Separation and purification of six isoflavones from Iris tectorum Maxim by macroporous resin-based column chromatography coupled with preparative high-performance liquid chromatography

ABSTRACT

A rapid and robust preparation method of six isoflavones from ethanol aqueous extract of Iris tectorum Maxim (I. tectorum) was established by using macroporous resin column chromatography and preparative high-performance liquid chromatography (Prep-HPLC). After separation by AB-8 resins, total flavonoids content increased from 10.60% in the crude extract to 54.20% with a recovery yield of 75.12%. Subsequently, the extracts were purified by Prep-HPLC, and the purities were more than 82.2% after assessment by analytical HPLC and characterization by mass spectrometry. The established method is expected to be used for preparing available quantities of pure isoflavones from I. tectorum.     

Separation of catalase from Amsonia orientalis with single step by aqueous two-phase partitioning system (ATPS)

Catalase from Amsonia orientalis was purified by ATPS, and its efficiency was compared against hydrophobic interaction chromatography. Activity recovery and purification fold of purified catalase by ATPS were examined under varying experimental conditions. The effects of various factors such as type of phase-forming salts, (PEG) mass, with their different concentrations, pH and temperature effects on partitioning were investigated. The highest activity recovery (156%) and purification fold (8.67) of catalase were obtained in the ATPS system containing 10% (g/g) PEG4000, 15% (g/g) Na₂SO₄ at pH 6.0 and room temperature. In hydrophobic interaction chromatography, the enzyme has been purified 12.54-fold with 57.5% recovery. The molecular weight of catalase was determined as 75 kDa by SDS-PAGE.     

Characterization of lipoxygenase extracts from Penicillum sp.

Biomasses of Penicillium camemberti and P. roqueforti strains were grown and harvested after 10 d of incubation, a period that corresponded to the maximal dry weight of mycelium as well as to lipoxygenase (LOX) activity. Partially purified LOX extracts were obtained by ammonium sulfate precipitation of the crude enzymatic extracts that had been recovered from the biomasses. The partially purified LOX extracts exhibited a preferential specificity toward free fatty acids, including linoleic, linolenic, and arachidonic acids, rather than fatty acid acylglycerols, including mono-, di-, and trilinolein. The K _m and V _max values of LOX activity were investigated. Normal-phase high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry analyses showed that the LOX activity of the microbial extracts converted linoleic acid mainly into the corresponding 9- and 13-hydroperoxides (HPOD). However, the production of a significant proportion of 10-HPOD, ranging from 4 to 9% of the total HPOD, was also demonstrated. In addition, the enantiomeric ratios of the 9- and 13-HPOD produced were determined at both pH optima by chiral-phase HPLC. The results indicated that an almost racemic mixture had been obtained which can be related to either low enantioselectivity of LOX or the presence of isozymes showing complementary enantioselectivity.     

Optimization of conditions for single-step purification of recombinant hepatitis B surface antigen produced in Pichia pastoris using ion exchange chromatography

ABSTRACT

The adsorption and release of rHBsAg extracted from the final dosage form on various ion exchange resins and under different pH conditions were investigated after its peptide map and isoelectric point (PI) determination. Efficient antigen adsorption to the anion exchange resins occurred when the pH value of the protein buffer was adjusted to 5.0. In purification of rHBsAg derived from the yeast crude extract using Q Sepharose FF column, with adjusting the pH value of the crude extract to 5.0 (i.e., near to the target protein PI) and using 2M NaCl, rHBsAg with high purity (up to >95%) was obtained.

