Induction of cell death in Caco-2 human colon carcinoma cells by ellagic acid rich fractions from muscadine grapes (Vitis rotundifolia)

Possible anticancer mechanisms exerted by polyphenolic compounds contained in fruits and vegetables include antioxidant activity, the inhibition of proliferation, and the induction of apoptosis in cancer cells. This study examined the effects of four isolated polyphenolic extracts from red muscadine grapes (Vitis rotundifolia) on vital cell parameters and the induction of apoptosis in Caco-2 colon carcinoma cells. The magnitude of effects in cell culture was then correlated to polyphenolic composition and antioxidant capacity. Whereas anticancer effects of individual polyphenolic compounds have been demonstrated multiple times, information relating to anticancer effects of polyphenolic extracts is not available in abundance. All four extracts induced apoptosis, decreased cell number, and caused alterations in cell cycle kinetics in a concentration-dependent manner. The efficacy of the polyphenolics on vital cell parameters correlated well to the presence of ellagic acid glycosides and flavonoids and also to the antioxidant capacity. This study demonstrated the anticancer properties of ellagic acid rich extracts from red muscadine juice.     

Phenolic compounds from blueberries can inhibit colon cancer cell proliferation and induce apoptosis

Research has shown that diets rich in phenolic compounds may be associated with lower risks of several chronic diseases including cancer. This study systematically evaluated the bioactivities of phenolic compounds in rabbiteye blueberries and assessed their potential antiproliferation and apoptosis induction effects using two colon cancer cell lines, HT-29 and Caco-2. Polyphenols in three blueberry cultivars, Briteblue, Tifblue, and Powderblue, were extracted and freeze-dried. The extracts were further separated into phenolic acids, tannins, flavonols, and anthocyanins using an HLB cartridge and LH20 column. Some individual phenolic acids and flavonoids were identified by HPLC with >90% purity in anthocyanin fractions. The dried extracts and fractions were added to the cell culture medium to test for antiproliferation activities and induction of apoptosis. Flavonol and tannin fractions resulted in 50% inhibition of cell proliferation at concentrations of 70-100 and 50-100 microg/mL in HT-29 and Caco-2 cells, respectively. The phenolic acid fraction showed relatively lower bioactivities with 50% inhibition at approximately 1000 microg/mL. The greatest antiproliferation effect among all four fractions was from the anthocyanin fractions. Both HT-29 and Caco-2 cell growth was significantly inhibited by >50% by the anthocyanin fractions at concentrations of 15-50 microg/mL. Anthocyanin fractions also resulted in 2-7 times increases in DNA fragmentation, indicating the induction of apoptosis. The effective dosage levels are close to the reported range of anthocyanin concentrations in rat plasma. These findings suggest that blueberry intake may reduce colon cancer risk.     

Purification of curcumin, demethoxycurcumin, and bisdemethoxycurcumin by high-speed countercurrent chromatography

Curcuminoids are substances of great interest because of their important pharmacological activities, particularly anti-inflammatory, anticarcinogenic, and anti-Alzheimer's activities. In this study, we report the first procedure and effect of processing for the high, efficient, and useful purification of curcumin, demethoxycurcumin, and bisdemethoxycurcumin from turmeric powder. Purification involves high-speed countercurrent chromatographic (HSCCC) separation of these curcuminoids using a simple two-phase solvent system composed of n-hexane/chloroform/methanol/water (5/10/7.5/2.5, v/v). The HSCCC-fractionated effluent peaks indicated that the peak resolutions were 1.7 between curcumin and demethoxycurcumin and 2.1 between demethoxycurcumin and bisdemethoxycurcumin for 25 mg of loaded turmeric powder. These purified substances were analyzed by liquid chromatography-tandem mass spectrometry with scan and daughter scan negative modes, and the wide absorbance from 200 to 500 nm was monitored by photodiode array detection. The separation yielded 1.1 mg of curcumin, 0.6 mg of demethoxycurcumin, and 0.9 mg of bisdemethoxycurcumin (>98% purity). Moreover, the antioxidant effect of curcuminoids was measured by a 1,1-diphenyl-2-picrylhydrazil assay. The order of antioxidant activity was purified curcumin > purified demethoxycurcumin > purified bisdemethoxycurcumin > turmeric powder. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin can be used for various evaluations of their pharmacological activities.     

