Modulation of cholesterol metabolism by the green tea polyphenol (-)-epigallocatechin gallate in cultured human liver (HepG2) cells

Epidemiological and animal studies have found that green tea is associated with lower plasma cholesterol. This study aimed to further elucidate how green tea modulates cholesterol metabolism. When HepG2 cells were incubated with the main green tea constituents, the catechins, epigallocatechin gallate (EGCG) was the only catechin to increase LDL receptor binding activity (3-fold) and protein (2.5-fold) above controls. EGCG increased the conversion of sterol regulatory element binding protein-1 (SREBP-1) to its active form (+56%) and lowered the cellular cholesterol concentration (-28%). At 50 microM, EGCG significantly lowered cellular cholesterol synthesis, explaining the reduction in cellular cholesterol. At 200 microM EGCG, cholesterol synthesis was significantly increased even though cellular cholesterol was lower, but there was a significant increase seen in medium cholesterol. This indicates that, at 200 microM, EGCG increases cellular cholesterol efflux. This study provides mechanisms by which green tea modulates cholesterol metabolism and indicates that EGCG might be its active constituent.     

Profiling the chlorogenic acids and other caffeic acid derivatives of herbal chrysanthemum by LC-MSn

Four samples of herbal chrysanthemum have been profiled qualitatively by LC-MS5 to identify their component chlorogenic acids and partially characterize other caffeic acid derivatives. The chlorogenic acids were minor components, and the four samples varied markedly in profile. Three p-coumaroylquinic acids, three feruloylquinic acids, four caffeoylquinic acids, six dicaffeoylquinic acids, and two tricaffeoylquinic acids were detected, 13 for the first time from this source. Partial characterization of minor components suggested the presence of five caffeoyl-hexose esters and caffeic acid-4-beta-d-glucose that have not previously been reported from this source, and eight caffeoylquinic acid glycosides and 16 dicaffeoylquinic acid glycosides that have not previously been reported in nature. Succinic acid-containing chlorogenic acids and chlorogenic acids based on epi-quinic acid, previously reported in Chrysanthemum spp., were not detected in these samples.     

A spectrophotometric study of the copigmentation of malvin with caffeic and ferulic acids

The process of copigmentation of the anthocyanin molecule malvidin 3, 5-diglucoside with two organic monocarboxylic phenolic acids, caffeic and ferulic acids, was studied via their absorption electronic spectra. The dependence of the copigmentation process on the pH of the medium, molecular concentration, and temperature was established. The process of copigmentation was observed at two pH values: 2.50 and 3.65. The stoichiometric ratio was 1:1 at both pH values. The copigmentation was characterized by approximately equal values of the equilibrium constant, K, within each of the pH values. The temperature was found to be a significant factor that determines the thermodynamic conditions of the copigmentation process, because the process is spontaneous (DeltaG degrees < 0), and results in entropy loss (DeltaS degrees < 0) at both pH values.     

Glycoalkaloids and metabolites inhibit the growth of human colon (HT29) and liver (HepG2) cancer cells

As part of an effort to improve plant-derived foods such as potatoes, eggplants, and tomatoes, the antiproliferative activities against human colon (HT29) and liver (HepG2) cancer cells of a series of structurally related individual compounds were examined using a microculture tetrazolium (MTT) assay. The objective was to assess the roles of the carbohydrate side chain and aglycon part of Solanum glycosides in influencing inhibitory activities of these compounds. Evaluations were carried out with four concentrations each (0.1, 1, 10, and 100 microg/mL) of the the potato trisaccharide glycoalkaloids alpha-chaconine and alpha-solanine; the disaccharides beta(1)-chaconine, beta(2)-chaconine, and beta(2)-solanine; the monosaccharide gamma-chaconine and their common aglycon solanidine; the tetrasaccharide potato glycoalkaloid dehydrocommersonine; the potato aglycon demissidine; the tetrasaccharide tomato glycoalkaloid alpha-tomatine, the trisaccharide beta(1)-tomatine, the disaccharide gamma-tomatine, the monosaccharide delta-tomatine, and their common aglycon tomatidine; the eggplant glycoalkaloids solamargine and solasonine and their common aglycon solasodine; and the nonsteroidal alkaloid jervine. All compounds were active in the assay, with the glycoalkaloids being the most active and the hydrolysis products less so. The effectiveness against the liver cells was greater than against the colon cells. Potencies of alpha-tomatine and alpha-chaconine at a concentration of 1 microg/mL against the liver carcinoma cells were higher than those observed with the anticancer drugs doxorubicin and camptothecin. Because alpha-chaconine, alpha-solanine, and alpha-tomatine also inhibited normal human liver HeLa (Chang) cells, safety considerations should guide the use of these compounds as preventative or therapeutic treatments against carcinomas.     

