Proanthocyanidins in common food products of plant origin.

The contents of extractable and unextractable proanthocyanidins were determined in a large number of commercial food products of plant origin available in Finland. Proanthocyanidins were extracted with aqueous acetone-methanol and quantified by normal phase high-performance liquid chromatography (HPLC) according to their degree of polymerization. Unextractable proanthocyanidins were analyzed from the extraction residue by reversed phase HPLC after acid-catalyzed depolymerization as free flavan-3-ols (terminal units) and benzylthioethers (extension units). Proanthocyanidins were detected in 49 of 99 selected food items. The highest contents per fresh weight were determined in chokeberries, rose hips, and cocoa products. Berries and fruits were generally the best sources of proanthocyanidins, whereas most of the vegetables, roots, and cereals lacked them completely. Many of the samples contained a significant proportion of insoluble proanthocyanidins, which need to be quantified as well if total proanthocyanidins are studied. Considerable variation was observed in proanthocyanidin contents in berries, which requires further research.

Characterization and fate of black currant and bilberry flavonols in enzyme-aided processing.

The fate of black currant ( Ribes nigrum L.) and bilberry ( Vaccinium myrtillus L.) flavonols in enzyme-aided processing was studied. The flavonols were quantified and characterized by high-performance liquid chromatography equipped with a diode array detector and an electrospray ionization mass spectrometer. A tentative identification for 14 black currant and 19 bilberry flavonols is presented representing 11 previously unpublished conjugates. For the first time in any berry, the presence of laricitrin conjugates is reported. The enzyme-aided processing affected the flavonol extractability, elevating the yield in juices and decreasing that in press residues. Importantly, no significant loss of the berry flavonols was observed during the experiments, although some hydrolysis of flavonol conjugates was recorded. To maximize the effect on flavonol extractability, higher enzyme dosages were needed for black currants than for bilberries. The data show that the flavonol extractability and hydrolysis are dependent on the texture of raw material, the glycosylation pattern of the conjugates, and the activity profile of the enzyme preparation.

Contents of anthocyanins and ellagitannins in selected foods consumed in Finland.

Numerous in vitro and in vivo studies have suggested that dietary anthocyanins and ellagitannins or ellagic acid might have beneficial health effects. Epidemiological evidence on the disease-preventing potential of these polyphenols is lacking, due to the absence of reliable data on their contents in foods. In this study was analyzed the content of anthocyanins and ellagitannins (as ellagic acid equivalents after acid hydrolysis) in foods consumed in Finland, including berries, fruits, vegetables, and processed products, using high-performance liquid chromatographic (HPLC) methods. Anthocyanins were detected in 41 of 54 selected food items. The total anthocyanin content varied in berries from 1 to 611 mg/100 g, in fruits from 2 to 66 mg/100 g, and in vegetables from 3 to 75 mg/100 g of fresh weight as the weight of the aglycone. Ellagitannins were screened in 33 food items, but were detected only in 5 species of berries, that is, in cloudberry, raspberry, rose hip, strawberry, and sea buckthorn, the content ranging from 1 to 330 mg/100 g. The results underscore the superiority of berries, especially dark blue or red berries, as excellent sources of anthocyanins and certain berries of the Rosaceae family as the major source of ellagitannins in the Finnish diet.

Berry phenolic extracts modulate the expression of p21(WAF1) and Bax but not Bcl-2 in HT-29 colon cancer cells.

