RESUMEN
Wedelia chinensis is a folk medicine used in many Asian countries to treat various ailments. Earlier investigations reported that the petroleum ether extract of the plant has potential biological activity, but the compounds responsible for activity are not yet completely known. Therefore, the current work was designed to isolate and characterize the compounds from the petroleum ether extract and to study their bioactivities. Four compounds including two diterepenes (-) kaur-16α-hydroxy-19-oic acid (1) and (-) kaur-16-en-19-oic acid (2), and two steroids ß-sitosterol (3), and cholesta-5,23-dien-3-ol (4) were isolated and characterized. Among the compounds, the diterpenes were found to have more biological activities than the steroidal compounds. Compound 1 showed the highest cytotoxicity with LC50 of 12.42 ± 0.87 µg/mL. Likewise, it possesses good antioxidant activity in terms of reducing power. On the contrary, compound 2 exerted the highest antiacetylcholinesterase and antibutyrylcholinesterase activity. Both the diterpenes showed almost similar antibacterial and antifungal activity. The identification of diterpenoid and steroid compounds with multifunctional activities suggests that W. chinensis may serve as an important source of bioactive compounds which should be further investigated in animal model for therapeutic potential in the treatment of different chronic diseases.
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Diterpenos , Wedelia , Animales , Inhibidores de la Colinesterasa/farmacología , Antioxidantes/farmacología , Antibacterianos , Extractos Vegetales/farmacologíaRESUMEN
Neurotoxicity is a serious health problem of patients chronically exposed to arsenic. There is no specific treatment of this problem. Oxidative stress has been implicated in the pathological process of neurotoxicity. Polyphenolics have proven antioxidant activity, thereby offering protection against oxidative stress. In this study, we have isolated the polyphenolics from Acacia nilotica and investigated its effect against arsenic-induced neurotoxicity and oxidative stress in mice. Acacia nilotica polyphenolics prepared from column chromatography of the crude methanol extract using diaion resin contained a phenolic content of 452.185 ± 7.879 mg gallic acid equivalent/gm of sample and flavonoid content of 200.075 ± 0.755 mg catechin equivalent/gm of sample. The polyphenolics exhibited potent antioxidant activity with respect to free radical scavenging ability, total antioxidant activity and inhibition of lipid peroxidation. Administration of arsenic in mice showed a reduction of acetylcholinesterase activity in the brain which was counteracted by Acacia nilotica polyphenolics. Similarly, elevation of lipid peroxidation and depletion of glutathione in the brain of mice was effectively restored to normal level by Acacia nilotica polyphenolics. Gallic acid methyl ester, catechin and catechin-7-gallate were identified in the polyphenolics as the major active compounds. These results suggest that Acacia nilotica polyphenolics due to its strong antioxidant potential might be effective in the management of arsenic induced neurotoxicity.
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Acacia , Antioxidantes/uso terapéutico , Intoxicación por Arsénico/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Polifenoles/uso terapéutico , Acacia/química , Animales , Antioxidantes/química , Arsénico/toxicidad , Intoxicación por Arsénico/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ratones , Polifenoles/químicaRESUMEN
Mistletoes are considered to be the potential medicinal herbs due to their rich traditional uses. Loranthus globosus is a Bangladeshi mango mistletoe that has been reported as folk medicine for various ailments and diseases. In an attempt to explore its effectiveness in Alzheimer's disease (AD), we investigated the antioxidant and acetylcholinesterase inhibitory activity of L. globosus. We report that the crude methanol extract (CME) of the plant contains a good amount of polyphenolics and possesses antioxidant and cholinesterase inhibitory activity. Fractionation of CME with solvents of varying polarity revealed the highest activity and polyphenolic content in the ethylacetate fraction (EAF). Correlation analysis revealed a significant (P < 0.05) association of polyphenolics with the antioxidant and cholinesterase inhibitory properties. Using column chromatography with diaion resin, the polyphenolics (EAF-PP) were isolated from the EAF that displayed the potent antioxidant and cholinesterase inhibitory activities. Kinetic analysis showed that EAF-PP exhibited a competitive type of inhibition. A total of thirty-six compounds including catechin and its different derivatives were identified in the EAF-PP by LC/MS analysis. Bioactivity-guided separation approach afforded the isolation of the two major active compounds catechin and catechin dimer from the EAF-PP. Hence, EAF-PP represents a potential source of antioxidants and cholinesterase inhibitors, which can be used in the management of AD.
