Multilayer Coil Countercurrent Chromatography Research Articles

Isolation of the glucose ester of ( E )-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid from Riesling wine

A glycosidic isolate of Riesling wine was separated with multilayer coil countercurrent chromatography (MLCCC). After acetylation and subsequent purification by high performance liquid chromatography (HPLC), the glucose ester of ( E )-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid (linalool-8-carboxylic acid) 1 was identified for the first time as natural wine constituent. The possible role of I as wine aroma precursor is discussed.

New Phenolic Grape Skin Products from Vitis vinifera cv. Pinot Noir

Anthocyanins and their related compounds were extracted from grape skins of Pinot noir, using 50% aqueous methanol, and purified by solid phase extraction chromatography using XAD-7 resin to obtain a pigment-rich fraction. This fraction was subjected to multilayer coil countercurrent chromatography (MLCCC) using a quaternary solvent system consisting of tert-butyl methyl ether/n-butanol/acetonitrile/water acidified with 0.01% trifluoroacetic acid (2:2:0.1-1.8:5) (v/v/v/v) in a step gradient elution to separate anthocyanin oligomers from grape anthocyanins. In the process of the characterization of the MLCCC fractions by electrospray mass spectrometry, two noncolored anthocyanin derivatives were found and characterized on the basis of their mass spectral data. As a result, these compounds have been tentatively identified as coupling products between both hydrated malvidin-3-glucoside and peonidin-3-glucoside, with 2-S-glutathionyl caffeoyl tartaric acid (GRP). It is therefore proposed that grape skins contain this new class of coupling product, and a possible chemical pathway for their formation is suggested.

Generation of (E)-1-(2,3,6-Trimethylphenyl)buta-1,3-diene from C13-Norisoprenoid Precursors

Three C(13)-norisoprenoid compounds, 3,6,9-trihydroxymegastigma-4,7-diene (6), 3,4,9-trihydroxymegastigma-5,7-diene (4), and the actinidols (8), have all been synthesized and subjected to acid hydrolysis. All three were shown to generate (E)-1-(2,3,6-trimethylphenyl)buta-1,3-diene (1) under wine conservation conditions. At 45 degrees C, approximately 4000-5000 ng/L of 1 was formed from 1.0 mg/L of precursor, after 173 days, while at 25 degrees C more wine-like amounts (200-600 ng/L) were observed. A glucoside, 4,5-dihydrovomifoliol-C(9)-beta-d-glucopyranoside (9b), was isolated from grapevine leaves by multilayer coil countercurrent chromatography (MLCCC), and its stereochemistry was deduced as being (5R, 6S, 9R) by NMR and CD spectroscopy. Hydrolysis of this glucoside produced 1, but in quantities insufficient to account for the levels observed in wine.

Mass Spectrometric Evidence for the Existence of Oligomeric Anthocyanins in Grape Skins

The fractionation of a grape skin extract by multilayer countercurrent chromatography coupled with step gradient elution allowed the preparation of a fraction almost devoid of free anthocyanins. This fraction appeared to be almost exclusively polymeric, as judged by liquid chromatographic-mass spectrometric (LC-MS) analysis, color-bleaching tests with sulfur dioxide, and thiolysis. Electrospray mass spectrometric analysis indicated that the pigmented material in this fraction was chiefly composed of direct condensation products of anthocyanin extending up to trimers. With regard to their linkages, the anthocyanin units in the oligomers were possibly linked by either an A-type (by both carbon-carbon and ether bonds) or B-type (by carbon-carbon bond) linkage, like proanthocyanidins. The terminal anthocyanin unit of the oligomers is consistently in the flavylium form but the extension units are in the flavan form for the A-type oligomers and in the flavene form for the B-type oligomers. Although their linkages still need to be defined rigorously, this is the first mass spectrometric evidence confirming the existence of anthocyanin oligomers in the grape skin extract.

Taste and mouth-feel properties of different types of tannin-like polyphenolic compounds and anthocyanins in wine

