Alyssa Williams December 8, 2012 Functional Food Lab Gallic Acid Trail Mix Purpose: The purpose of this

s lab was to showcase gallic acid as a phytonutrient in an original recipe that is both appealing and has health benefits due to the present of the phytonutrient.

Abstract: Phytonutrients are substances found in plants that have been found to have health benefits. Usually they are known for their antioxidant or ant carcinogenic properties. A functional food is an edible item that has a purpose to improve health in the form of disease prevention or overall physical/mental health maintenance. This is different than a nutraceutical because, a nutraceutical is usually a nutrient that is purified to take in the form of a supplement rather than incorporated into an edible item (Brown, 2011). Gallic acid is a phytonutrient that has been found to have health benefits as an antioxidant, anti-carcinogen and anti-obesity agent (Nabavi et al, 2012) (Elango et al, 2011) (Hsu and Yen, 2007). Including a phytonutrient in foods can have health benefits without having to supplement with concentrated pills. Gallic acid has a poor absorption rate due to it being a water soluble polyphenol. The supplementation in pill form would also be poorly absorbed as it would be in an isolated state. Background: The phytonutrient Gallic Acid is a polyphenol thought to have powerful health benefits. It is synthesized as a byproduct in the pathway responsible for producing tannins in plants such as tea, cocoa and walnuts. This polyphenol is a byproduct of the Shikamate pathway, responsible for producing tannins. This process in which tannins are produced contains the enzyme Shikamate Dehydrogenase (SDH) that is responsible for the step of the pathway that turns 3-dehydroshikamate (3DHS) into aromatic compounds such as L-tryptophan. 3DHS however, also has the ability to take a different route in the pathway and produce Gallic Acid via

NADP+, changing 3DHS from a ketone to an alcohol. In a study, the SDH enzyme was cloned from the walnuts SDH enzyme and used to produce Gallic Acid successfully in vitro. The same study tested several plans known for containing the SDH enzyme and producing Gallic Acid and found that the walnut showed one of the most active rates of producing GA (Muir et al 2011). As most phytonutrients, Gallic Acid is known mainly for its antioxidant effect. It has long been in question, however, if phytonutrient properties are influenced by their combination with other nutrients in their native state or if they are as effective when isolated. GA was used in an experiment its scavenging ability was tested both independent of other compounds and as part of a plant based sugar solution. The scavenging properties were tested on superoxide anion radical (O2-) and on hydroxyl radical (OH). The experiments showed that although GA alone had noticeable activity in the scavenging of the free radicals, the activity was increased when part of a sugar solution against hydroxyl radicals. This suggests that GA in the presence of sugars, as it would be found in its natural state in fruit, may be beneficial when fighting off hydroxyl radicals. This data supports a popular idea that obtaining nutrients from their source rather than a supplement would be more beneficial. Another piece of information concluded in this study was that when the GA was measured for its scavenging activity in high doses, it actually worked as a pro oxidant, creating more free radicals. Valuable information from this study suggests that GA is an effective antioxidant but as most antioxidants can have harmful effects in dangerously high doses. Consuming the nutrient through the consumption of plant products would be both a safe and effective way to obtain the benefits of Gallic Acid (Lo Scalzo, 2010). This polyphenol is also widely known for its antioxidant activity as one of the polyphenols found in tea. Although they are found in small amount, gallic acid can still be extracted from black tea types such as Uva and C. Ptilophylla leaves (Wang et al, 2010). Teas

