Novel α-glycosyl compounds from glycosylation of rubusoside.

In this work we investigated mixtures from α-glycosylation of rubusoside with cyclodextrin glycosyltransferases. In addition to the previously known α-1,4 glycosylated derivatives, nine new compounds with rare α-1,3-glycosidic bonds were identified based on nuclear magnetic resonance spectroscopy and mass spectrometric analysis. Furthermore, sensory properties of monoglycosylated rubusoside derivatives were investigated and compared to previously described monoglycosylated compounds. Additionally, digestion with α-amylase from human saliva was investigated for different glycosylated rubusoside derivatives.

Preparative isolation of procyanidins from grape seed extracts by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) has been applied to the separation of grape seed procyanidins. The isolation of dimeric to tetrameric procyanidins is achieved after removing the polymeric compounds by solvent precipitation. An additional clean-up by solid-phase extraction on polyamide improved the purities of the isolated compounds. The solvent systems ethyl acetate/2-propanol/water (40:1:40, v/v/v), ethyl acetate/2-propanol/water (20:1:20, v/v/v), and ethyl acetate/1-butanol/water (14:1:15, v/v/v) were successfully used for the fractionation. The combination of HPLC-MS, diode array detection, and NMR analysis, as well as phloroglucinolysis, confirmed the structures of the isolated compounds: B1 [EC-(4beta-->8)-C], B2 [EC-(4beta-->8)-EC], B3 [C-(4alpha-->8)-C], B4 [C-(4alpha-->8)-EC], B5 [EC-4beta-->6-EC], B7 [EC-(4beta-->8)-C], [ECG-(4beta-->8)-C], trimeric procyanidin C1 [EC-4beta-->8-EC-4beta-->8-EC], and the tetrameric procyanidin cinnamtannin A2 (where C: catechin, EC: epicatechin and ECG: epicatechin-3-O-gallate).

Large-scale isolation of flavan-3-ol phloroglucinol adducts by high-speed counter-current chromatography.

Flavan-3-ol phloroglucinol adducts were synthesised through acid catalysed degradation of a procyanidins-rich grape seed extract in the presence of phloroglucinol. The reaction mixture (3.3 g) was fractionated without further sample preparation using the all-liquid chromatographic technique of high-speed counter-current chromatography (HSCCC). Selected solvent systems were hexane-ethyl acetate-methanol-water (0.1:5:0.1:5, v/v/v/v) and (1.5:10:1.5:10, v/v/v/v). The fractions obtained were found to contain almost pure compounds, in some cases final purification was achieved by preparative HPLC. The so-obtained pure standards of (+)catechin-(4alpha-->2)-phloroglucinol, (-)epicatechin-(4beta-->2)-phloroglucinol, (+)catechine, (-)epicatechin-3-O-galloyl-(4beta-->2)-phloroglucinol, (-)epicatechin, and (-)epicatechin gallate are required for quantification of acid-catalysed phloroglucinol degradation products of procyanidins.

Isolation of flavanol-anthocyanin adducts by countercurrent chromatography.

Pigments of the flavanol-anthocyanin (F-A+) type detected earlier in wine are synthesized using a protocol adapted from the synthesis of procyanidin dimers. The F-A+ adduct thus obtained is purified by countercurrent liquid-liquid partition, currently referred to as countercurrent chromatography (CCC). The solvent system consists of tert-butyl methyl ether-n-butanol-acetonitrile-water (2:2:1:5, acidified with 0.1% trifluoroacetyl) with the light organic phase acting as a stationary phase and the aqueous phase as the mobile phase. Four fractions are recovered and analyzed by high-performance liquid chromatography coupled to a diode-array detector and electrospray ionization mass spectrometer. The multilayer CCC method allowed the separation of pigments in three different groups. The first group consists of hydrosoluble pigments present in fraction 1; the second group consists of the F-A+ adducts [catechin-malvidin 3 glucoside (Mv3glc), along with some (catechin)2-Mv3glc]; and the third group is their anthocyanin precursor, Mv3glc.

