Removal of polyphenolic compounds interfering with carbohydrate determinations in plant extracts with an insoluble polyphenol adsorbent.
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PURPOSE: Green tea and green tea polyphenols have been shown to possess cancer preventive activities in preclinical model systems. In preparation for future green tea intervention trials, we have conducted a clinical study to determine the safety and pharmacokinetics of green tea polyphenols after 4 weeks of daily p.o. administration of epigallocatechin gallate (EGCG) or Polyphenon E (a defined, decaffeinated green tea polyphenol mixture). In an exploratory fashion, we have also determined the effect of chronic green tea polyphenol administration on UV-induced erythema response. EXPERIMENTAL DESIGN: Healthy participants with Fitzpatric skin type II or III underwent a 2-week run-in period and were randomly assigned to receive one of the five treatments for 4 weeks: 800 mg EGCG once/day, 400 mg EGCG twice/day, 800 mg EGCG as Polyphenon E once/day, 400 mg EGCG as Polyphenon E twice/day, or a placebo once/day (8 subjects/group). Samples were collected and measurements performed before and after the 4-week treatment period for determination of safety, pharmacokinetics, and biological activity of green tea polyphenol treatment. RESULTS: Adverse events reported during the 4-week treatment period include excess gas, upset stomach, nausea, heartburn, stomach ache, abdominal pain, dizziness, headache, and muscle pain. All of the reported events were rated as mild events. For most events, the incidence reported in the polyphenol-treated groups was not more than that reported in the placebo group. No significant changes were observed in blood counts and blood chemistry profiles after repeated administration of green tea polyphenol products. There was a >60% increase in the area under the plasma EGCG concentration-time curve after 4 weeks of green tea polyphenol treatment at a dosing schedule of 800 mg once daily. No significant changes were observed in the pharmacokinetics of EGCG after repeated green tea polyphenol treatment at a regimen of 400 mg twice daily. The pharmacokinetics of the conjugated metabolites of epigallocatechin and epicatechin were not affected by repeated green tea polyphenol treatment. Four weeks of green tea polyphenol treatment at the selected dose and dosing schedule did not provide protection against UV-induced erythema. CONCLUSIONS: We conclude that it is safe for healthy individuals to take green tea polyphenol products in amounts equivalent to the EGCG content in 8-16 cups of green tea once a day or in divided doses twice a day for 4 weeks. There is a >60% increase in the systemic availability of free EGCG after chronic green tea polyphenol administration at a high daily bolus dose (800 mg EGCG or Polyphenon E once daily).
Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or -18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles x mg(-1) chlorophyll x hr(-1). Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes.Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30 minutes. No single peptide from the digested trypsin was found to be the sole activating factor. About 0.25 mug of trypsin activated 50% the polyphenol oxidase activity in a standard chloroplast assay containing 2.1 mug of chlorophyll. Treatment of spinach chloroplasts with tris buffer or ethylenediamine tetraacetate extracted the ATPase activity, but the polyphenol oxidase activity remained with the broken plastids. However these treatments increased the latent polyphenol oxidase activity 50- to 100-fold.Chloroplasts from a second group of plants, including alfalfa, wheat, oats, peas, and sugarcane leaves, oxidized dihydroxyphenylalanine at a rate of 11 to 120 mumoles x mg(-1) chlorophyll x hr(-1). Polyphenol oxidase in these chloroplasts required a low intensity of red light for activity. Fifty or 75% activation of the oxidase in wheat chloroplasts required 4 to 6 foot candles of light and more light was required for alfalfa chloroplasts. Blue or far red light were ineffective. Trypsin was inhibitory. Upon aging chloroplasts from wheat leaves, but not alfalfa or peas, for 5 to 7 days at 4 C the total polyphenol oxidase activity did not increase, but the activation characteristics changed to those of chloroplasts from the spinach group. Chloroplasts from a third group of plants, including bean, tomato, and corn leaves, slowly oxidized dihydroxyphenylalanine in the dark and exhibited no latency.
