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A comprehensive metabolomic profiling of Silybum marianum (L.) Gaernt cell cultures elicited with yeast extract or methyl jasmonate for the production of silymarin was carried out using one- and two-dimensional nuclear magnetic resonance spectroscopy. With these techniques we were able to detect both temporal quantitative variations in the metabolite pool in yeast extract-elicited cultures and qualitative differences in cultures treated with the two types of elicitors. Yeast extract and methyl jasmonate caused a metabolic reprogramming that affected amino acid and carbohydrate metabolism; upon elicitation sucrose decreased and glucose levels increased, these changes being dependent on "de novo" protein synthesis. Also dependent on protein synthesis were the increase seen in alanine and glutamine in elicited cultures. Yeast extract differentially acted on threonine and valine metabolism and promoted accumulation of choline and alpha-linolenic acid in cells thus suggesting its action on membranes and the involvement of the octadecanoid pathway in the induction of silymarin in S. marianum cultures. Phenylpropanoid metabolism was altered by elicitation but, depending on elicitor, different phenylpropanoid profile was produced. The results obtained in this study will permit in the future to identify candidate components of the signalling pathway involved in the stimulation of the constitutive pathway of silymarin.

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.