Previous studies have shown that anthocyanin-rich berry extracts inhibit the growth of cancer cells in vitro. The objective of this study was to compare the effects of berry extracts containing different phenolic profiles on cell viability and expression of markers of cell proliferation and apoptosis in human colon cancer HT-29 cells. Berry extracts were prepared with methanol extraction, and contents of the main phenolic compounds were analyzed using HPLC. Anthocyanins were the predominant phenolic compounds in bilberry, black currant, and lingonberry extracts and ellagitannins in cloudberry extract, whereas both were present in raspberry and strawberry extracts. Cells were exposed to 0-60 mg/mL of extracts, and the cell growth inhibition was determined after 24 h. The degree of cell growth inhibition was as follows: bilberry > black currant > cloudberry > lingonberry > raspberry > strawberry. A 14-fold increase in the expression of p21WAF1, an inhibitor of cell proliferation and a member of the cyclin kinase inhibitors, was seen in cells exposed to cloudberry extract compared to other berry treatments (2.7-7-fold increase). The pro-apoptosis marker, Bax, was increased 1.3-fold only in cloudberry- and bilberry-treated cells, whereas the pro-survival marker, Bcl-2, was detected only in control cells. The results demonstrate that berry extracts inhibit cancer cell proliferation mainly via the p21WAF1 pathway. Cloudberry, despite its very low anthocyanin content, was a potent inhibitor of cell proliferation. Therefore, it is concluded that, in addition to anthocyanins, also other phenolic or nonphenolic phytochemicals are responsible for the antiproliferative activity of berries.

Impact of sugar beet pectin and polydextrose on fasting and postprandial glycemia and fasting concentrations of serum total and lipoprotein lipids in middle-aged subjects with abnormal glucose metabolism.

OBJECTIVE: To examine the effects of sugar beet pectin (SBP) and polydextrose (PDX) on fasting plasma glucose concentration, serum lipid profile and postprandial glycemia in middle-aged subjects with abnormal glucose metabolism. DESIGN: A placebo-controlled, randomized, parallel double-blinded study. SUBJECTS AND SETTING: Subjects were recruited via newspaper announcements. Seventy subjects were recruited of which 66 completed the study. INTERVENTION: The intervention period lasted for 12 weeks during which the subjects consumed a drink enriched with either SBP (n=22) or PDX (n=22) or without fiber enrichment (control group, n=22). The daily dose of the drinks was 4 dl. The subjects were also given nutrition counseling. Postprandial glycemia was examined in 24 subjects (n=8 in each group) at 0 and 12 weeks after a standardized breakfast. RESULTS: Fasting plasma glucose concentration did not change in the SBP and PDX groups, whereas it increased in the control group (P=0.007). On the contrary, the glycosylated hemoglobin A1(c) increased marginally but significantly (P< or =0.05) in the intervention groups without a change in the control group. In postprandial glycemia, no differences between the groups were found. In both the SBP and PDX groups, fasting serum High-density lipoprotein (HDL)-cholesterol concentration increased (P< or =0.05) without a change in the control group. Total to HDL-cholesterol ratio decreased in all groups (P< or =0.05). CONCLUSIONS: It was found that SBP and PDX do not have positive effects on fasting or postprandial plasma glucose concentrations or serum lipid profile in subjects with abnormal glucose metabolism.

Catechins and procyanidins in berries of vaccinium species and their antioxidant activity.

The fractions of monomeric catechins and the fractions of dimeric and trimeric procyanidins were extracted and concentrated from wild berries of Vaccinium species to study their antioxidant activities. The compositions of the fractions were analyzed using high-performance liquid chromatography combined with diode-array and electrospray ionization mass spectrometric detection. Rare A-type dimers and trimers were identified as the predominant procyanidins in wild lingonberry, cranberry, bilberry, and bog whortleberry. Lingonberry and cranberry catechin and procyanidin fractions as well as bog whortleberry catechin fraction were good scavengers of radicals in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test and more efficient than the respective bilberry fractions. Bog whortleberry procyanidin fraction was less active, this being mainly due to the lower content of these compounds. Fractions from lingonberry, cranberry, and bilberry were equally efficient in inhibiting the oxidation of methyl linoleate emulsion, but differences among the berries were found in their abilities to inhibit low-density lipoprotein (LDL) oxidation. Catechins, the monomers, exhibited comparable activity to the fractions containing dimers and trimers in inhibiting the oxidation of methyl linoleate emulsion and human LDL. Bog whortleberry catechins were excellent antioxidants toward the oxidation of human LDL. Radical scavenging and antioxidant activities of Vaccinium berry fractions were attributable to the their composition of catechins and procyanidins. In conclusion, Vaccinium catechins as well as dimeric and trimeric procyanidins provide substantial antioxidant protection.

