Extraction of anthocyanins and other phenolics from black currants with sulfured water

Health benefits of fruits, vegetables, and red wine are attributed to anthocyanins and other phytochemicals. In this research, the extraction of phenolics from black currants was optimized using different SO(2) concentrations (28, 300, 700, 1100, and 1372 ppm), temperatures (6, 20, 40, 60, and 74 degrees C), and solvent to solid ratios (S/S) (6, 20, 40, 60, and 74 mL/g). Surface response methodology was used to optimize yields of anthocyanins and total phenolics, as well as their antiradical and antioxidant activities. The extraction of phenolics varied with the SO(2) concentration, S/S, and temperature. Maximum yields of total phenolics and anthocyanins were obtained at an SO(2) concentration of 1000-1200 ppm and 19 L of solvent/kg of milled frozen berries. The increase of extraction temperature increased the rate of extraction and, thus, times to reach equilibrium for the extraction of total phenolics and anthocyanins were reduced. However, for the extraction of anthocyanins it is recommended that temperatures of 30-35 degrees C be used, as higher temperatures will degrade these compounds. Antioxidant activity was affected by all three experimental variables evaluated; however, the main variable affecting it was S/S. The higher the S/S, the lower the antioxidant index.     

Interspecific variation in anthocyanins, phenolics, and antioxidant capacity among genotypes of highbush and lowbush blueberries (Vaccinium section cyanococcus spp.)

Recent interest in the possible protective effects of dietary antioxidant compounds against human degenerative disease has prompted investigation of foods such as blueberries (Vaccinium sp.), which have a high antioxidant capacity. Fruit obtained from genotypes of highbush blueberries (Vaccinium corymbosum L.) and lowbush blueberries (Vaccinium angustifolium Aiton) were analyzed for their antioxidant capacity, their content of anthocyanins, and total phenolic compounds, to evaluate the intraspecific and interspecific variation in these parameters. The method of extraction influenced the composition of fruit extracts; the highest anthocyanin and total phenolic contents and antioxidant capacity were found in extracts obtained using a solvent of acidified aqueous methanol. Regardless of the method, lowbush blueberries were consistently higher in anthocyanins, total phenolics, and antioxidant capacity, compared with highbush blueberries. There was no relationship between fruit size and anthocyanin content in either species.     

Anthocyanins in wild blueberries of Quebec: extraction and identification

Anthocyanins were extracted from a mixture of berries of Vaccinium angustifolium and Vaccinium myrtillo?des at 7.7 degrees C, 26 degrees C, and 79 degrees C using ethanol alone or ethanol acidified with hydrochloric, citric, tartaric, lactic, or phosphoric acids at a solvent to solid ratio of 10. The effect of these parameters on extracted anthocyanins stability was investigated. The pH-differential and HPLC-DAD methods were used to determine anthocyanin contents. Extracted anthocyanins were purified on a C-18 solid-phase extraction cartridge and characterized by HPLC/electrospray ionization/mass spectrometry (HPLC-ESI-MS/MS). Anthocyanins were identified according to their HPLC retention times, elution order, and MS fragmentation pattern and by comparison with standards and published data. Anthocyanin extractions gave different yields depending on the type of added acid and the extraction temperature. High yields of monomeric and total anthocyanins (26.3 and 28.9 mg/g of dry matter) were obtained at 79 degrees C using phosphoric acid. Extraction using tartaric acid at 79 degrees C provided the lowest degradation index (1.05). Anthocyanins were stable and browning by polyphenol oxidase was inhibited under these conditions. Of the six common anthocyanindins, five were identified in the extracts, namely, delpinidin, cyanidin, peonidin, petunidin, and malvidin; pelargonidin was not found. In addition to well-known major anthocyanins, new anthocyanins were identified for the first time in extracts of wild blueberries from Quebec.     

