Structure and function of the oxidation products of polyphenols and identification of potent lipoxygenase inhibitors from Fe-catalyzed oxidation of resveratrol

Polyphenols have recently attracted much attention as potent antioxidants and related bioactive substances. These potent antioxidative polyphenols are very oxidizable due to their chemical properties, and their oxidation products must accumulate in the oxidizing foods when they are contained as the active ingredients. In this investigation, 30 polyphenols and related phenolics were oxidized with oxygen in the presence of a catalytic amount of Fe ions. Piceatannol, catechin, epicatechin, hydroxytyrosol, carnosol, and carnosic acid were oxidized very quickly. Sinapic acid, caffeic acid, chlorogenic acid, rosmarinic acid, gallic acid, propyl gallate, α-tocopherol, quercetin, and nordihydroguaiaretic acid were moderately oxidized. Protocatechuic acid, syringic acid, taxifolin, resveratrol, gentisic acid, secoisolariciresinol, and ellagic acid were oxidized for 19-20 days; however, their oxidation was very slow and did not complete. The other phenolics were not oxidized. The obtained oxidation products were next subjected to a lipoxygenase inhibition assay and the results compared to those of the corresponding phenols. Very interestingly, the oxidation product from resveratrol showed a high inhibitory activity, whereas resveratrol itself had no activity and its oxidation efficiency was low. To clarify the inhibition principle of the oxidation product, an LC-MS analysis was carried out on the oxidation product. The analytical results showed that they are the oligomeric and degraded compounds of resveratrol. Among them, the structures of three dimeric compounds were successfully identified, and their activity data clarified that the closed ring dimers were potent lipoxygenase inhibitors, whereas the opened ring dimer was not. It should be noted that resveratrol had almost no lipoxygenase inhibitory activity, contrary to some researchers' findings.     

苹果渣多酚提取工艺的优化

为优化苹果渣中多酚提取工艺,并得到提取工艺和多酚组成之间的关系,利用微波辅助提取法设计了由物料颗粒、液料比、乙醇浓度、微波功率和微波提取时间5个因素构成的多酚提取优化试验流程,构建了涵盖黄酮、原花青素2种典型多酚物质和抗氧化能力的提取工艺评价指标体系。采用证据理论对不同工艺在评价指标下的焦元进行识别,并基于信度函数和似真函数得到了不同提取工艺的效用区间和优化方案。优化结果为：物料颗粒60目,液料比30?mL/g,乙醇体积分数60%,微波功率600?W,提取时间70?s,此条件下苹果渣多酚的提取量为213.83?mg/100g,黄酮提取量为83.21?mg/100g,原花青素提取量为52.79?mg/100g,抗氧化的EC50值为3.71?mg/100mL,验证了采用证据理论进行苹果渣多酚提取工艺优化的有效性。     

莲衣粉多酚提取工艺优化及体外抗氧化活性研究

为探究酸性乙醇溶液(乙酸+乙醇+水)提取莲衣粉(LSPW)多酚的最佳工艺,明确莲衣粉多酚提取物(PEL)作为天然食品抗氧化剂的应用潜力,本研究在单因素试验基础上,通过响应面分析法优化莲衣粉多酚的提取工艺,并对PEL进行体外抗氧化研究。结果表明,莲衣粉多酚的最佳提取工艺为乙醇浓度41%、液料比59 mL·g^-1、乙酸添加量1.56 mL·100mL^-1、提取温度70℃、提取时间20 min。在此条件下,莲衣粉多酚得率达9.65%。PEL的多酚含量达24.03%,且其具有较强的清除DPPH自由基(IC₅₀=0.032 mg·mL^-1)、还原Fe³⁺(eq=268.64 mg AAE·g^-1)的能力,抑制脂质氧化能力分别相当于叔丁基对苯二酚(TBHQ)、叔丁基羟基茴香醚(BHA)的37.12%、39.20%(硫氰酸铁法)与72.92%、67.60%(硫代巴比妥酸法)。本研究为莲衣粉多酚的开发利用及PEL作为潜在的天然食品抗氧化剂提供了理论基础。     

