Transcript S. aureus

Evaluation of the Antibacterial Activity of
the Traditional Medicinal Plant, Astragalus membranaceus
Katrina Cheung
Biology Department, Skyline College, San Bruno CA
Abstract
Methods
Results
Astragalus membranaceus (Fabaceae) is commonly used in Chinese medicine to treat a
wide variety of infections. However, there is a lack of information on the effectiveness of
A. membranaceus against microorganisms. The purpose of this research is to determine
whether A. membranaceus inhibits bacterial growth in vitro. Methanolic and ethanolic
extracts were prepared using dried root from Mt. Hengshan, Shanxi Province (China). The
extracts were heated to boiling, dried by evaporation, and resuspended in sterile distilled
water. The 167-mg/mL extracts were tested in well diffusion assays against gram-negative
Escherichia coli and gram-positive Staphylococcus aureus and Mycobacterium phlei
bacteria. Both alcoholic extracts inhibited bacterial growth. The inhibitory effect of
methanolic Astragalus extract against gram-positive organism is comparable to
streptomycin (10 µg disk). The ethanolic extract is 50% more effective at inhibiting E. coli
than penicillin (10 µg disk). Microdilutions were performed to determine the minimal
inhibitory concentrations and minimal bactericidal concentrations of the methanolic
Astragalus extract against E.coli (150.00-mg/mL, 166.70-mg/mL), S. aureus (83.33mg/mL, 166.700mg/mL), and M. phlei (16.67-mg/mL, 33.33-mg/mL). The minimal
inhibitory concentrations and minimal bactericidal concentrations of the ethanolic
Astragalus extract against E.coli (100.00-mg/mL, 116.67-mg/mL), S. aureus (50-mg/mL,
100-mg/mL) and M. phlei (16.67-mg/mL, 33.33-mg/mL) were determined using the same
technique. Methanolic Astragalus extract inhibited the growth of E. coli and S. aureus in
cottage cheese. The use of Astragalus membranaceus as a disinfectant or sanitizer will be
discussed.
Extract Preparation
1. Dried root of Astragalus membranaceus was obtained from Mt.
Hengshan, Shanxi province (China).
2. Root was ground into fine pieces using scissors, mortar, and pestle.
3. Ground root was mixed with alcohol (95% methanol or 95% ethanol), to
a final concentration of 143 mg/mL.
4. Mixture was heated to boiling (65°C or 78°C) for 60 minutes, to a final
concentration of 166.67 mg/mL.
5. Root extracts were filtered through two layers of cheesecloth and then
dried by evaporation.
1. Alcoholic extracts of A. membranaceus inhibited gram-positive (S.
aureus and M. phlei) and gram-negative (E. coli) bacteria.
2. The MIC and MBC against these organisms were determined
(Figure 1).
3. The inhibitory effect of the ethanolic extract against S. aureus is
comparable to that of a 10-µg streptomycin disk (Table 2).
4. The extracts were especially effective against M. phlei (Figure 2, Table
2).
5. The ethanolic extract is 50% more effective at inhibiting E. coli than a
10-µg penicillin disk (Table 2).
6. The bacterial doubling time in the control cottage cheese was 30 min.
• The death rate of S. aureus growth in the methanolic extract-cottage
cheese was 13 min (Figure 3).
• The death rate of E. coli in the methanolic extract-cottage cheese
was 46 min (Figure 4).
7. Both alcoholic extracts inhibited S. aureus harvested from human nose
and ears (Table 2).
Background
1. Humans have used plants for medicinal purposes since the
Neanderthals, 60,000 years ago (1).
2. The use of Chinese plants as medicine is believed to have first started
over 4000 years ago during the Xia dynasty (2).
3. Astragalus membranaceus (Figure 1) is utilized by Chinese herbalists
to treat a variety of infections (2,6,8).
4. Previous studies have shown that the plant has immune-enhancing
effects (6).
Control
30
Methanolic extract
25
20
5. Four medicinally beneficial components can be separated from the
plant: flavonoids, saponins, polysaccharides, and gamma amino butyric
acid (3,4).
Alcohol controls
Methanol
Ethanol
0.0
0.0
0.0
0.0
0.0
0.0
7.0
8.0
Discussion & Conclusions
1. Mycobacterium is difficult to kill because of the cell wall mycolic acids.
Low concentrations (33.33 mg/mL) of both alcoholic A. membranaceus
extracts were bactericidal against M. phlei.
2. Traditional medicine utilizes A. membranaceus to treat patients infected
with M. tuberculosis. Further testing needs to be done to determine
whether A. membranaceus is bactericidal against M. tuberculosis, and
whether it can be used to treat multi-drug resistant M. tuberculosis.
3. E. coli is a causative agent in foodborne illness (9). The methanolic
extract significantly decreased E. coli growth in cottage cheese
(Figure 3).
4. S. aureus is a common cause of foodborne intoxication (9). The
methanolic extract prevented growth of S. aureus in cottage cheese
(Figure 4).
5. There is potential for alcoholic A. membranaceus extract to be used as a
disinfectant, a food preservative, and tuberculocide.
Literature Cited
5
0
-5
0
200
400
600
800
1000
1200
1400
1600
-10
Time (min)
Figure 3. Methanolic Astragalus extract (166.7 mg/mL) prevented growth of S.
aureus in cottage cheese. Error bars=1 S.E.
7. Five different A. membranaceus saponins (AMS) can be extracted from
the root of the plant using methanolic and ethanolic solvents (3,4).