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Antioxidantes/aislamiento & purificación , Inhibidores de la Colinesterasa/aislamiento & purificación , Loranthaceae/química , Plantas Medicinales/química , Polifenoles/aislamiento & purificación , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Antioxidantes/farmacología , Bangladesh , Inhibidores de la Colinesterasa/farmacología , Humanos , Técnicas In Vitro , Ratones , Estrés Oxidativo/efectos de los fármacos , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacología , Fitoterapia , Polifenoles/farmacologíaRESUMEN
BACKGROUND: Wedelia chinensis has been reported as a folk medicine for the treatment of different diseases including neurodegenerative disease. Although the plant has been studied well for diverse biological activities, the effect of this plant in neurological disorder is largely unknown. The present study was undertaken to evaluate the cholinesterase inhibitory and antioxidant potential of W. chinensis. METHODS: The extract and fractions of the plant were evaluated for acetylcholinesterase and butyrylcholinesterase inhibitory activity by modified Ellman method. The antioxidant activity was assessed in several in vitro models/assays such as reducing power, total antioxidant capacity, total phenolic and flavonoid content, scavenging of 2,2'-diphenyl-1-picrylhydrazyl (DPPH) free radical and hydroxyl radical, and inhibition of brain lipid peroxidation. Chromatographic and spectroscopic methods were used to isolate and identify the active compound from the extract. RESULTS: Among the fractions, aqueous fraction (AQF) and ethylacetate fraction (EAF) exhibited high inhibition against acetylcholinesterase (IC50: 40.02 ± 0.16 µg/ml and 57.76 ± 0.37 µg/ml) and butyrylcholinesterase (IC50: 31.79 ± 0.18 µg/ml and 48.41 ± 0.05 µg/ml). Similarly, the EAF and AQF had high content of phenolics and flavonoids and possess strong antioxidant activity in several antioxidant assays including DPPH and hydroxyl radical scavenging, reducing power and total antioxidant activity. They effectively inhibited the peroxidation of brain lipid in vitro with IC50 values of 45.20 ± 0.10 µg/ml and 25.53 ± 0.04 µg/ml, respectively. A significant correlation was observed between total flavonoids and antioxidant and cholinesterase inhibitory activity. Activity guided chromatographic separation led to the isolation of a major active compound from the EAF and its structure was elucidated as apigenin by spectral analysis. CONCLUSIONS: The potential ability of W. chinensis to inhibit the cholinesterase activity and peroxidation of lipids suggest that the plant might be useful for the management of AD.
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Antioxidantes/farmacología , Apigenina/aislamiento & purificación , Inhibidores de la Colinesterasa/farmacología , Wedelia , Apigenina/farmacología , Peroxidación de Lípido/efectos de los fármacos , Fotoquímica , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacologíaRESUMEN
Vanda roxburghii has been used in traditional medicine to treat nervous system disorders including Alzheimer's disease (AD). We reported earlier a high acetylcholinesterase inhibitory and antioxidant activity in the chloroform fraction of this plant. Therefore, this study was designed to explore the compounds with acetylcholinesterase inhibitory and antioxidant activities from the chloroform fraction of Vanda roxburghii. Phytochemical investigation led to the isolation for the first time of a fatty acid ester: methyl linoleate (1), and three phenolics: syringaldehyde (2), vanillin (3), and dihydroconiferyl dihydro-p-coumarate (4) along with the previously reported compound gigantol (5). Among the isolates, vanillin (3) and dihydroconiferyl dihydro-p-coumarate (4) were found to significantly inhibit the activity of acetylcholinesterase, scavenge the free radicals, exhibit the reducing power and total antioxidant activity, and effectively reduce the peroxidation of lipid. Gigantol (5) and syringaldehyde (2), despite lacking the activity against acetylcholinesterase, exhibited antioxidant activity. Among the compounds, gigantol (5) appeared to be the most potent antioxidant. These findings revealed that V. roxburghii contained compounds with potential acetylcholinesterase inhibitory and antioxidant activity, which support its traditional use in the treatment of AD.
