1. Antibacterial Potential of Spilanthes Extract on Common Skin Bacteria Hayley Lonai, Department of Biology, York College Introduction The skin disease acne affects over 45 million Americans annually (The Lewin Group 2004). The development of acne is thought to be caused by the bacteria Propionibacterium acnes. Two other skin disease and infection causing bacteria Staphylococcus aureus and Streptococcus pyogenes also affect millions of others. These bacterium are commonly found as part of the natural microbiotic community on the human skin. However, in optimum conditions these bacteria can turn pathogenic and lead to disease and infection. Scalded skin syndrome, staph infections, and abscesses are produced in the presence of S.aureus and impetigo and cellulitis by S.pyogenes. There currently are broad spectrums of antibiotics that can control the potential infections of these bacteria. Concern has been raised that signs of resistance have been seen among the bacteria in both nosocomial and communicable instances. This concern has spurred new endeavors to find alternative antibacterial agents. Spilanthes acmella, commonly referred to as the toothache plant, is an herb found in the tropics and has been traditionally used in folk medicine. The herb has been used to treat an array of ailments comprising of stomatitis, periodontitis and other acute or long-term microbial infections (Dias 2012). Other medicinal properties S.acmella is claimed to posses include local anesthetic, immune modulatory affects, a diuretic, and larvicidal and insecticidal properties (Boonen 2010). The main isolated bioactive component is believed to be Spilanthol ((2E,6Z,8E)-N-Isobutyl-2,6,8- decatrienamide), an N-isobutylamide. The precise mechanism of Spilanthol is not fully understood. The structure of the compound, possessing a part that is 2E unsaturated, is thought to be toxic to bacteria by altering their growth cycle. Exploring and understanding the isolates within the Spilanthes acmella extract may lead to a new product with antibacterial potential against these common strains of bacteria. Methods Overall Conclusions Maximum Number of Inhibited Wells (MNIW) Bacteria From overnight cultures in TSB a 1:100 dilution of a 0.5 MacFarland standard was made for each bacteria Prepared Solutions 5% Ethanol solution Spilanthes Extract containing 5% Ethanol 500 ug/ml solution of Ampicillin Sterile TSB The Dilution(2X) Disk Diffusion Method A Trypitc Soy agar plate was inoculated with 200 ul of an overnight culture of bacteria in Tryptic Soy Broth (TSB) 3 sterile BBL™ 6mm blank paper disks were saturated into 1) Spilanthes extract, 2) 65% Ethanol solution, 3) 55% Ethanol solution and each disk placed evenly apart on the inoculated plate Incubated overnight at 37 o C. 18 replicates were performed for each bacteria Zone of Inhibition (ZI) diameter in mm was measured around each disk and recorded Disks with known Antibiotics were used as controls for each bacteria and the ZI also recorded ZI (mm) Figure 1. Shows the Zone of Inhibition around the disk saturated in Spilanthes extract on a plate inoculated with P.acnes H 1 : Spilanthes extract will inhibit the growth of the bacteria; S.aureus, S.pyogenes, and P.acnes H 0 : Spilanthes extract shows no antibacterial properties and does not inhibit the growth of the bacteria H 1 : Spilanthes extract will inhibit the growth of the bacteria; S.aureus, S.pyogenes, and P.acnes H 0 : Spilanthes extract shows no antibacterial properties and does not inhibit the growth of the bacteria Hypotheses Literature cited Acknowledgements I would like to thank Dr. Singleton, Dr. Kleiner and the York College Biology department for their guidance and support. The Lewin Group, Inc. The Burden of Skin Diseases 2004. The Society for Investigative Dermatology and The American Academy of Dermatology Association. 2004 p62. A.M.A. Dias, P. Santos, I.J. Seabra, R.N.C. Júnior, M.E.M. Braga, H.C. de Sousa. Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective supercritical carbon dioxide extraction. The Journal of Supercritical Fluids. Volume 61, January 2012, Pages 62-70 Boonen J, Baert B, Roche N, Burvenich C, De Spiegeleer B. Transdermal behaviour of the N-alkylamide spilanthol (affinin) from Spilanthes acmella (Compositae) extracts. Journal Of Ethnopharmacology. January 8, 2010;127(1):77-84 When tested against S.aureus, the Zone of Inhibition did not differ among the Spilanthes extract and the two ethanol solutions Spilanthes extract had a greater inhibitory affect against P.acnes than either of the two ethanol solvents In the testing of S.pyogenes, Spilanthes extract showed a greater inhibitory affect than either of the two ethanol solvents Figure 3. Shows Zone of Inhibition measurements (mm) of two alcohol solvents (65%-55%) and Spilanthes plant extract against three strains of bacteria; S.aureus, P.acnes, and S.pyogenes. Different letters above bars represent significant difference of P<.05. One way analysis of variance and a Tukey post test was done. Zone of Inhibition Figure 4. Displays the number of wells presenting the inhibition of bacteria growth on a 96 well microtiter plate. Spilanthes extract with a content of 5% ethanol, a 5% ethanol solution, and an Ampicillin solution were tested against three different bacteria; S.aureus, P.acnes, and S.pyogenes. Different letters above bars represent significant difference of P<.05. A Kruskal- Wallis and a Dunn’s Post test was performed. MNIW Objective: To test Spilanthes extract against several bacteria to determine any antibacterial potential S.pyogenes showed a greater susceptibility to the Spilanthes extract having a higher MNIW compared to the other two bacteria S.aureus, P.acnes, and S.pyogenes were more susceptible to the Spilanthes extract than the 5% ethanol solution, and also more susceptible to the ampicillin than the Spilanthes extract Spilanthes extract exhibited antibacterial properties against the tested bacteria, and appears to be an effective antibacterial agent Spilanthes extract was more effective at inhibiting S.pyogenes and P.acnes than S.aureus The magnitude of the the effect of Spilanthes extract is additive of the ethanol content in the extract solution Spilanthes extract exhibited antibacterial properties against the tested bacteria, and appears to be an effective antibacterial agent Spilanthes extract was more effective at inhibiting S.pyogenes and P.acnes than S.aureus The magnitude of the the effect of Spilanthes extract is additive of the ethanol content in the extract solution Future Research: Isolate the main Bioactive component, Spilanthol, from the Spilanthes acmella plant and test it against various bacteria strains, or other microorganisms Evaluate the mechanism between isolated Spilanthol and bacteria, or other microorganisms Results www.dshs.state.tx.us www.henriettesherbal.com MNIW was determined by counting the total number of wells in the row that inhibited bacteria growth 10 Microtiter plate trials were performed for each Bacteria Figure 2. Template of a Microtiter plate inoculated with S.pyogenes. Bacteria growth No growth MNIW = 7 = 6 = 0 = 6 100ul of the Bacteria was added to wells 1-11 all rows and then incubated at 37 o C overnight 12 3 5 4 7 6 9 8 11 10 12 H G F E D C B A 200 ul Spilanthes solution 200 ul Ethanol solution 20 ul Ampicillin solution + 180 ul TSB 100 ul TSB 12 3 5 4 7 6 9 8 11 10 12 C B A Tra nsfer 100 ul Discard 100 ul from well 10 No drug added