Small branches of olive tree: a source of biophenols complementary to olive leaves

The extraction of biophenols (BPs) from small branches (fibrous softwood) of olive tree accelerated by microwave assistance is proposed for the first time. Under optimal working conditions, no further extraction of the target analytes was achieved after 10 min, so complete removal of them within this interval was assumed (amounts ca. to 19000, 1000, 2000, 900, and 700 mg/kg of oleuropein, verbascoside, tyrosol, alpha-taxifolin, and hydroxytyrosol, respectively; the three last BPs are absent in branch-free olive leaves). The extracts required no cleanup or concentration prior to injection into a chromatograph-photodiode array detector assembly for individual separation-quantification. Extraction from this raw material was also implemented in continuous and discontinuous-continuous extractors using ultrasound assistance and superheated liquids, respectively, as auxiliary energies, and the results were compared with those obtained by microwave-assisted extraction. The simultaneous extraction of small branches and leaves from olive tree provided extracts with a higher variety of BPs, but either extracts richer in oleuropein and verbascoside without tyrosol, alpha-taxifolin, and hydroxytyrosol or rich in these three BPs can be obtained by separate extraction of leaves and branches, respectively.     

Novel secoiridoids with antioxidant activity from Australian olive mill waste

A new biophenolic secoiridoid was identified in Australian Frantoio olive mill waste (OMW) extracts. Isolation, purification, and structure elucidation were performed. Hydroxytyrosyl acyclodihydroelenolate, the first nonaldehydic acyclic secoiridoid, is reported. A second compound was identified as p-coumaroyl-6'-secologanoside (comselogoside), and although it has been identified recently in OMW and leaves, this is the first time it has been identified in both OMW and olive fruits. UV, mass spectral, and NMR data are given for both compounds. The two compounds were quantified by HPLC-DAD, and their antioxidant potential was assessed against the classical olive biophenols, hydroxytyrosol and oleuropein, by the in vitro DPPH radical scavenging assay.     

Static-dynamic superheated liquid extraction of hydroxytyrosol and other biophenols from alperujo (a semisolid residue of the olive oil industry)

Hydroxytyrosol and other olive biophenols (OBPs) such as tyrosol, verbascoside, apigenin-7-glucoside, and alpha-taxifolin have been extracted from alperujo by using static-dynamic superheated liquids. Multivariate methodology has been used to carry out a detailed optimization of the extraction. Under the optimal working conditions no further extraction of the target analytes was achieved after 27 min (up to 2800 and 1500 mg/kg of hydroxytyrosol and tyrosol, respectively), so complete removal of them within this interval was assumed. The extract was injected into a chromatograph-photodiode array detector assembly for individual separation-quantification. The efficacy of ethanol/water mixtures to extract OBPs from alperujo has been demonstrated and compared with that of a conventional stirring-based method. These less toxic extractant mixtures are of interest with a view to future human uses of OBPs.     

Rapid Isolation of Sorghum and Other Cereal Starches Using Sonication

High‐intensity ultrasound (sonication) was investigated as a method to rapidly purify starch from sorghum and other cereal grains. To improve the process, buffers were optimized to solubilize sorghum proteins in combination with the sonication. Protein content and starch color were determined to evaluate the efficiency of the extraction process. Sonication times, SDS concentration, different types and concentrations of reducing agents (sodium metabisulfite, dithiothreitol, and β‐mercaptoethanol), and centrifugation speeds of the starch washing procedure were tested. Protein content of isolated sorghum starch was reduced to 0–0.14% (db) after 2 min of sonication (using any of the reducing agents tested). Sodium metabisulfite was chosen as the preferred reducing agent because of its lower toxicity and odor compared with other reducing agents tested. The optimum conditions for producing high‐purity sorghum starches (0.06% protein) were obtained using the following conditions: 2 min of sonication time with 12.5 mM sodium borate buffer, pH 10, containing 0.5% SDS (w/v) and 0.5% sodium metabisulfite (w/v) using 1,500 rpm centrifugation speed during starch washing. Starches separated by this method showed significantly less protein content and b values (yellowness) compared with starches separated by enzymatic methods or methods using NaCl solutions and protein extraction buffers with multiple washing steps, both of which take several hours to complete. Differential scanning calorimetry thermogram values for starches isolated by three different methods showed similar patterns, except that starches obtained with the enzymatic method had slightly higher values of T_o, T_p, and ΔH. Other cereal starches from whole wheat meal, wheat flour, corn, rice, and barley were also obtained rapidly using sonication.     

