RESUMEN
Co-cultivation is a powerful emerging tool for awakening biosynthetic gene clusters (BGCs) that remain transcriptionally silent under artificial culture conditions. It has recently been used increasingly extensively to study natural interactions and discover new bioactive metabolites. As a part of our project aiming at the discovery of structurally novel and biologically active natural products from mangrove endophytic fungi, an established co-culture of a strain of Phomopsis asparagi DHS-48 with another Phomopsis genus fungus DHS-11, both endophytes in mangrove Rhizophora mangle, proved to be very efficient to induce the production of new metabolites as well as to increase the yields of respective target metabolites. A detailed chemical investigation of the minor metabolites produced by the co-culture of these two titled fungal strains led to the isolation of six alkaloids (1-6), two sterols (7, 8), and six polyketides (9-14). In addition, all the compounds except 8 and 10, as well as three new metabolites phomopyrazine (1), phomosterol C (7), and phomopyrone E (9), were not present in discrete fungal cultures and only detected in the co-cultures. The structures were elucidated on the basis of spectroscopic analysis, and the absolute configurations were assumed by electronic circular dichroism (ECD) calculations. Subsequently, the cytotoxic, immunosuppressive, and acetylcholinesterase inhibitory properties of all the isolated metabolites were determined in vitro. Compound 8 exhibited moderate inhibitory activity against ConA-induced T and LPS-induced B murine splenic lymphocytes, with IC50 values of 35.75 ± 1.09 and 47.65 ± 1.21 µM, respectively.
Asunto(s)
Técnicas de Cocultivo , Endófitos , Phomopsis , Rhizophoraceae , Animales , Ratones , Rhizophoraceae/microbiología , Endófitos/metabolismo , Phomopsis/metabolismo , Alcaloides/farmacología , Alcaloides/aislamiento & purificación , Alcaloides/química , Productos Biológicos/farmacología , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Metabolismo Secundario , Policétidos/metabolismo , Policétidos/farmacología , Policétidos/aislamiento & purificación , Policétidos/química , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/aislamiento & purificación , Pueblos del Este de AsiaRESUMEN
Vomifoliol, a natural sesquiterpene compound, is a secondary metabolite isolated from the mangrove plant Ceriops tagal. The present study aimed to determine the immunosuppressive effects and underlying mechanisms of vomifoliol on Jurkat cells in vitro. The results show that vomifoliol significantly inhibited calcineurin (CN) at concentrations resulting in relatively low cytotoxicity. Moreover, vomifoliol was found to exert an inhibitory effect on phorbol 12-myristate 13-acetate (PMA)/ ionomycin (Io) -induced Jurkat cells and the dephosphorylation of NFAT1. In addition, it reduced the expression of IL-2. Based on these results, we concluded that vomifoliol may inhibit the immune response of Jurkat cells, and vomifoliol may use CN as the target enzyme to inhibit NFAT signaling pathway. Therefore, vomifoliol may be promising as a low-toxic natural immunosuppressant.
Asunto(s)
Butanoles/farmacología , Ciclohexanonas/farmacología , Factores de Transcripción NFATC/antagonistas & inhibidores , Rhizophoraceae/química , Butanoles/química , Butanoles/aislamiento & purificación , Ciclohexanonas/química , Ciclohexanonas/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Humanos , Células Jurkat , Estructura Molecular , Factores de Transcripción NFATC/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Calcineurin (CN) is an important phosphatase that mediates many physiological and pathological processes. The regulators of calcineurin (RCAN1) and Cu, Zn superoxide dismutase (SOD1) are two endogenous modulators of CN activity. Cyclosporine A (CsA) is a well-known exogenous inhibitor of CN and used as an immunosuppressive drug after transplantation and for the treatment of immune diseases. The degree of CN inhibition by CsA varies among each tissue. The brain accumulates low levels of CsA due to the blood-brain barrier after oral administration. In our study, we investigated RCAN1 and SOD1 expression in long-term CsA-treated mouse brain. Using Western blot, we found that chronic CsA treatment had caused significant up-regulation of RCAN1-1L and RCAN1-4 protein isoforms after 25 days in mouse brain. At the same time, chronic CsA treatment also resulted in decreased expression of SOD1. We simultaneously found more dramatic CN inhibition in mouse brain. It was suspected that the significant reduction of CN activity in vivo resulted partially from up-regulated RCAN1 and down-regulated SOD1 expression. In contrast, CsA treatment in SY5Y cells affected SOD1 expression and CN activity significantly, but had no obvious effects on RCAN1-1 mRNA expression. The changes of RCAN1, SOD1, and CN activity may be part of maladaptive responses, resulting in neuropathological conditions. These data might partially explain CsA neurotoxicity despite the low concentration of CsA in brain.
