There are few flavonoid structures which do not show any hydroxyl group in the A-ring or a single hydroxyl group in position 6 [2]. Such rare structures seem to be formed particularly in the Primulaceae, Rutaceae and Thymelaeaceae families. The mechanism(s) of their biochemical formation, however, is still unknown. The vast majority of flavonoids possess a basic 5,7-hydroxylation pattern of the A-ring, which is built from malonyl-CoA during the formation of the chalcone (Figure 5).

Chalcones are the first C₁₅-structures of the flavonoid pathway (Figure 5). During the catalytic process, the A-ring of chalcones is assembled from 3 × 2 carbons from the three molecules of malonyl-CoA, whereas the three other carbons are removed by decarboxylation [41,53–55]. Chalcones are converted by CHI to (2S)-flavanones, which are the intermediates for all further flavonoid classes. CHIs of most plants show a distinct substrate specificity and accept only the common 6′-hydroxychalcones as substrates [3].

In some plants, particularly in members of the Leguminosae family, flavonoids lacking a 5-hydroxyl group are found [4]. Their formation occurs by co-action of CHS and CHR (synonyms: polyketide reductase PKR, chalcone ketide reductase). CHR catalyzes the reduction of the keto group of the coumaroyl-trione intermediate in position 6′ and after the loss of water the 6′-deoxychalcones are obtained [56], which are the precursors for the formation of all 5-deoxyflavonoid classes. The ability of plants to form 5-deoxyflavonoids primarily depends on the presence of CHR and a specific CHI, which accepts 6′-deoxychalcones as substrates beside the common 6′-hydroxychalcones. All further downstream enzymes from the flavonoid pathway seem to accept 5-deoxyflavonoids apart from their natural 5-hydroxyflavonoid substrates. Conversion rates, however, depend on the enzyme type and origin [57–59]. Because of the lacking hydroxyl group in position 5, 5-deoxyflavan 3,4-diols are much more stable than their corresponding 5-hydroxycounterparts.

Additional hydroxyl groups in ring A can be introduced at positions 6 and 8 (Figure 6). A 2-oxoglutarate-dependent dioxygenase (F6H) is responsible for the 6-hydroxylation of partially methylated flavonols in the semiaquatic weed plant Chrysosplenium americanum [60,61]. In contrast, F6Hs from Tagetes patula and Rudbeckia hirta require the aglycon quercetin as a substrate and were identified as a cytochrome P450-dependent monooxygenases [62,63]. Generally, the occurrence of two different enzyme systems catalyzing one and the same reaction has rarely been reported and is therefore of great biochemical interest. A cytochrome P450-dependent F6H from elicitor-treated soybean seems to be specifically involved in the biosynthesis of 6-hydroxyisoflavones. Flavanones were the preferred substrates, whereas the flavonol kaempferol was barely accepted [64].

An enzyme introducing a hydroxyl group in position 8 of flavonols and flavones was demonstrated with enzyme preparations from petals of Chrysanthemum segetum [65]. Flavanones, dihydroflavonols and glucosylated flavonoids were not accepted as substrates. The enzyme is localized in the microsomal fraction and requires NADPH and FAD as cofactors. F8H represents a novel type of hydroxylating enzyme in the flavonoid pathway as there seems to be no involvement of cytochrome P450 in the reaction [65].

The hydroxylation pattern of the B-ring is firstly determined by the C₆-C₃ precursor used by CHS. As a rule, p-coumaroyl-CoA (4-hydroxycinnamoyl-CoA) is the physiological standard precursor. The resulting basic C₁₅ chalcone intermediate, naringenin chalcone, carries a hydroxyl group in position 4 and all derived flavonoid structures have the hydroxyl group in position 4′, which is found in common flavonoid structures (Figure 5). Thus, the introduction of the hydroxyl group in position 4′ is a pre-flavonoid step, which is performed by cinnamate 4-hydroxylase, a cytochrome P450-dependent monooxygenase, which catalyzes the formation of p-coumaric acid [66,67].

