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Polyphenols as Supplements in Foods and Beverages
C H A P T E R
7 Polyphenols as Supplements in Foods and Beverages: Recent Methods, Benefits and Risks Andre´a Pittelli Boiago Gollu¨cke*, Daniel Araki Ribeiro† and Odair Aguiar Junior† *Nutrition Department/HEXALAB, Catholic University of Santos. Av. Conselheiro Ne´bias, SP, Brazil †Department of Biosciences, Federal University of Sa˜o Paulo, Santos, SP, Brazil
1. INTRODUCTION
industry. The survey asked 900 physicians and 277 nurses about their use of dietary supplements. The authors found that 72% of the physicians and 89% of the nurses used dietary supplements on a regular, occasional or seasonal basis. Regular use was reported by 51% of the physicians and 59% of the nurses. Moreover, 79% of the physicians and 82% of the nurses said they recommended supplements to their patients, including the ones who did not use supplements themselves. These findings are in accordance with national surveys, including the National Health and Nutrition Examination Survey (NHANES) of 1999 2000. The dietary supplement product most commonly used was the multivitamin, with or without minerals. Amongst the non-vitamin/mineral products, the physicians and nurses reported the consumption of green tea, fish oil, glucosamine, soy, flax seed, chondroitin and Echinacea as supplements. Interestingly, green tea and soy, two of the most cited supplements, contain polyphenols as bioactive compounds.
Polyphenols in foods and beverages are related to sensorial qualities such as color, bitterness, astringency, etc., which are relevant in products such as wine, tea and grape juice.1 5 These compounds occur naturally in forms varying from simple phenolic acids to complex polymerized tannins. Due to their inherent instability, polyphenols undergo transformations in the presence of light, oxygen, and as a result of heat processing, storage and extraction procedures.6 8 For that reason, daily consumption and dietary requirements for these compounds are difficult to measure. Although considered a powerful antioxidant, recently new mechanisms for polyphenols physiological effects have been proposed. For example, modulation of gene expression, induction of apoptosis, a decrease in platelet aggregation, an increase in blood vessel dilation, modulation of intercellular signaling, modulation of enzyme activities associated with carcinogen activation and detoxification and chelation of transition metals, such as iron.9 11 The alleged knowledge that polyphenols are associated with protection against diseases has raised a distinctive interest. Meanwhile, in western countries, the intake of fruits and vegetables, a main source of polyphenols, is considered to be insufficient. Consequently, supplementation appears to be a viable alternative. In 2009, Dickinson and colleagues12 published an online survey conducted in 2007 by Ipsos Public Affairs for the Council for Responsible Nutrition (CRN), a trade association representing the dietary supplement
Polyphenols in Human Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-398456-2.00007-4
2. METHODS FOR SUPPLEMENT PREPARATION The abundance of bioactive polyphenols in fruits, teas and their by-products such as pomace, skins and seeds brought new possibilities to food researchers and industries to develop new products/supplements. With that in mind, many new supplements have been developed using these products. In 2009, Alkayali Ahmad was
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granted a patent for inventing a method of preparing pomegranate extract (rich in ellagic acid) from pomegranate seeds.13 The product, in powder form, is obtained by ethanol extraction followed by concentration and drying. An interesting process for obtaining cocoa products rich in polyphenols was described by Pons-Andreu and colleagues.14 The method is based on producing liquid and powered cocoa polyphenol concentrate from unfermented cocoa beans. The possibility of increasing the polyphenols content in cocoa powder has a positive impact on its antioxidant content and also flavor. The total polyphenol content in unfermented, unroasted, defatted dried cocoa beans varies from 12 to 20% (in weight). The main polyphenols in cocoa beans under these conditions are catechins, dimers, and other oligomeric flavonoids. The extraction process involves blanching, followed by drying of unfermented beans up to 15% moisture content, grinding and finally polyphenol extraction with preferably polar solvents for human consumption (water, ethanol or both). In contrast, Jacob and colleagues15 proposed a method to obtain a fermented supplement from pomegranate, claiming that the aglycones resulting from the fermentation of polyphenol-glycosides possess enhanced physiological properties. The authors proposed the use of the yeast Saccharomyces boulardii and at least one species of lactobacilli followed by a freeze drying process to reduce water volume and obtain the final product. A more complex extraction procedure takes place to produce an algae extract rich in polyphenols to be used against inflammation processes. In the phylum of brown algae (Phaeophyceae), the phlorotannins are derived by polymerization from one and the same monomer: phloroglucinol (1,3,5-trihydroxybenzene). The extraction of polyphenols starts with the ground algae and is based on a solid/liquid extraction in the presence or not of added water. After centrifugation the solution undergoes membrane filtration or chromatographic purification of the desired polyphenols. Then the extract is eluted in alcohol and spray-dried to its final commercial powder. This pharmaceutical product, according to the invention, can be used as such or integrated in a food matrix.16 One unusual source of polyphenols to be used as supplements is a beer waste-product. Taidi and colleagues17 proposed a method of recovering the polyphenols removed from beer during process. Some specific polyphenols in beer are undesirable due to their association with proteins forming insoluble compounds causing permanent cloudiness after cold storage. In the brewing industry, these polyphenols are removed from the beer by a PVPP (polyvinyl polypyrrolidone) resin. The authors suggested a method for recovering these bioactive compounds and then applying the end product in the cosmetic/food/nutraceutic industry. The inventors found catechin, epicatechin, tyrosol and ferulic acid
as relevant polyphenols in this beer by-product extract. The application of grape seed oil in foods, food supplements, additives, medicaments and cosmetic products has also been recently reviewed. In 2007, Eckert and colleagues18 described a method for the preparation of cold-pressed grape oil, crushed grape and grape flour. Grape seed oils contain important amounts of polyphenols such as catechins and procyanidins. The concentration of polyphenols in grape seed oil is dependent on the extraction process, especially the pressing temperature. The crushed seeds obtained in the process are also a viable source of polyphenols, which could undergo further extraction with water or other solvent mixtures. Ibarra and Zagiary19 developed a process in which olive polyphenols concentrate is obtained by mixing the waste-product of pressing with a polar solvent. Polyphenols are further extracted from the mixture and then concentrated up to 10% (w/w) by the use of membranes. The main compounds found in this polyphenol extract are oleuropein, demethyuloleuropein and logstroside. Aware of the polyphenols presence in fruit processing residues, the above cited patents have presented advanced extraction methods for skins, seeds, etc. Methods using hot water extraction and an adsorbent resin followed by concentration and purification of polyphenols seem to be a promising challenge.