The taste and mouth-feel properties of three different types of tannin-like polyphenolic compounds, representative of some of the tannin-like polyphenolic compounds found in red wines, were determined using descriptive sensory analysis. Ethyl-bridged flavanols were produced by reaction of (+)-catechin with acetaldehyde under acidic conditions. Red coloured tannin-like polyphenolic compounds from wine and from wine pomace were isolated by multi layer coil counter-current chromatography (MLCCC). Mouth-feel attributes and bitterness of the fractions dissolved in a model wine (MW) medium were rated while the fractions were held in the mouth and after expectoration. The sensory properties of the fractions described above were compared to those of apple procyanidins (tannins) with degree of polymerisation (dp) 3 and 9. Both wine and pomace-derived coloured tannin-like polyphenolic compounds were rated as significantly less astringent when compared to both the apple tannin fractions but were similar to the MW. The ethyl-bridged flavanols (with mean dp of 5) were rated significantly more bitter than both the apple procyanidin fractions. Astringency of the ethyl-bridged flavanols was lesser than the apple procyanidin fraction with dp9 and more than that with dp3. Highly purified grape anthocyanidin monoglucosides and monoglucoside coumarates, prepared either by solid-phase extraction or by multi layer coil counter-current chromatography were rated similarly to the MW indicating that free anthocyanins, like the coloured tannin-like polyphenolic compounds from wine and pomace, do not contribute astringency nor bitterness to wine.

Fractionation of grape anthocyanin classes using multilayer coil countercurrent chromatography with step gradient elution.

Grape anthocyanins from rosé wine pomace and grape skins were fractionated by multilayer coil countercurrent chromatography (MLCCC). Tert-butyl methyl ether/n-butanol/acetonitrile/water acidified with trifluoroacetic acid (2/2/x/5) was chosen as the solvent system because of its demonstrated efficiency in separating anthocyanins. A method combining MLCCC and step gradient elution has been developed. This method enabled the fractionation of anthocyanins as series of glucosides, and the corresponding acetylated, coumaroylated, and caffeoylated derivatives. The different anthocyanins were identified on the basis of their UV spectra, their elution time on reversed-phase HPLC as well as by mass spectrometry. A polar-colored fraction free of anthocyanin monomers and corresponding to the material remaining in the stationary phase after the completion of the gradient was recovered.

Taste and mouth-feel properties of different types of tannin-like polyphenolic compounds and anthocyanins in wine

The taste and mouth-feel properties of three different types of tannin-like polyphenolic compounds, representative of some of the tannin-like polyphenolic compounds found in red wines, were determined using descriptive sensory analysis. Ethyl-bridged flavanols were produced by reaction of (+)-catechin with acetaldehyde under acidic conditions. Red coloured tannin-like polyphenolic compounds from wine and from wine pomace were isolated by multi layer coil counter-current chromatography (MLCCC). Mouth-feel attributes and bitterness of the fractions dissolved in a model wine (MW) medium were rated while the fractions were held in the mouth and after expectoration. The sensory properties of the fractions described above were compared to those of apple procyanidins (tannins) with degree of polymerisation (dp) 3 and 9. Both wine and pomace-derived coloured tannin-like polyphenolic compounds were rated as significantly less astringent when compared to both the apple tannin fractions but were similar to the MW. The ethyl-bridged flavanols (with mean dp of 5) were rated significantly more bitter than both the apple procyanidin fractions. Astringency of the ethyl-bridged flavanols was lesser than the apple procyanidin fraction with dp9 and more than that with dp3. Highly purified grape anthocyanidin monoglucosides and monoglucoside coumarates, prepared either by solid-phase extraction or by multi layer coil counter-current chromatography were rated similarly to the MW indicating that free anthocyanins, like the coloured tannin-like polyphenolic compounds from wine and pomace, do not contribute astringency nor bitterness to wine.

In vitro radical scavenging activity of essential oils from Columbian plants and fractions from oregano (Origanum vulgare L.) essential oil

AbstractAntioxidant activities of essential oils from anise (Pimpinella anisum L.), fennel (Foeniculum vulgare Mill.), coriander (Coriandrum sativum L.), rosemary (Rosmarinus officinalis L.), oregano (Origanum vulgare L.), ylang‐ylang (Cananga odorata Hook.), verbena (Lippia alba Mill.) and salvia negra (Lepechinia schiedeana (Schlecht) Vatke, syn. Stachys schiedeana Schlecht) were determined using the free radical α‐α‐diphenyl‐β‐picrylhydrazyl (DPPH·) and the preformed radical monocation 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) (ABTS·+). Some of the oils under investigation had antioxidant capacities in a range comparable to that of α‐tocopherol, BHA, ascorbic acid and Trolox, which were used as reference antioxidants. The all‐liquid chromatographic technique of multilayer coil countercurrent chromatography (MLCCC) was applied to the fractionation of oregano essential oil. For oregano, a high antioxidant activity was observed, which is mainly due to the presence of thymol and carvacrol in the essential oil. Copyright © 2002 John Wiley & Sons, Ltd.