The hydroxyl group in position 3′ can be generated in two different ways (Figure 7). First, the incorporation of caffeoyl-CoA (CoA-ester of the 3,4-dihydroxycinnamic acid) instead of p-coumaroyl-CoA (CoA-ester of the 4-hydroxycinnamic acid) in the CHS step results in the formation of 3,4-hydroxylated chalcones which are intermediates for the corresponding 3′,4′-hydroxylated flavonoids. In most plants, CHS accepts caffeoyl-CoA as a substrate to a sufficient extent in vitro. However, only in a few cases caffeoyl-CoA was shown to be physiologically relevant as precursor for chalcone formation [3, 68], e.g for scarlet Verbena hybrida lines accumulating 10% cyanidin in the petals despite the absence of F3′H activity [69]. Second, as a rule, the additional hydroxyl group in position 3′ of ring B is introduced by specific enzymes, which can act at different levels of the pathway.

The cytochrome P450-dependent monooxygenase flavonoid 3′-hydroxylase (F3′H) (Figure 8) was first demonstrated in microsomal fractions of cell suspension cultures from Haplopappus gracilis [70] and was then investigated in many different plant species [3]. The enzyme was shown to accept flavanones, flavones, flavonols and dihydroflavonols as substrates [71]. Anthocyanidins, however, cannot be hydroxylated by F3′H [3]. The first f3′h cDNA clone was isolated from Petunia hybrida and functionally expressed in yeast [72].

Many plants (e.g., Asteraceae species) show a corresponding B-ring hydroxylation pattern of chalcones and flavonoids [4]. An enzyme was demonstrated in D. variabilis petals that catalyzes the hydroxylation of chalcones in position 3 [73], but it remained unclear whether chalcone 3-hydroxylation is performed by the common F3′H or an unknown independent enzyme. The involvement of F3′H seemed to be likely, because (i) the same position in the B-ring of flavonoids and chalcones is affected, (ii) F3′H generally shows broad substrate acceptance and (iii) the hydroxylation of both flavonoids and chalcones is catalyzed by NADPH-dependent enzymes localized in the microsomal fraction. However, common F3′Hs do not hydroxylate chalcones at position 3 [74]. Recent research has shown that chalcone 3-hydroxylation can be catalyzed to some extent by special F3′Hs with distinct structural pre-requisites [75]. In addition, some plants possess beside the common F3′Hs additional specific enzymes, which have a better ability to hydroxylate chalcones compared to their F3′H activity [76]. Such enzymes can be regarded as chalcone 3-hydroxylases (CH3Hs).

Flavonoid 3′,5′-hydroxylase (F3′5′H) is responsible for the generation of the 3′,4′,5′ hydroxylation pattern (Figure 8). It can also act at different levels in the flavonoid pathway and catalyzes 3′,5′-hydroxylation of 4′-hydroxylated flavanones, flavonols, flavones and dihydroflavonols and 5′-hydroxylation of the corresponding 3′,4′-hydroxylated structures. F3′5′H is a cytochrome P450-dependent monooxygenase [77,78]. The presence of this enzyme is an essential pre-condition for the formation of delphinidin (anthocyanidin with three hydroxyl groups in the B-ring), which is responsible for blue and violet flower coloration attracting honey bees – the main pollinators in the temperate regions. F3′5′H was first demonstrated in microsomal preparations from Verbena hybrida [79] and has since been isolated from approximately 50 plants, mainly angiosperms. F3′5′H was apparently recruited from F3′H before the separation of angiosperms and gymnosperms [80] as an adaptation to the requirement of blue flower coloration to attract insect pollinators. Interestingly, Asteraceae show specific F3′5′Hs, which have evolved independently from Asteraceae specific F3′Hs.

The presence of an enzyme catalyzing the introduction of a hydroxyl group in position 2′ of flavonoids is expected for Heywoodiella oligocephala accumulating isoetin (2′-hydroxyluteolin) derivatives as main yellow flower pigment [81,82]. However, hydroxylation in position 2′ has not been investigated so far.

