Karel Vande Casteele

Separation of flavonoids by reversed-phase high-performance liquid chromatography

Abstract The ability of a reversed-phase high-performance liquid chromatographic system to separate flavonoids [LiChrosorb RP-18 and a combination of an isocratic and a gradient (5% aqueous formic acid and methanol) technique] has been studied. Retention times of some 141 flavonoids ranging from triglycosides to aglycones are reported. The correlations between structure and tR values are discussed.

Separation of phenolics (benzoic acids, cinnamic acids, phenylacetic acids, quinic acid esters, benzaldehydes and acetophenones, miscellaneous phenolics) and coumarins by reversed-phase high-performance liquid chromatography

Abstract The high-performance liquid chromatographic separation of a relative large series of simple phenolics and related substances by means of a reversed-phase system is described. The system consists of a LiChrosorb RP-18 (10 μm) Knauer column and a combination of isocratic and linear-gradient elution (solvent a, water—formic acid (95:5); solvent B, methanol; temperature, 35°C). The series of compounds studied embraces derivatives of benzoic acid (29 compounds), phenylacetic acid (11 compounds), cinnamic acid (26 compounds) and dihydrocinnamic acid (8 compounds), quinic acid esters of phenolic benzoic and cinnamic acid derivatives (9 compounds), benzaldehydes and acetophenones (25 compounds), miscellaneous phenolics (27 compounds) and coumarins (43 compounds). Amongst these substances, phenolic acids, lactones, aldehydes and ketones are represented. The possible use of the system for the analysis of complex mixtures of natural products as well as for the elucidation of the structures of more complex natural compounds is discussed.

Free and bound phenolic acids of lucerne (Medicago sativa cv europe)

Abstract The distribution of free and covalently-bound phenolic acids was studied in various fractions obtained from fresh lucerne shoots. p-Hydroxybenzoic, vanillic, p-coumaric and ferulic acids were present, both free and bound, in all the fractions. Salicylic and sinapic acids occurred only in a bound, alkali-labile state and were found almost entirely in the ‘aqueous phase’ fraction after treatment of methanol-chloroform-water extract according to Bligh and Dyer. Many other common phenolics were absent. Amounts of the phenolic acids much larger than those extracted by methanol-chloroform-water were extracted subsequently by phenol-acetic acid-water and passed into the ‘diffusate’ fraction on dialysis of this extract against 70% acetic acid. Small, though significant, quantities of phenolic acids remained with the bulk protein in the ‘bag contents’ fraction. The extent to which the phenolic acids in these last two fractions are held to protein by covalent bonds or by secondary-valence attractions is discussed, particularly in relation to the isolation of N-feruloylglycylphenylalanine after partial hydrolysis. Suggestions are made for improving analytical procedures.

Separation of some anthocyanidins, anthocyanins, proanthocyanidins and related substances by reversed-phase high-performance liquid chromatography

Abstract The separation of some anthocyanidins, anthocyanins, proanthocyanidins and related substances by means of reversed-phase high-performance liquid chromatography is described. The system consists of a LiChrosorb RP-18 (10 μm) Knauer column and a combination of isocratic and linear gradient elution (solvent A: water—formic acid (95:5 v/v); solvent B: methanol; 35°C). These results and those of previous studies on related compounds are employed to present a “review chromatogram” demonstrating the separatory power of the RP—HPLC technique. The possible use of this system for the analysis of complex mixtures of natural products is discussed.

Flavonoid biosynthesis in petals of Rhododendron simsii

Abstract The results of a comparative in vivo study of flavonoid biosynthesis in Rhododendron simsii petals, with [2- 14 C] p -coumaric acid and [2- 14 C]caffeic acid indicate that p -coumaric acid is the true precursor. Moreover, supplementary in vitro experiments with enzyme preparations show that p -coumaroyl-CoA is a much better substrate for chalcone synthase than caffeoyl-CoA. This suggests that in Rhododendron the 3′-hydroxylation of the B-ring occurs at the stage of a C 15 intermediate. In addition, flavonoid 3′-hydroxylase activity was demonstrated by means of comparative in vivo experiments with, respectively, [4a,6,8- 14 C]naringenin and [4a,6,8- 14 C]dihydrokaempferol as C 15 precursors. In both cases, the formation of radioactive 3′,4′-hydroxylated co-pigments and anthocyanins was established. Furthermore, co-pigment biosynthesis may proceed via the sequence: naringenin, eriodictyol, dihydroquercetin (3′-hydroxylation at the flavanone stage), whereas the formation of the anthocyanins may take place via naringenin, dihydrokaempferol and dihydroquercetin (3′-hydroxylation at the dihydroflavonol level).

