Tuba Esatbeyoglu

Betanin—A food colorant with biological activity

Betalains are water-soluble nitrogen-containing pigments that are subdivided in red-violet betacyanins and yellow-orange betaxanthins. Due to glycosylation and acylation betalains exhibit a huge structural diversity. Betanin (betanidin-5-O-β-glucoside) is the most common betacyanin in the plant kingdom. According to the regulation on food additives betanin is permitted quantum satis as a natural red food colorant (E162). Moreover, betanin is used as colorant in cosmetics and pharmaceuticals. Recently, potential health benefits of betalains and betalain-rich foods (e.g. red beet, Opuntia sp.) have been discussed. Betanin is a scavenger of reactive oxygen species and exhibits gene-regulatory activity partly via nuclear factor (erythroid-derived 2)-like 2-(Nrf2) dependent signaling pathways. Betanin may induce phase II enzymes and antioxidant defense mechanisms. Furthermore, betanin possibly prevents LDL oxidation and DNA damage. Potential blood pressure lowering effects of red beet seem to be mainly mediated by dietary nitrate rather than by betanin per se.

Free radical scavenging and antioxidant activity of betanin: Electron spin resonance spectroscopy studies and studies in cultured cells

Betanin is a red pigment present in red beetroot. Recently, potential health benefits of betanin-rich beetroot have been suggested. However, little is known regarding the free radical scavenging and antioxidant activity of betanin. Electron spin resonance spectroscopy (ESR) and spin trapping techniques were applied to evaluate the ability of betanin to scavenge hydroxyl, superoxide, 2,2 diphenyl-1-picrylhydrazyl (DPPH), and galvinoxyl free radicals. In addition, we tested in cultured cells the ability of betanin to prevent DNA damage and to induce the transcription factor Nrf2 (nuclear factor (erythroid-derived 2)-like 2) as well as its down-stream target heme oxygenase1 (HO-1), paraoxonase1 (PON1) and glutathione (GSH). Betanin dose-dependently scavenged DPPH-, galvinoxyl-, superoxide-, and hydroxyl-radicals in the ESR and spin trapping studies and prevented hydrogen peroxide induced DNA damage as determined by the Comet assay. Furthermore, betanin treatment induced the transcription factor Nrf2 and resulted in an increase of HO-1 protein levels, PON1-transactivation and cellular GSH. Present data suggest that betanin is both a free radical scavenger and an inducer of antioxidant defense mechanism in cultured cells.

Preparation of Dimeric Procyanidins B1, B2, B5, and B7 from a Polymeric Procyanidin Fraction of Black Chokeberry (Aronia melanocarpa)

A semisynthetic approach has been used for the preparative formation of dimeric procyanidins B1, B2, B5, and B7. As starting material for the semisynthesis, polymeric procyanidins from black chokeberry were applied. These polymers were found to consist almost exclusively of (-)-epicatechin units. Under acidic conditions the interflavanoid linkages of the polymeric procyanidins are cleaved and the liberated (-)-epicatechin can react with nucleophiles, such as (+)-catechin or (-)-epicatechin. In this way, the polymeric procyanidins are degraded while dimeric procyanidins are formed. During this reaction only dimeric procyanidins are formed that contain (-)-epicatechin in the upper unit, that is, B1 [(-)-EC-4beta-->8-(+)-C)], B2 [(-)-EC-4beta-->8-(-)-EC], B5 [(-)-EC-4beta-->6-(-)-EC], and B7 [(-)-EC-4beta-->6-(+)-C]. The reaction mixtures of the semisynthesis can be successfully fractionated with high-speed countercurrent chromatography (HSCCC), and it is possible to isolate pure procyanidins B1, B2, B5, and B7 on a preparative scale.

Isolation of dimeric, trimeric, tetrameric and pentameric procyanidins from unroasted cocoa beans (Theobroma cacao L.) using countercurrent chromatography.

