Purification of rat pro-atrial natriuretic factor: a simplified scheme using reversed-phase high-performance liquid chromatography

A simple scheme for the rapid and efficient isolation of rat pro-atrial natriuretic factor (pro-ANF) has been developed. An isolated rat adrenal cell bioassay for ANF was established to optimize heart tissue extraction and chromatography conditions. This assay is based on the ability of ANF to inhibit angiotensin II-stimulated aldosterone secretion. IC50 values for ANF were approximately 320 pM. The protocol that was established consisted of extraction of rat atria in 5 N acetic acid containing protease inhibitors. The extract was lyophilized, resolubilized in 0.1% trifluoroacetic acid containing 1% (w/v) sodium chloride, and subjected to RP-HPLC. Extraction of small batches of atria (i.e., from 10 or 20 rats) resulted generally in a yield of 2 nmol per rat (i.e., approximately 30 micrograms). The identity and purity of the pro-ANF were confirmed by the determination of both the amino acid composition and the amino-terminal sequence. Purified pro-ANF was radioiodinated and the efficiency of the extraction and purification procedure was assessed by adding labeled peptide to the initial tissue extract. The structural integrity and overall recovery of radioactivity were determined by RP-HPLC. The purification scheme provides undamaged pro-ANF of high purity. Purified pro-ANF was compared with synthetic rat ANF in the rat adrenal glomerulosa cell and isolated rat aortic strip bioassays. The peptides were apparently equally active in the adrenal cell system and approximately threefold less potent in relaxing aortic strips. The apparent equipotency in the adrenal cell bioassay may be due to the conversion of pro-ANF to ANF-like peptides during the bioassay incubation.     

Purification of recombinant rotavirus VP7 glycoprotein for the study of in vitro rotavirus-like particles assembly

Rotavirus VP7 is a glycoprotein that forms the viral capsid outerlayer and is essential to the correct assembly of triple-layered rotavirus-like particles (RLPs). In this work, a novel purification strategy was designed to allow obtaining highly pure monomeric VP7 required for the RLPs in vitro assembly. VP7 production kinetics in baculovirus-insect cells at cell concentration at infection (CCI) of 1x10(6)cellsmL(-1) was compared in terms of VP7/glycoprotein 64 (gp64) ratio at different multiplicity of infection (MOI). The best productivity was achieved at MOI of 0.1plaque forming unit (pfu)cell(-1) and time of harvest of 80h post-infection. After preliminary clarification steps, the proteins eluted from Concanavalin A were concentrated and loaded onto size exclusion chromatography. The polishing step was anion exchange chromatography with Mono Q. The high resolution of this column resulted in separation of monomers from dimers of VP7. Overall, the purification protocol yielded high level of purity (>90%). Purified VP7 was characterized by MALDI-TOF mass spectrometry and SDS-capillary gel electrophoresis. The MW and apparent MW were determined as 31.6 and 39kDa, respectively, confirming the efficacy of the proposed purification strategy that now enables RLPs assembly studies.     

The receptor for the basement membrane glycoprotein entactin is the integrin alpha 3/beta 1.

Entactin is a glycoprotein found in basement membranes in complex with laminin, and purified entactin can promote the attachment and spreading of cells. We report here the isolation and identification of the plasma membrane receptor for entactin from PC-3 human prostate carcinoma cells which attach and spread on entactin. The receptor was isolated by affinity chromatography on mouse recombinant entactin-Sepharose of 125I surface-labeled octyl glucoside cell extracts. The receptor, which consisted of two polypeptides of relative molecular masses of 150 and 116 kDa, bound to the entactin-Sepharose matrix in the presence of CaCl2, MgCl2, and MnCl2, and was eluted with EDTA, but not with Arg-Gly-Asp-containing peptides. Utilizing anti-integrin antibodies, the heterodimeric receptor was identified as the integrin alpha 3 beta 1. Purified alpha 3 beta 1 bound to entactin Sepharose in a divalent cation-dependent manner and liposomes prepared with fractions eluted from the entactin-Sepharose matrix, as well as purified alpha 3 beta 1 also bound to entactin. Liposomes prepared with other integrins such as alpha 2 beta 1 did not bind to entactin. Antibody inhibition assays demonstrated that an anti-alpha 3 antibody (P1B5) inhibited the attachment of PC-3 cells to entactin whereas this antibody did not inhibit the attachment of these cells to laminin. Attachment to laminin was, however, blocked by anti-alpha 6 antibody (G0H3). These data demonstrate that the cell surface receptor for entactin on these prostate carcinoma cells is the integrin alpha 3 beta 1 and that these cells utilize alpha 6 beta 1 as the receptor for laminin.     