3. FORMULATIONS USING POLYPHENOLS Recents patents describing polyphenol-rich supplements frequently use green tea or grape products. In 2009, the effects of a multi-phytonutrient supplement on DNA damage in skin cells exposed to UV rays was investigated. The supplement was composed of several vitamins, minerals and natural extracts such as green tea, pomegranate, citrus fruits, acai and different types of berries.20 Howard and colleagues21 produced a dry concentrate by distillation and concentration of either wine or grape juice. The concentrate was then filtered and eluted with a mixture of water and ethanol (50:50). The intake of 1 2 g of the powder for 2 weeks improved platelet aggregation indicators, suggesting a positive impact on cardiovascular health. Earlier, the main author had described a method of concentrating grape flavonols from wine, juice or dealcoholized wine, grape skins and fermented grape skins. In this invention, several solvents were tested as eluents. Although the organic solvents are eliminated at the drying stage, the use of water would be preferable. The stability of polyphenols in solutions is also a concern. In 2012, a method for dispersing microparticulated water-insoluble bioactive polyphenol in a beverage was described.22 In this form, the polyphenols
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4. BENEFITS OF POLYPHENOL CONSUMPTION: EXPERIMENTAL DATA
added to the drink are stabilized as suspended particles and do not separate from the water-based beverage. Mower and Brady23 had earlier described the invention of a beverage having resveratrol mixed in water with other ingredients such as energy agents, preservatives, pH balancing and colorant additives. The method uses nanosize water clusters and nanoparticles of resveratrol. With this system, it is possible to enhance polyphenols amounts in beverages and thus improve bioavailability. In an interesting case, Schiffelers and colleagues24 tested parenteral administration of a pharmaceutical composition containing polyphenols in mice inoculated with B16 murine melanoma and C26 murine colon carcinoma cells. The authors propose a method for targeting polyphenols to the tumor tissue using liposomes. After a week, effective inhibition in tumor growth was observed at dosages of 1.0 to 10 mg liposomal caffeic acid (per kg body weight per week) when compared with controls. The pharmaceutical composition comprised colloidal carriers where the polyphenols or polyphenol derivatives could be entrapped. Some supplements for foods and beverages have incorporated fruit and vegetable extracts to enhance health appeal. In 2006, Wild and Sass25 developed a concentrated food ingredient comprising extracts of green tea, grape skin and grape seed, yielding a polyphenol content of 1155 mg gallic acid/L in order to improve antioxidant appeal to commercial beverages. Shrikhande and colleagues26 maximized the extraction of monomeric and oligomeric procyanidins to obtain a final product with 15% of monomers and 20% of dimers and up to 30% of trimers, tetramers and pentamers, by weight. The extraction procedure was the use of hot water and pectolytic enzymes for at least 2 hours. Formulated dry mix products to be used in beverages, nutraceutical capsules and supplements containing vitamins and minerals were further proposed using this extract. In 2008, Perlman and colleagues27 presented an interesting use of the waste-products of the grape juice industry. The authors proposed the fortification of grape juice with the grape pomace polyphenol extract. The new beverage was tested at different extract concentrations for sensorial acceptance and antioxidant activity. An unacceptable astringency was noticed at 4% (by weight) with antioxidant activity increasing three times. Cyclodextrin, added at 0.2% by weight, was able to successfully reduce the astringent sensation. In 2009, Draijer and colleagues28 proposed a mixture of red wine and grape polyphenols (total 800 mg polyphenols) added to a soy drink (200 mL). The beverage was given daily to 35 males with a positive impact on the blood pressure. Recently, Rao and colleagues29 investigated the antioxidant potential of a commercial product (Greens1 ) using a
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liposome model for the in vitro assessment and 10 healthy human subjects for the in vivo observation. The product is a mixture of several ingredients from vegetable powders to bifidobacteria and including polyphenols. In this case, the sources of polyphenols (30 mg/8.5 g final product) are green tea, bilberry and grape extract and 500 ppm resveratrol. The results showed the supplementation with Greens1 increased the serum antioxidant potential and the presence of kaempferol in plasma. A reduction in lipid oxidation was observed indicating a possible positive effect in meliorating chronic diseases. Dietary supplements containing polyphenols have also been proposed for skincare. In combination with other ingredients such as lycopene, vitamin C and E, the mixture is intended to maintain and restore the biomechanical properties of keratinous materials such as connective tissue.30
4. BENEFITS OF POLYPHENOL CONSUMPTION: EXPERIMENTAL DATA The efforts in investigating and proposing new forms of polyphenol supplementation are justified by the encouraging results observed in experimental data in recent years. In spite of the debate in respect to human applicability, animal experimental data confirm the benefits of polyphenol-rich products.31 Resveratrol, for instance, has been considered as a “miracle molecule” for fighting cancer. According to Delmas and colleagues,32 in their extensive review, resveratrol protects cells from DNA adducts formation induced by various chemical agents. Such DNA alterations are responsible for the initiation phase of the tumor, when cells start growing autonomously. Suppression of the metabolic activation and/or increasing of detoxification rate are some of the mechanisms attributed to the resveratrol leading to its anti-initiation property. Also, decreasing the reactive oxygen species (ROS) production, and the consequent procarcinogens activations and oncogenes mutation, is attributed to resveratrol. Also credited to resveratrol is the stimulation of DNA repair, by increasing the p53 activity, the cell cycle progression blockage (in G1, S and G2/M phases), induction of apoptosis in malignant cells, inhibition of inducible nitric oxide synthase (iNOS) (with consequent blockage of the metastasis) and inhibition of angiogenesis; all of them making resveratrol a promising molecule to prevent and treat cancers.32 Treatments with resveratrol were also employed for liver disorders, simulated in several experimental designs.33 35 Bujanda and colleagues33 demonstrated that liver lesions and animal mortality were reduced in alcohol-exposed mice. According to the authors,
