Chalcone Scaffolds, Bioprecursors of Flavonoids: Chemistry, Bioactivities, and Pharmacokinetics

Chalcones are secondary metabolites belonging to the flavonoid (C6-C3-C6 system) family that are ubiquitous in edible and medicinal plants, and they are bioprecursors of plant flavonoids. Chalcones and their natural derivatives are important intermediates of the flavonoid biosynthetic pathway. Plants containing chalcones have been used in traditional medicines since antiquity. Chalcones are basically α,β-unsaturated ketones that exert great diversity in pharmacological activities such as antioxidant, anticancer, antimicrobial, antiviral, antitubercular, antiplasmodial, antileishmanial, immunosuppressive, anti-inflammatory, and so on. This review provides an insight into the chemistry, biosynthesis, and occurrence of chalcones from natural sources, particularly dietary and medicinal plants. Furthermore, the pharmacological, pharmacokinetics, and toxicological aspects of naturally occurring chalcone derivatives are also discussed herein. In view of having tremendous pharmacological potential, chalcone scaffolds/chalcone derivatives and bioflavonoids after subtle chemical modification could serve as a reliable platform for natural products-based drug discovery toward promising drug lead molecules/drug candidates.


Introduction
Chalcones (or 1,3-diaryl-2-propen-1-ones) are one of the major secondary metabolites of plants belonging to the flavonoid family. These metabolites are abundantly present in edible plants. A majority of naturally occurring chalcones is polyhydroxylated aromatic compounds, and they are considered the bioprecursors of open chain flavonoids, flavonoids, and isoflavonoids. Due to the presence of phenolic groups, chalcones have a radical quenching property, which has created interest among researchers to investigate chalcone-rich plant extracts in search for therapeutically useful compounds. The therapeutic applications of chalcones have been associated since time immemorial for the treatment of different diseases [1]. Chalconarngenin, phloretin, and its glucosidephloridzin (phloretin 2 -O-glucose) are some of the most common chalcones present in food [2].
Chalcones and their structural analogues, either natural or synthetic, are known to exhibit diverse therapeutic and pharmacological activities such as antioxidant, antiinflammatory, antiplasmodial (antimalarial), antileishmanial, antitubercular, antimicrobial,

Biosynthesis of Chalcones
Chalcone is one of the precursors in the biosynthesis of flavonoids, isoflavonoids, anthocyanidins, proanthocyanidins, and other polyphenolic compounds [7]. Chalcone synthase (CHS) is the major enzyme that plays a vital role in the biosynthesis of chalcones [8,9]. The effectiveness of chalcone synthase (CHS) as an enzyme for chalcone formation is brought about by the presence of two active sites in the enzyme. One of the active sites referred to as the upper domain consists of four amino acids. The second active site referred as the lower domain is also essential for chalcone formation [7]. Phenylalanine is the major precursor for chalcones biosynthesis (phenylalanine is formed from chorismate as a precursor). p-Coumaroyl CoA and malonyl CoA are other important biomolecules required for the formation of chalcones. However, p-coumaroyl CoA is formed from phenylalanine [9]. Phenylalanine undergoes deamination at the aliphatic chain to form cinnamic acid. This is catalyzed by phenylalanine ammonia-lyase (PAL), which is followed by hydroxylation at the para position of the phenylalanine aromatic ring mediated by cinnamate-4-hydroxylase to form p-coumaric acid. Succinyl-CoA substitution of the

Occurrence of Chalcones
Chalcones are secondary plant metabolites, belonging to the flavonoid family that are abundantly present in edible plants, particularly fruits and vegetables. Therefore, chalcones belong to an important class of plant flavonoids (C 6 -C 3 -C 6 system) (Figure 1c). Chalcones and their derivatives are important intermediates of the flavonoid biosynthetic pathway. Flavonoids are an important group of naturally occurring bioactive compounds. The majority of naturally occurring chalcones are polyhydroxylated aromatic compounds abundantly found in fruits, grains, legumes, vegetables, and beverages such as tea, coffee, red wine, beer, etc. The medicinal benefits of polyhydroxylated chalcones are mainly attributed due to their free radical scavenging activity (antioxidant property), which in turn mitigates oxidative stress-induced tissue damage associated with some chronic disorders such as cardiovascular diseases, inflammatory diseases and neurological disorders, and certain infectious diseases [7][8][9].