Use of microbore high-performance liquid chromatography for purifying subnanomole levels of polypeptides for microsequencing. Structural studies on the murine plasma cell antigen PC-1

A procedure for the purification of subnanomole levels of polypeptides has been developed. Reversed-phase high performance liquid chromatography on short (10 cm or less) microbore (1-2 mm internal diameter) columns has been used to fractionate and purify a number of tryptic peptides generated from approximately 600 pmol of purified murine plasma cell antigen PC-1, a major membrane glycoprotein on all cells secreting immunoglobulins. The use of reversed-phase microbore columns permits the recovery of subnanomole amounts of polypeptides from large volumes in high yield (greater than 90%) and in small eluent volumes (40-60 microL) which can be loaded directly onto the gas-phase sequencer without further concentration. This procedure avoids the severe sample loss which frequently occurs with other concentration procedures such as lyophilization and evaporation. The use of a photodiode-array detector for identifying tryptophan-containing peptides from on-the-fly, ultraviolet spectra is described. This procedure permits the selection of tryptophan-containing peptides from complex tryptic digests for use as candidate peptides for oligonucleotide probe construction. Automated Edman degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11% of the molecule.     

Solid-phase synthesis and biological activities of gastrointestinal hormones: Secretin and motilin

Many successful solid-phase syntheses of peptide chains in the region of 20–40 amino acid residues have now been routinely reported. Utilizing standard solid-phase synthetic methodologies but, particularly, new and powerful purification techniques we have been developing rapid and efficient preparative routes for the numerous neuro-gastrointestinal peptides. In the present study, secretin and motilin were obtained in 16% and 10% yields, respectively, after simplified two-step purification of hydrogen fluoride-cleaved peptides by gel filtration followed by preparative high performance liquid chromatography. Peptides were essentially homogeneous by TLC and analytical high performance liquid chromatography. Secretin was found to have full biological activity when tested against a standard sample of natural material for effects on pancreatic secretion in the dog. Motilin exhibited full biological activity on interdigestive motility in the dog. Secretin has been reported to undergo rearrangement with loss of bioactivity during purification and prolonged storage. We observed no obvious problems during our abbreviated purification schedule and have found no loss of purity of peptide which has been kept for 6 months as powder lyophilized from dilute acetic acid.     

Receptors on phaeochromocytoma cells for two members of the PP-fold family--NPY and PP

Pancreatic polypeptide (PP) and neuropeptide Y (NPY) belong to a family of regulatory peptides which hold a distinct tertiary structure, the PP-fold, even in dilute aqueous solution. High-affinity receptors, specific for both PP and NPY, are described on the rat phaeochromocytoma cell line, PC-12. The binding of [125I-Tyr36]PP to PC-12 cells was inhibited by concentrations of unlabeled PP which correspond to physiological concentrations of the hormone, 10(-11)-10(-9) mol/l. The affinity of the receptor for the neuropeptide, NPY, was 10(2)-times lower than that of the PP receptor. C-terminal fragments of both PP (PP24-36) and NPY (NPY13-36) were between 10(2)- and 10(3)-times less potent in displacing the radiolabeled 36-amino-acid peptides from their respective receptors. It is concluded that PC-12 cells are suited for structure-function studies of the PP-fold peptides and studies on the cellular events following cellular binding of PP-fold peptides.     

Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures : I. Induction and Purification

l-Tyrosine decarboxylase (EC 4.1.1.25) activity was induced in cell suspension cultures of Thalictrum rugosum Ait. and Eschscholtzia californica Cham. with a yeast polysaccharide preparation (elicitor). The highest l-tyrosine decarboxylase activity in extracts from 7-day-old cell cultures of E. californica was observed 5 hours after addition of 30 to 40 micrograms elicitor per gram cell fresh weight. The enzyme extracted from cells of E. californica was purified 1540-fold to a specific activity of 2.6 micromoles CO(2) produced per minute per milligram protein at pH 8.4 and 30 degrees C. Purified enzyme from T. rugosum showed a specific activity of 0.18 micromoles per minute per milligram protein. The purification procedure involved ammonium sulfate fractionation, anion-exchange fast protein liquid chromatography, ultrafiltration, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme from the two plant cell cultures had subunits of identical molecular weight (56,300 +/- 300 daltons.     

