conference.