Four species of plants were tested for antimicrobial properties. The plants included the Pomegranate, Punica granatum L, both organic and inorganic; Echinacea, Echinacea purpurea; and the Eastern White Spruce, Picea glauca. Out of the four plants the best results were seen with the Pomegranate plant. Negligible differences were evident between the organic and inorganic fruit, the zones of inhibition were almost identical. The organisms, which the plants were tested against included: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. The plants were dried and soaked in methanol. The methanol was evaporated through rotoevaporation technique. The crude products of plants were tested by bioassay. The pomegranate stunted the growth of S. aureus, P. aeruginosa and C. albicans. Next, column chromatography was performed to obtain different fractions of the Pomegranate compounds through the use of various solvent systems. The second bioassay on Punica granatum, tested second and fourth methanol fractions. This time, only S. aureus and C. albicans’ growth were inhibited by the pomegranate.
![Researching Possible Antimicrobial Compounds in the Rind of Punica granatum L. Fruit, Against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans Organisms ,[object Object],[object Object],[object Object],Agazatian, Diana; Sabol, Ashley; Nagy, M; Bamstauk, B; and KO, Pascoe; Department of Biology, Georgia State University Atlanta GA, 30303 OBJECTIVE The objective of this study was to discover antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans, from the Pomegranate fruit, Echinacea, and the EasternWhite Spruce. ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],RESULTS CONCLUSIONS The zones of inhibition for Echinacea and Eastern White Spruce were small, displaying poor antimicrobial properties. Though, these two plants should not be disregarded and further testing should be carried out. Future improvements on Echinacea testing, require the uses of different solvent systems. The experimentation used nonpolar solvents for extracting Echinacea. It may be of value to find a solvent mixture with polar components to extract compounds that may have antimicrobial properties in Echinacea. As for the Eastern White Spruce, it has more rigid cell walls and a brawny physique. Favorable results may be produced if the plant is initially broken and crushed into smaller fragments. This would enhance the solvents ability to extract the compounds with antimicrobial properties. Out of the four plants tested, only the pomegranate achieved successful results. Proving to contain antimicrobial properties against S. aureus and C. albicans. Since, the initial experimentation began with testing both organic and inorganic pomegranate it is significant to mention that the results for both organic and inorganic were similar in the first bioassay. The second bioassay was solely performed on the inorganic plant. This proves that Punica granatum L. does exhibit antimicrobial activity against S. aureus and C. albicans organisms. Further testing needs to be completed in order to determine consistency in the results. DISCUSSION In the TLC procedure, several solvent systems were used, though the most effective results were produced form the polar solvents. In Picture 2, the top two TLC plates were performed with methanol as the polar solvent. This proved to be ineffective, causing the sample to run up the plate too quickly with almost no separation. The bottom left plate used 65% acetone and 45% ethyl acetate. This mixture was ineffective due to the lack of a strong nonpolar solvent. Producing poorly defined borders between the components. The last attempt used 70% acetone and 30% hexane. The separation in the compounds is defined showing distinct five to six component dissociation. The M2 sample, which is the second methanol extraction in the chromatography, produced as many as six component dissociations. The other two, crude and M4 only produced five dissociations. The components closer to the start line are the nonpolar components and the layers at the top are the polar components. In the bio assay, the two methanol extractions displayed strong zones of inhibition. The second methanol fraction was more potent than the fourth methanol fraction. In Table 3 the quantitative results show the numbers in (mm). The antimicrobial properties were more powerful against S. aureus than C. albicans . As seen in the chart the average zone of inhibition is 20 mm for S. aureus and only 19 mm for C. albicans . A minor inconsistency is seen in the poor zones of inhibition for the positive controls of C. albicans . This could be due to poor disc inoculation in the disc preparation step. Still, the plant sample shows strong inhibition for C. albicans even if the positive control has small zones. Bioassay Results from Column Chromatography Samples of Inorganic Pomegranate Extracts Picture 1: Whole Pomegranate Fruit Table 2: Solvent systems used for TLC and Column