Silymarin Production Research Articles

Study of different Trichoderma strains on growth characteristics and silymarin accumulation of milk thistle plant

Biological control involves the use of beneficial organisms such as Trichoderma that promote positive responses by the plant. Trichoderma species produce and/or release a variety of compounds, including cell wall degrading enzymes and secondary metabolites, which enhance root development, crop productivity and resistance to biotic and abiotic stresses. This study examines the effects of the five Trichoderma strains (M7, KHB, G124-1, G46-3 and G46-7) on milk thistle including morphological characteristics and silymarin accumulation. This research indicated that treating milk thistle by M7 strain, significantly increased the highest branch length of plants, while strain KHB induced the production of silychristin, isosilybin and silymarin. Finally, strain G46-7 dramatically increased silydianin content in milk thistle plant. The findings suggested that certain Trichoderma strains are positive elicitors for promoting growth characteristics and silymarin accumulation in Silybum marianum (L.) Gaertn.

ENHANCED SILYMARIN ACCUMULATION AS INFLUENCE OF MEDIUM COMPOSITION IN CELL SUSPENSION CULTURES OF Silybum marianum (L.) GAERTN.

Silymarin production was examined in cell suspension cultures derived from hypocotyl of Silybum marianum (L.) Gaertn. MS medium supplemented with different concentrations of sucrose, glucose, KH2PO4, KH2PO4 + K2SO4, NH4NO3, CaCl2, L-Phenylalanine, L-Tyrosine and yeast extract was used. Tyrosine 10 mg/l, 0.5 KH2PO4 + K2SO4 con., 0.75 NH4NO3 con., 0.75 KH2PO4 + K2SO4 con., glucose 30 g/l, 0.5 CaCl2.2H2O, tyrosine 20 mg/l and yeast extract 40 mg/l (3 days incubation period) showed the highest production of silymarin in callus cells to reach 79%, 57.5%, 46%, 24.2%, 21.2%, 19.7%, 9.7% and 8.6% over the control respectively. The same trend was obtained by HPLC (qualitative assay).

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.

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.

Evaluation of the yeast-extract signaling pathway leading to silymarin biosynthesis in milk thistle hairy root culture

The biosynthesis of silymarin, a potent antihepatotoxic compound, from the dried fruits of Silybum marianum L. Gaertn in hairy root cultures can be stimulated by a yeast extract elicitor. These results correlated with culture time, and the biosynthesis reached a maximum of 0.47 mg g−1 DW by 72 h after culture (2-fold higher than the control). Lipoxygenase activity and linoleic acid content were stimulated by this treatment, suggesting that the jasmonate pathway may mediate the elicitor-induced accumulation of silymarin. The H2O2 content increased 24 h after elicitation and did not have marked changes between 48 and 72 h. In addition, the tocopherol content (especially α- and δ-tocopherols) increased 72 h after elicitation in comparison with non-treated cultures. Ascorbate had trace changes during feeding time and was lower than the control. The antioxidant activity was assayed by the 1-1-diphenyl-2-picrylhydrazyl stable free radical method and results were calculated base on an IC50 that increased upon treatment, especially 24 h after treatment, with changes related to H2O2 content. These observations suggested that reactive oxygen species may mediate elicitor signals to the jasmonate pathway that lead to the production of silymarin.

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.

Ag+-induced silymarin production in cell suspension culture of Silybum marianum L. Gaertn

Silymarin production in cell suspension culture of Silybum marianum treated with Ag+ was investigated. It is well known that elicitation is one of the strategies employed to increase accumulation of secondary metabolites (1). Callus induction was initiated from cotyledon segments of seedlings on MS medium supplemented with picloram (3mg l–1) and kinetin (0.4mg l–1). Cell suspensions were established from 1-month-old undifferentiated callus and maintained in darkness in MS liquid medium and were incubated on a rotary shaker cultures (150 rpm) and 25°C (1 and 2). Cell suspension cultures of S. marianum were exposed to various levels of Ag+ (0.2, 0.4, 0.8, 1 and 2 mM) and harvested 12, 24, 48, 72, 144 and 216 hr after treatment. The highest silymarin production reached about 60µg g-1Dw, 24 hours after treatment with Ag+ (0.8mM), witch was 30-fold that of the control. Therefore cell suspension culture of S. marianum are susceptible to elicitation by Ag+ and the information obtained in this work is useful for efficient large-scale production of silymarin by cell suspension culture of S.marianum.