Human intervention studies have provided clear evidence that dietary polyphenols (eg, flavonoids--eg, flavonols--and isoflavones) are at least partly absorbed and that they have the potential to exert biological effects. Biological activity of polyphenols is often assessed by using cultured cells as tissue models; in almost all such studies, cells are treated with aglycones or polyphenol-rich extracts (derived from plants and foods), and data are reported at concentrations that elicited a response. There are 2 inherent flaws in such an approach. First, plasma and tissues are not exposed in vivo to polyphenols in these forms. Several human studies have identified the nature of polyphenol conjugates in vivo and have shown that dietary polyphenols undergo extensive modification during first-pass metabolism so that the forms reaching the blood and tissues are, in general, neither aglycones (except for green tea catechins) nor the same as the dietary source. Polyphenols are present as conjugates of glucuronate or sulfate, with or without methylation of the catechol functional group. As a consequence, the polyphenol conjugates are likely to possess different biological properties and distribution patterns within tissues and cells than do polyphenol aglycones. Although deconjugation can potentially occur in vivo to produce aglycone, it occurs only at certain sites. Second, the polyphenol concentrations tested should be of the same order as the maximum plasma concentrations attained after a polyphenol-rich meal, which are in the range of 0.1-10 micromol/L. For correct interpretation of results, future efforts to define biological activities of polyphenols must make use of the available data concerning bioavailability and metabolism in humans.
Cocoa contains many different types of physiologically active components. It was shown that cocoa beans are rich in specific antioxidants such as flavonoids, catechins, epicatechins and proanthocyanidins. Additionally, beta-sitosterol, the most common phytosterol, may play a protective role in the development of cancer. The aim of this in-vitro study was to evaluate the inhibitory effect of different cocoa polyphenols extracts, alone or combined with beta-sitosterol, on two human prostate cancer cell lines (nonmetastatic 22Rv1 cells and metastatic DU145 cells) and a normal human prostate cell line (RWEP-1). A synergy between beta-sitosterol and cocoa polyphenols extract was also researched. Cells were treated independently with five products from 1 to 72 h: (1/) synthetic beta-sitosterol, (2/) a cocoa polyphenols extract supplemented with beta-sitosterol, (3/) three different cocoa polyphenols extracts naturally containing beta-sitosterol. In the experiment, beta-sitosterol was tested from 10(-6) to 10(-3)%; cocoa polyphenols extract supplementation was with 0.72% beta-sitosterol; finally cocoa polyphenols extracts were added to the cells at very low concentrations ranging from 0.001 to 0.2%. The growth and viability of cells were measured using colorimetric assay at 1, 3, 6, 24, 48 and 72 h of treatment. IC50 and IC100 corresponding to the concentration leading to a decrease of 50% and 100% of cell growth were determined. At the highest tested concentration, cocoa polyphenols extracts induced a complete inhibition of growth of metastatic and nonmetastatic cancer cell lines. In addition, cocoa polyphenols extracts were more active against local cancer cells than against metastatic cells. Moreover, at the highest tested concentration, cocoa polyphenols extracts are not effective on a normal prostate cell lines. Beta-sitosterol induced low growth inhibition of both cancer cell line. Cocoa polyphenols extracts, however, were significantly more active and showed a strong and fast inhibition of cell growth than beta-sitosterol alone. No synergy or addition was observed when beta-sitosterol was tested together with the cocoa polyphenols extract. Our results show that cocoa polyphenols extracts have an antiproliferative effect on prostate cancer cell growth but not on normal cells, at the highest tested concentration.
Polyphenols are a large and diverse class of compounds, many of which occur naturally in a range of food plants. The flavonoids are the largest and best-studied group of these. A range of plant polyphenols are either being actively developed or currently sold as dietary supplements and/or herbal remedies. Although, these compounds play no known role in nutrition (non-nutrients), many of them have properties including antioxidant, anti-mutagenic, anti-oestrogenic, anti-carcinogenic and anti-inflammatory effects that might potentially be beneficial in preventing disease and protecting the stability of the genome. However not all polyphenols and not all actions of individual polyphenols are necessarily beneficial. Some have mutagenic and/or pro-oxidant effects, as well as interfering with essential biochemical pathways including topoisomerase enzyme activities, prostanoid biosynthesis and signal transduction. There is a very large amount of in vitro data available, but far fewer animal studies, and these are not necessarily predictive of human effects because of differences in bacterial and hepatic metabolism of polyphenols between species. Epidemiological studies suggest that high green tea consumption in the Japanese population and moderate red wine consumption in the French population may be beneficial for heart disease and cancer, and these effects may relate to specific polyphenols. A small number of adequately controlled human intervention studies suggest that some, but not all polyphenol extracts or high polyphenol diets may lead to transitory changes in the antioxidative capacity of plasma in humans. However, none of these studies have adequately considered long-term effects on DNA or the chromosome and unequivocally associated these with polyphenol uptake. Furthermore, clinical trials have required intravenously administered polyphenols at concentrations around 1400mg/m(2) before effects are seen. These plasma concentrations are unlikely to be achieved using the dietary supplements currently available. More focused human studies are necessary before recommending specific polyphenolic supplements at specific doses in the human population.