The majority of flavonoid classes carry a hydroxyl group in position 3 (Figure 2). The hydroxyl group is introduced at the level of flavanones by the well studied FHT, a 2-oxoglutarate, Fe(II) and ascorbate-dependent dioxygenase [83–85]. The absence of this enzyme is a precondition for the formation of the rare 3-deoxyanthocyanidins [86] (Figure 9). Absent or reduced FHT activity is observed in natural mutants [87–90] or during transient downregulation of FHT [91,92]. Similar effects can be achieved by the application of chemical compounds [57,59] or via genetic engineering [93–95]. However, the absence of FHT alone does not ensure the formation of 3-deoxyanthocyanidins in plant tissues.

The majority of flavonoid classes carry an oxo group in position 4, but in the case of flavan-3,4-diols and flavan-4-ols, a hydroxyl group is found instead. This hydroxyl group results from the reduction of the 4-oxo group of ring C. The step is catalyzed by the well studied oxidoreductase DFR [3,58,86,96–98]. As dehydrogenase, DFR catalyzes also the reverse reaction, i.e. the conversion of flavan 3,4-diols into dihydroflavonols or flavan-4-ols into flavanones in the presence of NADP⁺ [58]. The physiological relevance of the reverse DFR reaction has not been studied so far, but an impact on metabolic fluxes in the flavonoid pathway is possible.

In some plants, e.g., petunia and tobacco, DFR shows a distinct substrate specificity in terms of the hydroxylation pattern in ring B and accepts dihydroquercetin (3′,4′-hydroxylation pattern) and dihydromyricetin (3′,4′,5′-hydroxylation pattern) but not dihydrokaempferol (4′-hydroxylation pattern) as substrates [3]. As dihydroflavonols are intermediates in the formation of anthocyanins, this DFR substrate specificity determines flower color. Such plants can accumulate derivatives of cyanidin (dark red pigment; 3′,4′-hydroxylation pattern) and delphinidin (blue pigment; 3′,4′,5′-hydroxylation pattern), but scarlet flower coloration, which would be caused by the accumulation of pelargonidin derivatives (scarlet pigment; 4′-hydroxylation pattern) does not occur in these plants.

In addition, DFR also catalyses the reduction of flavanones to the corresponding flavan-4-ols (flavanone-4-reductase activity, FNR-activity), which are intermediates in the 3-deoxyanthocyanidin formation (Figure 9) [86,99]. This was first investigated with plants producing 3-deoxyanthocyanidins under natural conditions [86,89]. Physiological relevance in planta of the FNR-activity of DFR is only observed in cases where FHT activity is reduced or absent. However, all DFR enzymes tested so far catalyze the reaction [3,57,86,89,96,100–102], although dihydroflavonols are the preferred substrates. Therefore, the common 3-hydroxyflavonoids are exclusively formed as long as dihydroflavonols are available as substrates.

The UV-VIS spectrum of flavonoids typically shows a band at 210–290 nm region (band I), which is due to the absorption of the benzoyl system (A-ring), and a second band in the 300–400 nm region (band II), which is associated with the cinnamoyl system (rings B and C) [117] (Figure 10). With increasing conjugation the absorbance shifts towards higher wavelengths, therefore, chalcones, aurones and anthocyanidins have maximal absorbance in the visible part of the spectrum. In the case of anthocyanidins, band II shows its maximum around 520 nm. For many pollinating insects absorption in the UV (band I) is also relevant for colour perception because their specific colour sense makes them blind to scarlet red coloration but sensitive to the UV-range of the spectrum [118–123]. The number and type of substituents affiliated to the basic flavonoid structure has an impact on light absorbance. Additional hydroxyl groups shift the absorption maxima. The hydroxyl group in position 3 of ring C has a higher impact on the absorption maximum of band II than hydroxyl groups in rings A and B. For anthocyanidins (compare a with e and b with d in Figure 11), this results in a colour shift of 3-deoxyanthocyanidins into the yellow region (Figure 12).