Impact of alkyl esters of caffeic and ferulic acids on tumor cell proliferation, cyclooxygenase enzyme, and lipid peroxidation

The antioxidant ferulic and caffeic acid phenolics are ubiquitous in plants and abundant in fruits and vegetables. We have synthesized a series of ferulic and caffeic acid esters and tested for tumor cell proliferation, cyclooxygenase enzymes (COX-1 and -2) and lipid peroxidation inhibitory activities in vitro. In the tumor cell proliferation assay, some of these esters showed excellent growth inhibition of colon cancer cells. Among the phenolics esters assayed, compounds 10 (C12-caffeate), 11 (C16-caffeate), 21 (C8-ferulate), and 23 (C12-ferulate) showed strong growth inhibition with IC50 values of 16.55, 13.46, 18.67, and 7.57 microg/mL in a breast cancer cell line; 9.65, 7.45, 17.05, and 4.35 microg/ mL in a lung cancer cell line; 5.78, 3.5, 4.29, and 2.46 microg/mL in a colon cancer cell line; 12.04, 12.21, 14.63, and 8.09 microg/ mL in a central nervous system cancer cell line; and 8.62, 7.76, 11.0, and 5.37 in a gastric cancer cell line. In COX enzyme inhibitory assays, ferulic and caffeic acid esters significantly inhibited both COX-1 and COX-2 enzymes. Caffeates 5-10 (C4-C12), inhibited COX-1 enzyme between 50% and 90% and COX-2 enzyme by about 70%, whereas ferulates 15-21 (C3-C8) inhibited COX-1 and COX-2 enzymes by 85-95% 25 microg/mL. Long-chain caffeates 11-14 (C16-C22) and short-chain ferulates 15-20 (C3-C5) were the most active in lipid peroxidation inhibition and showed 60-70% activity at 5 microg/mL concentration.     

Characterization by LC-MS(n) of four new classes of chlorogenic acids in green coffee beans: dimethoxycinnamoylquinic acids, diferuloylquinic acids, caffeoyl-dimethoxycinnamoylquinic acids, and feruloyl-dimethoxycinnamoylquinic acids

LC-MS4 has been used to detect and characterize in green coffee beans 12 chlorogenic acids not previously reported in nature. These comprise three isomeric dimethoxycinnamoylquinic acids (7-9) (Mr 382), three caffeoyl-dimethoxycinnamoylquinic acids (22, 24, and 26) (Mr 544), three diferuloylquinic acids (13-15) (Mr 544), and three feruloyl-dimethoxycinnamoylquinic acids (28, 30, and 32) (Mr 558). Structures have been assigned on the basis of LC-MS4 patterns of fragmentation and relative hydrophobicity and, in the case of the dimethoxycinnamoylquinic acids, by comparison with authentic standards. Several new structure-diagnostic fragmentations have been identified for use with diacyl-chlorogenic acids, for example, m/z 299 and 255 for C4 caffeoyl, m/z 313 and 269 for C4 feruloyl, nearly equal elimination of both cinnamoyl residues for vic-3,4-diacyl, and an increasing ratio of "dehydrated" ions to "non-dehydrated" ions at MS2 with increasing methylation of those cinnamoyl residues. Possible mechanisms have been proposed to account for the fragmentations observed. The mass spectrometric resolution of six isomeric chlorogenic acids (Mr 544) in a crude plant extract by fragment-targeted LC-MS2 and LC-MS3 experiments illustrates the analytical power and advantage of ion trap mass spectroscopy.     

