Anthocyanidin formation

Dihydroflavonol 4-reductase (DFR EC 1.1.1.219) is a member of the short-chain dehydrogenase/reductase family and catalyzes the stereospecific conversion of (+)-(2R,3R)-dihydroflavonols to the corresponding (2R,3S,4S) flavan-3,4-cw-diols (leucoanthocyanidins), with NADPH as a required cofactor. The enzyme activity was first identified in cell suspension cultures of Douglas fir (Pseudotsuga menziesii) and was shown to be related to the accumulation of flavan-3-ols and proanthocyanidins [96]. Leucoanthocyanidins and DFR were later shown to be required for anthocyanidin formation by complementation of Matthiola incana mutants blocked between dihydroflavonol and anthocyanidin biosynthesis [97, 98], DFR has been purified to apparent homogeneity and biochemically analyzed from flower buds of Dahlia variabilis [99]. DFR was shown to accept different substrates depending on the plant species from which it was isolated (reviewed in 100). 

With the knowledge that flavan-3,4-diols are freely associated with flavan-3-ols [e. g. (H-)-catechin, (—)-epicatechin, ( —)-fisetinidol, ( + )-epifisetinidol] in tannin mixtures comprised of these molecular entities, experimental conditions were sought which would permit formation of C-4 —C-6 or 8 interflavanoid links. The conditions selected had to be sufficiently mild to avoid self-condensation of the flavan-3,4-diol, to avoid anthocyanidin formation as a side-reaction, to permit stereochemical conclusions (i. e. the reaction should be under kinetic rather than under thermodynamic control), and to allow assessments regarding the re-gioselectivity of the condensation. 

The pKj, value (2.43) of the hydration constant of the cyanidin was found to be lower than the pKj, values of glycosylated and acylated cyanidins, meaning lower resistance of the anthocyanidin to hydration. The stability of nonacylated 3,5-diglucosides was lower compared to the 3-glucoside because the 5 position markedly lowered the hydration constant due to decreased electron density of the pyrilium ring that favors nucleophilic attack by water, enhancing hemiacetal formation. ... 

Chlorogenic acid forms a 1 1 complex with caffeine, which can be crystallized from aqueous alcohol and yields very little free caffeine on extraction with chloroform. Other compounds with which caffeine will complex in this way include isoeugenol, coumarin, indole-acetic acid, and anthocyanidin. The basis for this selection was the requirement for a substituted aromatic ring and a conjugated double bond in forming such a complex. This kind of complex does modify the physiological effects of caffeine.14 Complex formation will also increase the apparent aqueous solubility of caffeine in the presence of alkali benzoates, cinnamates, citrates, and salicylates.9... 

Bilberry Vaccinium myrtillus) Uses Prevent/treat visual problems such as cataract, retinopathy, myopia, glaucoma, macular degeuCTation treat vascular problems such as hemorrhoids, varicose veins Actions Contains anthocyanidin that X vascular permeability, inhibit pit aggregation thrombus formation, t antioxidant effects on LDLs liver, t regeneration of rhodopsin... 

FIGURE 3.2 General phenylpropanoid and flavonoid bios5mthetic pathways. The B-ring hydroxylation steps are not shown. For formation of anthocyanins from leucoanthocyanidins two routes are represented a simplified scheme via the anthocyanidin (pelargonidin) and the likely in vivo route via the pseudobase. Enzyme abbreviations are defined in the text and in Table 3.1. 

From extensive analysis of recombinant proteins, and the crystal structure of A. thaliana protein, detailed reaction mechanisms have been proposed. The ANS reaction likely proceeds via stereospecific hydroxylation of the leucoanthocyanidin (flavan-3,4-cA-diol) at the C-3 to give a flavan-3,3,4-triol, which spontaneously 2,3-dehydrates and isomerizes to 2-flaven-3,4-diol, which then spontaneously isomerizes to a thermodynamically more stable anthocyanidin pseudobase, 3-flaven-2,3-diol (Figure 3.2). The formation of 3-flaven-2,3-diol via the 2-flaven-3,4-diol was previously hypothesized by Heller and Forkmann. The reaction sequence, and the subsequent formation of the anthocyanidin 3-D-glycoside, does not require activity of a separate dehydratase, which was once postulated. Recombinant ANS and uridine diphosphate (UDP)-glucose flavonoid 3-D-glucosyltransferase (F3GT, sometimes... 

Finally, Weinges et al. (31, 33) postulated the formation of dimers such as 27 or 28, 29 or 30 directly from catechins without involving 3,4-flavandiols (leucoanthocyanidins). This process has never been demonstrated in fruits directly and specifically not in grapes. Only the proanthocyanin dimers have been positively identified through the formation of catechins and anthocyanidins during acid hydrolysis. Dimer formation proceeds by enzymatic oxidation of two molecules of catechin... 