Identification and quantification of phenolic compounds in berries of Fragaria and Rubus species (family Rosaceae).

High-performance liquid chromatography combined with diode array and electrospray ionization mass spectrometric detection was used to study soluble and insoluble forms of phenolic compounds in strawberries, raspberries (red and yellow cultivated and red wild), arctic bramble, and cloudberries. Hydroxycinnamic acids were present as free forms in cloudberries and mainly as sugar esters in the other berries. Quercetin 3-glucuronide was the typical flavonol glycoside in all of the berries studied. The composition of the predominant anthocyanins can be used to distinguish the studied red Rubus species from each other since cyanidin was glycosylated typically with 3-sophorose (56%) in cultivated red raspberry, with 3-sophorose (30%) and 3-glucose (27%) in wild red raspberry, and with 3-rutinose (80%) in arctic bramble. Ellagic acid was present as free and glycosylated forms and as ellagitannins of varying degrees of polymerization. Comparable levels of ellagitannins were obtained by the analysis of soluble ellagitannins as gallic acid equivalents and by the analysis of ellagic acid equivalents released by acid hydrolysis of the extracts.

Distribution and contents of phenolic compounds in eighteen Scandinavian berry species.

Berries contain a wide range of phenolic compounds in different conjugated forms, a fact that makes their simultaneous analysis a difficult task. In this work, soluble and insoluble phenolic compounds were identified and quantified in 18 species of berries by reversed phase high-performance liquid chromatography combined with diode array detection. The analytical results and literature data were used for the identification of the predominant conjugated hydroxycinnamic acids, flavonol glycosides, and anthocyanins in berries from six families, viz. Grossulariaceae, Ericaceae, Rosaceae, Empetraceae, Elaeagnaceae, and Caprifoliaceae. The study showed distinctive similarities among berry species of the same family in the distribution of conjugated forms of phenolic compounds but differences in chromatographic profiles of conjugates and compositions of aglycones especially in the case of anthocyanins. The chromatographic profiles of chokeberry and the related sweet rowanberry (Rosaceae) were exceptionally similar. These data are informative to studies on the authenticity of berry raw materials as well as to those on the evaluation of berries as sources of phenolic compounds.

High-performance liquid chromatography (HPLC) analysis of phenolic compounds in berries with diode array and electrospray ionization mass spectrometric (MS) detection: ribes species.

High-performance liquid chromatography combined with diode array and electrospray ionization mass spectrometric (MS) detection was used to study phenolic compounds in berries of black, green, red, and white currants (Ribes spp.). UV-visible spectrometry was a valuable tool for the identification of the class of the phenolic compound, whereas MS and MS-MS fragmentation data were useful for further structural characterization. Distinct similarities were found in the relative distribution of conjugated forms of phenolic compounds among the four currants. Phenolic acids were found mainly as hexose esters. Flavonol glycosides and anthocyanin pigments were mainly found as 3-O-rutinosides and second as 3-O-glucosides. However, cyanidin 3-O-sambubioside and quercetin hexoside-malonate were notable phenolic compounds in red currant. Flavonol hexoside-malonates were identified and quantified in the berries of currants for the first time.

Flavonol content varies among black currant cultivars.

Flavonoids and related plant compounds in fruits and vegetables are of particular importance as they have been found to possess antioxidant and free radical scavenging activity. The HPLC-based quantitative procedure, with improved extraction and hydrolysis, was used to analyze the content of the flavonols quercetin, myricetin, and kaempferol in 10 black currant cultivars from organic farms and in 5 cultivars from conventional farms. Myricetin was the most abundant flavonol, and its amount varied significantly among cultivars, from 8.9 to 24.5 mg x 100 g(-1) (fresh weight). The quercetin levels in black currant also varied widely among the cultivars, from 5.2 to 12.2 mg x 100 g(-1). The kaempferol levels in black currant cultivars were low, ranging from 0.9 to 2.3 mg x 100 g(-1). The sum of these major flavonols varied widely among black currant cultivars. No consistent differences in the contents of flavonols were found between the same black currant cultivars grown in organic and conventional ways. The high variability in the levels of flavonols in different cultivars offers possible avenues for identifying and selecting cultivars rich in certain flavonols for the special production of berries for industrial use.

