In the Laboratory
Effects of Storage Conditions on Lycopene Stability in Tomato Extracts An Undergraduate Experiment Amanda L. Miller and Pamela Vaughan Department of Chemistry, University of West Florida, Pensacola, FL 32514 Tara M. Sirvent* Department of Chemistry, Vanguard University, Costa Mesa, CA 92626; *
[email protected] Foods containing nutritional as well as medicinal value (often called nutraceuticals) are a hot commodity in today’s society. One such nutraceutical is the tomato (Lycopersicon esculentum L). Tomatoes are one of the principal sources of carotenoids in the Western diet (1) and are the fourth most commonly consumed fresh vegetable and the most frequently consumed canned vegetable in the United States (2). Responsible for its red color, lycopene is the prominent carotenoid in tomatoes and often represents an index of quality for tomato products (3, 4). Lycopene is the most abundant carotenoid in human tissues (5). Carotenoids are considered critical nutrients in the human diet and function as antioxidants (6, 7). Carotenoids such as lycopene and β-carotene are tetraterpenes characterized by a polyene chain containing multiple conjugated double bonds that are responsible for the characteristic absorption spectrum and photochemical properties (8, 9). Carotenoids play an important role in photosynthetic organisms by harvesting light for energy transfer to chlorophylls, providing photo-protection by quenching triplet-state chlorophyll molecules, and scavenging singlet-state chlorophyll (8, 9). Cis isomers of lycopene1 tend to be more bioactive than the all-trans isomer (8, 10–14); however, the all-trans isomer composes 80–95% of the total lycopene content in fresh red tomato varieties (14, 15). This may be a result of sample preparation but it appears that the cis isomers are more bioavailable in the human body. In fact, the pH of gastric juices degrades the all-trans-lycopene isomer while 13-cis-lycopene2 is more stable at the low pH in vitro (13). In vivo studies also support the conversion of an all-trans-lycopene isomer to the 5-cis-lycopene2 in ferrets (11). It is not clear what effect heat and light have on the stability of the all-trans-lycopene isomer. It is known that elevated temperatures, exposure to light or oxygen, pH changes, and exposure to active surfaces such as lipoproteins in the intestine have a catalytic effect on the isomerization and degradation of lycopene (16). Lycopene oxidative degradation not only affects the quality of the final product but also the health benefit of tomato-based foods. Factors such as bleaching, freezing, and heat-induced damage result in lycopene loss (17). Extraction of heat- and light-sensitive materials requires extra precaution in the laboratory, techniques with which many students are unfamiliar. This study explores the stability of lycopene through a simple laboratory exercise that introduces students to isolation of natural products from plant material by organic extraction, sample preparation, HPLC analysis, and compound stability. It has been designed for third- or fourth-year students as a guided independent project to be completed over 2–3 laboratory sessions (approximately 3 hours each). 1304
Experiment Chemicals Chloroform (or petroleum ether) may be used as an extraction solvent. Acetone is used to redissolve extracts prior to high-performance liquid chromatography (HPLC) analysis. The HPLC mobile phase consists of methanol (MeOH), acetonitrile (ACN), ethyl acetate (EtOAc). All solvents were purchased from Fisher Scientific, Inc. An all-trans-lycopene standard was purchased from MP Biomedicals, Inc. Airgas UHP nitrogen was used for solvent evaporation and evacuation of oxygen from sample vessels (standard grade nitrogen can also be used if available). Carotenoid Extraction If students work in pairs, a comparison can be made between fresh tomatoes and tomato paste. Carotenoid extraction from fresh tomatoes (or paste) is modified from the procedure used by Rodriguez-Amaya et al. (18). Briefly, the tomatoes are washed, cut into small squares, and homogenized using a glass tissue homogenizer. The tissue is then extracted with 2 volumes of chloroform. The extract is allowed to steep for 15 minutes and then filtered using a Büchner funnel (when using tomato paste, it may be more expedient for the students to centrifuge the extraction material at low speed, 0.0056).4,5 Levels of cis-lycopene were also elevated by a factor of 8 when comparing dark and nitrogen samples to light and oxygen samples (DF = 2, F = 66.7, P > 0.0008). Perhaps the rate of total lycopene degradation is slowed under these conditions but not isomerization. Lee reports that the all-trans-
light, oxygen
light, nitrogen
dark, nitrogen
Storage Condition Figure 2. Effect of light on the quantity of all-trans-lycopene (gray bars) and cis-lycopene (white bars) in lipophillic tomato extracts stored at 25 ˚C.
lycopene completely degrades within 160 hours in the presence of 2000–3000 lux of illumination at 25 °C in a linear fashion (16, 21). It is apparent (although not completely understood) from both our results and other literature that the degradation of all-trans-lycopene occurs at a faster rate than the isomerization of all-trans-lycopene to any of the cis isomers (21, 22). Oxygen can cause an 80% decrease in the levels of lycopene at room temperature as compared to oxygen-removed oil-inwater emulsions (23). With respect to tomato juice powder, airpacked samples retained the lowest levels of lycopene compared to CO2-, N2- or vacuum-packed samples (24). In these previous studies, however, the overall levels of lycopene decreased and the degree of isomerization of the all-trans-lycopene to any of the cis isomers is not known.