The phenolics and a hydrolysable tannin polyphenol oxidase of Medinilla magnifica

Abstract The production of viable meristem cultures of Medinilla magnifica has proved to be very difficult. This may be due, in part, to a pronounced ‘browning’ response of the tissues on cutting. For this reason the phenolic compounds and the hydrolysable-tannin polyphenol oxidase from Medinilla were studied. The distribution of the compounds was: simple phenols 19% , flavonoids 5% , hydrolysable tannins 69% , condensed tannins 7%. Amongst the simple phenols and phenolic acids, the following were identified: phloroglucinol, p -hydroxybenzoic acid, vanillic acid, protocatechuic acid, gallic acid (both in free and bound form the most abundant simple phenol), syringic acid, trans - p -coumaric acid, trans -ferulic acid and trans -caffeic acid. No kaempferol or quercetin or their derivatives were detected but condensed tannins are present. Methods for the extraction, fractionation and quantitative determination of phloroglucinol and the phenolic acids, as well as correction factors for losses during the extraction, alkali treatment and derivatization, are presented in a supplementary publication. With regard to the hydrolysable tannin polyphenol oxidase activity of Medinilla stems, the enzyme(s) is rather specific since at neither of its two pH optima (6 and 7) could a classical polyphenol oxidase activity be detected. The enzyme was strongly inhibited by 2-mercaptoethanol. Preliminary experiments have further shown that in addition to the hydrolysable tannins of the tissue, the ferrous ions of the medium, and oxygen together with the hydrolysable tannin polyphenol oxidase could play a role in the browning response. Ways to overcome this difficulty have been suggested.

Separation of some metabolically important aromatic nacylamino acids of the benzoyl and cinnamoyl series by thin-layer, gas—liquid and high-performance liquid chromatography

Abstract The separations of 30 different N-acylamino acids and peptides from the benzoyl and cinnamoyl series have been studied by thin-layer (TLC), gas—liquid (GLC) and high-performance liquid chromatography (HPLC). TLC separation on three different layers and with four different solvent systems are described. GLC separations fo the trimethylsilyl derivatives of some of the compounds were carried out on a glass column packed with Chromosorb W AW DMCS (80–100 mesh) coated with 1.5% SE-30 + 1.5% SE-52 and using a temperature program. HPLC separations on a reversed-phase column (LiChrosorb RP-18, 10 μm, Knauer) employed a combination of isocratic and a linear gradient elution [solvent A, water—formic acid (95:5 v/v); solvent B, methanol; 35°C]. The few compounds which not be separated by the latter system were separated on a second column (LiChrosorb Si 60, 7 μm, Knauer) by means of a combination of isocratic and linear gradient elution [solvent A, dichloromethane—cyclohexane—formic acid (55:45:2, v/v/v); solvent B, methanol; 30°C]. These methods can easily be combined, and allow the separation of N-acylamino acids and N-acylpeptides from biological fluids, extract an partial hydrolysates from phenolic acid-containing plant proteins.

N-Feruloylglycine amidohydrolase from barley seeds and isolated barley embryos

Abstract N -Feruloylglycine amidohydrolase, an enzyme catalysing the hydrolysis of a variety of N -acylated- l -amino acids from the cinnamoyl and substituted cinnamoyl series was isolated from barley seeds. The enzyme, which was localized within the embryo and the aleurone layer, increased in activity during the first 14 hr of seed imbibition. A 0.1 M Tris-HCl buffer pH 8.0–10% glycerol extract of germinated embryos furnished an active hydrolase preparation which was purified to ca 102-fold. Fractionation consisted of treatment with ammonium sulphate, gel filtration over Sepharose 6B and hydroxyapatite chromatography. Enzyme activity was measured by: (i) a radiobiochemical TLC liquid scintillation sampling method using N -feruloyl-2-[ 14 C]glycine-2-[ 3 H] as a substrate and (ii) an HPLC sampling assay using unlabelled N -feruloylglycine. The pH and temperature optima of the purified hydrolase were respectively pH 8.0 and 32°. The enzyme was very stable between pH 7.5 and 10.0, and upto a temperature of 35°. 10% Glycerol stabilized the enzyme and the activity of the crude preparation was further enhanced by 0.4 M ammonium sulphate which also provided stability during fractionation. The specific activity of the enzyme, which changed with the barley cultivar used as well as with the time of seed germination, fluctuated for the unpurified enzyme of germinated embryos from 3.6 pkat/mg (cv Zephyr; after 24 hr imbibition to 11 pkat/mg (cv Gitane after 14 hr imbibition). The specific activity of the 102-fold purified enzyme of germinated embryos from cv Zephyr was 0.37 nkat/mg.

Characteristics and specificity of purified N-feruloylglycine amidohydrolase from isolated barley embryos

Abstract N -Feruloylglycine amidohydrolase with an estimated M r of 155 000, showed with N -feruloylglycine at pH 8 and 30° a K m of 85 μM, a V m of 3.92 nmol/0.1 mg protein/min, a physiological efficiency ( V m / K m ) of 46.1.10 3 and an apparent activation energy of 43.5 kJ/mol. Sulphydryl reagents were shown to decrease enzyme activity and relative high concentrations (10 mM) of metal chelating reagents (EDTA, dithizone and o -phenanthroline) were also inhibitory. The effect produced by o -phenanthroline could be partially reversed by CuCl 2 , and CoCl 2 , and to a lesser extent by ZnCl 2 and MnCl 2 . N -Feruloyldipeptides such as N -feruloylglycyl- l -phenylalanine ( K i = 42 μM; α = 7.8 and β = O) and N -feruloylglycyl- l -leucine ( K i = 300 μM; α = 5.8; β = O) were potent reversible mixed type inhibitors. The purified enzyme did not show any deformylase or amidase activity and was thus totally different from N -acylamino acid amidohydrolase (EC 3.5.1.14). In addition, the pseudopeptide bond of N -feruloylglycine was not split by a series of peptidases and proteinases. N -Feruloylglycine amidohydrolase is a new acylase of plant origin and the name proposed for the enzyme is well supported by substrate specificity studies.