**3. Biosynthetic pathway of flavonoids**

After several decades of efforts, the pathway for flavonoid biosynthesis has been largely deciphered even though quite a number of details remain unknown (**Figure 2**). The flavonoids and their derivatives are biosynthesized by a variety of enzymes. These enzymes belong to different families [76], mainly including 2-oxoglutarate-dependent dioxygenase (2-ODD), cytochrome P450 hydroxylase, short-chain dehydrogenase/reductase (SDR), *O*-methyltransferase (OMT), and *O*-glycosyltransferase (GT). The 2-ODD, cytochrome P450, and SDR enzymes constitute the major pathway for flavonoid biosynthesis [76], and the OMT and GT enzymes are involved in modification of flavonoids. The involved 2-ODD enzymes mainly comprise flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), flavone synthase I (FSI), anthocyanidin synthase (ANS), and flavonol 6-hydroxylase (F6H) [17, 76–81]. The related cytochrome P450 enzymes contain cinnamate 4-hydroxylase (C4H), isoflavone synthase (IFS), flavanone 2-hydroxylase (F2H), flavone synthase II (FSII), flavonol 6-hydroxylase (F6H), flavonoid 3′-hydroxylase (F3'H), flavonoid 3′,5′-hydroxylase (F3′5′H), isoflavone 2′-hydroxylase (I2′H), and isoflavone 3′-hydroxylase (I3′H) [17, 18, 76, 80, 82, 83]. The SDR enzymes participating in flavonoid biosynthesis include dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANR) [76]. Interestingly, the flavone synthase (FS) activity is specified either by a 2-ODD (FSI) or a P450 (FSII) enzyme in a plant speciesdependent manner [84, 85]. Similarly, the flavonol 6-hydroxylase (F6H) activity is also endowed either by a 2-ODD [81, 86] or P450 [87, 88] enzyme in different plant species. These findings further increase the complexity of flavonoid biosynthesis.

Basically, biosynthesis of flavonoids can be arbitrarily divided into three major stages. The first stage (a.k.a the phenylpropanoid pathway) includes three successive chemical reactions catalyzed by phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumaroyl:CoA ligase (4CL), respectively, to convert l-phenylalanine to 4-coumroyl-CoA. In addition, l-tyrosine can also

#### **Figure 2.**

*Schematic of the biosynthetic pathway leading to the major subclasses of flavonoids. Adapted from [10, 12, 68]. 4CL, 4-coumaroyl:CoA ligase; ACC, acetyl CoA carboxylase; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; AS, aureusidin synthase; C4H, cinnamate 4-hydroxylase; CE: condensing enzyme; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; DMID, 7,2*′*-dihydroxy-4*′*-methoxyisoflavanol dehydratase; F3H, flavanone 3-hydroxylase; F3*′*H, flavonoid 3*′*-hydroxylase; F3*′*5*′*H, flavonoid 3*′*,5*′*-hydroxylase; FLS, flavonol synthase; FSI/FSII, flavone synthase I/II; I2*′*H, isoflavone 2*′*-hydroxylase; IFR, isoflavone reductase; IFS, isoflavone synthase; IOMT, isoflavone O-methyltransferase; LAR, leucoanthocyanidin reductase; LDOX, leucoanthocyanidin dioxygenase; OMT, O-methyltransferase; PAL, phenylalanine ammonia-lyase; PPO, polyphenol oxidase; RT, rhamnosyltransferase; TAL, tyrosine ammonia-lyase; UFGT, UDP flavonoid glucosyltransferase; VR, vestitone reductase.*

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*DOI: http://dx.doi.org/10.5772/intechopen.84960*

functional diversity of flavonoids.

**4. Derivation of flavonoids**

participate in the flavonoid biosynthesis via two successive enzymatic reactions catalyzed by tyrosine ammonia lyase (TAL) and 4CL, respectively. The second stage is crucial for the biosynthesis of flavonoids, in which the backbones of major subclasses of flavonoids are formed. This stage begins from the formation of chalcone by conversion of the 4-coumroyl-CoA from the first stage and the malonyl-CoA from carboxylation of acetyl-CoA. Chalcone synthase (CHS), an entry point enzyme into the pathway, catalyzes this chemical reaction by conversion of one molecule of 4-coumroyl-CoA and three molecules of malonyl-CoA to one molecule of chalcone (e.g., tetrahydroxychalcone). Then, the chalcone molecule is cyclized to form a flavanone (e.g., naringenin) by chalcone isomerase (CHI) and an aurone (e.g., aureusidin) by aureusidin synthase (AS). The flavanone can be further converted to dihydroflavonol by F3H and then flavonol by FLS. Alternatively, the flavanone molecule can also be converted to a flavone by FS, a flavanol by DFR, an isoflavone by IFS, and an anthocyanidin by a series of successive enzymatic reactions catalyzed by F3H, DFR, and leucoanthocyanidin dioxygenase (LDOX), respectively. The resulting anthocyanidin molecule can be further modified to form anthocyanins by a series of chemical modifications by OMT, UDP flavonoid glucosyltransferase (UFGT), and rhamnosyltransferase (RT). The third stage is mainly involved in various chemical decorations of flavonoids. Generally, natural flavonoids are often extensively modified by chemical reactions, including glycosylation and methylation [76], acylation [89], sulfonation [90, 91], prenylation [92, 93], and galloylation [94], which further contribute to the structural and

Due to the intrinsic health benefits possessed by flavonoids, numerous approaches have been developed during the past decades for the derivation of a wide range of flavonoids. Basically, these approaches can be divided into three major categories: traditional plant extraction, chemical synthesis, and biosynthesis.