The role of iron and the factors affecting off-color development of polyphenols

Iron deficiency affects over two billion people worldwide (Lotfi, M.; Venkatesh Mannar, M. G.; Merx, R. J.; Naber-van den Heuvel, P. Micronutrient Fortification of Foods: Current Practices, Research,and Opportunities; Micronutrient Initiative: Ottawa, Ontario, Canada, 1996). However, fortifying foods with highly bioavailable iron is technically challenging because of off-color and off-flavor development, catalytic degradation of vitamins, and oxidation of lipids. The role of highly bioavailable iron in the off-color development of foods and beverages is not well-understood. The goal of this research was to examine the interaction of iron with simple phenolics and polyphenols. Factors that may affect off-color development, such as pH, oxygen, temperature, and reducing and chelating agents, were evaluated as a model for food products. Our results demonstrated that the iron that reacts with the simple phenolic, catechol, to develop off-color must be in the oxidized state, and the iron is reduced in the presence of catechol. Because this is an oxidation/reduction reaction, the redox potential of all of the components is critical to the color development. Ferrous iron sources with low redox potentials and ferric iron sources with high redox potentials caused off-color development with catechol. Only polyphenols that contain ortho-hydroxyl groups cause off-color development with iron. All of the factors tested affect off-color development and redox potential of the system. Low pH, low oxygen, high temperature, and the presence of reducing and chelating agents inhibited off-color development. To confirm the model, foods that contained these polyphenols were evaluated for off-color development when iron was added. The foods tested reacted similarly to the models of polyphenols with iron. Off-color development was caused by oxidation-reduction interactions between ferric iron and polyphenols that contained ortho-dihydroxyl groups. Ferrous iron needed to be oxidized to participate in off-color development. In addition, methods identified in the models to prevent off-color development were effective in most of the food products examined. Using the ferrous form of iron and maintaining it in its reduced form by lowering pH, removing oxygen, and including reducing agents, it was possible to fortify foods with highly bioavailable iron.     

Chemical interaction between polyphenols and a cysteinyl thiol under radical oxidation conditions

Chemical interaction between polyphenols and thiols was investigated under radical oxidation conditions using a model cysteinyl thiol derivative, N-benzoylcysteine methyl ester. The radical oxidation was carried out with a stoichiometric amount of 2,2-diphenyl-1-picrylhydrazyl (DPPH), and the decreases in the amounts of polyphenols and the thiol were measured by HPLC analysis. Cross-coupling products between various polyphenols and the thiol were examined by LC-MS in reactions that showed decreases in both the polyphenols and the thiol. The LC-MS results indicated that three phenolic acid esters (methyl caffeate, methyl dihydrocaffeate, and methyl protocatechuate) and six flavonoids (kaempferol, myricetin, luteolin, morin, taxifolin, and catechin) gave corresponding thiol adducts, whereas three polyphenols (methyl ferulate, methyl sinapate, and quercetin) gave only dimers or simple oxidation products without thiol substituents. Thiol adducts of the structurally related compounds methyl caffeate and methyl dihydrocaffeate were isolated, and their chemical structures were determined by NMR analysis. The mechanism for the thiol addition was discussed on the basis of the structures of the products.     

