Catechin contents of foods commonly consumed in The Netherlands. 1. Fruits, vegetables, staple foods, and processed foods

Catechins, compounds that belong to the flavonoid class, are potentially beneficial to human health. To enable epidemiological evaluation of these compounds, data on their contents in foods are required. HPLC with UV and fluorescence detection was used to determine the levels of (+)-catechin, (-)-epicatechin, (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg) in 24 types of fruits, 27 types of vegetables and legumes, some staple foods, and processed foods commonly consumed in The Netherlands. Most fruits, chocolate, and some legumes contained catechins. Levels varied to a large extent: from 4.5 mg/kg in kiwi fruit to 610 mg/kg in black chocolate. (+)-Catechin and (-)-epicatechin were the predominant catechins; GC, EGC, and ECg were detected in some foods, but none of the foods contained EGCg. The data reported here provide a base for the epidemiological evaluation of the effect of catechins on the risk for chronic diseases.     

Inhibition of radical reaction of apolipoprotein B-100 and alpha-tocopherol in human plasma by green tea catechins

(-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-epigallocatechin gallate (EGCg), and Trolox inhibited the decreases of apolipoprotein B-100 (apoB) and alpha-tocopherol in a radical reaction of human plasma initiated by Cu(2+). The concentrations of EC, EGC, ECg, EGCg, and Trolox for 50% inhibition (IC50) of apoB fragmentation were 39.1, 42.2, 14.6, 21.3, and 36.2 microM, respectively. Similar IC50 values were observed for alpha-tocopherol consumption, indicating the close relationship between apoB fragmentation and alpha-tocopherol consumption. These results demonstrate that tea catechins serve as an effective antioxidant in plasma and that the gallate group has a strong antioxidative activity.     

Antioxidative activity of green tea treated with radical initiator 2, 2'-azobis(2-amidinopropane) dihydrochloride

This study investigated the antioxidative activity of green tea extract, and a green tea tannin mixture and its components, under conditions of radical generation using the hydrophilic azo compound, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) to generate peroxyl radicals at a constant and measurable rate in the cultured renal epithelial cell line, LLC-PK(1), which is susceptible to oxidative damage. Treatment with AAPH decreased cell viability and increased the formation of thiobarbituric acid-reactive substances. However, green tea extract, and the tannin mixture and its components, comprising (-)-epigallocatechin 3-O-gallate (EGCg), (-)-gallocatechin 3-O-gallate (GCg), (-)-epicatechin 3-O-gallate (ECg), (-)-epigallocatechin (EGC), (+)-gallocatechin (GC), (-)-epicatechin (EC), and (+)-catechin (C), showed protective activity against AAPH-induced cellular damage. The tannin mixture and its components exhibited higher antioxidative activity than the green tea extract. Furthermore, EGCg and GCg had higher activity than EGC and GC, respectively. In particular, EGCg exerted the most significant cellular protective activity against AAPH. These results indicate that green tea tannin may inhibit cellular loss and lipid peroxidation resulting from the peroxyl radical generated by AAPH, and that the chemical structure of tannin is also involved in the activity, suggesting that the O-dihydroxy structure in the B ring and the galloyl groups are important determinants for radical scavenging and antioxidative potential.     

Capillary electrophoretic determination of theanine, caffeine, and catechins in fresh tea leaves and oolong tea and their effects on rat neurosphere adhesion and migration