Separation with Multi-Layer Coil Counter-Current Chromatography (MLCCC)

Counter-current chromatography (CCC) is a low-pressure, all-liquid chromatographic technique which requires no solid support. [ 1 ] Commonly it utilizes two immiscible solvent phases plus an auxiliary solvent which is miscible in both phases. The mobile phase is pumped through the stationary phase, causing the compounds to partition between two immiscible solvents, based on the partition coefficients of the solutes. MLCCC is an advanced form of CCC and employs teflon tubing wound onto a bobbin, which is centrifugally rotated in a planetary fashion [ 2 ] (Fig. 1). The planetary motion sets up alternating zones of mixing and settling which travel along the length of the coil and result in the partitioning of the components. The main advantages of CCC over conventional chromatography techniques are the absence of a solid support, the use of inexpensive solvents, and complete recovery of highly functional and reactive compounds. In contrast to other forms of CCC, MLCCC can be used on both an analytical and preparative scale. Due to high centrifugal forces, an increase in the number oftheoretical plates is achieved, thereby allowing up to 50000 mixing zones to occur per hour. This results in efficient partitioning and separation of the components in a complex mixture. Hence MLCCC is beginning to be more widely accepted as a method of choice for the separation and isolation of highly polar natural products.

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.

Free and bound volatile composition and characterization of some glucoconjugates as aroma precursors in melón de olor fruit pulp (Sicana odorifera).

Free and glycosidically bound volatiles obtained from the fruit pulp of Sicana odorifera by liquid-liquid extraction and by chromatography, followed by enzymatic hydrolysis with Rohapect D5L, respectively, were analyzed by capillary gas chromatography (HRGC), HRGC-mass spectrometry (HRGC-MS), and HRGC-Olfatometry (HRGC-O) analyses. A total of 37 free volatiles was detected, with the major components being 3-methyl-2-butanol, 3-hydroxy-2-butanone, ethyl 3-hydroxybutanoate, and (Z)-3-hexenol. Among the 22 detected glycosidically bound compounds, 4-hydroxybenzyl methyl ether, 4-hydroxybenzyl alcohol, and 2-phenylethanol were found to be the major constituents. Additionally, two glucoconjugates were isolated in pure form by multilayer coil countercurrent chromatography (MLCCC) of the glycosidic extrac and further purification. Their structures were elucidated by MS and NMR analyses to be the novel [4-(beta-D-glucopyranosyloxy)benzyl] 2,3-dihydroxy-3-methylbutanoate 2, and the known 4-(beta-D-glucopyranosyloxy)benzyl alcohol 1. Compounds 1 and 2 are precursors of 4-hydroxybenzyl alcohol, one of the major volatiles generated by enzymatic hydrolysis of the glycosidic fraction.

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.

C13-Norisoprenoid Glucoconjugates from Lulo (Solanum quitoense L.) Leaves

With the aid of multilayer coil countercurrent chromatography, subsequent acetylation, and liquid chromatographic purification of a glycosidic mixture obtained from lulo (Solanum quitoense L.) leaves, three C(13)-norisoprenoid glucoconjugates were isolated in pure form. Their structures were elucidated by NMR, MS, and CD analyses to be the novel (6R,9R)-13-hydroxy-3-oxo-alpha-ionol 9-O-beta-D-glucopyranoside (4a), the uncommon (3S,5R,8R)-3, 5-dihydroxy-6,7-megastigmadien-9-one 5-O-beta-D-glucopyranoside (citroside A) (5a), and the known (6S,9R)-vomifoliol 9-O-beta-D-glucopyranoside (6a). Enzymatic treatment of compound 5a showed the formation of 3-hydroxy-7,8-didehydro-beta-ionone (7), an important lulo peeling volatile, which in its turn after chemical reduction and heated acid catalyzed rearrangement generates beta-damascenone (9) and 3-hydroxy-beta-damascone (10).

Three Novel Eugenol Glycosides From Rose Flowers, Rosa Damascena Mill

Three novel eugenol disaccharides have been isolated from a methanolic extract of rose flowers using multilayer coil countercurrent chromatography. Their structures were unambiguously determined by two-dimensional NMR techniques (HSQC, HMBC, H-H-COSY, TOCSY, NOESY) as eugenyl-β-xylopyranosyl-(1→6)-β-glucopyranoside 1, eugenyl-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside 2 and eugenyl-α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside aroma precursors, flowers, Rosa damascena Mill.

Identification of Novel Glycosidic Aroma Precursors in Saffron (Crocus sativus L.)

The methanolic extract of saffron was separated with the aid of multilayer coil countercurrent chromatography. After purification by HPLC, the following seven novel carotenoid metabolites were iden...

(S)-3,7-Dimethyl-5-octene-1,7-diol and Related Oxygenated Monoterpenoids from Petals of Rosa damascena Mill.

