RESUMEN
Adipogenesis is a complex process in which cell commitment and mitotic clonal expansion (MCE) are in-sequence crucial events leading to terminal adipocyte differentiation. The molecules able to block some key signals in this cascade can hamper adipogenesis becoming promising agents to counteract hyperplasia and hypertrophy of adipose tissue. Mono- and di-caffeoylquinic acid isomers are biologically active polyphenols, displaying in vitro and in vivo antioxidant, hepatoprotective, anti-diabetic and anti-obesity properties. Among these isomers, 3,5-dicaffeoylquinic acid (DCQA) has been reported to inhibit lipid accumulation in adipose cells more successfully than others. Thus, we investigated DCQA effects and molecular mechanisms on 3T3-L1 pre-adipocytes induced to differentiate with a hormonal cocktail (MDI). Oil Red O incorporation assessed that DCQA pre-treatment inhibited lipid accumulation in 3T3-L1 cells induced to differentiate for 10 days. At this time, an increased phosphorylation of both AMP-activated kinase and acetyl-CoA carboxylase, as well as a strong decrease in fatty acid synthase protein level, were registered by immunoblotting, thereby suggesting that DCQA treatment can reduce fatty acid anabolism in 3T3-L1 adipocytes. Furthermore, BrdU incorporation assay, performed 48 h after hormonal stimulation, revealed that DCQA treatment was also able to hinder the 3T3-L1 cell proliferation during the MCE, which is an essential step in the adipogenic process. Thus, we focused our attention on early signals triggered by the differentiation stimuli. In the first hours after hormonal cocktail administration, the activation of ERK1/2 and Akt kinases, or CREB and STAT3 transcription factors, was not affected by DCQA pre-treatment. Whereas 24 h after MDI induction, DCQA pre-treated cells showed increased level of the transcription factor Nrf2, that induced the expression of the antioxidant enzyme heme oxygenase 1 (HO-1). In control samples, the expression level of HO-1 was reduced 24 h after MDI induction in comparison with the higher amount of HO-1 protein found at 2 h. The HO-1 decrease was functional by allowing reactive oxygen species to boost and allowing cell proliferation induction at the beginning of MCE phase. Instead, in DCQA-treated cells the HO-1 expression was maintained at high levels for a further 24 h; in fact, its expression decreased only 48 h after MDI stimulation. The longer period in which HO-1 expression remained high led to a delay of the MCE phase, with a subsequent inhibition of both C/EBP-α expression and adipocyte terminal differentiation. In conclusion, DCQA counteracting an excessive adipose tissue expansion may become an attractive option in obesity treatment.
Asunto(s)
Adipocitos/citología , Adipocitos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Ácido Clorogénico/análogos & derivados , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Mitosis/efectos de los fármacos , Células 3T3-L1 , Adipocitos/metabolismo , Adipogénesis/efectos de los fármacos , Animales , Ácido Clorogénico/farmacología , RatonesRESUMEN
Echinacea angustifolia cell suspension cultures are usually grown and maintained in the dark, but we also exposed cells to light for one culture cycle (14 days) and then compared the metabolomes of dark-grown and illuminated cells by liquid chromatography-mass spectrometry. Among 256 signals, we putatively identified 159 molecules corresponding to 56 different metabolites plus their fragments, adducts and isotopologs. The E. angustifolia metabolome consisted mainly of caffeic acid derivatives, comprising (a) caffeic acid conjugated with tartaric, quinic and hexaric acids; and (b) caffeic acid conjugated with hydroxytyrosol glycosides (e.g., echinacoside, verbascoside and related molecules). Many of these metabolites have not been previously described in E. angustifolia, which currently lacks detailed metabolic profiles. Exposure to light significantly increased the levels of certain caffeic acid derivatives (particularly caffeoylquinic acids and hydroxytyrosol derivatives lacking rhamnose residues) and reduced the level of hydroxytyrosol derivatives with rhamnose residues, revealing that light specifically inhibits the rhamnosylation of caffeoyl phenylethanoid glycosides. These results are significant because they suggest that the metabolic profile of cell cultures can be manipulated by controlling simple environmental variables such as illumination to modulate the levels of potentially therapeutic compounds.