Theanine, caffeine, and catechins in fresh tea leaves and oolong tea were determined by using capillary electrophoresis (CE). CE separated these tea polyphenols from three other tea ingredients, namely, caffeine, theophylline, and theanine, within 8 min. The young leaves (apical bud and the two youngest leaves) were found to be richer in caffeine, (-)-epigallocatechin gallate (EGCg), and (-)-epicatechin gallate (ECg) than old leaves (from 5th to 7th leaves). On the other hand, the old leaves (from 8th to 10th leaves) contained higher levels of theanine, (-)-epigallocatechin (EGC), and (-)-epicatechin (EC). Results from a comparison of fresh young tea and oolong tea compositions indicated oolong tea contained more theanine and catechins than fresh young tea. Furthermore, it was found that the levels of theanine, EGC, and EGCg in young leaves rose markedly with the withering process. Caffeine did not markedly change. However, fully or partially fermented teas (oolong tea or pauchong tea) have a common initial step in the withering process. Fresh tea leaves or oolong tea extract (0.1%, w/v) markedly inhibited neurosphere adhesion, cell migration, and neurite outgrowth in rat neurospheres. Theanine (348 micrograms/mL) and caffeine at high concentration (50 micrograms/mL) did not inhibit neurosphere adhesion or migration activities, but EGCg at 20 micrograms/mL effectively inhibited neurosphere adhesion for 24 h. These results indicated that EGCg might affect neural stem cell survival or differentiation.     

Polyphenol-induced inhibition of the response of na(+)/glucose cotransporter expressed in Xenopus oocytes

To study the effects of polyphenols on the Na(+)/glucose cotransporter (SGLT1) response, SGLT1 was expressed in Xenopus oocytes by injecting cRNA synthesized from the cloned cDNA of the small intestine cotransporter of rats, and the electrical response elicited by glucose or galactose was measured by a voltage clamping method. Most phenol derivatives had no effect on the response. However, the polyphenols (+)-catechin, (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg), which are components of green tea, caused an inhibition of the response, which was almost independent of glucose concentration. The inhibition constants were estimated to be 2.3 mM for (+)-catechin and 0.45 mM for both ECg and EGCg, assuming the noncompetitive inhibition mechanism. Saponin prepared from tea seeds also inhibited the response significantly. Tannic acid and aqueous extracts of teas induced nonspecific electrical responses in both cRNA-injected and noninjected oocytes at lower concentrations than those that caused an inhibition of the SGLT1 response when their dose-dependent effects were examined. These results are possibly helpful in the development of a dietary supplement for diabetic patients.     

Effect of 3-O-octanoyl-(+)-catechin on the responses of GABA(A) receptors and Na+/glucose cotransporters expressed in xenopus oocytes and on the oocyte membrane potential

Recently, 3-O-octanoyl-(+)-catechin (OC) was synthesized from (+)-catechin (C) by incorporation of an octanoyl chain into C in the light of (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg), which are the major polyphenols found in green tea and have strong physiological activities. OC was found to inhibit the response of ionotropic gamma-aminobutyric acid (GABA) receptors (GABA(A) receptors) and Na+/glucose cotransporters expressed in Xenopus oocytes in a noncompetitive manner more strongly than does C. OC also induced a nonspecific membrane current and decreased the membrane potential of the oocyte, and thus death of the oocyte occurred even at lower concentrations than that induced by C or EGCg. Although EGCg produced H2O2 in aqueous solution, OC did not. This newly synthesized catechin derivative OC possibly binds to the lipid membrane more strongly than does C, Ecg, or EGCg and as a result perturbs the membrane structure.     

A tea catechin suppresses the expression of the high-affinity IgE receptor Fc epsilon RI in human basophilic KU812 cells

Human basophilic KU812 cells express the high-affinity IgE receptor Fc epsilon RI, which plays a central role in the IgE-mediated allergic response. The effect of several major tea catechins, (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, and (-)-epigallocatechin gallate (EGCg), on the cell surface expression of Fc epsilon RI in KU812 cells was studied. Flow cytometric analysis showed that only EGCg was able to decrease the cell surface expression of Fc epsilon RI after a 24 h treatment in a dose-dependent manner. Moreover, immunoblot analysis revealed that the total cellular expression of the Fc epsilon RI alpha chain decreased upon treatment with EGCg. Fc epsilon RI is a tetrameric structure comprising one alpha chain, one beta chain, and two gamma chains. The level of mRNA production of each subunit in KU812 cells was investigated. KU812 cells treated with EGCg expressed lower levels of Fc epsilon RI alpha and gamma mRNA than nontreated cells. These results suggest that EGCg has an ability to down-regulate Fc epsilon RI expression, and this suppressive effect may be due to the down-regulation of Fc epsilon RI alpha and gamma mRNA levels.     

