Acknowledgements

1. Antibacterial Activity Of The Spice Elettaria cardamomum Shervin Afghani and Imad Mugatash Biology Department, Skyline College, San Bruno CA Abstract Elettaria cardamomum, green cardamom, has been long used in south Asia both for medical purposes and as a seasoning. This plant is used in traditional botanic medicine to treat infectious disease such as pulmonary tuberculosis and lung congestion. Additionally, green cardamom is used for its important positive effects on the gastric system, blood pressure, and “gut modulatory”. The high rate of natural selection for mutations and genome rearrangement in bacteria resulting in antibiotic resistance, has forced a search for new antibiotics. Thus, after years of extensive research on soil microorganisms able to produce antibiotics, we need to look for new sources of antibiotics such as plants. Our hypothesis is that E. cardamomum can inhibit bacterial growth. We prepared aqueous, methanolic, ethanolic, and acetone extracts of E. cardamomum seeds and seed shells. The extracts were filtered, dried, and reconstituted to 833 mg/mL in their respective solvent. Each extract was tested against Escherichia coli (gram-negative), Staphylococcus aureus (gram-positive), Bacillus subtilis (gram-positive), Streptococcus mutans (gram-positive) and Pseudomonas aeruginosa (gram-negative) bacteria. The well diffusion assay method was used to test for antimicrobial activity. After incubation at 35°C for 48-72 hours, the 100% methanolic extracts and the 70% ethanolic and acetone extracts inhibited the growth of all five bacteria and inhibition of S. aureus, E.coli, B. subtilis, S.mutans bacteria with 100 % methanolic and 70% ethanolic and acetone extracts was observed. The acetone, methanol, and ethanol extracts killed all five bacteria with a MBC of 833mg/ml and inhibited their growth with an MIC of 417 mg/mL. These results are comparable to reported results for other spices and suggest that green cardamom could be useful as an antibacterial for food preservation. Methods 1. Dried Elettaria cardamomum seeds were purchased in bulk from Abu Kameel store. The manufacturer was M.R. Corp. and they were grown in Bangladesh. 2. 10 g dried E, cardamomum seeds, was finely ground in an electric mixer and soaked for 48-72 hr in 13 mL of seven different solvents: water, 100% methanol, 100% ethanol, acetone, 70% methanol, 70% ethanol, and 70% acetone for 48 to 72 hr. 3. The mixtures were filtered through Whatman No.1 paper. The supernatant was diluted to final concentrations of 1667 and 833 mg/mL. 4. 20 µL of each extract and its respective solvent was tested in the well diffusion assay against the following gram-negative bacteria: Escherichia coli and Pseudomonas aeruginosa; and against the gram-positive bacteria: Staphylococcus aureus, Bacillus subtilis, Streptococcus mutans. 5. The minimum inhibitory concentration (MIC) and minimum bacterial concentration MBC of extract was determined by preparing serial dilutions of the cardamom extract reconstituted with their respective solvents in nutrient broth and inoculating each with 100 µL of E. coli, S. aureus, B. subtilis, Str. mutans, and P. aeruginosa. • Results • The alcoholic and acetone extracts were most effective against gram-positive bacteria. The increasing effectiveness in polar solvents suggests a non-water soluble protein or lipid (Figure 2). • The methanolic extracts were most effective against S. aureus (Figure 3). • The extracts were 50% less effective against gram-negative bacteria. The aqueous extract was only effective against Pseudomonas, suggesting there is a different, water-soluble anti-pseudomonas compounds (Figure 4). • The minimal inhibitory concentration and the minimal bacterial concentration of the both alcoholic and acetones extracts against all five test bacteria was 417 mg/mL and 833 mg/mL respectively. • Discussion & Conclusion • Antibiotics are valuable drugs; however, their overuse has created worldwide antimicrobial resistance. Therefore, new