Polyphenols are abundant micronutrients in our diet, and evidence for their role in the prevention of degenerative diseases is emerging. Bioavailability differs greatly from one polyphenol to another, so that the most abundant polyphenols in our diet are not necessarily those leading to the highest concentrations of active metabolites in target tissues. Mean values for the maximal plasma concentration, the time to reach the maximal plasma concentration, the area under the plasma concentration-time curve, the elimination half-life, and the relative urinary excretion were calculated for 18 major polyphenols. We used data from 97 studies that investigated the kinetics and extent of polyphenol absorption among adults, after ingestion of a single dose of polyphenol provided as pure compound, plant extract, or whole food/beverage. The metabolites present in blood, resulting from digestive and hepatic activity, usually differ from the native compounds. The nature of the known metabolites is described when data are available. The plasma concentrations of total metabolites ranged from 0 to 4 mumol/L with an intake of 50 mg aglycone equivalents, and the relative urinary excretion ranged from 0.3% to 43% of the ingested dose, depending on the polyphenol. Gallic acid and isoflavones are the most well-absorbed polyphenols, followed by catechins, flavanones, and quercetin glucosides, but with different kinetics. The least well-absorbed polyphenols are the proanthocyanidins, the galloylated tea catechins, and the anthocyanins. Data are still too limited for assessment of hydroxycinnamic acids and other polyphenols. These data may be useful for the design and interpretation of intervention studies investigating the health effects of polyphenols.
Polyphenol interactions with both cellulose and collagen in the solid state have been studied by using chromatography on cellulose and by evaluating the hydrothermal stability of the polyphenol treated sheepskin collagen. Twenty-four polyphenolic compounds were studied, including seven glucose-based gallotannins, five polyalcohol-based gallotannins, and twelve ellagitannins. In the cellulose-polyphenols systems, the polyphenol's affinity to cellulose is positively correlated with their molecular masses, the number of galloyl groups, and their hydrophobicity (logP). The polyphenol treatment increased the hydrothermal stability of collagen samples, and such effects are also positively correlated with the molecular masses, total number of galloyl groups and the hydrophobicity of polyphenols. Ellagitannins showed much weaker interactions with both biopolymers than gallotannins having similar molecular mass, the same number of galloyl groups, and the same number of phenolic hydroxyl groups. It is concluded that, for the polyphenol interactions with both cellulose and collagen, (1) the galloyl group of polyphenols is the functional group; (2) the strength of interactions are positively correlated with molecular size, the number of galloyl groups and the hydrophobicity of polyphenols; (3) the hydrophobic interactions are of great significance; and (4) the interactions are strongly dependent on the flexibility of galloyl groups.
Nutrition is thought to play an essential role in the pathogenesis of inflammatory and malignant gastrointestinal diseases. It is well known that plant ingredients such as polyphenols and flavonoids show anticarcinogenic effects both in vitro and in animal experiments, and may thus reduce the risk of colorectal cancer in man. The aim of the study was to determine the amount of polyphenols reaching the colon after oral intake of apple juice. After consumption of a polyphenol-free diet 11 healthy ileostomy volunteers drank 1 L of a polyphenol-rich cloudy apple juice. Ileostomy effluent was collected immediately before and 1, 2, 4, 6, and 8 h after consumption of apple juice. A broad spectrum of polyphenols was identified using HPLC-diode array detection (HPLC-DAD) as well as HPLC-ESI-MS/MS; quantitation was performed with HPLC-DAD. Most of the orally administered apple polyphenols were absorbed from or metabolized in the small intestine. Between 0 and 33% of the oral dose was recovered in the ileostomy bags with a maximum of excretion after 2 h. Phloretin glucuronide as product of polyphenol metabolism was detected in the ileostomy effluent. The present results show that most of the apple juice polyphenols are absorbed in the small intestine. Minor amounts of unmetabolized polyphenols are recovered in the ileostomy effluent, which would reach the colon under physiologic circumstances. These data have to be considered when polyphenols are used in model systems to show preventive effects in colorectal carcinogenesis.