Optimization of microwave-assisted extraction for the characterization of olive leaf phenolic compounds by using HPLC-ESI-TOF-MS/IT-MS(2)

In the present work, a simple and rapid method for the extraction of phenolic compounds from olive leaves, using microwave-assisted extraction (MAE) technique, has been developed. The experimental variables that affect the MAE process, such as the solvent type and composition, microwave temperature, and extraction time, were optimized using a univariate method. The obtained extracts were analyzed by using high-performance liquid chromatography (HPLC) coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion trap tandem mass spectrometry (ESI-IT-MS(2)) to prove the MAE extraction efficiency. The optimal MAE conditions were methanol:water (80:20, v/v) as extracting solvent, at a temperature equal to 80 °C for 6 min. Under these conditions, several phenolic compounds could be characterized by HPLC-ESI-MS/MS(2). As compared to the conventional method, MAE can be used as an alternative extraction method for the characterization of phenolic compounds from olive leaves due to its efficiency and speed.     

Murta leaves (Ugni molinae Turcz) as a source of antioxidant polyphenols

Extracts from Murta leaves are used by Chilean natives for their benefits on health and cosmetic properties, which are mainly due to the presence of polyphenolic compounds. Extraction of such compounds is strongly influenced by several variables, the effects of which are studied in this work; the antioxidant power of the resulting extracts was measured by two different methods [2,2-diphenyl-1-picrylhydrazyl (DPPH) and thiobarbituric acid reactive substances (TBARS)]. On the whole, maximum values of polyphenolic yields and antiradical power (DPPH method) were attained at 50 degrees C (from 25 to 50 degrees C) and a solvent-to-solid ratio (v/w) of 15:1 (15:1-25:1). The solvents assayed were ethanol, methanol, and water. The highest polyphenolic yield values (2.6% expressed as gallic acid) were reached with methanol, whereas maximum EC50 was attained by the ethanol extract (0.121 mol gallic acid/mol DPPH). Contact time was shown to have only a slight influence in alcoholic extraction, while in water a remarkable effect of increasing contact times (30-90 min) was observed. Just water was the solvent that offered the best result when the antioxidant power was measured by the TBARS method. High-performance liquid chromatography-mass spectrometry analysis revealed the presence of polyphenols, basically flavonols and flavanols, sometimes glycosilated; myricetin and quercetin glycosides were detected in all extracts, whereas epicatechin was present in alcoholic extracts and gallic acid was only present in water.     

Colorimetric evaluation of phenolic content and GC-MS characterization of phenolic composition of alimentary and cosmetic argan oil and press cake

The global phenolic content of argan oil and press cake samples (alimentary and cosmetic) was evaluated using the Folin-Ciocalteu colorimetric method and the phenolic composition of argan oil (alimentary and cosmetic) and press cake (alimentary) samples were analyzed by GC-MS after extraction with 80:20 (v/v) methanol:water and silylation. Identification of chromatographic peaks was made by mass selective detection. Nineteen simple phenols were detected, 16 in press cake, 6 in the alimentary oil, and 7 in the cosmetic oil, among which 15 compounds [3-hydroxypyridine (3-pyridinol), 6-methyl-3-hydroxypyridine, catechol, resorcinol, 4-hydroxybenzyl alcohol, vanillin, 4-hydroxyphenylacetic acid, vanillyl alcohol, 3,4-dihydroxybenzyl alcohol, 4-hydroxy-3-methoxyphenethyl alcohol, methyl 3,4-dihydroxybenzoate, hydroxytyrosol, protocatechuic acid, epicatechin, and catechin] were identified for the first time in such materials.     

