Amberlite XAD-2 Column Research Articles

Evaluation of Inhibitory Effects of Vegetables and Herbs on Hyaluronidase and Identification of Rosmarinic Acid as a Hyaluronidase Inhibitor in Lemon Balm(Melissa officinalis L.).

The inhibitory effects of 46 vegetable and herb extracts on hyaluronidase (EC 3.2.1.35), which is known to be related to inflammation and involved in migration of tumor cells, were examined. Twenty-two extracts showed inhibitory activity, and the extracts of 8 Labiatae plants and borage demonstrated potent inhibition of hyaluronidase. The hyaluronidase-inhibitory compound was isolated from the MeOH extract of leaves of lemon balm, which showed the strongest inhibition among them, by a combination of partition between solvents, Amberlite XAD-7 column chromatography and ODS-HPLC. Mostly on the basis of spectrometric techniques, the hyaluronidase inhibitor in lemon balm was identified with rosmarinic acid.

Frontal analysis of aqueous phenol solutions in amberlite XAD-4 columns: Implications on the operation and design of solid phase extraction systems

Frontal analysis of aqueous phenol solutions in Amberlite XAD-4 columns was carried out at different experimental conditions. Operating variables such as the concentration, pH and ionic strength of the influent, the presence of competitor solutes, the fluid flow-rate and the column length were considered and their effects on the front profile, the phenol breakthrough volume and the equilibrium parameters were determined. The obtained results may explain some contradictory reported data concerning the recovery of hydrophilic compounds in solid phase extraction (SPE) systems using Amberlite XAD-2 or XAD-4 columns. Furthermore, it is demonstrated that the adsorption parameters derived from a frontal analysis in an XAD-4 column are directly transposable to columns of different sizes (at the analytical level). Therefore, the results of this study may also be used for prediction and/or design of a phenol SPE system adapted to a particular problem.

Datura metel: in vitro production of tropane alkaloids.

Hairy root lines of Datura metel were established following infection of aseptic stem segments with Agrobacterium rhizogenes strain A4 and cultured in hormone-free B5 solid medium. The growth and production of hyoscyamine and scopolamine (mg/g dry wt.) of these root cultures was encouraged by using B5 liquid medium with half-strength salts. In these culture conditions, the capacity of the highest productive root line 25 to excrete scopolamine into the culture medium rose from 8.7% to 70% when the permeabilizing agent Tween 20 was added for 24 h to the medium, after 2 and 4 weeks of culture. Using an airlift bioreactor (41) with modifications in order to increase root anchorage, the Tween 20 treatment encouraged both growth and alkaloid productivity of cultured root line 25. After 4 weeks their biomass yield was 2.3 mg/l/day and 0.84 mg/l/day of scopolamine was produced (70% extracellular). The scopolamine released into the culture medium was separated with an Amberlite XAD-2 column located in the media exit.

Extraction and Identification of an Antioxidative Component from Jue Ming Zi (Cassia tora L.)

The antioxidant activity of two cultivars of “jue ming zi” (Cassia tora L. and Cassia occidentalis L.) was investigated. The results indicated that methanolic extracts from C. tora L. (MECT) and C. occidentalis L. (MECO) produced stronger antioxidant activity and gave higher yields of extract than other organic solvents. The MECT showed stronger antioxidant activity than did the MECO on peroxidation of linoleic acid. MECT at 200 ppm was stronger than 200 ppm of α-tocopherol, but weaker than 200 ppm of butylated hydroxyanisole. Amberlite XAD-2 column chromatography separated MECT into eight fractions. Of the eight fractions, fraction V possessed significant antioxidant activity and showed 85.8% inhibition on peroxidation of linoleic acid. Subsequently, fraction V was separated into two subfractions, Va and Vb, by Toyoperal HW-40 F gel filtration chromatography. The subfraction Vb exhibited stronger antioxidant activity than did subfraction Va and was identified as 1,3,8-trihydroxy-6-methyl-9,10-anthracened...

Preparative Chromatography of Furosemide 1-O-Acyl-Glucuronide from Urine Using Micronized Amberiite XAD-2 and its Application to Other 1-O-Acyl-Glucuronides

Furosemide 1-O-acyl glucuronide (Fgnd) was extracted from the urine following oral administration of furosemide. The crude Fgnd was applied to micronized Amberlite XAD-2 column (2.5 cm i.d. × 90 cm length, 75–500 μm particle size). The purified Fgnd was identified by mass spectrometry and β-glucuronidase treatment. This method was also applicable to the purification of glucuronide of tolmetin (nonsteroidal anti-inflammatory drug, NSAID), suggesting that it was applicable to the other NSAIDs, most of which were known to be metabolized to acyl glucuronides.