The main procyanidins, including dimeric B2 and B5, trimeric C1, tetrameric and pentameric procyanidins, were isolated from unroasted cocoa beans (Theobroma cacao L.) using various techniques of countercurrent chromatography, such as high-speed countercurrent chromatography (HSCCC), low-speed rotary countercurrent chromatography (LSRCCC) and spiral-coil LSRCCC. Furthermore, dimeric procyanidins B1 and B7, which are not present naturally in the analysed cocoa beans, were obtained after semisynthesis of cocoa bean polymers with (+)-catechin as nucleophile and separated by countercurrent chromatography. In this way, the isolation of dimeric procyanidin B1 in considerable amounts (500mg, purity>97%) was possible in a single run. This is the first report concerning the isolation and semisynthesis of dimeric to pentameric procyanidins from T. cacao by countercurrent chromatography. Additionally, the chemical structures of tetrameric (cinnamtannin A2) and pentameric procyanidins (cinnamtannin A3) were elucidated on the basis of (1)H NMR spectroscopy. Interflavanoid linkage was determined by NOE-correlations, for the first time.

Dimeric Procyanidins: Screening for B1 to B8 and Semisynthetic Preparation of B3, B4, B6, and B8 from a Polymeric Procyanidin Fraction of White Willow Bark (Salix alba)

Fifty-seven samples have been analyzed with regard to the occurrence of dimeric procyanidins B1-B8 as well as the composition of polymeric procyanidins. Fifty-two samples were found to contain polymeric procyanidins. In most of the samples, (-)-epicatechin was the predominant unit present. In white willow bark (Salix alba), however, large amounts of (+)-catechin (81.0%) were determined by means of phloroglucinolysis. White willow bark has therefore been used for the semisynthetic formation of dimeric procyanidins B3 [(+)-C-4alpha --> 8-(+)-C)], B4 [(+)-C-4alpha --> 8-(-)-EC)], B6 [(+)-C-4alpha --> 6-(+)-C)], and B8 [(+)-C-4alpha --> 6-(-)-EC)]. The reaction mixtures of the semisynthesis were successfully fractionated with high-speed countercurrent chromatography (HSCCC), and dimeric procyanidins B3, B4, B6, and B8 were obtained on a preparative scale.

Free Radical Scavenging and Cellular Antioxidant Properties of Astaxanthin

Astaxanthin is a coloring agent which is used as a feed additive in aquaculture nutrition. Recently, potential health benefits of astaxanthin have been discussed which may be partly related to its free radical scavenging and antioxidant properties. Our electron spin resonance (ESR) and spin trapping data suggest that synthetic astaxanthin is a potent free radical scavenger in terms of diphenylpicryl-hydrazyl (DPPH) and galvinoxyl free radicals. Furthermore, astaxanthin dose-dependently quenched singlet oxygen as determined by photon counting. In addition to free radical scavenging and singlet oxygen quenching properties, astaxanthin induced the antioxidant enzyme paroxoanase-1, enhanced glutathione concentrations and prevented lipid peroxidation in cultured hepatocytes. Present results suggest that, beyond its coloring properties, synthetic astaxanthin exhibits free radical scavenging, singlet oxygen quenching, and antioxidant activities which could probably positively affect animal and human health.

Structure elucidation of procyanidin oligomers by low-temperature 1H NMR spectroscopy.

Procyanidin dimers and trimers, needed as reference compounds for biological studies, have been synthesized from various natural sources using a semisynthetic approach and purified by high-speed countercurrent chromatography (HSCCC). In the past, it has been difficult to elucidate the structure of these compounds, especially the determination of the interflavanoid bond. Here, the structure of two B-type procyanidin dimers, with (+)-catechin ((+)-C) in the upper unit, and eight C-type procyanidin trimers, with (-)-epicatechin ((-)-EC) in the upper unit, have been elucidated using low-temperature (1)H NMR spectroscopy, as well as circular dichroism (CD) spectroscopy. This is the first time NOE interactions have been used to characterize the interflavanoid linkage in underivatized procyanidin trimers. Complete analyses of procyanidin C1 (-)-EC-4β→8-(-)-EC-4β→8-(-)-EC, (-)-EC-4β→8-(-)-EC-4β→8-(+)-C, (-)-EC-4β→6-(-)-EC-4β→8-(-)-EC, (-)-EC-4β→6-(-)-EC-4β→8-(+)-C, (-)-EC-4β→8-(-)-EC-4β→6-(-)-EC, (-)-EC-4β→8-(-)-EC-4β→6-(+)-C, (-)-EC-4β→8-(+)-C-4α→8-(-)-EC, procyanidin C4 (-)-EC-4β→8-(+)-C-4α→8-(+)-C, and procyanidin dimers B6 (+)-C-4α→6-(+)-C and B8 (+)-C-4α→6-(-)-EC are presented.