Elicitor-enhanced Silymarin production in hairy root cultures of Silybum marianum L. Gaertn

Silymarin is an antihepatotoxic polyphenolic substance isolated from the milke thistle (Silybum marianum) plant [1]. Hairy roots cultures of S. marianum produce silymarin. In an attempt to increase productivity, the effect of yeast extract was tested. The hairy roots induced by inoculation of leaf explants with A. rhizogenes (AR15834). One month old hairy roots were dissected from the explants and growth in the Erlenmeyer flask containing 50ml MS liquid medium [2]. PCR analysis using rolb gene and GUS assay were used for identification of transformed hairy roots [3]. The presences of flavonolignans in the hairy roots were determined by high-performance liquid chromatography (HPLC). There were five isolated components; taxifolin, silychristin, silydianin, silybin and isosilybin and the major flavonolignan was silybin that has not been reported in previous published work. For increasing the growth rate of hairy roots, effects of NAA, IBA and IAA (0.1, 0.5 and 1mg/L) were tested. The best media was achieved by 0.5mg/L NAA. Elicitation was carried out with yeast extract (0, 0.5, 1, 2.5 and 5mg/L) and different exposure times (24, 48 and 72h). The highest silymarin content (0.476mg/g DW) was obtained by 2.5mg/mL yeast extract, after 72h. This study indicates that elicitor treatment promotes silymarin production in hairy root culture of S. marianum.

Some common signal transduction events are not necessary for the elicitor-induced accumulation of silymarin in cell cultures of Silybum marianum

A variety of pharmacological effectors of signal transduction pathways were used to investigate the elicitor-activated sequence of cellular responses by which yeast extract (YE) or methyljasmonate (MeJA) enhanced production of silymarin in cell cultures of Silybum marianum. As we recently showed that inhibition of external and internal calcium fluxes significantly increased flavonolignan production in S. marianum cultures, we examined whether calcium mediates signaling events leading to enhancement of silymarin production upon YE or MeJA elicitation. Pre-treatment of cultures with calcium chelators, calcium blockers or intracellular antagonists enhanced the elicitor effect of YE or MeJA. The increase of intracellular-free Ca(2+) level also promoted the elicitor effect, suggesting that an external source of calcium or alterations in internal calcium fluxes were not required for the elicitation to occur. Activation of phosphorylation/dephosphorylation cascades did not appear to mediate the elicitation mechanism; the increase in silymarin induced by elicitation was not suppressed by inhibitors of protein phosphatases or by protein kinase inhibitors. No H(2)O(2) generation was detected at any time after elicitation. Also, diphenyleneiodonium, a potent inhibitor of NAD(P)H-oxidase, did not block silymarin production in elicited cultures. From these results, we conclude that S. marianum cell cultures do not appear to employ conserved signaling components in the transduction of the elicitor signal to downstream responses such as silymarin production.

Flavonolignan Production in Cell Suspension Culture of Silybum marianum

The fruits of Silybum marianum (L.) Gaertn (Compositae) contain silymarin, an isomeric mixture of flavonolignans (silybin, silychristin, and silydianin). Silymarin acts as a strong anti-hepatotoxic. Silybum marianum cell cultures represent an alternate source of flavonolignans, but the yields of these compounds in such cultures are very low. In this study, in vitro cell culture systems of Silybum marianum were used to monitor the effect of elicitation (picloram, jasmonic acid, and light) on cell growth and production of silymarin. The presence of silymarin was established by high performance liquid chromatography and the greatest growth rates dry weight (32 mg) and silymarin content (0.413 mg g− 1 DW) were obtained with 3 mg l− 1 picloram and 2 mg l− 1 jasmonic acid in darkness after 28 days. The light grown cell suspension cultures showed reduced growth in term of dry weight (24 mg) and silymarin content. The data presented in this study demonstrate that media supplemented with 3 mg l− 1 picloram with JA in dark conditions can be useful for production of silymarin and growth in cell suspension cultures of Silybum marianum.

Metabolomic alterations in elicitor treated Silybum marianum suspension cultures monitored by nuclear magnetic resonance spectroscopy

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.

Self-mating effect on growth traits and silymarin production for some selected lines among milk thistle (Silybum marianum L.) varieties

Ten selected lines for each purple and white head flower varieties of milk thistle, Silybum marianum were assessed for five growth traits and silymarin production among three generations: open parents, selfing progenies and selfing offspring. Highly significant variations existed between lines, varieties and generations as well as their interactions in all tested traits. The line characters for each variety were subjected to analysis of variance only for open parents opposite to selfing offspring, and seemed highly significant variablities. The selfing offspring generation produced higher mean value in all purple variety traits except no. of flower heads. Contrarily, the parent generation produced higher values in all white variety traits except fruit yield (FY). Lines 34 and 9 for purple and white varieties respectively were the best lines in both open and selfing generations. Coefficient of variation, genotypic and phenotypic coefficients of variation as well as broad sense heritabilities and genetic advance for most of studied growth traits were improved in the selfing offspring generation to indicate that milk thistle traits were governed with additive gene effects. Fruit yield trait had the highest parent offspring regression and narrow sense heritability in both varieties. On the other hand lines 34, 22 and 28 in the purple as well as 9, 2 and 13 in the white variety were the highest, medium and lowest fruit yield, respectively and subjected for fruit content of silymarin using HPLC. Concentration and total yield of six detected silymarin compounds showed wide variations between lines, varieties and generations and ranged from 11.92 to 62.85mg/g and between 329.8 to 2121.3mg/plant, respectively. Selfing improved the silymarin contents in the purple lines, but reduced the content of white lines. Interesting to notice is that silymarin production has the same pattern of fruit yield trait, so selection should be based on this trait to produce new improved high yielding silymarin genotypes.

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.