Fractionation of an anthocyanin-rich bilberry extract and in vitro antioxidative activity testing.

The incidence of chronic diseases increases with advancing age of the population. A commonly discussed cause of chronic diseases is oxidative stress, which occurs in the body when there is an imbalance between the formation and inactivation of so-called reactive oxygen species (ROS). Epidemiological data suggest that a 'healthy diet', with a high content of flavonoids indicates preventive properties and correlates with an inverse effect with respect to the risk of chronic diseases. Berries (especially bilberries, Vaccinium myrtillus L.) are an important source of these flavonoids. In this study, we investigated, in vitro, the antioxidative properties of fractions obtained from a commercially available anthocyanin-rich bilberry extract (BE). As markers for antioxidative activity, the intracellularly generated ROS levels, oxidative DNA damage, and total glutathione (tGSH) levels were determined in the human colon cell lines Caco-2 and HT-29. In Caco-2 cells, the ROS levels and, in both cell lines, the oxidative DNA damage, were significantly reduced in the presence of the original BE and phenolcarbonic acid-rich fraction. Total GSH levels were slightly increased after pretreatment with BE, phenolcarbonic acid and the polymeric fractions, but not with the anthocyanin fraction. In summary, the BE and the therefrom-isolated phenolcarbonic acid-rich fraction, showed the most potent antioxidative activity whereas the polymeric and anthocyanin-rich fraction, in total, were less active.

A C13-norisoprenoid gentiobioside from quince fruit.

The beta-D-gentiobioside [beta-D-glucopyranosyl(1----6)-beta-D-glucopyranoside] of 3-hydroxy-beta-ionol has been isolated and characterized in quince (Cydonia oblonga) fruit through spectral and chemical studies. Model experiments carried out with this new natural compound revealed its important role as precursor of a number of C13-norisoprenoid flavour compounds of quince essential oil.

Evaluation of different tubing geometries for high-speed counter-current chromatography.

Different tubing geometries were evaluated for use in high-speed counter-current chromatography. Standard round tubing was compared to rectangular and twisted rectangular tubing. The number of theoretical plates for a standard anthocyanin mixture from black currant was determined for each experiment. The results of twisted rectangular tubing were superior to a standard setup.

Isolation and characterization of novel stilbene derivatives from Riesling wine.

Besides the already known stilbenes trans-resveratrol as well as isomeric piceids seven novel stilbene derivatives have been isolated from a commercial Riesling wine. The newly identified compounds included the monostilbene 2,4,6-trihydroxyphenanthrene-2-O-glucoside, as well as two isomeric resveratrol-2-C-glucosides. In addition, four dimeric stilbenes, i.e., cis- and trans-epsilon-viniferin diglucoside as well as pallidol glucoside and pallidol diglucoside, have also been obtained for the first time from Riesling wine.

Isolation and characterization of novel benzoates, cinnamates, flavonoids, and lignans from Riesling wine and screening for antioxidant activity.

A German Riesling wine has been fractionated with the aid of countercurrent chromatography. After purification by HPLC, the structures of 101 compounds were established by mass spectrometry and NMR spectroscopy. Seventy-three of the isolated compounds exhibited a phenolic or benzylic structure. Fifty-four compounds were reported for the first time as Riesling wine constituents. New compounds identified in this work included twelve benzoic and cinnamic acid derivatives. In addition to two isomeric (E)-caffeoyl ethyl tartrates, the glucose esters of (E)-cinnamic, (E)-p-coumaric, and (E)-ferulic acid, as well as the 4-O-glucosides of (E)- and (Z)-ferulic acid, have been identified for the first time in Riesling wine. The structures of two additional phenylpropanoids were elucidated as 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one and 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one. Moreover, two ethyl esters, i.e., ethyl protocatechuate and ethyl gallate, as well as the glucose ester of vanillic acid, were newly detected in Riesling wine. Novel representatives in the flavonoid group were dihydrokaempferol, dihydroquercetin, and four dihydroflavonol glycoconjugates, i.e., the 3-O-glucosides of dihydrokaempferol and dihydroquercetin, as well as the 3-O-xyloside and the 3'-O-glucoside of dihydroquercetin. Additionally, six novel lignans, i.e., lariciresinol 4-O-glucoside, three isolariciresinol derivatives, and two secoisolariciresinols, as well as three neolignans were isolated. Structural elucidation of the newly isolated wine constituents is reported together with the determination of their antioxidant activity.

