RESUMEN
Hyperforin is a major active constituent of Hypericum perforatum (St. John's wort). It has amazing pharmacological activities, such as antidepressant properties, but it is labile and difficult to synthesize. Its sensitivity and lipophilicity are challenges for processing and formulation. Its chemical complexity provokes approaches of biotechnological production and modification. Dedifferentiated H. perforatum cell cultures lack appropriate storage sites and hence appreciable hyperforin levels. Shoot cultures are capable of forming hyperforin but less suitable for biomass up-scaling in bioreactors. Roots commonly lack hyperforin but a recently established adventitious root line has been demonstrated to produce hyperforin and derivatives at promising levels. The roots also contained lupulones, the typical constituents of hop (Humulus lupulus). Although shear-sensitive, these root cultures provide a potential production platform for both individual compounds and extracts with novel combinations of constituents and pharmacological activities. Besides in vitro cultivation techniques, the reconstruction of hyperforin biosynthesis in microorganisms is a promising alternative for biotechnological production. The biosynthetic pathway is under study, with omics-technologies being increasingly implemented. These biotechnological approaches may not only yield hyperforin at reasonable productivity but also allow for modifications of its chemical structure and pharmacological profile.
Asunto(s)
Composición de Medicamentos/métodos , Hypericum , Floroglucinol/análogos & derivados , Extractos Vegetales/síntesis química , Tecnología Farmacéutica/métodos , Terpenos/síntesis química , Biotecnología , Floroglucinol/síntesis química , Floroglucinol/aislamiento & purificación , Componentes Aéreos de las Plantas , Extractos Vegetales/aislamiento & purificación , Raíces de Plantas , Terpenos/aislamiento & purificaciónRESUMEN
Hyperforin is a major metabolite of the medicinal plant Hypericum perforatum (St. John's Wort) and has recently been found in hormone induced root cultures. The objective of this study is to identify a downstream process for the production of a hyperforin-rich extract with maximum extraction efficiency and minimal decomposition. The maximum extraction time was found to be 60min. The comparison of two equipment concepts for the extraction and solvent evaporation was performed employing two different solvents. While the rotary mixer showed better results for the extraction efficiency than a stirred vessel, the latter set-up was able to handle larger volumes but did not meet all process requirements. For the evaporation the prompt evaporation of the extraction agent using nitrogen stripping led to minor decomposition. In a 5L stirred vessel, the highest specific extraction of hyperforin was 4.3mg hyperforin/g dry weight bio material. Parameters for the equipment design for extraction and solvent evaporation were determined based on the experimental data.
Asunto(s)
Química Farmacéutica/métodos , Hypericum , Floroglucinol/análogos & derivados , Extractos Vegetales/síntesis química , Raíces de Plantas , Terpenos/síntesis química , Cromatografía Líquida de Alta Presión/métodos , Floroglucinol/análisis , Floroglucinol/síntesis química , Floroglucinol/aislamiento & purificación , Extractos Vegetales/análisis , Extractos Vegetales/aislamiento & purificación , Terpenos/análisis , Terpenos/aislamiento & purificaciónRESUMEN
Extracts of the medicinal plant Hypericum perforatum are used to treat depression and skin irritation. A major API is hyperforin, characterized by sensitivity to light, oxygen and temperature. Total synthesis of hyperforin is challenging and its content in field-grown plants is variable. We have established in vitro cultures of auxin-induced roots, which are capable of producing hyperforin, as indicated by HPLC-DAD and ESI-MS analyses. The extraction yield and the productivity upon use of petroleum ether after solvent screening were â¼5 mg/g DW and â¼50 mg/L culture after six weeks of cultivation. The root cultures also contained secohyperforin and lupulones, which were not yet detected in intact plants. In contrast, they lacked another class of typical H. perforatum constituents, hypericins, as indicated by the analysis of methanolic extracts. Hyperforins and lupulones were stabilized and enriched as dicyclohexylammonium salts. Upon up-scaling of biomass production and downstream processing, H. perforatum root cultures may provide an alternative platform for the preparation of medicinal extracts and the isolation of APIs.
