Silymarin is a pharmacologically active structural complex extracted from the seeds of milk thistle and is present in a range of 1.5% to 3% of the seed weight (Flora et a., 1998; Martin et al., 2006; Abenavoli et al., 2010).

Structural complexes are composed of flavonolignan isomers, and the major isomers include silybin (silibinin), isosilybin (isosilibinin), silychristin, isosilychristin, and silydianin (Deep et al., 2008; Valková et al., 2021). Of the isomers, the principal active compound is silybin, representing approximately 50% - 60% (Saller et al., 2001). Silybin, isosilybin, and silychristin form two diastereoisomers, namely A and B (Smith et al., 2005). Silymarin is a flavonolignans amalgamated with phenylpropane and flavonoid units (Althagafy et al., 2013; Bijak, 2017).

Phenylpropane and flavonoid units are structurally related to coniferyl alcohol and taxifolin, respectively. The structures of silymarin components are shown in Fig. 1.

Fig. 1.  Chemical structure of various silymarin components.

(A) silybin A, (B) silybin B, (C) isosilybin A, (D) isosilybin B, (E) silychristin A, (F) silychristin B, (G) isosilychristin, and (H) silydianin. Representative examples of silymarin containing flavonid (retangle by straight lines) and phenylpropane moiety (retangle by dotted lines).

One of the silymarin precursors, coniferyl alcohol, is synthesized via the monolignol biosynthetic pathway, which plays a major role in producing source materials for lignin biosynthesis (Wang et al., 2019).

The overall biosynthetic pathway of coniferyl alcohol is presented in Fig. 2. Monolignol belongs to one of the phenylpropanoid classes, and its pathway starts with the phenylpropanoid biochemical pathway (Vogt, 2010).

Fig. 2.  This schematic view present coniferyl alcohol biosynthetic pathway.

The aromatic acids (L-phenylalanine and L-tyrosine) are converted to hydroxycinnamic acids (p-coumaric acid, caffeic acid, and ferulic acid), hydroxycinnamoyl-CoA thioesters (p-coumaroyl, caffeoyl, and feruloyl-CoA), hydroxycinnamaldehydes (p-coumaryl, caffeyl, and coniferyl-aldehyde), and hydroxycinnamyl alcohols (p-coumaryl, caffeyl, and coniferyl alcohol) by several enzymes. The enzymes involved in this pathway are phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), cinnamate 4-hydroxylase (C4H), p-coumarate 3-hydroxylase (C3H), p-coumaroylester 3′-hydroxylase (C3′H), caffeic acid 3-O-methyltransferase (COMT), caffeoyl-CoA 3-O-methyltransferase (CCOMT), 4-coumaric acid:coenzyme A ligase (4CL), hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), caffeoyl shikimate esterase (CSE), cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD).

The phenylpropanoid pathway begins with the use of the aromatic amino acids phenylalanine and tyrosine, which are the end products of the shikimic acid pathway (Tzin and Galili, 2010; Santos-Sánchez et al., 2019).

L-Phenylalanine is catalyzed into trans-cinnamic acid by the deamination of phenylalanine ammonia-lyase (Koukol and Conn, 1961). Cinnamate 4-hydroxylase, which belongs to the CYP73A family of cytochrome P450 monooxygenases, catalyzes the p-hydroxylation of trans-cinnamic acid to yield p-coumaric acid (Hahlbrock and Scheel, 1989; Duan et al., 2004; Zhang et al., 2020). In several studies, tyrosine ammonia-lyase has also been shown to convert L-tyrosine to p-coumaric acid by deamination (Beaudoin-Eagan and Thorpe, 1985; Rosler et al., 1997; Nishiyama et al., 2010). This part, from aromatic amino acids to p-coumaric acid, is the general phenylpropanoid pathway.

The activity of p-coumarate 3-hydroxylase (C3H), which is a cytochrome P450 monooxygenase belonging to the CYP98 family, and caffeic acid 3-O-methyltransferase can transform p-coumaric acid into caffeic acid and ferulic acid, respectively (Inoue et al., 1998; Franke et al., 2002). 4-Coumaric acid: Coenzyme A ligase can convert hydroxycinnamic acids (p-coumaric acid, caffeic acid, and ferulic acid) to hydroxycinnamoyl-CoA thioesters (p-coumaroyl, caffeoyl, and feruloyl-CoA) by ligation of coenzyme A (CoA) (Chen et al., 2013).

p-Coumaroyl-CoA is treated as a precursor for the production of secondary plant metabolites, including flavonoids, and is catalyzed to caffeoyl-CoA by the two enzymes p-coumaroylester 3′-hydroxylases, which are cytochrome P450s belonging to the CYP98A3 family, and hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyl transferase with shikimate/quinate, leading to p-coumaroylquinic/p-coumaroylshikimic acid and caffeoylquinic/caffeoylshikimic acid (Hoffmann et al., 2004; Boudet, 2007; Mahesh et al., 2007).

