RESUMEN
Imbalance in production and clearance of amyloid beta (Aß) is the primary reason for its deposition in Alzheimer disease. Macroautophagy/autophagy is one of the important mechanisms for clearance of both intracellular and extracellular Aß. Here, through screening, we identified alborixin, an ionophore, as a potent inducer of autophagy. We found that autophagy induced by alborixin substantially cleared Aß in microglia and primary neuronal cells. Induction of autophagy was accompanied by up regulation of autophagy proteins BECN1/Beclin 1, ATG5, ATG7 and increased lysosomal activities. Autophagy induced by alborixin was associated with inhibition of the phosphoinositide 3-kinase (PI3K)-AKT pathway. A knock down of PTEN and consistent, constitutive activation of AKT inhibited alborixin-induced autophagy and consequent clearance of Aß. Furthermore, clearance of Aß by alborixin led to significant reduction of Aß-mediated cytotoxicity in primary neurons and differentiated N2a cells. Thus, our findings put forward alborixin as a potential anti-Alzheimer therapeutic lead. Abbreviations: Aß: amyloid beta; ALB: alborixin; ATG: autophagy-related; BECN1: beclin 1; DAPI: 4, 6-diamidino-2-phenylindole; DCFH-DA: 2,7-dichlorodihydrofluorescein diacetate; fAß: fibrillary form of amyloid beta; GFAP: glial fibrillary acidic protein; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule-associated protein 2; MTOR: mechanistic target of rapamycin kinase; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species; SQSTM1: sequestosome 1; TMRE: tetramethylrhodamine, ethyl ester.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Autofagia/efectos de los fármacos , Fosfohidrolasa PTEN/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Péptidos beta-Amiloides/efectos de los fármacos , Animales , Autofagia/fisiología , Células Cultivadas , Embrión de Mamíferos , Humanos , Ratones , Ratones Endogámicos C57BL , Fosfohidrolasa PTEN/genética , Proteolisis/efectos de los fármacos , Piranos/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
The ethyl acetate extract from the fermentation broth of an actinomycete strain, identified as Streptomyces scabrisporus isolated from soil of Kashmir Himalayas - India, exhibited significant cytotoxic activity against a panel of human cancer cell lines. The active fraction subjected to column chromatography led to the isolation of pharmacologically potent anticancer compound whose structure was established to be alborixin on the basis of spectral data analysis. The compound exhibited antiproliferative activity against panel of cell lines N2a, MCF-7, MiaPaca-2, PC-3, HCT-116, MDA-MB-231, HL-60 and A-549 cells with IC50 of 9.7, 15.4, 7.2, 8.1, 3.2, 9.7, 7.5 and 11.5 µM respectively. Alborixin displayed the maximum cytotoxic activity against HCT-116 human colon carcinoma cells and therefore further studies were carried on this cell line. Alborixin decreased the clonogenic potential of HCT-116 cells in a dose dependent manner. It induced apoptotic cell death in HCT116 cells that were confirmed by Flow cytometric analysis of Annexin V/PI staining and microscopic examination of cellular morphology through DAPI-stained cells. Biochemical evidence of apoptosis came from elevating the intracellular ROS level that was accompanied by mitochondrial membrane potential loss, decreasing the expression profile of anti-apoptotic protein Bcl-2, whereas it augments cleavage of caspase-3 and PARP-1, activates caspase-8 and 9 with concomitant increase in expression of proapoptotic protein Bax in a dose dependent manner. These results indicate that alborixin obtained from Streptomyces scabrisporus IIIM55 induces apoptotic cell death in colon cancer cells HCT-116 and can be further evaluated for its potential as an anticancer agent.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Streptomyces/química , Antineoplásicos/química , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células HCT116 , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Piranos/química , Piranos/aislamiento & purificación , Piranos/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
ETHNO-PHARMACOLOGICAL RELEVANCE: The roots of Thalictrum minus are traditionally used in the treatment of inflammation and infectious diseases such as bovine mastitis. However, there are no reports available in literature till date regarding the antibacterial studies of T. minus against bovine mastitis. AIM OF THE STUDY: The present study was undertaken to evaluate the antibacterial potential of crude extract of T. minus (root) and some of its isolated constituents against bovine mastitis in order to scientifically validate its traditional use. MATERIALS AND METHODS: A total of three alkaloid compounds were isolated from the DCM: MeOH extract of roots of T. minus using silica gel column chromatography. Structural elucidation of the isolated compounds was done by using spectroscopic techniques like mass spectrometry and NMR spectroscopy. Pathogens were isolated from cases of bovine mastitis and identified by using 16S rRNA gene sequencing. The broth micro-dilution method was used to evaluate the antibacterial activities of DCM: MeOH extract and isolated compounds against mastitis pathogens. RESULTS: The three isolated compounds were identified as benzylisoquinoline alkaloids (1) 5'-Hydroxythalidasine, (2) Thalrugosaminine and (3) O-Methylthalicberine. Compounds (2) and (3) are reported for the first time from the roots of T. minus. Five mastitis pathogens viz., Staphylococcus xylosus, Staphylococcus lentus, Staphylococcus equorum, Enterococcus faecalis and Pantoea agglomerans were identified on the basis of sequence analysis of isolates using the nucleotide BLAST algorithm. This study reports for the first time the isolation and molecular characterization of mastitis pathogens from Kashmir valley, India. The DCM: MeOH extract exhibited broad spectrum antibacterial activities that varied between the bacterial species (MIC=250-500µg/ml). 5'-Hydroxythalidasine and Thalrugosaminine showed promising antibacterial activity with MIC values of 64-128µg/ml while Staphylococcus species were found to be the most sensitive strains. CONCLUSIONS: The antibacterial activities of the DCM: MeOH extract and isolated compounds support the traditional use of T. minus in the treatment of bovine mastitis.