Metabolism of the olive oil phenols hydroxytyrosol, tyrosol, and hydroxytyrosyl acetate by human hepatoma HepG2 cells

To study the potential hepatic metabolism of olive oil phenols, human hepatoma HepG2 cells were incubated for 2 and 18 h with hydroxytyrosol, tyrosol, and hydroxytyrosyl acetate, three phenolic constituents of olive oil. After incubation, culture media and cell lysates were hydrolyzed with beta-glucuronidase and sulfatase and analyzed by LC-MS. In vitro methylation, glucuronidation, and sulfation of pure phenols were also performed. Methylated and glucuronidated forms of hydroxytyrosol were detected at 18 h of incubation, together with methylglucuronidated metabolites. Hydroxytyrosyl acetate was largely converted into free hydroxytyrosol and subsequently metabolized, yet small amounts of glucuronidated hydroxytyrosyl acetate were detected. Tyrosol was poorly metabolized, with <10% of the phenol glucuronidated after 18 h. Minor amounts of free or conjugated phenols were detected in cell lysates. No sulfated metabolites were found. In conclusion, olive oil phenols can be metabolized by the liver as suggested by the results obtained using HepG2 cells as a hepatic model system.     

Humic substances can modulate the allelopathic potential of caffeic, ferulic, and salicylic acids for seedlings of lettuce (Lactuca sativa L.) and tomato (Lycopersicon esculentum Mill.)

The capacity of a leonardite humic acid (LHA), a soil humic acid (SHA), and a soil fulvic acid (SFA) in modulating the allelopathic potential of caffeic acid (CA), ferulic acid (FA), and salicylic acid (SA) on seedlings of lettuce (Lactuca sativa L.) and tomato (Lycopersicon esculentum Mill.) was investigated. Lettuce showed a sensitivity greater than that of tomato to CA, FA, and SA phytotoxicity, which was significantly reduced or even suppressed in the presence of SHA or SFA, especially at the highest dose, but not LHA. In general, SFA was slightly more active than SHA, and the efficiency of the action depended on their concentration, the plant species and the organ examined, and the allelochemical. The daily measured residual concentration of CA and FA decreased drastically and that of SA slightly in the presence of germinating seeds of lettuce, which were thus able to absorb and/or enhance the degradation of CA and FA. The adsorption capacity of SHA for the three allelochemicals was small and decreased in the order FA > CA > SA, thus suggesting that adsorption could be a relevant mechanism, but not the only one, involved in the "antiallelopathic" action.     

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.     

Transepithelial transport of chlorogenic acid, caffeic acid, and their colonic metabolites in intestinal caco-2 cell monolayers

Both chlorogenic and caffeic acids exhibited nonsaturable transport in Caco-2 cells, whereas caffeic acid also showed proton-coupled polarized absorption. Thus, the absorption efficiency of caffeic acid was greater than that of chlorogenic acid. Polarized transport of caffeic acid was inhibited by substrates of MCT such as benzoic and acetic acids. Almost all of the apically loaded chlorogenic and caffeic acid was retained on the apical side, and the transepithelial flux was inversely correlated with the paracellular permeability of Caco-2 cells. These results indicate that transport was mainly via paracellular diffusion, although caffeic acid was absorbed to a lesser extent by the monocarboxylic acid transporter (MCT). Furthermore, m-coumaric acid and 3-(m-hydroxyphenyl)propionic acid, the main metabolites of chlorogenic and caffeic acid by colonic microflora, competitively inhibited the transport of fluorescein, a known substrate of MCT. This suggests that their absorption could also be mediated by MCT. These findings have exemplified the physiological importance of MCT-mediated absorption in both phenolic acids per se and their colonic metabolites.     