Biochanin A and prunetin improve epithelial barrier function in intestinal CaCo-2 cells via downregulation of ERK, NF-κB, and tyrosine phosphorylation.

The single-layered gut epithelium represents the primary line of defense against environmental stressors; thereby monolayer integrity and tightness are essentially required to maintain gut health and function. To date only a few plant-derived phytochemicals have been described as affecting intestinal barrier function. We investigated the impact of 28 secondary plant compounds on the barrier function of intestinal epithelial CaCo-2/TC-7 cells via transepithelial electrical resistance (TEER) measurements. Apart from genistein, the compounds that had the biggest effect in the TEER measurements were biochanin A and prunetin. These isoflavones improved barrier tightness by 36 and 60%, respectively, compared to the untreated control. Furthermore, both isoflavones significantly attenuated TNFα-dependent barrier disruption, thereby maintaining a high barrier resistance comparable to nonstressed cells. In docking analyses exploring the putative interaction with the tyrosine kinase EGFR, these novel modulators of barrier tightness showed very similar values compared to the known tyrosine kinase inhibitor genistein. Both biochanin A and prunetin were also identified as potent reducers of NF-κB and ERK activation, zonula occludens 1 tyrosine phosphorylation, and metalloproteinase-mediated shedding activity, which may account for the barrier-improving ability of these isoflavones.

Methylation of Catechins and Procyanidins by Rat and Human Catechol-O-Methyltransferase: Metabolite Profiling and Molecular Modeling Studies

Catechins and procyanidins are major polyphenols in plant-derived foods. Despite intensive studies in recent years, neither their biochemical nor their toxicological properties have been clarified sufficiently. This study aimed to compare the methylation of catechins and procyanidins by the enzyme catechol-O-methyltransferase (COMT) in vitro. We conducted incubations with rat liver cytosol and human placental cytosol including S-adenosyl-l-methionine. The set of substrates comprised the catechins (−)-epicatechin (EC) and (+)-catechin (CAT), the procyanidin dimers B1, B2, B3, B4, B5, and B7 as well as procyanidin trimer C1. After extraction, metabolites were analyzed by means of liquid chromatography-electrospray ionization-mass spectrometry and liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. EC and CAT were converted to two monomethylated metabolites each by human and rat COMT, with the 3′-O-methyl derivatives being consistently the main metabolites. Furthermore, the flavanyl units of procyanidins were methylated consecutively, leading to monomethylated and dimethylated dimeric metabolites as well as monomethylated, dimethylated, and trimethylated C1 metabolites. The methylation status of each flavanyl unit was determined by means of mass spectrometric quinone-methide fragmentation patterns. In addition, molecular modeling studies were performed with the aim to predict the preferred site of methylation and to verify the experimental data. In conclusion, our results indicate that the degree and position of methylation depend clearly on the three-dimensional structure of the entire substrate molecule.

Rapid Method for Glutathione Quantitation Using High-Performance Liquid Chromatography with Coulometric Electrochemical Detection

A rapid, sensitive, and direct method (without derivatization) was developed for the detection of reduced glutathione (GSH) in cultured hepatocytes (HepG2 cells) using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The method was validated according to the guidelines of the U.S. Food and Drug Administration in terms of linearity, lower limit of quantitation (LOQ), lower limit of detection (LOD), precision, accuracy, recovery, and stabilities of GSH standards and quality control samples. The total analysis time was 5 min, and the retention time of GSH was 1.78 min. Separation was carried out isocratically using 50 mM sodium phosphate (pH 3.0) as a mobile phase with a fused-core column. The detector response was linear between 0.01 and 80 μmol/L, and the regression coefficient (R(2)) was >0.99. The LOD for GSH was 15 fmol, and the intra- and interday recoveries ranged between 100.7 and 104.6%. This method also enabled the rapid detection (in 4 min) of other compounds involved in GSH metabolism such as uric acid, ascorbic acid, and glutathione disulfite. The optimized and validated HPLC-ECD method was successfully applied for the determination of GSH levels in HepG2 cells treated with buthionine sulfoximine (BSO), an inhibitor, and α-lipoic acid (α-LA), an inducer of GSH synthesis. As expected, the amount of GSH concentration-dependently decreased with BSO and increased with α-LA treatments in HepG2 cells. This method could also be useful for the quantitation of GSH, uric acid, ascorbic acid, and glutathione disulfide in other biological matrices such as tissue homogenates and blood.