Gene Expression Analysis and Metabolite Profiling of Silymarin Biosynthesis during Milk Thistle (Silybum marianum (L.) Gaertn.) Fruit Ripening.

Mature fruits (i.e., achenes) of milk thistle (Silybum marianum (L.) Gaertn., Asteraceae) accumulate high amounts of silymarin (SILM), a complex mixture of bioactive flavonolignans deriving from taxifolin. Their biological activities in relation with human health promotion and disease prevention are well described. However, the conditions of their biosynthesis in planta are still obscure. To fill this gap, fruit development stages were first precisely defined to study the accumulation kinetics of SILM constituents during fruit ripening. The accumulation profiles of the SILM components during fruit maturation were determined using the LC-MS analysis of these defined developmental phases. The kinetics of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and peroxidase (POX) activities suggest in situ biosynthesis of SILM from l-Phenylalanine during fruit maturation rather than a transport of precursors to the achene. In particular, in contrast to laccase activity, POX activity was associated with the accumulation of silymarin, thus indicating a possible preferential involvement of peroxidase(s) in the oxidative coupling step leading to flavonolignans. Reference genes have been identified, selected and validated to allow accurate gene expression profiling of candidate biosynthetic genes (PAL, CAD, CHS, F3H, F3'H and POX) related to SILM accumulation. Gene expression profiles were correlated with SILM accumulation kinetic and preferential location in pericarp during S. marianum fruit maturation, reaching maximum biosynthesis when desiccation occurs, thus reinforcing the hypothesis of an in situ biosynthesis. This observation led us to consider the involvement of abscisic acid (ABA), a key phytohormone in the control of fruit ripening process. ABA accumulation timing and location during milk thistle fruit ripening appeared in line with a potential regulation of the SLIM accumulation. A possible transcriptional regulation of SILM biosynthesis by ABA was supported by the presence of ABA-responsive cis-acting elements in the promoter regions of the SILM biosynthetic genes studied. These results pave the way for a better understanding of the biosynthetic regulation of SILM during the maturation of S. marianum fruit and offer important insights to better control the production of these medicinally important compounds.

Interactive Effects of Light and Melatonin on Biosynthesis of Silymarin and Anti-Inflammatory Potential in Callus Cultures of Silybum marianum (L.) Gaertn.

Silybum marianum (L.) Gaertn. is a well-known medicinal herb, primarily used in liver protection. Light strongly affects several physiological processes along with secondary metabolites biosynthesis in plants. Herein, S. marianum was exploited for in vitro potential under different light regimes in the presence of melatonin. The optimal callogenic response occurred in the combination of 1.0 mg/L α-naphthalene acetic acid and 0.5 mg/L 6-benzylaminopurine under photoperiod. Continuous light associated with melatonin treatment increased total flavonoid content (TFC), total phenolic content (TPC) and antioxidant potential, followed by photoperiod and dark treatments. The increased level of melatonin has a synergistic effect on biomass accumulation under continuous light and photoperiod, while an adverse effect was observed under dark conditions. More detailed phytochemical analysis showed maximum total silymarin content (11.92 mg/g dry weight (DW)) when placed under continuous light + 1.0 mg/L melatonin. Individually, the level of silybins (A and B), silydianin, isolsilychristin and silychristin was found highest under continuous light. Anti-inflammatory activities were also studied and highest percent inhibition was recorded against 15-lipoxygenase (15-LOX) for cultures cultivated under continuous light (42.33%). The current study helps us to better understand the influence of melatonin and different light regimes on silymarin production as well as antioxidant and anti-inflammatory activities in S. marianum callus extracts.

The study of flavonolignan association patterns in fruits of diverging Silybum marianum (L.) Gaertn. chemotypes provides new insights into the silymarin biosynthetic pathway.