Asunto(s)
Echinacea/citología , Echinacea/metabolismo , Luz , Metabolómica/métodos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Echinacea/efectos de la radiación , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
We here report the results of our investigations carried out on verbascoside, a phenylpropanoid glycoside known for its antioxidant, anti-inflammatory and photoprotective actions. Verbascoside was obtained from Buddleia davidii meristematic cells, obtained in turn using a sustainable biotechnology platform which employs an in vitro plant cell culture technology. Verbascoside was first investigated to assess the behaviour of the active ingredient in solution or in finished preparations, in view of its potential topical use, especially in skin protection. Stability studies were performed by HPLC, and a PCL assay was adopted to determine the radical scavenging activity toward superoxide anion. The high hydrophilic character of verbascoside, suggested in a somewhat limited range of possible applications, leading us to explore its derivatization to obtain the semi-synthetic derivative VPP, an acyl derivative of verbascoside, with an improved range of applications due to its lower hydrophilic profile. Alone, VPP revealed increased antioxidant activity, both as an active ingredient and in dermocosmetic preparations. Stability studies showed a greater stability of VPP in lipophilic vehicles, whereas the parent verbascoside proved more stable in an O/W emulsions. Verbascoside was also stable in suppositories, an interesting pharmaceutical form for possible applications in treatment of inflammation of the intestinal mucosa.
Asunto(s)
Antioxidantes , Buddleja/química , Química Farmacéutica/métodos , Cosméticos , Glucósidos , Fenoles , Administración Tópica , Antioxidantes/química , Antioxidantes/farmacología , Cromatografía Líquida de Alta Presión , Cosméticos/química , Cosméticos/farmacología , Estabilidad de Medicamentos , Emulsiones/química , Emulsiones/farmacología , Excipientes/química , Glucósidos/química , Glucósidos/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inflamación/tratamiento farmacológico , Inflamación/patología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Luminiscencia , Mediciones Luminiscentes , Fenoles/química , Fenoles/farmacología , Piel/efectos de los fármacos , Piel/patología , Enfermedades de la Piel/tratamiento farmacológico , Enfermedades de la Piel/patología , Solubilidad , Superóxidos/antagonistas & inhibidores , Superóxidos/metabolismo , Supositorios/químicaRESUMEN
The blood-brain barrier (BBB) maintains the homeostasis of the brain microenvironment, which is crucial for neuronal activity and function. Under pathological conditions, the BBB may fail due to yet unknown mechanisms. BBB failure is accompanied by an increase in the transendothelial permeability to substances such as sucrose that are normally extruded. Furthermore, altered BBB function may also lead to development of abnormal drug extrusion mechanisms including expression of multiple drug resistance proteins. Therefore, it is not surprising that strategies have been developed to "repair" the BBB in order to restore normal brain homeostasis and penetration/extrusion of pharmacologically active (noxious) substances. To this end, steroidal hormones and synthetic analogues such as dexamethasone (DEX) have been used to counteract BBB failure. However, several side effects limit the usefulness of steroid treatment in humans leading to the quest for developing novel strategies for BBB repair. We here show that, in an in vitro model of the BBB based on a co-culture of endothelial cells (EC) and glia, the natural compound glycerophosphoinositol (GPI) may replicate the effects of DEX. Thus, GPI in concentrations ranging from 3 to 100 microM promoted both BBB formation and repair in a dose dependent fashion. Similar effects were obtained with an elevated dose of DEX (10 microM); at higher concentrations (100 microM), DEX was cytotoxic. We conclude that the endogenous anti-inflammatory agent GPI may ameliorate BBB function with efficacy comparable to that of steroids, but with significantly fewer side effects. Further experiments will confirm the efficacy of this treatment in vivo and elucidate the pathways that lead to BBB repair after exposure to GPI.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Dexametasona/farmacología , Células Endoteliales/efectos de los fármacos , Fosfatos de Inositol/farmacología , Animales , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/fisiología , Bovinos , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Impedancia Eléctrica , Células Endoteliales/citología , Células Endoteliales/fisiología , Ratas , Células Tumorales CultivadasRESUMEN