8. A. membranaceus is used to treat Mycobacterium tuberculosis in China.
AMS has been shown to have macrophage-promoting effects in
tuberculosis patients (7).
1. Cowan, M. 1999. “Plant Products as Antimicrobial Agents” Clinical Microbiology
Reviews 12(4): 564-582.
2. Tan, B. et al. 2004. “Immunomodulatory and antimicrobial effects of some traditional
Chinese medicinal herbs: A review” Current Medicinal Chemistry 11: 1423-1430.
3. Yu, Q. et al. 2007. “Determination of seventeen main flavonoids and saponins in the
medicinal plant Huang-qi (Radix Astragali) by HPLC-DAD-ELSD” Journal of
Separation Science 30: 1292-1299.
4. Ma, X. et al. 2002. “Chemical analysis of radix Astragali (Huangqi) in China: A
comparison with its adulterants and seasonal variations” Journal of Agriculture and
Food Chemistry 50: 4861-4866.
5. Gao, X. et al. 2009. “Saponin fraction from Astragalus membranaceus roots protects
mice against polymicrobial sepsis induced by cecal ligation and puncture by inhibiting
inflammation and upregulating protein C pathway” Journal of Natural Medicines 63:
421-429.
6. Wong, C. et al. 1988.”Immunotherapy with Chinese medicinal herbs II. Reversal of
cyclophasphamide-induced immune suppression by administration of fractionated
Astragalus membranaceus in vivo.” Journal of Clinical Laboratory Immunology 25:
125-129.
7. Xu, H. et al. 2007. “Effects of Astragalus polysaccharides and astragalosides on the
phagocytosis of Mycobacterium tuberculosis by macrophages” The Journal of
International Medical Research 35: 84-90.
8. Louie, Ken. Chinese Herbalist. Personal Interview. 2 February 2010.
9. Mead P. et al. 1999. “Food-Related Illness and Death in the United States.” Emerging
Infectious Diseases 5(5): 608-625.
0 S.E.
Figure 1. Sliced roots of
Astragalus membranaceus
(huang-qi, 黃耆) (Fabaceae) are
sold as herbal remedies. This
plant is grown commercially in
China’s Shanxi, Gansu, and
Hellongjiang provinces. Common
names include locoweed and milk
vetch.
Acknowledgements
Table 1. MIC and MBC of Astragalus extracts
Bacterium
E. coli
S. aureus
M. phlei
Ethanolic Extract
MIC (mg/mL)
MBC (mg/mL)
100.00
116.67
50.00
100.00
16.67
33.33
Methanolic Extract
MIC (mg/mL)
MBC (mg/mL)
150.00
166.70
83.33
166.70
16.67
33.33
Antibiotic disks
Penicillin Streptomycin
47.0
20.0
10.0
18.0
0.0
10.0
43.0
19.0
10
Figure 2. Ethanolic Astragalus extract
strongly inhibited the growth of M.
phlei. Control well contains 95%
ethanol. Penicillin disk (10 µg) had no
effect on M. phlei growth.
6. Observed success of A. membranaceus against a number of infections
in vivo has been attributed to the saponin component (5,7).
Bacterium
S. aureus
E. coli
M. phlei
Wild-type S. aureus
Astragalus extracts
Methanol Ethanol
15.0
20.0
9.0
15.0
27.0
37.0
13.0
15.0
15
0 S.E.
Bacterial Growth in Cottage Cheese
1. 10 g cottage cheese was placed into sterile Petri dishes.
2. Two plates were inoculated with 100 µL of a 24-hour E. coli culture and
two plates with 100 µL of a 24-hour S. aureus culture.
3. Methanolic extract (16.67 mg/mL) was added to one plate of each
organism.
4. Plates were incubated at 37°C for 25 hours.
5. Plate counts were used to determine the number of bacteria.
Zone of inhibition (mm)
106
Extracts prepared from the root of Astragalus membranaceus will inhibit
bacterial growth.
Minimal Inhibitory (MIC)/Bactericidal Concentration (MBC)
1. Serial dilutions (166.67-16.67 mg/mL) of extracts were made in sterile
water.
2. Dilutions were placed in separate wells in a tissue culture plate along
with nutrient broth or tryptic soy broth.
3. Each set of dilutions were inoculated with E. coli, S. aureus, or M. phlei
and incubated for 24-48 hours at 37°C.
4. Dilutions with no growth were then subcultured.
35
CFU/g x
Hypothesis
Well Diffusion Assay
1. Nutrient agar or Tryptic soy sgar plates were inoculated with test
bacteria: Escherichia coli (ATCC 11775), Staphylococcus aureus
(ATCC 27659), Mycobacterium phlei (Wards 85 W 1691), and
mannitol-positive, coagulase-positive staphylococci isolated from
human skin.
2. 6-mm wells were made in the agar with a cork borer.
3. Wells5 were filled with 40 µL extract or solvent (negative control).
4. Antibiotic disks were placed on each plate as positive controls.
• Penicillin and Streptomycin, 10 µg/disk (Hardy Diagnostics)
5. Plates were incubated at 37°C for 24-72 hours.
Table 2. Inhibition by Astragalus extracts in the well diffusion assay.
Figure 4. Methanolic Astragalus extract (166.7 mg/mL) prevented growth of E.
coli in cottage cheese.
•Dr. Christine Case, Skyline College Professor of Biology
•Patricia Carter, Skyline College Biology Lab Technician
•Stephen Fredricks, Skyline College MESA Director
•Ken Louie, Chinese Herbalist
•Casey Fortier, Skyline College Student Researcher
•Skyline College SACNAS Chapter