The branch pathway for anthocyanin biosynthesis starts with the enzymatic reduction of dihydrofiavonols to their corresponding flavan 3,4-diols (leucoanthocyanidins) by substrate-specific dihydroflavonol 4-reductases (DFR). Flavan 3,4-diols are the immediate precursors for the synthesis of catechins and proanthocyanidins. Catechins are formed by enzymatic reduction of the flavan 3,4-diols in the presence of NADPH to leucoanthocyanidins, which are subsequently converted to anthocyanidins by the 2-oxoglutarate-dependant dioxygenase, anthocyanidin synthase. Further glycosylation, methylation, and/or acylation of the latter lead to the formation of the more stable, colored anthocyanins (Scheme 1.1). The details of the individual steps involved in flavonoid and isoflavonoid biosynthesis, including the biochemistry and molecular biology of the enzymes involved, have recently appeared in two excellent reviews.7,8... 

The flavonoid hydroxylases (F3 H, F3 ,5 H) and the phenylpropanoid enzyme cinnamate 4-hydroxylase (C4H) are cytochrome P-450s that are anchored to the cytoplasmic face of the ER.14 F3 H and F3 ,5 H add hydroxyl groups to the 3 and/or 5 of the B ring bringing about chemical and spectral diversity to the flavonoids. Dihydroflavonol 4-reductase (DFR) is the committed step to the production of the precursor of the colored compounds - the leucoanthocyanidins (Fig. 3.2). It is upon the reduction of the 4 keto of the C -ring by DFR (Fig. 3.1) and its further reduction by leucoanthocyanidin dioxygenase/anthocyanidin synthase (LDOX/AS) that the de-localization of the electrons necessary for the formation of the planar flavylium ion is permitted.2... 

A different cyclization leads to the flavones and anthocyanidins. Reaction of the stable enol from a 1,3-diketone with the thiol ester as electrophile results in acylation at carbon in the manner of the Claisen ester condensation (Chapter 28) with loss of CoASH and the formation of a trihydroxyben-zene ring. 

Nonhydrolyzable or condensed tannins are also named proanthocyanidins. These are polymers of flavan-3-ols, with the flavan bonds most commonly between C4 and C8 or C6 (Figure 6-23) (Macheix et al. 1990). Many plants contain tannins that are polymers of (+)-catechin or (-)-epicatechin. These are hydrogenated forms of flavonoids or anthocyanidins. Other monomers occupying places in condensed fruit tannins have trihydroxylation in the B-ring (+)-gallocat-echin and (-)-epigallocatechin. Oligomeric and polymeric procyanidins are formed by addition of more flavan-3-ol units and result in the formation of helical structures. These structures can form bonds with proteins. 

The biosynthesis of the flavonoids and anthocyanidins involves a union of a C -C unit with a C mit. It is of interest that the Cr-Ca intermediate is derived from the S-deoxy-n-arobino-heptulosonic acid 7-phos-phate pathway, while the C6 intermediate is derived from the acetate pathwayIn the formation of quercetin (XXVI) by the buckwheat plant,... 

In general, the radicals dimerize rapidly unless inhibited by pyranyl ring substitution 2-(4-nitrophenyl)benzopyranyl is reported not to dimerize, however.The kinetics of the formation of a series of 36 by reduction of the precursor flavylium ions by Cr(II) have been investigated a Hammett plot of the second-order rate constants versus has p = 1.03. Most work has been concerned with the polarography of the parent cations there appear to be no ESR results for benzopyranyl radicals. The mediation of benzopyranyl radicals in the oxidation of 2-phenyl-2//-benzopyrans by potassium permanganate has been suggested.(See Section II,B,5,b for radicals from anthocyanidins.)... 

The antioxidant activity of a compound depends upon which free radical or oxidant is used in the assay (Halliwell and Gutteridge, 1995), and a different order of antioxidant activity is therefore to be expected when analyses are performed using different methods. This has been demonstrated by Tsuda et al. (1994) in their study of antioxidative activity of an anthocyanin (cyanidin-3-O-p-D-glucosidc) and an anthocyanidin (cyanidin) in four different lipophilic assay systems. Both compounds had antioxidative activity in all four systems, but the relative activity between them and their activity, compared with Trolox, varied with the method used. Fukumoto and Mazza (2000) reported that antioxidant activity of compounds with similar structures gave the same trends, although not always the same results, when measured by P-carotene bleaching, DPPH and HPLC detection of malonaldehyde formation in linoleic acid emulsion. 

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