RESUMEN
Silybum marianum (L.) Gaertn is a rich source of antioxidants and anti-inflammatory flavonolignans with great potential for use in pharmaceutical and cosmetic products. Its biotechnological production using in vitro culture system has been proposed. Chitosan is a well-known elicitor that strongly affects both secondary metabolites and biomass production by plants. The effect of chitosan on S. marianum cell suspension is not known yet. In the present study, suspension cultures of S. marianum were exploited for their in vitro potential to produce bioactive flavonolignans in the presence of chitosan. Established cell suspension cultures were maintained on the same hormonal media supplemented with 0.5 mg/L BAP (6-benzylaminopurine) and 1.0 mg/L NAA (α-naphthalene acetic acid) under photoperiod 16/8 h (light/dark) and exposed to various treatments of chitosan (ranging from 0.5 to 50.0 mg/L). The highest biomass production was observed for cell suspension treated with 5.0 mg/L chitosan, resulting in 123.3 ± 1.7 g/L fresh weight (FW) and 17.7 ± 0.5 g/L dry weight (DW) productions. All chitosan treatments resulted in an overall increase in the accumulation of total flavonoids (5.0 ± 0.1 mg/g DW for 5.0 mg/L chitosan), total phenolic compounds (11.0 ± 0.2 mg/g DW for 0.5 mg/L chitosan) and silymarin (9.9 ± 0.5 mg/g DW for 0.5 mg/L chitosan). In particular, higher accumulation levels of silybin B (6.3 ± 0.2 mg/g DW), silybin A (1.2 ± 0.1 mg/g DW) and silydianin (1.0 ± 0.0 mg/g DW) were recorded for 0.5 mg/L chitosan. The corresponding extracts displayed enhanced antioxidant and anti-inflammatory capacities: in particular, high ABTS antioxidant activity (741.5 ± 4.4 µM Trolox C equivalent antioxidant capacity) was recorded in extracts obtained in presence of 0.5 mg/L of chitosan, whereas highest inhibitions of cyclooxygenase 2 (COX-2, 30.5 ± 1.3 %), secretory phospholipase A2 (sPLA2, 33.9 ± 1.3 %) and 15-lipoxygenase (15-LOX-2, 31.6 ± 1.2 %) enzymes involved in inflammation process were measured in extracts obtained in the presence of 5.0 mg/L of chitosan. Taken together, these results highlight the high potential of the chitosan elicitation in the S. marianum cell suspension for enhanced production of antioxidant and anti-inflammatory silymarin-rich extracts.
Asunto(s)
Antiinflamatorios , Antioxidantes , Quitosano , Lignanos , Células Vegetales/metabolismo , Silybum marianum/metabolismo , Animales , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Antioxidantes/química , Antioxidantes/metabolismo , Antioxidantes/farmacología , Quitosano/química , Quitosano/metabolismo , Quitosano/farmacología , Humanos , Lignanos/biosíntesis , Lignanos/química , Lignanos/farmacología , Silybum marianum/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , OvinosRESUMEN
Currently, the evolution of green chemistry in the synthesis of nanoparticles (NPs) with the usage of plants has captivated a great response. In this study, in vitro plantlets and callus of Silybum marianum were exploited as a stabilising agent for the synthesis of zinc oxide (ZnO) NPs using zinc acetate and sodium hydroxide as a substitute for chemical method. The contemporary investigation defines the synthesis of ZnO NPs prepared by chemical and bio-extract-assisted methods. Characterisation techniques such as X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy and energy dispersive X-ray were used to confirm the synthesis. Although chemical and bio-assisted methods are suitable choices for NPs synthesis, the bio-assisted green assembly is advantageous due to superior stability. Moreover, this report describes the antibacterial activity of the synthesised NPs against standard strains of Klebsiella pneumonia and Bacillus subtilis.
Asunto(s)
Antibacterianos/química , Antibacterianos/síntesis química , Nanopartículas del Metal/química , Nanotecnología/métodos , Silybum marianum/metabolismo , Óxido de Zinc/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Tecnología Química Verde , Silybum marianum/química , Silybum marianum/citología , Extractos Vegetales/química , Extractos Vegetales/metabolismo , Óxido de Zinc/farmacologíaRESUMEN
Elicitation with methyl jasmonate (MeJA) or/and cyclodextrin (CD) strongly induced silymarin (Sm) accumulation in suspensions of Silybum marianum, with most of Sm isomers being detected in the culture medium. This induction provides a model platform to characterize the regulation of flavonolignan accumulation and release in response to elicitors and, with this aim, changes in the S. marianum cell proteome were investigated. The DIGE technique was used to detect statistically significant changes in the cell's proteome. A total number of 1269 unique spots were detected, 67 of which were de-regulated upon elicitation. Nineteen spots were identified by nLC-MS/MS database search analysis. Identified proteins belong to a few categories, including metabolism, stress and defense responses and transport processes. The most abundant group was represented by pathogenesis-related (PR) proteins and heat shock proteins. Two proteins related to transport process were identified and both were upregulated by elicitation. One was identified as Ras-related protein Rab11C of the Rab family of small ATPase superfamily. A second protein was identified as an ABC transporter. Some of the identified proteins are discussed with respect to their putative role in the extracellular flavonolignan accumulation in S. marianum cultures. BIOLOGICAL SIGNIFICANCE: Most approaches to increase secondary metabolite yields using plant cell cultures have been focused on the optimization of its biosynthesis. The study of other post biosynthetic events, like chemical or enzymatic modifications, transport, storage/secretion and catabolism/degradation are also biotechnologically relevant. Secretion is of particular interest since if cell cultures are to be used routinely for the commercial production, they must release the targeted metabolites into the extracellular medium. Elicitor-induced silymarin accumulation and release in S. marianum cell cultures provide a responsive model system to profile both alterations in proteins related to monolignol/flavonoid biosynthesis and to identify potential systems involved in secretion of secondary metabolites. The proteomic approach undertaken in this work has permitted identify some of the events occurring in elicited S. marianum cell cultures. One attainment of this study is that a vesicular transport mechanism could be involved in the release of this class of secondary metabolites to the extracellular compartment. This finding forms a baseline for future research on a non-sequenced medicinal plant S. marianum at molecular level.