polyphenols including Gallic Acid have also been shown to have protective benefits for human liver cells. A study published in the Journal of Medical Plants Research tested its effects against liver cells exposed to hydrogen peroxide and carbon tetrachloride damage. Treatment of these exposed cells with antioxidants of tea polyphenols, gallic acid or alpha tocopherol significantly decreased amounts of markers of apoptosis and lipid peroxidation such as levels of lactate dehydrogenase. Since hydrogen peroxide and carbon tetrachloride are compounds related to oxidative stress, the protective effect gallic acid and tea polyphenols have against their damage showcases the phytonutrients health benefits (Li, Zhang, Zhao, 2010). Antioxidants can play an important role in the protection of many of our tissues, including brain tissue. Damage to our brain tissue can cause serious neurological conditions that can change our lives forever. Elements such as fluoride have been found to have toxic effects on our brain tissue. As other compounds can induce oxidative stress in body tissue, fluoride has been found to be one cause of oxidative stress in brain tissue. Antioxidants such as gallic acid and vitamin c were both used in experiments to measure their protective effects against the damage done by fluoride. Both gallic acid and vitamin c proved worthy soldiers in the battle against lipid peroxidation in the brain but, gallic acid displayed a stronger effect (Nabavi et al, 2012). Neurological disorders such as Parkinsons disease also have shown beneficial effects of intake of antioxidants such as gallic acid since the death of neurons in the brain are related to oxidative stress. Gallic acid derivatives were tested in their ability to inhibit catalepsy in the same manner in which drugs used to treat Parkinsons work. The derivative Triacetoxy Butyl gallate displayed the most promising results (Kasture et al, 2012). The evidence of this study only adds to the neurological benefits of gallic acid as an antioxidant.

Gallic Acid has been known as a phytonutrient displaying antioxidant properties but a study published in the British Journal of Nutrition experimented with the theory that it also has antiobesity properties. Those designing the experiment went off the idea conclude from previous experiments that GA has the ability to prevent adipogenesis in human cell lines. In this study rats that were fed high fat diets were broken up into three categories in which one received no GA supplementation and the other two groups received either high or low dose supplements. The results of the study showed promising evidence for GA as a supplement to improve the health condition of those consuming a high fat diet. The rats that were fed a high fat diet with supplemented GA had lower overall body weight, hepatic weight, adipose tissue weight, serum triacylglyceride levels, serum phospholipids levels and serum cholesterol levels than those who received the high fat diet not supplemented with GA. These discoveries could open doors for more research into GA as beneficial in terms of lipid related disease such as fatty liver disease (Chin-Lin, Gow-Chin 2007). The discovery of Gallic Acid as an anti-obesity agent triggered many studies on the subject. An article in The Journal of Olea Science discussed similar studies involving phytonutrients of tea having similar effects the study from the British Journal of Nutrition. With the consumption of these phytonutrients it was found that excretion rates were higher suggesting that absorption of cholesterol was decreased. The mechanism by which this occurred was said to be due to the inhibition of digestive enzymes. Gallic Acids effect in this process was studied both as an isolated compound and as a synthetic compound created as a glycerol ester when it was combined with 1,2 dioleoylglycerol. It was found that GA displayed more benefits in the high fat diet scenario when supplemented as a synthetic ester compound rather than an independent

molecule. This may be related to solubility aspect of GA. Both GA and glycerol are water soluble but glycerol also has the ability to bind with fatty acids (Totani et al 2011). The solubility of Gallic Acid has also been a topic of discussion because it is known for having a low bioavailability for this reason. Due to the many health benefits of GA intake, the quest to improve absorption when supplemented has led some scientists to the creation of synthetic forms of the polyphenol. A study published in the International Journal of Research in Pharmacy and Science tested the absorption and effectiveness of Gallic Acid as a complex with phospholipids in efforts to overcome this barrier. When the correct ratio of GA and the phospholipid 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) was determined to create the complex, its scavenging activity was then tested against independent Gallic Acid and a mixture of GA and DPPH. Not only was the solubility of the GA and DPPH complex noticeably higher but, the scavenging activity was also higher than the mixture or isolated Gallic Acid. This discovery makes much leeway in the use of natural compounds such as GA in treatment of disease (Shyam et al 2012). Natural treatment of disease is an important area of research because much medicine used today has undesirable side effects. In study in which rats were supplemented the GA glycerol ester, they noted health benefits without negative side effects (Totani et al 2011). Many other disease states have also been correlated with health benefits from Gallic Acid. With chemotherapy being a less than pleasant treatment for cancer, GA is also being considered as one alternative. Four groups of cancerous human cells (known as A549 cells) were tested amongst three different concentrations of Gallic Acid with one group being a control. After twenty four hours, the four groups of cells were tested for various levels of apoptosis by measuring lactate dehydrogenase, DNA fragmentation and caspase enzyme. All of these are markers of cell death.