Asunto(s)
Reactores Biológicos , Hypericum/metabolismo , Floroglucinol/análogos & derivados , Raíces de Plantas/metabolismo , Terpenos/metabolismo , Hypericum/química , Floroglucinol/análisis , Floroglucinol/química , Floroglucinol/metabolismo , Extractos Vegetales , Raíces de Plantas/química , Terpenos/análisis , Terpenos/químicaRESUMEN
Acute mu and kappa opioids activate the ERK/MAPK phosphorylation cascade that represents an integral part of the signaling pathway of growth factors in astrocytes. By this cross-talk, opioids may impact neural development and plasticity among other basic neurobiological processes in vivo. The mu agonist, [D-ala2,mephe4,glyol5]enkephalin (DAMGO), induces a transient stimulation of ERK phosphorylation, whereas kappa agonist, U69,593, engenders sustained ERK activation. Here we demonstrate that acute U69,593 and DAMGO stimulate ERK phosphorylation by utilization of different secondary messengers and protein kinase C (PKC) isoforms upstream of the growth factor pathway. Immortalized astrocytes transfected with either antisense calmodulin (CaM), a mutant mu opioid receptor that binds CaM poorly or a dominant negative mutant of PKCepsilon were used as a model system to study mu signaling. Evidence was gained to implicate CaM and PKCepsilon in DAMGO stimulation of ERK. DAMGO activation of PKCepsilon and/or ERK was insensitive to selective inhibitors of Ca2+ mobilization, but it was blocked upon phospholipase C inhibition. These results suggest a novel mechanism wherein, upon DAMGO binding, CaM is released from the mu receptor and activates phospholipase C. Subsequently, phospholipase C generates diacylglycerides that activate PKCepsilon. In contrast, U69,593 appears to act via phosphoinositide 3-kinase, PKCzeta, and Ca2+ mobilization. These signaling components were implicated based on studies with specific inhibitors and a dominant negative mutant of PKCzeta. Collectively, our findings on acute opioid effects suggest that differences in their mechanism of signaling may contribute to the distinct outcomes on ERK modulation induced by chronic mu and kappa opioids.
Asunto(s)
Astrocitos/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Proteína Quinasa C/química , Receptores Opioides kappa/fisiología , Receptores Opioides mu/fisiología , Analgésicos/farmacología , Analgésicos Opioides/farmacología , Animales , Astrocitos/citología , Bencenoacetamidas/farmacología , Calcio/metabolismo , Calmodulina/metabolismo , Carbazoles/farmacología , Membrana Celular/metabolismo , Células Cultivadas , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Activación Enzimática , Receptores ErbB/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Indoles , Maleimidas , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Unión Proteica , Isoformas de Proteínas , Proteína Quinasa C/metabolismo , Proteína Quinasa C-epsilon , Pirrolidinas/farmacología , Ratas , Receptores Opioides kappa/agonistas , Transducción de Señal , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
Mitogenic signaling of G protein-coupled receptors (GPCRs) can proceed via sequential epidermal growth factor receptor (EGFR) transactivation and extracellular signal-regulated kinase (ERK) phosphorylation. Although the mu-opioid receptor (MOR) mediates stimulation of ERK via EGFR transactivation in human embryonic kidney 293 cells, the mechanism of acute MOR signaling to ERK has not been characterized in rat C6 glioma cells that seem to contain little EGFR. Herein, we describe experiments that implicate fibroblast growth factor (FGF) receptor (FGFR) transactivation in the convergence of MOR and growth factor signaling pathways in C6 cells. MOR agonists, endomorphin-1 and morphine, induced a rapid (3-min) increase of ERK phosphorylation that was abolished by MOR antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2. By using selective inhibitors and overexpression of dominant negative mutants, data were obtained to suggest that MOR signaling to ERK is transduced by Gbetagamma and entails Ca2+- and protein kinase C-mediated steps, whereas the FGFR branch of the pathway is Ras-dependent. An intermediary role of FGFR1 transactivation was suggested by MOR- but not kappa-opioid receptor (KOR)-induced FGFR1 tyrosine phosphorylation. A dominant negative mutant of FGFR1 attenuated MOR- but not KOR-induced ERK phosphorylation. Thus, a novel transactivation mechanism entailing secreted endogenous FGF may link the GPCR and growth factor pathways involved in MOR activation of ERK in C6 cells.