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.     

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.     

Evaporative light-scattering analysis of sulforaphane in broccoli samples: Quality of broccoli products regarding sulforaphane contents

Broccoli sulforaphane has received attention as a possible anticarcinogen. Sulforaphane analysis is difficult due to the lack of a chromophore for spectrometric detection. Hence, we developed a method for determining sulforaphane by using high-performance liquid chromatography (HPLC) coupled with an evaporative light-scattering detector (ELSD). Sulforaphane was extracted from acid-hydrolyzed broccoli samples, followed by solid-phase extraction and reversed-phase HPLC. Sulforaphane was detected by ELSD and concurrently identified by electrospray ionization time-of-flight mass spectrometry. The recovery of sulforaphane from broccoli samples was above 95%. The detection limit was 0.5 mug. The present method was sensitive enough to determine sulforaphane in mature broccoli, broccoli sprouts, and commercial broccoli products. Sulforaphane concentration in broccoli sprout (1153 mg/100 g dry weight) was about 10 times higher than that of mature broccoli (44-171 mg/100 g dry weight). Therefore, the broccoli sprout is recommended as a source of sulforaphane-rich products. In contrast, we found that sulforaphane could not be detected in most of broccoli products, suggesting present commercial broccoli products having low quality.     

Separation and purification of sulforaphane from broccoli seeds by solid phase extraction and preparative high-performance liquid chromatography

A novel, rapid, and economical method to isolate and purify natural sulforaphane from broccoli seeds is described. The procedure involves solvent extraction of autolyzed seed meal, followed by separation by solid phase extraction (SPE) and purification by preparative high-performance liquid chromatography (HPLC). The SPE method provides higher yield of sulforaphane from crude extracts compared to conventional liquid-liquid extraction. High purity and recovery of sulforaphane product can be obtained by preparative HPLC with a C 18 column and 30% methanol in water as the mobile phase. The purified compound was characterized by MS and (1)H and (13)C NMR. The techniques described here are useful tools in the preparative-scale isolation of sulforaphane in a fast, cost-effective, and waste-conscious manner.     

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.     

Purification of curcumin, demethoxycurcumin, and bisdemethoxycurcumin by high-speed countercurrent chromatography

Curcuminoids are substances of great interest because of their important pharmacological activities, particularly anti-inflammatory, anticarcinogenic, and anti-Alzheimer's activities. In this study, we report the first procedure and effect of processing for the high, efficient, and useful purification of curcumin, demethoxycurcumin, and bisdemethoxycurcumin from turmeric powder. Purification involves high-speed countercurrent chromatographic (HSCCC) separation of these curcuminoids using a simple two-phase solvent system composed of n-hexane/chloroform/methanol/water (5/10/7.5/2.5, v/v). The HSCCC-fractionated effluent peaks indicated that the peak resolutions were 1.7 between curcumin and demethoxycurcumin and 2.1 between demethoxycurcumin and bisdemethoxycurcumin for 25 mg of loaded turmeric powder. These purified substances were analyzed by liquid chromatography-tandem mass spectrometry with scan and daughter scan negative modes, and the wide absorbance from 200 to 500 nm was monitored by photodiode array detection. The separation yielded 1.1 mg of curcumin, 0.6 mg of demethoxycurcumin, and 0.9 mg of bisdemethoxycurcumin (>98% purity). Moreover, the antioxidant effect of curcuminoids was measured by a 1,1-diphenyl-2-picrylhydrazil assay. The order of antioxidant activity was purified curcumin > purified demethoxycurcumin > purified bisdemethoxycurcumin > turmeric powder. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin can be used for various evaluations of their pharmacological activities.     

Nonvolatiles of commercial lime and grapefruit oils separated by high-speed countercurrent chromatography

The nonvolatile fractions of cold-pressed peel oils of Key and Persian lime as well as grapefruit were separated by high-speed countercurrent chromatography (HS-CCC). In addition to the isolation of the main coumarins, psoralens and polymethoxyflavones, a number of minor constituents were enriched and successfully characterized by GC-MS and HPLC-UV. 5,7,8-Trimethoxycoumarin and the cyclical acetals of oxypeucedanin hydrate with citral were determined as new nonvolatile trace constituents of lime oils and confirmed by NMR spectroscopy. The citral oxypeucedaninyl acetals were found particularly in Key lime oil type A, which as a result of the juice-oil contact, is exposed to acidic conditions during industrial processing. Some of the confirmed minor constituents, such as pabulenol, isooxypeucedanin, and oxypeucedanin methanolate in lime as well as auraptenol in grapefruit, may have been generated by hydrolysis-sensitive precursors during CCC separation or their respective industrial processing techniques.     