Silymarin is the phytochemical with medicinal properties extracted from Silybum marianum (L.) Gaertn. fruits. Yet, little information is available about silymarin biosynthesis. Moreover, the generally accepted pathway, formulated thus far, is not in agreement with actual experimental measurements on flavonolignan contents. The present work analyses flavonolignan and taxifolin content in 201 S. marianum samples taking into consideration a wide phenotypic variability. Two stable chemotypes were identified: one characterized by both high silychristin and silybin content (chemotype A) and another by a high silydianin content (chemotype B). Through the correlation analysis of samples divided according to chemotype, it was possible to construct a simplified silymarin biosynthetic pathway that is sufficiently versatile in explaining experimental results responding to the actually unresolved questions about this process. The proposed pathway highlights that three separate and equally sized metabolite pools exist, namely: diastereoisomers A (silybin A plus isosilybin A), diastereoisomers B (silybin B plus isosilybin B) and silychristin. In both A and B diastereoisomers pools, isosilybin A and isosilybin B always represent a given amount of the metabolite flux through the specific metabolite pool suggesting the possible involvement of dirigent protein-like enzymes. We suggest that chemotype B possesses a complete silymarin biosynthetic pathway in which silydianin biosynthesis is enzymatically controlled. On the contrary, chemotype A is probably a natural mutant unable to biosynthesize silydianin. The present simplified pathway for silymarin biosynthesis will constitute an important tool for the further understanding of the reactions that drive flavonolignan biosynthesis in S. marianum.

Enhanced production of silymarin by Ag+ elicitor in cell suspension cultures of Silybum marianum.

Cell suspension cultures of Silybum marianum L. Gaertn (Compositae) produce silymarin, a mixture of flavonolignans. In an attempt to increase cell growth and silymarin production, we exposed cell cultures to various levels of Ag+ (0.2, 0.4, 0.8, 1, and 2 mM) for different exposure times (12, 24, 48, 72, 144, and 216 h). A dramatic increase in cell growth was observed after 12 h in media supplemented with 0.2, 0.4, 0.8, and 1 mM Ag+, and the value in medium treated by 1 mM Ag+ after 72 h was 7.21 g, which was about two-fold that of the control (3.32 g). The highest silymarin production reached about 56 microg g(-1) DW, 24 h after treatment with Ag+ (0.8 mM), which was 30-fold that of the control. Silybin, isosilybin, silychristin, silydianin, and taxifolin were abundant flavonolignans. Ag+ in low concentrations is a positive elicitor for cell growth and silymarin production in cell suspension cultures of Silybum marianum.

Silymarin secretion and its elicitation by methyl jasmonate in cell cultures of Silybum marianum is mediated by phospholipase D-phosphatidic acid.

The flavonolignan silymarin is released to the extracellular medium of Silybum marianum cultures and its production can be stimulated by the elicitor methyljasmonate (MeJA). The sequence of the signalling processes leading to this response is unknown at present. It is reported in this work that MeJA increased the activity of the enzyme phospholipase D (PLD). Treatment with mastoparan (Mst), a PLD activity stimulator, also enhanced PLD and caused a substantial increase in silymarin production. The application of the product of PLD activity, phosphatidic acid (PA) promoted silymarin accumulation. Altering PLD activity by introducing in cultures n-butanol (nBuOH), which inhibits PA production by PLD, prevented silymarin elicitation by MeJA or Mst and also impeded its release in non-elicited cultures. Treatment with iso-, sec- or tert- butanol had no effect on silymarin production. The exogenous addition of PA reversed the inhibitory action of nBuOH, both in control and MeJA-treated cultures. These results suggest that the enzyme PLD and its product PA mediate silymarin secretion to the medium of S. marianum cultures.

Elicitation of silymarin in cell cultures of Silybum marianum: effect of subculture and repeated addition of methyl jasmonate.

Production of silymarin and the effect of the elicitor, methyl jasmonate (MeJA), was monitored in cell cultures of Silybum marianum over 4 years. Silymarin concentrations gradually declined after prolonged subculture, making the success of elicitor strategy limited in long-term cultures. The continuous presence of MeJA in cultures for an extended period was necessary for induction of silymarin accumulation. A repeated elicitor strategy was not a good option for improving silymarin productivity in batch cultures. Removal of medium from elicited cultures and addition of fresh medium avoided the toxic effects of elicitor accumulation, allowing the system to respond to a repeated MeJA treatment without loss of productivity.

Influence of exogenous salicylic acid on flavonolignans and lipoxygenase activity in the hairy root cultures of Silybum marianum.