Protection of human HepG2 cells against oxidative stress by cocoa phenolic extract

Cocoa is a rich source of flavanols and procyanidin oligomers with antioxidative properties, providing protection against oxidation and nitration. The present study investigated the potential protective effect of a polyphenolic extract from cocoa on cell viability and antioxidant defenses of cultured human HepG2 cells submitted to oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Pretreatment of cells with 0.05-50 microg/mL of cocoa polyphenolic extract (CPE) for 2 or 20 h completely prevented cell damage and enhanced activity of antioxidant enzymes induced by a treatment with t-BOOH. Moreover, lower levels of GSH caused by t-BOOH in HepG2 cells were partly recovered by a pretreatment with CPE. Increased reactive oxygen species (ROS) induced by t-BOOH was dose-dependently prevented when cells were pretreated for 2 or 20 h with CPE. These results show that treatment of HepG2 in culture with CPE (within the physiological range of concentrations) confers a significant protection against oxidation to the cells.     

Absorption, disposition, metabolism, and excretion of [3-(14)C]caffeic acid in rats

Male Sprague-Dawley rats ingested 140 × 10(6) dpm of [3-(14)C]trans-caffeic acid, and over the ensuing 72 h period, body tissues, plasma, urine, and feces were collected and the overall levels of radioactivity determined. Where sufficient radioactivity had accumulated, samples were analyzed by HPLC with online radioactivity and tandem mass spectrometric detection. Nine labeled compounds were identified, the substrate and its cis isomer, 3'-O- and 4'-O-sulfates and glucuronides of caffeic acid, 4'-O-sulfates and glucuronides of ferulic acid, and isoferulic acid-4'-O-sulfate. Four unidentified metabolites were also detected. After passing down the gastrointestinal tract, the majority of the radiolabeled metabolites were excreted in urine with minimal accumulation in plasma. Only relatively small amounts of an unidentified (14)C-labeled metabolite were expelled in feces. There was little or no accumulation of radioactivity in body tissues, including the brain. The overall recovery of radioactivity 72 h after ingestion of [3-(14)C]caffeic acid was ～80% of intake.     

Comparative effects of food-derived polyphenols on the viability and apoptosis of a human hepatoma cell line (HepG2)

Consumption of fruits and vegetables, which are rich in polyphenols, has been associated with a reduced risk of chronic diseases such as cancer. Dietary polyphenols have antioxidant and antiproliferative properties that might explain their beneficial effect on cancer prevention. The aim of this study was to investigate the effects of different pure polyphenols [quercetin, chlorogenic acid, and (-)-epicatechin] and natural fruit extracts (strawberry and plum) on viability or apoptosis of human hepatoma HepG2 cells. The treatment of cells for 18 h with quercetin and fruit extracts reduced cell viability in a dose-dependent manner; however, chlorogenic acid and (-)-epicatechin had no prominent effects on the cell death rate. Similarly, quercetin and strawberry and plum extracts, rather than chlorogenic acid and (-)-epicatechin, induced apoptosis in HepG2 cells. Moreover, quercetin and fruit extracts arrested the G1 phase in the cell cycle progression prior to apoptosis. Quercetin and strawberry and plum extracts may induce apoptosis and contribute to a reduced cell viability in HepG2 cells.     

Uptake,transformation and fixation of Se(VI) by a mixed selenium-tolerant ecosystem

The uptake, transformation and fate of Se, added as selenate to a mixed microbial ecosystem containing Se-tolerant bacteria and cyanobacteria, has been studied in simulated laboratory ponds. There was evidence of concerted activities of the microbial community in the system. Motile bacteria were responsible for transport into a surface mat and the combined actions of the microbial consortium provide chemical conditions under which the Se could be reduced to the elemental form and physically entrained in the mat. Strong qualitative indications of the formation of volatile alkylselenium compounds were also observed. The removal of Se from the water column was rapid and essentially quantitative. The advantages of such biological ecosystems for remediation of Se-contaminated aquatic ecosystems are discussed.     

