Rebecca Belloso and Christabel CausapinBiology Department, Skyline College, San Bruno CA

Rebecca Belloso and Christabel CausapinBiology Department, Skyline College, San Bruno CA

AcknowledgementsChristine Case, Biology Professor, Skyline CollegePat Carter, Biology Lab Technician, Skyline CollegeTiffany Reardon, Assistant Director, California MESAStephen Fredricks, Director, Skyline MESABardo Castro, Colleague, Skyline CollegePamela Rios, Colleague, Skyline College

Literature Cited1. Duke, J. A. 1985. CRC Handbook of Medicinal Herbs. Boca Raton, FL: CRC Press.

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4. Serkedjieva, J, N. Manolova, I. Zgórniak-Nowosielska et al. 1990. “Antiviral activity of the infusion (SHS-174) from flowers of Sambucus nigra L., aerial parts of Hypericum perforatum L., and roots of Saponaria officinalis L. against influenza and herpes simplex viruses.” Phytother. Res. 4:97-100.

5. Zakay-Rones, Z. N. Varsano, M. Zlotnik et al. 1995. “Inhibition of several strains of influenza virus in vitro and reduction of symptoms by an elderberry extract (Sambucus nigra L.) during an outbreak of influenza B Panama.” J. Alt. Compl. Med. 1:361-9.

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7. Newall, C., L. Anderson, and J. Phillipson. 1996. Herbal Medicines: A Guide for Health-Care Professionals. London: The Pharmaceutical Press.

8. Jimenez-Arellanes, A., M, Meckes, R. Ramirez, J. Torres, and J. Luna-Herrera. 2003. “Activity against multidrug-resistant Mycobacterium tuberculosis in Mexican plants used to treat respiratory diseases.” Phytother Res. 17:903-8.

9. Belloso, R. 2009. “Ethnobotany: The Antimicrobial Properties of Sambucus mexicana (Elderberry), A Native American Medicinal Plant.” SACNAS National Conference Abstracts 2009: 113.

Background• 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.

AbstractHistorically, 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.

Materials and MethodsPlantsFresh 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 bactericidalconcentration (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 (56C for 30 minutes)• 0.9% saline (control)

• Plate counts were performed to determine the number of surviving bacteria.

Results• 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 acetone-flower 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 acetone-berry 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).

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.

HypothesisElderberries and elderflowers kill or inhibit bacteria.

ANTIBACTERIAL ACTIVITY OF SAMBUCUS MEXICANA(ELDERBERRY), A NATIVE AMERICAN MEDICINAL PLANT

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.

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.

Figure 4. Rf values and zones of inhibition from acetone-berry extract in paper chromatography against S. aureus.

Table 1. Disk diffusion assay Zone of inhibition (mm)

Extract S. aureus M. phlei E. coli Flower Acetone 14.13 15.30 0 Berry Acetone 10.50 13.00 0 Berry Ethanol 10.50 11.50 0 Berry Methanol 10.50 0 0

Table 2. Bactericidal and bacteriostatic concentrations MBC (mg/mL) MIC (mg/mL)

Extract S. aureus M. phlei S. aureus M. phlei Flower Acetone 1000 250 625 250 Berry Acetone 125 250 250 125 Berry Ethanol 1000 500 250 250 Berry Methanol 1000 500 250 250

Figure 2. The acetone-flower extract inhibited M. phlei in a disk diffusion assay.