Asunto(s)
Acetatos/farmacología , Ciclopentanos/farmacología , Oxilipinas/farmacología , Células Vegetales/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Silybum marianum/metabolismo , beta-Ciclodextrinas/farmacología , Flavonolignanos/biosíntesis , Silybum marianum/citologíaRESUMEN
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⻳ 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 × 10â»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 × 10â»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 × 10â»6 mol/L).
Asunto(s)
Amidas , Fabaceae/química , Flavonoides/biosíntesis , Flavonolignanos/biosíntesis , Pirazinas , Silybum marianum/química , Amidas/química , Amidas/farmacología , Células Cultivadas , Fabaceae/citología , Fabaceae/metabolismo , Flavonoides/química , Flavonolignanos/química , Humanos , Silybum marianum/citología , Silybum marianum/metabolismo , Estructura Molecular , Extractos Vegetales/química , Extractos Vegetales/farmacología , Pirazinas/química , Pirazinas/farmacologíaRESUMEN
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.
Asunto(s)
Acetatos/farmacología , Ciclopentanos/farmacología , Oxilipinas/farmacología , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/metabolismo , Silybum marianum/citología , Silybum marianum/enzimología , Silimarina/biosíntesis , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Extractos Celulares , Células Cultivadas , Silybum marianum/efectos de los fármacos , Ácidos Fosfatidicos/farmacología , Fosfatidilcolinas/metabolismo , Factores de TiempoRESUMEN
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.
Asunto(s)
Antioxidantes/metabolismo , Ácido Salicílico/farmacología , Silybum marianum/efectos de los fármacos , Silybum marianum/metabolismo , Silimarina/biosíntesis , Técnicas de Cultivo de Célula , Flavonolignanos/metabolismo , Ácido Linoleico/agonistas , Ácido Linoleico/metabolismo , Lipooxigenasa/efectos de los fármacos , Lipooxigenasa/metabolismo , Silybum marianum/citología , Raíces de Plantas/citología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Quercetina/análogos & derivados , Quercetina/biosíntesis , Silibina , Silimarina/análogos & derivadosRESUMEN
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.
Asunto(s)
Transducción de Señal , Silybum marianum/citología , Silimarina/metabolismo , Acetatos/farmacología , Calcio/antagonistas & inhibidores , Células Cultivadas , Ciclopentanos/farmacología , Inhibidores Enzimáticos/farmacología , Silybum marianum/efectos de los fármacos , Silybum marianum/enzimología , Oxilipinas/farmacología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , LevadurasRESUMEN
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.
Asunto(s)
Acetatos/farmacología , Ciclopentanos/farmacología , Resonancia Magnética Nuclear Biomolecular , Reguladores del Crecimiento de las Plantas/farmacología , Silybum marianum/citología , Silybum marianum/metabolismo , Alanina/biosíntesis , Aminoácidos/metabolismo , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Técnicas de Cultivo de Célula , Células Cultivadas , Colina/biosíntesis , Cicloheximida/farmacología , Glucosa/metabolismo , Ácido Glutámico/biosíntesis , Oxilipinas , Inhibidores de la Síntesis de la Proteína/farmacología , Silimarina/metabolismo , Sacarosa/metabolismo , Levaduras/química , Ácido alfa-Linolénico/biosíntesisRESUMEN
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.
Asunto(s)
Peroxidasas/metabolismo , Silybum marianum/citología , Silybum marianum/enzimología , Silimarina/metabolismo , Acetatos/farmacología , Células Cultivadas , Ciclopentanos/farmacología , Silybum marianum/efectos de los fármacos , Oxilipinas , Silimarina/biosíntesisRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Silybum marianum/metabolismo , Silimarina/metabolismo , Calcimicina/farmacología , Células Cultivadas , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Lignanos/metabolismo , Silybum marianum/citología , Rojo de Rutenio/farmacología , Tapsigargina/farmacologíaRESUMEN
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.