The results of the study showed that the two higher doses of Gallic Acid ad 10 and 20 ug/mL were effectively displaying ant carcinogenic properties by inducing apoptosis as evidence by increased lactate dehydrogenase and increased DNA fragmentation (Elango et al, 2011). Aside from being found as an effective treatment for cancer cells and decreased absorption of fat, Gallic Acid is also widely studied for its cardiovascular benefits. Gallic Acid was tested against the effects of isoproterenol (ISO), a catecholamine known for its relation to myocardial infarction, in rats. Four groups of rats were created with a control group, a group receiving Gallic Acid supplementation, a group receiving ISO via saline, and a group receiving both Gallic Acid supplementation and ISO via saline. Two doses of ISO were administered to the appropriate groups and after twelve hours the data was collected. Classic serum markers for myocardial infarction such as CK-MB and Lactate Dehydrogenase were measured in all groups. The administration of isoproterenol significantly increased the serum markers of MI in the rats receiving only ISO in comparison to groups that did not receive ISO. In the group receiving both ISO and Gallic Acid the serum markers were significantly decreased, suggesting that these rats risk of myocardial infarction was decreased. With the only difference in the groups being supplementation of Gallic Acid, it can be concluded that the polyphenol has cardiovascular benefits in protection against myocardial infarction (Priscilla et al, 2010)

Materials: One 8 oz bag halved walnuts Two 3.5 oz 85% Cacao Bars Two 5.3 oz Pomegranate Seeds

Two black tea bags 1 Tbsp pomegranate extract 4 oz water Double boiler Dehydrater Sheet pan Wax paper Large spoon Tablespoon

Procedure: Place pomegranate seeds into dehydrator for about 6 hours or until raisin like consistency. Set up double boiler with water over very low heat. Chop both cacao bars into small pieces Gradually place pieces of cacao bar in double boiler, stirring constantly. Continue adding and stirring until all the chocolate is melted and a smooth consistency. As chocolate is melting, boil 4 oz of water and steep two black tea bags. Once chocolate is smooth, velvety consistency stir in 1 Tbsp pomegranate extract and 1 Tbsp black tea. On sheet pan place wax paper and spread walnuts and dried pomegranate seeds Drizzle melted chocolate over walnuts and pomegranate seeds Refrigerate until chocolate is hard

Results: The gallic acid trail mix came out both aesthetically appealing and delicious. The pomegranate concentrate used to infuse the dark chocolate was powerful in taste which sweetened the bitterness of the chocolate effectively even with the small amount used. The walnuts and pomegranate seeds also gave a satisfying variety of texture. The more bitter taste of the walnuts complimented the sweetness of the chocolate and pomegranate seeds. Fear that the chocolate would not harden properly because liquid was added was disproved. This leaves potential room for more tea or concentrate to be added to enhance the nutritional value. Another concern was the result of dehydrating the pomegranate seeds. Their high water content brought up the potential issue that dehydration would strip them of any sort of flavor. Fortunately, they were able to be dried enough to achieve a raisin-like texture without losing all of their contents. This could potentially cause them to lose some of their gallic acid due to its water soluble properties.

Product Gallic Acid Trail Mix

Appearance Overall fresh, chocolate looks velvety, pomegranate seeds look wrinkled, sticky