Effect of preparation conditions on protein secondary structure and biofilm formation of kafirin

Various extraction and drying conditions for the isolation of kafirin from dry-milled, whole grain sorghum have been investigated, with a view to optimizing extraction of the protein for commercial food coatings and packaging films. The addition of sodium hydroxide to an aqueous ethanol extractant increased the yield and solubility of kafirin. Subsequent heat drying at 40 degrees C was shown to cause the kafirin to aggregate as indicated by an increase in intermolecular beta-sheets. Extraction of the flour using ethanol (70%, w/w) with 0.5% (w/w) sodium metabisulfite and 0.35% (w/w) sodium hydroxide at 70 degrees C followed by freeze-drying of the protein was found to produce a yield of 54% kafirin with good film-forming properties. The kafirin films were assessed for their sensory properties, tensile strength, strain, and water vapor permeability. Fourier transform infrared spectroscopy was used to study the secondary structure of the extracted kafirins. The best films were made with kafirin containing a large proportion of nativelike alpha-helical structures with little intermolecular beta-sheet content as indicated by the Fourier transform infrared reflectance peak intensity ratios associated with these secondary structures. The principal factor affecting the secondary structure of the protein appeared to be the temperature at which the protein was dried. Heat drying resulted in a greater proportion of intermolecular beta-sheets. Any industrial-scale extraction must therefore minimize protein aggregation and maximize native alpha-helical structures to achieve optimal film quality.     

Identification and quantification of carotenoids including geometrical isomers in fruit and vegetable juices by liquid chromatography with ultraviolet-diode array detection

A method was established for the identification and quantification of carotenoids including geometrical isomers in fruit and vegetable juices by liquid chromatography with an ultraviolet-diode array detector, using a C(18) Vydac 201TP54 column. The mobile phase used was the ternary methanol mixture (0.1 M ammonium acetate), tert-butyl methyl ether and water, in a concentration gradient, and a temperature gradient was applied. Retinol palmitate was added as an internal standard. An extraction process (ethanol/hexane, 4:3, v/v) was performed, followed by saponification with diethyl ether/methanolic KOH (0.1%, w/v, BHT) (1:1, v/v) for 0.5 h at room temperature. Seventeen different (cis and trans) carotenoids were identified by UV-vis spectra and retention times in HPLC in the juices analyzed. The analytic parameters show that the method proposed is sensitive, reliable, accurate, and reproducible.     

Comparison of accelerated methods for the extraction of phenolic compounds from different vine-shoot cultivars

Most research on the extraction of high-priced compounds from vineyard/wine byproducts has traditionally been focused on grape seeds and skins as raw materials. Vine-shoots can represent an additional source to those materials, the characteristics of which could depend on the cultivar. A comparative study of hydroalcoholic extracts from 18 different vineyard cultivars obtained by superheated liquid extraction (SHLE), microwave-assisted extraction (MAE), and ultrasound-assisted extraction (USAE) is here presented. The optimal working conditions for each type of extraction have been investigated by using multivariate experimental designs to maximize the yield of total phenolic compounds, measured by the Folin-Ciocalteu method, and control hydroxymethylfurfural because of the organoleptic properties of furanic derivatives and toxicity at given levels. The best values found for the influential variables on each extraction method were 80% (v/v) aqueous ethanol at pH 3, 180 °C, and 60 min for SHLE; 140 W and 5 min microwave irradiation for MAE; and 280 W, 50% duty cycle, and 7.5 min extraction for USAE. SHLE reported better extraction efficiencies as compared to the other two approaches, supporting the utility of SHLE for scaling-up the process. The extracts were dried in a rotary evaporator, reconstituted in 5 mL of methanol, and finally subjected to liquid-liquid extraction with n-hexane to remove nonpolar compounds that could complicate chromatographic separation. The methanolic fractions were analyzed by both LC-DAD and LC-TOF/MS, and the differences in composition according to the extraction conditions were studied. Compounds usually present in commercial wood extracts (mainly benzoic and hydroxycinnamic acids and aldehydes) were detected in vine-shoot extracts.     