Free and Glycosidically Bound Volatile Compounds from Two Banana Cultivars: Valery and Pequeña Enana

Headspace composition and glycosidically bound volatile compounds from two banana cultivars, Valery and Pequeña Enana, have been studied. Glycosides were isolated from aqueous extracts of banana pulp by means of an Amberlite XAD-2 column. Volatile compounds were released by enzymatic hydrolysis using almond β-glucosidase and further analyzed by HRGC/MS. Twenty-five main aglycons were analyzed for the first time in banana fruit pulp. These aglycons can be grouped in two biogenetically different groups: fatty acids and shikimic acid-derived compounds. Alcohols, such as decan-1-ol and 2-phenylethanol, and acids, such as 3-oxo-pentanoic acid, 3-methylbutanoic acid, and benzoic acid, were quantitatively the most important aglycons in glycosides isolated from both banana cultivars. Keywords: Aroma; flavor; banana; headspace; enzymatic hydrolysis; glycosides; volatiles

Large volume preconcentration of dissolved hydrocarbons and polychlorinated biphenyls from seawater. Intercomparison between C 18 disks and XAD-2 column

The extraction efficiency of XAD-2 and 90 mm C 18 Empore disk for trace level determination (ng/L) of organic pollutants (i.e. hydrocarbons, chlorinated compounds) has been evaluated. Whereas C 18 Empore extraction disk exhibited higher extraction efficiency for aliphatic hydrocarbons from spiked Milli Q water and seawater, Amberlite XAD-2 column was more efficient for the extraction of individual PCB congeners and most of parent PAHs from spiked Milli Q water. However, in case of seawater analysis, the C 18 disk exhibited a higher extraction efficiency for the 3–4 aromatic ring PAHs. Only labile PAHs (i.e. benzo[ a]pyrene) and other analytes associated with fine particles were better recovered in XAD-2. Breakthrough volumes for the C 18 ( φ=90 mm) disks were compound dependent but they were at least 5–10 times lower than typical volumes for XAD-2. However, the C 18 disk application enables a solvent usage reduction leading to procedural blank improvement compared to XAD-2 columns.

Determination of mycotoxins produced by Fusarium isolates from banana fruits by capillary gas chromatography and high-performance liquid chromatography

A method of analysis for trichothecenes (nivalenol, deoxynivalenol, 3- and 15-acetyldeoxynivalenol, diacetoxyscirpenol, neosolaniol, T-2 tetraol, T-2 and HT-2 toxins), zearalenone and zearalenols, and another method for determination of fumonisin B 1 are described and applied to cultures of Fusarium isolated from bananas. Both methods were adapted from different techniques of extraction, clean-up and determination of these mycotoxins. The first method involves extraction with methanol-1% aqueous sodium chloride, clean-up of extracts by partition with hexane and dichloromethane, additional solid reversed-phase clean-up and analysis of two eluates by both high-performance liquid chromatography with ultraviolet detection and capillary gas chromatography. The method for fumonisin B 1 implies extraction with aqueous methanol, concentration, clean-up with water and methanol on Amberlite XAD-2 column, formation of a fluorescent 4-fluoro-7-nitrobenzofurazan derivative and analysis by high-performance liquid chromatography with fluorescence detection. Both procedures give good limits of detection and recoveries, and are considered suitable for the detection and quantification of the studied toxins in corn and rice cultures of Fusarium spp. isolated from banana fruits.

Identification of the metabolites of a new oxazolidinone MAO-A inhibitor in rat

1. Six metabolites present in rat urine after the oral administration of E2011 ((5R)3-[2-((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidinone) were isolated with an Amberlite XAD-4 column and hplc, and termed HPM-1, HPM-2, HPM-31, HPM-32, HPM-33 and HPM-4. 2. To determine the correspondence of the findings of the metabolites between tlc (which was used in our previous study) and hplc, the six metabolites were isolated from rat urine after the administration of 14C-labelled E2011 with an Amberlite XAD-4 column and hplc, and then analysed by tlc. HPM-1, HPM-2, HPM-31, HPM-32, HPM-33 and HPM4 were identified as IM7, IM3, IM4, IM2, IM1 and E2011, respectively. 3. The structures of the metabolites were identified with nmr and mass spectrometry. One of the compounds identified, HPM-4, was the unchanged drug, E2011, and HPM-2 was O-desmethyl-E2011. Another metabolite (HPM-33), the main metabolite in the urine, was identified as (4S)-hydroxy-E2011, and the others were (4S)-hydroxy-O-desmethylE2011 (HPM-1), 2′′-hydroxy-E2011 (HPM-31) and (4R)-hydroxy-E2011 (HPM-32). 4. In conclusion, the main metabolic pathway of E2011 in the rat consisted of O-demethylation and hydroxylation.