Phenylpropanoids have several highly significant biological properties in both plants and animals. Four phenylpropanoid glycosides (PPGs), verbascoside (VB), forsythoside B (FB), echinacoside (EC) and campneoside I (CP), were purified and tested for their capability to activate NRF2 and induce phase II cytoprotective enzymes in a human keratinocyte cell line (HaCaT). All four substances showed similar strong antioxidant and radical-scavenging activities as determined by diphenylpicrylhydrazyl assay. Furthermore, in HaCaT cells, FB and EC are strong activators of NRF2, the nuclear transcription factor regulating many phase II detoxifying and cytoprotective enzymes, such as heme oxygenase 1 (HMOX1). In HaCaT cells, FB and EC (200 µM) induced nuclear translocation of NRF2 protein after 24 h and reduced nuclear protein levels of BACH1, a repressor of the antioxidant response element. FB and EC greatly HMOX1 mRNA levels by more than 40-fold in 72 h. Cytoplasmic HMOX1 protein levels were also increased at 48 h after treatment. VB was less active compared to FB and EC, and CP was slightly active only at later times of treatment. We suggest that hydroxytyrosol (HYD) could be a potential bioactive metabolite of PPGs since HYD, in equimolar amounts to PGGs, is able to both activate HO-1 transcription and modify Nrf2/Bach1 nuclear protein levels. This is in agreement with the poor activity of CP, which contains a HYD moiety modified by an O-methyl group. In conclusion, FB and EC from plant cell cultures may provide long-lasting skin protection by induction of phase II cytoprotective capabilities.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Glicósidos/farmacología , Hemo-Oxigenasa 1/genética , Queratinocitos/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Extractos Vegetales/farmacología , Regulación hacia Arriba/efectos de los fármacos , Antioxidantes/química , Antioxidantes/farmacología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Línea Celular , Citocinas/inmunología , Citoprotección/efectos de los fármacos , Echinacea/química , Echinacea/citología , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Glicósidos/química , Hemo-Oxigenasa 1/inmunología , Hemo-Oxigenasa 1/metabolismo , Humanos , Queratinocitos/inmunología , Queratinocitos/metabolismo , Factor 2 Relacionado con NF-E2/genética , Fenoles/química , Fenoles/farmacología , Alcohol Feniletílico/análogos & derivados , Alcohol Feniletílico/farmacología , Extractos Vegetales/química , ARN Mensajero/genética , Syringa/química , Syringa/citologíaRESUMEN
3,5-O-dicaffeoyl-4-O-malonilquinic acid (1) (irbic acid) has been isolated for the first time from cell cultures of Centella asiatica and till now it has never been reported to be present in the intact plant. Evidence of its structure was obtained by spectroscopic analyses (MS/NMR). Besides 1, cell cultures produce also the known 3,5-O-dicaffeoylquinic acid, chlorogenic acid, and the triferulic acid 2 (4-O-8'/4'-O-8â³-didehydrotriferulic acid). Biological activities were evaluated for compound 1, which showed to have a strong radical scavenging capacity, together with a high inhibitory activity on collagenase. This suggests a possible utilization of this substance as a topical agent to reduce the skin ageing process.
Asunto(s)
Centella/química , Colagenasas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Extractos Vegetales/farmacología , Ácido Quínico/análogos & derivados , Técnicas de Cultivo de Célula , Colagenasas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Molecular , Extractos Vegetales/química , Ácido Quínico/química , Ácido Quínico/aislamiento & purificación , Ácido Quínico/farmacologíaRESUMEN
The previous results suggest that peroxisome proliferator-activated receptor-alpha (PPAR)-alpha, an intracellular transcription factor activated by fatty acids, plays a role in control of inflammation. There is persuasive epidemiological and experimental evidence that dietary polyphenols have anti-inflammatory activity. In this regard, it has been demonstrated that verbascoside (VB) functions as intracellular radical scavenger and reduces the microscopic and macroscopic signs of experimental colitis. With the aim to characterize the role of PPAR-alpha in VB-mediated anti-inflammatory activity, we tested the efficacy of VB in an experimental model of inflammatory bowel disease induced by dinitrobenzene sulfonic acid, comparing mice lacking PPAR-alpha (PPAR-alphaKO) with wild type (WT) mice. Results indicate that VB-mediated anti-inflammatory activity is weakened in PPAR-alphaKO mice, compared to WT controls, especially in the inhibition of neutrophil infiltration, intestinal permeability and colon injury. These results indicate that PPAR-alpha can contribute to the anti-inflammatory activity of VB in inflammatory bowel disease.