Purification of alkylamides from Echinacea angustifolia (DC.) Hell. Roots by high-speed countercurrent chromatography

High-speed countercurrent chromatography (HSCCC) was used for the separation of alkylamides from the roots of Echinacea angustifolia (DC.) Hell. For this purpose, the alkylamides were extracted with hexane and subjected to semipreparative HSCCC using a two-phase solvent system consisting of n-hexane, ethyl acetate, methanol, and water (4:1:2:1). The lower aqueous phase was used as the mobile phase at a flow rate of 3 mL/min and a rotary speed of 1000 rpm. This procedure led to the isolation of four pure alkylamides, that is, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide (38.9 mg, 97% purity), dodeca-2E,4E,8Z-trienoic acid isobutylamide (4.4 mg, 92% purity), dodeca-2E,4E-dienoic acid isobutylamide (3.2 mg, 99% purity), and dodeca-2E,4E-dienoic acid 2-methylbutylamide (0.3 mg, 92% purity). The identity and purity of the isolated alkylamides were confirmed by LC-ESI-MS and (1)H NMR and (13)C NMR data. To the best of the authors' knowledge, this is the first report of dodeca-2E,4E-dienoic acid 2-methylbutylamide in E. angustifolia roots.     

Comparison of the bioactivity of two glucoraphanin hydrolysis products found in broccoli, sulforaphane and sulforaphane nitrile.

Epidemiological and laboratory studies suggest that dietary broccoli may prevent or delay a variety of cancers. Broccoli and other crucifers contain a relatively unique family of secondary metabolites called glucosinolates. Glucoraphanin, the major glucosinolate in broccoli, is hydrolyzed by an endogenous plant myrosinase to form either the potent anticarcinogen sulforaphane (SF) or sulforaphane nitrile (SF nitrile). The bioactivities of SF and SF nitrile were compared in rats and in mouse hepatoma cells. Male, 4-week-old, Fischer 344 rats were administered SF or SF nitrile (200, 500, or 1000 micromol/kg) by gavage daily for 5 days. Hepatic, colonic mucosal, and pancreatic quinone reductase and glutathione S-transferase activities were induced by high doses of SF, but not by SF nitrile. When Hepa 1c1c7 cells were exposed to increasing levels of each compound for 24 h, quinone reductase showed a 3-fold maximal induction over control at 2.5 microM SF and a 3.5-fold maximal induction over control at 2000 microM SF nitrile, the highest concentration tested. These results demonstrate that SF nitrile is substantially less potent than SF as an inducing agent of phase II detoxification enzymes. Therefore, glucoraphanin hydrolysis directed toward the production of SF rather than SF nitrile could increase the potential chemoprotective effects of broccoli.     

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).     

Rapid isolation and purification of 1-cyano-2-hydroxy-3-butene (crambene) from Crambe abyssinica seed meal using immiscible solvent extraction and high-performance liquid chromatography

1-Cyano-2-hydroxy-3-butene (crambene) is a nitrile found in cruciferous vegetables that causes significant upregulation of quinone reductase and glutathione S-transferases in vivo and in vitro, making it a likely candidate as a cancer chemopreventive compound. To investigate further the putative anticarcinogenic mechanisms of crambene, a compound of the highest possible purity is vital. Therefore, a rapid and effective method of purification of crambene is necessary to continue studies of its beneficial health effects. A rapid method to isolate and purify natural crambene from either Crambe abyssinica (crambe) seed or commercially processed crambe seed meal was developed using immiscible solvent extraction followed by high-performance liquid chromatography. Use of this methodology eliminated the need for time-consuming and relatively inefficient column chromatography, improved extraction efficiency, and resulted in higher purity than previously used methodologies. Elimination of trace amounts of fatty acid residues, unachievable with previous methodologies, also was accomplished.     

Epithiospecifier protein from broccoli (Brassica oleracea L. ssp. italica) inhibits formation of the anticancer agent sulforaphane

In some cruciferous plants, epithiospecifier protein (ESP) directs myrosinase (EC 3.2.3.1)-catalyzed hydrolysis of alkenyl glucosinolates toward epithionitrile formation. Here, for the first time, we show that ESP activity is negatively correlated with the extent of formation of the health-promoting phytochemical sulforaphane in broccoli (Brassica oleracea L. ssp. italica). A 43 kDa protein with ESP activity and sequence homology to the ESP of Arabidopsis thaliana was cloned from the broccoli cv. Packman and expressed in Escherichia coli. In a model system, the recombinant protein not only directed myrosinase-dependent metabolism of the alkenyl glucosinolate epi-progoitrin [(2S)-2-hydroxy-3-butenyl glucosinolate] toward formation of an epithionitrile but also directed myrosinase-dependent hydrolysis of the glucosinolate glucoraphanin [4-(methylsulfinyl)butyl glucosinolate] to form sulforaphane nitrile, in place of the isothiocyanate sulforaphane. The importance of this finding is that, whereas sulforaphane has been shown to have anticarcinogenic properties, sulforaphane nitrile has not. Genetic manipulation designed to attenuate or eliminate expression of ESP in broccoli could increase the fractional conversion of glucoraphanin to sulforaphane, enhancing potential health benefits.     