Effect of Albizia julibrissin water extracts on low-density lipoprotein oxidization

High-value phytochemicals could be extracted from biomass prior to the current cellulosic pretreatment technologies (i.e., lime, ammonia, dilute acid, or pressurized hot water treatments) provided that the extraction is performed with a solvent that is compatible with the pretreatment. This work reports on the extraction of flavonoids from Albizia julibrissin biomass. While extracting A. julibrissin foliage with 50 degrees C water, 2.227 mg/g of hyperoside and 8.134 mg/g quercitrin were obtained, which is in the realm of what was obtained with 60% methanol. A. julibrissin foliage, flower, and whole plant extracts were tested in terms of their potential to inhibit low-density lipoprotein (LDL) oxidization. The highest inhibition was obtained with foliage water extracts, which were standardized at 2.5 microM of flavonoids. Also, the 2.5 microM foliage water extract resulted in a reduction from 43% to only 1% of the observed monocyte adherence. To have commercial application, A. julibrissin water extracts should be devoid of toxicity. The A. julibrissin foliage, flower, and whole plant water extracts were not toxic to Vero 76 cells. In summary, A. julibrissin biomass can be extracted with 50 degrees C water to yield an antioxidant stream, showing that it may be possible to couple extraction of valuable phytochemicals to the cellulosic pretreatment step.     

Optimization of solid-liquid extraction of resveratrol and other phenolic compounds from milled grape canes (Vitis vinifera)

Optimization of the solid-liquid extraction conditions for trans-resveratrol, trans--viniferin, ferulic acid, and total phenolics from milled grape canes has been investigated. The temperature and ethanol concentration were found to be major process variables for all responses, whereas the solvent to solid ratio was found not to be significant for any of the responses studied. The yields of trans-resveratrol, trans--viniferin, and total phenolics increased with increasing temperature. Maximum yields of trans-resveratrol (4.25 mg/g dw), trans--viniferin (2.03 mg/g), and total phenolics (9.28 mg/g dw) were predicted from the combination of a moderate ethanol concentration (50-70%) and the highest temperature (83.6 degrees C), whereas an ethanol concentration of 35% at the lowest temperature studied (16.4 degrees C) was optimal for the extraction of ferulic acid (1.05 mg/g dw). Effective diffusivity values of resveratrol in the solid phase, D eff for different extraction conditions, were calculated by fitting the experimental results to a model derived from the Fick's second law. Effective diffusivity of resveratrol in the solid phase varied from 3.1 x 10 (-13) to 26.6 x 10 (-13) m (2) s (-1) with changing extraction conditions. The increase in effective diffusivity of resveratrol was observed with increasing temperature, and the highest predicted level was obtained when using 54% ethanol/water mixture at 83.6 degrees C. The increase in ethanol concentration exhibited the favorable effect up to 50-55%, thereafter effective diffusivity decreased with a further increase in concentration.     

Identification of ellagic acid conjugates and other polyphenolics in muscadine grapes by HPLC-ESI-MS

Ellagic acid, ellagic acid glycosides, and ellagitannins found in various fruits and nuts, including muscadine grape, are reported to have potential health-promoting benefits and antioxidant properties. This study isolated and identified several ellagic acid derivatives present in muscadine grapes and determined their relative antioxidant properties (AOX). Compounds were extracted from grape skins and pulp using methanol, and the solvent was evaporated. Isolates were dissolved in citric acid buffer (pH 3.5) and absorbed onto C18 cartridges. Nonretained polyphenolics were collected separately and again partitioned from Sephadex LH-20, whereas retained polyphenolics were first eluted with ethyl acetate followed by methanol. Ellagic acid derivatives were identified on the basis of UV and mass spectra, and the presence of ellagitannins was confirmed by a significant increase in free ellagic acid with HPLC followed by acid hydrolysis. Muscadine grapes contained phenolic acids, flavonols, anthocyanins, ellagic acid, and numerous ellagic acid derivatives. AOX varied in the order ethyl acetate > methanol > C18 nonretained fractions; each correlated to both total phenolics (r = 0.90) and total ellagic acid (r = 0.99) contents. Results of this study revealed previously unidentified ellagic acid derivatives in muscadine grapes.     

