*2.2.2 Methylation*

Methylated flavonoids are a form of natural flavonoid derivative with possibly many health advantages, including enhanced bioavailability when compared to flavonoid precursors [21]. According to studies, methylating these flavonoids might boost their promise as pharmacological agents, leading to innovative uses [22]. Flavonoids have been shown to have a wide range of bioactivities, including anticancer, immunomodulation, and antioxidant activities, which can be enhanced to some extent by methylation [21]. Methylation of flavonoids through their free hydroxyl groups or C atoms significantly boosts their metabolic stability and improves membrane transport, resulting in easier absorption and significantly enhanced oral bioavailability [22]. Although cinnamic acid derivatives are methylated at the phenylpropanoid level in certain circumstances, and feruloyl-CoA serves as a poor substrate for chalcone synthase in others, methylation mainly happens at the C15 level. Poulton presents an overview of plant transmethylation and demethylation processes [23]. Grisebach documented the substrate specificities of several O-methyltransferases from parsley and soybean cell cultures, as well as shoots of *Chrysosplenium americanum* [24], while Heller and Forkmann added further test characteristics [25]. The methyl donor in all of these reactions is S-adenosyl-L-methionine. Ibrahim's group has now identified five different O-methyltransferases in the flavonol pathway from *C. americanum* [26].

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

#### *2.2.3 Glycosylation*

The stability of flavonoids under glycosylation reaction circumstances is an important element to consider. For some flavonoids, direct glycosylation might result in the degradation of the changed molecules. It should also be noted that glycosylation is more than just attaching the carbohydrate residue to the flavonoid component of the molecule; it also involves the removal of the protecting groups. Some flavonoids may be partially destroyed as a result of this procedure [27]. It is generally agreed that glycosylation is a late or terminal step in flavonoid glycoside biosynthesis, except for acylation and prenylation reactions. Since the glycosylation step converts the flavonoid into a more water-soluble constituent, a step necessary for the retention of some flavonoids in the vacuole, the site of the glycosylation might be expected to act at the tonoplast boundary during transfer via the cisternae of, or vesicles derived from the endoplasmic reticulum [16]. The process of direct glycosylation for some classes of flavonoids can lead to the destruction of the modified compounds. There are two major types of linkages that form either O-glycosides or C-glycosides. Parsley preparations contain both types. There is a strict specificity for the position of the hydroxyl group, generally at the 3, 5, and 7 positions of the C- and A-rings. Both 3′ and 4' B-ring glycosides are known. Recently, rarer 2′ and 5′ glycosides of highly methylated flavonol glucosides have been identified in Ibrahim's laboratory [26].

#### *2.2.4 Acylation*

Many flavonoid families include acylated sugars. They exhibit a variety of physicochemical characteristics and biological activity; however, they have limited solubility and stability. To make use of these features, various publications have indicated that enzymatic acylation of these molecules with fatty and aromatic acids by protease and lipase under varied working conditions is a potential strategy. However, it is critical to strike a balance between increasing stability and solubility while maintaining biological activity. In fact, the acylation site (regioselectivity) can significantly alter these features [28]. The acyl groups are often aromatic acids like hydroxycinnamic acids or aliphatic acids like malonic acid. They appear to be position-specific for glucoside. Malonyl glucosides, which are catalyzed by malonyl transferases, are found in isoflavonoids, flavones, flavonols, and potentially anthocyanins. O-Malonyltransferases were isolated from parsley, which included malonated flavones and flavonols. Aromatic acylation, particularly of anthocyanins, has been observed in Silene and Matthiola sp. In both cases, the acyl groups transferred were either 4-coumaroyl or caffeoyl. Acylation has been observed to promote flavonoid absorption into parsley vacuoles; alterations in the molecular symmetry of the malonylglucosides are thought to be responsible for flavonoid vacuolar entrapment inside the vacuole [29].