Asunto(s)
Antibacterianos/uso terapéutico , Bencilisoquinolinas/uso terapéutico , Mastitis Bovina/tratamiento farmacológico , Animales , Antibacterianos/química , Bencilisoquinolinas/química , Espectroscopía de Resonancia Magnética con Carbono-13 , Bovinos , Femenino , Espectroscopía de Protones por Resonancia Magnética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
ETHNO-PHARMACOLOGICAL RELEVANCE: The underground parts of Aquilegia fragrans are traditionally used for the treatment of wounds and various inflammatory diseases like bovine mastitis. However, there are no reports on the phytochemical characterization and antibacterial studies of A. fragrans. AIM OF THE STUDY: To isolate compounds from the methanol extract of the underground parts of A. fragrans and determine their antibacterial activity against the pathogens of bovine mastitis. The study was undertaken in order to scientifically validate the traditional use of A. fragrans. MATERIALS AND METHODS: Five compounds were isolated from the methanol extract of the underground parts of A. fragrans using silica gel column chromatography. Structural elucidation of the isolated compounds was done using spectral data analysis and comparison with literature. High performance liquid chromatography (HPLC) was used for the qualitative and quantitative determination of isolated compounds in the crude methanol extract. The methanol extract and isolated compounds were evaluated for antibacterial activities against mastitis pathogens using broth micro-dilution technique. RESULTS: The five isolated compounds were identified as (1) 2, 4-dihydroxyphenylacetic acid methyl ester (2) ß-sitosterol (3) Aquilegiolide (4) Glochidionolactone-A and (5) Magnoflorine. A quick and sensitive HPLC method was developed for the first time for qualitative and quantitative determination of four isolated marker compounds from A. fragrans. The crude methanol extract and compound 5 exhibited weak antibacterial activities that varied between the bacterial species (MIC=500-3000 µg/ml). CONCLUSIONS: The above results show that the crude methanol extract and isolated compounds from A. fragrans exhibit weak antibacterial activities. Further phytochemical and pharmacological studies are required for proper scientific validation of the folk use of this plant species in the treatment of various inflammatory diseases like bovine mastitis.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Aquilegia/química , Mastitis Bovina/tratamiento farmacológico , Extractos Vegetales/química , Extractos Vegetales/farmacología , Animales , Aporfinas/química , Benzofuranos/química , Bovinos , Cromatografía Líquida de Alta Presión , Femenino , Metanol/química , Pruebas de Sensibilidad Microbiana/métodos , Sitoesteroles/químicaRESUMEN
A Psychrotolerant alkaline protease producing bacterium IIIM-ST045 was isolated from a soil sample collected from the Thajiwas glacier of Kashmir, India and identified as Stenotrophomonas sp. on the basis of its biochemical properties and 16S ribosomal gene sequencing. The strain could grow well within a temperature range of 4-37°C however, showed optimum growth at 15°C. The strain was found to over-produce proteases when it was grown in media containing lactose as carbon source (157.50 U mg(-1)). The maximum specific enzyme activity (398 U mg(-1)) was obtained using soya oil as nitrogen source, however, the inorganic nitrogen sources urea, ammonium chloride and ammonium sulphate showed the lowest production of 38.9, 62.2 and 57.9 U mg(-1). The enzyme was purified to 18.45 folds and the molecular weight of the partially purified protease was estimated to be ~55 kDa by SDS-PAGE analysis. The protease activity increased as the increase in enzyme concentration while as the optimum enzyme activity was found when casein (1% w/v) was used as substrate. The enzyme was highly active over a wide range of pH from 6.5 to 12.0 showing optimum activity at pH 10.0. The optimum temperature for the enzyme was 15°C. Proteolytic activity reduced gradually with higher temperatures with a decrease of 56% at 40°C. The purified enzyme was checked for the removal of protein containing tea stains using a silk cloth within a temperature range of 10-60°C. The best washing efficiency results obtained at low temperatures indicate that the enzyme may be used for cold washing purposes of delicate fabrics that otherwise are vulnerable to high temperatures.