Trapping of methylglyoxal by curcumin in cell-free systems and in human umbilical vein endothelial cells

Curcumin, the most active compound of curcuminoids, has been shown to inhibit formation of advanced glycation end products (AGEs) in streptozotocin-induced diabetic rats. However, little is known on whether curcumin may trap methylglyoxal (MGO), a major reactive dicarbonyl compound, to inhibit AGE formation. We found that one molecule of curcumin effectively trapped one molecule of MGO at a 1:3 ratio at 24 h of incubation under physiological conditions (pH 7.4, 37 °C). Curcumin decreased N(ε)-(carboxymethyl)lysine (CML) expression in human umbilical vein endothelial cells. We further used two curcumin analogues, dimethoxycurcumin (DIMC) and ferulic acid, to investigate the possible MGO-trapping mechanism of curcumin. Results reveal that DIMC, but not ferulic acid, exhibited MGO-trapping capacity, indicating curcumin traps MGO at the electron-dense carbon atom (C10) between the two keto carbon groups. Thus, curcumin may prevent MGO-induced endothelial dysfunction by directly trapping MGO.     

Storage elevates phenolic content and antioxidant activity but suppresses antiproliferative and pro-apoptotic properties of colored-flesh potatoes against human colon cancer cell lines

Colored-flesh potatoes are an excellent source of health-benefiting dietary polyphenols, but are stored for up to 3-6 months before consumption. This study investigated the effect of simulated commercial storage conditions on antioxidant activity (DPPH, ABTS), phenolic content (FCR) and composition (UPLC-MS), and anticancer properties (early, HCT-116 and advanced stage, HT-29 human colon cancer cell lines) of potato bioactive compounds. Extracts from seven potato clones of differing flesh colors (white, yellow, and purple) before and after 90 days of storage were used in this study. The antioxidant activity of all clones increased with storage; however, an increase in total phenolic content was observed only in purple-fleshed clones. Advanced purple-fleshed selection CO97227-2P/PW had greater levels of total phenolics, monomeric anthocyanins, antioxidant activity and a diverse anthocyanin composition as compared with Purple Majesty. Purple-fleshed potatoes were more potent in suppressing proliferation and elevating apoptosis of colon cancer cells compared with white- and yellow-fleshed potatoes. The extracts from both fresh and stored potatoes (10-30 μg/mL) suppressed cancer cell proliferation and elevated apoptosis compared with the solvent control, but these anticancer effects were more pronounced with the fresh potatoes. Storage duration had a strong positive correlation with antioxidant activity and percentage of viable cancer cells and a negative correlation with apoptosis induction. These results suggest that although the antioxidant activity and phenolic content of potatoes were increased with storage, the antiproliferative and pro-apoptotic activities were suppressed. Thus, in the assessment of the effects of farm to fork operations on the health-benefiting properties of plant foods, it is critical to use quantitative analytical techniques in conjunction with in vitro and/or in vivo biological assays.     

Curcumin bioavailability from enriched bread: the effect of microencapsulated ingredients

Human bioavailability of curcumin from breads enriched with 1 g/portion of free curcumin (FCB), encapsulated curcumin (ECB), or encapsulated curcumin plus other polyphenols (ECBB) was evaluated. Parental and metabolized curcuminoids and phenolic acids were quantified by HPLC/MS/MS in blood, urine, and feces collected over 24 h. The concentrations of serum curcuminoids were always below 4 nmol/L and those of glucuronides 10-fold less. Encapsulation delayed and increased curcuminoid absorption as compared to the free ingredient. Serum and urinary concentrations of ferulic and vanillic acid were between 2- and 1000-fold higher than those of curcuminoids, with ECBB eliciting the highest amounts. Fecal curcuminoids were 6-fold more abundant after ECB than FCB, while phenolic acids after ECBB quadruplicated those after ECB. Curcuminoid encapsulation increased their bioavailability from enriched bread, probably preventing their biotransformation, with combined compounds slightly reducing this effect. Phenolic acids are the major metabolites of curcuminoids and may contribute to their biological properties.     