Asunto(s)
Encéfalo/efectos de los fármacos , Calcineurina/metabolismo , Ciclosporina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Administración Oral , Animales , Calcineurina/genética , Proteínas de Unión al Calcio , Línea Celular Tumoral , Humanos , Inmunosupresores/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Proteínas Musculares/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , Regulación hacia Arriba/efectos de los fármacosRESUMEN
In the present study, we developed a modified protocol for the basic comet assay that increased efficiency without sacrificing assay reliability. A spreader was used to spread agarose-embedded cells on a slide, making the manipulation and processing of multiple samples easier. Using this technique, we are able to rapidly prepare five or more comet assay samples on one slide. To demonstrate the effect of the protocol modifications on assay reliability, we present an example of how the comet assay was used in our laboratory to analyze the effect of melatonin (N-acetyl-5-methoxitryptamine; MEL) on the DNA repair ability of Gentiana macrophylla Pall. protoplasts after irradiation with different doses of ultraviolet-B radiation. A slight, but statistically significant (P<0.01), dose-related protective effect of MEL was observed in our experiments. The first use of the comet assay was to confirm the antioxidant and DNA repair functions of MEL in plants. The modified protocol is cost-effective and provides substantial advantages over the conventional comet assay.
Asunto(s)
Antioxidantes/farmacología , Ensayo Cometa/métodos , Reparación del ADN , Gentiana/genética , Melatonina/farmacología , Rayos Ultravioleta/efectos adversos , Daño del ADN , Relación Dosis-Respuesta a Droga , Gentiana/efectos de los fármacos , Reproducibilidad de los ResultadosRESUMEN
Tyrosine decarboxylase (TYDC) can catalyze tyrosine into tyramine. Several studies demonstrated its roles in the acidity, salidroside and defense response. Here we found that TYDC from Violaâ¯×â¯wittrockiana Gam (VwTYDC) may contribute to the formation of cyaninc blotches in the petal. VwTYDC gene were cloned from Violaâ¯×â¯wittrockiana and the cDNA full-length sequences were 1634 bp encoding 494 amino acids. Gene expression of VwTYDC in different tissues and developmental stages showed that they were significantly higher expressed in flowers than stems, leaves and roots. In addition, VwTYDC expression were higher in cyanic blotches than those observed in acyanic blotches of petal. Metabolites analysis showed the contents of tyramine in cyanic blotches were also higher than that in acyanic areas. Furthermore, in vitro assay revealed the absorption peak of anthocyanins had a red shift and an increase when fed tyramine. We speculated that tyramine might contribute to flower color expression of pansy as co-pigment. Our study demonstrated for the first time that the contents of tyramine led to flower blotches formation in cyanic blotches of the petals in plant flowers, and this may due to the higher expression of VwTYDC gene.
Asunto(s)
Quimera , Flores , Pigmentación/fisiología , Proteínas de Plantas , Tiramina/farmacología , Tirosina Descarboxilasa , Viola , Antocianinas/biosíntesis , Antocianinas/genética , Quimera/genética , Quimera/metabolismo , Flores/genética , Flores/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tirosina Descarboxilasa/genética , Tirosina Descarboxilasa/metabolismo , Viola/genética , Viola/metabolismoRESUMEN
The antioxidant activity and genotoxicity of isogarcinol were assessed by several in vitro tests. Its IC50 values for DPPH and ABTS were 36.3⯱â¯3.35⯵M and 16.6⯱â¯3.98⯵M, respectively, which were all lower than those of VC and BHT. Isogarcinol had no cyctotoxic or promotional activities at 1-10⯵M in the CCK-8 assay, and negligible genotoxic effects at 50-500⯵M on HepG2 cells by the single-cell gel electrophoresis assay. Pre-incubation of the cells with 0.5-1.5⯵M isogarcinol, before exposure to H2O2, significantly increased cell viability in a concentration-dependent manner. Isogarcinol also reduced intercellular reactive oxygen species accumulation, lactate dehydrogenase release and malondialdehyde levels, and increased superoxide dismutase activity and glutathione levels. Western-blot analysis revealed that it up-regulated pro-caspase-3 and reduced cleaved caspase-3 during H2O2-induced oxidative stress. All the above results indicate that isogarcinol promises to be useful as a natural antioxidant.