9-Hydroxypiperitone beta-D-glucopyranoside and other polar constituents from dill (Anethum graveolens L.) herb.

A methanolic extract from dill (Anethum graveolens) herb was subjected to XAD-2 adsorption chromatography. The methanolic eluate was fractionated with the all liquid chromatographic technique of multilayer coil countercurrent chromatography (MLCCC). After acetylation of MLCCC subfractions and flash chromatography, final purification of dill herb constituents was achieved by preparative and/or analytical HPLC. Nine compounds were obtained in pure form, including the beta-D-glucopyranosides of 9-hydroxypiperitone, p-menth-2-ene-1,6-diol, and 8-hydroxygeraniol. Structure elucidation is based on electrospray ionization ion trap multiple mass spectrometry (ESI-MS/MS) as well as one- and two-dimensional nuclear magnetic resonance spectroscopy.

Separation and purification of anthocyanins by high-speed countercurrent chromatography and screening for antioxidant activity.

The all-liquid chromatographic technique of high-speed countercurrent chromatography (HSCCC) has been applied for separations of anthocyanins. The biphasic mixture of tert-butyl methyl ether/n-butanol/acetonitrile/water (2:2:1:5) acidified with trifluoroacetic acid was found to be a suitable solvent system for anthocyanin separation. In some cases, enrichment of the pigments on Amberlite XAD-7 resin prior to HSCCC has been carried out. The anthocyanin mixtures from red cabbage, black currant, black chokeberry, and roselle were successfully fractionated using HSCCC. Peak purity control was done by nuclear magnetic resonance spectroscopy as well as electrospray ionization ion trap multiple mass spectrometry. Finally, antioxidant activity of the purified pigments was determined using the Trolox equivalent antioxidant capacity test.

Preparative isolation of anthocyanins by high-speed countercurrent chromatography and application of the color activity concept to red wine.

Red pigments were isolated from wine and grape-skin extracts using preparative high-speed countercurrent chromatography (HSCCC) and identified by NMR and MS techniques. Four solvent systems were developed in order to separate anthocyanins with different polarities. Malvidin-3-glucoside was the major component present in young red wines, and up to 500 mg of pure malvidin-3-glucoside could be obtained from a single bottle of a red wine. Other isolated pigments were the malvidin- and peonidin-3,5-diglucosides, as well as acetyl-, coumaroyl-, and caffeoyl-derivatives of anthocyanins. Furthermore, condensed red wine pigments formed from malvidin-3-glucoside (vitisin A and acetylvitisin A) were isolated on a preparative scale. Isolated compounds were used as standards for quantification of anthocyanins in a range of red wines. The "color activity concept" was applied to red wine, and visual detection thresholds were determined for some of the isolated anthocyanins. Mono-glucosides were found to exhibit lower visual detection thresholds than di-glucosides and acylated anthocyanins.

Glycosidically bound flavor compounds of cape gooseberry (Physalis peruviana L.).