Human lung mast cells: purification and characterization

Detailed studies of the biochemistry and pharmacology of mast cell-mediated inflammatory disorders have been hampered by the inability to purify human mast cells. We now report techniques to purify human lung mast cells to apparent homogeneity. The major purification steps are: 1) dispersion of lung fragments into a single-cell suspension with enzyme combinations (pronase-chymopapain, collagenase-elastase); 2) partial purification by countercurrent centrifugation elutriation (CCE); and 3) affinity column chromatography. Enzymatic dispersion yielded suspensions with congruent to 10(6) mast cells per gram of lung parenchyma in purities of 1.2 to 9.7%. Dispersed mast cells responded comparably to those in parent lung fragments to challenge with anti-human IgG and pharmacologic agonists. Elutriation of lung cell suspensions yielded mast cell-enriched fractions with purities up to 70%. High purity mast cell fractions were combined, passively sensitized with purified human penicillin (BPO)-specific IgE, and purified by a BPO-affinity column chromatography procedure. Post elutriation mast cell purities of 29 +/- 3.5% were increased to 84 +/- 3% (range 65 to 98%) by the affinity column. Short-term (24 hr) culture of column-purified mast cells allowed adherence of non-mast cell contaminants to tissue culture plates, further increasing purity (up to 100%). Purified mast cells were intact and functional as assessed by dye exclusion, survival in short-term culture, IgE-mediated histamine release, and modulation of release by the pharmacologic agonists adenosine, IBMX, prostaglandin E2, and fenoterol.     

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a “discovery” assay, the latter as a “monitoring” assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique (“Hi3” method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.     

Purification and characterization of the lymphocyte function-associated-2 (LFA-2) molecule

The lymphocyte function-associated-2 (LFA-2) molecule, equivalent to CD2 and the E rosette receptor, was purified by MAb affinity chromatography from the Jurkat T lymphoma cell line. Jurkat was selected for its high level of expression of 1.0 X 10(5) sites/cell. A two-site radioimmunometric assay was developed to monitor purification. From 50 g of packed cells, 230 micrograms of LFA-2 was obtained with 65% yield of antigenic activity with a purification factor of 13,000. A major component of 58,000 and 54,000 was obtained that corresponded to LFA-2 antigenic activity as shown by immunoblotting and immunoprecipitation. The doublet was resolved by 2D IEF-SDS-PAGE into components of pI = 5.5 and 5.6. Smaller amounts of lower Mr components were also seen. All these components appeared related by processing or proteolytic breakdown, as shown by Cleveland peptide mapping. The LFA-2 deoxycholate complex had an apparent Mr of 68,000 by gel filtration, suggesting it was monomeric. Purified LFA-2 inhibited rosetting of T lymphocytes with sheep E, and addition to preformed rosettes caused their disruption. Inhibitory activity was absorbed by sheep E. This is the first evidence that the CD2/LFA-2 molecule can directly bind to sheep E. Purified LFA-2 should be useful for the further biochemical and functional characterization of this molecule.     

Purification and partial amino acid sequence of proteins from human epidermal keratinocyte conditioned medium

Keratinocytes are the main cell type of the epidermis. They secrete a variety of proteins and peptides that have diverse roles in epidermal physiology. In this report, we present purification and partial amino acid sequence of LEKTI, a serine proteinase inhibitor, and DAN (NO₃) zinc-finger protein, a tumor suppressor protein of neuroblastoma, from human keratinocyte conditioned medium. Epidermal keratinocytes were isolated from human foreskin and serially passaged in a defined medium (MSBM). At confluence of the fourth passage, MSBM medium was replaced with protein-free Dulbecco's modified Eagle medium/F12 (DMEM:F12) 3:1 base medium and collected every 24 h for 4 days. Medium was pooled and concentrated using a stirred cell concentrator. Concentrated medium was diluted 1:1 in 50 mM sodium phosphate, pH 8 buffer, and loaded onto a preparative heparin affinity column. Proteins/peptides were purified from heparin column passthrough by the combination of preparative and analytical FPLC-based gel filtration chromatography and reversed-phase HPLC. Samples electroblotted onto a PVDF support were sequenced by Edman degradation in a gas-phase sequencing system.     

Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms

Manufacturing practices for recombinant adeno‐associated viruses (AAV) have improved in the last decade through the development of new platforms in conjunction with better production and purification methods. In this review, we discuss the advantages and limitations of the most popular systems and methods employed with mammalian cell platforms. Methods and systems such as transient transfection, packaging and producer cells and adenovirus and herpes simplex virus are described. In terms of best production yields, they are comparable with about 10⁴–10⁵ vector genomes produced per cell but transient transfection of HEK293 cells is by far the most commonly used. For small‐scale productions, AAV can be directly purified from the producing cell lysate by ultracentrifugation on a CsCl or iodixanol‐step gradient whereas large‐scale purification requires a combination of multiple steps. Micro/macrofiltration (i.e. including tangential flow filtration and/or dead‐end filtration) and chromatography based‐methods are used for large‐scale purification. Purified AAV products must then be quantified and characterized to ensure quality. Recent purification methods and current analytical techniques are reviewed here. Finally, AAV technology is very promising, but manufacturing improvements are still required to meet the needs of affordable, safe and effective AAV vectors essential for licensing of gene therapy clinical protocols.     

