Flavonolignan Production Research Articles

Production of phenylpropanoids and flavonolignans from glycerol by metabolically engineered Escherichia coli.

Phenylpropanoids are a group of plant natural products with medicinal importance derived from aromatic amino acids. Here, we report the production of two representative phenylpropanoids-coniferyl alcohol (CA) and dihydroquercetin (DHQ)-from glycerol by engineered Escherichia coli. First, an E. coli strain capable of producing 187.7 mg/L of CA from glycerol was constructed by the introduction of hpaBC from E. coli and OMT1, 4CL4, and CCR1 from Arabidopsis thaliana to the p-coumaric acid producer. Next, an E. coli strain capable of producing 239.4 mg/L of DHQ from glycerol was constructed by the introduction of F3H, TT7, and CPR from A. thaliana to the naringenin producer, followed by engineering the signal peptide of a cytochrome P450 TT7. Furthermore, to demonstrate the production of flavonolignans, a group of heterodimeric phenylpropanoids, from glycerol, ascorbate peroxidase 1 from Silybum marianum was employed and engineered to produce 0.04 μg/L of silybin and 1.29 μg/L of isosilybin from glycerol by stepwise culture. Finally, a single strain harboring all the 16 necessary genes was constructed, resulting in 0.12 μg/L of isosilybin production directly from glycerol. The strategies described here will be useful for the production of pharmaceutically important yet complex natural products.

Enrichment of Therapeutically Significant Flavonolignans of Silybum Marianum in Vegetative Parts by Applying Fungal Elicitors, Methyl Jasmonate and Silver Nanoparticles as Elicitor in Hydroponic Culture

Introduction: Medicinal plants are being used to treat several diseases for many decades and this is an ancient method to treat the patients. Herbal plant Silybum marianum found most effective one to cure liver disorders. This plant produces silymarin which is a secondary metabolite and have hepatoprotective properties. Silymarin is a mixture of flavonolignans (silybin A, silybin B, isosilybin A isosilybin B, silychristin, silydianin apigenin 7-D glucose and taxifolin) that has antiviral, antibacterial, antifungal and antiallergenic properties. Therefore, the excessive production of silymarin is necessary to cure different types liver disorders, so the present study executed to boost up production of Silymarin flavonolignans in vegetative parts of the Silybum marianum plant by using different elicitors.
 Materials and Methods: In the present study, elicitation technique in hydroponics system was used to enhance the production of pharmacologically active flavonolignans in Silybum marianum. Fungal elicitors prepared from lyophilized Aspergillus niger biomass, methyl jasmonate, silver nanoparticles and combination of silver nanoparticles and methyl jasmonate were added (0.2g/l), (100µM/l), (1 ppm) and (100µM/lppm) in hydroponics with Hoagland’s solution in hydroponics to enhance the production of flavonolignans of Silybum marianum. Controls were also set for each treatment. Plants were harvested after 72 hours of introduction of elicitors. High performance liquid chromatography technique was used for analytical purpose. Four solvents (methanol, acetonitrile, chloroform and 2% Trifloro acetic acid) were used in HPLC. Column was C18 and run time of sample was 1 hour. Silybum marianum’s seed extract was used as a standard. Extract of control and treated plants were run on the same polarity in HPLC.
 Results: Results showed that after elicitation significant increase was observed in production of silymarin’s flavonolignans (silybin A, silybin B, isosilybin A, isosilybin B and apigenin 7-D glucose) in vegetative part of the plant but rate of production was different for each elicitor, fungal elicitors prepared from lyophilized Aspergillus niger biomass proved best among all treatments.

Gene expression and flavonolignan production in fruits and cell cultures of Silybum marianum