Protection by polyphenols of postprandial human plasma and low-density lipoprotein modification: the stomach as a bioreactor

Recent studies dramatically showed that the removal of circulating modified low-density lipoprotein (LDL) results in complete prevention of atherosclerosis. The gastrointestinal tract is constantly exposed to food, some of it containing oxidized compounds. Lipid oxidation in the stomach was demonstrated by ingesting heated red meat in rats. Red wine polyphenols added to the rats' meat diet prevented lipid peroxidation in the stomach and absorption of malondialdehyde (MDA) in rat plasma. In humans, postprandial plasma MDA levels rose by 3-fold after a meal of red meat cutlets. MDA derived from meat consumption caused postprandial plasma LDL modification in human. The levels of plasma MDA showed a 75% reduction by consumption of red wine polyphenols during the meat meal. Locating the main biological site of action of polyphenols in the stomach led to a revision in the understanding of how antioxidants work in vivo and may help to elucidate the mechanism involved in the protective effects of polyphenols in human health.     

Influence of thermal treatments simulating cooking processes on the polyphenol content in virgin olive oil

Virgin olive oils were subjected to simulated common domestic processing, including frying, microwave heating, and boiling with water in a pressure cooker. The impact of these processes on polyphenol content and physicochemical characteristics of oils was assessed. Thermal oxidation of oils at 180 degrees C caused a significant decrease in hydroxytyrosol- and tyrosol-like substances. In contrast, oils heated for 25 h still retained a high proportion of the lignans 1-acetoxypinoresinol and pinoresinol. Thermal oxidation also resulted in a rapid degradation of alpha-tocopherol and the glyceridic fraction of oils. Microwave heating of oils for 10 min caused only minor losses in polyphenols, and the oil degradation was lower than that in thermoxidation assays. Again, lignans were the least affected polyphenols and did not change during microwave heating. Boiling a mixture of virgin olive oil and water in a pressure cooker for 30 min provoked the hydrolysis of the secoiridoid aglycons and the diffusion of the free phenolics hydroxytyrosol and tyrosol from the oil to the water phase. Losses of polyphenols were detected only at pH lower than 6. Moreover, alpha-tocopherol and the glyceridic fraction of oils were not modified during this process. It is worth noting that all the heating methods assayed resulted in more severe polyphenols losses and oil degradation for Arbequina than for Picual oil, which could be related to the lower content in polyunsaturated fatty acids of the latter olive cultivar. These findings may be relevant to the choice of cooking method and olive oil cultivar to increase the intake of olive polyphenols.     

Treatment of cork process wastewater by a successive chemical-physical method

In cork processing, the operation of boiling the raw cork generates large volumes of wastewater which are more often than not released directly into the environment untreated. Even when the wastewater is treated, this is usually by retention in evaporation ponds. This procedure, however, causes bad odors and may pollute surface water and groundwater. The present study evaluates a physicochemical method involving Fenton oxidation and coagulation/flocculation for the removal of chemical oxygen demand (COD), total polyphenols (TP), and aromatic compounds (A) from cork manufacturing process wastewater. The experimental variables studied were the dosages of iron salts (from 0.001 to 0.2 mol/L) and hydrogen peroxide (between 0.06 and 1 mol/L). The integrated Fenton-coagulation/flocculation process reduced the COD of the effluent by from 22% to 85%. The removal of total polyphenols ranged from 4% to 98%, and of aromatic compounds from 2% to 97%. A further two experiments were performed modifying the manner in which the reagents were added, splitting the reagent dose (of hydrogen peroxide and ferrous salt) into two and three fractions. Finally, an economic study was made of the chemical costs deriving from the application of this purification system. The cost of a treatment with an [H2O2](o)/COD(o) ratio of 1.8 g/g (splitting the reagent dose into three fractions) that yields a COD removal of 73% was estimated to be 11.5 euros/m(3) of wastewater.     

Treatment of High Ammonium–Nitrogen Wastewater from Composting Facilities by Air Stripping and Catalytic Oxidation

Composting municipal wastewater sludge may generate composting wastewater (acid washer water and tunnel wastewater) with high ammonium–nitrogen (NH₄–N) concentration; this kind of wastewater is usually generated in a rather small daily amount. A procedure of air stripping with catalytic oxidation was developed and tested with pilot-scale and full-scale units for synthetic disposal of the high NH₄–N wastewaters from composting facilities. In air stripping, around 90% NH₄–N removal efficiency was reliably achieved with a maximum of 98%. A model to describe the stripping process efficiency was constructed, which can be used for process optimization. After catalytic oxidation, the concentrations in the outlet gas were acceptable for NH₃, NO_X, NO₂, and N₂O, but the NH₃ and N₂O concentrations limited the feasible loading range. The treatment costs were estimated in detail. The results indicate that air stripping with the catalytic oxidation process can be applied for wastewater treatment in composting facilities.     