Biosynthesis of Chalcones
Chalcone is one of the precursors in the biosynthesis of flavonoids, isoflavonoids, anthocyanidins, proanthocyanidins, and other polyphenolic compounds [7]. Chalcone synthase (CHS) is the major enzyme that plays a vital role in the biosynthesis of chalcones [8,9]. The effectiveness of chalcone synthase (CHS) as an enzyme for chalcone formation is brought about by the presence of two active sites in the enzyme. One of the active sites referred to as the upper domain consists of four amino acids. The second active site referred as the lower domain is also essential for chalcone formation [7]. Phenylalanine is the major precursor for chalcones biosynthesis (phenylalanine is formed from chorismate as a precursor). p-Coumaroyl CoA and malonyl CoA are other important biomolecules required for the formation of chalcones. However, p-coumaroyl CoA is formed from phenylalanine [9]. Phenylalanine undergoes deamination at the aliphatic chain to form cinnamic acid. This is catalyzed by phenylalanine ammonia-lyase (PAL), which is followed by hydroxylation at the para position of the phenylalanine aromatic ring mediated by cinnamate-4-hydroxylase to form p-coumaric acid. Succinyl-CoA substitution of the hydroxyl group occurs at the aliphatic carboxyl group of the p-coumaric acid to yield p-coumaroyl CoA by the enzyme 4-coumaroyl-coenzyme A ligase. CHS catalyzes the condensation of three molecules of malonyl CoA and p-coumaroyl CoA (one molecule) successively. The process also involves the decarboxylation, cyclization, and aromatization of malonyl CoA, which is mediated by  [9]. The biosynthesis of chalcones is depicted in Figure 2. hydroxyl group occurs at the aliphatic carboxyl group of the p-coumaric acid to yield p-coumaroyl CoA by the enzyme 4-coumaroyl-coenzyme A ligase. CHS catalyzes the condensation of three molecules of malonyl CoA and p-coumaroyl CoA (one molecule) successively. The process also involves the decarboxylation, cyclization, and aromatization of malonyl CoA, which is mediated by the four amino acids (Asn 336, His 303, Phe 215, and Cys 164) present in the active site of CHS [9]. The biosynthesis of chalcones is depicted in Figure 2. The chalcone formed is a biosynthetic precursor for various polyphenolic classes of natural products such as flavanones, flavonols, flavanols, dihydroflavonols, isoflavones, flavones, isoflavonoids, aurone, and anthocyanidins [4]. The biosynthesis of various chalcone bioprecursors is represented in Figure 3.
The formation of prenylated chalcones has been reported to be mediated by prenyltransferase, which plays a significant role in transferring prenyl units to an acceptor molecule from an isoprenyl source, which is usually dimethylallyl pyrophosphate (DMAPP) (Figure 4a) [10].
In the formation of methoxylated chalcones, methylation takes place through a catalytic mediation of S-adenosyl-L-methionine-dependent-O-methyltransferase (OMTs) [11]. It mediates the transfer of a methyl group from a donor (S-adenosyl-L-methionine) to an acceptor molecule. Methylenedioxy chalcone is generated through the formation of methylenedioxy bridges and catalyzed by cytochrome P450-dependent enzymes alongside NADPH, which acts as a cofactor (Figure 4b) [12,13]. Retro chalcones have been reported to be formed by the inversion of α, β-unsaturated ketone during the biosynthesis of 6′-deoxychalconeisoliquiritigenin ( Figure 4c) [14]. It has been reported that the presence of CHS and a polyketide reductase (CHR) as the active enzymes in a biosynthetic process generates 6′-deoxychalcones ( Figure 4d) [13].
During chalcone biosynthesis, the linkage of a sugar molecule catalyzed by the enzyme uridine diphosphate glycosyltransferase yields glycosylated chalcones. In this case, a nucleophilic substitution reaction is used to transfer the sugar molecule from a donor molecule (UDP-glycoside) to an acceptor molecule [15,16]. The chalcone formed is a biosynthetic precursor for various polyphenolic classes of natural products such as flavanones, flavonols, flavanols, dihydroflavonols, isoflavones, flavones, isoflavonoids, aurone, and anthocyanidins [4]. The biosynthesis of various chalcone bioprecursors is represented in Figure 3.
The formation of prenylated chalcones has been reported to be mediated by prenyltransferase, which plays a significant role in transferring prenyl units to an acceptor molecule from an isoprenyl source, which is usually dimethylallyl pyrophosphate (DMAPP) (Figure 4a) [10].
In the formation of methoxylated chalcones, methylation takes place through a catalytic mediation of S-adenosyl-L-methionine-dependent-O-methyltransferase (OMTs) [11]. It mediates the transfer of a methyl group from a donor (S-adenosyl-L-methionine) to an acceptor molecule. Methylenedioxy chalcone is generated through the formation of methylenedioxy bridges and catalyzed by cytochrome P450-dependent enzymes alongside NADPH, which acts as a cofactor (Figure 4b) [12,13]. Retro chalcones have been reported to be formed by the inversion of α, β-unsaturated ketone during the biosynthesis of 6deoxychalconeisoliquiritigenin ( Figure 4c) [14]. It has been reported that the presence of CHS and a polyketide reductase (CHR) as the active enzymes in a biosynthetic process generates 6 -deoxychalcones ( Figure 4d) [13].
During chalcone biosynthesis, the linkage of a sugar molecule catalyzed by the enzyme uridine diphosphate glycosyltransferase yields glycosylated chalcones. In this case, a nucleophilic substitution reaction is used to transfer the sugar molecule from a donor molecule (UDP-glycoside) to an acceptor molecule [15,16].