Influence of domestic processing and storage on flavonol contents in berries.

Effects of domestic processing and storage on the flavonols quercetin, myricetin, and kaempferol in five berries were studied using an optimized RP-HPLC method with UV and diode array detection after an acid hydrolysis of the corresponding glycosides. In fresh berries, the total content of flavonols was highest in lingonberry (169 mg/kg) and black currant (157 mg/kg), intermediate in bilberry (41 mg/kg) and strawberry (17 mg/kg), and lowest in red raspberry (9.5 mg/kg). Cooking strawberries with sugar to make jam resulted in minor losses (quercetin 15%, kaempferol 18%). During cooking of bilberries with water and sugar to make soup, 40% of quercetin was lost. Traditional preservation of crushed lingonberries in their own juice caused a considerable (40%) loss of quercetin. Only 15% of quercetin and 30% of myricetin present in unprocessed berries were retained in juices made by common domestic methods (steam-extracted black currant juice, unpasteurized lingonberry juice). Cold-pressing was superior to steam-extraction in extracting flavonols from black currants. During 9 months of storage at 20 C, quercetin content decreased markedly (40%) in bilberries and lingonberries, but not in black currants or red raspberries. Myricetin and kaempferol were more susceptible than quercetin to losses during storage.

Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli.

DsbC is one of five Escherichia coli proteins required for disulfide bond formation and is thought to function as a disulfide bond isomerase during oxidative protein folding in the periplasm. DsbC is a 2 x 23 kDa homodimer and has both protein disulfide isomerase and chaperone activity. We report the 1.9 A resolution crystal structure of oxidized DsbC where both Cys-X-X-Cys active sites form disulfide bonds. The molecule consists of separate thioredoxin-like domains joined via hinged linker helices to an N-terminal dimerization domain. The hinges allow relative movement of the active sites, and a broad uncharged cleft between them may be involved in peptide binding and DsbC foldase activities.

Content of the flavonols quercetin, myricetin, and kaempferol in 25 edible berries.

The amounts of quercetin, myricetin, and kaempferol aglycons in 25 edible berries were analyzed by an optimized RP-HPLC method with UV detection and identified with diode array and electrospray ionization mass spectrometry detection. Sixteen species of cultivated berries and nine species of wild berries were collected in Finland in 1997. Quercetin was found in all berries, the contents being highest in bog whortleberry (158 mg/kg, fresh weight), lingonberry (74 and 146 mg/kg), cranberry (83 and 121 mg/kg), chokeberry (89 mg/kg), sweet rowan (85 mg/kg), rowanberry (63 mg/kg), sea buckthorn berry (62 mg/kg), and crowberry (53 and 56 mg/kg). Amounts between 14 and 142 mg/kg of myricetin were detected in cranberry, black currant, crowberry, bog whortleberry, blueberries, and bilberry. Kaempferol was detected only in gooseberries (16 and 19 mg/kg) and strawberries (5 and 8 mg/kg). Total contents of these flavonols (100-263 mg/kg) in cranberry, bog whortleberry, lingonberry, black currant, and crowberry were higher than those in the commonly consumed fruits or vegetables, except for onion, kale, and broccoli.

Functional conformational changes of endo-1,4-xylanase II from Trichoderma reesei: a molecular dynamics study.