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BACKGROUND: Alzheimer's disease (AD), one of the major causes of dementia, is an overwhelming neurodegenerative disease that particularly affects the brain, leading to memory loss and impairment of language and judgment capacity. The aim of the present study was to investigate the antioxidant and anticholinesterase properties of the leaves of Elatostema papillosum (EPL) and correlate with their phytochemical profiles, which are relevant to the treatment of AD. METHODS: The dried coarse powder of EPL was extracted with 80% methanol (EPL-M80) by cold extraction method. The resultant EPL-M80 was assessed for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity by the Ellman method. The antioxidant activity was determined by DPPH (1, 1-diphenyl-2-picrylhydrazyl) and hydroxyl radical scavenging assays. Quantitative phytochemical (phenolic and flavonoid contents) analysis of endogenous substances in EPL-M80 was performed by standard spectrophotometric methods. RESULTS: EPL-M80 significantly (p < 0.05) inhibited AChE and BChE activity with IC50 of 165.40 ± 4.01 and 213.81 ± 3.57 µg/mL, respectively in a dose-dependent manner. Additionally, EPL-M80 exhibited strong radical scavenging activity against DPPH (IC50 = 32.35 ± 0.68 µg/mL) and hydroxyl radical (IC50 = 19.67 ± 1.42 µg/mL) when compared to that of standards. EPL-M80 was found to be rich in phenolic (23.74 mg gallic acid equivalent/g of dry extract) and flavonoid (31.18 mg quercetin equivalent/g of dry extract) content. Furthermore, a positive correlation (p < 0.001) was observed between the total phenolics and antioxidant as well as the anticholinesterase potential. CONCLUSIONS: The marked inhibition of AChE and BChE, and potent antioxidant activity of the leaves of Elatostema papillosum highlight its potential to provide an effective treatment for AD.
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Antioxidantes , Encéfalo , Inhibidores de la Colinesterasa , Fitoquímicos , Extractos Vegetales , Urticaceae/química , Enfermedad de Alzheimer , Animales , Antioxidantes/química , Antioxidantes/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Femenino , Flavonoides , Masculino , Fitoquímicos/química , Fitoquímicos/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , Ratas , Ratas WistarRESUMEN
BACKGROUND: The marine invertebrate starfish was found to contain a novel α-N-acetylgalactosaminidase, α-GalNAcase II, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc), in addition to a typical α-N-acetylgalactosaminidase, α-GalNAcase I, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc) and, to a lesser extent, galactose. The interrelationship between α-GalNAcase I and α-GalNAcase II and the molecular basis of their differences in substrate specificity remain unknown. RESULTS: Chemical and structural comparisons between α-GalNAcase I and II using immunostaining, N-terminal amino acid sequencing and peptide analysis showed high homology to each other and also to other glycoside hydrolase family (GHF) 27 members. The amino acid sequence of peptides showed conserved residues at the active site as seen in typical α-GalNAcase. Some substitutions of conserved amino acid residues were found in α-GalNAcase II that were located near catalytic site. Among them G171 and A173, in place of C171 and W173, respectively in α-GalNAcase were identified to be responsible for lacking intrinsic α-galactosidase activity of α-GalNAcase II. Chemical modifications supported the presence of serine, aspartate and tryptophan as active site residues. Two tryptophan residues (W16 and W173) were involved in α-galactosidase activity, and one (W16) of them was involved in α-GalNAcase activity. CONCLUSIONS: The results suggested that α-GalNAcase I and II are closely related with respect to primary and higher order structure and that their structural differences are responsible for difference in substrate specificities.