Polyphenols are abundant micronutrients in our diet, and evidence for their role in the prevention of degenerative diseases such as cancer and cardiovascular diseases is emerging. The health effects of polyphenols depend on the amount consumed and on their bioavailability. In this article, the nature and contents of the various polyphenols present in food sources and the influence of agricultural practices and industrial processes are reviewed. Estimates of dietary intakes are given for each class of polyphenols. The bioavailability of polyphenols is also reviewed, with particular focus on intestinal absorption and the influence of chemical structure (eg, glycosylation, esterification, and polymerization), food matrix, and excretion back into the intestinal lumen. Information on the role of microflora in the catabolism of polyphenols and the production of some active metabolites is presented. Mechanisms of intestinal and hepatic conjugation (methylation, glucuronidation, sulfation), plasma transport, and elimination in bile and urine are also described. Pharmacokinetic data for the various polyphenols are compared. Studies on the identification of circulating metabolites, cellular uptake, intracellular metabolism with possible deconjugation, biological properties of the conjugated metabolites, and specific accumulation in some target tissues are discussed. Finally, bioavailability appears to differ greatly between the various polyphenols, and the most abundant polyphenols in our diet are not necessarily those that have the best bioavailability profile. A thorough knowledge of the bioavailability of the hundreds of dietary polyphenols will help us to identify those that are most likely to exert protective health effects.
The objective of this study was to create a French database on the polyphenol content of fruit and vegetables as uncooked fruits and vegetables and then to evaluate polyphenol intake through fruit and vegetable consumption in France. To achieve this, we used the Folin-Ciocalteu method adapted to fruit and vegetable polyphenol quantitation (1). Vegetables with the highest polyphenol concentration were artichokes, parsley, and brussels sprouts [>250 mg of gallic acid equivalent (GAE)/100 g fresh edible portion (FEP)]; fruits with the highest concentrations were strawberries, lychees, and grapes (>180 mg of GAE/100 g FEP). Conversely, melons (Cantaloupe cv.) and avocados had the lowest polyphenol concentration for fruits and vegetables, respectively. Based on fruit consumption data, apples and strawberries are the main sources of polyphenols in the French diet, whereas potatoes, lettuces, and onions are the most important vegetable sources. Total polyphenol intake from fruit is about 3 times higher than from vegetables, due to the lower polyphenol concentration in vegetables. The calculation of polyphenol intake, based on both assessment methods used [(Société d'Etudes de la Communication, Distribution et Publicité (SECODIP) and Supplémentation en Vitamines et Minéraux Antioxydants (SUVIMAX)], showed that apples and potatoes provide approximatively half of the total polyphenol intake from fruit and vegetables in the French diet.
Investigations indicate that an extract of green tea, polyphenol, can significantly increase the culture survival rate of rat islets without deteriorating their functionality. In this study, we examined the effect of adding polyphenol to islets isolated from human pancreata and nonhuman primate pancreata. Islets were isolated from human pancreata that did not meet criteria for clinical transplantation (n = 6) and from nonhuman primate pancreata (n = 5). The islets were cultured in CMRL-1066 + 10% FCS with the addition of 0, 30, 60, 125, 250, or 500 microg/ml of polyphenol. After 24 or 48 h of culture, islet yield, viability, purity, morphology, and stimulation index was assessed. RT-PCR and Western blot analysis were also performed to assess the expression levels of the apoptotic related genes, Bcl-2 and BAX. After 24 h of culture, islet yields were significantly higher in cultures supplemented with 30-250 microg/ml of polyphenol than in cultures without polyphenol. After 48 h of culture, significant differences in islet numbers were observed with polyphenol concentrations of 125 microg/ml (p < 0.01) and 250 microg/ml (p < 0.01). However, no significant differences were noted in islet viability, purity, morphology, and stimulation index at each time point with or without polyphenol. RT-PCR and Western blot analysis of the islets indicated that Bcl-2 levels increased by 2.5-fold and BAX levels decreased by twofold in cultures supplemented with polyphenol. This resulted in BAX/Bcl-2 ratios that were lower in polyphenol-supplemented cultures than with control cultures. Polyphenol increases culture recovery rates by precluding islet apoptosis.