Effect of cysteinyl caffeic acid, caffeic acid, and L-dopa on the oxidative cross-linking of feruloylated arabinoxylans by a fungal laccase

To study a way to covalently link arabinoxylans and proteins using a fungal laccase from the fungus Pycnoporus cinnabarinus, the effect of cysteinyl caffeic acid on the cross-linking of wheat arabinoxylans was investigated by means of capillary viscometry and RP-HPLC of alkali labile phenolic compounds. Cysteinyl caffeic acid provoked a delay in gelation and in the consumption of the esterified ferulic acid on arabinoxylans. When reacting free ferulic acid and cysteinyl caffeic acid with laccase, the ferulic acid consumption and the dehydrodimers production were also diminished. These results suggest that cysteinyl caffeic acid is oxidized while reducing the semiquinones of ferulic acid produced by laccase. Thus, ferulic acid could not be oxidized into dimers until all cysteinyl caffeic acid was consumed, preventing the cross-linking of feruloylated arabinoxylan chains. A similar mechanism is proposed in the case of caffeic acid and of L-Dopa.     

Oxidative metabolism of the mammalian lignans enterolactone and enterodiol by rat, pig, and human liver microsomes.

Hepatic microsomes from aroclor-treated male Wistar rats biotransform enterolactone to 12 metabolites, six of which carry an additional hydroxy group at the aromatic and six at the aliphatic moiety according to HPLC/MS and GC/MS analysis. The aromatic hydroxylation products were identified with the help of synthesized reference compounds as enterolactone monohydroxylated in the para position and in both ortho positions of the original phenolic hydroxy group of either aromatic ring. The synthesis of the reference compounds and their spectroscopic characterization is described. Enterodiol is metabolized by hepatic microsomes from aroclor-treated male rats to three aromatic and four aliphatic monohydroxylated metabolites. Aromatic hydroxylation occurs in the para position and the two ortho positions of the original phenolic hydroxy group. Most of the metabolites of enterolactone and enterodiol were also formed with microsomes from uninduced rat, pig, and human liver, suggesting that oxidative metabolism is a common feature in the disposition of these lignans in the mammalian organism.     

Characterization by LC-MS(n) of four new classes of p-coumaric acid-containing diacyl chlorogenic acids in green coffee beans

LC-MS4 has been used to detect and characterize in green coffee beans 15 quantitatively minor p-coumaric acid-containing chlorogenic acids not previously reported in nature. These comprise 3,4-di-p-coumaroylquinic acid, 3,5-di-p-coumaroylquinic acid, and 4,5-di-p-coumaroylquinic acid (Mr 484); 3-p-coumaroyl-4-caffeoylquinic acid, 3-p-coumaroyl-5-caffeoylquinic acid, 4-p-coumaroyl-5-caffeoylquinic acid, 3-caffeoyl-4-p-coumaroyl-quinic acid, 3-caffeoyl-5-p-coumaroyl-quinic acid; and 4-caffeoyl-5-p-coumaroyl-quinic acid (Mr 500); 3-p-coumaroyl-4-feruloylquinic acid, 3-p-coumaroyl-5-feruloylquinic acid and 4-p-coumaroyl-5-feruloylquinic acid (Mr 514); and 4-dimethoxycinnamoyl-5-p-coumaroylquinic acid and two isomers (Mr 528) for which identities could not be assigned unequivocally. Structures have been assigned on the basis of LC-MS4 patterns of fragmentation. Forty-five chlorogenic acids have now been characterized in green Robusta coffee beans.     

Comparison of the effectiveness of fatty acids, chlorogenic acids, and elements for the chemometric discrimination of coffee (Coffea arabica L.) varieties and growing origins

The objective of this work was to compare the effectiveness of three chemical families, namely, chlorogenic acids, fatty acids, and elements, for the discrimination of Arabica varieties (traditional versus modern introgressed lines) and potential terroirs within a given coffee-growing area. The experimental design included three Colombian locations in full combination with five (one traditional and four introgressed) Arabica varieties and two field replications. Chlorogenic acids, fatty acids, and elements were analyzed in coffee bean samples by HPLC, GC, and ICP-AES, respectively. Principal component analysis and discriminant analysis were carried out to compare the three methods. Although elements provided an excellent classification of the three locations studied, this chemical class was useless for Arabica variety discrimination. Chlorogenic acids gave satisfactory results, but fatty acids clearly offered the best results for the determination of both varieties and environments, with very high percentages of correct classification (79 and 90%, respectively).