Asunto(s)
Antioxidantes/farmacología , Antioxidantes/toxicidad , Mutágenos/farmacología , Mutágenos/toxicidad , Terpenos/farmacología , Terpenos/toxicidad , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glutatión/metabolismo , Células Hep G2 , Humanos , Peróxido de Hidrógeno/farmacología , Concentración 50 Inhibidora , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacosRESUMEN
Isogarcinol (YDIS), a natural compound extracted from Garcinia mangostana L., has a significant immunosuppressive effect on systemic lupus erythematosus and rheumatoid arthritis. This paper reports that it reduced imiquimod-induced psoriasis-like skin lesions in mice. It strongly attenuated the aberrant proliferation and differentiation of keratinocytes. Moreover, the expression of genes involving the interleukin-23 (IL-23)/T-helper 17 (Th17) axis was significantly inhibited in the dorsal skin of the YDIS-treated mice, as was that of the other pro-inflammatory factors TNF-α, IL-2, and even interferon (IFN)-γ. Furthermore, YDIS prevented the abnormal distribution of T cell types and suppressed the differentiation of CD4+ T cells into Th17 cells in the spleens of mice exposed to imiquimod. Interestingly, it elevated numbers of regulatory T cells (Tregs) in the spleen and boosted IL-10 expression in the skin. In agreement with the above, YDIS increased serum IL-10 and reduced serum IL-17. It also caused less damage to the liver and, especially, kidneys of mice than cyclosporine A (CsA). In vitro, YDIS caused more death of HaCaT keratinocytes than CsA. It also strongly inhibited inflammatory factor expression in lipopolysaccharide (LPS)-stimulated HaCaT cells. These findings suggest that YDIS is a promising immunosuppressive agent for treating psoriasis.
Asunto(s)
Aminoquinolinas/administración & dosificación , Garcinia mangostana/química , Extractos Vegetales/administración & dosificación , Psoriasis/tratamiento farmacológico , Piel/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Imiquimod , Interleucina-2/genética , Interleucina-2/inmunología , Interleucina-23/genética , Interleucina-23/inmunología , Ratones , Ratones Endogámicos C57BL , Psoriasis/genética , Psoriasis/inmunología , Piel/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Factor de Necrosis Tumoral alfaRESUMEN
Isogarcinol is a new natural immunosuppressant that was extracted from Garcinia mangostana L. in our laboratory. Knowledge of its effects on treatable diseases and its mechanism of action is still very limited. In this study, we explored the therapeutic effect of isogarcinol in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). Treatment with oral 100 mg/kg isogarcinol markedly ameliorated clinical scores, alleviated inflammation and demyelination of the spinal cord, and reduced intracranial lesions in EAE mice. The percentages of Th cells and macrophages were also strongly reduced. Isogarcinol appeared to act by inhibiting T helper (Th) 1 and Th17 cell differentiation via the janus kinase/signal transducers and activators of transcription pathway and by impairing macrophage function. Our data suggest that isogarcinol has the potential to be an effective therapeutic agent of low toxicity for treating MS and other autoimmune diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Garcinia mangostana/química , Inmunosupresores/farmacología , Extractos Vegetales/farmacología , Terpenos/farmacología , Animales , Antiinflamatorios/farmacología , Diferenciación Celular/efectos de los fármacos , Femenino , Inflamación/tratamiento farmacológico , Quinasas Janus/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Células Th17RESUMEN
Isogarcinol, a bioactive polyisoprenylated benzophenone derivative isolated from Garcinia mangostana L., has been shown previously to exert a strong inhibitory effect on calcineurin and is thus a potential oral, low-toxicity immunomodulatory drug. In the present study, enzyme kinetic analysis showed that inhibition of calcineurin by isogarcinol was competitive. Fluorescence spectroscopy indicated that isogarcinol bound to calcineurin. Isothermal titration calorimetry showed that binding was mainly driven by enthalpy, and was exothermic because the enthalpy change exceeded the entropy reduction. The interaction force is either hydrogen bonding or Van der Waals forces. Fluorescence resonance energy transfer and molecular docking experiments indicated that there were two potential binding sites for isogarcinol in the catalytic domain of calcineurin. In summary, isogarcinol binds directly to calcineurin in vitro, unlike the classical calcineurin inhibitors cyclosporin A and tacrolimus.