A new process for the management of olive oil mill waste water and recovery of natural antioxidants

The high polyphenol content of the wastewater is the major environmental problem caused by the olive mills. A pilot scale system for the treatment of the olive oil mills wastewater was developed aiming at the recovery of high added value-contained polyphenols and the reduction of the environmental problems. The treatment system consists of three main successive sections: The first one includes successive filtration stages aiming at the gradual reduction of the wastewater suspended solids up to a limit of 25 microm. The second section includes passing of the filtered wastewater through a series of adsorbent resins (XAD16 and XAD7HP) in order to achieve the de-odoring and decolorization of the wastewater and the removal/ recovery of the polyphenol and lactone content. The third section of the procedure includes the thermal evaporation and recovery of the organic solvents mixture, which has been used in the resin regeneration process, and finally the separation of the polyphenols and other organic substance contents using fast centrifuge partition chromatography. The final outcome of the whole procedure is (i) an odorless yellowish wastewater with a 99.99% reduced content in polyphenols and 98% reduced COD, (ii) an extract rich in polyphenols and lactones with high antioxidant activity and high added value, (iii) an extract containing the coloring substances of the olive fruit, and (iv) pure hydroxytyrosol.     

New investigation of the isothermal oxidation of extra virgin olive oil: determination of free radicals, total polyphenols, total antioxidant capacity, and kinetic data

As a follow-up of the research programs carried out by our group concerning the artificial isothermal rancidification process in extra virgin olive oil (EVOO), in the present work the trends of both the total antioxidant capacity and the total polyphenols concentration as well as the main kinetic parameters of the process during the thermal oxidation of EVOO were studied and compared. In addition, the possibility of evaluating the increase in radicals concentration during the thermal oxidation process using a superoxide dismutase biosensor was also studied. The present investigation concerning this important food product is highly topical as it refers to the state of alteration of the EVOO used for cooking or frying, as a function of the temperature reached.     

Determination of peroxyl radical scavenging activity of flavonoids and plant extracts using an automatic potentiometric titrator

A novel potentiometric method for evaluation of peroxyl radical scavenging activity of flavonoids and plant extracts was developed. The oxidation of potassium iodide (KI) was performed in acetonitrilephosphate buffer (1:1) containing antioxidant using 2,2'-azobis(2-amidinopropane) dihydrochloride as a peroxyl radical generator. The amount of iodine released from KI during a 20-min free radical oxidation was determined quantitatively using an automatic potentiometric titrator with sodium thiosulfate. The radical scavenging activity of the sample was expressed as the inhibition ratio for iodine release of the control group mediated by the radical. The results obtained from some authentic polyphenols correlated well with those of previous reports. This is a simple, time-saving method requiring less than 30 min and is useful in assessing the radical scavenging activity of antioxidants in plant extracts. We describe the radical scavenging activities of various flavonoids including 21 kinds of tea catechins and vegetable extracts by this method.     