Metabolism of curcuminoids in tissue slices and subcellular fractions from rat liver

Curcumin and its natural congeners are of current interest because of their putative anti-inflammatory and anticarcinogenic activities, but knowledge about their metabolic fate is scant. In the present study conducted with precision-cut liver slices from male and female Sprague-Dawley rats, five reductive but no oxidative metabolites of curcumin and its demethoxy and bis-demethoxy analogues were observed and identified by HPLC and GC-MS analysis, mostly by comparison with authentic reference compounds. The major reductive metabolites were the hexahydrocurcuminoids in both male and female rat liver slices, whereas male rats formed more octahydro than tetrahydro metabolites and female rats more tetrahydro- than octahydrocurcuminoids. Tetrahydro, hexahydro, and octahydro metabolites were predominantly present as glucuronides, but a significant proportion of sulfate conjugates was also observed. The lack of formation of oxidative metabolites of curcumin and the ready generation of reductive metabolites were confirmed using rat liver microsomes and cytosol, respectively. Results of enzymatic hydrolysis studies conducted under various conditions revealed that curcumin and demethoxycurcumin are chemically less stable than bis-demethoxycurcumin, whereas the reductive metabolites of all three curcuminoids are stable compounds. This is the first report on the metabolism of demethoxycurcumin and bis-demethoxycurcumin. In view of the chemical instability of the parent curcuminoids, it is proposed to use their major phase I metabolites, that is, the stable hexahydro products, as biomarkers for exposure in clinical studies.     

Improved HPLC method for the determination of curcumin,demethoxycurcumin, and bisdemethoxycurcumin

Commercially available curcumin, a bright orange-yellow color pigment of turmeric, consists of a mixture of three curcuminoids, namely, curcumin, demethoxycurcumin, and bisdemethoxycurcumin. These were isolated by column chromatography and identified by spectroscopic studies. The purity of the curcuminoids was analyzed by an improved HPLC method. HPLC separation was performed on a C(18) column using three solvents, methanol, 2% AcOH, and acetonitrile, with detection at 425 nm. Four different commercially available varieties of turmeric, namely, Salem, Erode, Balasore, and local market samples, were analyzed to detect the percentage of these three curcuminoids. The percentages of curcumin, demethoxycurcumin, and bisdemethoxycurcumin as estimated using their calibration curves were found to be 1.06 +/- 0.061 to 5.65 +/- 0.040, 0.83 +/- 0.047 to 3.36 +/- 0.040, and 0.42 +/- 0.036 to 2.16 +/- 0.06, respectively, in four different samples. The total percentages of curcuminoids are 2.34 +/- 0.171 to 9.18 +/- 0.232%.     

Induction of apoptosis by garcinol and curcumin through cytochrome c release and activation of caspases in human leukemia HL-60 cells

Garcinol, a polyisoprenylated benzophenone, was purified from Garcinia indica fruit rind. The effects of garcinol and curcumin on cell viability in human leukemia HL-60 cells were investigated. Garcinol and curcumin displayed strong growth inhibitory effects against human leukemia HL-60 cells, with estimated IC(50) values of 9.42 and 19.5 microM, respectively. Garcinol was able to induce apoptosis in a concentration- and time-dependent manner; however, curcumin was less effective. Treatment with garcinol caused induction of caspase-3/CPP32 activity in a dose- and time-dependent manner, but not caspase-1 activity, and induced the degradation of poly(ADP-ribose) polymerase (PARP). Pretreatment with caspase-3 inhibitor inhibited garcinol-induced DNA fragmentation. Treatment with garcinol (20 microM) caused a rapid loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 processing. The cleavage of D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, occurred simultaneously with the activation of caspase-3 but preceded DNA fragmentation and the morphological changes associated with apoptotic cell death. Of these, Bcl-2, Bad, and Bax were studied. The level of expression of Bcl-2 slightly decreased, while the levels of Bad and Bax were dramatically increased in cells treated with garcinol. These results indicate that garcinol allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA and induces DFF-45 (DNA fragmentation factor) degradation. It is suggested that garcinol-induced apoptosis is triggered by the release of cytochrome c into the cytosol, procaspase-9 processing, activation of caspase-3 and caspase-2, degradation of PARP, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by garcinol may provide a pivotal mechanism for its cancer chemopreventive action.     