Abbreviations: rHBsAg, recombinant hepatitis B surface antigen; Alhydrogel, aluminum hydroxide; IEF, isoelectric focusing; PI, isoelectric point; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RP-HPLC, reversed-phase high-performance liquid chromatography; SE-HPLC, size-exclusion high-performance liquid chromatography; PBS, phosphate-buffered saline     

Characteristics of the thiobarbituric acid reactivity of human urine as a possible consequence of lipid peroxidation

A 532 nm red pigment formed in the thiobarbituric acid (TBA) assay of human urine was characterized after separation of the pigment by high-performance liquid chromatography. The yield of the red pigment was some-what higher at pH 2 than at pH 5; its development was not inhibited by ethylenediaminetetraacetic acid. The characteristics of the pigment were similar to those of the pigment derived from standard malonaldehyde. The amount of the pigment formed was roughly equal to the content of malonaldehyde derivatives estimated as 1-(2,4-dinitrophenyl)pyrazole. Pigment formation was significantly enhanced byt-butyl hydroperoxide (t-BuOOH) and ferric ions, which may be due to pigment formed from aldehydes other than malonaldehyde; the presence of these aldehydes was confirmed by the formation of the corresponding 2,4-dinitrophenylhydrazones. The amount of pigment produced from 24-h urine samples of 12 healthy subjects was estimated to be 26–95 nmol/kg, and 65–182 nmol/kg in the presence oft-BuOOH. These values are lower than those for urine of rabbit or rat. The TBA reactivity in the absence and presence oft-BuOOH of human urine was not related to age or sex. The TBA reactivity of human urine collected in the afternoon and in the evening was higher than that of urine collected in the morning.     

A novel process for scopolamine separation from Hindu Datura extracts by liquid–liquid extraction,macroporous resins,and crystallization

ABSTRACT

An efficient separation method of scopolamine from Hindu Datura extracts was successfully developed by combining liquid–liquid extraction, macroporous resins, and crystallization. First, the extraction solution was successively performed with liquid–liquid extraction by acid water and carbon tetrachloride. Secondly, D151 resin was selected from six tested resins, and the separation parameters were optimized. Finally, scopolamine hydrobromide was obtained after crystallization at ?20°C overnight. Through one run of above combined treatments, the content of scopolamine increased from 3.02% (w/w) to 99.12% (w/w). In addition, the products were assessed by high-performance liquid chromatography (HPLC).     

Determination of the conjugated linoleic acid-containing triacylglycerols in New Zealand bovine milk fat

Reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection at 233 nm was used to separate, quantify, and identify the triacylglycerols (TAG) of milk fat that contain conjugated linoleic acid (CLA). The absorbance at 233 nm was substantially due to CLA-TAG (chromatography of some representative TAG devoid of CLA, such as tripalmitin and triolein, showed poor responses at 233 nm, 1/800th that of CLA-TAG). A CLA molar extinction coefficient at 233 nm of 23 360 L mol⁻¹ cm⁻¹ and an HPLC UV response factor were obtained from a commercially available cis-9, trans-11-CLA standard. This molar extinction coefficient was only 86% reported literature values. Summation of all chromatographic peaks absorbing at 233 nm using the corrected response factor gave good agreement with independent determinations of total CLA by gas chromatography and UV spectrophotometry. This agreement allowed quantification of individual CLA-TAG peaks in the HPLC separation of a typical New Zealand bovine milk fat. Three CLA-containing TAG, CLA-dipalmitin, CLA-oleoyl-palmitin and CLA-diolein, were prepared by interesterification of tripalmitin with the respective fatty acid methyl esters and used to assign individual peaks in the reversed-phase chromatography of total milk fat, of which CLA-oleoyl-palmitin was coincident with the largest UV peak. Band fractions from argentation thin-layer chromatography of total milk fat were similarly employed to identify five predominant CLA-TAG groups in total milk fat: CLA-disaturates, CLA-oleoyl-saturates, CLA-vaccenyl-saturates, CLA-vaccenyl-olein, and CLA-diolein.     