Caffeoyl CoA 3-O-methyltransferase activates caffeoyl-CoA to produce feruloyl-CoA (Inoue et al., 1998). The production of hydroxycinnamaldehydes (p-coumaryl, caffeyl, and coniferyl-aldehyde) from hydroxycinnamoyl-CoA thioesters is achieved via activation of cinnamoyl-CoA reductase (Lauvergeat et al., 2001; Vanholme et al., 2019). Cinnamyl alcohol dehydrogenase converts hydroxycinnamaldehydes to hydroxycinnamyl alcohols (p-coumaryl, caffeyl, and coniferyl alcohol) (Liu et al., 2018). Caffeic acid 3-O-methyltransferase has high activity for caffeyl-aldehyde and caffeyl-alcohol to catalyze the conversion of coniferyl-aldehyde and coniferyl-alcohol, respectively (Parvathi et al., 2001).

In the biosynthesis of silymarin, the phenylpropane unit (coniferyl alcohol) and flavonoid unit (taxifolin) synthesis require p-coumaryl CoA, which activates both synthesis pathways (Torres and Corchete, 2016). The taxifolin biosynthetic pathway is shown in Fig. 3.

Fig. 3.  This schematic view present taxifolin biosynthetic pathway.

The p-coumaroyl-CoA and caffeoyl-CoA are converted to taxifolin by several enzymes. The enzymes involved in this pathway are chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), and homoeriodictyol/eriodictyol synthase (HEDS/HvCHS2).

Chalcone synthase, which is a key enzyme in the flavonoid biosynthesis pathway and a member of the plant polyketide synthase family, uses one p-coumaroyl CoA as a substrate and forms naringenin chalcone by a condensation reaction with three malonyl-CoA (Flores-Sanchez, 2008; Dao et al., 2011).

Chalcone isomerase, also known as chalcone-flavanone isomerase and belonging to the class of intramolecular lyases, catalyzes the conversion of naringenin chalcone into its corresponding flavanone, naringenin (Sun et al., 2019).

Flavonoid 3′-hydroxylase (F3′H) and flavanone 3-hydroxylase (F3H) are oxoglutarate-dependent dioxygenases and cytochrome P450 hydroxylases, respectively (Dixon and Steele, 1999; Winkel-Shirley, 2001). The sequential reaction of F3′H and F3H allows the catalysis of naringenin to eriodictyol and taxifolin (dihydroquercetin) (Hammerbacher et al., 2019). In addition, the other sequential reaction of F3H and F3′H catalyze the conversion of naringenin into dihydrokaempferol and taxifolin (Brugliera et al., 1999).

In other routes to taxifolin biosynthesis, homoeriodictyol/eriodictyol synthase uses one caffeoyl-CoA as a substrate to form eriodictyol chalcone as the reaction of the chalcone synthase catalyst, and the activity of chalcone isomerase and F3H catalyzes eriodictyol chalcone into taxifolin (Christensen et al., 1998; Flores-Sanchez and Verpoorte, 2008; Morita et al., 2010; Meinert et al., 2021).

Coniferyl alcohol and taxifolin are amalgamated via oxidative coupling for silymarin biosynthesis (flavonolignan). The oxidative coupling reaction is mediated by the formation of free radicals and is catalyzed by a peroxidase enzyme called a radical generator (AbouZid and Ahmed, 2013).

In addition, laccases (benzenediol: oxygen oxidoreductases) mediate oxidative coupling for dimerization and production of silybin (Setti et al., 1999; Gažák et al., 2008; Gavezzotti et al., 2014). A study by Lv et al. (2017) reported that silybin components of silymarin were catalyzed by ascorbate peroxidase 1 (APX1), one of the candidates for peroxidase, and APX1 showed a distinct peroxidase activity and the capacity to synthesize silybin.

Schrall and Becker (1977) reported that horseradish-peroxidase and a cell-free extract of milk thistle suspension cultures could synthesize silybin starting from coniferyl alcohol and taxifolin. In some studies, peroxidases of APX1 and horseradish-peroxidase have been used to demonstrate a green process for silybin and isosilybin production (Yang et al., 2020).

The contents and components of silymarin biosynthesis are influenced by some factors. The study of Martin et al. (2006), use various milk thistle cultivars to present the factors influencing on contents and components of silymarin. This study show that each plant parts have different total contents and components of silymarin. The part of root have only silychristin B and silybin B components, and the two components are major in flowers. But, the contents have very low levels. In the seeds and seed heads, silychristin A, silydianin, and silybin B are the dominant components, and it have the highest total silymarin content. In addition to the difference of contents and components in each plant parts, the study show difference of contents and components depending on growth stage between the cultivars.