Antitumor activity of capsaicin on human colon cancer cells in vitro and colo 205 tumor xenografts in vivo

Capsaicin was reported to inhibit cancer cell growth. The aim of this study was to evaluate the antitumor potential of capsaicin by studying antitumor activity in vitro as well as in vivo. The in vitro studies are to examine the effects of capsaicin on human colon cancer colo 205 cells after exposure to capsaicin. The results showed that capsaicin induced cytotoxic effects in a time- and dose-dependent manner and increased reactive oxygen species (ROS) and Ca(2+) but decreased the level of mitochondrial membrane potential (ΔΨ(m)) in colo 205 cells. Data from Western blotting analysis indicated that the levels of Fas, cytochrome c, and caspases were increased, leading to cell apoptosis. Capsaicin decreased the levels of anti-apoptotic proteins such as Bcl-2 and increased the levels of pro-apoptotic proteins such as Bax. Capsaicin-induced apoptosis in colo 205 cells was also done through the activations of caspase-8, -9 and -3. In vivo studies in immunodeficient nu/nu mice bearing colo 205 tumor xenografts showed that capsaicin effectively inhibited tumor growth. The potent in vitro and in vivo antitumor activities of capsaicin suggest that capsaicin might be developed for the treatment of human colon cancer.     

Açai (Euterpe oleracea Mart.) polyphenolics in their glycoside and aglycone forms induce apoptosis of HL-60 leukemia cells

The effects of a?ai polyphenolics on the antiproliferation and induction of apoptosis in HL-60 human leukemia cells were investigated. Interactions between anthocyanins and non-anthocyanin-polyphenolics in both their glycosidic and their aglycone forms were also investigated to determine additive or nonadditive responses. Polyphenolic fractions at 0.17-10.7 microM were found to reduce cell proliferation from 56 to 86% likely due to caspase-3 activation (apoptosis). Anthocyanin and polyphenolic fractions were nonadditive in their contribution to the cell antiproliferation activity. At equimolar concentrations, the glycosidic forms of phenolic acids and flavonoids induced a higher magnitude of change in cell parameters (proliferation and apoptosis) than their respective aglycone forms, while the opposite trend was observed for anthocyanin aglycones. This study demonstrated that a?ai offers a rich source of bioactive polyphenolics and confirmed the importance of investigating whole food systems when evaluating the potential health benefits of individual phytochemical compounds.     

Curcuminoids form reactive glucuronides in vitro

Curcumin is of current interest because of its putative anti-inflammatory, anticarcinogenic, and anti-Alzheimer's activity, but its pharmacokinetic and metabolic fate is poorly understood. The present in vitro study has therefore been conducted on the glucuronidation of curcumin and its major phase I metabolite, hexahydro-curcumin, as well as of various natural and artificial analogs. The predominant glucuronide generated by rat and human liver microsomes from curcumin, hexahydro-curcumin, and other analogs with a phenolic hydroxyl group was a phenolic glucuronide according to LC-MS/MS analysis. However, a second glucuronide carrying the glucuronic acid moiety at the alcoholic hydroxyl group was formed from the same curcuminoids, but not hexahydro-curcuminoids, by human microsomes. Curcuminoids without a phenolic hydroxyl group gave rise to the aliphatic glucuronide only. The phenolic glucuronides of curcuminoids, but not of hexahydro-curcuminoids, were rather lipophilic and, in part, unstable in aqueous solution, their stability depending strongly on the type of aromatic substitution. The phenolic glucuronide of curcumin and of its natural congeners, but not the parent compounds, clearly inhibited the assembly of microtubule proteins under cell-free conditions, implying chemical reactivity of the glucuronides. These novel properties of the major phase II metabolites of curcuminoids deserve further investigation.     