Traditionally, flavonoids are extracted from various plant species, which currently remains the most commonly used methods. During the past decades, researchers have developed plenty of methods to improve the yield and purity of flavonoids derived from plants. Generally, the plant tissues are air-dried and ground into powder for extraction via organic solvents (most commonly methanol and ethanol), and the extracts are then subjected to successive fractionation with other organic solvents (most commonly petroleum ether, chloroform, ethyl acetate, and n-butyl alcohol), followed by repeated silica gel and Sephadex LH-20 column chromatographies [44, 95]. The yield of plant-derived flavonoids can be improved by ultrasonic wave- [96], microwave- [97], and enzyme-assisted extraction [98]; aqueous two-phase extraction [99]; and a combination of these modifications [100]. The isolated flavonoids are then subjected to polyamide thin plate chromatography (TLC), high performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR) analyses to determine their identity and purity [2, 3]. Due to the high solubility of most flavonoids in organic solvents, this strategy often demonstrates a high efficiency in the derivation of flavonoids from plant tissues. However, the disadvantage of the plant extraction is obvious. Due to the very low content of most flavonoids in plant tissues, the extraction and isolation of flavonoids often requires multiple

**4.1 Traditional plant extraction via organic solvents**

#### *Flavonoids and Pectins DOI: http://dx.doi.org/10.5772/intechopen.84960*

*Pectins - Extraction, Purification, Characterization and Applications*

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**Figure 2.**

*Schematic of the biosynthetic pathway leading to the major subclasses of flavonoids. Adapted from [10, 12, 68]. 4CL, 4-coumaroyl:CoA ligase; ACC, acetyl CoA carboxylase; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; AS, aureusidin synthase; C4H, cinnamate 4-hydroxylase; CE: condensing enzyme; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; DMID, 7,2*′*-dihydroxy-4*′*-methoxyisoflavanol dehydratase; F3H, flavanone 3-hydroxylase; F3*′*H, flavonoid 3*′*-hydroxylase; F3*′*5*′*H, flavonoid 3*′*,5*′*-hydroxylase; FLS, flavonol synthase; FSI/FSII, flavone synthase I/II; I2*′*H, isoflavone 2*′*-hydroxylase; IFR, isoflavone reductase; IFS, isoflavone synthase; IOMT, isoflavone O-methyltransferase; LAR, leucoanthocyanidin reductase; LDOX, leucoanthocyanidin dioxygenase; OMT, O-methyltransferase; PAL, phenylalanine ammonia-lyase; PPO, polyphenol oxidase; RT, rhamnosyltransferase; TAL, tyrosine* 

*ammonia-lyase; UFGT, UDP flavonoid glucosyltransferase; VR, vestitone reductase.*

participate in the flavonoid biosynthesis via two successive enzymatic reactions catalyzed by tyrosine ammonia lyase (TAL) and 4CL, respectively. The second stage is crucial for the biosynthesis of flavonoids, in which the backbones of major subclasses of flavonoids are formed. This stage begins from the formation of chalcone by conversion of the 4-coumroyl-CoA from the first stage and the malonyl-CoA from carboxylation of acetyl-CoA. Chalcone synthase (CHS), an entry point enzyme into the pathway, catalyzes this chemical reaction by conversion of one molecule of 4-coumroyl-CoA and three molecules of malonyl-CoA to one molecule of chalcone (e.g., tetrahydroxychalcone). Then, the chalcone molecule is cyclized to form a flavanone (e.g., naringenin) by chalcone isomerase (CHI) and an aurone (e.g., aureusidin) by aureusidin synthase (AS). The flavanone can be further converted to dihydroflavonol by F3H and then flavonol by FLS. Alternatively, the flavanone molecule can also be converted to a flavone by FS, a flavanol by DFR, an isoflavone by IFS, and an anthocyanidin by a series of successive enzymatic reactions catalyzed by F3H, DFR, and leucoanthocyanidin dioxygenase (LDOX), respectively. The resulting anthocyanidin molecule can be further modified to form anthocyanins by a series of chemical modifications by OMT, UDP flavonoid glucosyltransferase (UFGT), and rhamnosyltransferase (RT). The third stage is mainly involved in various chemical decorations of flavonoids. Generally, natural flavonoids are often extensively modified by chemical reactions, including glycosylation and methylation [76], acylation [89], sulfonation [90, 91], prenylation [92, 93], and galloylation [94], which further contribute to the structural and functional diversity of flavonoids.