The bound volatile fraction of cape gooseberry (Physalis peruviana L.) fruit harvested in Colombia has been examined by HRGC and HRGC-MS after enzymatic hydrolysis using a nonselective pectinase (Rohapect D5L). Forty bound volatiles could be identified, with 21 of them being reported for the first time in cape gooseberry. After preparative isolation of the glycosidic precursors on XAD-2 resin, purification by multilayer coil countercurrent chromatography and HPLC of the peracetylated glycosides were carried out. Structure elucidation by NMR, ESI-MS/MS, and optical rotation enabled the identification of (1S,2S)-1-phenylpropane-1,2-diol 2-O-beta-D-glucopyranoside (1) and p-menth-4(8)-ene-1,2-diol 1-O-alpha-L-arabinopyranosyl-(1-6)-beta-D-glucopyranoside (2). Both glycosides have been identified for the first time in nature. They could be considered as immediate precursors of 1-phenylpropane-1,2-diol and p-menth-4(8)-ene-1,2-diol, typical volatiles found in the fruit of cape gooseberry.

Nickel peroxide induced oxidation of canthaxanthin.

Treatment of canthaxanthin (beta,beta-carotene-4,4'-dione) (1) with nickel peroxide in dichloromethane yielded a series of cleavage products, i.e., 4-oxo-beta-ionone (2), (7E, 9E)-4-oxo-beta-apo-11-carotenal (3a), (7E, 9Z)-4-oxo-beta-apo-11-carotenal (3b), 4-oxo-beta-apo-13-carotenone (4), 4-oxo-beta-apo-14'-carotenal (5), 4-oxo-beta-apo-12'-carotenal (6), and 4-oxo-beta-apo-10'-carotenal (7). In addition, oxidized canthaxanthin derivatives, i.e., isomeric ketols all-trans-9, 10-dihydro-9-hydroxy-10-oxo-canthaxanthin (8a), (9'Z)-9, 10-dihydro-9-hydroxy-10-oxo-canthaxanthin (8b), and (13'Z)-9, 10-dihydro-9-hydroxy-10-oxo-canthaxanthin (8c) were obtained together with the tentatively identified (9'Z)-canthaxanthin-20-al (9). Structure elucidation of the reaction products was achieved by mass spectrometry and two-dimensional NMR spectroscopy.

Isolation of black tea pigments using high-speed countercurrent chromatography and studies on properties of black tea polymers.

Isolation of theaflavins and epitheaflavic acids from black tea using high-speed countercurrent chromatography (HSCCC) on a preparative scale is demonstrated. HSCCC also enabled the isolation of a polymeric fraction from black tea. According to Roberts' classification, the polymeric fraction mainly consisted of SII thearubigins (TR). HPLC analysis showed that the isolated material is free of any known chromatographically resolved tea constituents and eluted from reversed-phase packings as a convex "hump" (a broad signal). The antioxidant activity of the TR fraction was 3.6 mmol of Trolox equivalents per gram. The total phenolic content of this fraction was determined to be 34.7 g/100 g (as gallic acid equivalents).

Preparative separation of polyphenols from tea by high-speed countercurrent chromatography.

High-speed countercurrent chromatography (HSCCC) was applied to the separation of polyphenols from tea leaves (Camellia sinensis L.). The capability of HSCCC to isolate pure tea polyphenols from complex mixtures on a preparative scale was demonstrated for catechins, flavonol glycosides, proanthocyanidins, and strictinin from green and black tea. The purity and identity of isolated compounds was confirmed by (1)H NMR and HPLC-ESI-MS/MS. Gram quantities of polyphenols from tea can be isolated with the procedure described.

Centrifugal precipitation chromatography -- a novel chromatographic system for fractionation of polymeric pigments from black tea and red wine.

A novel chromatographic system was developed and first applied to the fractionation of polymeric pigments from black tea and red wine. Centrifugal precipitation chromatography (CPC) generates solvent gradients through a long separation channel under a centrifugal force field. Tea and wine extracts are precipitated in a hexane- or methyl tert-butyl ether-rich environment and are exposed to a gradually increasing ethanol concentration. This causes a repetitive precipitation and dissolution of the biopolymers along the channel. Consequently, they are eluted in the order of their solubility in the organic solvent. It is shown by HPLC analysis of the separated fractions that monomers elute first, whereas fractionated polymers can be found at the end of the chromatographic run. This novel method allows gentle fractionation of polymeric tea and wine constituents and also has potential for use in preparative-scale separations.