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resveratrol might have diminished the release of proinflammatory cytokines (such as IL-1), protecting the liver from damage. Alcohol-induced fatty liver was ameliorated by resveratrol treatment in studies by Ajmo and colleagues,34 who found that this polyphenol was able to reduce lipid synthesis and increase the rates of fatty acid oxidation, preventing alcoholic liver steatosis. The increment in rates of fatty acid oxidation seems to be modulated by increased mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ) coactivator α (PGC-1α) target genes, which encode the fatty acid oxidative enzymes.34 The role of resveratrol was also investigated by Chan and colleagues35 in animal models of cholestatic liver injury. As in the studies by Ajmo and colleagues,34 the survival of mice after resveratrol treatment was higher. Also, inflammatory markers (TNF- α and IL-6) were reduced, hepatic fibrosis also decreased and the number of Ki671 hepatocytes increased, indicating that resveratrol stimulated hepatocyte proliferation.35 Also, when applied to hyperlipidemic rats, resveratrol was shown to be effective in restoring normal physiological conditions. As demonstrated by Zhu and colleagues,36 oral administration of resveratrol to cholesterol-fed rats resulted in lowered serum lipid, hepatic cholesterol and triglycerides, besides an increment of the antioxidant capacity expressed by significantly increased levels of superoxide dismutase (SOD), catalase and glutathione peroxidase. Also, hepatic thiobarbituric acid reactive substances (TBARS) were lowered, possibly by inhibition of the oxidation of LDL, maintaining antioxidant efficacy and denoting the antihyperlipidemic effects of resveratrol. Quercetin, considered the major flavonoid found in the human diet, has also been vastly studied. Relevant results have been found by Tieppo and colleagues37 when working with hepatopulmonary syndrome (HPS), a complication of liver cirrhosis. In an animal model of hepatic cirrhosis (through bile duct ligation), quercetin treatment showed decreased oxidative stress, by restoration of plasma TBARS, SOD and nitric oxide (NO), besides a lower severity of the consequent HPS and less pronounced evolution of the hepatic injuries.37 A slowing down in the cirrhotic process was also observed by Hamed and colleagues38 using quercetin in a model of thioacetamide-induced cirrhosis. The amelioration in the hepatic biochemical, morphological and functional aspects was attributed to a potentiation of the antioxidant defense, breaking the “vicious circle” between oxidative stress and oxidative damage. Pro-apoptotic properties have also been ascribed to quercetin by Bulzomi and colleagues.39 This polyphenol was demonstrated to mimic the apoptotic effect of 1λ-estradiol (E2) in two transformed cell lineages, leading to the activation of the p38
MAP-kinase which, in turn, is responsible for caspase3 activation and apoptosis. Such a role for quercetin, acting at the estrogen receptor (ER), ensures an anticarcinogenic potential to this molecule. Also, when applied to diabetic mice (by induction with streptozotocin), quercetin was shown to result in the lowering of blood glucose and improving plasma insulin levels.40 The authors suggested that quercetin might improve liver and pancreas functions by suppressing the streptozotocin-induced expression of the cell-cycle inhibitor CDKN1A, restoring the cell proliferative capacity. The interference of quercetin with the cell cycle regulation in cancer cells has also been very well documented in the extensive review by Gibellini and colleagues.41 According to those authors, quercetin has many molecular targets within the cycle including cyclin-dependent kinases (CDKs) and cyclins, making this polyphenol able to block the cell cycle progression at various transition points, depending on the cancer cell lineage investigated. Quercetin also acts in multiple ways to influence the p53 activity, leading cancer cells to apoptosis and also contributing to improve the cell fighting against ROS, since p53 has been found to regulate a series of genes related to oxidative stress mitigation.41 A pro-oxidant property to quercetin was also proposed by Vargas and Burd.42 When at high concentrations (greater than 40 μM) quercetin is cytotoxic leading cells to apoptosis. Such a property puts this molecule in a good status to be used as an adjuvant to current chemotherapies. Curcumin has well-known benefits for a series of pathological conditions. According to Chiu and colleagues,43 diabetic nephropathy is a consequence of the high glucose levels (and concomitant increased level of ROS), leading to an increase of the extracellular matrix proteins which, in turn, is responsible for the thickening of the glomerular basement membrane. When applied to diabetic rats, curcumin prevented renal lesions and mesangial matrix expansion. The authors attributed to curcumin, besides a neutralization of the oxidative stress, an inhibition of p300, a histone acetiltransferase, which upregulates specific genes in association with nuclear factor-κB (NF-κB).43 As with the other polyphenols cited above, curcumin has a pleotropic effect, which includes pro-apoptotic properties, gene regulation, cell cycle control (proliferation arrest), which indicates that curcumin is now used in phases II and III clinical trials for a variety of cancer types.44 According to Aggarwall and colleagues,45 curcumin can suppress tumor initiation, promotion and metastasis, being also a potent antiinflammatory agent. Tea is one of the most consumed beverages in the world. Green tea (GT) polyphenols have been found to
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5. NOXIOUS ACTIVITIES INDUCED BY POLYPHENOLS: AN INTRIGUING ISSUE
decrease oxidative stress, which has been implicated in the pathogenesis of some neurodegenerative diseases.46 The authors showed that GT polyphenols protect the neural cells against oxidative stress-induced NO toxicity, attenuating NO-induced apoptotic cell death by modulating pro-apoptotic gene expression. GT polyphenols have also been found to maintain the bone architecture in females with and without ovariectomy.47 It has been described that estrogen is a reactive oxygen species scavenger and that its decreasing with advanced age accelerates bone loss by increasing the oxidative stress and reducing thiol antioxidant defenses in osteoclasts.47 These authors have found that GT consumption was able to maintain bone microarchitecture by increasing bone formation and suppressing bone erosion. Also, GT polyphenols have been found to have a beneficial role in neurodegenerative diseases.48 Besides the antioxidant properties, GT seems to modulate various protein-kinase signaling pathways, mainly involving protein-kinase C (PKC) whose activation generates cytoprotection by apoptosis prevention, so preventing or delaying the neuronal loss in the neurodegenerative diseases.48
5. NOXIOUS ACTIVITIES INDUCED BY POLYPHENOLS: AN INTRIGUING ISSUE Few studies have discussed the injurious effects closely related to the toxic potential of polyphenols. In 2010, Keith and colleagues49 published a review about the possible toxicity of high-dose intakes of polyphenols through supplementation. The consumption of natural compounds such as polyphenols, found in innumerable foods, is commonly considered safe. The doses used in supplements, however, are often much higher than those found in normal daily intake. Halliwell50 summarized the pro-oxidant effects observed in in vitro studies. In the presence of iron, high pH and depending on concentration, polyphenols can begin auto-oxidation. Other mechanisms may also be involved in the toxic effects of polyphenols in high-dose supplementation; for example, soy foods and breast cancer association. There has been considerable investigation on the potential for soy foods to reduce risks of breast cancer due to the presence of isoflavones, compounds that bind to estrogen receptors, exhibiting weak estrogen-like effects. Recently, however, concerns have been raised suggesting that isoflavones could stimulate the growth of estrogen-dependent breast tumors.51 The contradiction is supported by other evidence. In an in vivo rodent model, ovariectomized athymic mice fed genistein and genistin both displayed enhanced growth of mammary tumors.52,53 Again, the dosage seems to play a fundamental role. Hepatoxicity by high dose intake of tea polyphenol supplementation