RESUMEN
The glycosylated phenylpropanoid verbascoside (VB), isolated from cultured cells of the medicinal plant Syringa vulgaris (Oleaceae), has previously been characterized as an effective scavenger of biologically active free radicals and an inhibitor of lipid peroxidation. The aim of the present study was to evaluate in a rat glioma cell line (C6) the effect of VB biotechnologically produced by S. vulgaris plant cell cultures in the regulation of the inflammatory response. We used a model of central nervous system inflammation induced by bacterial endotoxin/cytokine (lipopolysaccharide (LPS)/interferon (IFN)-gamma, 1 microg/ml and 100 U/ml, respectively). Our results show that the treatment with LPS/IFN-gamma for 24 h elicited the induction of inducible nitric oxide synthase (iNOS) activity as determined by NO(x) accumulation in the culture medium. Preincubation with VB (10-100 microg/ml) abrogated the mixed cytokine-mediated induction of iNOS. The effect was concentration-dependent. Our studies also showed an inhibitory effect of VB on neuronal nitric oxide synthase expression. Moreover, Western blot analysis showed that this glycoside prevents specifically the activation of the proinflammatory enzyme cyclooxygenase (COX)-2 in glioma cells without simultaneous inhibition of COX-1 enzyme. Moreover, we found that VB reduced the expression of proinflammatory enzymes in LPS/IFN-gamma through the inhibition of the activation of nuclear factor kappa B and mitogen-activated protein kinase signaling pathway. The mechanisms underlying in vitro the neuroprotective properties of VB involve modulation of transcription factors and consequent altered gene expression, resulting in downregulation of inflammation. These findings provide support that VB may provide a promising approach for the treatment of oxidative-stress-related neurodegenerative diseases.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Glioma/patología , Glioma/prevención & control , Glucósidos/uso terapéutico , Fenoles/uso terapéutico , Syringa , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Glioma/metabolismo , Ratones , Extractos Vegetales/uso terapéutico , Ratas , VerbenaceaeRESUMEN
The aim of the present study was to examine the effects of verbascoside (VB) in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of 2,4 dinitrobenzene sulfonic acid (DNBS; 25 mg/rat). VB was administered daily per os (0.2 and 2 mg/kg) 4 days after DNBS administration in the colon. Treatment with VB significantly (P < 0.01) reduced macroscopic damage score, loss of body weight, myeloperoxidase activity and thiobarbituric acid-reactant substances. Moreover, the intensity of the positive staining for tumor necrosis factor-alpha, interleukin-1beta, intercellular adhesion molecule-1, P-selectin, inducible nitric oxide synthase, and poly(ADP ribose) was also significantly (P < 0.01) reduced by VB treatment. Therefore, VB treatment significantly (P < 0.01) reduced the degree of NF-kappaB p65 and activation of the pro-active form metalloproteinase (MMP)-2 and pro-MMP-9 activity. The results of this study suggested that VB functions as an intracellular radical scavenger and so reduces the microscopic and macroscopic signs of colitis in the rat. Therefore, administration of VB may be beneficial for the treatment of inflammatory bowel disease.
