Purificación Corchete

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

Abstract 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 2 PO 4 , iron and calcium. Only the removal of calcium ions promoted flavonolignan accumulation, although under this condition, growth was significantly reduced.

Antifungal activity of scopoletin and its differential accumulation in Ulmus pumila and Ulmus campestris cell suspension cultures infected with Ophiostoma ulmi spores

Abstract Inoculation of cell suspension cultures derived from Ulmus pumila , a species resistant to Dutch elm disease (DED) and U. campestris , a susceptible elm, with spores of Ophiostoma ulmi , differentially induced the accumulation of the hydroxycoumarin scopoletin. This coumarin was induced rapidly and accumulated mainly in the medium of the challenged resistant culture. In in vitro bioassays, scopoletin showed a direct antifungal activity against O. ulmi spore germination being more sensitive to this inhibitory activity than mycelial growth. The significance of scopoletin in vivo is also discussed.

Diarylmethyloxime and hydrazone derivatives with 5-indolyl moieties as potent inhibitors of tubulin polymerization.

We describe the synthesis and biological evaluation of a series of diarylmethyloxime and diarylmethylhydrazone analogues that contain an indole ring and different modifications on the nitrogen of the bridge. Several compounds showed potent tubulin polymerization inhibitory action as well as cytotoxic activity against cancer cell lines. The N-methyl-5-indolyl substituted analogues are more potent than ethyl substituted ones. The most potent inhibitors of tubulin polymerization are the diarylketones and the diaryloximes. The cytotoxicity against several cancer cell lines is lower for the oximes than for the ketones. Other substitutions on the imine nitrogen greatly reduce the tubulin inhibitory and/or cytotoxic potencies.

Exploring the effect of 2,3,4-trimethoxy-phenyl moiety as a component of indolephenstatins

A new family of phenstatin analogues has been synthesized and assayed. This family simultaneously incorporates modifications of the A-ring (replacement of the 3,4,5-trimethoxyphenyl by the 2,3,4-trimethoxyphenyl arrangement), B-ring (N-alkyl-5-indolyl) and conversion of the Oxygen keto group into a substituted nitrogen (oximes, hydrazones, and their acetylderivatives). The conjunction of all this changes greatly diminishes the antimitotic and antiproliferative activities, but the maintenance of the keto bridge produces a potent analogue with the unusual 2,3,4-trimethoxyphenyl moiety.

Naphthylphenstatins as tubulin ligands : Synthesis and biological evaluation

A new family of naphthalenic analogues of phenstatins with modifications on the ketone-bridge has been synthesised. The synthesised compounds have been assayed for tubulin polymerisation inhibitory activity as well as for cytotoxic activity against cancer cell lines. The naphthalene has been confirmed as a good surrogate for the isovanillin moiety (3-hydroxy-4-methoxyphenyl) of phenstatin, when combined with the 3,4,5-trimethoxyphenyl ring, but not when combines with the 2,3,4-trimethoxyphenyl ring. Binding models for the synthesised compounds have been generated and analysed in terms of a pharmacophore proposed for colchicine site ligands. The ketone is the optimal bridge substitution but E-acetyloximes or acetylhydrazones are also tolerated. Significant differences with indole substituted phenstatins are observed and discussed.

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.

New trends in biotechnological production of rosmarinic acid

Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyl lactic acid, is widely distributed in the plant kingdom. Interest in it is growing due to its promising biological activities, including cognitive-enhancing effects and slowing the development of Alzheimer’s disease, cancer chemoprotection or anti-inflammatory activity, among others. In order to meet the increasing demand for this compound, several biotechnological approaches to its production based on plant cell and hairy root cultures have been developed. Empirical strategies are currently being combined with metabolic engineering tools to increase RA production in plant cell platforms in a more rational way. Discussed here are the latest advances in the field, together with recent trends in plant biotechnology, such as the application of single use technology and the use of biosensors in downstream processes.

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.

Calcium restriction induces cardenolide accumulation in cell suspension cultures of Digitalis thapsi L.

SummaryThe removal of calcium ions from Murashige and Skoog culture medium induced a marked increase in the accumulation of cardenolides in cell suspension cultures of Digitalis thapsi. Cell viability was not affected although growth was slightly reduced. Strontium ions could substitute for calcium in inhibiting cardenolide production, this effect of calcium being reversed by the addition of LaCl3 or ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid. The results suggest that calcium, apart from its general effects on growth, may play a role in the regulation of cardenolide metabolism in a concentration dependent manner.

Silybum marianum (L.) Gaertn: the Source of Silymarin

The milk thistle Silybum marianum(L.) Gaernt, a member of the Asteraceae family, is an herb whose fruits have been used medicinally for over 2000 years. Their properties are due to the presence of silymarin, an isomeric mixture of the flavonolignans silydianin, silychristin, present in two diastereoisomeric forms, A and B, silybin and isosilybin, which also exist as two diastereoisomers: silybin A and B, and isosilybin A and B. The biosynthesis of these compounds is carried out by oxidative coupling catalysed by peroxidase enzymes between the flavonoid taxifolin and the phenylpropanoid coniferyl alcohol. The silymarin content in fruits depends on the milk thistle variety and geographic and climatic conditions in which they grow; however, the relative proportions of individual components is a genetic characteristic associated with specific chemoraces. Extracts of the fruits have traditionally been employed for treating liver disorders. Studies performed in vitro and in vivo have demonstrated the antioxidant activity of silymarin and its ability to stimulate protein synthesis and cell regeneration; thus, silymarin is being used for the treatment of toxic liver damage and for therapy of chronic inflammatory liver diseases and liver cirrhosis.Silymarin also inhibits chemically induced carcinogenesis and shows direct anticarcinogenic activity against several human carcinoma cells; in addition, silymarin shows antidiabetic, hipolipidaemic, anti-inflammatory, cardioprotective, neurotrophic and neuroprotective effects. Further studies with pure components of silymarin will extend its applications. Tissue cultures have been derived from several organs of this species. Silymarin accumulation in cell cultures is lower than in the fruit and can be stimulated by elicitation with yeast extract and/or methyl jasmonate. An extensive metabolic reprogramming occurred upon elicitation: phenylpropanoid, carbohydrate and amino acid metabolism was altered and probably redirected to support the biosynthesis of flavonolignans. Yeast extract promotes the accumulation of choline and α-linolenic acid in cells, suggesting an action on membranes and the involvement of the octadecanoid pathway in the induction of silymarin in S. marianum cultures.