Influence of sample preparation on assay of phenolic acids from eggplant

Sample preparation is often overlooked and is frequently considered as "a means to an end". This systematic study with a phenolic-enriched substrate, eggplant (Solanum melongena L.), was undertaken to evaluate the substantial variations in the extraction techniques, solvents, and parameters as described in the published literature. Direct comparison of over 10 extraction procedures or conditions was performed to show the importance and influence of sample preparation on the assay of phenolic compounds. Chlorogenic acid (CA) was the most abundant phenolic acid accounting for >75% of the total phenolic acids content extracted from the eggplant sample. Optimum extraction of CA and total phenolics (TP) from Black Bell cultivar of eggplant were obtained when extractions were performed with a mixture of MeOH/H2O at a ratio of 80:20% v/v using a pressurized liquid extractor (PLE) at 100 degrees C. The amount of CA and TP extracted from eggplant by the previously reported procedures using a wrist shaker, rotary shaker, stirring, sonication, or reflux with different extraction solvents (acetone or varying composition of MeOH/H2O solvent mixtures) varied significantly between 5 and 95% as compared to PLE. The predominant phenolic acids in the free phenolic acid fraction of Black Beauty cultivar of eggplant were CA isomers. However, caffeic acid isomers were the major phenolic acids extracted from the base-hydrolyzed fraction. The total amount of caffeic acid extracted from the Italian Neon cultivar was more that twice that of four other eggplant cultivars (Orient Express, Calliope Zebra Stripe, Orient Charm Neon, and Black Beauty).     

Antioxidant capacity in cranberry is influenced by cultivar and storage temperature

Ten cranberry (Vaccinium macrocarpon Aiton) cultivars were evaluated for oxygen radical absorbance capacity (ORAC), anthocyanins, and total phenolics contents after three months of storage at 0, 5, 10, 15, and 20 degrees C. The antioxidant capacity of cranberry was affected by cultivars and storage temperatures. Among the 10 cranberry cultivars used in this study, Early Black, Crowley, and Franklin had higher antioxidant capacities than the other cultivars. ORAC values, anthocyanins, and total phenolics contents increased during storage. The highest increases in antioxidant activity, anthocyanin, and phenolics contents occurred at 15 degrees C storage. Fruit stored at 20 degrees C had lower ORAC values than those stored at 15 degrees C. A positive relationship existed between ORAC values and anthocyanin or phenolic content in all 10 cranberry cultivars at different storage temperatures.     

Purification of lactulose from mixtures with lactose using pressurized liquid extraction with ethanol-water at different temperatures

The viability of the purification of lactulose from a mixture with lactose [70:30 (w/w)] using pressurized liquid extraction (PLE) at 1500 psi for 30 min was studied. Different temperatures (from 40 to 130 degrees C) and proportions of ethanol:water (70:30, 80:20, 90:10, 95:5, and 100:0) as the extraction solvent were assayed. Lactose and lactulose were measured by gas chromatographic analysis as their trimethylsilyl derivatives. Data were fitted through multiple linear regressions to different quadratic models to describe both the extraction yield (in terms of mg of lactulose) and the purity of the lactulose extracted. The optimum extraction conditions provided by the model were as follows: extraction temperature, 40 degrees C; and solvent composition, 70:30 ethanol:water. PLE extraction under the optimized conditions was also applied to purify lactulose from lactose in a synthesis mixture. To our knowledge, this is the first time that PLE has been tested for extraction and purification of lactulose from its mixture with lactose; this technique showed several advantages over classical methods such as the short extraction time and the low solvent consumption.     

