Anti-cancer properties of watercress ’ s ( Nasturtium officinale ) gluconasturtiin

1. Anti-cancerpropertiesofwatercress’s (Nasturtiumofficinale) gluconasturtiin Brad Frate and Marianne Stopperof SUNY Oswego Supervisors: Dr. Eric FuchsCastillo & Dr. Rodriguez of UNIBE Introduction Objective Cancer is the leading cause of death in most developed countries, causing an increase in anti-cancer research to potentially find a cure. Many plants contain anti-cancer molecules, such as gluconasturtiin in watercress (Nasturtium officinale) leaves. Gluconasturtiin is a precursor to an ITC called phenethylisothiocyanate (PEITC) which blocks degradation of structural proteins in cancer cells and reduces tumor cell survival which decreases the action of hypoxia-inducible factor (HIF), which is a molecule that stimulates angiogenesis (blood vessel development), allowing a tumor to obtain a blood supply. When humans consume watercress, PEITC levels elevate, and HIF activity is reduced, confirming in humans the anti-cancer effects of watercress. The goal of the experiment was to extract the anti-cancer compound, Gluconasturtiin, from watercress (Nasturtium officinale) leaves using ethanol, hexane, and chloroform. And, one of these solvents, based on thin-layer chromatography results, would be selected for further tests and examinations to validate it’s anti-cancer properties. Theory and Methods In order to test the anti-cancer properties of watercress, organic watercress samples were obtained, and the leaves were removed from the stems. After obtaining the leaves they were divided into three 1000mL flasks, and 50.26g of watercress were placed into each of the three beakers. They were then mixed separately with 360mL of ethanol, hexane, and chloroform. Reflux, shown in figure 1, was performed on the three samples for three hours using a condenser. The hot plate was set at 200oC and 600rpm for stirring. The solution was then cooled down using a cold water bath. Once cooled, vacuum filtration was performed on the three samples with a Hirsh funnel. The watercress extracts were then concentrated by putting the ethanol extract, hexane extract, and chloroform extract in a rotary evaporator (figure 3). After the samples were collected from the rotary evaporator, they were tested using Thin-Layer Chromatography (TLC). TLC was performed with different solutions to find the best solvent to do a column separation. The solvents tested were chloroform, hexane, ethanol, and ethyl acetate, and it was found that none of those solvents alone were the best solvents. In order to solve this problem “trial and error” was used, and some of the solvents were mixed to see which mixture gave the best result. It was found that a 1:3 ratio of hexane to ethyl acetate solution was the best solvent to continue the experiment. And, according to the TLC, the ethanol extract, shown in figure 2, proved to be the best because it separated the best in the 1:3 hexane to ethyl acetate solution. Ethanol was then mixed with silica gel and placed into the rotary evaporator, which produced a green powdered solid sample. The next step was column chromatography with the green powder watercress sample. In order to do this, powdered silicon was mixed with hexane to form a mixture between them. A cotton ball was placed at the bottom of the column, and then the mixture of silicon and hexane was poured on top of the cotton ball until the column was about 2/3 full. Another cotton ball was placed on top of the silicon and hexane mixture, and then additional hexane was poured to make sure the cotton ball on top was moist. The watercress green powder (solid sample) was then put into a column that was set up on a stand for column chromatography. Hexane was added in 10mL increments until the watercress solid sample reached the bottom of the silicon mixture. After the solid sample reached the bottom of the column a 9:1 hexane to ethyl acetate solution was poured on top six times in 10mL increments. The step used for the 9:1 ratio of hexane to ethyl acetate solution was repeated with the following ratios of 8:2, 7:3, 6:4, and 5:5 six times in 10mL increments. The drips of the solvent that came out of the column were collected in vials, which were labeled with the certain ratios. In order to determine what sample was the best to continue testing, we took the samples in the vials and performed TLC with ethyl acetate solution in the chambers. To make the separations visible “trial and error” was used again. It was found that an iodine chamber was unsuccessful in making the separations of the samples, on the TLC plates, visible. A second attempt using a UV light 9, shown in figure 4, proved to be successful, and it was found that the 7:3 ratio vials of hexane to ethyl acetate solution were the best to continue testing. The five vials of 7:3 hexane to ethyl acetate, were then poured into a 200mL round bottomed flask to be concentrated in the rotary evaporator. The sample was then rinsed, after the rotary evaporator, with chloroform and was placed into a vial to be tested for gas chromatography-mass spectroscopy, infrared spectroscopy, and ultra-violet spectroscopy. Figure 1. Ethanol Reflux Figure 2. Ethanol Watercress Extract Figure 3. Rotary Evaporator Figure 4. TLC Plate Under UV Light Results Figure 6 (left): GC-MS (gas chromatography-mass spectroscopy) According to the GC-MS results, figures 6 and 7 the compound extracted from watercress leaves has a 99% chance of being 6- Octadecenoic Acid. The IR results in figure 5, also suggests 6- Octadecenoic Acid with corresponding peaks to the results on the Spectral Database for Organic Compounds, agreeing with the results from figures 6 and 7. Figure 7 (right): GC-MS (gas chromatography-mass spectroscopy) Figure 5: IR spectroscopy results Conclusion References Acknowledgements Burns, S. (2011). Wonderful Watercress: Pungent, peppery, and good for your skin. Skin Deep, 9(3), 11 Engelen-Eigles, G., Holden, G., Cohen, J., & Gardner, G. (2006). The effect of temperature, photoperiod, and light quality on gluconasturtiin concentration in watercress (Nasturtium officinale R. Br.). Journal Of Agricultural And Food Chemistry, 54(2), 328-334. Grosso, C., Vinholes, J., Silva, L., Guedes, P., Gonçalves, R., Valentão, P., et al. (2007). Chemical composition and biological screening of Capsella bursa-pastoris. Scielo. Jemal, A., Bray, F., Center, M. M., Ferlay, J., Ward, E. and Forman, D. (2011). Global cancer statistics. A cancer journal for clinicians, 62(2), doi: 10.3322/caac. 20107 Ozen, T. (2009). Investigation of antioxidant properties of Nasturtium officinale (watercress) leaf extracts. Acta PoloniaePharmaceutica, 66(2), 187-193. Precht, D., & Molkentin, J. (1999). C18:1, c18:2 and c18:3 trans and cis fatty acid isomers including conjugated cis delta 9, trans delta 11 linoleic acid (cla) as well as total fat composition of german human milk lipids.Nahrung, 43(4), 233-44. Spectral Database for Organic Compounds SDBS. (2012). C18H34O2 http:// riodb01.ibase.aist.go.jp/sdbs/cgi-bin/direct_frame_top.cgi Varricchio, C. G. (2004). A cancer source book for nurses. Boston: Jones and Bartlett Publishers. Although, the anti-cancer compound Gluconasturtiin, wasn’t found during the experiment, a similar compound, 6- Octadecenoic acid, was found. This compound is very similar to Gluconasturtiin with its anti-cancer and medicinal properties. 6- Octadecenoic acid is a global central nervous system depressant that decreases cerebral oxygen consumption, reduces intracranial pressure and has potent anti-convulsing properties. It is also a potent antioxidant and bronchodilator, and has anti-inflammatory properties along with anti-cancer tumor suppressing properties. Thus, through the results and data obtained it was concluded that watercress is indeed a medicinal and anti-cancer plant, with more than one anti-cancer and medicinal compound.