RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Salvia mirzayanii Rech. f. & Esfand. is an endemic plant, which is only distributed in the south of Iran. In traditional Iranian medicine, the aerial parts of Salvia mirzayanii have been used for infections, inflammatory diseases, and as a tonic. From this plant, the sesquiterpene teuclatriol was isolated by bioactivity-guided fractionation due to its anti-proliferative actions on human lymphocytes. The guaiane sesquiterpene is lacking the methylene-γ-lactone function that is typically involved in the inhibiting properties of sesquiterpenes on NF-κB, a pivotal transcription factor in inflammatory processes. We here investigated anti-inflammatory effects of teuclatriol on human macrophage-like and endothelial cells. MATERIALS AND METHODS: Non-toxic doses of teuclatriol were determined for both cell types by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-assay. The effect of teuclatriol on the activity of NF-κB in LPS-stimulated human monocytic THP-1 cells was studied using infrared electrophoretic mobility shift assay (IR-EMSA) using curcumin as positive control (32µM). THP-1 were differentiated into macrophage-like cells and evaluated for TNF-α secretion by L929 bioassay following stimulation with LPS and treatment with teuclatriol. Inflammatory gene expression in human umbilical vein endothelial cells (HUVEC), modeling target cells for TNF-α-induced inflammatory gene activation, was investigated by real-time RT-PCR. RESULTS: The LPS-induced DNA binding activity of NF-κB in THP-1 was significantly decreased by non-toxic doses of teuclatriol (312 and 390µM). Teuclatriol reduced the production of TNF-α in a dose-dependent manner. mRNA levels of both monocyte chemoattractant protein (MCP)-1 and toll-like receptor (TLR)2 were decreased in TNF-α-activated HUVEC. CONCLUSION: These data show an inhibitory effect of teuclatriol on NF-κB signaling at doses of 312µM and higher, validating the traditional use of Salvia mirzayanii in the treatment of inflammatory diseases. Future work on the mode of action of teuclatriol may provide new lead structures with NF-κB inhibiting properties, lacking possible side effects mediated via alkylating centers of sesquiterpene lactones.
Asunto(s)
Mediadores de Inflamación/metabolismo , FN-kappa B/metabolismo , Salvia/química , Sesquiterpenos de Guayano/farmacología , Sesquiterpenos/farmacología , Antiinflamatorios/aislamiento & purificación , Antiinflamatorios/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quimiocina CCL2/biosíntesis , Curcumina/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Lipopolisacáridos/farmacología , Sesquiterpenos/aislamiento & purificación , Sesquiterpenos de Guayano/aislamiento & purificación , Receptor Toll-Like 2/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Salvia mirzayanii, a native plant to Iran, is shown to have immunomodulatory effects on lymphocyte proliferation. OBJECTIVE: To identify the bioactive immunomodulatory compound(s) present in S. mirzayanii. METHODS: The crude extract was fractionated to five fractions in two steps using different solvents. The fractions were subjected to bioassay-guided fractionation. All the fractions were tested for bioactivity on human activated-peripheral blood lymphocytes (PBLs) using cell proliferation assay. RESULTS: The methanol fraction (Fr. M) showed the highest inhibitory effect on PBLs compared to other fractions. Fr. M was applied on a gravity column chromatography for further fractionation. Resultant fractions, demonstrated inhibitory effects at higher concentrations. Fr. 4 with an 18.9 ± 0.2% inhibitory activity at 200 µg/ml and with the highest quantity was applied on preparative TLC plates for further purification. The final purified compound was identified as teuclatriol, a guaiane sesquiterpene, by NMR analysis. This compound showed a significant anti-proliferative effect on human activated-peripheral blood lymphocytes (IC50, 72.8 ± 5.4 µg/ml). CONCLUSION: Teuclatriol was found to be one of the compounds responsible for the immunoinhibitory effect of Salvia mirzayanii. We suggest further studies on teuclatriol, exploring its mechanism of action as an immunomodulatory compound.
Asunto(s)
Cicloparafinas/farmacología , Inmunosupresores/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Fitoterapia , Salvia/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Fraccionamiento Químico , Humanos , Leucocitos Mononucleares/inmunología , Espectroscopía de Resonancia Magnética , Extractos Vegetales/farmacología , Sesquiterpenos de GuayanoRESUMEN
The methanol extract of Salvia mirzayanii has shown an immunomodulatory effect on peripheral blood lymphocytes. Bioassay-guided fractionation using a lymphocyte proliferation assay on Salvia mirzayanii was performed in order to purify and identify the active compounds. Fractionation of the methanol extract and purification of the components using normal column chromatography and preparative thin layer chromatography resulted in identification of the bioactive compound, spathulenol, with an immunoinhibitory effect. Identification of this compound was performed by 1D and 2D NMR methods and HRMS. Treatment of activated lymphocytes with a concentrated fraction containing 62% of spathulenol (SP) showed a decrease in the proliferation of lymphocytes with an IC(50) of 85.4 ± 11.08 µg/mL. Flow cytometry analysis using annexin V and propidium iodide staining of the stimulated peripheral blood lymphocytes in the presence of SP demonstrated a dose dependent increase in the percentage of apoptotic cells (IC(50) ; 77.2 ± 5.31 µg/mL). No significant increase in caspase 3 activity in a 20 h treatment of stimulated lymphocytes compared with the control was observed. In conclusion, this study identified the possible activity of spathulenol as one of the immunomodulatory compounds present in Salvia mirzayanii. SP showed the capacity to inhibit proliferation in the lymphocytes and to induce apoptosis in these cells possibly through a caspase-3 independent pathway.
Asunto(s)
Adyuvantes Inmunológicos/análisis , Salvia/química , Sesquiterpenos/análisis , Adyuvantes Inmunológicos/farmacología , Caspasa 3/metabolismo , Cromatografía en Capa Delgada , Citometría de Flujo , Humanos , Linfocitos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Sesquiterpenos/farmacologíaRESUMEN
In this study, the immunomodulatory effects of Salvia mirzayanii were investigated. Study of the effect of this plant on activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p < 0.01). In flow cytometry analysis, accumulation of apoptotic cells in the sub-G1 phase of the cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. DNA fragmentation analysis of these cells showed a typical DNA laddering. Immunisation of extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titre at higher concentrations (p < 0.007). These results indicated the presence of immunomodulatory compounds in the extract of S. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract that was observed at higher concentrations.