Dual action of sulforaphane in the regulation of thioredoxin reductase and thioredoxin in human HepG2 and Caco-2 cells

We have previously demonstrated that sulforaphane is a potent inducer for thioredoxin reductase in HepG2 and MCF-7 cells (Zhang et al. Carcinogenesis 2003, 24, 497-503; Wang et al. J. Agric. Food Chem. 2005, 53, 1417-1421). In this study, we have shown that sulforaphane is not only an inducer for thioredoxin reductase but also an inducer for its substrate, thioredoxin in HepG2, and undifferentiated Caco-2 cells. Sulforaphane acts at two levels in the regulation of thioredoxin reductase/thioredoxin system by the upregulation of the expression of both the enzyme and the substrate. In human hepatoma HepG2 cells, sulforaphane induced thioredoxin reductase mRNA and protein by 4- and 2-fold, respectively, whereas thioredoxin mRNA was induced 2.9-fold and thioredoxin protein was unchanged in whole cell extracts, but an increase in nuclear accumulation (1.8-fold) was observed. Moreover, the induction of thioredoxin reductase was found faster than that of thioredoxin. The effects of PI3K and MAPK kinase inhibitors, LY294002, PD98059, SP600125, and SB202190, have been investigated on the sulforaphane-induced expression of thioredoxin reductase and thioredoxin. PD98059 abrogates the sulforaphane-induced thioredoxin reductase at both mRNA and protein levels in HepG2 cells, although other inhibitors were found less effective. However, both PD98059 and LY294002 significantly decrease thioredoxin mRNA expression in HepG2 cells. None of the inhibitors tested were able to modulate the level of expression of either thioredoxin reductase mRNA or protein in Caco-2 cells suggesting that there are cell-specific responses to sulforaphane. In summary, the dietary isothiocyanate, sulforaphane, is important in the regulation of thioredoxin reductase/thioredoxin redox system in cells.     

Apple polyphenols and fibers attenuate atherosclerosis in apolipoprotein E-deficient mice

Atherosclerosis, which is closely linked to nutritional habits, is a major cause of mortality in Western countries. Most of the previous investigations carried out on health effects of apples have been focused on their capacity to lower lipid concentration as well as on their antioxidant effects. The aim of the present study was to investigate the antiatherosclerotic effects of apple polyphenols and fibers. A crude apple polyphenol extract and low-viscosity apple fibers isolated from cider apples were administered separately or in association with the diet of apo E-deficient mice. After 4 months of supplementation, lipemia and oxidative stress biomarkers were measured and atheroslerotic lesions assessed by histomorphometry. Total plasmatic cholesterol and triacylgycerol levels were not affected by supplementation, and hepatic cholesterol level was lower in the group supplemented with both fibers and polyphenols. Uric acid concentrations and antioxidant capacity (FRAP) in plasma were reduced in all groups supplemented with polyphenols or fibers. The mean lesion area was reduced by 17, 38, and 38%, respectively, for the polyphenol, fiber, and polyphenol + fiber groups. Apple constituents supplied at nutritional doses therefore limit the development of atherosclerotic lesions in the aorta of apo E-deficient mice. On the basis of the results, we hypothesize that apple fibers and polyphenols may play a role in preventing atherosclerosis disease by decreasing uric acid plasma level.     

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.     

Mycelia from Antrodia camphorata in Submerged culture induce apoptosis of human hepatoma HepG2 cells possibly through regulation of Fas pathway

The objective of this study was to investigate the antiproliferative effect and the mechanism of the methanol extracts of mycelia (MEM) form Antrodia camphorata in submerged culture toward HepG2 cells. The results showed that MEM-induced cell apoptosis involved up-regulation of Fas and down-regulation of Bcl-2, DR3, DR4, TNFRI, and TNFRII in HepG2 cells, while no changes on the levels of Bax, Bid, Bad, and Bak protein were observed. On the basis of these results, the involvement of the Fas/Fas ligand (FasL) death-receptor pathway, in MEM-induced apoptosis in HepG2 cells, was investigated. The apoptosis inducing activity was significantly enhanced by a Fas activator and inhibited by a Fas antagonist. To know about the effect of MEM on the activation of the apoptotic pathway, the adenovirus transfected with Bcl-2 was infected on HepG2 cells. The data showed that the percentage of apoptotic cells induced by MEM in Bcl-2-infected HepG2 (Bcl-2 overexpression) was not significantly different from that of uninfected HepG2. These results demonstrate that MEM induces HepG2 apoptosis through inhibition of cell growth and up-regulation of Fas/FasL to activate the pathway of caspase-3 and -8 cascades.     