The hepatoprotectant flavonolignan silymarin (Sm) is synthesized through 4-coumaroyl-CoA, which enters both the flavonoid and the monolignol pathway giving the two immediate precursors taxifolin (Tx) and coniferyl alcohol (CA), respectively. Sm formation occurs via oxidative radicalization of Tx and CA and is accumulated at high levels at final stages of maturation of Silybum marianum fruits. By contrast, Sm production is severely reduced in cell cultures of this species, although suspensions are able to excrete Sm compounds into the medium upon elicitation with methyl jasmonate (MeJA) or cyclodextrins (CD). Knowledge of gene expression is important to understand Sm dynamics and to develop strategies aimed at increasing production by means of cell cultures but, to date, only one gene of the pathway (chalcone synthase, SmCHS) has been cloned. Therefore, to elucidate the relationship between expression of Sm pathway genes and production of these metabolites, four cDNA fragments of genes putatively involved in flavonolignan biosynthesis, chalcone isomerase, flavanone 3-hydroxylase, flavonol 3′-hydroxylase and cinnamyl alcohol dehydrogenase, were isolated from Sm producing S. marianum fruits and their expression, together with that of the SmCHS, were studied both in fruits at different maturation stages and in elicited cell suspensions. Combined results at both transcript expression and metabolite levels at three different stages of fruit maturation revealed that the formation of the flavonoid moiety precedes flavonolignan biosynthesis, being Sm accumulation associated to expression of the monolignol pathway. There was not detectable accumulation of transcripts in cell suspensions, however, elicitation with MeJA or CD notably induced expression of the studied fragments. These results indicate that the five genes expressed during maturation of S. marianum fruits may contribute to observed increases in flavonolignan accumulation upon treatment of cell cultures with elicitors.

Weed infestation in the stand of milk thistle and infestation in sustainable crop rotation

Weed infestation in the stand of milk thistle and infestation in sustainable crop rotation

Interactive effect of deficit irrigation and soil organic amendments on seed yield and flavonolignan production of milk thistle (Silybum marianum L. Gaertn.)

Milk thistle is a recognized medicinal plant cultivating for production of silymarin and oil. Responses of milk thistle to deficit irrigation and organic soil amendments were studied in a 2-year field experiment. Three levels of irrigation regimes including normal irrigation (I100), moderate deficit (I75) and severe deficit irrigation (I50) and two sources of soil amendments consisted of vermicompost (VM) and poultry manure (PM) plus no amendments comprised nine treatments laid out in a factorial pattern in a randomized complete block design. Moderate and severe deficit irrigation resulted in 7 and 27% seed yield reduction, respectively. Among yield components, number of seeds head−1 showed the highest sensitivity to deficit irrigation followed by number of heads plant−1 while seed weight remained stable. As irrigation deficiency intensified, Irrigation water use efficiency followed an increasing trend. Among the two soil organic amendments, only PM significantly improved the seed yield where plots amended with PM produced 8, 12 and 17% higher seed yield under I100, I75 and I50, respectively. Application of PM also improved irrigation water use efficiency. Implementation of deficit irrigation improved silymarin content of the seeds; however, silymarin yield declined due to the lower productivity in stressed conditions. Oil yield was also adversely affected by both moderate and severe deficit irrigation and reduced by 9 and 32%, respectively. Application of soil amendments had no significant effect on silymarin and oil content of the seeds, but PM improved silymarin and oil yield due to its beneficial effect on seed yield. The results of this study indicated that milk thistle can be grown under moderate deficit irrigation (saving 25% of irrigation water) and amending soil with PM can improve the yield and minimize the negative impact of drought stress.

Flavonolignans from Aspergillus iizukae, a Fungal Endophyte of Milk Thistle (Silybum marianum)

Silybin A (1), silybin B (2), and isosilybin A (3), three of the seven flavonolignans that constitute silymarin, an extract of the fruits of milk thistle (Silybum marianum), were detected for the first time from a fungal endophyte, Aspergillus iizukae, isolated from the surface-sterilized leaves of S. marianum. The flavonolignans were identified using a UPLC-PDA-HRMS-MS/MS method by matching retention times, HRMS, and MS/MS data with authentic reference compounds. Attenuation of flavonolignan production was observed following successive subculturing of the original flavonolignan-producing culture, as is often the case with endophytes that produce plant-based secondary metabolites. However, production of 1 and 2 resumed when attenuated spores were harvested from cultures grown on a medium to which autoclaved leaves of S. marianum were added. The cycle of attenuation followed by resumed biosynthesis of these flavonolignans was replicated in triplicate.