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has been described in human studies.54 Other possible toxic effects have been reported.55 57 Nevertheless, to date, there is not enough evidence to support that a daily intake compatible with the Asian soy food habit is detrimental to health. Harmful effects of some polyphenols can be dubious. Studying the luminal surface of the gastrointestinal tract, covered by a protective mucus gel layer, D’Agostino and colleagues58 have demonstrated that epigallocatechin gallate was very toxic to the HT29 cells. However, the substance was less toxic to the HT29-MTX-E12 cells, suggesting that the mucus gel layer on the HT29-MTX-E12 cells is able to protect the cells against epigallocatechin gallate toxicity. In contrast, epicatechin had no effect on the viability of either the HT29 or HT29-MTX-E12 cells, suggesting that proteins within the mucus gel layer on the apical surface of gut epithelial cells may bind to the galloyl ring of epigallocatechin gallate. In the same way, some authors have postulated that the antioxidant capacity of the tested polyphenols quercetin and epigallocatechin gallate is due to their stabilizing effect on the cell membranes, thus contributing to cell protection in various pathologies and as adjuvant therapy in highly toxic treatment regimens.59 Epigallocatechin gallate also inhibited β-glucuronidase activity in native Hepa 1c1c7 mouse hepatoma cells, while it failed to affect the enzyme in alamethicin-permeabilized cells, where the endoplasmic membrane barrier was eliminated. Such findings indicate that tea flavanols inhibit deglucuronidation in the endoplasmic reticulum at the glucuronide transport stage.60 Epigallocatechin gallate induced apoptosis in the carcinoma HSC-2 cells, but not in the normal HGF-2 fibroblasts.61 This research supports those studies suggesting that GT is an effective chemopreventive agent of oral carcinoma cells in vitro. Mice exposed for 28 days at doses of 0, 30, 300, and 3000 mg/kg body weight/day of quercetin, which are equivalent to 5, 50, and 500 times, respectively the estimated mean human intake of these polyphenols (25 mg/ day), revealed no mortality during the experimental period.62 No significant body weight gain in the male or female groups was also observed. Red blood cell numbers and the hematocrit increased after polyphenol administration compared to control groups. Biochemical parameters were not affected. Histopathological examination revealed no alterations in clinical signs or organ weight at any dose.62 Resveratrol at a concentration of 10 μM or more (up to 100 μM) led to a significant dose-dependent increase in the population of dead cells, shrunken living cells, annexin V-positive cells and cells with hypodiploidal DNA. In the presence of benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK), a pan-inhibitor of caspases, the resveratrol-induced increase in the population of cells with hypodiploidal DNA was partially
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inhibited.63 Overall, it is suggested that resveratrol at a concentration of 10 μM or more induces apoptosis in normal cells as well as cancer cells. Thus, at concentrations that are suitable for chemopreventive and chemotherapeutic actions, resveratrol may exert a cytotoxic effect on normal cells.63
6. CONCLUDING REMARKS AND FUTURE CHALLENGES Many epidemiological and experimental studies support the action of polyphenols or polyphenol-rich foods on health, but there are still many gaps in current knowledge. More adequately powered, randomized, placebo controlled human studies as well as animal studies are needed on polyphenols. There are a large number of structurally different polyphenols that are relevant for health, and obtaining enough information to set a DRI for each of these will not be feasible in the foreseeable future.64 Therefore, this area warrants further investigation as a new way of thinking, which would apply not only to polyphenols but also to other phytochemicals used as promising therapeutic agents against human diseases. The future challenge in this field may be to develop new ingredients and/or products as similar as possible to the polyphenols found in foods. Supplementation, however, should be carried out responsibly with great attention to the doses used. The challenge in this field is to provide “right on the target” doses for the different polyphenols and the most adequate form of supplementation (i.e., beverages, foods, capsules, etc.). Delivery of polyphenols directly to human tissues so that local concentrations are increased seems an interesting future approach.
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30. Manissier P, Montastier C, Piccirilli A. Combination of lycopene, polyphenols and vitamins for the care of keratin materials. Patent US 2011/0281941 A1, 2011. 31. Delmas D, Lanc¸on A, Colin D, Jannin B, Latruffe N. Resveratrol as a chemopreventive agent: a promising molecule for fighting cancer. Curr Drug Targets 2006;7(4):423 42. 32. Scalbert A, Manach C, Morand C, Re´me´sy C, Jime´nez L. Dietary polyphenols and the prevention of diseases. Crit Rev Food Sci Nutr 2005;45:287 306. 33. Bujanda L, Garcı´a-Barcina M, Gutie´rrez-de Juan V, Bidaurrazaga J, Luco MF, Gutie´rrez-Stampa M, et al. Effect of resveratrol on alcohol-induced mortality and liver lesions in mice. BMC Gastroenterol 2006;6:35. 34. Ajmo JM, Liang X, Rogers CQ, Pennock B, You M. Resveratrol alleviates alcoholic fatty liver in mice. Am J Physiol Gastrointest Liver Physiol 2008;295:G833 42. 35. Chan CC, Cheng LY, Lin CL, Huang YH, Lin HC, Lee FY. The protective role of natural phytoalexin resveratrol on inflammation, fibrosis and regeneration in cholestatic liver injury. Mol Nutr Food Res 2011;55(12):1841 9. 36. Zhu L, Luo X, Jin Z. Effect of resveratrol on serum and liver lipid profile and antioxidant activity in hyperlipidemia rats. Asian Aust J Anim Sci 2008;21:890 5. 37. Tieppo J, Cuevas MJ, Vercelino R, Tun˜o´n MJ, Marroni NP, Gonza´lez-Gallego J. Quercetin administration ameliorates pulmonary complications of cirrhosis in rats. J Nutr 2009;139 (7):1339 46. 38. Hamed GM, Bahgat NM, Mottaleb FIA, Emara MM. Effect of flavonoid quercetin supplement on the progress of liver cirrhosis in rats. Life Sci J 2011;8(1):1 11. 39. Bulzomi P, Galluzzo P, Bolli A, Leone S, Acconcia F, Marino M. The pro-apoptotic effect of quercetin in cancer cell lines requires ERβ-dependent signals. J Cell Physiol 2012;227:1891 8. 40. Kobori M, Masumoto S, Akimoto Y, Takahashi Y. Dietary quercetin alleviates diabetic symptoms and reduces streptozotocininduced disturbance of hepatic gene expression in mice. Mol Nutr Food Res 2009;53(7):859 68. 41. Gibellini L, Pinti M, Nasi M, Montagna JP, De Biasi S, Roat E, et al. Quercetin and cancer chemoprevention. Evid Based Complement Alternat Med 2011;2011:1 15. 42. Vargas AJ, Randy Burd R. Hormesis and synergy: pathways and mechanisms of quercetin in cancer prevention and management. Nutr Rev 2010;68(7):418 28. 43. Chiu J, Khan ZA, Farhangkhoee H, Chakrabarti S. Curcumin prevents diabetes-associated abnormalities in the kidneys by inhibiting p300 and nuclear factor-κB. Nutrition 2009;25 (9):964 72. 44. Shehzad A, Wahid F, Lee YS. Curcumin in cancer chemoprevention: molecular targets, pharmacokinetics, bioavailability, and clinical trials. Arch Pharm 2009;343(9):489 99. 45. Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res 2003;23 (1A):363 98. 46. Chung JH, Kim M, Kim HK. Green tea polyphenols suppress nitric oxide-induced apoptosis and acetylcholinesterase activity in human neuroblastoma cells. Nutr Res 2005;25:477 83. 47. Shen CL, Yehb JK, Stoecker BJ, Chyu MC, Wang JS. Green tea polyphenols mitigate deterioration of bone microarchitecture in middle-aged female rats. Bone 2009;44(4):684 90.