Dietary polyphenolics in fruits, vegetables, wines, spices and herbal medicines have beneficial antioxidant, anti-inflammatory and anticancer effects. However, we have observed that dietary polyphenolics with phenol rings were metabolized by peroxidase to form prooxidant phenoxyl radicals which, in some cases were sufficiently reactive to cooxidize GSH or NADH accompanied by extensive oxygen uptake and reactive oxygen species formation. The order of catalytic effectiveness found for oxygen activation when polyphenolics were metabolized by peroxidase in the presence of GSH was phloretin>phloridzin>4,2'-dihydroxy chalcone>p-coumaric acid>naringenin>apigenin>curcumin>resveratrol>isoliquiritigenin>capsaicin>kaempferol. Ascorbate was also cooxidized by the phenoxyl radicals but without oxygen activation. Polyphenolics with catechol rings also cooxidized ascorbate, likely mediated by semiquinone radicals. The order of catalytic effectiveness found for ascorbate cooxidation was fisetin luteolin, quercetin, >eriodictyol, caffeic acid, nordihydroguaiaretic acid>catechin>taxifolin, catechol. NADH was stoichiometrically oxidized without oxygen uptake which, suggests that o-quinone metabolites were responsible. GSH was not cooxidized and GSH conjugates were formed, likely mediated by the o-quinone metabolites. Incubation of hepatocytes with dietary polyphenolics containing phenol rings was found to partially oxidize hepatocyte GSH to GSSG while polyphenolics with a catechol ring were found to deplete GSH through formation of GSH conjugates. Dietary polyphenolics with phenol rings also oxidized human erythrocyte oxyhemoglobin and caused erythrocyte hemolysis more readily than polyphenolics with catechol rings. It is concluded that polyphenolics containing a phenol ring are generally more prooxidant than polyphenolics containing a catechol ring.
Polyphenols in cereals and legumes have been receiving considerable attention largely because of their adverse influence on color, flavor, and nutritional quality. These compounds belong to the flavonoid and tannin groups and are mostly located in the seed coat or pericarp of the grains. The pearl millet flavonoids have been identified as C-glycosylflavones by the combined use of paper chromatography and UV spectroscopy. Although nontoxic, physiological and nutritional significance of these compounds occurring in high amounts in the pearl millet grain are still not clearly understood. In view of aesthetic quality, bleaching of the millet grains in acidic solution is recommended. A large proportion of current assays involves spectrophotometry of tannin or its chromogen and tannin-protein interaction. Sorghum and legume tannins have been characterized as condensed tannins. Several factors such as plant type, age of the plant or plant parts, stage of development, and environmental conditions govern the polyphenol contents in plants. Polyphenols are known to interact with proteins and form tannin-protein complexes leading to either inactivation of enzymes or making proteins insoluble. These are implicated in decreasing the activities of digestive enzymes, protein and amino acid availabilities, mineral uptake, vitamin metabolism, and depression of growth. Polyphenols are known to cause certain ultrastructural changes in the different parts of experimental animals. A correlation between dietary tannins and occurrence of esophageal cancer has been established. Bird resistance and seed germination in food crops have been correlated to high contents of polyphenols. The antinutritional activity of polyphenols can be reduced by removing polyphenols from the grains by chemical treatments or removing pericarp and testa by pearling. Treatment of alkaline reagents and ammonia can remove 90% of the polyphenols. Supplementation of polyphenols-rich diet with protein can alleviate the growth-depressing effect of polyphenols.
Oxidized low-density lipoprotein (LDL) is considered a risk factor in atherosclerosis, and polyphenols are the potential agents to inhibit the oxidation of LDL. We determined the polyphenol contents and the antioxidant activities of commonly consumed vegetables in Vietnam and assessed the quantity of the polyphenol intake from vegetables in the current Vietnamese diet. The polyphenol contents in 30 kinds of vegetables was determined by the Folin-Ciocalteu method. The antioxidant activities of vegetables were evaluated by measuring the oxidation of LDL and the reduction of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. In this study, some herbs and edible wild vegetables possessed high contents of polyphenols and antioxidant activities. Among green vegetables, sweet potato leaves showed both a high polyphenol content and antioxidant activity. The mean polyphenol daily intake of the Vietnamese was a 595 mg catechin equivalent. Water spinach, a kind of green vegetable, contributed the highest amount (45%) of the total polyphenol intake, followed by other green vegetables. Neither herbs nor edible wild vegetables contributed significantly to the total polyphenol intake due to their low consumption. Green vegetables are therefore considered very important sources of polyphenol intake for the Vietnamese.