Synthesis and Chromatographic Separation of Monomethoxypolyethylene Glycol Modified Insulin

Abstract

Insulin was modified with monomethoxypolyethylene glycol (MPEG)‐succinimidyl succinate and succinimidyl ester of carboxymethyl MPEG. Effects of reaction solvents, initial molar ratio of MPEG derivative to insulin and reaction time on PEGylation of insulin were investigated by 2,4,6‐trinitrobenzenesulfonic acid spectrophotometric assay and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Sephadex G75 size exclusion chromatography (SEC), ion exchange chromatography (IEC) and reversed phase‐high performance liquid chromatography (RP‐HPLC) were applied to separate PEGylated insulin. IEC and RP‐HPLC were proved to be efficient tools on separation of different PEGylated insulin species.     

Rapid screening and separating two radical scavengers in Lycium barbarum L. by DPPH-HPLC analysis-combined dual-mode high-speed countercurrent chromatography

A target-guidance separation strategy composed of activity screening process with high-performance liquid chromatography and separating process with high-speed countercurrent chromatography (HSCCC) has been developed and used to screen and separate radical scavengers from Lycium barbarum extract. Two compounds were found to be radical scavengers and dual-mode HSCCC was used to separate these two defined compounds due to the big gap in polarity between them. Rutin and quercetin were separated with purities of 96.5% and 95.0%. Their structures were identified by nuclear magnetic resonance and both of them exhibit potent radical scavenging activity with the IC₅₀ values being 20.07 ± 0.10 and 3.11 ± 0.03 μg/mL, respectively.     

Characterization of triacylglycerols in the seeds ofAquilegia vulgaris by chromatographic and mass spectrometric methods

A combination of analytical techniques is generally necessary to properly characterize complex lipid materials. Chromatographic separation in conjunction with spectroscopic characterization was utilized for the analysis of the triacylglycerols in the seeds ofAquilegia vulgaris. Reversed-phase high-performance liquid chromatography (HPLC), micropacked argentation supercritical fluid chromatography (SFC), and combinations of the two techniques were used. The fatty acid profile was determined by gas chromatography/mass spectrometry of the picolinyl esters and by gas chromatography/flame-ionization detection of the methyl esters. The major components were also identified by direct inlet mass spectrometry. The excellent selectivity of packed fused silica argentation SFC for the separation of triacylglycerols was demonstrated.     

Silver ion high-performance liquid chromatography of the triacylglycerols ofCrepis alpina seed oil

The triacylglycerols ofCrepis alpina oil were characterized because this oil has a high concentration of crepenynic (cis-9-octadecen-12-ynoic) acid, a fatty acid useful in the chemical synthesis of deuterated fats for human metabolism studies. The triacylglycerols were separated from the crude oil by solid-phase extraction. Resolution, quantitation and isolation of the individual triacylglycerols were performed by silver ion high-performance liquid chromatography on a commercial column, an acetonitrile in hexane isocratic mobile phase and flame-ionization detection. Isolated triacylglycerols were identified by capillary gas chromatography of their fatty acid methyl esters. Of the eleven eluted triacylglycerols ofCrepis alpina oil, 85% included 35% tricrepenynoyl, 34% linoleoyldicrepenynoyl and 16% dilinoleoylcrepenynoyl glycerols. Triacylglycerols eluted according to the numbers of alkene and alkyne bonds. Elution times, resolution and quantitation were reproducible over a three-month period. The flame-ionization detector response required no response factors for quantitation of the triacylglycerols present inCrepis alpina oil. The silver ion chromatography system permitted the identification of 95% of the triacylglycerols compared to 70% of the triacylglycerols previously identified with reversed-phase high-performance liquid chromatography.     

Comparison of analyses of surfactants in cosmetics using high-performance liquid chromatography and high-performance capillary electrophoresis

Separation of anionic, cationic, and amphoteric surfactants containing n-dodecyl groups and hydrophilic moieties was done by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE), and an ultraviolet-visible detector. Quantitation of surfactants in commercial cosmetic and toiletry products was also done using similar methods. Conditions used to separate mixtures of surfactants by HPLC are as follows: stationary phase: ODS 2; mobile phase: MeOH/H₂O (80∶20, vol/vol) containing 1.0M NH₄Cl, 0.003 M tetrabutylammonium hydrogen sulfate, and 0.005 M diammonium phosphate buffer solution at pH=6.0. Conditions for HPCE are as follows: an uncoated capillary (100 μm i.d., 110 cm in length, effective length of 75 cm) with 25 kV of applied voltage, and an aqueous buffer containing 80 mM sodium borate/20 mM NaOH at pH=9.2. Surfactants were eluted within 15 min. The accuracy of both techniques was evaluated by analyzing the recovery ratio of surfactants in the mixture.     