Induction of autophagy and apoptosis by the extract of Solanum nigrum Linn in HepG2 cells

Solanum nigrum L. (SN) has been used in traditional folk medicine to treat different cancers. It is also used as a hepatoprotective and anti-inflammatory agent. In this study, we demonstrated that the extract of SN (SNE) induced a strong cytotoxic effect toward HepG2 cells but much less to Chang liver and WRL-68 cells. The mechanisms of the cytotoxic effect were concentration-dependent. High doses of SNE (2 and 5 mg/mL) induced apoptotic cell death in HepG2 cells, as evidenced by increases in the expressions of p-JNK and Bax, mitochodrial release of cytochrome c, and caspase activation. On the other hand, cells treated with low concentrations of SNE (50-1000 microg/mL) revealed morphological and ultrastructural changes of autophagocytic death under electron microscopic observation. Furthermore, these cells showed increased levels of autophagic vacuoles and LC3-I and LC3-II proteins, specific markers of autophagy. The levels of Bcl-2 and Akt that have been implicated in the down-regulation of autophagy were decreased upon SNE treatment. Taken together, these findings indicate that SNE induced cell death in hepatoma cells via two distinct antineoplastic activities of SNE, the ability to induce apoptosis and autophagocytosis, therefore suggesting that it may provide leverage to treat liver cancer.     

Alkyl hydroxytyrosyl ethers show protective effects against oxidative stress in HepG2 cells

Alkyl hydroxytyrosyl ethers (methyl, ethyl, propyl, and butyl ethers) have been synthesized from hydroxytyrosol (HTy) in response to the increasing food industry demand of new lipophilic antioxidants. Having confirmed that these compounds reach portal blood partially unconjugated and thus are effectively absorbed, their potential antioxidant activity was evaluated in the human hepatocarcinoma cell line (HepG2). The effects of 0.5-10 μM alkyl hydroxytyrosyl ethers on HepG2 cell integrity and redox status were assessed as well as the protective effect against oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Cell viability (Crystal violet) and cell proliferation (BrdU assay) were measured as markers of cell integrity, concentration of reduced glutathione (GSH), generation of reactive oxygen species (ROS), and activity of antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR) as markers of redox status and determination of malondialdehyde (MDA) as a marker of lipid peroxidation. Direct treatment of HepG2 with alkyl hydroxytyrosyl ethers induced slight changes in cellular intrinsic antioxidants status, reducing ROS generation and inducing changes in GPx and GR activities. Pretreatment of HepG2 cells with alkyl hydroxytyrosyl ethers counteracted cell damage induced by t-BOOH, partially after 2 h and completely after 20 h, by increasing GSH and decreasing ROS generation, MDA levels, and antioxidant enzyme (GPx and GR) activity. According to these results the alkyl hydroxytyrosyl ethers show clear protective effects against oxidative stress, related to their lipophilic nature, that are similar to or even higher than those of their precursor, HTy.     

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.     

Isolation and identification of phlorotannins from Ecklonia stolonifera with antioxidant and hepatoprotective properties in tacrine-treated HepG2 cells

Four kinds of phlorotannins having antioxidant activity were isolated from the ethyl acetate fraction of Ecklonia stolonifera ethanolic extract. The structures of the phlorotannins were determined on the basis of spectroscopic evidence, including 1D and 2D nuclear magnetic resonance. The isolated phlorotannins showed potential radical-scavenging activities against 2,2-diphenyl-1-picrylhydrazyl and suppressed the intracellular reactive oxygen species in tacrine-treated HepG2 cells. Among them, eckol and 2-phloroeckol showed hepatoprotective activity in tacrine-treated HepG2 cells; however, phlorofucofuroeckol B and 6,6'-bieckol did not show the activity, even though having high antioxidant activity. Both eckol and 2-phloroeckol inhibited the expression of Fas-mediated cell-death proteins, including tBid, caspase-3, and poly(ADP-ribose) polymerase, and suppressed the release of cytochrome c from mitochondria to cytosol in a dose-dependent manner in tacrine-treated HepG2 cells. These results suggest that eckol and 2-phloroeckol are the principal hepatoprotective constituents of the ethyl acetate fraction of E. stolonifera ethanolic extract.     

