Scientific Poster

1
Evaluation of the Antibacterial Activity of the
Traditional Medicinal Plant, Astragalus
membranaceus
Katrina Cheung Biology Department, Skyline
College, San Bruno CA

• Methods
• 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 (65C or 78C)
  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.
• 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 37C for 24-72 hours.
• 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 37C.
• 4. Dilutions with no growth were then
  subcultured.

• Results
• 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).
• 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).

Abstract 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 (83.33-mg/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.
Table 2. Inhibition by Astragalus extracts in the well diffusion assay. Table 2. Inhibition by Astragalus extracts in the well diffusion assay. Table 2. Inhibition by Astragalus extracts in the well diffusion assay. Table 2. Inhibition by Astragalus extracts in the well diffusion assay. Table 2. Inhibition by Astragalus extracts in the well diffusion assay. Table 2. Inhibition by Astragalus extracts in the well diffusion assay. Table 2. Inhibition by Astragalus extracts in the well diffusion assay.
Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm)
Astragalus extracts Astragalus extracts Alcohol controls Alcohol controls Antibiotic disks Antibiotic disks
Bacterium Methanol Ethanol Methanol Ethanol Penicillin Streptomycin
S. aureus 15.0 20.0 0.0 0.0 47.0 20.0
E. coli 9.0 15.0 0.0 0.0 10.0 18.0
M. phlei 27.0 37.0 0.0 0.0 0.0 10.0
Wild-type S. aureus 13.0 15.0 7.0 8.0 43.0 19.0
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.
Hypothesis Extracts prepared from the root of
Astragalus membranaceus will inhibit bacterial
growth.
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). 5. Four medicinally beneficial components
can be separated from the plant flavonoids,
saponins, polysaccharides, and gamma amino
butyric acid (3,4). 6. Observed success of A.
membranaceus against a number of infections in
vivo has been attributed to the saponin component
(5,7). 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).
Literature Cited 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.
Figure 3. Methanolic Astragalus extract (166.7
mg/mL) prevented growth of S. aureus in cottage
cheese. Error bars1 S.E.
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.
0 S.E.

• Acknowledgements
• 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

Table 1. MIC and MBC of Astragalus extracts Table 1. MIC and MBC of Astragalus extracts Table 1. MIC and MBC of Astragalus extracts Table 1. MIC and MBC of Astragalus extracts Table 1. MIC and MBC of Astragalus extracts
Bacterium Ethanolic Extract Ethanolic Extract Methanolic Extract Methanolic Extract
Bacterium MIC (mg/mL) MBC (mg/mL) MIC (mg/mL) MBC (mg/mL)
E. coli 100.00 116.67 150.00 166.70
S. aureus 50.00 100.00 83.33 166.70
M. phlei 16.67 33.33 16.67 33.33
Figure 4. Methanolic Astragalus extract (166.7
mg/mL) prevented growth of E. coli in cottage
cheese.