Heat-epimerized tea catechins rich in gallocatechin gallate and catechin gallate are more effective to inhibit cholesterol absorption than tea catechins rich in epigallocatechin gallate and epicatechin gallate

It has been known that tea catechins, (-)-epicatechin (1), (-)-epigallocatechin (2), (-)-epicatechin gallate (3), and (-)-epigallocatechin gallate (4) are epimerized to(-)-catechin (5), (-)-gallocatechin (6), (-)-catechin gallate (7), and (-)-gallocatechin gallate (8), respectively, during retort pasteurization. We previously reported that tea catechins, mainly composed of 3 and 4, effectively inhibit cholesterol absorption in rats. In this study, the effect of heat-epimerized catechins on cholesterol absorption was compared with tea catechins. Both tea catechins and heat-epimerized catechins lowered lymphatic recovery of cholesterol in rats cannulated in the thoracic duct and epimerized catechins were more effective than tea catechins. The effect of purified catechins on micellar solubility of cholesterol was examined in an in vitro study. The addition of gallate esters of catechins reduced micellar solubility of cholesterol by precipitating cholesterol from bile salt micelles. Compounds 7 and 8 were more effective to precipitate cholesterol than 3 and 4, respectively. These observations strongly suggest that heat-epimerized catechins may be more hypocholesterolemic than tea catechins.     

Different effects of five catechins on 6-hydroxydopamine-induced apoptosis in PC12 cells.

Five catechins [(-)-epigallocatechins gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC), and (+)-catechin (C)] were compared with regard to their effects on 6-hydroxydopamine (OHDA)-induced apoptosis in PC12 cells--the vitro model of Parkinson's disease. Measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 6-OHDA inhibited cell viability in a time- and concentration-dependent manner. When PC12 cells were pretreated with the five catechins for 30 min before exposure to 250 microM 6-OHDA, MTT results showed that the five catechins had different effects: EGCG and ECG had obvious concentration-dependent protective effects at 50-400 microM; EC and (+)-C had almost no effects; and EGC especially decreased cell viability. Catechins also had different effects on apoptotic morphology. Only 200-400 microM EGCG and ECG kept cells adhering well. When pretreated with other catechins at any concentration, PC12 cells became round and some of them were detached as when treated with 6-OHDA. In addition, typical apoptotic characteristics of PC12 cells were determined by fluorescence microscopy, flow cytometry, and DNA fragment electrophoresis after the cells were treated with 250 microM 6-OHDA for 24 h or pretreated with catechins before it. Preincubation with 200-400 microM EGCG and ECG led to significant inhibitory effects against PC12 cell apoptosis, as shown by flow cytometry. The other catechins have little protective effect. Therefore, at 200-400 microM, the classified protective effects of the five catechins were in the order ECG > EGCG > EC > (+)-C > EGC. The data also indicated that EGCG and ECG might be potent neuroprotective agents for Parkinson's disease. The results of fluorescence microscopy and DNA fragment analysis supported the conclusion.     

Inhibitory effects of green tea polyphenols on the production of a virulence factor of the periodontal-disease-causing anaerobic bacterium Porphyromonas gingivalis

The effect of polyphenolic compounds isolated from green tea (Camellia sinensis) on the production of toxic end metabolites of Porphyromonas gingivalis was investigated. Green tea polyphenols completely inhibited the production of n-butyric acid and propionic acid at a concentration of 1.0-2.0 mg/mL in general anaerobic medium (GAM). (-)-Epigallocatechin gallate (EGCg), which is a major component of tea polyphenols also inhibited the production of phenylacetic acid at 0.5 mg/mL in GAM broth. In the experiment using resting cells of P. gingivalis, phenylacetic acid was produced from l-phenylalanine and phenylpyruvic acid, but this reaction was also inhibited by EGCg, (-)-epicatechin gallate, and (-)-gallocatechin gallate. However, (+)-catechin, (+)-gallocatechin, (-)-epicatechin, and (-)-epigallocatechin did not inhibit those reactions. These results indicate that the inhibitory effect on the production of toxic end metabolites of P. gingivalis can be attributed to the presence of the galloyl moiety, which is ester-linked with the 3-OH of the catechin moiety in the polyphenolic compounds. This study shows that continuous application of tea polyphenols on a daily basis can be considered as a useful and practical method for the prevention of periodontal diseases.     