antibiotics are vital. • Green cardamom has antimicrobial compounds that inhibit the growth of medically important microorganisms. • The use of E. cardamomum as a medicine and flavoring in food and drinks for centuries has shown the seeds lack of toxicity for humans. Thus, E. cardamomum can be a potential source for new antibiotics that are effective against a broad collection of bacteria. • Further investigation will consist of isolation of the active compound(s), studying the antimicrobial activity of more concentrated extracts, and testing against other microorganism. • Literature Cited • Brooks, J. D., H. Corke, Y-Z. Cai, and B. Shan. 2007. “Antibacterial Properties and Major Bioactive Components of Cinnamon Stick (Cinnamomum burmannii): Activity against Foodborne Pathogenic Bacteria.” Journal of Agricultural and Food Chemistery 55(14):5484-5490. • Buhner, S. 1999. Herbal Antibiotics. Pownal VT: Storey Books. • Delaha, E. C. and V. F. Garagusi. 1985. “Inhibition of Mycobacteria by Garlic Extract.” Antimicrobial Agents and Chemotherapy 27(4):485-486. • Gilani, A. H., Q. Jabeen, A-u Khan, and A. J. Shah. 2007. “Gut modulatory, blood pressure lowering, diuretic and sedative activities of cardamom.” Journal of Ethnopharmacology 115(3):463-472. • Guilfoile, P. 2007. Antibiotic-Resistant Bacteria. New York: Infobase Publishing. • Hausler, T. 2006. Viruses vs. Superbugs: A Solution to the Antibiotics Crisis? New York: Macmillan. • Jamal, A., K. Javed, M. Aslam, and M. A. Jafri. 2006. “Gastroprotective effect of cardamom, Elettaria cardamomum Maton. fruits in rats.” Journal of Ethnopharmacology 103(2):149-153. • Sinu, P. A., and K. R. Shivanna. 2007. “Pollination ecology of cardamom (Elettaria cardamomum) in the Western Ghats, India.” Journal of Tropical Ecology 23(4):493-497. Figure 1.Elettaria cardamomum, green cardamom, is in the Zingiberaceae and is native to Western Ghats, India (8). The seeds are harvested and dried for use in food as a spice, in drinks as flavoring, and in medicine (4, 7). Hypothesis Green cardamom has medical uses in folk medicine. It is expected that the green cardamom will inhibit growth of bacteria tested in this experiment. Figure 2. Inhibition of gram-positive bacteria by 0.36 g/mL extracts in the well diffusion assay. Background • The availability of antibiotics is crucial. Antibiotics are molecules that are produced by other bacteria, fungi, plants, and other organisms that kill the infectious bacteria. • New antibiotics are needed. There has been a rise in antibiotic resistance over the past decades because of the misuse of antibiotics. One case is the emergence of antibiotic-resistant Staphylococcus aureus (5, 6). • Ancient medicine involved the use of many plants to treat diseases and infections (2). Cinnamon is an effective antimicrobic against gram-positive and gram-negative bacteria (1). Garlic is another effective antimicrobial that inhibits 16 mycobacteria (3). These observations led us to question whether other commonly used spices, such as green cardamom, have antimicrobial activity. • Elettaria cardamomum, green cardamom (Figure 1), is widely used in Asia to treat gum and tooth infections, tuberculosis, and digestive disorders (4, 7). However, its value has not been tested scientifically. • The purpose of our work is to evaluate whether E. cardamomum has antibacterial activity. Figure 3. Well diffusion assay showing inhibition of S. aureus by extracts in 100% methanol. The control well contains 100% methanol. Acknowledgements Genuine appreciation and particular thanks to Dr. Christine Case for her helpful guidance throughout this project. Also a special thanks to the Skyline College Biology Department, Patricia Carter and Ruth Arce for providing laboratory equipments, as well as wise guidance, to conduct this research. And finally a thank you to MESA directors Stephen Fredricks and Tiffany Reardon. Figure 4. Inhibition of gram-negative bacteria by 0.77 g/mL extracts in the well diffusion assay.