Chromatography Picture 2: Above are the top four solvent systems that best separated the components of P. granatum . Picture 3 : The above plates are of the species where it appeared that P. granatum exhibited antimicrobial activity (top - S. aureus ; bottom - C. albicans ). ,[object Object],[object Object],REFERENCES Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli . Kenneth Todar University of Wisconsin-Madison Department of Bacteriology. Todar's Online Textbook of Bacteriology . http://www.textbookofbacteriology.net/staph.html “ The Candida Albicans Report: Yeast Infection is a Killer Running Loose in Your Body.” The Colon Therapists Network. April 6, 2008. http://www.colonhealth.net/free_reports/candirpt.htm Morton, J. 1987. Pomegranate. p. 352–355. In: Fruits of warm climates. Julia F. Morton, Miami, FL. http://www.hort.purdue.edu/newcrop/morton/pomegranate.html Evelyn Dean. Pomegranate Seeds: Latin American Jewish Tales Folklore Forum 37.1 (2007). Indiana University, Department of Anthropology. Retrieved 30 March, 2008. https://www.indiana.edu/~folkpub/forum/journal.php?action=articledisplay&articleid=38 Nagy, Maria. Professor. Theme-lab Biology 4905. “Medicinal Plants”. Spring of 2008. Georgia State University Atlanta GA, 30303. Duke. J. A. and Ayensu. E. S. Medicinal Plants of China Reference Publications, Inc. 1985 ISBN 0-917256-20-4 CU Boulder Department of Chemistry. TLC – Retention Factor (R f ). Spring 2008. http://orgchem.colorado.edu/hndbksupport/TLC/TLCrf.html. ACKNOWLEDGEMENTS Howard Hughes Medical Institute Georgia State University Maria Nagy ABSTRACT Four species of plants were tested for antimicrobial properties. The plants included the Pomegranate , Punica granatum L, both organic and inorganic ; Echinacea, Echinacea purpurea; and the Eastern White Spruce , Picea glauca. Out of the four plants the best results were seen with the Pomegranate plant. Negligible differences were evident between the organic and inorganic fruit, the zones of inhibition were almost identical. The organisms, which the plants were tested against included: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. The plants were dried and soaked in methanol. The methanol was evaporated through rotoevaporation technique. The crude products of plants were tested by bioassay. The pomegranate stunted the growth of S. aureus, P. aeruginosa and C. albicans . Next, column chromatography was performed to obtain different fractions of the Pomegranate compounds through the use of various solvent systems. The second bioassay on Punica granatum, tested second and fourth methanol fractions. This time, only S. aureus and C. albicans’ growth were inhibited by the pomegranate . Zones of Inhibition for Methanol Extractions After Bioassay Table 3: Quantitative results for the zones of inhibition from each column chromatography fractions against each microbial organism. R f Values for Measured Components from Different Solvent Systems of TLC Table 4 : The R f values indicate the polarity of a compound. The larger the R f the less polar because it interacts less strongly with the polar absorbent. Above, the M2 samples has better values in the acetone/hexane mixture and the acetone/ethyl acetate mixture . TLC Results for Inorganic Pomegranate ,[object Object],The R f (Retention Factor) is a value that means the given values reported are relative to a standard, or the multiple compounds being run on a plate at the same time are being compared together. The larger the R f value, the larger distance it has traveled on the TLC plate. When comparing two compounds that are running on the same plate at the same time, then the one with the larger R f is less polar because it interacts less strongly with the polar solvent on the TLC plate. The R f value can also be used to corroborate evidence as to the identity of a compound. If there is a suspected known compound that an unknown compound is considered to be, then the two samples can be spotted on a single plate. R f values are not definitive, but if the R f values of the two compounds, (known compound and unknown), are the same then the unknown is most likely that which it was compared against, the known compound. It is assumed that the compounds M2 and M4 are very close in relation due to there very similar R f values. TLC and Column Chromatography Solvent Systems/Amounts TLC (%) Column Chromatography (mL) Hexane N/A 250mL Methanol N/A Extraction 2 – 200mL Extraction 4 – 200mL Acetone N/A 200mL Dichloromethane N/A 200mL Hexane/Methanol 50:50 30:70 40:60 N/A Hexane/Acetone 40:60 20:80 30:70 N/A Acetone/Ethyl Acetate 65:35 N/A Scientific Name Punica granatum L. Common Name Pomegranate Folklore ,[object Object],[object Object],[object Object],Reason for Selection Based on an Armenian Family Remedy](https://image.slidesharecdn.com/inorganicpomegranateposter2-12522595217544-phpapp01/85/analysis-of-pomegranate-rind-for-antibacterial-properties-in-both-the-organic-and-conventionally-grown-fruit-1-320.jpg)