Pressurized liquid extraction of capsaicinoids from peppers

A method has been developed for the extraction of capsaicinoids from peppers by pressurized liquid extraction (PLE); these compounds are determined by reverse phase high-performance liquid chromatography (HPLC), with detection by fluorescence spectrophotometry and mass spectrometry (MS). The stability of capsaicin and dihydrocapsaicin has been studied at different temperatures (50-200 degrees C), and several extraction variables have been assayed: solvent (methanol, ethanol, and water), different percentages of water in the methanol (0-20%) and in the ethanol (0-20%), and the number of extraction cycles. The study has evaluated the repeatability (RSD < 7%) and the reproducibility (RSD < 7%) of the method. Finally, the PLE method developed has been applied to quantify the capsaicinoids present in three varieties of hot peppers cultivated in Spain, quantifying five capsaicinoids: nordihydrocapsaicin, capsaicin, dihydrocapsaicin, an isomer of dihydrocapsaicin, and homodihydrocapsaicin.     

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.     

Lettuce and chicory byproducts as a source of antioxidant phenolic extracts

A process to obtain enriched antioxidant phenolic extracts from lettuce (baby, romaine, and iceberg cultivars) and chichory byproducts as a way to valorize these byproducts was developed. Two extraction protocols using water and methanol as solvent were used. Amberlite XAD-2 nonionic polymeric resin was used to purify the extracts. The extraction yield, phenolic content, and phenolic yield were evaluated as well as the antioxidant capacity of the extracts (DPPH, ABTS, and FRAP assays). Baby and romaine lettuce byproducts showed the highest water extract yields [27 and 26 g of freeze-dried extracts/kg of byproduct fresh weight (fw), respectively], whereas baby and iceberg lettuce showed highest methanol extract yields (31 and 23 g of freeze-dried extracts/kg of byproduct fw, respectively). Methanol extraction yielded a raw extract with a high phenolic content, the baby and chicory extracts being the richest with approximately 50 mg of phenolics/g of freeze-dried extract. Regarding the purified extracts, water extraction yielded a higher phenolic content, baby and chicory being also the highest with mean values of approximately 190 and 300 mg of phenolics/g of freeze-dried extract, respectively. Both raw and purified extracts from baby and chicory showed the higher antioxidant contents (DPPH, ABTS, and FRAP assays). The antioxidant capacity was linearly correlated with the phenolic content. The results obtained indicate that lettuce byproducts could be, from the industrial point of view, an interesting and cheap source of antioxidant phenolic extracts to funcionalize foodstuffs.     

Effect of solvent, temperature, and solvent-to-solid ratio on the total phenolic content and antiradical activity of extracts from different components of grape pomace

Grape byproducts were subjected to an extraction process under various different experimental conditions (namely, solvent type, temperature, solvent-to-solid ratio, time contact, and raw material) in order to study the effect of these conditions on the yield of phenolic compounds and the corresponding antiradical activity of extracts. Although the order of decreasing capacity to extract soluble materials was ethanol > methanol > water, methanol was the most selective for extracting phenolic compounds. Temperature and solvent-to-solid ratio were found to have a critical role in extraction efficiency; values of 50 degrees C (between 25 and 50 degrees C) and 1:1 (between 1:1 and 5:1) maximized the antiradical activity of phenolic extracts. In addition, extracts from grape samples previously subjected to distillation reached higher antiradical values in comparison to those coming directly from pressing; in both cases, seed extracts showed better results than those of stem when ethanol or water was employed, whereas the opposite occurred in the case of methanol. These differences were attributed to the different phenolic compositions of the considered fractions.     