Naturally Occurring Chalcones
Chalcones occurring in nature have plants as their major source. They are usually found either in medicinal plants or in dietary plants. In nature, chalcones can be found as chalcone derivatives and flavonoids [17]. Chalcone derivatives of medicinal importance can be chemically synthesized in the laboratory by chemical modification of the parent chalcone scaffolds with a diverse range of structural substitutions [18].
Other chalcone constituents such as phloretin-3 ,5 -di-C-glucoside present in tomatoes have been reported to possess antioxidant properties [96]. Panduratin A, boesenbergin A, and pinostrobin chalcone in tomatoes have been reported for their aphrodisiac properties [110].
The bioactivities of chalcones obtained from medicinal plants are illustrated in Table 1. 7

Humulus lupulus L. Xanthohumol B and dihydroxanthohumol
Anti-inflammatory activity by inhibition of production of NO due to the suppression of iNOS [136] 9

Pharmacokinetics and Toxicities of Chalcones
Although chalcones have a wide range of pharmacological activities, the unavailability of sufficient bioavailability and bioaccessibility data in humans is a major challenge toward their development as therapeutic agents [159]. Synthetic chalcones have been widely studied, whereas the bioavailability of chalcones from natural sources is limited. The expected level of in vivo efficacy in preclinical evaluation has not been reached yet due to poor bioavailability profile. However, optimization of the physiochemical properties of chalcone derivatives could be an important step in their further development as lead molecules or drug candidates. The adsorption, distribution, metabolism, excretion, and toxicity (ADMET) of some naturally occurring chalcones have been studied, but the data do not satisfactorily support their ADMET profile [160,161] (Figure 7).

Pharmacokinetics and Toxicities of Chalcones
Although chalcones have a wide range of pharmacological activities, the unavailability of sufficient bioavailability and bioaccessibility data in humans is a major challenge toward their development as therapeutic agents [159]. Synthetic chalcones have been widely studied, whereas the bioavailability of chalcones from natural sources is limited. The expected level of in vivo efficacy in preclinical evaluation has not been reached yet due to poor bioavailability profile. However, optimization of the physiochemical properties of chalcone derivatives could be an important step in their further development as lead molecules or drug candidates. The adsorption, distribution, metabolism, excretion, and toxicity (ADMET) of some naturally occurring chalcones have been studied, but the data do not satisfactorily support their ADMET profile [160,161] (Figure 7).
Studies have shown that amongst many natural chalcones, prenylated derivatives are bioavailable, but they exhibit low bioaccessibility. One such chalcone is xanthohumol obtained in hop plant (Humulus lupulus), which upon oral administration by force feeding at extremely higher dosage to rodents (1 g/kg body weight) produces good oral bioavailability, but it does not obtain appreciable accessibility at the site of action. Xanthohumal 4 -O-glucoronide has been found to be the major metabolite in plasma, and unmetabolized xanthohumol has also been detected ten times less concentration after 4 h post administration [162]. In vitro metabolism studies indicate that xanthohumal in human and rat liver microsomes can be freely converted to glucuronides [163]. Gil-Izquierdo et al. (2001) studied the bioavailability of diversely processed juice of Citrus sinensis (L.) by mimicking in vitro digestion in stomach as well as the small intestine [164]. They have reported that in mild alkaline medium, 50-60% of the dissolved flavanones (mainly hesperidine) becomes converted to chalcones (hisperidin chalcone). Due to the poor solubility of these chalcones, the bioequivalence is not achieved to the expected level [165,166]. Another chalcone derivative is cardamonin, which is obtained from plants belonging to the Zingiberacea family, which has been reported to be poorly absorbed upon oral administration exhibiting 18% oral bioavailability in mice. It exhibited a high volume of distribution, short mean residence, high clearance, and was excreted in bile in its conjugated and unchanged form.