Recent crystallographic studies have revealed a range of structural changes in the three-dimensional structure of endo-1,4-xylanase (XYNII) from Trichoderma reesei. The observed conformational changes can be described as snapshots of an open-close movement of the active site of XYNII. These structures were further analyzed in this study. In addition, a total of four 1 ns molecular dynamics (MD) simulations were performed representing different states of the enzyme. A comparison of the global and local changes found in the X-ray structures and the MD runs suggested that the simulations reproduced a similar kind of active site opening and closing as predicted by the crystal structures. The open-close movement was characterized by the use of distance difference matrixes and the Hinge-find program (Wriggers and Schulten, Proteins 29:1-14, 1997) to be a 'hinge-bending' motion involving two large rigidly-moving regions and an extended hinge. This conformational feature is probably inherent to this molecular architecture and probably plays a role in the function of XYNII.

Structural and functional properties of low molecular weight endo-1,4-beta-xylanases.

There are currently four crystal structures of low molecular weight endo-1,4-beta-xylanases (E.C.3.2.1.8), i.e. family G/11 xylanases, available at the Brookhaven Data Bank: 2 xylanases from Trichoderma reesei (Törrönen et al., 1994; Törrönen and Rouvinen, 1995) and one from Bacillus circulans and another from Trichoderma harzianum (Campbell et al., 1993). They consist of two beta-sheets and one alpha-helix and have been described to resemble a partly-closed right hand. The catalytic residues are two conserved glutamate residues, which are located opposite to each other in an open active site cleft. The catalytic mechanism is thought to resemble that of the widely-studied enzyme lysozyme. The role of one glutamate is to act as an acid/base catalyst whereas the other is a nucleophile and stabilizes the reaction intermediate. Complex structures of partly-bound xylotetraose in mutated XYN from Bacillus circulans (Wakarchuck et al., 1994a) and three recently-obtained structures of XYNII from Trichoderma reesei with epoxyalkyl-xylose derivatives (Havukainen et al., 1996) have provided important information on substrate binding. Family G/11 xylanases show clear amino acid homology and thus have a common fold. However, variations in their functional properties, such as catalytic activity, substrate cleaving patterns, pH optima and thermostabilities, exist.

Crystallization of DsbC, the disulfide bond isomerase of Escherichia coli.

DsbC is a 2 x 23 kDa soluble dimeric protein molecule involved in protein disulfide bond formation in the E. coli periplasm, primarily catalyzing disulfide bond rearrangements. Crystals of both the native and selenomethione protein suitable for structure determination were obtained using the hanging-drop vapour-diffusion method. The best crystals were obtained using 18-22% (v/v) polyethylene glycol 550 monomethyl ether in 100 mM Tris-HCl (pH 8.9). Seeding methods were used to produce large crystals diffracting to 2 A resolution, and the detergent n-octyl-beta-glucoside was used to improve crystal quality. Significant variation in cell dimensions and crystal order was observed. Cell dimensions obtained for frozen crystals were in the range a = 58.8 (0.3), b = 78.9 (0.5), c = 95.2 (5.0) A. The lattice is orthorhombic and systematic absences indicate that the space group is P2(1)2(1)2(1).

Glucose-induced secretion of Trichoderma reesei xylanases.

To produce two xylanases with Trichoderma reesei grown on glucose, recombinant strains which carry either the xyn1 or the xyn2 (xylanase I and II [XYN I and XYN II]-encoding) structural genes under the expression signals of the homologous pki1 (pyruvate kinase-encoding) gene were constructed. The two types of transformants secreted XYN I or II, respectively, during growth on glucose, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining. The corresponding specific xylanase activities of the best transformants on glucose were 76 and 145 U/mg of protein for XYN I and XYN II, respectively, as opposed to that obtained by the parent strain (26 U/mg of protein). When related to the amount of biomass formed, however, they produced only about 4 to 5 U/g, in contrast to much higher activities (10 to 12 U/g) during growth on xylan. The ultrastructural location of XYN II in the transformant strain producing the highest constitutive XYN II formation (ATX2-12) was investigated by immunoelectron microscopy and compared with that in the wild-type strain growing on xylan. Cell extracts from both types of transformants grown on glucose exhibited a higher intracellular xylanase activity than did the parent strain grown on xylan. By using electron microscopy and immunogold labelling, XYN II was detected in the endoplasmic reticulum, Golgi-like vesicles, secretory vesicles, vacuoles, and cell walls. The immunolabel in the vacuoles was detected preferentially in subapical cells. When a recombinant strain which expressed xyn2 from the pki1 promoter was compared with the parent strain during growth on xylan, the former exhibited a less proliferated endoplasmic reticulum and a smaller number of secretory vesicles; however, a higher density of labelling was observed. The relationship of these findings to the efficacy of protein secretion during growth on glucose is discussed.