Asunto(s)
Calcineurina/química , Ciclosporina/química , Garcinia/química , Inmunosupresores/química , Inmunosupresores/aislamiento & purificación , Tacrolimus/química , Benzofenonas/química , Humanos , Unión ProteicaRESUMEN
Isogarcinol is a new immunosuppressant that we extracted from Garcinia mangostana L. In the present study, we elucidate its beneficial effect in chronic graft-versus-host disease (cGVHD) in mice -- a model for systemic lupus erythematosus (SLE) in human. The oral administration of 60 mg/kg isogarcinol significantly reduced proteinuria, corrected the abnormal serum biochemical indicator, and decreased the amount of serum antibodies and lowered the renal histopathology score. In addition, isogarcinol alleviated the abnormal activation of CD4 T cells and decreased the expression of inflammatory genes and cytokines in the kidneys and peritoneal macrophages. The mechanism of action of isogarcinol is associated with downregulation of CD4 T cells and inflammatory effects. Therefore, we believe that isogarcinol may be a potential therapeutic drug candidate for future treatment of SLE.
Asunto(s)
Garcinia mangostana/química , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Lupus Eritematoso Sistémico/tratamiento farmacológico , Extractos Vegetales/administración & dosificación , Terpenos/administración & dosificación , Animales , Citocinas/genética , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/química , Inmunosupresores/aislamiento & purificación , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Terpenos/química , Terpenos/aislamiento & purificaciónRESUMEN
Isogarcinol is a natural compound that we extracted from Garcinia mangostana L., and we were the first to report that it is a new immunosuppressant. In the present study, we investigated the immune regulation and anti-inflammatory effects of isogarcinol on collagen-induced arthritis (CIA) and explored its potential mechanism in the treatment of rheumatoid arthritis. The oral administration of isogarcinol significantly reduced clinical scores, alleviated cartilage and bone erosion, and reduced the levels of serum inflammatory cytokines in CIA mice. Isogarcinol inhibited xylene-induced mouse ear edema in vivo. In vitro, isogarcinol decreased iNOS and COX-2 mRNA expression and NO content by inhibiting NF-κB expression. Furthermore, isogarcinol decreased the activity of NFAT and inhibited IL-2 expression. The mechanism of action of isogarcinol is associated with down-regulation of both autoimmune and inflammatory reactions.
Asunto(s)
Antiinflamatorios/administración & dosificación , Artritis Experimental/tratamiento farmacológico , Garcinia mangostana/química , Extractos Vegetales/administración & dosificación , Animales , Artritis Experimental/genética , Artritis Experimental/inmunología , Modelos Animales de Enfermedad , Humanos , Factores Inmunológicos/genética , Factores Inmunológicos/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Endogámicos ICR , FN-kappa B/genética , FN-kappa B/inmunología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunologíaRESUMEN
Calcineurin (CN), a unique protein phosphatase, plays an important role in immune regulation. In this study we used CN as a target enzyme to investigate the immunosuppressive properties of a series of natural compounds from Garcinia mangostana L., and discovered an active compound, isogarcinol. Enzymatic assays showed that isogarcinol inhibited CN in a dose-dependent manner. At concentrations resulting in relatively low cytotoxicity isogarcinol significantly inhibited proliferation of murine spleen T-lymphocytes induced by concanavalin A (ConA) and the mixed lymphocyte reaction (MLR). In addition, it performed much better in acute toxicity tests and via oral administration in mice than cyclosporin A (CsA), with few adverse reactions and low toxicity in experimental animals. Oral administration of isogarcinol in mice resulted in a dose-dependent decrease in delayed type hypersensitivity (DTH) and prolonged graft survival in allogeneic skin transplantation. These findings suggest that isogarcinol could serve as a new oral immunomodulatory drug for preventing transplant rejection, and for long-term medication in autoimmune diseases.