**1. Introduction**

Peels represent a large percentage of the total weight of fruits, for example, 50–65% of *Citrus* fruits (lemon, lime, orange, and grapefruit) [1]. During processing of fruits for juice and oil extractions, the peels remain as the primary byproducts and become waste if not processed further, which can lead to serious environmental pollution [1]. Therefore, the fruit-processing industries are also interested in making use of these wastes.

The peels are also a good commercial source of pectins (polygalacturonic acid) and flavonoids [1]. The pectins are polysaccharide macromolecules contained in the primary cell wall of plants and involved in controlling cell wall ionic status, cell expansion, and separation [1]. Usually, the pectins are commercially extracted and isolated from *Citrus* peels and apple pomace. They are not only used as a gelling agent, dessert filling, or juice and milk stabilizer in food industry but also as a source of dietary fiber. Flavonoids are a large group of small secondary metabolites contained in the vacuoles and possess a wide range of biological activities, especially those with human health benefits [2, 3]. In the *Citrus* peels, flavonoids mainly include flavones (e.g., rhoifolin, isorhoifolin, diosmin, and neodiosmin), flavanones (e.g., eriocitrin, neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, poncirin, and neoponcirin), and flavonols (e.g., rutin) [4]. It has been reported that the highest concentrations of *Citrus* flavonoids occur in the peels [1]. Due to the importance of pectins and flavonoids in food, cosmetic, and medicinal industries,

quite a number of studies have been focused on these two groups of compounds. Accordingly, a variety of approaches have been developed for efficient isolation of pectins and flavonoids from fruit peels and pomace. For example, to make a better use of yellow passion fruit rind, de Souza and colleagues have developed a strategy for sequential extraction of flavonoids and pectin [5].

As we know, pectins are abundant in the middle lamella of the plant cell walls with a gradual decrease in the content toward the plasma membrane, whereas flavonoids are naturally located within the cells [6]. Generally, flavonoids within the cells do not come into contact with the cell wall materials, such as pectins, celluloses, and hemicelluloses, prior to food processing. When fruits are processed and eaten, intracellular flavonoids can be released from the cells, leading to their interaction with substances like metal ions and plant cell wall materials [7, 8]. For example, procyanidins and anthocyanins can spontaneously bind to water-, chelator-, and sodium carbonate-soluble pectins. It is believed that the binding of flavonoids to cell wall materials results from noncovalent, hydrophobic, hydrogen bonding, and ionic interactions [9–11]. Recently, Chirug and colleagues have presented a novel possible mechanism that iron ions mediate the interaction between pectins and quercetin [6]. Such interaction might affect their shelf-life stability and functionality, as well as their bioavailability and bioaccessibility [6, 12]. Therefore, it could be of high importance to study their interaction. Since there are several reviews on the interaction [8, 13], we will not discuss it in this chapter. Instead, we will concentrate on understanding the current opinions on flavonoids, including the classification, biological activities, and biosynthetic pathway of these secondary compounds. We will then review the general strategies for derivation of these compounds, including the traditional plant extraction, chemical synthesis, and biosynthesis of these important small bioactive molecules in a microbial cell factory or an *in vitro* multienzyme synthetic platform. We will also discuss the advantages and disadvantages of these strategies and the future research directions in the field of flavonoid biosynthesis.

### **2. Classification and biological activities of flavonoids**

Flavonoids belong to a class of secondary metabolites and comprise a large group of natural products that are widespread in higher plants but also found in mosses and liverworts [14, 15]. Chemically, flavonoid compounds have the basic structure of 15-carbon atoms with two phenolic rings connected by a 3-carbon chain [16], forming a C6-C3-C6 carbon framework (**Figure 1**). Generally, these small molecules can be divided into six major subclasses on the basis of the variations on the

**149**

*Flavonoids and Pectins*

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

glycosylation in turn increases their water solubility [19].

phosphatase and acetylcholinesterase [75], and tyrosinase [44, 64].

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

**3. Biosynthetic pathway of flavonoids**

heterocyclic C-ring and the degree of oxidation: the flavanols, flavones, flavonols, flavanones, anthocyanidins, and isoflavones [2, 16–18]. The flavonoids can exist in a free aglycone form but are often glycosylated (most commonly glucose), and the

The flavonoids are involved in the formation of plant pigments [20] and protect plants against pathogens, herbivores, and UV radiation [21]. However, the study of flavonoids, like that of most natural products, has emerged from the search of new compounds with promising pharmacological properties. After decades of endeavors, scientists have found that flavonoids possess a wide variety of biological and pharmacological properties, which leads to numerous studies on these secondary metabolites. These health-beneficial properties include antiangiogenic [22], antibacterial [23–27], anti-cancer [24, 28], anti-inflammatory [28–33], antiglycating [34], antimalarial [35], antimicrobial [36–42], anti-oxidant [26, 36, 38, 42–51], anti-platelet [48], anti-proliferation [52], agonistic/antagonistic [53], ammonialowering and regulation of urea cycle [54], anxiolytic [55], atheroprotective [56], cardioprotective and hypouricemic [57], cytotoxic [51, 58], endocrine disrupting [59], free radical-scavenging [31–33, 39, 40, 46, 51, 52, 58, 60–66], hepatoprotective [67], leishmanicidal [68], neuroprotective [69], photoprotective [43], and trypanocidal activities [68, 70]. In addition, the flavonoids can inhibit eukaryotic protein synthesis [71] and a variety of important enzymes such as aggrecanase [72], aldose reductase [30, 73], alpha-glucosidase [60], cholinesterase [26, 74], protein tyrosine