Covalent binding of three epoxyalkyl xylosides to the active site of endo-1,4-xylanase II from Trichoderma reesei.

The three-dimensional structures of endo-1,4-xylanase II (XYNII) from Trichoderma reesei complexed with 4,5-epoxypentyl beta-D-xyloside (X-O-C5),3,4-epoxybutyl beta-D-xyloside (X-O-C4), and 2,3-epoxypropyl beta-D-xyloside (X-O-C3) were determined by X-ray crystallography. High-resolution measurement revealed clear electron densities for each ligand. Both X-O-C5 and X-O-C3 were found to form a covalent bond with the putative nucleophile Glu86. Unexpectedly, X-O-C4 was found to bind to the putative acid/base catalyst Glu177. In all three complexes, clear conformational changes were found in XYNII compared to the native structure. These changes were largest in the X-O-C3 complex structure.

Large-scale purification and preliminary x-ray diffraction studies of human aspartylglucosaminidase.

Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that takes part in the ordered degradation of glycoproteins and a deficiency of which results in a lysosomal accumulation disease aspartylglucosaminuria in human. The mature enzyme consists of 24-kDa and 17-kDa subunits, which are both heterogeneously glycosylated. Activation of the enzyme from a single precursor polypeptide into two subunits is accomplished in the endoplasmic reticulum (ER). The relative lack of this proteolytic capacity in several tested high-producing expression systems has complicated the production of active recombinant enzyme in high quantities, which would be an alternative for purification of this molecule for crystallization. Consequently, the AGA enzyme has to be purified directly from cellular or tissue sources for crystallographic analysis. Here we describe a large-scale purification method to produce milligram amounts of homogeneous AGA from human leukocytes. The purified AGA enzyme represents a heterogeneous pool of molecules not only due to glycosylation, but also heterogeneity at the polypeptide level, as demonstrated here. We were able to isolate a homogeneous peptide pool that was successfully crystallized and preliminary X-ray data collected from the crystals. The crystals diffract well to 2.0 angstroms and are thus suitable for determination of the crystal structure of AGA.

Comparison of expression of aldehyde dehydrogenase 3 and CYP1A1 in dominant and recessive aryl hydrocarbon hydroxylase-deficient mutant mouse hepatoma cells.

The mouse hepatoma cell line Hepa-1 is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for both CYP1A1 (aryl hydrocarbon hydroxylase, AHH) and class 3 aldehyde dehydrogenase (ALDH3) enzymes. To test the hypothesis of a common regulatory mechanism, several AHH deficient mutants of Hepa-1 were studied for their ALDH3 activities and specific mRNA levels before and after TCDD treatment. The recessive (with respect to the wild-type Hepa-1) mutants have defects in Cypla-1 structural gene (mutant c1) or in the Ah (aryl hydrocarbon) receptor (mutants c2 and c6 with decreased levels of Ah receptor; mutant c4 defective in the DNA binding of the Ah receptor). The results with these mutants suggested that Ah receptor nuclear translocator protein, ARNT, is needed for ALDH3 expression. Two dominant mutants, one of which is characterized by preventing the binding of the Ah receptor complex to DNA, were also studied. Surprisingly, these mutants possessed elevated levels of ALDH3 mRNA and enzyme activities which were also inducible by TCDD. The binding of Ah receptor-ligand complex to DNA was thus not needed for the expression of ALDH3. A dominant repressor for Cypla-1 gene transcription did not prevent the derepression or induction of ALDH3. The results thus suggest that Aldh-3 gene is regulated by a mechanism independent of the Ah receptor.