Preparative HPLC method for the purification of sulforaphane and sulforaphane nitrile from Brassica oleracea

An extraction and preparative HPLC method has been devised to simultaneously purify sulforaphane and sulforaphane nitrile from the seed of Brassica oleracea var. italica cv. Brigadier. The seed was defatted with hexane, dried, and hydrolyzed in deionized water (1:9) for 8 h. The hydrolyzed seed meal was salted and extracted with methylene chloride. The dried residue was redissolved in a 5% acetonitrile solution and washed with excess hexane to remove nonpolar contaminants. The aqueous phase was filtered through a 0.22-microm cellulose filter and separated by HPLC using a Waters Prep Nova-Pak HR C-18 reverse-phase column. Refractive index was used to detect sulforaphane nitrile, and absorbance at 254 nm was used to detect sulforaphane. Peak identification was confirmed using gas chromatography and electron-impact mass spectrometry. Each kilogram of extracted seed yielded approximately 4.8 g of sulforaphane and 3.8 g of sulforaphane nitrile. Standard curves were developed using the purified compounds to allow quantification of sulforaphane and sulforaphane nitrile in broccoli tissue using a rapid GC method. The methodology was used to compare sulforaphane and sulforaphane nitrile content of autolyzed samples of several broccoli varieties.     

Characterization of pigments from different high speed countercurrent chromatography wine fractions

A red wine, made from Cabernet Sauvignon (60%) and Tannat (40%) cultivars, was fractionated by high speed countercurrent chromatography (HSCCC). The biphasic solvent system consisting of tert-butyl methyl ether/n-butanol/acetonitrile/water (2/2/1/5, acidified with 0.1% trifluoroacetic acid) was chosen for its demonstrated efficiency in separating anthocyanins. The different native and derived anthocyanins were identified on the basis of their UV-visible spectra, their elution time on reversed-phase high-performance liquid chromatography (HPLC), and their mass spectra, before and after thiolysis. The HSCCC method allowed the separation of different families of anthocyanin-derived pigments that were eluted in different fractions according to their structures. The hydrosoluble fraction was almost devoid of native anthocyanins. Further characterization (glucose quantification, UV-visible absorbance measurements) indicated that it contained flavanol and anthocyanin copolymers in which parts of the anthocyanin units were in colorless forms. Pigments in the hydrosoluble fraction showed increased resistance to sulfite bleaching and to the nucleophilic attack of water.     

Bioavailability and kinetics of sulforaphane in humans after consumption of cooked versus raw broccoli

The aim of this study was to determine the bioavailability and kinetics of the supposed anticarcinogen sulforaphane, the hydrolysis product of glucoraphanin, from raw and cooked broccoli. Eight men consumed 200 g of crushed broccoli, raw or cooked, with a warm meal in a randomized, free-living, open cross-over trial. Higher amounts of sulforaphane were found in the blood and urine when broccoli was eaten raw (bioavailability of 37%) versus cooked (3.4%, p ) 0.002). Absorption of sulforaphane was delayed when cooked broccoli was consumed (peak plasma time ) 6 h) versus raw broccoli (1.6 h, p ) 0.001). Excretion half-lives were comparable, 2.6 and 2.4 h on average, for raw and cooked broccoli, respectively (p ) 0.5). This study gives complete kinetic data and shows that consumption of raw broccoli results in faster absorption, higher bioavailability, and higher peak plasma amounts of sulforaphane, compared to cooked broccoli.     

Analysis of antioxidants from orange juice obtained by countercurrent supercritical fluid extraction,using micellar electrokinetic chromatography and reverse-phase liquid chromatography

Antioxidants from orange juice were determined by the combined use of countercurrent supercritical fluid extraction (CC-SFE) prior to reverse-phase liquic chromatography (RP-LC) or micellar electrokinetic chromatography (MEKC). The separation of antioxidants found in the SFE fractions was achieved by using a new MEKC method and a published LC procedure, both using diode array detection. The characterization of the different antioxidants was further done by LC-mass spectrometry. Advantages and drawbacks of LC and MEKC for analyzing the antioxidants found in the different orange extracts are discussed. Although LC yields higher peak area and slightly better reproducibility than MEKC, the latter technique provides information about the CC-SFE extracts in analysis times 7 times faster than by LC. This analysis advantage can be used for the quick adjustment of CC-SFE conditions, thus providing a fast way to obtain orange fractions of specific composition.     