Quantification of the predominant monomeric catechins in baking chocolate standard reference material by LC/APCI-MS

Catechins are polyphenolic plant compounds (flavonoids) that may offer significant health benefits to humans. These benefits stem largely from their anticarcinogenic, antioxidant, and antimutagenic properties. Recent epidemiological studies suggest that the consumption of flavonoid-containing foods is associated with reduced risk of cardiovascular disease. Chocolate is a natural cocoa bean-based product that reportedly contains high levels of monomeric, oligomeric, and polymeric catechins. We have applied solid-liquid extraction and liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry to the identification and determination of the predominant monomeric catechins, (+)-catechin and (-)-epicatechin, in a baking chocolate Standard Reference Material (NIST Standard Reference Material 2384). (+)-Catechin and (-)-epicatechin are detected and quantified in chocolate extracts on the basis of selected-ion monitoring of their protonated [M + H](+) molecular ions. Tryptophan methyl ester is used as an internal standard. The developed method has the capacity to accurately quantify as little as 0.1 microg/mL (0.01 mg of catechin/g of chocolate) of either catechin in chocolate extracts, and the method has additionally been used to certify (+)-catechin and (-)-epicatechin levels in the baking chocolate Standard Reference Material. This is the first reported use of liquid chromatography/mass spectrometry for the quantitative determination of monomeric catechins in chocolate and the only report certifying monomeric catechin levels in a food-based Standard Reference Material.     

Stability of tea catechins in the breadmaking process

A green tea extract (GTE) was incorporated into bread as a source of tea catechins. The stability of tea catechins in the breadmaking process including unfrozen and frozen dough was studied. A method was developed for the separation and quantification of tea catechins in GTE, dough, and bread samples using a RP-HPLC system. The separation system consisted of a C18 reversed-phase column, a gradient elution system of water/methanol and formic acid, and a photodiode array UV detector. Tea catechins were detected at 275 nm. GTEs at 50, 100, and 150 mg per 100 g of flour were formulated. The results obtained showed that green tea catechins were relatively stable in dough during freezing and frozen storage at -20 degrees C for up to 9 weeks. There were no further detectable losses of tea catechins in bread during a storage of 4 days at room temperature. It was also revealed that (-)-epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) were more susceptible to degradation than (-)-epicatechin gallate (ECG) and (-)-epicatechin (EC). (-)-EGCG and (-)-ECG were normally selected as the quality indices of green tea catechins, and their retention levels in freshly baked bread were ca. 83 and 91%, respectively. One piece of bread (53 g) containing 150 mg of GTE/100 g of flour will provide 28 mg of tea catechins, which is approximately 35% of those infused from one green tea bag (2 g).     

Dynamic behavior of tea catechins interacting with lipid membranes as determined by NMR spectroscopy

Interaction between tea catechins, such as epicatechin gallate (ECg) and epigallocatechin gallate (EGCg), and isotropic bicelle model lipid membranes was investigated by solution NMR techniques. (1)H NMR measurements provided signals from the B-ring and the galloyl moiety in ECg and EGCg that were obviously shifted, and whose proton T1 relaxation times were shortened upon interaction of the catechins with the bicelles. These results indicate that the B-ring and the galloyl moiety play an important role in this interaction. Nuclear Overhauser effect spectrometry experiments demonstrated that the B-ring and the galloyl moiety are located near the gamma-H in the phospholipid trimethylammonium group. On the basis of these findings, we propose that ECg and EGCg interact with the surface of lipid membranes via the choline moiety.     