Extraction of polyphenols from vine shoots of Vitis vinifera by superheated ethanol-water mixtures

A study of the nonvolatile fraction of extracts from vine shoots obtained by superheated ethanol-water mixtures is presented. The influence of the temperature, extraction time, and percentage of ethanol on extraction was investigated by a multivariate experimental design to maximize the yield of total phenolic compounds, measured by using the Folin-Ciocalteu method. The best values found for these variables were 80% (v/v) ethanol, 240 degrees C, and 60 min. Under these conditions, the effect of pH was also investigated, and a strong improvement of yield was observed by decreasing the pH. The extracts were subject to liquid-liquid extraction with n-hexane. The remaining polar phase was dried in a rotary evaporator and then reconstituted in 10 mL of water. The insoluble residue was dissolved in 10 mL of methanol. Both fractions (aqueous and methanolic) were analyzed by HPLC, and the differences in composition according to the extraction conditions were studied. Compounds usually present in commercial wood extracts were identified (mainly benzoic and hydroxycinnamic acids and aldehydes); the most abundant were quantified, and the stability of the identified phenolic families under different extraction conditions was also investigated. Finally, the superiority of the superheated liquid extraction over conventional solid-liquid extraction was demonstrated.     

Ultrasonic extraction of phenols from olive mill wastewater: comparison with conventional methods

Recovery of phenols from olive mill wastewater (OMWW) was studied, comparing five sample preparation methods: filtration, solid-phase (SPE), liquid-liquid (LLE) and ultrasonic (US)-assisted extraction of liquid and solid (freeze-dried) OMWW. Results showed that ultrasonication is a good alternative to conventional solvent extractions, providing higher recoveries at both levels of individual and total phenol yields. Sonication of liquid OMWW in organic solvent was more efficient vs its nonassisted counterpart (agitation), but did not provide a representative phenol chromatogram due to ethyl acetate use. By contrast, the US-assisted extraction of freeze-dried OMWW (3 × 20 min) in 100% methanol (1.5 g/25 mL, w/v) offered the highest qualitative-quantitative phenol yields without any US-induced alterations. Moreover, freeze-drying is an excellent preservation of initial liquid OMWW, holding a great potential for delayed analysis. This study is also the first report that Slovenian OMWW may be utilized as a valuable source of phenols, especially hydroxytyrosol and tyrosol.     

Total Phenolic Content and Antioxidant Capacity of Germinated,Popped, and Cooked Huauzontle (Chenopodium berlandieri spp. nuttalliae) Seeds

Raw, germinated, popped, and cooked huauzontle (Chenopodium berlandieri spp. nuttalliae) seeds were analyzed for the contents of phenolics extracted with water (WE), methanol, 1:1 (v/v) methanol/water (MWE), and 1.2M HCl in 1:1 (v/v) methanol/water (HMWE); radical scavenging capacity measured by the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) (ABTS) methods was studied. The effect of the system solvents used for the accurate quantification of antioxidant content and capacity showed that for raw, germinated, and cooked extracts, water gave the highest yield of total phenolic content, and MWE could recover the highest yield in popped extracts. Thermal treatments increased the flavonoid content more in all extracts than did the germinating process, with values ranging from 10 to 620 μg/g db of quercetin equivalents. However, all treatments significantly decreased (P < 0.05) the total phenolics (from 3,010 μg of gallic acid equivalents/g db in raw seeds WE to 710 μg/g db in germinated seeds MWE). HMWE in all treatments showed the highest values (up to 95.41%) by the DPPH method. With the ABTS method, germinated and popped MWE showed the highest values (up to 2,740mM Trolox/kg db). Based on these results, huauzontle seeds represent a useful potential ingredient for consumer health, because it has been shown to be a good source of total phenolic content having high antioxidant activity; moreover, for further studies, water appears to be effective as an extraction solvent of phenolic compounds.     

Production of triacetylhydroxytyrosol from olive mill waste waters for use as stabilized bioantioxidant

A hydroxytyrosol triacetyl derivative was very efficiently produced as a highly pure stabilized antioxidant compound by a short treatment of olive mill waste water (OMWW) organic extracts, rich in hydroxytyrosol, with an acetylating mixture composed of HClO4-SiO2 and Ac2O (Chakborti and Gulhane reaction), in mild and safe conditions. A successive single step of middle pressure liquid chromatography (MPLC) purification of the reaction product was performed, with an overall yield of 35.6%. (This process, including both the Chakborti and Gulhane reaction and the MPLC purification, is protected by an international patent under PCT/IT2005/000781.) The o-diphenol triacetyl derivative was also produced by direct reaction of hydroxytyrosol, previously purified by MPLC, with HClO4-SiO2 and Ac2O, with an overall yield of 29.5%. A further procedure for the production of the hydroxytyrosol triacetyl derivative was consistent with the direct treatment of raw OMWW with the acetylating agent and a single step of MPLC purification, with an overall yield of 27.6%. The purified natural triacetylhydroxytyrosol confirmed the same strong protective effects against the oxidative stress in human cells as the corresponding synthetic compound, likely because of the biochemical activation of the acetyl derivative into the active parent hydroxytyrosol by esterases. We therefore propose the utilization of OMWW for recovering hydroxytyrosol as a natural antioxidant in a chemically stabilized form, with a good yield, which can be potentially used as a nontoxic functional component in nutritional, pharmaceutical, and cosmetic preparations.     

