Liquid-liquid/solid three-phase high-speed counter-current chromatography, a new technique for separation of polyphenols from Geranium wilfordii Maxim.

High-speed counter-current chromatography using a new liquid-liquid/solid three-phase system was used for the separation of the polyphenols corilagin and geraniin from a crude extract of Geranium wilfordii Maxim in one step. The optimized three-phase system was composed of n-hexane/ethyl acetate/methanol/acetic acid/water and to which was added 10-μm average diameter microspheres of cross-linked 12% agarose at the ratio of 0.2:10:2:1:5 and 0.

Separation of polyphenols using porous polyamide resin and assessment of mechanism of retention.

A porous polyamide resin is shown to possess hydrogen bond acceptor properties suitable for the separation of polyphenolic solutes such as phenolic acids, flavonols and flavonoids. The separation is achieved in the presence of solvent mixtures of acetic acid and ethanol. The extent of hydrogen bond adsorption is reviewed based on data obtained from the elution behaviour of a variety of simple polyphenolic solutes.

One-step separation and purification of hydrolysable tannins from Geranium wilfordii Maxim by adsorption chromatography on cross-linked 12% agarose gel.

The hydrolysable tannins corilagin and geraniin, the major active components of the traditional Chinese medicine Geranium wilfordii Maxim, have been separated and purified from crude extracts in one step by adsorption chromatography on cross-linked 12% agarose gel (Superose 12 10/300 GL). The separation was achieved by gradient elution using mobile phase A composed of 5% ethanol and 5% acetic acid and mobile phase B composed of 30% ethanol and 30% acetic acid. The gradients were composed as follows: 0-240 mL, 0-25% B; 240-480 mL, 25-40% B; after 480 mL, 100% B.

Cross-linked agarose for separation of low molecular weight natural products in hydrophilic interaction liquid chromatography.

Following its market introduction in 1982, the cross-linked 12% agarose gel media Superose 12 has become widely known as a tool for size exclusion chromatography of proteins and other biological macromolecules. In this review it is shown that, when appropriate mobile phases are used, Superose possesses adsorption properties similar to that of traditional media for hydrophilic interaction liquid chromatography (HILIC). This is illustrated by the separation and purification of low molecular weight compounds such as polyphenols including active components of traditional Chinese medicinal herbs and green tea.

Investigation of the adsorption behavior of glycine peptides on 12% cross-linked agarose gel media.

The highly cross-linked 12% agarose gel Superose 12 10/300 GL causes retardation of glycine peptides when mobile phases containing varying concentrations of acetonitrile in water are used. An investigation has been made into the retention mechanism behind this retardation using the glycine dipeptide (GG) and tripeptide (GGG) as models. The dependence of retention times of analytical-size peaks under different experimental conditions was interpreted such that the adsorption most probably was caused by the formation of hydrogen bonds but that electrostatic interactions cannot be ruled out.

One-step rapid determination and purification of puerarin from Radix puerariae by n-octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monolith.

n-Octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monoliths were prepared for rapid screening, determination and one-step purification of puerarin from Radix puerariae (a crude extract of the root of Pueraria lobata). The modified monolith showed a specific surface area of 17.8 m(2) g(-1), an average pore size of 0.

One-step purification of epigallocatechin gallate from crude green tea extracts by mixed-mode adsorption chromatography on highly cross-linked agarose media.

(-)-Epigallocatechin gallate (EGCG) was purified in one step from a green tea polyphenol (GTP) crude extract by adsorption chromatography on a Superose 12 HR 10/30 column. The mobile phase used was a mixture of acetonitrile and water with an optimum mobile phase compositions regarding purity, recovery and separation time of 78/22 (v/v). Maximum practical sample loading was 100 mg GTP per run (corresponding to 4.

NMR studies on the interaction between (-)-epigallocatechin gallate and cyclodextrins, free and bonded to silica gels.