Profiling and Qualitative Assessment of Enzymatically and Thermally Oxidized Egg Yolk Phospholipids using a Two-Step High-Performance Liquid Chromatography Protocol

A two-step high-performance liquid chromatography (HPLC) method for the profiling and qualitative assessment of oxidized phospholipids (oxPL) present in foods was developed. The applicability of the investigated two-step HPLC protocol was verified for separation of enzymatically and thermally oxidized hen egg yolk phospholipids (PL) as a relevant food model. In the first step, seven individual PL classes were separated using hydrophilic interaction liquid chromatography (HILIC). The collected fractions of two main egg yolk PL classes—phosphatidylethanolamines and phosphatidylcholines—were further directed to the second step of separation aimed at profiling and qualitative assessment of their oxidized species. For this purpose, the reverse phase (RP) chromatography coupled to charged aerosol, ultraviolet detection (UV) and mass spectrometry detection were employed. A database of potential oxPL including primary (hydroperoxides) and long-chain secondary PL oxidation products (epoxides, alcohols, and ketones) as well as some of their possible combinations was created. Additionally, the results were compared with the profiles of PL hydroperoxides obtained using thin layer chromatography (TLC) with N,N-dimethyl-p-phenylenediamine visualization.     

Autotoxic Compounds from Fresh Alfalfa Leaf Extracts: Identification and Biological Activity

Many investigators have attempted to identify the allelochemicals in alfalfa (Medicago sativa), that cause autotoxicity. The autotoxic compounds from fresh alfalfa leaves were separated and quantified, and their biological activity was determined. Chemical separation procedures involved an 80% methanol extract of fresh alfalfa leaves, treatment with activated charcoal, microcrystalline cellulose thin-layer chromatography (MCTLC), and finally separation by Sephadex LH-20 column chromatography. The various fractions were examined further by high-performance liquid chromatography (HPLC). Preliminary identification by HPLC analysis resulted in peaks with retention times close to those of chlorogenic (m/z = 354) and salicylic acid (m/z = 138) standards, and these compounds were confirmed with GC-MS. Several other peaks remain unidentified. Chlorogenic acid occurs in relatively large amounts (0.39 mg/g) in alfalfa aqueous extracts as compared to salicylic acid (0.03 mg/g), and bioassays suggest that chlorogenic acid is involved in alfalfa autotoxicity.     

Reinvestigation of positional distribution of fatty acids in docosahexaenoic acid-rich fish oil triacyl-sn-glycerols

Positional distribution of fatty acids in triacyl-sn-glycerols of docosahexaenoic acid (DHA)-rich tuna orbital and bonito head oils has been reanalyzed by a method based on chromatographic separation of isomeric and enantiomeric monoacyl-sn-glycerol (MAG) derivatives. When boric acid thinlayer chromatography (TLC) was used for separation of 1(3)- and 2-MAG analytical intermediates, the stereospecific analysis showed the preferential association of DHA to the sn-2 position followed by the sn-3 position. This distribution pattern differed from that obtained by silicic acid LTC of their bis-3,5-dinitrophenylurethane (DNPU) derivatives. Reversed-phase high-performance liquid chromatography elution profiles of 1(3)- and 2-MAG intermediates revealed that 1(3)- and 2-MAG made up of both short- and long-chain lengths cannot be clearly resolved by TLC after preparation of the DNPU derivatives. The 1(3)- and 2-MAG must be resolved by boric acid TLC prior to derivatization.     