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48. Mandel SA, Amit T, Kalfon L, Reznichenko L, Youdim MBH. Targeting multiple neurodegenerative diseases etiologies with multimodal-acting green tea catechins. J Nutr 2008;138:1578S 83S. 49. Martin KR, Appel CL. Polyphenols as dietary supplements: a double-edged sword. Nutr Diet Suppl 2010;2:1 12. 50. Halliwell B. Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies?. Arch Biochem Biophys 2008;476(2):107 12. 51. Messina MJ, Wood CL. Soy isoflavones, estrogen therapy, and breast cancer risk: analysis and commentary. Nutrition J 2008;7:17. 52. Hsieh CY, Santell RC, Haslam SZ, Helferich WG. Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo. Cancer Res 2001;58(17):3833 8. 53. Allred CD, Ju HY, Allred KF, Chang J, Helferich WG. Dietary genistin stimulates growth of estrogen-dependent breast cancer tumors similar to that observed with genistein. Carcinogenesis 2001;22:1667 73. 54. Bonkovsky HL. Hepatotoxicity associated with supplements containing Chinese green tea (Camellia sinensis). Ann Intern Med 2006;144(1):68 71. 55. Halliwell B. Establishing the significance and optimal intake of dietary antioxidants: the biomarker concept. Nutr Rev 1999;57 (4):104 13. 56. Halliwell B. Dietary polyphenols: good, bad, or indifferent for your health? Cardiovasc Res 2007;73(2):341 7. 57. Lambert JD, Sang S, Yang CS. Possible controversy over dietary polyphenols: benefits vs risks. Chem Res Toxicol 2007;20:583 5. 58. D’Agostino EM, Rossetti D, Atkins D, Ferdinando D, Yakubov GE. Interaction of tea polyphenols and food constituents with model gut epithelia: the protective role of the mucus gel layer. J Agric Food Chem 2012;60(12):3318 28. 59. Margina D, Ilie M, Manda G, Neagoe I, Mocanu M, Ionescu D, et al. Quercetin and epigallocatechin gallate effects on the cell membranes biophysical properties correlate with their antioxidant potential. Gen Physiol Biophys 2012;31(1):47 55. 60. Re´ve´sz K, Tu¨tto A, Margittai E, Ba´nhegyi G, Magyar JE, Mandl J, et al. Glucuronide transport across the endoplasmic reticulum membrane is inhibited by epigallocatechin gallate and other green tea polyphenols. Int J Biochem Cell Biol 2007;39(5):922 30. 61. Babich H, Krupka ME, Nissim HA, Zuckerbraun HL. Differential in vitro cytotoxicity of (2)-epicatechin gallate (ECG) to cancer and normal cells from the human oral cavity. Toxicol In Vitro 2005;19(2):231 42. 62. Ruiz MJ, Ferna´ndez M, Pico´ Y, Man˜es J, Asensi M, Carda C, et al. Dietary administration of high doses of pterostilbene and quercetin to mice is not toxic. J Agric Food Chem 2009;57 (8):3180 6. 63. Fujimoto A, Sakanashi Y, Matsui H, Oyama T, Nishimura Y, Masuda T, et al. Cytometric analysis of cytotoxicity of polyphenols and related phenolics to rat thymocytes: potent cytotoxicity of resveratrol to normal cells. Basic Clin Pharmacol Toxicol 2009;104(6):455 62. 64. Williamson G, Holst B. Dietary reference intake (DRI) value for dietary polyphenols: are we heading in the right direction? Br J Nutr 2008;99(Suppl. 3):S55 8.
1. OVERVIEW OF POLYPHENOLS AND HEALTH
7 Polyphenols as Supplements in Foods and Beverages: Recent Methods, Benefits and Risks Andre´a Pittelli Boiago Gollu¨cke*, Daniel Araki Ribeiro† and Odair Aguiar Junior† *Nutrition Department/HEXALAB, Catholic University of Santos. Av. Conselheiro Ne´bias, SP, Brazil †Department of Biosciences, Federal University of Sa˜o Paulo, Santos, SP, Brazil
1. INTRODUCTION
industry. The survey asked 900 physicians and 277 nurses about their use of dietary supplements. The authors found that 72% of the physicians and 89% of the nurses used dietary supplements on a regular, occasional or seasonal basis. Regular use was reported by 51% of the physicians and 59% of the nurses. Moreover, 79% of the physicians and 82% of the nurses said they recommended supplements to their patients, including the ones who did not use supplements themselves. These findings are in accordance with national surveys, including the National Health and Nutrition Examination Survey (NHANES) of 1999 2000. The dietary supplement product most commonly used was the multivitamin, with or without minerals. Amongst the non-vitamin/mineral products, the physicians and nurses reported the consumption of green tea, fish oil, glucosamine, soy, flax seed, chondroitin and Echinacea as supplements. Interestingly, green tea and soy, two of the most cited supplements, contain polyphenols as bioactive compounds.