Asunto(s)
Antioxidantes/uso terapéutico , Colitis/tratamiento farmacológico , Glucósidos/uso terapéutico , Fenoles/uso terapéutico , Syringa/química , Animales , Antioxidantes/administración & dosificación , Antioxidantes/aislamiento & purificación , Peso Corporal/efectos de los fármacos , Células Cultivadas , Colitis/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Precursores Enzimáticos/efectos de los fármacos , Precursores Enzimáticos/metabolismo , Gelatinasas/efectos de los fármacos , Gelatinasas/metabolismo , Glucósidos/administración & dosificación , Glucósidos/aislamiento & purificación , Masculino , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Peroxidasa/efectos de los fármacos , Peroxidasa/metabolismo , Fenoles/administración & dosificación , Fenoles/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Factor de Transcripción ReIA/efectos de los fármacos , Factor de Transcripción ReIA/metabolismoRESUMEN
The aim of the present study was to examine the effects of phenylpropanoid glycoside, teupolioside, biotechnologically produced by IRBN22 Ajuga reptans cell line, in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). Teupolioside was administered daily orally (0.2 or 2mgkg(-1)). On Day 4, animals were sacrificed and tissues were taken for histological and biochemical analysis. Four days after DNBS administration, colon TNF-alpha and IL-1beta productions were increased, associated with colon damage. Neutrophil infiltration, by myeloperoxidase activity, in the mucosa was associated with up-regulation of ICAM-1 and P-selectin and high levels of malondialdehyde. Immunohistochemistry for nitrotyrosine and poly (ADP-ribose) polymerase (PARP) showed an intense staining in the inflamed colon. Biochemical methods and zymography were used to analyze MMP-9 and -2 activities in colon tissues from DNBS-injured rats. Treatment with teupolioside significantly reduced the appearance of diarrhoea and the loss of body weight. This was associated with a remarkable amelioration in the disruption of the colonic architecture and a significant reduction in colonic myeloperoxidase activity and malondialdehyde levels. Teupolioside also reduced the pro-inflammatory cytokines release, the appearance of nitrotyrosine and PARP immunoreactivity in the colon and reduced the up-regulation of ICAM-1 and the expression of P-selectin. Therefore, teupolioside also reduced proMMP-9 and -2 activity induced in the colon by DNBS administration. The results of this study suggested that administration of teupolioside may be beneficial for treatment of inflammatory bowel disease.
Asunto(s)
Ajuga/química , Ácidos Cafeicos/uso terapéutico , Colitis/tratamiento farmacológico , Modelos Animales de Enfermedad , Trisacáridos/uso terapéutico , Ajuga/citología , Animales , Bencenosulfonatos/administración & dosificación , Ácidos Cafeicos/aislamiento & purificación , Línea Celular , Cromatografía Líquida de Alta Presión , Colitis/inducido químicamente , Colitis/metabolismo , Colon/enzimología , Colon/metabolismo , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-1beta/biosíntesis , Peroxidación de Lípido/efectos de los fármacos , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Selectina-P/metabolismo , Ratas , Ratas Sprague-Dawley , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Trisacáridos/aislamiento & purificación , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The glycosylated phenylpropanoid verbascoside isolated from cultured cells of the medicinal plant Syringa vulgaris (Oleaceae) has previously been characterized as an effective scavenger of biologically active free radicals such as hydroxyl, superoxide, and nitric oxide, as a chelator of redox active transition metal ions (Fe (2+), Fe (3+), Cu (2+), and Ni (2+)), and an inhibitor of lipid peroxidation. In the present work, we have compared the cytoprotective effects of the biotechnologically produced verbascoside with two commercially available polyphenols (the glycosylated flavonoid rutin and its aglycone quercetin) against free radical-mediated UVC-induced cellular death in cultures of human keratinocytes (HaCaT) and breast cancer cells (MCF 7). We have shown that all the polyphenols studied afforded effective protection against UVC-induced necrosis and did not prevent UVC-induced apoptosis in both normal and tumor cell lines. The cytoprotection did not correlate either with UVC absorbance by polyphenols or with their superoxide radical scavenging properties. However, UVC protection strongly depended on the lipid peroxidation inhibiting and Fe (2+) chelating properties of polyphenols. We suggest that these plant polyphenols could be feasible for a photoprotection of human skin.