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.     

Inhibition of low-density lipoprotein oxidation and up-regulation of low-density lipoprotein receptor in HepG2 cells by tropical plant extracts

Twelve edible plant extracts rich in polyphenols were screened for their potential to inhibit oxidation of low-density lipoprotein (LDL) in vitro and to modulate LDL receptor (LDLr) activity in cultured HepG2 cells. The antioxidant activity (inhibition of LDL oxidation) was determined by measuring the formation of conjugated dienes (lag time) and thiobarbituric acid reagent substances (TBARS). Betel leaf (94%), cashew shoot (63%), Japanese mint (52%), semambu leaf (50%), palm frond (41%), sweet potato shoot, chilli fruit, papaya shoot, roselle calyx, and maman showed significantly increased lag time (>55 min, P < 0.05) and inhibition of TBARS formation (P < 0.05) compared to control. LDLr was significantly up-regulated (P < 0.05) by Japanese mint (67%), semambu (51%), cashew (50%), and noni (49%). Except for noni and betel leaf, most plant extracts studied demonstrated a positive association between antioxidant activity and the ability to up-regulate LDL receptor. Findings suggest that reported protective actions of plant polyphenols on lipoprotein metabolism might be exerted at different biochemical mechanisms.     

From plums to prunes: influence of drying parameters on polyphenols and antioxidant activity

Prunes, which are industrially obtained by dehydrating fresh plums at 85-90 degrees C for 18 h, contain higher levels of phenolic compounds than most other fruits. Prune phenolics have shown beneficial effects on human health. Reports are available in the literature on ascorbic acid, phenol composition, and antioxidant activity of fresh plums and prunes, but there is a lack of publications on the influence of drying parameters on the phenolic compounds and antioxidant activity. A study was carried out on two plum cultivars using two sets of air-drying temperatures: (i) air temperature at 85 degrees C until 50% of prune moisture level and then the temperature was lowered to 70 degrees C; (ii) air temperature at 60 degrees C. Whole fresh and dried fruits were assessed for phenolics (catechins, hydroxycinnamic acids, anthocyanins, and flavonols), ascorbic acid, and antioxidant activity (all parameters were calculated on a dry matter basis). Analysis of data shows that chlorogenic and neochlorogenic acid changes were affected by both process parameters and cultivar. Drying destroyed anthocyanins, and there was a significant decrease in flavonols. Ascorbic acid was drastically reduced in relation to process temperature. The most striking result was that drying at 85 degrees C doubled antioxidant activity in both cultivars, while contradictory results were found for 60 degrees C processed plums.     

Inositol phosphates influence iron uptake in Caco-2 cells.

Phytate, inositol hexaphosphate (InsP(6)), may be hydrolyzed to inositol phosphates with lower degree of phosphorylation, i.e., inositol penta- to monophosphates (InsP(5)-InsP(1)), during food processing. Each of these lower inositol phosphates exists in different isomeric forms. The objective of this study was to determine if different isomers of InsP(3)-InsP(5) (Ins(1,2,4)P(3), Ins(1,2,3)P(3), Ins(1,2,6)P(3), Ins(1,3,4)P(3), Ins(1,2,3,4)P(4), Ins(1,2,5,6)P(4), Ins(1,2,4,5,6)P(5), and Ins(1,3,4,5,6)P(5)) and InsP(6) affect the uptake of iron. We studied the iron absorption in vitro using the human intestinal epithelial cell line, Caco-2. Addition of a 2-fold molar excess of InsP(6) or InsP(5) in proportion to Fe (1 h incubation at 37 degrees C) reduced iron uptake by 46-52% (p < 0.001). Neither InsP(4) isomers nor InsP(3) isomers affected iron uptake significantly at 1 h incubation with a molar InsP:Fe level of 2:1. Iron uptake was shown to not be a function of the isomeric form of inositol phosphates. The inositol phosphate isomers did not seem likely to interact with each other through iron to form more stable iron complexes. At a molar InsP:Fe level of 20:1 an inhibitory effect of InsP(4) was found, while InsP(3) did not affect the iron absorption even at a 20-fold molar excess.     