Rapid Screening,Identification, Separation,and Purification of Four Bioactive Compounds from Swertia mussotii Franch

Gentiopicroside, mangiferin, sweroside, and isoorientin, the bioactive constituents of Swertia mussotii Franch, have various pharmacological effects, and are used in particular for treating liver disorders. However, efficient methods for their separation are not currently available. In this study, these bioactive compounds were detected in S. mussotii extracts using high-performance liquid chromatography coupled with mass spectrometry, and separated using a combination of high-speed counter-current chromatography and Sephadex LH-20 column chromatography. Their structures were determined by using ¹H and ¹³C nuclear magnetic resonance spectroscopies. The results show that this method is effective for the separation and purification of bioactive compounds from S. mussotii.     

Preparation of malvalic and sterculic acid methyl esters from Bombax munguba and Sterculia foetida seed oils

A method has been developed for the preparation of highly pure malvalic (cis-8,9-methyleneheptadec-8-enoic) and sterculic (cis-9,10-methyleneoctadec-9-enoic) acid methyl esters starting from Bombax munguba and Sterculia foetida seed oils. The methyl esters of these oils were prepared by sodium methylate-catalyzed transmethylation followed by cooling (6°C) the hexane solution of crude methyl esters and separation of insoluble fatty acid methyl esters by centrifugation in the case of B. munguba and by column chromatography in the case of S. foetida. Subsequently, the saturated straight-chain fatty acid methyl esters were almost quantitatively removed by urea adduct formation. Finally, methyl malvalate and methyl sterculate were separated from the remaining unsaturated fatty acid methyl esters, in particular methyl oleate and methyl linoleate, by preparative high-performance liquid chromatography on C₁₈ reversed-phase using acetonitrile isocratically. Methyl malvalate and methyl sterculate were obtained with purities of 95–97 and 95–98%, respectively.     

Purification and characterization of antioxidative peptides from protein hydrolysate of lecithin-free egg yolk

The protein extracted from lecithin-free egg yolk, normally discarded by lecithin processing plants, was hydrolyzed with the aid of Alcalase, a commercial enzyme. The hydrolysate was separated through a series of ultrafiltration membranes with molecular weight cutoffs of 10, 5, and 1 kDa; and three types of permeates including 10 K (permeate from 10 kDa), 5 K (permeate from 5 kDa), and 1 K (permeate from 1 kDa) were obtained. The antioxidative efficacy of hydrolysates so obtained was investigated and compared with α-tocopherol. Furthermore, two different peptides showing strong antioxidative activity were isolated from the hydrolysates by using consecutive chromatographic methods including ion exchange chromatography on a SP-Sephadex C-25 column, gel filtration on a Sephadex G-25 column, and high-performance liquid chromatography on an octadecylsilane column. The purity of the peptides was identified using capillary electrophoresis. The isolated peptides were composed of 10 and 15 amino acid residues, and both contained a leucine residue at their N-terminal positions.     

Determination of sorbitan tristearate in vegetable oils and fats

A new method for analysis of Sorbitan Tristearate (STS) in vegetable oils and fats has been developed. The method is based on isolation and hydrolysis of STS compounds in a silica cartridge. The polyalcohols are eluted from the silica cartridge and the final separation and quantitation are done by high-performance liquid chromatography and refractive index detection. Linearity, precision, and recovery satisfy general demands on quantitative methods. The detection limit and the quantitation limit are well below the concentrations normally used to attain functional effects of STS in vegetable oils and fats.     

Enantiomeric resolution of diacylglycerol derivatives by high-performance liquid chromatography on a chiral stationary phase at low temperatures

Separation of 1,2-diacylglycerol (DG) enantiomers as their 3,5-dinitrophenylurethane derivatives by high-performance liquid chromatography on a chiral column (Sumichiral OA-4100) was significantly improved at low temperature,e.g., at −30°C. A linear relationship between logarithmic retention volumes and the number of carbons and olefinic bonds of the DG enantiomers was obtained. The effects of temperature on the separation are discussed.