The constituents ofTaraxacum hallaisanensis roots

Three sesquiterpene lactone compounds, two novel(1β, 3β-dihydroxy-6β, 11β, 4α, 5α, 7αH-eudesm-12,6-olide-1-O-β-D-glucopyranoside, 1β, 3β-dihydroxy-6β, 4β, 5α, 7αH-eudesm-11 en-12,6-olide-1-O-β-D-glucopyranoside) and 1β, 3β-dihydroxy-6β, 11β, 4α, 5α, 7αH-eudesm-12,6-olide were isolated from the aqueous fraction of MeOH extract of the roots fromTaraxacum hallaisanensis (Compositae) employing Amberlite XAD-2, ODS-gel, silica gel and Sephadex LH-20 column chromatographics. Another known compound, (−)-epicatechin, was isolated from the aqueous fraction of the MeOH extract. The total MeOH extract also contained phytosterol and a mixture of β-amyrin acetate, α-amyrin acetate and lupeol acetate. Structures of isolated compounds were elucidated by spectroscopic parameters of IR, Mass,13C-NMR,1H-NMR,1H-1H COSY,13C-1H COSY and HMBC.

A partially automated pretreatment module for routine analyses for seventeen non-steroid antiinflammatory drugs in race horses using gas chromatography/mass spectrometry.

A partially automated module for the routine determination of illicit non-steroid antiinflammatory drugs (NSAIDs) in biological fluids from race horses was built, tested, refined, and shown to work. This pretreatment module retains 17 NSAIDs on an Amberlite XAD-2 column before back-elution derivatization with methyl iodide in acetonitrile. Methylated derivatives are manually injected into a gas chromatograph connected to a mass spectrometer. The quantification limits thus achieved are 50-100 ng/mL in 1 mL of urine or plasma. The proposed method is more expeditious than its manual liquid-liquid and liquid-solid extraction counterparts and is similar in speed to a typical gas chromatographic program.

Synthesis of neohesperidin glycosides and naringin glycosides by cyclodextrin glucanotransferase from an alkalophilic Bacillus species.

Cyclodextrin glucanotransferase from an alkalophilic Bacillus species produced neohesperidin monoglucoside and a series of its maltooligoglucosides by transglycosylation with neohesperidin as an acceptor and soluble starch as a donor. As the reaction using beta-CD as a donor at an alkaline pH was very effective for solubilizing neohesperidin, the amount of glycosides formed was increased. As a result, its amount with beta-CD at pH 10 was about 7 times greater than that with soluble starch at pH 5. Neohesperidin monoglucoside was purified from the reaction mixture by glucoamylase and naringinase treatments, an Amberlite XAD-16 column, a Sephadex LH20 column, and HPLC on an ODS column. The structure of the purified monoglucoside was identified as 3G-alpha-D-glucopyranosyl neohesperidin by FAB-MS, methylation analysis, and 1H- and 13C-NMR. The solubility of neohesperidin monoglucoside in water was approximately 1500 times higher than that of neohesperidin, and the bitterness of the monoglucoside was about 10 times less than that of neophesperidin. In addition, naringin was also glycosylated by the same method as neohesperidin, and its monoglucoside was identified as 3G-alpha-D-glucopyranosyl naringin. The solubility of naringin monoglucoside in water was also at least 1000 times higher than that of naringin without altering its bitterness.