The Effect of Pyridinecarbothioamides on Isoflavonoid Production in Genista tinctoria Cultures in Vitro

Previous studies showed that derivatives of pyrazinecarboxylic acid used as elicitors increased either flavonoid or flavonolignan production. The effect of 2-(3-bromobenzylsulfanyl) pyridine-4-carbothioamide on isoflavonoids production in Genista tinctoria culture in vitro was tested. The highest isoflavonoid content (0.3 mg.g−1 DW) in callus culture was found after elicitor application at a concentration of 2.96 × 10−5 mol.L−1 after 24 h of treatment. The maximum daidzein production (0.2 mg.g−1 DW) was detected after 48 h of elicitor treatment at a concentration of 2.96 × 10−3 mol.L-1 and after 24 h of elicitor treatment at a concentration of 2.96 × 10−5 mol.L−1. Suspension culture produced the highest amount of isoflavonoids (0.3 mg.g−1 DW) after elicitor application at concentrations of 2.96 × 10−4 mol.L−1 after 24 and 168 h of treatment, and 2.96 × 10−5 mol.L−1 after 168 h of treatment. The highest content of genistin (0.3 mg. g−1 DW) was detected after 24 h of elicitor treatment at a concentration of 2.96 × 10−4 mol.L−1. The maximum content of daidzein (3.80 mg.L−1) released into the suspension culture medium was found after elicitor application at a concentration of 2.96 × 10−4 mol.L−1 after 12 h of treatment.

Enhanced production of flavonolignans in hairy root cultures of Silybum marianum by over-expression of chalcone synthase gene

In the present study, metabolic engineering approach was used through over-expressing the Petunia chalcone synthase (chsA) gene in order to enhance the silymarin production level in the hairy root cultures of Silybum marianum. Molecular analysis confirmed the presence and integration of chsA transgene in transgenic hairy roots. Chemical analysis indicated that the over-expression of chsA gene enhanced the silymarin production level in the transgenic line as much as 7-folds than the non-transgenic hairy roots. Moreover, the silybin content, the main active component of silymarin, was proved to be 10 times higher in transgenic hairy roots than those of the non-transgenic ones. Therefore, the over-expression of petunia chsA gene in S. marianum hairy roots did not result in gene silencing, but led to an enhanced biosynthesis of the flavonolignans.

Pyrazinecarboxamides as Potential Elicitors of Flavonolignan and Flavonoid Production in Silybum marianum and Ononis arvensis Cultures In Vitro

The effect of new synthetic pyrazinecarboxamide derivatives as potential elicitors of flavonolignan and flavonoid production in Silybum marianum and Ononis arvensis cultures in vitro was investigated. Both tested elicitors increased the production of flavonolignans in S. marianum callus and suspension cultures and flavonoids in O. arvensis callus and suspension cultures. Compound I, 5-(2-hydroxybenzoyl)-pyrazine-2-carboxamide, has shown to be an effective elicitor of flavonolignans and taxifoline production in Silybum marianum culture in vitro. The maximum content of silydianin (0.11%) in S. marianum suspension culture was induced by 24 h elicitor application in concentration of 1.159 × 10−3 mol/L. The maximum content of silymarin complex (0.08%) in callus culture of S. marianum was induced by 168 h elicitor application of a concentration 1.159 × 10−4 mol/L, which represents contents of silydianin (0.03%), silychristin (0.01%) and isosilybin A (0.04%) compared with control. All three tested concentrations of compound II, N-(2-bromo-3-methylphenyl)-5-tert-butylpyrazin-2-carboxamide increased the flavonoid production in callus culture of O. arvensis in a statistically significant way. The best elicitation effect of all elicitor concentrations had the weakest c3 concentration (8.36 × 10−6 mol/L) after 168 h time of duration. The maximum content of flavonoids (about 5,900%) in suspension culture of O. arvensis was induced by 48 h application of c3 concentration (8.36 × 10−6 mol/L).

Methyl jasmonate increases silymarin production in Silybum marianum (L.) Gaernt cell cultures treated with β-cyclodextrins

Silymarin (Sm) from the fruit of Silybum marianum is an isomeric mixture of pharmacologically active flavonolignans which are formed by oxidative coupling of taxifolin (Tx) and coniferyl alcohol (CA). Suspension cultures of this plant constitutively secrete small amounts of Sm into the extracellular medium. Production can be increased by inclusion of cyclodextrins (CDs) in cultures. Both hydroxylated (RHCD) and dimethylated (RMCD) CDs strongly induced prompt accumulation of CA in the medium followed by a late production of flavonolignans. Simultaneous addition of methyl jasmonate (MJ) and RMCD to cells did not significantly modify CA release or flavonolignan accumulation. Delayed addition of MJ to cultures subcultivated in medium containing RMCD markedly influenced Sm production by promoting conversion of the previously formed CA precursor.