With increasing numbers of hydroxyl groups at rings A and B, the absorbance shifts slightly towards higher wavelengths. Thus, pelargonidin (one hydroxyl group in the B-ring) has its maximum at 510 nm, cyanidin (two hydroxyl groups in the B-ring) at 520 nm and delphinidin (three hydroxyl groups in the B-ring) at 530 nm (Figures 11 and 13). To the human eye, this shifts the colour of the pure substances as well as of tissues in which the substances are accumulated, from bright red in the case of pelargonidin to the lilac hues of delphinidin (Figure 13). However tissue colouration by anthocyanidins may also be influenced by other factors, such as pH, co-pigmentation and metal complexation [12,14].

Cyanidin is regarded as the most primitive anthocyanidin pigment, because it is (i) the most common type found in gymnosperms, which are the anchestors of angiosperms, (ii) the most common anthocyanidins formed in leaves and stems, and (iii) the most common pigment of wind-pollinated flowers [18]. The other anthocyanidin types are regarded as being more advanced and seem to have evolved during the adaptation of angiosperms to different pollinators. The regular occurrence of bright-red pelargonidin (a in Figure 11), orange luteolinidin (d in Figure 11) and yellow apigeninidin (f in Figure 11) in tropical plants and their almost complete absence in temperate floras reflect the need for adaptation to birds as the most active pollinators in tropical habitats [14].

In contrast, flowers in temperate regions are pollinated by insects rather than by birds. This resulted in the formation of purple and blue delphinidin-based pigments (c in Figure 11) as an adaptation to the specific colour sense of insects [14]. Flavonols with additional hydroxyl groups in ring A are responsible for the formation of UV-honey guides in Rudbeckia hirta [14,119], but recent research has shown that the glycosylation pattern is also decisive [63]. The pigments accumulate in the inner part of the petals and this is perceived as colour pattern by pollinating insects whereas the flowers appear uniformly yellow to the human eye.

Most of the beneficial health effects of flavonoids are ascribed to their antioxidant abilities [124]. Antioxidant activity is influenced by a mixture of properties including the capacity for reactive oxygen species (ROS) scavenging, metal ions chelation, the regeneration of other antioxidative compounds such as α-tocopherol, activation of antioxidant enzymes and the inhibition of radical-producing enzymes [124–126].

Although flavonoids are widely accepted as being potent antioxidant compounds, there is a controversy discussion about their relative contribution and SARs [126]. One of the reasons for this might be the broad spectrum of different methods used to measure antioxidant capacities and the fact that the different components influencing antioxidative behaviour cannot easily be measured separately. However, the antioxidant capacity of a compound primarily depends on the presence of dissociable protons, vicinal hydroxyl groups and conjugated double bonds [125,127,128].

In the case of flavonoids, the structural indications for high antioxidant capacity seem to be (i) an ortho 3′, 4′-dihydroxy moiety in the B-ring (Figure 14 red), (ii) a 2,3-double bond in combination with the 4-oxo group and a hydroxyl group in position 3 (Figure 14 blue), and (iii) a meta 5, 7-dihydroxy moiety in the A-ring in combination with the 4-oxo group (Figure 14 green), because these ensure the capacity for good radical stabilization, which is achieved by delocalization of the unpaired electron in the aroxyl radical structure [129].

In general, the B-ring hydroxylation pattern is regarded as being the most significant determinant [130–133], whereas the rest of the molecule seems to become more important only in cases in which only one hydroxyl group in ring B is present or if the hydroxyl group at position 4′ is inactivated by methylation [126]. The A-ring hydroxylation pattern in contrast seems to correlate little with antioxidant activity [124]. A free hydroxyl group in position 3 (ring C) increases the antioxidant activity by the formation of intramolecular hydrogen bonds with the B-ring. This influences the torsion angle of the B-ring with respect to the free molecule and results in a planar molecule, which allows conjugation, electron dislocation and an increased radical stability [124,134]. Flavonols seem to have the highest antioxidant activities and are also easily available commercially, which is well-reflected by the fact that they represent the best studied compounds [27].

Some flavonoids also show prooxidant activities and these also depend on the number of hydroxyl substituents and on the presence of a conjugation system between rings A and B [135]. Pyrogallol structures (3 adjacent hydroxyl groups) in rings A and B seem to be particularly effective in promoting pro-oxidant activity [124]. Thus, antioxidant and pro-oxidant activity of flavonoids are triggered to some extent by the same structural attributes; however, their pro-oxidant activity largely depends on the presence of divalent Cu or Fe ions.