南瓜籽粕酶法制备血管紧张素转化酶抑制肽工艺优化

用中性蛋白酶水解南瓜籽粕,制备血管紧张素转换酶（angiotensin-converting enzyme,ACE）抑制肽,是南瓜籽粕蛋白深度开发的途径之一。为了探寻南瓜籽粕酶法制备ACE抑制肽的最佳工艺,该文以ACE抑制率为响应值,用响应面分析法研究酶浓度、底物质量浓度和水解时间等因素对酶解产物的ACE抑制活性的影响,优化制备工艺。结果表明,各因素对制备ACE抑制肽的活性具有极显著影响（P<0.001）。获得中性蛋白酶水解南瓜籽粕制备ACE抑制肽的最佳工艺条件为：酶体积分数为4.8%、底物质量浓度为4.0g/100 mL、水解时间为320 min。在此条件下,南瓜籽粕蛋白酶解产物对ACE的理论抑制率最高可达80.0％,验证值为80.56％±0.23%,预测模型可靠性高,可应用于南瓜籽粕ACE抑制肽的酶法制备。通过优化,提高了南瓜籽粕ACE抑制肽的活性。     

Determination of ethalfluralin in canola seed, meal, and refined oil by capillary gas chromatography with mass selective detection

Ethalfluralin is a herbicide that is effective for weed control on a wide variety of crops, including canola. A method is described for the determination of ethalfluralin residues in canola seed, meal, and refined oil. Residues are extracted from canola sample matrixes with acetonitrile. An aliquot of the extract is diluted with water and purified by C(18) solid-phase extraction prior to analysis by capillary gas chromatography with mass selective detection. For all three sample matrixes, the method has a validated limit of quantitation of 0.02 microg/g and a limit of detection of 0.006 microg/g. Recoveries averaged 96 +/- 7% for canola seed, 87 +/- 6% for canola meal, and 89 +/- 5% for refined oil. In a magnitude-of-residue study, canola seed from field plots that had been treated with ethalfluralin at one to three times the maximum label rate for weed control were found to contain no detectable residue of the herbicide.     

Impact of isolation method on the antioxidant activity of rapeseed meal phenolics

Rapeseed meal is the byproduct of the rapeseed deoiling process. Among oilseed plants, rapeseed contains the greatest amount of phenolic compounds. In this study, the rapeseed phenolics were isolated with aqueous methanol, aqueous ethanol, hot water, and enzymatically with ferulic acid esterase. These isolates were tested for radical scavenging and for liposome and low-density lipoprotein (LDL) model systems. The radical scavenging activities of all isolates were >60% at a concentration of 1.5 mg/mL. In the liposome model system the formation of hexanal was inhibited by all rapeseed meal isolates by >90% and the formation of conjugated diene hydroperoxides by >80% (8.4 microg/mL concentration). All rapeseed meal isolates also inhibited oxidation of LDL particles by >90% inhibition (4.2 microg/mL concentration). Isolation of rapeseed meal phenolics with either water or enzyme is a very suitable method devoid of organic solvents. Thus, rapeseed meal phenolics constitute an interesting source for food and cosmetic applications with antioxidant effect.     

LC-MS/MS quantification of sulforaphane and indole-3-carbinol metabolites in human plasma and urine after dietary intake of selenium-fortified broccoli

This study aimed at developing a sensitive LC-MS/MS method for the quantification of sulforaphane (SFN) and indole-3-carbinol metabolites in plasma and urine after dietary intake of regular and selenium-fertilized broccoli using stable isotope dilution analysis. In a three-armed, placebo-controlled, randomized human intervention study with 76 healthy volunteers, 200 g of regular (485 μg of total glucosinolates and <0.01 μg of selenium per gram fresh weight) or selenium-fertilized broccoli (589 μg of total glucosinolates and 0.25 μg of selenium per gram fresh weight) was administered daily for 4 weeks. Glucoraphanin and glucobrassicin metabolites quantified in plasma and urine were SFN-glutathione, SFN-cysteine, SFN-cysteinylglycine, SFN-acetylcysteine, and indole-3-carboxaldehyde, indole-3-carboxylic acid, and ascorbigen, respectively. Dietary intake of selenium-fertilized broccoli increased serum selenium concentration analyzed by means of atomic absorption spectroscopy by up to 25% (p < 0.001), but affected neither glucosinolate concentrations in broccoli nor their metabolite concentrations in plasma and urine compared to regular broccoli.