Solubilization and Functional Reconstitution of the Tonoplast H +-ATPase from Citrus in Liposomes

Summary Proteoliposomes were formed after solubilization of tonoplast proteins from Citrus with octylglucoside in the presence of soybean phospholipids (phosphatidylcholine), with removal of detergent by passage through an Amberlite XAD-2 column. The specific ATP-hydrolysis activity of the proteoliposomes was increased 4-fold over the activity of the native tonoplast vesicles. Pumping of protons by the reconstituted ATPase was demonstrated by quinacrine-fluorescence quenching. Neither vanadate nor azide inhibited ATP-hydrolysis and transport activity. Both the ATP-hydrolysis and the H + -translocating activities were inhibited by nitrate, the first activity being the more sensitive one. Both activities were completely inhibited by bafilomycin A 1 , an inhibitor of vacuolar type ATPase, at concentrations of 20 nM. A potent inhibitor of H + -transport, DCCD, completely inhibited both activities at 100 gM and with halfmaximal inhibition at concentrations of 40 pM and 60 μM for the H + -transport and the ATP-hydrolysis activities, respectively. The reconstituted H+-ATPase was stimulated by anions (Cl - > Br - > SO 4 2− ), inhibited by NO 3 − and I − and largely insensitive to monovalent cations. The proteoliposomes showed no PP i ase activity. SDS-PAGE showed that the reconstituted H + -ATPase from Citrus contained polypeptides of apparent molecular masses of 63, 52, 37, 33 and 16 kDa that cross-reacted with an antiserum against the tonoplast ATPase from Kalanchoe daigremontiana . Freeze-fracture electron microscopy of the proteoliposomes showed that proteins were incorporated into the bilayer membrane. The proteoliposomes from Citrus were mainly vesicles intramembraneous with particles whose diameter ranged from 6 to 7 nm. The results conclusively demonstrate a selective reconstitution of a functional H + -ATPase from Citrus .

Flavonoids from Portuguese heather honey

The flavonoids present in twenty samples of Portuguese heather honey were analysed by reversed-phase HPLC. The flavonoid fraction was purified by a combination of chromatography on Amberlite XAD-2 and Sephadex LH-20 columns. The total amount of flavonoids ranged between 60 and 500 μg/100g honey, values which are much smaller than those previously found for Spanish rosemary honey samples (500-2000 μg/100 g honey). The main flavonoids present in Portuguese heather honey were the flavanones pinocembrin and pinobanksin and the flavones chrysin and galangin. These analyses showed that all the samples contained a similar flavonoid pattern composed of at least 22 compounds. The most characteristic substances were myricetin, myricetin 3-methyl ether, myricetin 3'-methyl ether and tricetin. These four flavonoids have in common the structural feature of having a trioxygenated ring B (3',4',5'-trioxygenation), this being an attribute not found in the flavonoids detected in any other honey sample of different plant origin analysed so far, suggesting that these substances could probably be used as markers for the botanical origin of heather honey.

Thermoregulatory Effects of Ergovaline Isolated from Endophyte-Infected Tall Fescue Seed on Rats

Ergovaline (EV) was isolated from Acremonium coenophialum-infected tall fescue seed. Seed (2 kg) was extracted with 20 L of 5% lactic acid in water/methanol (4:1), filtered, and passed over an Amberlite XAD-2 column. Ergovaline was eluted with MeOH, concentrated, and chromatographed on two reversed-phase C 18 columns (purity >95%, 40% overall recovery). It was administered to rats (15 μg/kg of BW, ip) at 22 o C to determine time-related acute effects on thermoregulatory ability

Determination of ceftriaxone in aqueous humor and serum samples of differential-pulse adsorptive stripping voltammetry.

Differential-pulse adsorptive stripping voltammetry was used to determine ceftriaxone in serum and aqueous humour samples. The method involved extraction of the ceftriaxone from serum samples with an Amberlite XAD-2 column followed by elution with methanol. The recovery was 97.6% with a relative standard deviation of 3.3% at a ceftriaxone concentration of 90.9 microg 1(-1). Peak currents of ceftriaxone were measured with a hanging mercury drop electrode at -0.78 V versus an Ag-AgCl reference electrode in pH 3.0 Britton-Robinson buffer. The calibration graph was linear from 0.02 to 1300 microg 1(-1). The method was applied to cataract cases and ceftriaxone levels were measured in aqueous humour and serum samples from patients who had received 1 or 2 g of ceftriaxone intravenously. Aqueous humour was added to the polarographic cell directly. The amounts of ceftriaxone in the aqueous humour and serum samples with respect to time were measured. The pharmacokinetic profiles for 1 and 2 g were compared.

Trans- and cis-linalool 3,6-oxide 6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides isolated as aroma precursors from leaves for oolong tea.

New glycosidic aroma precursors (1 and 2) of the main volatile constituents, trans- and cis-linalool 3,6-oxides (linalool oxides I and II), were isolated from oolong tea leaves (Camellia sinensis var. sinensis cv. Maoxie). The isolation was guided by an enzymatic hydrolysis with acetone powder prepared from fresh tea leaves (cv. Yabukita) followed by GC or GC-MS analyses. Chromatographic purification of hot water extracts of the tea leaves on active charcoal, Amberlite XAD-2, and Sephadex LH-20 columns as well as HPLC gave two new glycosides, trans- and cis-linalool 3,6-oxide 6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides (1 and 2).