Biphasic effect of falcarinol on caco-2 cell proliferation, DNA damage, and apoptosis

The polyacetylene falcarinol, isolated from carrots, has been shown to be protective against chemically induced colon cancer development in rats, but the mechanisms are not fully understood. In this study CaCo-2 cells were exposed to falcarinol (0.5-100 microM) and the effects on proliferation, DNA damage, and apoptosis investigated. Low-dose falcarinol exposure (0.5-10 microM) decreased expression of the apoptosis indicator caspase-3 concomitantly with decreased basal DNA strand breakage. Cell proliferation was increased (1-10 microM), whereas cellular attachment was unaffected by <10 microM falcarinol. At concentrations above 20 microM falcarinol, proliferation of CaCo-2 cells decreased and the number of cells expressing active caspase-3 increased simultaneously with increased cell detachment. Furthermore, DNA single-strand breakage was significantly increased at concentrations above 10 microM falcarinol. Thus, the effects of falcarinol on CaCo-2 cells appear to be biphasic, inducing pro-proliferative and apoptotic characteristics at low and high concentrations of falcarinol, respectively.     

Curcuminoids, curcumin, and demethoxycurcumin reduce lead-induced memory deficits in male Wistar rats

This study investigated the neuroprotective effects of the curcuminoids against lead-induced neurotoxicity. The results show that lead significantly increases lipid peroxidation and reduces the viability of primary hippocampal neurons in culture. This lead-induced toxicity was significantly curtailed by the co-incubation of the neurons with the curcuminoids. In a whole animal experiment, rats were trained in a water maze and thereafter dosed with lead and/or curcumin (CURC), demethoxycurcumin (DMC), or bisdemethoxycurcumin (BDMC) for 5 days. Animals treated with curcumin and demethoxycurcumin but not bisdemethoxycurcumin had more glutathione and less oxidized proteins in the hippocampus than those treated with lead alone. These animals also had faster escape latencies when compared to the Pb-treated animals indicating that CURC- and DMC-treated animals retain the spatial reference memory. The findings of this study indicate that curcumin, a well-established dietary antioxidant, is capable of playing a major role against heavy metal-induced neurotoxicity and has neuroprotective properties.     

Quantitation of curcuminoids in curcuma rhizome by near-infrared spectroscopic analysis

This study investigated a nondestructive and rapid quantitation method for the curcuminoids, including curcumin, demethoxycurcumin, and bisdemethoxycurcumin, present in turmeric using near-infrared (NIR) spectroscopy and multivariate statistics. In the second derivatives of the NIR spectra of turmeric samples, two characteristic absorptions of curcuminoids were detected around 1700 and 2300-2320 nm. Partial least-squares regression (PLS-R) analysis was applied to the NIR spectra obtained from 34 turmeric samples, and PLS models for the quantitation of curcumin, demethoxycurcumin, bisdemethoxycurcumin, and total curcuminoid contents in the pulverized turmeric samples were constructed. Combination usage of the Standard Normal Variate (SNV) and second derivatives was obviously superior to other preprocessing methods. The lowest root mean squared error of cross-validation (RMSECV) values were detected at 6, 6, 6, and 6 PLS factors, for the quantitative subjects curcumin, demethoxycurcumin, bisdemethoxycurcumin, and total curcuminoid contents. It was clarified that the prediction of the composition by PLS-R analysis showed high correlation with the results of HPLC quantitations.     

Chemical studies on antioxidant mechanism of curcuminoid: analysis of radical reaction products from curcumin

In the course of studies on the antioxidant mechanism of curcumin, its radical reaction was investigated. Curcumin was reacted with radical species, which were generated from the pyrolysis of 2, 2'-azobis(isobutyronitrile) under an oxygen atmosphere, and the reaction products from curcumin were followed by HPLC. The reaction at 70 degrees C gave several products, three of which were structurally identified to be vanillin, ferulic acid, and a dimer of curcumin after their isolation. The dimer was a newly identified compound bearing a dihydrofuran moiety, and its chemical structure was elucidated using spectroscopic analyses, especially 2D NMR techniques. A mechanism for the dimer production is proposed and its relation to curcumin's antioxidant activity discussed. The time course and gel permeation chromatography studies of the reaction were also investigated, and the results indicate that the dimer is a radical-terminated product in the initial stage.     

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.