**Figure 1.** *Structure and atom numbering of flavonoid backbone.*

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

*Pectins - Extraction, Purification, Characterization and Applications*

for sequential extraction of flavonoids and pectin [5].

**2. Classification and biological activities of flavonoids**

Flavonoids belong to a class of secondary metabolites and comprise a large group of natural products that are widespread in higher plants but also found in mosses and liverworts [14, 15]. Chemically, flavonoid compounds have the basic structure of 15-carbon atoms with two phenolic rings connected by a 3-carbon chain [16], forming a C6-C3-C6 carbon framework (**Figure 1**). Generally, these small molecules can be divided into six major subclasses on the basis of the variations on the

quite a number of studies have been focused on these two groups of compounds. Accordingly, a variety of approaches have been developed for efficient isolation of pectins and flavonoids from fruit peels and pomace. For example, to make a better use of yellow passion fruit rind, de Souza and colleagues have developed a strategy

As we know, pectins are abundant in the middle lamella of the plant cell walls with a gradual decrease in the content toward the plasma membrane, whereas flavonoids are naturally located within the cells [6]. Generally, flavonoids within the cells do not come into contact with the cell wall materials, such as pectins, celluloses, and hemicelluloses, prior to food processing. When fruits are processed and eaten, intracellular flavonoids can be released from the cells, leading to their interaction with substances like metal ions and plant cell wall materials [7, 8]. For example, procyanidins and anthocyanins can spontaneously bind to water-, chelator-, and sodium carbonate-soluble pectins. It is believed that the binding of flavonoids to cell wall materials results from noncovalent, hydrophobic, hydrogen bonding, and ionic interactions [9–11]. Recently, Chirug and colleagues have presented a novel possible mechanism that iron ions mediate the interaction between pectins and quercetin [6]. Such interaction might affect their shelf-life stability and functionality, as well as their bioavailability and bioaccessibility [6, 12]. Therefore, it could be of high importance to study their interaction. Since there are several reviews on the interaction [8, 13], we will not discuss it in this chapter. Instead, we will concentrate on understanding the current opinions on flavonoids, including the classification, biological activities, and biosynthetic pathway of these secondary compounds. We will then review the general strategies for derivation of these compounds, including the traditional plant extraction, chemical synthesis, and biosynthesis of these important small bioactive molecules in a microbial cell factory or an *in vitro* multienzyme synthetic platform. We will also discuss the advantages and disadvantages of these strategies and the future research directions in the field of flavonoid biosynthesis.

**148**

**Figure 1.**

*Structure and atom numbering of flavonoid backbone.*

heterocyclic C-ring and the degree of oxidation: the flavanols, flavones, flavonols, flavanones, anthocyanidins, and isoflavones [2, 16–18]. The flavonoids can exist in a free aglycone form but are often glycosylated (most commonly glucose), and the glycosylation in turn increases their water solubility [19].

The flavonoids are involved in the formation of plant pigments [20] and protect plants against pathogens, herbivores, and UV radiation [21]. However, the study of flavonoids, like that of most natural products, has emerged from the search of new compounds with promising pharmacological properties. After decades of endeavors, scientists have found that flavonoids possess a wide variety of biological and pharmacological properties, which leads to numerous studies on these secondary metabolites. These health-beneficial properties include antiangiogenic [22], antibacterial [23–27], anti-cancer [24, 28], anti-inflammatory [28–33], antiglycating [34], antimalarial [35], antimicrobial [36–42], anti-oxidant [26, 36, 38, 42–51], anti-platelet [48], anti-proliferation [52], agonistic/antagonistic [53], ammonialowering and regulation of urea cycle [54], anxiolytic [55], atheroprotective [56], cardioprotective and hypouricemic [57], cytotoxic [51, 58], endocrine disrupting [59], free radical-scavenging [31–33, 39, 40, 46, 51, 52, 58, 60–66], hepatoprotective [67], leishmanicidal [68], neuroprotective [69], photoprotective [43], and trypanocidal activities [68, 70]. In addition, the flavonoids can inhibit eukaryotic protein synthesis [71] and a variety of important enzymes such as aggrecanase [72], aldose reductase [30, 73], alpha-glucosidase [60], cholinesterase [26, 74], protein tyrosine phosphatase and acetylcholinesterase [75], and tyrosinase [44, 64].
