Transcript M. phlei

ANTIBACTERIAL ACTIVITY OF SAMBUCUS MEXICANA
(ELDERBERRY), A NATIVE AMERICAN MEDICINAL PLANT
Rebecca Belloso and Christabel Causapin
Abstract
Historically, Native Americans used Sambucus mexicana (elderberry)
flowers and berries to treat urinary tract infections. Now, elderberry
flower tea is available commercially as a health elixir. The antioxidant and
antiviral effects of the berries have been demonstrated. The objective of
this study was to isolate and characterize the antibacterial activity in fresh
S. mexicana flowers and berries. Alcoholic and acetonic extracts of
various plant parts do not inhibit gram-negative Escherichia coli bacteria.
The extracts inhibit gram-positive Staphylococcus aureus and
Mycobacterium phlei bacteria in disk-diffusion assays. The minimum
bactericidal concentrations (berries, 125 mg/mL; flowers, 250 mg/mL)
were determined using microdilutions. The bactericidal compound is
being isolated and characterized. The results do not explain the traditional
use of Sambucus but may lead to a novel antibacterial compound useful
against antibiotic-resistant S. aureus and other gram-positive bacteria.
Hypothesis
Elderberries and elderflowers kill or inhibit bacteria.
Background
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Sambucus flowers and berries (Figure 1) have been used in traditional
medicine.
European herbalists used elderberry as a pain reliever and Native
American herbalists used elderberry against coughs, infections and
skin conditions (1).
Elderflowers and elderberries contain flavonoids (2), which are
synthesized by plants in response to infection and may have
antibacterial properties (3).
An extract of elderberry leaves combined with soapwort roots inhibits
herpes simplex virus and influenza virus in vitro (4).
An extract of black elderberries (S. nigra), Sambucol, is sold
commercially as a viral and cancer treatment. Test subjects who used
Sambucol claim to have recovered from influenza faster than those
using a placebo as the control (5).
Elderflowers have shown anti-inflammatory properties in animal
studies (6).
Ingestion of root, leaf, stem and unripe berries may cause nausea,
diarrhea or vomiting (7).
Medicinal plants are being investigated as an alternate treatment for
tuberculosis since the emergence of antibiotic-resistant strains (8).
S. mexicana berries and flowers inhibit growth of gram-positive
bacteria (9).
The purpose of this study is to characterize the antibacterial activity of
S. mexicana flowers and S. nigra berries.
Biology Department, Skyline College, San Bruno CA
Materials and Methods
Results
Plants
Fresh S. mexicana flowers were collected from Sweeney Ridge in San
Bruno, CA. Commercial S. nigra dried berries purchased from Lhasa
Karnak Herb (Berkeley, CA).
Preparation of extracts
• Flowers or berries were ground with in a mortar.
• Extracts were centrifuged at 3360 g for 4 min. The supernatant was
used for disk diffusion assays.
• The flower extracts were 1.0 g/mL in acetone. The berry extracts were
1.0 g/mL in acetone, 95% methanol and 95% ethanol.
Disk diffusion assay
• Nutrient agar plates were inoculated with Staphylococcus aureus
(ATCC 27659), Mycobacterium phlei (Wards 85W 1691) and
Escherichia coli (ATCC 11775) bacteria.
• Sterile 10-mm filter paper disks saturated with plant extracts or
solvents were placed on the cultures.
• Cultures were incubated at 35°C for 24-48 hrs.
Minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) determination
• Serial dilutions (500-31.25 mg/mL) were made in nutrient broth and
inoculated with S. aureus and M. phlei in cell well plates.
• Cell-well plates were incubated at 35°C for 24-48 hrs.
• Wells with no growth were subcultured in nutrient broth to determine
the MBC.
Isolation of active compound
• Paper chromatography was used to separate extracts using petroleum
ether as the chromatography solvent.
• Rf values were measured.
