Wolfgang Barz

Enzymatic transformation of flavonols with a cell-free preparation from Cicer arietinum L.

Abstract 1. 1. Crude enzyme preparations from Cicer arietnum L. were shown to convert the flavonols kaempferol, quercetin, datiscetin and morin in an oxygen-dependent reaction to compounds possessing ultraviolet spectra similar to dihydroflavonols. 2. 2. The enzyme reation only occurred with flavonol aglycones. 3. 3. The enzymatically formed product derived from kaempferol readily regenerated kaempferol when warmed or treated with acid and gave rise to phloroglucinol and p-hydroxybenzoic acid under alkaline conditions. These results indicated that the enzymatically formed product is based upon the flavonoid skeleton. 4. 4. Analysis of the ultraviolet, infrared and NMR spectra of the flavonol conversion products from the four flavonols proved that the new compounds are substituted 2,3-dihydroxyflavanones. 5. 5. Electrophoretic studies indicated that the hydroxyl groups at C-2 and C-3 are cis to each other.

Uber den Abbau aromatischer Verbindungen durch Fusarium oxysporum Schlecht

Summary0725 091.A strain of Fusarium oxysporum Schlecht has been isolated from the rhizosphere of hydroponically grown Cicer arietinum L. plants using 2′,4,4′-trihydroxychalcone as a sole source of carbon.2.The organism has been shown to degrade a wide variety of aromatic plant constituents (benzoic acids, cinnamic acids, flavonoids, isoflavones).3.o-coumaric acid is converted to 4-hydroxy coumarin, while melilotic acid is not metabolized.4.The flavonols, kaempferol and quercetin when degraded yield phloroglucinol carboxylic acid, carbon monoxide, p-hydroxy benzoic acid and protocatechuic acid, respectively. The benzoic acids formed from ring B of flavonols are in turn rapidly metabolized.5.Upon degradation of flavanones benzoic acid arise from ring B, thus hesperetin (3′,5,7-trihydroxy-4′-methoxyflavanon) gives rise to isovanillic acid.6.Fusarium oxysporum Schlecht contains considerable β-glucosidase activity, because β-D-glucosides (salicin, prunin, apigenin-7-O-β-D-glucoside) are hydrolyzed without a lag-phase.7.The results are compared with published data from other fungi.Zusammenfassung1.Aus der Rhizosphäre hydroponisch gezogener Kichererbsenpflanzen, Cicer arietinum L., wurde mit 2′,4,4′-Trihydroxychalkon als einziger Kohlenstoffquelle ein Stamm von Fusarium oxysporum Schlecht in Reinkultur erhalten.2.Der Oraanismus ist in der Lage, sehr unterschiedliche aromatische Pflanzeninhaltsstoffe (Benzoesäuren, Zimtsäuren, Flavonoide, Isoflavone) vollständig abzubauen.3.o-Cumarsäure wird in 4-Hydroxycumarin umgewandelt, während Melilotsäure nicht metabolisiert wird.4.Die Flavonole Kämpferol und Quercetin werden in Phloroglucincarbonsäure, Kohlenmonxid und p-Hydroxybenzoesäure bzw. Protocatechusäure gespalten. Die aus dem B-Ring der Flavonole gebildeten Benzoesäuren werden ihrerseits in schneller Reaktion weiter abgebaut.5.Beim Abbau von Flavanonen werden aus dem B-Ring Benzoesäuren gebildet; so entsteht aus Hesperetin (3′,5,7-Trihydroxy-4′-methoxyflavanon) Isovanillinsäure.6.Fusarium oxysporum Schlecht enthält eine hole β-Glucosidaseaktivität, da β-Glucoside (Salicin, Prunin, Apigenin-7-O-β-D-glucosid) ohne lag-Phase hydrolysiert werden.7.Die Ergebnisse werden mit bekannten Daten anderer Pilze verglichen.

Untersuchungen über den Einbau methylierter Vorstufen in die Isoflavone der Kichererbse