Purification and characterization of dipeptidyl peptidase I from human spleen.

The lysosomal hydrolase, dipeptidyl peptidase I (DPPI), was purified from human spleen and its enzymatic activity characterized. The enzyme was purified to apparent homogeneity by a combination of differential pH solubility, heat-treatment, affinity chromatography on concanavalin A-agarose and p-hydroxymercuribenzoate-agarose, and gel filtration chromatography on Sephacryl S-300. This procedure resulted in a 1100-fold purification of DPPI protein with a yield of approximately 2% of the total DPPI activity. The enzyme was characterized as a glycoprotein with a pI of 5.4, a molecular mass of 200,000 Da as determined by gel filtration under nondenaturing conditions, and a subunit size of 24,000 Da. Amino acid sequence analysis of peptides isolated from cyanogen bromide and trypsin digests of the 24,000-Da subunit revealed extensive sequence similarity between human and rat DPPI. Purified DPPI exhibited both hydrolytic and transpeptidase (polymerase) activity. DPPI exhibited activity against a variety of dipeptide substrates including peptides with either non-polar or polar residues in the P1 position. In contrast to the reported substrate specificity of bovine and murine DPPI, the human enzyme exhibited a modest preference for peptides with nonpolar residues in the P1 position. DPPI content was found to be highest among cytotoxic lymphocytes and myeloid cells. The high level of DPPI expression in these cell populations correlates with their sensitivity to the toxic effects of leucyl-leucine methyl ester, a substrate for DPPI.     

Isolation and characterization of ciliary neurotrophic factor from rabbit sciatic nerves

Ciliary neurotrophic factor (CNTF) has been purified 35,000-fold to homogeneity from rabbit sciatic nerves using its ability to promote the survival of chick embryo ciliary ganglion neurons as the bioassay. The purification involved a combination of acid treatment, ammonium sulfate fractionation, hydrophobic interaction chromatography, chromatofocusing, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reversed-phase high performance liquid chromatography. Overlapping peptide sequences were obtained which accounted for 49% of the primary structure of the molecule. This information was used to prepare synthetic peptides in order to elicit antibodies. Purified CNTF exhibited two major and several minor bands between 24 and 22 kDa on silver-stained sodium dodecyl sulfate-polyacrylamide electrophoresis gels. All of the molecular forms were immunostained in Western blots by antiserum to synthetic peptides. The peptide sequences also provided a basis for cloning and expression of the rabbit CNTF gene (Lin, L-F. H., Mismer, D., Lile, J. D., Armes, L. G., Butler, E. T., III, Vannic, J. L., and Collins, F. (1989) Science 246, 1023-1025) confirming that the protein purified as reported here is CNTF.     

Synthesis of peptide fragments of influenza virus A/Aichi/2/68 (H3N2) hemagglutinins

Peptides corresponding to sequences 122-133, 136-147, and 154-164 of the heavy chain of hemagglutinin of the A/Aichi/2/68 (H3N2) influenza virus have been synthesized by stepwise elongation of the peptide chain with Boc-amino acid activated esters or by condensation of peptide blocks by DCC/HOBt-method. A coloured C-protecting group, 2-[4-(phenylazo)-benzylsulfonyl]ethyl (PSE), was used, which is convenient in purification of synthetic peptides. After removal of terminal N-and C-protecting groups the side-protecting residues were cleaved off with 1 M trifluoromethanesulfonic acid in trifluoroacetic acid containing 10% thioanisole. Crude products were purified by preparative reversed-phase liquid chromatography. Synthesized peptides were conjugated with BSA.     

Purification methods for the sequence-specific DNA-binding protein nuclear factor I (NFI) — Generation of protein sequence information

The paper describes a potent purification method, preparative gel retention, for the purification of sequence-specific DNA-binding proteins. This procedure exploits the sequence-specific DNA-binding affinity of such proteins for their enrichment, comparable to recognition site DNA affinity chromatography. The method was employed to obtain a pure preparation of nuclear factor I (NFI) from porcine liver from which sequences of partial peptides could be obtained. Oligonucleotide probes derived from these amino-acid sequences were used to identify genomic and cDNA clones of NFI.     