Flavonoids are well-investigated chelators that frequently bind metal ions in the bidentate mode. Chelation of transition metal ions is an important characteristic contributing to a high antioxidant activity. The most common flavonoids have two to three potential metal-binding sites (I–III in Figure 15), but rare structures with additional hydroxyl groups in the A-ring may have up to six (IV-VI in Figure 15). Keto groups show a lower metal affinity, therefore site I is preferred over II and III. However, site II is the most likely chelation site for Fe ions [136]. Methylated or glucosylated phenol groups are not able to bind metals. Flavonoids with more than one metal-binding site can simultaneously chelate the corresponding number of metal ions, but vicinal sites, e.g., II and III, cannot be used at the same time. Depending on the pH and the concentration of both metal and ligand 1:1, 2:1 and 3:1 complexes can be formed. Thus, oligomeric and polymeric structures can occur in the presence of suitable flavonoid ligands and metal ions, which results in aggregation effects and a decreased ability of to pass through membranes [128]. Complexes of flavonoids with transition metal ions (Fe²⁺, Fe³⁺, and Cu²⁺) have significantly higher radical scavenging properties e.g., superoxide dismutase activity [137,138].

The capacity for metal chelation also results in the inhibition of enzymes with metal ion(s) in the catalytic domain, e.g., the copper-containing metalloenzyme polyphenol oxidase [130]. The highest inhibition (lowest IC₅₀ values) is observed with 8-hydroxyluteolin, which can simultaneously interact with metal ions at sites I and IV (Figure 15). In contrast, flavonoids carrying an additional hydroxyl group in position 6 and therefore possessing a comparable dual binding site (I/V and I/VI respectively), show only a low inhibitory activity [130]. Moreover, the hydroxyl group in position 3 drastically reduces the inhibitory activity and therefore flavones are better polyphenol oxidase inhibitors than flavonols. Thus, the inhibitory effect depends on the number of hydroxyl groups on the ether side (see Figure 15) of the flavonoid molecule rather than on the total number of hydroxyl groups.

The antiinfective activity of flavonoids has been largely investigated [35]. It seems to be based on a diverse range of effects, and there is no clear and general correlation between the hydroxylation pattern of flavonoids and their antiinfective activity [35]. However, in some cases such a relation was reported. Thus, the antibacterial activity of flavanones against methicillin-resistant Staphylococcus aureus and cariogenic Streptococcus sp. depends on a m-dihydroxy pattern in rings B and A [35,139]. The antimicrobial activity of isoflavonoids and dihydrochalcones was reported to be linked to the number of hydroxyl groups [140,141]. Likewise, monomeric and condensed flavanols with a higher degree of hydroxylation in their B-rings showed increased anthelminitic effects [114,116].

In the pathogen defence of plants, there is a prominent example for flavonoid-type phytoalexins with a specific hydroxylation pattern – particularly in ring C. These flavonoids are characterised by a lack of hydroxyl group in position 3. 3-Deoxyanthocyanidins are formed de novo after pathogen attack in a limited number of plants, e.g., sorghum and sugar cane [142–145]. As a rule, the tissues accumulate common 3-hydroxyanthocyanins, which are formed by a light-dependent gene set, whereas 3-deoxyanthocyanins are synthesized only in the case of infection by a separate gene set which is regulated independently of light [143,144,146–149]. The possible contribution of flavan-4-ols, which are the immediate precursors for 3-deoxyanthocyanidin formation, to the observed antimicrobial activity of 3-deoxyanthocyanidin based phytoalexins in vivo has not yet been investigated. Strong antimicrobial activity was reported for luteoforol (flavan-4-ol with a 3′,4′-hydroxylation pattern, Figure 9) [150,151]. The lack of comparable data for the corresponding flavan-3,4-diols is mainly based on the instability of these compounds. Compared with catechin, 3-deoxycatechin (luteoliflavan) did not show enhanced antimicrobial activity with respect to Erwinia amylovora [152,153].