In order to determine at what stage in the biosynthesis of biochanin A (5.7-dihydroxy-4′-methoxyisoflavone) and formononetin (7-hydroxy-4′-methoxyisoflavone) the methylation of the 4′-hydroxygroup occurs, the incorporation of p-methoxy-cinnamic acid-[3-14C-methyl-T], 4-methoxy-2′.4′-dihydroxychalcone- [β-14C-methyl-T] and a number of unmethylated precursors into the isoflavones of chana seedlings (Cicer arietinum L.) was compared. p-Methoxy-cinnamic acid and 4.2′.4′-Trihydroxychalcone were found to be the best precursors. Although the incorporation of p-methoxy-cinnamic acid was very good (incorporation rate 6.2%) the T/14C ratio dropped by 95% upon incorporation into the isoflavones, a fact which showed that a rapid demethylation takes place in the plants. In addition, kinetic experiments were carried out with p-methoxycinnamic acid- [3-14C-methyl-14C-methyl-T] and 4-methoxy-2′.4′-dihydroxychalcone- [β-14C-methyl-14C-methyl-T]. But in this case also the rapid demethylation did not permit a definite conclusion to be drawn as to the stage in the biosynthesis at which the methylation step occurs. The very good incorporation of 4.2′.4′-trihydroxycalcone- [β-14C] into the isoflavones (5,3% incorporation, dilution 61), however, shows that methylation can occur at a late stage in the biosynthesis.

Über die Bedeutung von 3.5.7.4′-Tetrahydroxyflavanon (Dihydrokaempferol) für die Biosynthese von Isoflavonen

In parallel experiments 3,5,7,4′-tetrahydroxyflavanone- [T] (dihydrokaempferol- [T]) (I) or 3,5,7,4′-tetrahydroxyflavone- [T] (kaempferol- [T]) (II) with or without the simultaneous addition of DL-phenylalanine-[1-14C] was fed to chana seedlings (Cicer arietinum) and the 14C and tritium incorporation into the isoflavones biochanin-A und formononetin was determined. Furthermore a competition experiment between I and phenylalanine-[1-14C] was carried out. The results demonstrate that the incorporation of I and II into the isoflavones is insignificant and unspecific. It must be concluded that dihydrokaempferol is not a precursor for the biosynthesis of biochanin-A in the chana seedlings. In connection with our earlier investigations it can be postulated, that the aryl migration leading to isoflavones occurs at the flavanone stage.

Stereospezifischer Einbau von (—)5.7.4′-Trihydroxyflavanon in Flavonoide und Isoflavone

(±)5,7,4′-Trihydroxyflavanone-5-glucoside-[2-14C] was synthesized and the diastereomeric (+) and (-) glucosides were separated by paper chromatography. In buckwheat seedlings the incorporation rate of the (-) enantiomer into quercetin was 16 times higher and into cyanidin 3.6 times higher than that of the (+) compound. In chana seedlings (Cicer arietinum L.) the (-) compound was incorporated into biochanin A (5,7-dihydroxy-4′-methoxy-isoflavone) 14 times better than the (+) compound, whereas in agreement with earlier results the incorporation of both enantiomers into formononetin (7-hydroxy-4′-methoxy-isoflavone) was very low. The results prove the stereospecific incorporation of the naturally occuring (-) · 2 · S · 5.7.4′-trihydroxyflavanone into flavonoids and isoflavonoids. The incorporation of the (+) enantiomer is probably due to a racemization of this compound via the chalcone.

Über den Abbau von Flavonolen in pflanzlichen Zellsuspensionskulturen / Degradation of Flavonols in Plant Suspension Cultures

The flavonols kaempferol, quercetin and isorhamnetin were labelled with 14C by keeping seven day old Cicer arietinum L. plants in an atmosphere of 14CO2 for five days. The purified (U-14C) flavonols were applied to cell suspension cultures of Cicer arietinum L., Phaseolus aureus Roxb., Glycine max and Petroselinum hortense. Based on the rates of 14CO2 formation and distribution of radioactivity after fractionation of the cells, the flavonols were shown to be catabolized to a very high extent. All four cell suspension cultures possess the enzymatic activity transforming flavonols to the recently discovered 2,3-dihydroxyflavanones. Upon incubation of the flavonols datiscetin and kaempferol with enzyme preparations from Cicer arietinum L. cell suspension cultures, it was demonstrated that the enzymatically formed 2,3-dihydroxyflavanones are further transformed in an enzyme catalyzed reaction. Salicylic acid was found as a degradation fragment of ring B of the 2,3,5,7,2′-pentahydroxyflavanone derived from datiscetin. Neither phloroglucinol nor phloroglucinol carboxylic acid were observed as metabolites of ring A. These in vitro findings were further substantiated by in vivo data because the flavonols kaempferol, quercetin and datiscetin when applied to cell suspension cultures of Cicer arietinum L. and Glycine max gave rise to para-hydroxybenzoic acid, protocatechuic acid and salicylic acid, respectively. It was thus concluded that flavonols are catabolized via 2,3-dihydroxyflavanones with the B-ring liberated as the respective benzoic acid. The data are discussed in connection with earlier findings on the catabolism of chalcones, cinnamic and benzoic acids.