β-carotene radical cation addition to green tea polyphenols. Mechanism of antioxidant antagonism in peroxidizing liposomes

Green tea polyphenols, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG), all showed antioxidative effect in liposomes for lipid oxidation initiated in the lipid phase (antioxidant efficiency EC > EGCG > ECG > EGC) or in the aqueous phase (EC ? EGC > EGCG > ECG) as monitored by the formation of conjugated dienes. For initiation in the lipid phase, β-carotene, itself active as an antioxidant, showed antagonism with the polyphenols (EC > ECG > EGCG > EGC). The Trolox equivalent antioxidant capacity (TEAC EGC > EGCG > ECG > EC) correlates with the lowest phenol O-H bond dissociation enthalpy (BDE) as calculated by density functional theory (DFT). Surface-enhanced Raman spectroscopy (SERS) was used to assess the reducing power of the phenolic hydroxyls in corroboration with DFT calculations. For homogeneous (1:9 v/v methanol/chloroform) solution, the β-carotene radical cation reacted readily with each of the polyphenol monoanions (but not with the neutral polyphenols) with a rate approaching the diffusion limit for EC as studied by laser flash photolysis at 25 °C monitoring the radical cation at 950 nm. The rate constant did not correlate with polyphenol HOMO/LUMO energy gap (DFT calculations), and β-carotene was not regenerated by an electron transfer reaction (monitored at 500 nm). It is suggested that the β-carotene radical cation is rather reacting with the tea polyphenols through addition, as further evidenced by steady-state absorption spectroscopy and liquid chromatography-mass spectroscopy (LC-MS), in effect preventing regeneration of β-carotene as an active lipid phase antioxidant and leading to the observed antagonism.     

脉冲强光处理对菜籽油储藏稳定性的影响

为探索脉冲强光(IPL)处理对富含油脂食品的影响,采用化学滴定和分光光度法研究了IPL处理对菜籽油储藏稳定性及其酚类物质的影响。结果表明,在试验储藏期内IPL处理5 次和15 次的菜籽油储藏稳定性与对照组相比显著降低(P<0.05),但IPL处理25 次的菜籽油储藏稳定性显著优于对照组(P<0.05)。同时,油脂氧化与酚类物质含量的相关性研究表明,在试验储藏期内IPL处理对菜籽油储藏稳定性的影响与酚类物质含量呈良好的相关性(r²>0.89),可推知IPL处理25 次使菜籽油中酚类化合物的稳定性增加,从而使得油脂储藏稳定性增加。本研究结果为IPL技术在富含油脂食品中的应用提供了一定的理论依据。     

Comparison of natural polyphenol antioxidants from extra virgin olive oil with synthetic antioxidants in tuna lipids during thermal oxidation

Polyphenols extracted from extra virgin olive oil (EVOO) were tested for their ability to inhibit lipid oxidation of canned tuna. Hydroperoxide formation during oxidation was monitored by measurement of peroxide value and decomposition of hydroperoxides by static headspace gas chromatographic analysis of volatiles. In tuna oxidized at 40 and 100 degrees C, 400 ppm of the EVOO polyphenols was an effective antioxidant as compared with 100 ppm of a 1:1 mixture of the synthetic antioxidants butylated hydroxytoluene and butylated hydroxyanisole. However, at concentrations <100 ppm, the EVOO phenolic compounds promoted hydroperoxide formation and decomposition. The EVOO polyphenols were effective antioxidants when added to heated tuna muscle in the presence of either brine or refined olive oil. The oxidation rate in tuna muscle packed in brine was higher than that of tuna packed in refined olive oil. The EVOO polyphenols had higher antioxidant activity in the brine samples than in the refined olive oil. The higher antioxidant activity of EVOO polyphenols in tuna packed in brine may be explained by their greater affinity toward the more polar interface between water and the fish oil system.     

Studies on the acetaldehyde-induced condensation of (-)-epicatechin and malvidin 3-O-glucoside in a model solution system.