Silymarin is one of the most potent antioxidant so far developed from plant sources used as hepatoprotectants. Influence of different concentrations (0, 1, 2, 4, 6 and 8mg/50ml culture) and exposure time (24, 48, 72, 96 and 120h) of salicylic acid on lipoxygenase activity, linoleic acid content, growth and production of silymarin in hairy root cultures of S. marianum were investigated. Detection and identification of flavonolignans was carried out by high performance liquid chromatograph method. Salicylic acid enhanced silymarin production (1.89mgg(-1) DW). The optimal feeding condition was the addition of salicylic acid (6 mg/50 ml culture) after 24h in which the silymarin content was 2.42 times higher than the control (0.78mgg(-1) DW). The content of silybin, isosilybin, silychristin, silydianin and taxifolin were 0.703, 0.017, 0.289, 0.02 and 0.863mgg(-1) DW respectively in these samples, while in non-treated hairy roots were 0.027, 0.046, 0.23, 0.022 and 0.453 respectively. Lipoxygenase activity also affected by elicitation. lipoxygenase activity increased 24h after treatment by approximately 1.57- fold (0.21 Delta OD(234)/mgproteinmin(-1)). Upon elicitation with salicylic acid, linoleic acid content of hairy roots (38.26mgg(-1) DW) were also elevated after 24h, in which the linoleic acid content was 2.37 times higher than the control (16.1mgg(-1) DW). It is feasible that elicitation with salicylic acid regulates the jasmonate pathway, which in turn mediates the elicitor-induced accumulation of silymarin.

Silymarin synthesis and degradation by peroxidases of cell suspension cultures of Silybum marianum.

Treatment of Silybum marianum cell cultures with methyl jasmonate elicits the production of the antihepatotoxic drug silymarin and its release into the culture medium. In this work, we investigated the involvement of peroxidases (EC 1.11.1.7; donor hydrogen peroxidase oxido-reductase) in silymarin turnover in cell cultures as well as the influence of elicitation on the activity towards several substrates. Peroxidases from cell extracts and, to a higher degree from the spent medium, used the silymarin precursors taxifolin and coniferyl alcohol as substrates. Silymarin compounds were also degraded by suspension culture peroxidases; however, the oxidation efficiency was not modified by elicitation. S. marianum peroxidases were able to catalyse the oxidative coupling of taxifolin and coniferyl alcohol to silybinins. The synthetic activity was mainly associated with the extracellular compartment and as before, methyl jasmonate did not modify oxidative coupling activity. Changes in the isoenzyme profiles were not observed in elicited cultures.

Yeast extract and methyl jasmonate-induced silymarin production in cell cultures of Silybum marianum (L.) Gaertn.

The biosynthesis of the flavonolignan silymarin, a constitutive compound of the fruits of Silybum marianum with strong antihepatotoxic and hepatoprotective activities, is severely reduced in cell cultures of this species. It is well known that elicitation is one of the strategies employed to increase accumulation of secondary metabolites. Our study here reports on the effect of several compounds on the production of silymarin in S. marianum cultures. Yeast extract (YE), chitin and chitosan were compared with respect to their effects on silymarin accumulation in S. marianum suspensions and only yeast extract stimulated production. Jasmonic acid (JA) potentiated the yeast extract effect. One of the jasmonic acid derivatives, methyl jasmonate (MeJA), strongly promoted the accumulation of silymarin. Methyl jasmonate acted in a number of steps of the metabolic pathway of flavonolignans and its stimulating effect was totally dependent of "de novo" protein synthesis. Chalcone synthase (CHS) activity was enhanced by methyl jasmonate; however there did not appear to be a temporal relationship between silymarin accumulation and increase in enzyme activity. Also, this increase was not blocked by the protein synthesis inhibitor cycloheximide (CH). This study indicates that elicitor treatment promotes secondary metabolite production in S. marianum cultures and that jasmonic acid and its functional analogue plays a critical role in elicitation.

[Silybum marianum in vitro]. / Silybum marianum in vitro.

Callus and suspension cultures of Silybum marianum were derived and the growth and production characteristics were assayed. The maximal production of flavonolignans was achieved at the beginning of cultivation, i.e. on day 10 after inoculation in MS medium with alpha-NAA and on day 15 after inoculation in MS medium with 2.4-D with continuous growth of the culture. The content of silymarin in the plant drug was 3.05%, in the in vitro culture it was just tenths of percents. TLC analysis revealed that the spectrum of flavonolignans of the drug differed from the spectrum of flavonolignans produced by tissue cultures. No differences in the spectra of flavonolignans produced by the callus and suspension cultures were observed. The test for antioxidative activity against the DPPH-radical demonstrated marked antioxidative properties of substances produced by the culture of Silybum marianum.