Polyphenols in foods and beverages are related to sensorial qualities such as color, bitterness, astringency, etc., which are relevant in products such as wine, tea and grape juice.1 5 These compounds occur naturally in forms varying from simple phenolic acids to complex polymerized tannins. Due to their inherent instability, polyphenols undergo transformations in the presence of light, oxygen, and as a result of heat processing, storage and extraction procedures.6 8 For that reason, daily consumption and dietary requirements for these compounds are difficult to measure. Although considered a powerful antioxidant, recently new mechanisms for polyphenols physiological effects have been proposed. For example, modulation of gene expression, induction of apoptosis, a decrease in platelet aggregation, an increase in blood vessel dilation, modulation of intercellular signaling, modulation of enzyme activities associated with carcinogen activation and detoxification and chelation of transition metals, such as iron.9 11 The alleged knowledge that polyphenols are associated with protection against diseases has raised a distinctive interest. Meanwhile, in western countries, the intake of fruits and vegetables, a main source of polyphenols, is considered to be insufficient. Consequently, supplementation appears to be a viable alternative. In 2009, Dickinson and colleagues12 published an online survey conducted in 2007 by Ipsos Public Affairs for the Council for Responsible Nutrition (CRN), a trade association representing the dietary supplement
Polyphenols in Human Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-398456-2.00007-4
2. METHODS FOR SUPPLEMENT PREPARATION The abundance of bioactive polyphenols in fruits, teas and their by-products such as pomace, skins and seeds brought new possibilities to food researchers and industries to develop new products/supplements. With that in mind, many new supplements have been developed using these products. In 2009, Alkayali Ahmad was
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granted a patent for inventing a method of preparing pomegranate extract (rich in ellagic acid) from pomegranate seeds.13 The product, in powder form, is obtained by ethanol extraction followed by concentration and drying. An interesting process for obtaining cocoa products rich in polyphenols was described by Pons-Andreu and colleagues.14 The method is based on producing liquid and powered cocoa polyphenol concentrate from unfermented cocoa beans. The possibility of increasing the polyphenols content in cocoa powder has a positive impact on its antioxidant content and also flavor. The total polyphenol content in unfermented, unroasted, defatted dried cocoa beans varies from 12 to 20% (in weight). The main polyphenols in cocoa beans under these conditions are catechins, dimers, and other oligomeric flavonoids. The extraction process involves blanching, followed by drying of unfermented beans up to 15% moisture content, grinding and finally polyphenol extraction with preferably polar solvents for human consumption (water, ethanol or both). In contrast, Jacob and colleagues15 proposed a method to obtain a fermented supplement from pomegranate, claiming that the aglycones resulting from the fermentation of polyphenol-glycosides possess enhanced physiological properties. The authors proposed the use of the yeast Saccharomyces boulardii and at least one species of lactobacilli followed by a freeze drying process to reduce water volume and obtain the final product. A more complex extraction procedure takes place to produce an algae extract rich in polyphenols to be used against inflammation processes. In the phylum of brown algae (Phaeophyceae), the phlorotannins are derived by polymerization from one and the same monomer: phloroglucinol (1,3,5-trihydroxybenzene). The extraction of polyphenols starts with the ground algae and is based on a solid/liquid extraction in the presence or not of added water. After centrifugation the solution undergoes membrane filtration or chromatographic purification of the desired polyphenols. Then the extract is eluted in alcohol and spray-dried to its final commercial powder. This pharmaceutical product, according to the invention, can be used as such or integrated in a food matrix.16 One unusual source of polyphenols to be used as supplements is a beer waste-product. Taidi and colleagues17 proposed a method of recovering the polyphenols removed from beer during process. Some specific polyphenols in beer are undesirable due to their association with proteins forming insoluble compounds causing permanent cloudiness after cold storage. In the brewing industry, these polyphenols are removed from the beer by a PVPP (polyvinyl polypyrrolidone) resin. The authors suggested a method for recovering these bioactive compounds and then applying the end product in the cosmetic/food/nutraceutic industry. The inventors found catechin, epicatechin, tyrosol and ferulic acid
as relevant polyphenols in this beer by-product extract. The application of grape seed oil in foods, food supplements, additives, medicaments and cosmetic products has also been recently reviewed. In 2007, Eckert and colleagues18 described a method for the preparation of cold-pressed grape oil, crushed grape and grape flour. Grape seed oils contain important amounts of polyphenols such as catechins and procyanidins. The concentration of polyphenols in grape seed oil is dependent on the extraction process, especially the pressing temperature. The crushed seeds obtained in the process are also a viable source of polyphenols, which could undergo further extraction with water or other solvent mixtures. Ibarra and Zagiary19 developed a process in which olive polyphenols concentrate is obtained by mixing the waste-product of pressing with a polar solvent. Polyphenols are further extracted from the mixture and then concentrated up to 10% (w/w) by the use of membranes. The main compounds found in this polyphenol extract are oleuropein, demethyuloleuropein and logstroside. Aware of the polyphenols presence in fruit processing residues, the above cited patents have presented advanced extraction methods for skins, seeds, etc. Methods using hot water extraction and an adsorbent resin followed by concentration and purification of polyphenols seem to be a promising challenge.
3. FORMULATIONS USING POLYPHENOLS Recents patents describing polyphenol-rich supplements frequently use green tea or grape products. In 2009, the effects of a multi-phytonutrient supplement on DNA damage in skin cells exposed to UV rays was investigated. The supplement was composed of several vitamins, minerals and natural extracts such as green tea, pomegranate, citrus fruits, acai and different types of berries.20 Howard and colleagues21 produced a dry concentrate by distillation and concentration of either wine or grape juice. The concentrate was then filtered and eluted with a mixture of water and ethanol (50:50). The intake of 1 2 g of the powder for 2 weeks improved platelet aggregation indicators, suggesting a positive impact on cardiovascular health. Earlier, the main author had described a method of concentrating grape flavonols from wine, juice or dealcoholized wine, grape skins and fermented grape skins. In this invention, several solvents were tested as eluents. Although the organic solvents are eliminated at the drying stage, the use of water would be preferable. The stability of polyphenols in solutions is also a concern. In 2012, a method for dispersing microparticulated water-insoluble bioactive polyphenol in a beverage was described.22 In this form, the polyphenols
1. OVERVIEW OF POLYPHENOLS AND HEALTH
4. BENEFITS OF POLYPHENOL CONSUMPTION: EXPERIMENTAL DATA