The methanolic extract obtained from rose flowers was subjected to XAD-2 adsorption chromatography. Prefractionation of the methanolic eluate using multilayer coil countercurrent chromatography (MLCCC) yielded five subfractions. From the least polar subfraction V, a major amount of the key odorants of rose oil, that is, isomeric rose oxides 1a/b, was liberated upon heat treatment at pH 2.5. Further chromatographic workup of fraction V led, for the first time, to the identification of the genuine rose oxide precursor (S)-3,7-dimethyl-5-octene-1,7-diol (2). In addition to diol 2, the following monoterpene diols have been identified: 3,7-dimethyl-7-octene-1,6-diol (3), 2,6-dimethyl-1,7-octadiene-3,6-diol (4), (2E,5E)-3,7-dimethyl-2,5-octadiene-1,7-diol (5), (2E)-3,7-dimethyl-2,7-octadiene-1,6-diol (6), (2Z,5E)-3,7-dimethyl-2,5-octadiene-1,7-diol (7), (2Z)-3,7-dimethyl-2,7-octadiene-1,6-diol (8), (Z)-2,6-dimethyl-2-octene-1,8-diol (9), (E)-2,6-dimethyl-2-octene-1,8-diol (10), (Z)-2,6-dimethyl-2,7-octadiene-1...

Isolation of Two Novel Terpenoid Glucose Esters from Riesling Wine

A glycosidic isolate of Riesling wine was separated with multilayer coil countercurrent chromatography (MLCCC). After acetylation and subsequent purification by high-performance liquid chromatography (HPLC), the glucose esters of (E)-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid (1) and (2E,6E)-10,11-dihydroxy-3,7,11-trimethyl-2,6-dodecadienoic acid (2) were identified for the first time as wine constituents. The identification was achieved by mass spectrometry (DCI-MS) as well as nuclear magnetic resonance spectroscopy (1H NMR, 13C NMR, COSY, HMBC). In biomimetic studies carried out with (E)-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid the potent wine aroma compound 3a,4,5,7a-tetrahydro-3,6-dimethylbenzofuran-2(3H)-one (wine lactone) was formed, thus revealing an important aroma precursor function for glucoconjugate 1. Keywords: (E)-2,6-Dimethyl-6-hydroxyocta-2,7-dienoic acid glucose ester; (2E,6E)-10,11-dihydroxy-3,7,11-trimethyl-2,6-dodecadienoic acid glucose ester; aroma precursor; wine lactone; multilay...

Two Kaempferol Sophorosides from Crocus Sativus

The 7-O-glucopyranoside-3-O-sophoroside and the 7-O-sophoroside of kaempferol were isolated from a methanolic extract of saffron. After separation with multilayer coil countercurrent chromatography (MLCCC), the flavonol glycosides 1 and 2 were acetylated and purified by HPLC.

Novel Glycosidic Constituents from Saffron

The methanolic extract of saffron was prefractionated with the aid of multilayer coil countercurrent chromatography (MLCCC). After subsequent purification of certain fractions by high-performance liquid chromatography, the following glycoconjugates were isolated and identified on the basis of their spectral (UV, MS, NMR) data: the β-d-glucosides of (4R)-4-hydroxy-3,5,5-trimethylcyclohex-2-enone (5), (4S)-4-hydroxy-3,5,5-trimethylcyclohex-2-enone (6), and (4S)-4-(hydroxymethyl)-3,5,5-trimethylcyclohex-2-enone (7), as well as the β-d-gentiobiosyl ester of 2-methyl-6-oxohepta-2,4-dienoic acid (4). In addition to these new natural products, known saffron constituents, i.e., the di-β-d-gentiobiosyl and β-d-gentiobiosyl-β-d-glucopyranosyl esters of crocetin, have also been isolated and separated using MLCCC. Keywords: (4R)-4-Hydroxy-3,5,5-trimethylcyclohex-2-enone 4-O-β-d-glucopyranoside; (4S)-4-hydroxy-3,5,5-trimethylcyclohex-2-enone 4-O-β-d-glucopyranoside; (4S)-4-(hydroxymethyl)-3,5,5-trimethylcyclohex-2-en...

Uncommon Glycoconjugates of 2-Phenylethanol from Rose Flowers and Riesling Wine

The β-D-galactopyranoside and the β-D-glucopyranoside of 2-phenylethanol 1 have been identified in glycosidic XAD-2 isolates obtained from rose flowers and Riesling wine, respectively. In both cases, the crude glycosidic mixtures were fractionated using ODS column chromatography and/or multi-layer coil countercurrent chromatography (MLCCC). After acetylation and purification by HPLC, the novel glycosidic aroma precursors were characterized as their tetraacetates.