Substituted Pyrazinecarboxamides as Abiotic Elicitors of Flavolignan Production in Silybum marianum (L.) Gaertn Cultures in Vitro

Substituted pyrazinecarboxamides markedly influenced production of flavonolignans in Silybum marianum callus and suspension cultures. In this study the effect of two compounds, N-(3-iodo-4-methylphenyl)pyrazine-2-carboxamide (1) and N-(3-iodo-4-methylphenyl)-5-tert-butyl-pyrazine-2-carboxamide (2), as abiotic elicitors on flavono-lignan production in callus culture of S. marianum was investigated. Silymarin complex compounds have hepatoprotective, anticancer and also hypocholesterolemic activity. In vitro flavonolignan concentration in cells is very low and the elicitation is one of the methods to increase production. Elicitors were tested at three concentrations and at different culture times. In the case of elicitation with 1, the greatest increase of flavonolignan and taxifoline production was observed at concentration c1a after 6-hours of elicitation and after 24 and 72-hours at concentration c1b. However, increased production of silychristin, one of the compounds in the silymarin complex, was achieved after only 6-hours elicitation with c1a (2.95 × 10-4 mol/L). The content of silychristin was 2-times higher compared to the control sample. An increased production of silychristin was reached with compound 2 at the concentration c2 (2.53 × 10-3 mol/L) after 72 h of elicitation. The production of silychristin in this case was increased 12-times compared to control.

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.

The effect of substituted amides of pyrazine-2-carboxylic acids on flavonolignan production in Silybum marianum culture in vitro

In vitro plant tissue and cell cultures were used to study herbicide effects on growth, selected metabolic activities and other phenomena. The effect of abiotic elicitors, two newly synthesized substituted amides of pyrazine-2-carboxylic acids (synthesized at the Department of Pharmaceutical Chemistry and Drug Control, School of Pharmacy in Hradec Kralove), on the flavonolignan accumulation in callus and suspension culture of Silybum marianum (L.) Gaertn. was investigated. The compounds markedly influenced production of flavonolignans in an in vitro culture. Particularly after the elicitation with a solution of compound 3-methylamide 5-tert-butylpyrazine-2-carboxylic acid at a concentration of 3.71×10−7 mol 1−1 and within 72 h of elicitation, an increase in flavonolignan production by 893 % in suspension culture versus control took place. The flavonolignan accumulation in callus culture after the elicitation with a solution of 5-brom-2-hydroxyphenylamide of 5-tert-butyl-6-chloropyrazine-2-carboxylic acid was also increased by about 1039% (24 hour elicitation and concentration of 2.59×10−4 mol 1−1).

Enhanced Silymarin accumulation is related to calcium deprivation in cell suspension cultures of Silybum marianum (L.) Gaertn

Elimination of calcium ions from the medium of cell cultures of Silybum marianum (L.) Gaertn increased flavonolignan production. Silymarin accumulation was not altered by treatment of cultures with the calcium ionophore A23187. The specific Ca2+ chelator, EGTA, enhanced the silymarin content in cells by 200%, and its secretion by 3-4 times. The inorganic ion La3+, as well as the calcium channel inhibitor verapamil, also stimulated production. Several reagents known to block intracellular calcium movement, such as ruthenium red, thapsigargin and TMB-8 appreciably increased silymarin accumulation. These results suggest that inhibition of external and internal calcium fluxes plays a significant role in flavonolignan metabolism of S. marianum cell cultures.

Flavonolignan production from Silybum marianum transformed and untransformed root cultures

Explants from seeds of Silybum marianum were used to produce either transformed (‘hairy’) or untransformed root cultures. Silybin, isosilybin, silychristin and silydianin were found in untransformed root cultures, by means of TLC and HPLC. In contrast, only isosilybin and traces of silychristin and silydianin were identified in the ‘hairy’ root cultures.

Influence of medium composition on the accumulation of flavonolignans in cultured cells of Silybum marianum (L.) Gaertn

Flavonolignan production was examined in callus and cell suspension cultures derived from cotyledons of Silybum marianum (L.) Gaertn. The flavonolignan content in 2-month-old calluses was lower than the values obtained for fruits (1.5%) and progressively decreased over repeated subcultures. Higher accumulation was observed in cell suspension cultures than in callus but continued to be very low with respect to the values obtained for fruits (14%). The growth and flavonolignan production of suspensions were tested using different concentrations of KNO 3, KH 2PO 4, iron and calcium. Only the removal of calcium ions promoted flavonolignan accumulation, although under this condition, growth was significantly reduced.