added to the drink are stabilized as suspended particles and do not separate from the water-based beverage. Mower and Brady23 had earlier described the invention of a beverage having resveratrol mixed in water with other ingredients such as energy agents, preservatives, pH balancing and colorant additives. The method uses nanosize water clusters and nanoparticles of resveratrol. With this system, it is possible to enhance polyphenols amounts in beverages and thus improve bioavailability. In an interesting case, Schiffelers and colleagues24 tested parenteral administration of a pharmaceutical composition containing polyphenols in mice inoculated with B16 murine melanoma and C26 murine colon carcinoma cells. The authors propose a method for targeting polyphenols to the tumor tissue using liposomes. After a week, effective inhibition in tumor growth was observed at dosages of 1.0 to 10 mg liposomal caffeic acid (per kg body weight per week) when compared with controls. The pharmaceutical composition comprised colloidal carriers where the polyphenols or polyphenol derivatives could be entrapped. Some supplements for foods and beverages have incorporated fruit and vegetable extracts to enhance health appeal. In 2006, Wild and Sass25 developed a concentrated food ingredient comprising extracts of green tea, grape skin and grape seed, yielding a polyphenol content of 1155 mg gallic acid/L in order to improve antioxidant appeal to commercial beverages. Shrikhande and colleagues26 maximized the extraction of monomeric and oligomeric procyanidins to obtain a final product with 15% of monomers and 20% of dimers and up to 30% of trimers, tetramers and pentamers, by weight. The extraction procedure was the use of hot water and pectolytic enzymes for at least 2 hours. Formulated dry mix products to be used in beverages, nutraceutical capsules and supplements containing vitamins and minerals were further proposed using this extract. In 2008, Perlman and colleagues27 presented an interesting use of the waste-products of the grape juice industry. The authors proposed the fortification of grape juice with the grape pomace polyphenol extract. The new beverage was tested at different extract concentrations for sensorial acceptance and antioxidant activity. An unacceptable astringency was noticed at 4% (by weight) with antioxidant activity increasing three times. Cyclodextrin, added at 0.2% by weight, was able to successfully reduce the astringent sensation. In 2009, Draijer and colleagues28 proposed a mixture of red wine and grape polyphenols (total 800 mg polyphenols) added to a soy drink (200 mL). The beverage was given daily to 35 males with a positive impact on the blood pressure. Recently, Rao and colleagues29 investigated the antioxidant potential of a commercial product (Greens1 ) using a
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liposome model for the in vitro assessment and 10 healthy human subjects for the in vivo observation. The product is a mixture of several ingredients from vegetable powders to bifidobacteria and including polyphenols. In this case, the sources of polyphenols (30 mg/8.5 g final product) are green tea, bilberry and grape extract and 500 ppm resveratrol. The results showed the supplementation with Greens1 increased the serum antioxidant potential and the presence of kaempferol in plasma. A reduction in lipid oxidation was observed indicating a possible positive effect in meliorating chronic diseases. Dietary supplements containing polyphenols have also been proposed for skincare. In combination with other ingredients such as lycopene, vitamin C and E, the mixture is intended to maintain and restore the biomechanical properties of keratinous materials such as connective tissue.30
4. BENEFITS OF POLYPHENOL CONSUMPTION: EXPERIMENTAL DATA The efforts in investigating and proposing new forms of polyphenol supplementation are justified by the encouraging results observed in experimental data in recent years. In spite of the debate in respect to human applicability, animal experimental data confirm the benefits of polyphenol-rich products.31 Resveratrol, for instance, has been considered as a “miracle molecule” for fighting cancer. According to Delmas and colleagues,32 in their extensive review, resveratrol protects cells from DNA adducts formation induced by various chemical agents. Such DNA alterations are responsible for the initiation phase of the tumor, when cells start growing autonomously. Suppression of the metabolic activation and/or increasing of detoxification rate are some of the mechanisms attributed to the resveratrol leading to its anti-initiation property. Also, decreasing the reactive oxygen species (ROS) production, and the consequent procarcinogens activations and oncogenes mutation, is attributed to resveratrol. Also credited to resveratrol is the stimulation of DNA repair, by increasing the p53 activity, the cell cycle progression blockage (in G1, S and G2/M phases), induction of apoptosis in malignant cells, inhibition of inducible nitric oxide synthase (iNOS) (with consequent blockage of the metastasis) and inhibition of angiogenesis; all of them making resveratrol a promising molecule to prevent and treat cancers.32 Treatments with resveratrol were also employed for liver disorders, simulated in several experimental designs.33 35 Bujanda and colleagues33 demonstrated that liver lesions and animal mortality were reduced in alcohol-exposed mice. According to the authors,
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resveratrol might have diminished the release of proinflammatory cytokines (such as IL-1), protecting the liver from damage. Alcohol-induced fatty liver was ameliorated by resveratrol treatment in studies by Ajmo and colleagues,34 who found that this polyphenol was able to reduce lipid synthesis and increase the rates of fatty acid oxidation, preventing alcoholic liver steatosis. The increment in rates of fatty acid oxidation seems to be modulated by increased mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ) coactivator α (PGC-1α) target genes, which encode the fatty acid oxidative enzymes.34 The role of resveratrol was also investigated by Chan and colleagues35 in animal models of cholestatic liver injury. As in the studies by Ajmo and colleagues,34 the survival of mice after resveratrol treatment was higher. Also, inflammatory markers (TNF- α and IL-6) were reduced, hepatic fibrosis also decreased and the number of Ki671 hepatocytes increased, indicating that resveratrol stimulated hepatocyte proliferation.35 Also, when applied to hyperlipidemic rats, resveratrol was shown to be effective in restoring normal physiological conditions. As demonstrated by Zhu and colleagues,36 oral administration of resveratrol to cholesterol-fed rats resulted in lowered serum lipid, hepatic cholesterol and triglycerides, besides an increment of the antioxidant capacity expressed by significantly increased levels of superoxide dismutase (SOD), catalase and glutathione peroxidase. Also, hepatic thiobarbituric acid reactive substances (TBARS) were lowered, possibly by inhibition of the oxidation of LDL, maintaining antioxidant efficacy and denoting the antihyperlipidemic effects of resveratrol. Quercetin, considered the major flavonoid found in the human diet, has also been vastly studied. Relevant results have been found by Tieppo and colleagues37 when working with hepatopulmonary syndrome (HPS), a complication of liver cirrhosis. In an animal model of hepatic cirrhosis (through bile duct ligation), quercetin treatment showed decreased oxidative stress, by restoration of plasma TBARS, SOD and nitric oxide (NO), besides a lower severity of the consequent HPS and less pronounced evolution of the hepatic injuries.37 A slowing down in the cirrhotic process was also observed by Hamed and colleagues38 using quercetin in a model of thioacetamide-induced cirrhosis. The amelioration in the hepatic biochemical, morphological and functional aspects was attributed to a potentiation of the antioxidant defense, breaking the “vicious circle” between oxidative stress and oxidative damage. Pro-apoptotic properties have also been ascribed to quercetin by Bulzomi and colleagues.39 This polyphenol was demonstrated to mimic the apoptotic effect of 1λ-estradiol (E2) in two transformed cell lineages, leading to the activation of the p38
MAP-kinase which, in turn, is responsible for caspase3 activation and apoptosis. Such a role for quercetin, acting at the estrogen receptor (ER), ensures an anticarcinogenic potential to this molecule. Also, when applied to diabetic mice (by induction with streptozotocin), quercetin was shown to result in the lowering of blood glucose and improving plasma insulin levels.40 The authors suggested that quercetin might improve liver and pancreas functions by suppressing the streptozotocin-induced expression of the cell-cycle inhibitor CDKN1A, restoring the cell proliferative capacity. The interference of quercetin with the cell cycle regulation in cancer cells has also been very well documented in the extensive review by Gibellini and colleagues.41 According to those authors, quercetin has many molecular targets within the cycle including cyclin-dependent kinases (CDKs) and cyclins, making this polyphenol able to block the cell cycle progression at various transition points, depending on the cancer cell lineage investigated. Quercetin also acts in multiple ways to influence the p53 activity, leading cancer cells to apoptosis and also contributing to improve the cell fighting against ROS, since p53 has been found to regulate a series of genes related to oxidative stress mitigation.41 A pro-oxidant property to quercetin was also proposed by Vargas and Burd.42 When at high concentrations (greater than 40 μM) quercetin is cytotoxic leading cells to apoptosis. Such a property puts this molecule in a good status to be used as an adjuvant to current chemotherapies. Curcumin has well-known benefits for a series of pathological conditions. According to Chiu and colleagues,43 diabetic nephropathy is a consequence of the high glucose levels (and concomitant increased level of ROS), leading to an increase of the extracellular matrix proteins which, in turn, is responsible for the thickening of the glomerular basement membrane. When applied to diabetic rats, curcumin prevented renal lesions and mesangial matrix expansion. The authors attributed to curcumin, besides a neutralization of the oxidative stress, an inhibition of p300, a histone acetiltransferase, which upregulates specific genes in association with nuclear factor-κB (NF-κB).43 As with the other polyphenols cited above, curcumin has a pleotropic effect, which includes pro-apoptotic properties, gene regulation, cell cycle control (proliferation arrest), which indicates that curcumin is now used in phases II and III clinical trials for a variety of cancer types.44 According to Aggarwall and colleagues,45 curcumin can suppress tumor initiation, promotion and metastasis, being also a potent antiinflammatory agent. Tea is one of the most consumed beverages in the world. Green tea (GT) polyphenols have been found to
