Evaluation of the Antibacterial Activity of the Traditional Medicinal Plant, Astragalus membranaceus

1. Evaluation of the Antibacterial Activity of the Traditional Medicinal Plant, Astragalus membranaceus 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. Katrina Cheung Biology Department, Skyline College, San Bruno CA 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. • 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 (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. • 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. • 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. • 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 µLof a24-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. • 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). 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 extractto 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 bars=1 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 Figure 4. Methanolic Astragalus extract (166.7 mg/mL) prevented growth of E. coli in cottage cheese.