Scientific Poster

1
The Antimicrobial Activity of Sapindus mukorossi
Raya Mesias, Anand Singh, and Christine
Case Biology Department, Skyline College, San
Bruno CA
Abstract The emergence of bacterial resistance to
existing antibiotics makes the need for new
antibiotics essential. After years of extensive
research on soil microorganisms, we need to look
for new sources of antimicrobics. There are
limited reports of antimicrobics from plants.
Soapnut, Sapindus mukorossi, has been used in
traditional South Asian culture as a skin
cleanser and a laundry detergent. Therefore, we
hypothesize that the soapnut seed has
antibacterial activity. Soapnut seeds were ground
in 95 ethanol or acetone to a final
concentration of 430 mg/mL. The extracts were
tested against the gram-negative
bacteria Salmonella enterica and Escherichia
coli and gram-positive Staphylococcus
aureus bacteria in a disk-diffusion test. The
ethanolic extract inhibits Salmonella. The
minimum bacteriostatic concentration
against Salmonella is 500 mg/mL no lethal
concentration was found. Bacteriostatic compounds
will be isolated by paper chromatography. The
method of action will be reported.

• Methods Materials
• Preparation of Soapnut Extract
• Dried Sapindus mukorossi fruits were purchased
  whole from local markets.
• The soapnut extract was prepared by first
  breaking the soapnut with a hammer. The contents
  were then separated into the pericarp, endocarp,
  and seed (Figure 1). 3 g soapnut seed was ground
  in 4 mL of 95 ethanol or acetone to a final
  concentration of 430 mg/mL.
• Screening
• Antibacterial testing was done by disk-diffusion
  assays (Figure 2) on nutrient agar against
  gram-negative Salmonella enterica (ATCC 14028)
  and Escherichia coli (ATCC 11775), and
  gram-positive Staphylococcus aureus (ATCC 27659).
  
• The minimum inhibitory concentration and the
  minimum bactericidal concentration were
  determined by serial dilutions using the
  ethanolic extract (430 to 500 mg/mL) in nutrient
  broth in a cell well plate. Each well was
  inoculated with 50 µL of Sa. enterica. Wells
  with no growth were subcultured into nutrient
  broth.
• Isolating Active Compounds
• The ethanolic extract was separated by paper
  chromatography in isopropanol-ammonium hydroxide.
  
• The dried chromatography paper was cut into 1 cm
  pieces and used in a disk diffusion assay against
  Sa. enterica (Figure 3).
• Determination of a Bacterial Growth Curve
• Nutrient broth with 340 mg/mL ethanolic soapnut
  seed extract in a Nephalo culture flask was
  inoculated with 10 mL Sa. enterica. The control
  flask contained nutrient broth ethanol and was
  also inoculated with 10 mL of Sa. enterica.
  Flasks were incubated at 37C. The absorbance was
  recorded at 540 nm every 30 minutes.

• Results
• The ethanolic extract of soapnut seed (340 mg/mL)
  inhibited the growth of the gram-negative
  bacterium, Sa. enterica (Figure 2). There was no
  inhibition of E. coli and St. aureus.
• The MIC of the ethanolic extract against Sa.
  enterica is 500 mg/mL no lethal concentration
  was found.
• Antibacterial compounds were found at Rf values
  of 0.85 and 0.97 (Figure 3).
• The growth rate of S. enterica in 340 mg/mL
  ethanoic soapnut extract was not affected (Figure
  4).

• Future Studies
• The effects of a higher concentration of the
  ethanolic seed extract on bacterial growth will
  be studied.
• The method of action of the ethanolic extract
  will be determined.

Hypothesis The soapnut seed contains
antibacterial activity.

• Literature Cited
• Aneja, K., R. Joshi, and C. Sharma. "In Vitro
  Antimicrobial Activity of Sapindus mukorossi and
  Emblica officinalis Against Dental Caries
  Pathogens." Ethnobotanical Leaflets 14
  402-412 (2010) 1-9.
• Kamra, D.N., A.K. Patra, P.N. Chatterjee, R.
  Kumar, N. Agarwal, and L.C. Chaudhary. 2008.
  Effect of Plant Extracts on Methanogenesis and
  Microbial Profile of the Rumen Buffalo.
  Australian Journal of Experimental Agriculture
  48(2) 175-178.
• Livermore, D. M. 2004. The Need for New
  Antibiotics. Clinical Microbiology and Infection
  10 (Supplement 4) 1-9
• Dhar, J.P., V. K. Bajpai, B. S. Setty. and V. P.
  Kamboj. 1989. Morphological changes in human
  spermatozoa as examined under scanning electron
  microscope after in vitro exposure to saponins
  isolated from S. mukorossi. Contraception 39(5)
  563-568.
• Kane W. Charles. Herbal Medicine of the American
  Southwest. Lincoln Town Press. 2006.
• Patra, A.K. and J. Saxena. 2009. The Effect of
  Mode of Action of Saponins on the Microbial
  Popluations and Fermentation in the Rumen and
  Ruminant Production. Nutrition Research Reviews
  22 204219.
• Rakesh, M., K. Ashok, S. Kumar, and T. Amitabh.
  2010. Formulation of Herbal Shampoos from
  Asparagus racemosus, Acacia concin, Sapindus
  mukorosii. International Journal of
  Pharmaceutical Sciences Review and Research 4
  39-44.
• Yang, C., Y. Huang, Y. Chen and M. Chang. 2010.
  Foam Properties, Detergent Abilities and
  Long-term Preservative Efficacy of the Saponins
  from Sapindus mukorossi. Journal of Food and
  Drug Analysis 18 155-160.

• Background
• Antibiotic resistance is a major threat to human
  health (3).
• The pericarp of the soapnut, Sapindus mukorossi
  (Figure 1), has been traditionally used as a
  washing agent in Southern Asia due to its
  saponins (8). The outer shell contains saponins
  that are natural surfactants thus providing
  soapy qualities to aqueous mixtures.
• Plants rich in secondary metabolites such as
  saponins are known to have antimicrobial activity
  (2).
• Currently, S. mukorossi is a key component in
  making natural shampoos in order to help replace
  detergents (7).
• Ground S. mukorossi seeds are used in making skin
  cleansers to prevent oil secretion (4).
• Studies have shown that S. mukorossi has been
  used as a natural additive in modulating rumen
  fermentation due to the fact that S. mukorossi
  contain saponins that inhibit protozoa (6).
• In the American Southwest, S. saponaria has been
  used to treat constipation (5).
• Extracts of soapnut inhibit Saccharomyces
  cerevisiae fungus (1).
• The purpose of our work is to determine the
  antibacterial activity of soapnut extracts.

.

• Discussion Conclusion
• In Southern Asia, S. mukorossi, has been used as
  a washing agent and S. saponaria has been used in
  the American Southwest to treat gastrointestinal
  ailments (5).
• Plants that contain saponins are known to have
  antimicrobial activity (2).
• Our research shows that an ethanoic extract of
  soapnut seed inhibits the gram-negative bacterium
  Sa. enterica.

Acknowledgements We are grateful to SACNAS for
giving us the opportunity to share our project
Pat Carter, for her advice and providing us with
the necessary material and equipment and Dr.
Christine Case, for her guidance and motivation.