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5. NOXIOUS ACTIVITIES INDUCED BY POLYPHENOLS: AN INTRIGUING ISSUE
decrease oxidative stress, which has been implicated in the pathogenesis of some neurodegenerative diseases.46 The authors showed that GT polyphenols protect the neural cells against oxidative stress-induced NO toxicity, attenuating NO-induced apoptotic cell death by modulating pro-apoptotic gene expression. GT polyphenols have also been found to maintain the bone architecture in females with and without ovariectomy.47 It has been described that estrogen is a reactive oxygen species scavenger and that its decreasing with advanced age accelerates bone loss by increasing the oxidative stress and reducing thiol antioxidant defenses in osteoclasts.47 These authors have found that GT consumption was able to maintain bone microarchitecture by increasing bone formation and suppressing bone erosion. Also, GT polyphenols have been found to have a beneficial role in neurodegenerative diseases.48 Besides the antioxidant properties, GT seems to modulate various protein-kinase signaling pathways, mainly involving protein-kinase C (PKC) whose activation generates cytoprotection by apoptosis prevention, so preventing or delaying the neuronal loss in the neurodegenerative diseases.48
5. NOXIOUS ACTIVITIES INDUCED BY POLYPHENOLS: AN INTRIGUING ISSUE Few studies have discussed the injurious effects closely related to the toxic potential of polyphenols. In 2010, Keith and colleagues49 published a review about the possible toxicity of high-dose intakes of polyphenols through supplementation. The consumption of natural compounds such as polyphenols, found in innumerable foods, is commonly considered safe. The doses used in supplements, however, are often much higher than those found in normal daily intake. Halliwell50 summarized the pro-oxidant effects observed in in vitro studies. In the presence of iron, high pH and depending on concentration, polyphenols can begin auto-oxidation. Other mechanisms may also be involved in the toxic effects of polyphenols in high-dose supplementation; for example, soy foods and breast cancer association. There has been considerable investigation on the potential for soy foods to reduce risks of breast cancer due to the presence of isoflavones, compounds that bind to estrogen receptors, exhibiting weak estrogen-like effects. Recently, however, concerns have been raised suggesting that isoflavones could stimulate the growth of estrogen-dependent breast tumors.51 The contradiction is supported by other evidence. In an in vivo rodent model, ovariectomized athymic mice fed genistein and genistin both displayed enhanced growth of mammary tumors.52,53 Again, the dosage seems to play a fundamental role. Hepatoxicity by high dose intake of tea polyphenol supplementation
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has been described in human studies.54 Other possible toxic effects have been reported.55 57 Nevertheless, to date, there is not enough evidence to support that a daily intake compatible with the Asian soy food habit is detrimental to health. Harmful effects of some polyphenols can be dubious. Studying the luminal surface of the gastrointestinal tract, covered by a protective mucus gel layer, D’Agostino and colleagues58 have demonstrated that epigallocatechin gallate was very toxic to the HT29 cells. However, the substance was less toxic to the HT29-MTX-E12 cells, suggesting that the mucus gel layer on the HT29-MTX-E12 cells is able to protect the cells against epigallocatechin gallate toxicity. In contrast, epicatechin had no effect on the viability of either the HT29 or HT29-MTX-E12 cells, suggesting that proteins within the mucus gel layer on the apical surface of gut epithelial cells may bind to the galloyl ring of epigallocatechin gallate. In the same way, some authors have postulated that the antioxidant capacity of the tested polyphenols quercetin and epigallocatechin gallate is due to their stabilizing effect on the cell membranes, thus contributing to cell protection in various pathologies and as adjuvant therapy in highly toxic treatment regimens.59 Epigallocatechin gallate also inhibited β-glucuronidase activity in native Hepa 1c1c7 mouse hepatoma cells, while it failed to affect the enzyme in alamethicin-permeabilized cells, where the endoplasmic membrane barrier was eliminated. Such findings indicate that tea flavanols inhibit deglucuronidation in the endoplasmic reticulum at the glucuronide transport stage.60 Epigallocatechin gallate induced apoptosis in the carcinoma HSC-2 cells, but not in the normal HGF-2 fibroblasts.61 This research supports those studies suggesting that GT is an effective chemopreventive agent of oral carcinoma cells in vitro. Mice exposed for 28 days at doses of 0, 30, 300, and 3000 mg/kg body weight/day of quercetin, which are equivalent to 5, 50, and 500 times, respectively the estimated mean human intake of these polyphenols (25 mg/ day), revealed no mortality during the experimental period.62 No significant body weight gain in the male or female groups was also observed. Red blood cell numbers and the hematocrit increased after polyphenol administration compared to control groups. Biochemical parameters were not affected. Histopathological examination revealed no alterations in clinical signs or organ weight at any dose.62 Resveratrol at a concentration of 10 μM or more (up to 100 μM) led to a significant dose-dependent increase in the population of dead cells, shrunken living cells, annexin V-positive cells and cells with hypodiploidal DNA. In the presence of benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK), a pan-inhibitor of caspases, the resveratrol-induced increase in the population of cells with hypodiploidal DNA was partially
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inhibited.63 Overall, it is suggested that resveratrol at a concentration of 10 μM or more induces apoptosis in normal cells as well as cancer cells. Thus, at concentrations that are suitable for chemopreventive and chemotherapeutic actions, resveratrol may exert a cytotoxic effect on normal cells.63
6. CONCLUDING REMARKS AND FUTURE CHALLENGES Many epidemiological and experimental studies support the action of polyphenols or polyphenol-rich foods on health, but there are still many gaps in current knowledge. More adequately powered, randomized, placebo controlled human studies as well as animal studies are needed on polyphenols. There are a large number of structurally different polyphenols that are relevant for health, and obtaining enough information to set a DRI for each of these will not be feasible in the foreseeable future.64 Therefore, this area warrants further investigation as a new way of thinking, which would apply not only to polyphenols but also to other phytochemicals used as promising therapeutic agents against human diseases. The future challenge in this field may be to develop new ingredients and/or products as similar as possible to the polyphenols found in foods. Supplementation, however, should be carried out responsibly with great attention to the doses used. The challenge in this field is to provide “right on the target” doses for the different polyphenols and the most adequate form of supplementation (i.e., beverages, foods, capsules, etc.). Delivery of polyphenols directly to human tissues so that local concentrations are increased seems an interesting future approach.
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