**2. Overview of chemistry and metabolism of flavonoid**

Flavonoids are phytonutrients that belong to the polyphenol class. Polyphenols have been employed in Chinese and Ayurvedic medicine for centuries. A novel chemical was extracted from oranges in 1930. It was thought to be a member of a novel class of vitamins at the time and was labeled as vitamin P. Later, it was discovered that this chemical was a flavonoid (rutin), and over 4000 other flavonoids have been found [14].

#### **2.1 Basic chemistry**

#### *2.1.1 General characteristics of the C15 unit*

Flavonoids exist in the form of aglycones, glycosides, and methylated derivatives. Flavonoids have a diphenyl propane skeleton with 15 carbon atoms in their main nucleus: two six-membered rings coupled with a three-carbon unit that may or may

*Application of Liquid Chromatography in the Analysis of Flavonoid Metabolism in Plant DOI: http://dx.doi.org/10.5772/intechopen.107182*

**Figure 1.** *General structure of flavonoid.*

not be part of a third ring. Two benzene rings (A and B in **Figure 1**) are primarily connected together by a third heterocyclic oxygen-containing pyrene ring (C) [15].

Isoflavones are flavonoids in which the B ring is connected in position 3 of the C ring. Those with the B ring linked in position 4, are referred to as neoflavonoids whereas those with the B ring linked in position 2 can be further classified into many subgroups based on the structural characteristics of the C ring. Flavones, flavonols, flavanones, flavanonols, flavanols or catechins, anthocyanins, and chalcones are the subclasses [1].

#### *2.1.2 Hydroxylation patterns of A-, B-, and C-rings*

Positions 3, 5, 7, 2, 3′, 4′, and 5′ are frequently hydroxylated in flavonoids. The most prevalent A-ring hydroxylation pattern is 5,7-hydroxylation; however, a 7-hydroxy ring (also known as a 5-deoxy ring) is seen in isoflavonoid subgroups and several proanthocyanidins. On rare occasions, a 5,7,8 or 5.6.7-hydroxylation pattern is discovered. The B-ring is often 4′-, 3,4′-, or 3′,4,5′-hydroxylation. Rare flavonoids do not have B-ring oxygenation. A 2′-hydroxylation pattern is present in isoflavonoids. In isoflavonoids, the C ring is commonly hydroxylated at the carbon 3 position and occasionally at the carbon 6a position. This six-membered ring can have a carbonyl group, a hydroxyl group, a double bond between positions 2 and 3, or it can be totally unsubstituted, as in unsubstituted flavans. The isoflavonoid pterocarpans have extra rings as a consequence of 2′-hydroxylation of the original B-ring or cyclization of the added prenyl groups (**Figure 2**) [16].

#### **2.2 Basic substitution**

#### *2.2.1 Hydroxylation*

There are just a few flavonoid structures with no hydroxyl groups in the A-ring or one hydroxyl group in position 6 [17]. Such unusual structures appear to occur most frequently in the Primulaceae, Rutaceae, and Thymelaeaceae groups. However, the mechanisms of their biochemical synthesis remains unclear. The great majority of flavonoids have a basic 5,7-hydroxylation pattern of the A-ring, which is formed from malonyl-CoA during chalcone synthesis.

The C6-C3 precursor employed by chalcone synthase determines the hydroxylation pattern of the B-ring first. The physiological standard precursor is typically p-coumaroyl-CoA (4-hydroxycinnamoyl-CoA). The resultant basic C15 chalcone

#### **Figure 2.**

*Structure backbone of the main flavonoid group.*

intermediate, naringenin chalcone, has a hydroxyl group in position 4′, which is seen in typical flavonoid structures, and all derived flavonoid structures have the hydroxyl group in position 4′. Thus, cinnamate 4-hydroxylase, a cytochrome P450-dependent monooxygenase that catalyzes the production of p-coumaric acid [18, 19], performs a pre-flavonoid step by introducing the hydroxyl group in position 4′.

The majority of flavonoid families have a hydroxyl group in C-ring position 3. (**Figure 1**). The well-studied flavanone 3-hydroxylase, a 2-oxoglutarate, Fe(II), and ascorbate-dependent dioxygenase [20], introduces the hydroxyl group at the flavanone level. The soluble dioxygenase catalyzes the 3-hydroxylation of the flavanone C-ring to create 3-hydroxyflavanone (flavanol). The same dioxygenase has also been linked to the catalysis of flavone synthases in several plants, with a 2-hydroxylation of the flavanone C-ring proposed as an intermediary step [16].