Soil Aluminum Effects on Growth and Nutrition of Cacao

In acid soils, Al toxicity and nutrient deficiencies are main constraints for low yield of cacao (Theobroma cacao L.). A controlled growth chamber experiment was conducted to evaluate the effect of three Al saturations (0.2, 19, and 26%) adjusted by addition of dolomitic lime on growth and nutrient uptake parameters of cacao. Overall, increasing soil Al saturation decreased shoot and root dry weight, stem height, root length, relative growth rate, and net assimilation rate. However, increasing soil Al saturation increased leaf area, specific leaf area (total leaf area/total leaf dry wt), and leaf area ratio (total leaf area/shoot+root wt). Increasing soil Al saturation decreased uptake of elements. Nutrient influx (IN) and transport (TR) decreased significantly for K, Ca and Mg, and showed an increasing trend for S and P as soil Al saturation increased. However, increasing soil Al saturation significantly increased nutrient use efficiency ratio (ER, mg of shoot weight produced per mg of element in shoot) of Ca, Mg and K and decreased ER for other elements. Reduction of soil acidity constraints with addition of lime and fertilizers appear to be key factors in improving cacao yields in infertile, acidic, tropical soils.     

猕猴桃皮多酚提取及其对B16细胞黑色素的影响

植物多酚类物质对皮肤增白和美容有着重要的作用,是美容护肤保健品中的主要成分,猕猴桃皮中多酚类物质含量丰富,可成为美容保健品的原料。为了探究猕猴桃皮中多酚类物质的提取及其美白作用,该研究采用深共熔溶剂（Deep-Eutectic Solvents,DESs）提取猕猴桃皮多酚,通过单因素试验和响应面试验确定优化提取工艺,用高效液相色谱分析猕猴桃皮多酚的成分,并研究猕猴桃皮多酚对酪氨酸酶的影响及对B16细胞黑色素的作用机制。结果表明：深共熔溶剂是提取猕猴桃皮多酚的较佳溶剂,提取猕猴桃皮多酚优化工艺为DES-1含水率18%、料液比1:29 g/mL、提取时间22 min、提取温度69 ℃,猕猴桃皮多酚得率55.53 mg/g。猕猴桃皮多酚中的主要成分有(+)-儿茶素、绿原酸、咖啡酸和表儿茶素,并且猕猴桃皮多酚对体外酪氨酸酶活性有抑制作用。此外,当猕猴桃皮多酚浓度为100 μg/mL时对B16细胞内黑色素生成有明显的抑制作用,能使细胞内黑色素相对含量降为75.08%,细胞内酪氨酸酶活性降至69.45%,且优于阳性对照组曲酸（P<0.05）。结果表明,猕猴桃皮多酚可作为一种天然美白剂应用于化妆品及其他保健品中。     

Soil Aluminum Effects on Growth and Nutrition of Cacao

In acid soils, Al toxicity and nutrient deficiencies are main constraints for low yield of cacao ( Theobroma cacao L.). A controlled growth chamber experiment was conducted to evaluate the effect of three Al saturations (0.2, 19, and 26%) adjusted by addition of dolomitic lime on growth and nutrient uptake parameters of cacao. Overall, increasing soil Al saturation decreased shoot and root dry weight, stem height, root length, relative growth rate, and net assimilation rate. However, increasing soil Al saturation increased leaf area, specific leaf area (total leaf area/total leaf dry wt), and leaf area ratio (total leaf area/shoot+root wt). Increasing soil Al saturation decreased uptake of elements. Nutrient influx (IN) and transport (TR) decreased significantly for K, Ca and Mg, and showed an increasing trend for S and P as soil Al saturation increased. However, increasing soil Al saturation significantly increased nutrient use efficiency ratio (ER, mg of shoot weight produced per mg of element in shoot) of Ca, Mg and K and decreased ER for other elements. Reduction of soil acidity constraints with addition of lime and fertilizers appear to be key factors in improving cacao yields in infertile, acidic, tropical soils.     

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.