Effects of extraction solvent mixtures on antioxidant activity evaluation and their extraction capacity and selectivity for free phenolic compounds in barley (Hordeum vulgare L.)

Four kinds of solvent extracts from three Chinese barley varieties (Ken-3, KA4B, and Gan-3) were used to examine the effects of extraction solvent mixtures on antioxidant activity evaluation and their extraction capacity and selectivity for free phenolic compounds in barley through free radical scavenging activity, reducing power and metal chelating activity, and individual and total phenolic contents. Results showed that extraction solvent mixtures had significant impacts on antioxidant activity estimation, as well as different extraction capacity and selectivity for free phenolic compounds in barley. The highest DPPH* and ABTS*+ scavenging activities and reducing power were found in 80% acetone extracts, whereas the strongest *OH scavenging activity, O2*- scavenging activity, and metal chelating activity were found in 80% ethanol, 80% methanol, and water extracts, respectively. Additionally, 80% acetone showed the highest extraction capacity for (+)-catechin and ferulic, caffeic, vanillic, and p-coumaric acids, 80% methanol for (-)-epicatechin and syringic acid, and water for protocatechuic and gallic acids. Furthermore, correlations analysis revealed that TPC, reducing power, DPPH* and ABTS*+ scavenging activities were well positively correlated with each other (p < 0.01). Thus, for routine screening of barley varieties with higher antioxidant activity, 80% acetone was recommended to extract free phenolic compounds from barley. DPPH* scavenging activity and ABTS*+ scavenging activity or reducing power could be used to assess barley antioxidant activity.     

Critical assessment of various techniques for the extraction of carotenoids and co-enzyme Q10 from the Thraustochytrid strain ONC-T18

A variety of techniques for extracting carotenoids from the marine Thraustochytrium sp. ONC-T18 was compared. Specifically, the organic solvents acetone, ethyl acetate, and petroleum ether were tested, along with direct and indirect ultrasonic assisted extraction (probe vs bath) methods. Techniques that used petroleum ether/acetone/water (15:75:10, v/v/v) with 3 h of agitation, or 5 min in an ultrasonic bath, produced the highest extraction yields of total carotenoids (29-30.5 microg g-1). Concentrations up to 11.5 microg g-1 of canthaxanthin and 17.5 microg g-1 of beta;-carotene were detected in extracts stored for 6 weeks. Astaxanthin and echinenone were also detected as minor compounds. Extracts with and without antioxidants showed similar carotenoid concentration profiles. However, total carotenoid concentrations were approximately 8% higher when antioxidants were used. Finally, an easy-to-perform and inexpensive method to detect co-enzymes in ONC-T18 was also developed using silica gel TLC plates. Five percent methanol in toluene as a mobile phase consistently eluted co-enzyme Q10 standards and could separate the co-enzyme fractions present in ONC-T18.     

Solanidine hydrolytic extraction and separation from the potato (Solanum tuberosum L.) vines by using solid-liquid-liquid systems