Optimization of simultaneous flavanol, phenolic acid, and anthocyanin extraction from grapes using an experimental design: application to the characterization of champagne grape varieties

Optimization of polyphenol extraction from grape skin, seed, and pulp was performed on Vitis vinifera L. cv. Pinot Noir, by response surface methodology using a Doehlert design. An acidified mixture of acetone/water/methanol was the best solvent for simultaneous extraction of major polyphenol groups from all berry parts, while optimum extraction times and solid-to-liquid ratios varied according to the part. The determined composition from the model agreed with independent experimental results. Analysis of the three Champagne grape varieties showed that proanthocyanidins were the major phenolic compounds in each part (60-93%). The total berry proanthocyanidin content was highest in Pinot Meunier (11 g kg(-1)) and lowest in Chardonnay (5 g kg(-1)), but Pinot Meunier pulp contained lower amounts of proanthocyanidins and phenolic acids (210 and 127 mg kg(-1) berry, respectively) than that of the other two varieties. The berry anthocyanin content was equivalent in both Pinot Noir and Pinot Meunier (632 and 602 mg kg(-1), respectively).     

Prediction of wine color attributes from the phenolic profiles of red grapes (Vitis vinifera)

Knowledge about the relation between grape and wine phenolics is of key interest for the wine industry with respect to being able to predict wine quality from analyses of grapes. Prediction of the phenolic composition and color of experimentally produced red wines from the detailed phenolic composition of the corresponding grapes was investigated using a multivariate approach. Grape extracts and wines were produced from 55 different grape samples, covering 8 different Vitis vinifera cultivars: Alicante, Merlot, Syrah, Cinsault, Grenache, Carignan, Cabernet Sauvignon, and Mourvedre. The phenolic composition of the grapes and wines showed that the average ratios between wine and grape phenolics ranged from 0.25 to 7.9 for the different phenolic compounds. Most interestingly, the average ratios were low for anthocyanins (0.31) and tannins (0.32), intermediate for (+)-catechin (0.75) and polymeric pigments (0.98), and high for gallic acid (7.9). Individual wine phenolics in general correlated well with several grape phenolics, indicating that a multivariate approach might be advantageous for prediction of wine phenolics from grape phenolics analysis. However the use of multivariate prediction of individual wine phenolics from the complete grape phenolic composition only improved the prediction of wine polymeric pigments, whereas wine anthocyanins were predicted with the same precision as from the direct relation with grape anthocyanins. Prediction of color attributes of pH normalized experimental wines from the phenolic profiles of grapes was accomplished using a multivariate approach. The correlation between predicted and measured total wine color was high ( r = 0.958) but was very similar to the correlation coefficient obtained for the direct relation between grape anthocyanins and total wine color ( r = 0.961). Color due to copigmentation, color due to anthocyanins, and color intensity were also predicted well.     

Extraction of polyphenols from processed black currant (Ribes nigrum L.) residues

The total phenol and anthocyanin contents of black currant pomace and black currant press residue (BPR) extracts, extracted with formic acid in methanol or with methanol/water/acetic acid, were studied. Anthocyanins and other phenols were identified by means of reversed phase HPLC, and differences between the two plant materials were monitored. In all BPR extracts, phenol levels, determined by the Folin-Ciocalteu method, were 8-9 times higher than in the pomace extracts. Acid hydrolysis liberated a much higher concentration of phenols from the pomace than from the black currant press residue. HPLC analysis revealed that delphinidin-3-O-glucoside, delphinidin-3-O-rutinoside, cyanidin-3-O-glucoside, and cyanidin-3-O-rutinoside were the major anthocyanins and constituted the main phenol class ( approximately 90%) in both types of black currant tissues tested. However, anthocyanins were present in considerably lower amounts in the pomace than in the BPR. In accordance with the total phenol content, the antioxidant activity determined by scavenging of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation, the ABTS(*)(+) assay, showed that BPR extracts prepared by solvent extraction exhibited significantly higher (7-10 times) radical scavenging activity than the pomace extracts, and BPR anthocyanins contributed significantly (74 and 77%) to the observed high radical scavenging capacity of the corresponding extracts.     