• Pieces cut from the paper strips were used in a disk diffusion assay
against S. aureus and M. phlei.
Characterization of active compound
• S. aureus and M. phlei were incubated at 35°C for 1 hr with
• Unheated plant extract
• Heated plant extract (56C for 30 minutes)
• 0.9% saline (control)
• Plate counts were performed to determine the number of surviving
bacteria.
• None of these extracts inhibited gram-negative E. coli bacteria (Table
1). This agrees with previous work (9).
• Alcoholic and acetone extracts inhibited gram-positive S. aureus and M.
phlei bacteria. Elderberries have not previously been tested against
mycobacteria.
• The acetone-flower extract worked best against M. phlei (Figure 2) in
the disk diffusion assays, with an average zone of inhibition of 15.25
mm (Figure 3).
• The acetone-berry extract is bactericidal (Table 2).
• The MIC of the acetone-berry extract against S. aureus is 250
mg/mL and against M. phlei is 125 mg/mL. The MIC of the
acetone-flower extracts against S. aureus and M. phlei is 250
mg/mL.
• The MBC of the acetone-berry extracts is 125 mg/mL against S.
aureus and 250 mg/mL against M. phlei. The MBC of the acetoneflower extracts is 250 mg/mL against both S. aureus and M. phlei.
• MIC and MBC alcohol extracts were equal to or less than the acetone
extracts.
• The bactericidal S. aureus compound was separated from the acetoneberry extract by paper chromatography in petroleum ether (Figure 4).
• Unheated acetone-flower extract killed all (106) S. aureus cells; 105 cells
grew after treatment with heated extract and 106 grew after no treatment
(control).
Table 1. Disk diffusion assay
Extract
Flower Acetone
Berry Acetone
Berry Ethanol
Berry Methanol
Zone of inhibition (mm)
S. aureus
M. phlei
E. coli
14.13
15.30
0
10.50
13.00
0
10.50
11.50
0
10.50
0
0
Table 2. Bactericidal and bacteriostatic concentrations
Extract
Flower Acetone
Berry Acetone
Berry Ethanol
Berry Methanol
MBC (mg/mL)
S. aureus
M. phlei
1000
250
125
250
1000
500
1000
500
MIC (mg/mL)
S. aureus
M. phlei
625
250
250
125
250
250
250
250
Figure 4. Rf values and zones of inhibition from acetone-berry
extract in paper chromatography against S. aureus.
Discussion and Conclusions
• Alcohol extracts showed minimal inhibition of S.aureus in disk
diffusion assays (9).
• The MIC and MBC of the acetone extracts against S. aureus is 125 to
250 mg/mL (9).
• The elderberry plant kills gram-positive bacteria, including
mycobacteria. This may provide an alternative antibiotic to treat
methicillin-resistant S. aureus and multidrug-resistant M. tuberculosis.
• The bactericidal compound is soluble in alcohols and acetone.
• The results indicate that the bactericidal compound in the flowers is a
protein.
• The bactericidal compound in the acetone-berry extract was isolated in
a paper chromatography fraction.
• Mass spectroscopy of HPLC fractions may identify the active
compound.
Literature Cited
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5.
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8.
9.
Figure 1. Sambucus
mexicana, blue
elderberry. This shrub, in the
Adoxaceae, is native to the
California coastal scrub and can
be found in other parts of North
America. Inset: Berries ripen in
the summer.
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Acknowledgements
Figure 2. The acetone-flower extract inhibited M. phlei in a
disk diffusion assay.
Figure 3. Average zone of inhibition of all extracts. Flower extract is
out of four trials. Berry extracts are out of two trials. Error bars=1 S.D.
Christine Case, Biology Professor, Skyline College
Pat Carter, Biology Lab Technician, Skyline College
Tiffany Reardon, Assistant Director, California MESA
Stephen Fredricks, Director, Skyline MESA
Bardo Castro, Colleague, Skyline College
Pamela Rios, Colleague, Skyline College