Purification methods for the sequence-specific DNA-binding protein nuclear factor I (NFI)--generation of protein sequence information

The paper describes a potent purification method, preparative gel retention, for the purification of sequence-specific DNA-binding proteins. This procedure exploits the sequence-specific DNA-binding affinity of such proteins for their enrichment, comparable to recognition site DNA affinity chromatography. The method was employed to obtain a pure preparation of nuclear factor I (NFI) from porcine liver from which sequences of partial peptides could be obtained. Oligonucleotide probes derived from these amino-acid sequences were used to identify genomic and cDNA clones of NFI.     

Human eosinophil cytotoxicity-enhancing factor. Purification, physical characteristics, and partial amino acid sequence of an active polypeptide

Medium conditioned by PMA/LPS-stimulated U937 cells was processed for the purification of an eosinophil cytotoxicity-enhancing factor (ECEF) by the following sequence: 1) phenyl-Sepharose chromatography; 2) DEAE-cartridge chromatography; 3) preparative SDS-gel electrophoresis; and 4) reversed-phase HPLC. This resulted in the isolation of a 10 kDa polypeptide with ECEF activity. Purified material from 21 different preparations enhanced eosinophil killing of antibody-coated Schistosoma mansoni schistosomula by a mean of 206% (increase from 13.2 +/- 7.9% to 40.4 +/- 20.2% of targets killed, p less than 0.0001). Activity was maximal at a concentration of 20 ng ECEF polypeptide/ml and half-maximal between 0.8 and 4 ng/ml. Antibody specific for the 10 kDa polypeptide precipitated ECEF activity from a crude preparation and, by Western blot analysis, reacted only with a 10 kDa species in that preparation. The following N-terminal amino acid sequence was determined for the purified polypeptide: Val-Lys-Gln-Ile-Glu-Ser-Lys-Thr-Ala-Phe-Gln-Lys-Ala-Leu- -Ala- Gly- -Lys-Leu....Computer search showed that this sequence is unrelated to other known protein sequences. Thus, the ECEF polypeptide is a newly defined monokine, with the ability to enhance eosinophil cytotoxic function in vitro. This monokine may be an important regulator of eosinophil function in inflammation in vivo.     

Glucose 1- and 2-oxidases from fungal strains: isolation and production of monoclonal antibodies.

Monoclonal antibodies (Mabs) against purified glucose 2-oxidase (EC 1.1.3.10) from Coriolus versicolor were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. Hybrid growth was observed in 42% of culture wells and 30% of these (i.e. 30 culture wells) contained anti-glucose 2-oxidase activity. Three positive wells containing hybrid cell lines were selected and cloned twice by the limiting dilution method and two hybridoma clones (E1A5 and E1A6) secreting Mabs were selected at random for purification and characterisation purposes. Both cell lines secreted Mabs of IgM class which were purified by gel filtration chromatography on a Sephacryl S-200 column with a final recovery of 80% and a purification factor of 16. The purified preparations were apparently homogeneous on native PAGE running with a M(r) of 950 kDa. Mabs were highly specific for glucose 2-oxidase as determined by Western blotting. These Mabs also crossreacted with glucose 1- and 2-oxidases from other fungal sources (Phanerochaeta chrysosporium, Penicillium amagasakiense and Aspergillus niger) as determined by Western blotting and by ELISA. Both glucose 1- and 2-oxidases from C. versicolor, P. chrysosporium, P. amagasakiense and A. niger were purified by hydrophobic interaction chromatography on Sepharose 4B-triazine dye with a recovery of enzyme activity in the range 85-92%. Purified preparations of glucose oxidases from fungal strains were apparently homogeneous on native PAGE. Glucose 2-oxidases were more hydrophobic than glucose 1-oxidases as determined by their chomatographic behaviour on Sepharose 4B-Cibacron Red G-E which could be used to study their roles in lignin biodegradation.     

Cysteinyl-flavan-3-ol conjugates from grape procyanidins. Antioxidant and antiproliferative properties

New bio-based antioxidant compounds have been obtained by depolymerisation of grape polymeric flavanols in the presence of cysteine. Their preparation and purification, as well as their antiradical/antioxidant and antiproliferative properties are reported. 4beta-(S-cysteinyl)epicatechin 5, 4beta-(S-cysteinyl)catechin 6 and 4beta-(S-cysteinyl)epicatechin 3-O-gallate 7 were efficiently purified from the crude depolymerised mixture by cation-exchange chromatography and preparative reversed-phase chromatography. The new compounds were more efficient than the underivatised (-)-epicatechin 1 as scavengers of the 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) and weak growth inhibitors of human colon carcinoma HT29 cells. The order of antiradical and antiproliferative efficiency was 7 >5 approximately 6 >1, the same for both assays.