Aroma nutty, slightly sweet

Flavor somewhat bitter, sweet aftertaste from pomegranate

Texture crunch from nuts, soft/chewy pomegranate seeds,

Consistency crunchy nuts, smooth chocolate, juicy/sticky pomegranate

smooth/creamy seeds chocolate

Discussion/Conclusion: My recipe is a success in being a tasty treat that is rich in in the polyphenol, gallic acid. Three of the elements of the recipe are well known sources of the phytonutrient featured. The pomegranate concentrate itself is marketed as a polyphenol supplement rich in gallic acid with as much as 165 mg of polyphenols per tablespoon. The only ingredient in the extract however, is pomegranate juice. Also, as the studies showed gallic acid activity improved when bound to sugars or lipids, both macronutrients are also present in the recipe. This allows for ample opportunity for nutrient absorption and utilization. A food product is a better choice of nutrient intake rather than a supplement. This product is both appealing to the senses and a valuable source of gallic acid. Supplements can be a chore for most people and something than can be forgotten but, when obtaining nutrients is a pleasure it is more likely one would keep up with it. A functional food such as this trail mix can serve as an enjoyable way for one to increase their phytonutrient intake. The sweetness of the recipe with variety of textures also gains positive feedback from the senses. Although ingredients such as the 85% cacao and walnuts are energy dense due to their high fat content, the overall dish is rich in nutrients such as gallic acid. Any improvements made upon this dish could include using a lower fat cacao. More black tea could possibly be added to the chocolate as well. Only a small amount of tea was incorporated into the recipe. If the resources were available, it would be insightful to measure the absorption gallic acid from this functional food. Gallic acid has been shown to be absorbed more readily in the form of a complex with a lipid. Analyzing the chocolate mixture in the lab may display if any

sort of complex was formed when the extract was mixed with the cacao as it has a bountiful amount of fat. If so it could be beneficial to consumer absorption.

References: Brown, A. (20100) Understanding Food Principles & Preparation. California: Wadsworth. Elango, S., Balwas, R., Padma, V.V., (2011) Gallic acid isolated from pomegranate peel extract induces reactive oxygen species mediated apoptosis in a549 cell line Journal of Cancer Therapy 2, 638-645 Hsu, C., and Yen, G. (2007). Effect of gallic acid on high fat diet-induced duslipdaemia, heptosteatosis and oxidative stress in rats. British Journal of Nutrition , 98 pp 727-735 doi:10.1017/S000711450774686X Kasture, V.S., Kasture, S.B., Sahu, R.K., Kumar, A., Musmade, D.S. (2012) Synthesis and antioarpkinson activity of gallic acid derivatives Medicinal Chemistry & Drug Discovery 3(2) 134-139 Li, T., Zhang, X., Zhao, X., (2010) Powerful protective effects of gallic acid and tea polyphenols on human hepatocytes injury induced by hydrogen peroxide or carbon tetrachloride in vitro Journal of Medicinal Plants Research 4(3) 247-254 Lo Scalzo, R. (2010) Measurement of free radical scavenging activity of gallic acid and unusual antioxidants as sugars and hydroxyacids Electronic Journal of Environmental, Agricultural and Food Chemistry ISSN 1579-4377 Muir, R., Ibanez, A., Uratsu, S., Ingham, E., Leslie, C., McGranahan, G., Neelu, B., Goyal, S., Joseph, J.,Jemmis, E., Dandekar, A. (2011) Mechanism of Gallic Acid Biosynthesis in Bacteria (Escherichia coli) and Walnut (Juglans Regia) Plant Molecular Biology 75:555-565 Nabavi, S.F., Habtemariam, S., Jafari, M., Sureda, A., Nabavi, S.M. (2012) Protective role of gallic acid on sodium fluoride induced oxidative stress in rat brain Bulletin of Environmental Contamination and Toxicology 89; 73-77 Priscilla, D.H., Prince, P.S.M., (2010) Gallic acid pretreatment ameliorates lipids, lipoproteins and HMG-CoA reductase in normal and isoproterenol induced myocardial infarction in male Wistar rats Journal of Pharmacy Research 3(5) 960-964 Shyam, K.R., K, M., Kumar, G.M., (2012) Preparation, characterization and antioxidant activities of gallic acid-phospholipids complex International Journal of Research in Pharmacy and Science 2(1) 138-148 Totani, Nagao., Tateishi, S., Takimoto, T., Madea, Y., Sasaki, H. (2011) Gallic acid glycerol ester promotes weight-loss in rats Journal of Oleo Science (9) 457-462

Wang, Y., Yang, X., Li, K., Li, C., Li, L., Li, J., Huang, H., He, Y., Ye, C., Song, X. (2010) Simultaneous determination of theanine, gallic acid, purine alkaloids, catechins, and theoflavins in black tea using HPLC International Journal of Food Science & Technology 45 1263-1269