The interaction between (-)-epigallocatechin-3-gallate (EGCG) and beta- or gamma-cyclodextrin (CD), in free solution and bonded to silica beads, has been studied by (1)H HR-MAS NMR spectroscopy. The chromatographic retardation of EGCG on columns packed with CD-silica beads was shown to be due to the interaction of EGCG with the CD ligands because no nonspecific interaction with the silica gel could be observed. EGCG forms a tighter complex with beta-CD than with gamma-CD and NMR data obtained from hydroxy protons together with MM2 calculations suggest that for beta-CD intermolecular hydrogen bonding, in addition to hydrophobic interaction, stabilizes the complex.

Aryl ketones from Acronychia pedunculata with cyclooxygenase-2 inhibitory effects.

1-[2,4-Dihydroxy-6-methoxy-3,5-bis(3-methylbut-2-en-1-yl)phenyl]ethanone (1), and a new aryl ketone, named acrovestenol (2), were isolated as cyclooxygenase-2 (COX-2) inhibitory principles from a CH2Cl2 extract of the bark of Acronychia pedunculata by a bioassay-guided fractionation procedure. Compound 2 inhibited COX-2 with an IC50 value of 142.0+/-2.

Assessment of 4-nitro-1,8-naphthalic anhydride reductase activity in homogenates of bakers' yeast by reversed-phase high-performance liquid chromatography.

A simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed for the simultaneous determination of yield and conversion ratio of 4-nitro-1,8-naphthalic anhydride to 4-amino-1,8-naphthalic anhydride following incubation with a crude bakers' yeast homogenate. The analytes were separated on a C18 column in gradient mode. The detection limit of 4-amino-1,8-naphthalic anhydride is 10ng/microl when using a 10microl sample injection volume.

Mixed-mode retention mechanism for (-)-epigallocatechin gallate on a 12% cross-linked agarose gel media.

The adsorption behaviour of (-)-epigallocatechin gallate (EGCG), the major polyphenolic substance in green tea extracts, on the cross-linked agarose gel Superose 12 HR 10/30, has been studied using a variety of solvent systems and shown to be based on a mixture of hydrogen bonding and hydrophobic interaction. The hydrogen bonding was studied in acetonitrile in the presence of different co-solvents possessing varying hydrogen bond donor (HBD) and/or hydrogen bond acceptor (HBA) characteristics. The HBA-value of the co-solvent had the highest effect whereas the HBD-value played a subordinate role.

Molecular characterization of recombinant Hepatitis B surface antigen from Chinese hamster ovary and Hansenula polymorpha cells by high-performance size exclusion chromatography and multi-angle laser light scattering.

The molecular weight and size of recombinant Hepatitis B surface antigen (HBsAg) derived from Chinese hamster ovary (CHO) and the Hansenula polymorph have been characterized by high-performance size exclusion chromatography with multi-angle laser light scattering (HPSEC-MALLS). The average molecular weight of CHO-derived HBsAg particle (CHO-rHBsAg) (4921 kDa) was higher than that of H. polymorpha yeast strain (Hans-rHBsAg) (3010 kDa).

Three-dimensional crystal structure and enzymic characterization of beta-mannanase Man5A from blue mussel Mytilus edulis.

Endo-beta-1,4-d-mannanase is the key depolymerizing enzyme for beta-1,4-mannan polymers present in the cell walls of plants and some algae, as well as in some types of plant seeds. Endo-1,4-beta-mannanase from blue mussel Mytilus edulis (MeMan5A) belongs to the glycoside hydrolase (GH) family 5 enzymes. The MeMan5A structure has been determined to 1.

Size characterization of green fluorescent protein inclusion bodies in E. coli using asymmetrical flow field-flow fractionation-multi-angle light scattering.

The goal of this study was to investigate the applicability of asymmetrical flow field-flow fractionation-multi-angle light scattering (AsFlFFF-MALS) for size analysis of green fluorescent protein inclusion bodies (GFPIBs). The size distributions of GFPIBs prepared by various culture conditions were determined. For GFPIBs prepared at 37 degrees C the peak maximum hydrodynamic diameter (d(H)) first increased and then decreased with the increase of the induction times in the presence of 0.

Ion-exchange chromatography of hepatitis B virus surface antigen from a recombinant Chinese hamster ovary cell line.