In the study of Liava et al. (2022), it present effects of fertilization regimes on growth, fruit, and silymarin yield in two cultivars of milk thistle. Sheep manure and calcium ammonium nitrate are used to exhibit the difference of plants growth depending on fertilization regimes. The use of manure and calcium ammonium nitrate fertilizer increase plant rosette diameter, biomass, fruit yield, and silymarin content. In this study, it is noticed that the plants growth with silymarin content can be increased based on treatment of fertilization regimes, and that the difference of silymarin content as well as silymarin composition between the two cultivars.

According to this two studies, we need to consider suitable milk thistle cultivars selection to produce silymarin as commercial viewpoint and crop management guidelines. Futhermore, the studies of Lv et al. (2017) and Torres et al. (2016) reported genes expression for silymarin biosynthetic pathway. It is regarded that molecular study for genetics is requiered using the understanding for segmented biosynthetic pathway and milk thistle cultivars which have difference of the contents and components of silymarin.

Metabolic engineering of biosynthetic pathways to produce high-value secondary metabolites as pharmaceuticals and food additives, have industrial importance using plant cell cultures, shoot cultures, root cultures, and transgenic hairy root cultures acquired through biotechnological means (Rao et al., 2002). These plant tissue cultures can be potential alternative sources for the secondary metabolites production.

The studies of Alikaridis et al. (2000) and Rahnama et al. (2008) show that production of silymarin and the components produced by the hairy root and root cultures of milk thistle. In the studies of Elwekeel et al. (2012a) and El Sherif et al. (2013), these show that silymarin accumulated through the root and shoot cultures of milk thistle can be improved by various elicitors. In addition to enhancement of silymarin production through addition of elicitors, the Elwekeel et al. (2012b) study present that the cultured cells of milk thistle have comparable cytotoxic, antioxidant, and hepato-protective effects to that of the fruits.

The study of plant tissue cultures for producing silymarin may be necessary to pharmaceutical industries. Thus, the overall understanding about silymarin biosynthetic pathways will be helpful to define efficient ways of silymarin production to utilize the various elicitors and tissue cultures.

The overall biosynthetic pathway for the silymarin components is shown in Fig. 4. The processing of the precursors (coniferyl alcohol and taxifolin) for biosynthetic pathways cannot be simply explained by one route, and the activating enzymes involved vary. In particular, the biosynthetic pathway of the precursor coniferyl alcohol, which is included in monolignol-specific pathways, has multiple routes than biosynthetic pathway of taxifolin.

Fig. 4.  This schematic view present overall biosynthetic pathway of silymarin.

In general phenylpropanoid pathway, the phenylalanine and tyrosine are converted to p-coumaric acid, and the involved enzymes are phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), cinnamate 4-hydroxylase (C4H). In biosynthetic pathway of coniferyl alcohol, the p-coumaric acid is converted to coniferyl alcohol, and the involved enzymes are p-coumarate 3-hydroxylase (C3H), p-coumaroylester 3′-hydroxylase (C3′H), caffeic acid 3-O-methyltransferase (COMT), caffeoyl-CoA 3-O-methyltransferase (CCOMT), 4-coumaric acid:coenzyme A ligase (4CL), hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), caffeoyl shikimate esterase (CSE), cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD). The p-coumaroyl-CoA and caffeoyl-CoA are converted to naringenin chalcone and eriodictyol chalcone by chalcone synthase (CHS) and homoeriodictyol/eriodictyol synthase (HEDS/HvCHS2), respectively. In biosynthetic pathway of taxifolin, the eriodictyol chalcone and naringenin chalcone are converted to taxifolin, and the involved enzymes are chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H). Coniferyl alcohol and taxifolin are amalgamated via oxidative coupling to formation for component of silymarin (silybin, isosilybin, silychristin, isosilychristin, and silydianin) by peroxidase and laccase.

In a study by Ha et al. (2016), caffeoyl shikimate esterase activity converted caffeoyl shikimic acid to caffeic acid in Medicago truncatula. Furthermore, the study by Liu et al. (2018) reported that cinnamyl alcohol dehydrogenases, cloned and characterized from Asarum sieboldii Miq., displayed efficient catalytic activity and substrate preference with the capability of converting aldehydes (p-coumaryl, coniferyl, and sinapyl aldehydes) to their corresponding alcohols. Regarding substrate preference, Franke et al. (2002) also reported that C3H, which is encoded by the REF8 gene isolated from Arabidopsis, had different levels of substrate activity for p-coumaric acid, p-coumaryl-aldehyde, and p-coumaryl-alcohol.

Thus, enzymes used in biosynthetic pathways in plants have substrate preferences that are activated differently on each substrate, and all plant species may not use identical enzymes for specific biosynthetic pathways. The study of biosynthetic pathways for specific secondary metabolites requires the recognition of the overall pathways with related factors (substrates, production, and activating enzymes), and an efficient activity pathway may be required in individual plants considering these factors.

Therefore, the overall biosynthetic pathway presented in this review can be utilized to study the biosynthesis of silymarin components.