Characterization of Hemolytic and Antifungal Substance, Cepalycin, from Pseudomonas cepacia

Hemolytic and antifungal substances, cepalycin I and cepalycin II, have been isolated from Pseudomonas cepacia JN106. A large amount of cepalycins were produced by growing the cells on 1% glycerin-nutrient agar medium covered with a cellophane membrane. The cell-washed supernatant was applied to an Amberlite XAD2 column, and cepalycins were eluted with 70% ethanol containing 1mM HCl. Cepalycins were separated by reverse phase HPLC in two fractions which were designated as cepalycin I and cepalycin II. The two cepalycins have indistinguishable UV absorption spectra but have different levels of hemolytic activity relative to the UV absorption. From the inhibition of hemolytic activity of cepalycin by sterols, both cepalycins were suggested to interact with cholesterol in erythrocyte membrane. Such an interaction may contribute to their hemolytic and antifungal activities.

Isolation and characterization of an endogenous Na+,K(+)-ATPase-specific inhibitor from pig urine.

A Na+,K(+)-ATPase inhibitor was purified from 88.6 l pig urine with a yield of approximately 10 micrograms. It inhibits the ouabain-sensitive uptake of 86Rb into human erythrocytes and the specific binding of ouabain to Na+,K(+)-ATPase. It also exhibits cross-reactivity to anti-ouabain serum. The purification procedure consisted of adsorption chromatography on an Amberlite XAD-2 column, preparative scale C18 low-pressure liquid chromatography (LPLC), and five steps of HPLC with five different types of reverse-phase columns. The dose dependence of the purified substance for the inhibition of ouabain-sensitive Na+,K(+)-ATPase activity and 86Rb uptake into human erythrocytes, and for the ouabain-displacing activity, paralleled those of ouabain, spanning two orders of magnitude in concentration range. However, the curve obtained from the cross-immunoreactivity with anti-ouabain serum did not parallel that of ouabain. The inhibitory potencies of the purified substance against the Na(+)-pump and ouabain-binding were diminished with increasing K+ concentration, exhibiting characteristics typical of cardiac glycosides. This substance had no effect on Ca(2+)-ATPase activity in human erythrocyte plasma membrane and skeletal-muscle sarcoplasmic reticulum, nor on Mg(2+)-ATPase activity. Acid treatment with 6 M HCl at 115 degrees C for less than 1 min destroyed approximately 82% of the inhibitory activity of the purified substance against Na(+)-pump activity. Alkaline treatment with 0.2 M NaOH at 23 degrees C for 2 h and heat treatment at 150 degrees C for 30 min partially destroyed the inhibitory activity. Boiling for 10 min and digestion by various enzymes did not affect the activity. Molecular mass was estimated to be 620 Da by gel-filtration column chromatography. Preliminary MS analysis suggested that the purified substance has a molecular mass of 625 Da. An 1H-NMR study revealed that this substance does not contain a tertiary methyl group. The results suggest that the purified Na+,K(+)-ATPase inhibitor is not a peptide and is distinct from any of the known cardiotonic steroids or various substances previously reported to exhibit Na+,K(+)-ATPase inhibitory activity. Thus, the purified substance may be a novel endogenous regulator of Na+,K(+)-ATPase.

Scopolamine release into media by Duboisia leichhardtii hairy root clones

The hairy root clones of Duboisia leichhardtii were found to release scopolamine into the medium. Among media examined, a modified Heller's medium that had 37 mm nitrate and no ammonium was suitable for scopolamine release. Scopolamine in the medium was efficiently recovered by the use of an Amberlite XAD-2 column. A reactor system consisting of a 2-1 airlift reactor and a 25-ml column packed with Amberlite XAD-2 was constructed for production of scopolamine by the culture of the hairy root clone. The culture medium was passed through the column and the eluent from the column was back into the reactor continuously by using a low-pressure pump. When the hairy root clone DL47-1 was cultured in the reactor, 245 mg/l of scopolamine was released into the medium during 6 weeks and 97% scopolamine in the medium was recovered by the column. The scopolamine production was about five times higher in the column-combined reactor than in the reactor without the column. Scopolamine was recovered as the hydrobromide salt with more than 90% purity.