Conclusions and Future Directions
Chalcone scaffolds considered as the key bioactive precursors of plant flavonoids possess huge chemical and biological potential with significance in medicinal chemistry and pharmacology in current times. The chemistry and biological importance of naturally occurring chalcones have not been extensively explored. However, regardless of its versatile medicinal importance, the pharmacokinetics of plant-derived/dietary chalcones is a major challenge. Moreover, there is a lack of preclinical or clinical data on naturally occurring chalcones in the current literature. Further in-depth research studies are required to be carried out to address the pharmacokinetic issues and toxicological aspects related to naturally occurring chalcones and chalcone-derived flavonoids. There are ample scopes for the discovery of lead molecules or drug candidates from naturally occurring bioactive chalcones. Therefore, the proper chemical derivatization of natural chalcones is necessary to obtain novel flavonoid molecules that would play a vital role in the chalcone scaffolds-based discovery of drug molecules.  Zhao et al. (2020) studied the pharamacokinetics of phloretin, a naturally occurring dihydrochalcone flavonoid found in apple, pear, roots peels, and juicy fruits peels, by orally administering it to Sprague-Dawley rats. Absorption mechanisms have been investigated in a Caco-2 cell monolayer and by a single pass intestinal perfusion in rats [167]. Phloretin is transported through active transport, efflux protein transport, and by cell bypass. It has been reported to be a substrate of P-glycoprotein (P-gp) and multi-drug resistance protein (MRP2) and found to have low oral bioavailability (8.676%) with colon as the best absorption site.
Naturally occurring chalcones have also been found to affect the pharmacokinetic parameters of drugs when administered simultaneously. Choi et al. (2014) investigated the effect of licochalcone A on the pharmacokinetics of nifedipine and its metabolite dehydronifedipine in rats. Hepatic CYP3A4 metabolizes nifedipine. Oral administration of nifedipine with licochalcone A has been found to inhibit CYP3A4 as well as exhibit the cellular accumulation of rhodamine-123 in MCF-7/ADR cells overexpressing P-gp, leading to a higher peak plasma concentration (Cmaxs) [168]. Boonnop et al. (2017) proposed that the co-administration of Boesenbergia rotunda extract with therapeutic drug may cause herb-drug interaction, leading to an alteration of the efficacy and toxicity of the drug. Panduratin A isolated from the Boesenbergia rotunda has been reported to cause herb-drug interaction and alter renal cationic drug clearance by inhibiting organic cation transporters (OCT2), which are responsible for the renal excretion of cationic drugs [169].
In view of the above facts, to design a chalcone derivative with acceptable ADMET properties, the maximization of its physiochemical properties with modification in the chemical structure would play a crucial role.

Conclusions and Future Directions
Chalcone scaffolds considered as the key bioactive precursors of plant flavonoids possess huge chemical and biological potential with significance in medicinal chemistry and pharmacology in current times. The chemistry and biological importance of naturally occurring chalcones have not been extensively explored. However, regardless of its versatile medicinal importance, the pharmacokinetics of plant-derived/dietary chalcones is a major challenge. Moreover, there is a lack of preclinical or clinical data on naturally occurring chalcones in the current literature. Further in-depth research studies are required to be carried out to address the pharmacokinetic issues and toxicological aspects related to naturally occurring chalcones and chalcone-derived flavonoids. There are ample scopes for the discovery of lead molecules or drug candidates from naturally occurring bioactive chalcones. Therefore, the proper chemical derivatization of natural chalcones is necessary to obtain novel flavonoid molecules that would play a vital role in the chalcone scaffoldsbased discovery of drug molecules.