A compositional study of Moringa stenopetala leaves.

OBJECTIVE: To investigate nutrient composition in moringa leaves and compare with those of kale (Brassica carinata) and Swiss chard (Beta vulgaris). DESIGN: Laboratory based study, nutrient composition of fresh and cooked leaves of M. stenopetala were analyzed. SETTING: Gama-Gofa, south-western Ethiopia. RESULTS: Raw M. stenopetala leaves contain 9% dry matter as crude protein, about 3-fold lower than in kale and swiss chard. M. stenopetala leaves contain higher percentage of carbohydrate, crude fiber and calcium compared to both raw and cooked kale and swiss chard. Vitamins are present at nutritionally significant levels averaging 28 mg/100g of vitamin C and 160 microg/100g of beta-carotene. Minerals such as potassium, iron, zinc, phosphorus and calcium also exist in significant concentrations with the average values of 3.08 mg/100g iron and 792.8 mg/100g calcium. CONCLUSION: Although the nutrient composition of M. stenopetala leaves in most cases is lower compared to kale and swiss chard they can be a good source of nutrients in dry season potentially when other vegetables are scarce. However, the presence of small amount of cyanogenic glucosides in M. stenopetala leaves may have a health risk in areas of high incidence of endemic goitre as an exacerbating factor if consumed more for a long period of time.

The growth of the canine glioblastoma cell line D-GBM and the canine histiocytic sarcoma cell line DH82 is inhibited by the resveratrol oligomers hopeaphenol and r2-viniferin.

Vineatrol(®) 30 is a grapevine-shoot extract, which contains resveratrol as well as considerable amounts of so-called resveratrol oligomers such as hopeaphenol and r2-viniferin. In this study, we analysed whether the two above-mentioned resveratrol oligomers were able to inhibit the growth of the canine glioblastoma cell line D-GBM and the canine histiocytic sarcoma cell line DH82, compared their potency to inhibit tumour cell growth with that of resveratrol and determined whether the induction of apoptosis via caspase 9 and 3/7 activation underlies the tumour cell growth-inhibiting effect of hopeaphenol and r2-viniferin. Vineatrol(®) 30, resveratrol, hopeaphenol and r2-viniferin inhibited the growth of D-GBM and DH82 cells in a concentration-dependent manner, whereby hopeaphenol and r2-viniferin were more potent than resveratrol itself in inhibiting the growth of the canine tumour cell lines. Moreover, the anti-proliferative effect of both resveratrol oligomers in D-GBM cells is based on their capacity to induce caspase 9 and 3/7 activation.

Nutritional assessment of processing effects on major and trace element content in sea buckthorn juice (Hippophaë rhamnoides L. ssp. rhamnoides).

Processing effects on the mineral content were investigated during juice production from sea buckthorn (Hippophaë rhamnoides L. ssp. rhamnoides, Elaeagnaceae) using berries from 2 different growing areas. The major and trace elements of sea buckthorn berries and juices were determined by atomic absorption spectroscopy (AAS)--(calcium, iron, magnesium, potassium, sodium) and inductively coupled plasma-mass spectrometry (ICP-MS)--(arsenic, boron, chromium, copper, manganese, molybdenum, nickel, selenium, zinc). Potassium is the most abundant major element in sea buckthorn berries and juices. The production process increased the potassium content in the juice by about 20%. Moreover, the processing of juice increased the value of manganese up to 32% compared to the content in berries. During industrial juice production, the technological steps caused a loss of about 53% to 77% of the chromium concentration, 50% of the copper content, 64% to 75% of the molybdenum amount, and up to 45% of the iron concentration in the final juice product. Consumption of sea buckthorn juice represents a beneficial source of chromium, copper, manganese, molybdenum, iron, and potassium for the achievement of the respective dietary requirements.