Antioxidant activity of extracts of black sesame seed (Sesamum indicum L.) by supercritical carbon dioxide extraction

Antioxidant activities of extracts derived from sesame seed by supercritical carbon dioxide (SC-CO(2)) extraction and by n-hexane were determined using alpha,alpha-diphenyl-beta-picylhydrazyl (DPPH) radical scavenging and linoleic acid system methods. The highest extracted yield was given at 35 degrees C, 40 MPa, and a CO(2) flow rate of 2.5 mL min(-1) by an orthogonal experiment. The yields of extracts increased with increasing pressure, and yields at 40 and 30 MPa were higher than that by solvent extraction at 46.50%. Results from the linoleic acid system showed that the antioxidant activity follows the order: extract at 35 degrees C, 20 MPa > BHT > extract at 55 degrees C, 40 MPa > extract at 55 degrees C, 30 MPa > Trolox > solvent extraction > alpha-tocopherol. The SC-CO(2) extracts exhibited significantly higher antioxidant activities comparable to that by n-hexane extraction. The extracts at 30 MPa presented the highest antioxidant activities assessed in the DPPH method. At 20 MPa, the EC(50) increased with temperature, which indicated that the antioxidant activity was decreased in a temperature-dependent manner. The significant differences of antioxidant activities were found between the extracts by SC-CO(2) extraction and n-hexane. However, no significant differences were exhibited among the extracts by SC-CO(2) extraction. The vitamin E concentrations were also significantly higher in SC-CO(2) extracts than in n-hexane extracts, and its concentrations in extracts corresponded with the antioxidant activity of extracts.     

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.     

Design and optimization of a semicontinuous hot-cold extraction of polyphenols from grape pomace

Grape pomace contains appreciable amounts of polyphenolic compounds such as anthocyanins and procyanidins which can be recovered for use as food supplements. The extraction of these polyphenols from the pomace is usually accomplished at slightly elevated temperatures, frequently employing hydroethanolic solvents. Due to governmental regulations and the cost involved in using ethanol as a solvent, as well as the loss in polyphenolics due to thermal degradation, improved extraction techniques are required. In this study, a semicontinuous extraction apparatus employing only water was developed to maximize the recovery of anthocyanins and procyanidins from red grape pomace (Vitis vinifera). Water is preheated prior to its entry to the extraction cell containing the grape pomace sample, where it is allowed to then flow continuously through the unheated extraction vessel prior to its collection at ambient conditions. Extraction variables that impacted the polyphenolic recovery included pomace moisture content (crude or dried), sample mass, water flow rate, and extraction temperature. A response surface method was used to analyze the results from the extraction, and the optimal conditions were found to be 140 °C and 9 mL/min water flow rate. These conditions can produce an extract containing 130 mg/100 g DW of anthocyanins and 2077 mg/100 g DW of procyanidins. Higher yields of polyphenolics were observed using crude (wet) rather than dried pomace, hence avoiding the need to dry the pomace prior to extraction. The described semicontinuous extraction method using only water as the extraction solvent under subcritical conditions allowed the efficient extraction of polyphenols from red grape pomace without the attendant loss of polyphenolic content due to having to heat the extraction vessel prior to commencement of extraction.     

Fractionation of honey carbohydrates using pressurized liquid extraction with activated charcoal

This article describes the development of a new procedure that combines the use of activated charcoal and pressurized liquid extraction (PLE) to obtain enriched fractions of di- and trisaccharides from honey. Honey was adsorbed onto activated charcoal and packed into a PLE extraction cell. Optimum results were obtained at 10 MPa and 40 degrees C using two consecutive PLE cycles: first, 1:99 (v/v) ethanol/water for 5 min and second, 50:50 (v/v) ethanol/water for 10 min. Di- and trisaccharide fractions were enriched after PLE treatment, accounting for 73% and 8% of total carbohydrates, respectively. This procedure was also compared with other methodologies reported in the literature for the fractionation of honey carbohydrates (yeast treatment and extraction from activated charcoal). While the removal of monosaccharides was more efficient with yeast treatment, recovery of di- and trisaccharides was higher when either the PLE or the activated charcoal treatment was used. PLE was found to be the faster technique; it also required less solvent volume and minimized handling of the sample.