The reaction between (-)-epicatechin, malvidin 3-O-glucoside, and acetaldehyde was studied in a model solution system. Ethyl-linked flavanol oligomers and anthocyanin-flavanol derivatives were observed, showing that the two polyphenols competed in the condensation process. Among the anthocyanin-ethyl-flavanol adducts, dimeric compounds in which the flavanol was linked to the anthocyanin with CH(3)-CH bridges were observed. In addition, trimeric and tetrameric products containing one anthocyanin and one, two, or three flavanols units were detected. A tetrameric product containing two anthocyanin and two flavanol units was also found as a doubly charged ion. No compound containing more than two malvidin 3-O-glucosides was detected, suggesting that only one anthocyanin A ring summit can be included in the polymerization process, which thus stops when both ends are occupied by an anthocyanin moiety. Thioacidolysis of the two isolated anthocyanin-ethyl-flavanol dimeric derivatives showed that anthocyanin-ethyl linkage was not sensitive to such reactants, whereas the flavanol-ethyl one was. In addition, flavanol-ethyl linkages involved in anthocyanin-ethyl-flavanol adducts were found to be less sensitive to those involved in flavan-ethyl dimers.     

Protection of lipids from oxidation by epicatechin, trans-resveratrol, and gallic and caffeic acids in intestinal model systems

Consumption of polyphenols is associated with health promotion through diet, although many are poorly absorbed in animals and humans alike. Lipid peroxides may reach the intestine and initiate deleterious oxidation. Here we measured inhibition of the oxidation of linoleic acid (LA) in authentic fluid from rat small intestine (RIF) by two dietary polyphenols, a flavonoid, epicatechin (EC), and a stilbene, resveratrol (RV), and by gallic (GA) and caffeic (CA) acids, and their partition coefficients. Both polyphenols inhibited 80%, and CA inhibited 65%, of the production of hexanal. GA was the weakest antioxidant in this assay. Interestingly, measuring peroxides production in RIF showed that only epicatechin inhibited the first stage of oxidation. The oxidizing agent, the antioxidant comound, the solution pH and lipophilicity are known to affect the total antioxidative activity. We suggest that the mechanism of this activity changes in accord with the environment: i.e., RV may act as a free radial scavenger, but here, in protecting lipids in intestinal fluid from oxidation, it acts as a hydrogen atom donor. Since the concentration of phenolics is much higher in the intestinal fluid than is ever achieved in plasma or other body tissues, it is suggested that their antioxidant activity could be exerted in the gastrointestinal tract (GIT), breaking the propagation of lipid peroxides oxidation and production of toxic compounds.     

Phenolic antioxidants and the protection of low density lipoprotein from peroxynitrite-mediated oxidations at physiologic CO2

Dietary phenolic antioxidants have been shown to prevent LDL modifications mediated by several physiologic oxidants including peroxynitrite. However, more recent data demonstrated that CO(2) affected the fate of peroxynitrite in biological fluids and significantly reduced peroxynitrite scavenging by polyphenols, raising doubts concerning their antioxidant activity. We found that the oxidation of LDL lipids mediated by peroxynitrite decreased in the presence of bicarbonate, while Trp oxidation and 3-nitroTyr formation increased, suggesting a redirection of peroxynitrite reactivity toward the protein moiety. We therefore evaluated the protective activity of some phenolic antioxidants (quercetin, oleuropein, resveratrol, (+)-catechin, (-)-epicatechin, tyrosol, alpha- and gamma-tocopherol, ascorbate) on peroxynitrite-mediated oxidation of LDL aromatic residues. Some of these phenols protected LDL Trp from oxidation better than ascorbate or alpha-tocopherol, although protection at 100 microM did not exceed 30-40%. However, the same phenolic antioxidants were more active in inhibiting 3-nitroTyr formation and those with a catechin structure provided significant protection (IC(50%) 40-50 microM). Red wine, a polyphenol-rich beverage, showed a protective effect comparable to that of the most active phenolic antioxidants. Direct EPR studies showed that bicarbonate significantly increased the peroxynitrite-dependent formation of O-semiquinone radicals in red wine, supporting the hypothesis that polyphenols are efficient scavengers of radicals formed by peroxynitrite/CO(2). Ascorbate was a poor inhibitor of peroxynitrite/CO(2)-induced LDL tyrosine nitration, but the simultaneous addition to the most active polyphenols halved their IC(50%). In conclusion, although cooperation with other antioxidants can further decrease the IC(50%) of polyphenolics, as demonstrated for ascorbate, their antioxidant activity appears to occur at concentrations at least 1 order of magnitude higher than their bioavailability.     