Effects of tea polyphenols on the invasion and matrix metalloproteinases activities of human fibrosarcoma HT1080 cells

The effects of tea polyphenols on the invasion of highly metastatic human fibrosarcoma HT1080 cells through a monolayer of human umbilical vein endothelial cells (HUVECs) and the accompanying basal membrane were investigated. Among the tea polyphenols tested, epicatechin gallate (ECg), epigallocatechin gallate (EGCg), and theaflavin strongly suppressed the invasion of HT1080 cells into the monolayer of HUVECs/gelatin membrane, whereas epicatechin, epigallocatechin, tea flavonols, tea flavones, and gallate derivatives had no effect. Both theaflavin-digallate and theasinensin D showed a weak invasion inhibitory effect. ECg significantly inhibited the invasion without cytotoxicity against cancer cells and HUVECs. Ester-type catechins (ECg and EGCg) and theaflavin strongly suppressed the gelatin degradation mediated by matrix metalloproteinase (MMP) 2 and MMP-9, which were secreted into the conditioned medium of HT1080 cells. In conclusion, among the tea polyphenols tested, ECg was considered to be the agent with the most potential antimetastasis activity because it inhibited invasion in the absence of cytotoxicity.     

Green tea polyphenols inhibit metalloproteinase activities in the skin,muscle, and blood of rainbow trout

We have investigated the inhibitory effects of polyphenols from natural products, such as green tea, bilberry, grape, ginkgo, and apple, on rainbow trout gelatinase activities. Gelatinases from the skin, muscle, and blood of rainbow trout contained serine proteinase, metalloproteinase, and other proteinase activities as measured by gelatin zymography. The polyphenols of green tea caused the strong inhibition of some gelatinase activities when compared with those of the other products. This inhibition was quite similar to that of metalloproteinase by ethylenediaminetetraacetic acid, suggesting that the effects of green tea polyphenols on proteinase activities are specific for metalloproteinases. The major catechins of green tea polyphenols were then separated and identified by reverse-phase chromatography to be (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin, (-)-epicatechin gallate, and (-)-epicatechin. The effects of these catechins on gelatinase activities were examined; the most potent inhibitor of metalloproteinase activities was found to be EGCG. These results have indicated that green tea polyphenols including EGCG are useful for regulating metalloproteinase activities of fish meat.     

Determination of tea polyphenols and caffeine in tea flowers (Camellia sinensis) and their hydroxyl radical scavenging and nitric oxide suppressing effects

The native occurrence of tea polyphenols, namely, (-)-epicatechin, (+)-catechin, (-)-epigallocatechin 3-gallate, (-)-epicatechin, and (-)-epicatechin 3-gallate, and caffeine in tea flowers was assessed by an isocratic HPLC procedure. The levels of total catechins and caffeine were determined in tea flowers collected from 10 different species of Camellia sinensis. The results showed the levels of total catechin ranged from 10 to 38 mg/g, whereas the level of caffeine ranged from 3 to 8 mg/g. Levels of catechins and caffeine in tea leaves and various teas were also determined and ranged from 2 to 126 mg/g and from 23 to 49 mg/g, respectively. Both tea flower and tea leaf extracts exert their strong hydroxyl radical scavenging effects in the Fenton reaction system and nitric oxide suppressing effects in LPS-induced RAW 264.7 cells. Most tea flowers contain less caffeine, but comparable amounts of total catechins, compared to tea leaves and teas. The present study demonstrates that both tea flowers and tea leaves contain appreciable amounts of catechins and caffeine. It is likely that tea flowers might be useful for making alternative tea beverages.     