About 10% of the Chinese population are chronic carriers of hepatitis B virus (HBV). Thus, the development of a highly efficient process for the preparation of a vaccine based on a recombinant hepatitis B surface antigen (HBsAg) is very important to the Chinese national immunization program. To this end, the ion exchange chromatography recovery of CHO-HBsAg from a recombinant Chinese hamster ovary cell line was shown to increase from about 55 to 80% by the addition of 1% poly(ethylene glycol) (PEG 10,000) to the mobile phase.

One-step purification of epigallocatechin gallate from crude green tea extracts by isocratic hydrogen bond adsorption chromatography on beta-cyclodextrin substituted agarose gel media.

An oligomerized beta-cyclodextrin ligand coupled to brominated allyl-group substituted Sepharose HP has been used for the one-step purification of polyphenolic epigallocatechin gallate (EGCG), an important antioxidant, by isocratic hydrogen bond adsorption chromatography. With a sample load of 1.33 mg crude green tea polyphenolic extract per ml column packing and with water/ethanol/acetonitrile (57/30/13, v/v) as the optimum mobile phase, an EGCG purity of about 98% with a recovery of approximate 73% could be achieved by proper peak cutting.

Immobilized beta-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle.

A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized beta-cyclodextrin (beta-CD) polymer coupled to an agarose gel, was introduced to refold recombinant Staphylococcus aureus elongation factor-G (EF-G). Pre-mixing of 10% Triton X-100 and unfolded EF-G at 24 mg/ml followed by a 20-fold dilution into refolding buffer led to successful capturing of EF-G by Triton X-100 resulting in formation of a detergent-protein complex at 1.2mg/ml of final protein concentration.

A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G.

Recombinant Staphylococcus aureus elongation factor G (EF-G) is difficult to refold by dilution due to the formation of large amounts of misfolded structures. However, refolding of EF-G by adsorption to a chromatographic column packed with immobilized polyethylene glycol 20,000 (PEG 20 K) followed by pulse elution with 8 M urea resulted in 88% mass recovery and 80% of correctly refolded structure. The PEG 20 K was coupled to brominated allyl group derivatized Sepharose High Performance to construct a mild hydrophobic adsorbent.

Purification of the isoflavonoid puerarin by adsorption chromatography on cross-linked 12% agarose.

The isoflavonoid puerarin in extracts of the well-known traditional Chinese drug Radix puerariae (root of the plant Pueraria lobata) can be separated from other isoflavonoids by adsorption chromatography on the cross-linked 12% agarose gel Superose 12 equilibrated in distilled water. The adsorption is totally quenched by the addition of 50% acetic acid. The separation of the isoflavonoids is tentatively ascribed to interaction with the residues of the cross-linking reagents used in the manufacturing process of Superose 12.

Isolation of high purity 1-[2',4'-dihydroxy-3',5'-di-(3"-methylbut-2"-enyl)-6'-methoxy] phenylethanone from Acronychia pedunculata (L.) Miq. by high-speed counter-current chromatography.

Following an initial clean-up step on silica, high-speed counter-current chromatography (HSCCC) was used to purify an aryl ketone, 1-[2',4'-dihydroxy-3',5'-di-(3"-methylbut-2"-enyl)-6'-methoxy] phenylethanone from an extract of the stem bark of the shrub Acronychia pedunculata. The two-phase solvent system used was composed of n-heptane-ethyl acetate-methanol-water at an optimized volume ratio of 4:1:4:1 (v/v/v/v). Target compound (58.

Separation and purification of puerarin using beta-cyclodextrin-coupled agarose gel media.

The isoflavonoid puerarin, a well-known traditional Chinese drug, has been purified in one step from an extract of Radix puerariae (root of the plant Pueralria lobata) by adsorption chromatography on an epichlorohydrin polymerized beta-cyclodextrin ligand coupled to brominated allyl-group substituted Sepharose HP. Acetic acid (10%) was used as the mobile phase and the optimum loading capacity was around 1.2 mg crude extract/ml packed gel.