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 11 November 2009 • Journal of Chemical Education
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In the Laboratory
Conclusions Many biochemistry experiments employ the use of HPLC to teach students about chromatographic separations of biomolecules. As demonstrated in this study, biological samples can be complex and storage conditions can greatly affect the ratio of components within the mixture and the rate of product degradation. This experiment emphasizes the importance of storage conditions when working with biological samples. Students write a report that details their experimental protocol, results, and conclusions. They report the quantity of all-trans-lycopene originally isolated, quantity remaining after each storage treatment, and the quantity of cis-lycopene present after storage. This experiment employs a concise method to extract, purify, and analyze carotenoid content of tomatoes. The HPLC method can resolve cis- or trans-lycopene isomers efficiently using a C18 column. Although our experiment compared the effects of light and dark in varied oxygen environments to stability of lycopene isomers it is recommended that students only examine one variable (i.e., light versus dark). This experiment can be expanded to include analyzing carotenoid content of other fresh and processed fruits and vegetables, as well as investigating industrial food preparations and their effect on trans- or cis-lycopene content by extracting tomato juice, tomato paste, and different varieties of spaghetti sauces and salsa. Notes 1. There are many cis isomers that can be formed when isomerizing the all-trans isomer. For this experiment we do not specify the actual isomers, but refer to the isomers as a general group called cis isomer or cis-lycopene. 2. These isomers have one cis bond and the rest are trans bonds. 3. Stability of lycopenes were not determined under dark, oxygen storage conditions. 4. Statistics performed with JMP Statistical software package using an ANOVA analysis with a Tukey–Kramer post-hoc test. 5. Visually from the plot the error bars do not overlap but when statistical analysis was performed using the ANOVA analysis with a Tukey–Kramer post-hoc test they were found be “statistically similar”.
6. Krinsky, N. I.; Rock, C. Carotenoids: Chemistry, Sources and Physiology. In Encyclopedia of Human Nutrition, Sadler, M. J., Starin, J. J., Caballero, B., Eds.; Academic Press: London, 1998; pp 304–314. 7. Takeoka, G. R.; Dao, L.; Flessa, S. J. Agric. Food. Chem. 2001, 49, 3713–3717. 8. Britton, G. FASEB J. 1995, 9, 1551–1558. 9. Ronen, G.; Cohen, M.; Zamir, D.; Hirschberg, J. Plant. J. 1999, 17, 341–351. 10. Agostinis, P. A.; Vandenbogaerde, A.; Donella, D.; Pinna, L.; Lee, K.; Goris, J.; Merlevede, W.; Vandenheede, J. R.; de Witte, P. Biochem. Pharmacol. 1995, 49, 1615–1622. 11. Boileau, A. C.; Merchen, N. R.; Wasson, K.; Atkinson, C. A.; Erdman, J. J. W. J. Nutr. 1999, 129, 1176–1181. 12. Boileau, T. W.; Boileau, A. C.; Erdman, J. W. Exp. Biol. Med. 2002, 227, 914–919. 13. Moraru, C.; Lee, T.-C. J. Agric. Food. Chem. 2005, 53, 8997–9004. 14. Unlu, N. Z.; Bohn, T.; Francis, D.; Clinton, S. K.; Schwartz, S. J. J. Agric. Food. Chem. 2007, 55, 1597–1603. 15. Hakala, S. H.; Heinonen, I. M. J. Agric. Food. Chem. 1994, 42, 1314–1316. 16. Xianquan, S.; Shi, J.; Kakunda, Y.; Yueming, J. J. Med. Food. 2005, 8, 413–422. 17. Shi, J.; Le Maguer, M. Crit. Rev. Biotechnol. 2000, 20, 293–334. 18. Rodriguez-Amaya, D. B. J. Chrom. Sci. 1988, 26, 624–629. 19. Barba, A.; Hurtado, M.; Mata, M.; Ruiz, V.; Tejada, M. Food Chem. 2006, 95, 328–336. 20. Young, J. A. J. Chem. Educ. 2008, 85, 628. 21. Lee, M. T.; Chen, B. H. Food Chem. 2002, 78, 425–432. 22. Shi, J.; Wu, Y.; Bryan, M.; Maguer, M. L. Nutr. Food. 2002, 7, 179–183. 23. Ax, K.; Mayer-Miebach, E.; Limnk, B.; Schuchmann, H.; Schubert, H. Eng. Life. Sci. 2003, 4, 199–201. 24. Wong , F.; Francis, G.; Bohart, S. G. Food Technol. 1957, 293–296.
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2009/Nov/abs1304.html Abstract and keywords
Literature Cited
Full text (PDF) with links to cited JCE article
1. Fraser, P.; Bramley, P.; Seymour, G. Phtyochem. 2001, 58, 75–79. 2. Canene-Adams, K.; Campbell, J. K.; Zaripheh, S.; Jeffery, E. H.; Erdman, J. W. J. J. Nutr. 2005, 135, 1226–1230. 3. Sakar, M. K. Fitoterapia 1990, 61, 478. 4. Djuric, Z.; Powell, L. C. Int. J. Food Sci. Nutr. 2007, 52, 143–149. 5. Kakunda, Y.; Shi, J.; Yueming, J.; Xianquan, S. J. Med. Food. 2005, 8, 413–422.
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Instructor notes
JCE Featured Molecules for February 2008 Structures of some of the molecules discussed in this article are available in fully manipulable Jmol format in the JCE Digital Library at http://www.jce.divched.org/JCEWWW/Features/ MonthlyMolecules/2008/Feb/index.html.
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