Oxidation of tea extracts and tea catechins by molecular oxygen

Tea polyphenols (PP) are known as potent antioxidants. At the same time, PP have been repeatedly reported to oxidize by molecular oxygen with the formation of active forms of oxygen. In this work, the Clark electrode technique was applied to study the kinetics of the autoxidation of tea extracts and individual tea PP as well as model PP, catechol, gallic acid, and pyrogallol. Aqueous extracts of both green and black teas were found to undergo extensive autoxidation under physiological conditions. The addition of superoxide dismutase (SOD) and milk resulted in a significant decrease in the rate of oxidation. Studied individually, PP were found to autoxidize at a rate, which increased with pH, proportional to PP concentration and nearly proportional to oxygen concentration. The collected data were used for the extrapolation/interpolation of the starting rates of oxidation to the standard conditions (at pH 7.40, 100 microM PP, 200 microM O2). PP oxidizability is basically determined by that of the key PP fragment (pyrogallol > gallate > catechol). Meta-OH groups do not contribute to the oxidation even at pH 13.0. Similar to tea brew, the oxidation of individual PP was inhibited by milk and SOD addition, with catechol being the only exception (the oxidation of catechol was accelerated when SOD was added). Comparison of the autoxidation of PP (o-hydroquinones) with that of p-hydroquinones (Roginsky, V.; Barsukova, T. K. J. Chem. Soc., Perkin Trans. 2 2000, 1575-1582) displays the dramatic difference both in the oxidizability and in the kinetic regularities. The difference in the kinetics has been suggested to be due to the difference in the initiation of the chain process. Whereas for p-hydroquinones the oxidation is initiated by the reaction between hydroquinone and a corresponding quinone, the oxidation of o-hydroquinones is likely started by direct interaction between substrate and molecular oxygen. As the second process is much slower, this may explain the relatively low oxidizability of PP as compared to p-hydroquinones.     

Physico-chemical characteristics of nanovesicle-carbohydrate complexes in grape juice concentrate

It is generally assumed that polyphenols, such as anthocyanins in fruit juice, exist in a free soluble state and are readily available for absorption in the gastro-intestinal tract. In the present study, we have investigated the interaction of polyphenols with soluble carbohydrate polymers, such as pectin and lipid nanovesicles, that are generated during homogenization of the fruit tissue during juice extraction. A commercially available grape juice concentrate contained nearly 25% of polyphenol fraction bound to macromolecules that were nondialyzable. Treatment of dialyzed juice with cellulase, pectinase, and beta-galactosidase did not cause the release of bound polyphenols; however, treatment with triton X-100 caused an increased release of bound polyphenols. The dialyzate contained relatively more -3-O glucosides and -3-O-acetoyl glucosides in comparison to the bound fraction which was enriched in -3-O-coumaroyl glucosides, suggesting qualitative differences in the bound and the free anthocyanin composition. Electron microscopic analysis of the juice fractions revealed the presence of electron-dense nanovesicle-fiber complexes ranging from 10 to 200 nm in diameter. Such complexes were absent in the dialyzate fraction. Cellulase treatment did not change the morphology of the complexes; however, treatment with pectinase and beta-galactosidase disrupted the complexes, releasing vesicular structures, suggestive of the pectin nature of the fibrous matrix. The dialyzed and the dialyzate fractions also showed differences in their 1H NMR and fluorescence spectral characteristics. The dialyzed fraction containing polyphenol-pectin complexes showed no superoxide scavenging capacity, reduced hydroxyl radical scavenging activity, and high 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, indicating potential changes in functionality because of the complex formation.