Solanidine is a steroidal aglycon of potato (Solanum tuberosum L.) glycoalkaloids and a very important precursor for the synthesis of hormones and some pharmacologically active compounds. Glycoalkaloids are hydrolyzed by mineral acid, yielding solanidine. This paper deals with the kinetics of solanidine hydrolytic extraction in different solid-liquid-liquid systems. The dried and milled potato (S. tuberosum L.) vines were used as a source of glycoalkaloids and as the solid phase. The solutions of hydrochloric acid in 2 and 10% (w/v) aqueous acetic acid, in 50% (volume) aqueous methanol, and in 50% (volume) aqueous ethanol were first liquid phase, and the medium for glycoalkaloid extraction from potato vines and their hydrolysis to solanidine. The chloroform, trichloroethylene, or carbon tetrachloride were the second, organic, liquid phase and the medium for solanidine extraction. This procedure combines three different processes: extraction of glycoalkaloids from potato vines, their hydrolysis to solanidine, and the extraction of solanidine, in a single step. The term hydrolytic extraction of solanidine was used for these processes. The purpose of the paper was to choose an optimal solid-liquid-liquid system for solanidine extraction and to define the procedure for its isolation from the organic liquid phase. The best degree of solanidine hydrolytic extraction (DHE) of more than 98% was achieved when 10% (w/v) hydrochloric acid in 50% (volume) methanol were the first liquid phase and chloroform was the second liquid phase, after 90 min. The yield of solanidine (q(S)) under these conditions is calculated to be 0.24 g/100 g of potato vines. Approximately 78% of the maximal possible yield of solanidine was isolated from chlorofom liquid phase. The IR and MS spectra of isolated solanidine were recorded.     

Literature data may underestimate the actual antioxidant capacity of cereals

Several recent articles have reported a significant antioxidant capacity of cereal products, determined in methanolic and ethanolic extracts. The aim of this work was to conduct an assessment of the antioxidant capacity of cereals using both chemical and in vitro digestive enzymatic extraction of antioxidants. Ferric reducing power (FRAP) and free radical scavenging capacity (DPPH) methods were used to determine the antioxidant capacity in wheat flour, bread, raw and boiled rice, wheat bran, and oat bran. The most efficient antioxidant extraction was achieved by using successively acidic methanol/water (50:50 v/v, pH 2) and acetone/water (70:30 v/v). The antioxidant capacity in these extracts ranged from 1.1 to 4.4 micromol Trolox/g dw. A significant amount of hydrolyzable phenolics with a high antioxidant capacity (from 5 to 108 micromol Trolox/g dw) was found in the residues of this aqueous-organic extraction. The antioxidant capacities of these nonextractable polyphenols are usually ignored in the literature, although they may have an antioxidant role in the gastrointestinal tract, especially after colonic fermentation, and may be fermentated to active metabolites. On the other hand, in vitro digestive enzymatic extracts obtained by enzymatic treatments that mimic conditions in the gastrointestinal tract showed that the amount of antioxidants released by the cereal matrix into the human intestine may be higher than the one that can be expected from measurements in the usual aqueous-organic extracts.     

Simultaneous determination of ginsenosides and polyacetylenes in American ginseng root (Panax quinquefolium L.) by high-performance liquid chromatography

A method for simultaneous determination of ginsenosides and polyacetylenes in Panax quinquefolium L. (American ginseng) roots was developed. The ginsenosides Rb1, Rb2, Rc, Rd, Re, Rg1, Ro, malonyl-Rb1, malonyl-Rc, and malonyl-Rd and the polyacetylenes falcarinol and panaxydol were extracted from fresh ginseng roots in a sequential extraction process with 100% methanol followed by 80% aqueous methanol and quantified simultaneously in extracts by high-performance liquid chromatography using diode array detection. Separations were achieved with a phosphate buffer-acetonitrile gradient system using an RP-C18 column. Except for Rd, the present extraction method resulted in similar or significantly higher concentrations of both ginsenosides and polyacetylenes in comparison to commonly used extraction methods for these compounds. The contents of polyacetylenes and ginsenosides were determined in the root hairs, lateral roots, and main roots of 6 year old ginseng plants. The total mean concentrations of ginsenosides and polyacetylenes in root hairs were 31.0 g/kg fresh weight (FW) and 2.6 g/kg FW, respectively, whereas the concentrations of these bioactive compounds in the main roots were significantly lower with total mean concentrations of 17.8 g/kg FW for ginsenosides and 0.6 g/kg FW for polyacetylenes. The concentration of individual and total ginsenosides and polyacetylenes did not differ significantly between main roots of different sizes. Consequently, it is possible to do quantitative screening for ginsenosides and polyacetylenes to breed ginseng roots with higher levels of bioactive compounds.     

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.