Electrofocusing of methanolic extracts for identification of individual flavonol biomolecules in Camellia species

An effort has been made to isolate individual catechin compounds from green tea leaves in their pure form by electrophoresis. In the present study total polyphenol extraction was carried out initially and estimated through spectrophotometric and HPLC methods. Extracted polyphenol was separated on 0.7% agarose gel and visualized at 360 nm. Fragmented individual compounds were gel eluted with methanol and confirmed as (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) by HPLC. The method developed describes a suitable method for the isolation of valuable molecules in tea.     

A novel convenient process to obtain a raw decaffeinated tea polyphenol fraction using a lignocellulose column

Lignocellulose prepared from sawdust was investigated for its potential application in obtaining a raw decaffeinated tea polyphenol fraction from tea extract. Tea polyphenols having gallate residues, namely, (-)epigallocatechin gallate (EGCg) and (-)epicatechin gallate (ECg), were adsorbed on the lignocellulose column, while caffeine was passed through it. Adsorbed polyphenols were eluted with 60% ethanol, and the elute was found to consist mainly of EGCg and ECg. The caffeine/EGCg ratio was 0.696 before lignocellulose column treatment, but it became 0.004 after the column treatment. These results suggest that the lignocellulose column provides a useful and convenient process of purification of tea polyphenol fraction accompanied by decaffeination.     

Regeneration of alpha-tocopherol in human low-density lipoprotein by green tea catechin.

Oxidative modification of low-density lipoproteins (LDL) may play an important role in the development of atherosclerosis. alpha-Tocopherol functions as a major antioxidant in human LDL. The present study was to test whether green tea catechins (GTC) would protect or regenerate alpha-tocopherol in human LDL. The oxidation of LDL incubated in sodium phosphate buffer (pH 7.4, 10 mM) was initiated by addition of 1.0 mM of 2,2'-azobis(2-amidinopropane) dihydrochloride at 40 degrees C. It was found that alpha-tocopherol was completely depleted within 1 h. Under the same experimental conditions, the longjing GTC extracts demonstrated a dose-dependent protective activity to alpha-tocopherol in LDL at concentrations ranging from 2 to 20 microM. Four pure epicatechin derivatives showed varying protective activity against depletion of alpha-tocopherol in LDL with (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG) being less effective than (-)-epicatechin (EC) and (-)-epicatechin gallate (ECG). The results showed that addition of longjing GTC extracts, EC, ECG, and EGCG at 5, 10, and 15 min to the incubation mixture demonstrated a gradual regeneration of alpha-tocopherol in human LDL.     

Determination of tea components with antioxidant activity

Levels of essential elements with antioxidant activity, as well as catechins, gallic acid, and caffeine levels, in a total of 45 samples of different teas commercialized in Spain have been evaluated. Chromium, manganese, selenium, and zinc were determined in the samples mineralized with HNO(3) and V(2)O(5), using ETAAS as the analytical technique. The reliability of the procedure was checked by analysis of a certified reference material. Large variations in the trace element composition of teas were observed. The levels ranged from 50.6 to 371.4 ng/g for Cr, from 76.1 to 987.6 microg/g for Mn, from 48.5 to 114.6 ng/g for Se, and from 56.3 to 78.6 ng/g for Zn. The four major catechins [(-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC)], gallic acid (GA), and caffeine were simultaneously determined by a simple and fast HPLC method using a photodiode array detector. In all analyzed samples, EGCG ranged from 1.4 to 103.5 mg/g, EGC from 3.9 to 45.3 mg/g, ECG from 0.2 to 45.6 mg/g, and EC ranged from 0.6 to 21.2 mg/g. These results indicated that green tea has a higher content of catechins than both oolong and fermented teas (red and black teas); the fermentation process during tea manufacturing reduces the levels of catechins significantly. Gallic acid content ranged from 0.039 to 6.7 mg/g; the fermentation process also elevated remarkably gallic acid levels in black teas (mean level of 3.9 +/- 1.5 mg/g). The amount of caffeine in the analyzed samples ranged from 7.5 to 86.6 mg/g, and the lower values were detected in green and oolong teas. This study will be useful for the appraisal of trace elements and antioxidant components in various teas, and it will also be of interest for people who like drinking this beverage.