Asunto(s)
Asterina/enzimología , alfa-N-Acetilgalactosaminidasa/química , Animales , Dominio Catalítico , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , alfa-Galactosidasa/metabolismo , alfa-N-Acetilgalactosaminidasa/metabolismoRESUMEN
Objective: To investigate the comparative effects of Diospyros blancoi (Ebenaceae) leaves (DBL), root bark (DBRB) and stem bark (DBSB) on free radicals and cancer. Methods: The polyphenol contents, antioxidant and free radical scavenging properties were determined using standard spectrophotometric methods. Cytotoxicity and anticancer activ-ities were performed on brine shrimp nauplii and Ehrlich ascite carcinoma cells, respectively. Results: Among the extracts, DBSB showed the highest total antioxidant capacity and reducing capacity on ferrous ion. Based on 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radical scavenging activities, DBSB showed (95.760 ± 0.343)%and (67.460 ± 2.641)%scavenging with IC50 of (3.10 ± 0.17) and (50.00 ± 3.11) mg/mL, respectively. The IC50 values of standard butylated hydroxytoluene and catechin (CA) for 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radicals were (8.50 ± 0.25) and (75.00 ± 0.14) mg/mL, respectively suggesting that DBSB had a significant (P DBRB > CA > DBL. Also, the phenolic [(139.91 ± 3.924) mg gallic acid equivalent/g] and flavonoid contents [(412.00 ± 16.70) mg catechin equivalent/g)] of DBSB were higher than that of other extracts. In addition, the DBSB showed the moderate cytotoxic and anticancer properties. Conclusions: Our results indicate that Diospyros blancoi stem bark had the significant highest antioxidant and free radical scavenging properties as well as moderate anticancer activity. Hence, we assume that the anticancer activity of this plant can be, at least in part, attributed to its content in phenolic compounds as well as its significant free radical scavenging properties.
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BACKGROUND: Ethnopharmacological approach has explored several leads from plant sources to identify potential new drugs for various diseases including pain. Vanda roxburghii R. Br., an epiphytic orchid is widely distributed throughout Bangladesh. The root of this plant has a folkloric reputation to treat inflammations, fever, dyspepsia, bronchitis, hiccough, piles, snake bites, and diseases of the nervous system. In this study therefore, we aimed to investigate antinociceptive and cytotoxic properties of the extracts from the root of V. roxburghii. METHODS: Dried powder of aerial root of V. roxburghii was extracted with methanol (VRM) and the resultant was fractionated into petroleum ether (VRP), chloroform (VRC), ethyl acetate (VRE), and residual aqueous fraction (VRA). The antinociceptive effect of the extracts was evaluated in mice using acetic acid-induced writhing, formalin injection, and hot plate tests. The brine shrimp lethality bioassay in vitro was used to evaluate cytotoxic activity of the plant extracts. RESULTS: In the acetic acid-induced writhing test, mice treated with different fractions (12.5, 25, and 50 mg/kg, i.p.) exhibited reduced number of writhing. Amongst, VRE showed the highest activity at all three concentrations (43.65, 71.34, and 80.23%, respectively) in a dose-dependent manner. Secondly, VRC (12.5, 25, and 50 mg/kg, i.p.) displayed the highest reduction of paw licking time in mice during the first phase of the formalin test (by 15.00, 37.05, and 56.44%, respectively) as well as during the second phase of the test (by 20.55, 49.08, and 59.81%, respectively). In hot plate test, VRE treatment at doses of 25 and 50 mg/kg both increased the highest latency time after 30 min. All fractions showed lower cytotoxicity compared with the standard drug vincristine sulfate in the brine shrimp bioassay. CONCLUSION: Overall studies suggest that the root of V. roxburghii is effective as a potent analgesic with lower toxicity. Our findings support previous claims of traditional uses of V. roxburghii in different inflammatory disorders.
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Analgésicos , Orchidaceae/química , Extractos Vegetales , Analgésicos/administración & dosificación , Analgésicos/química , Analgésicos/farmacología , Animales , Artemia/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Ratones , Modelos Animales , Dimensión del Dolor , Extractos Vegetales/administración & dosificación , Extractos Vegetales/química , Extractos Vegetales/farmacologíaRESUMEN
BACKGROUND: Alzheimer's disease (AD) is a progressively developing neurodegenerative disorder of the brain in the elderly people. Vanda roxburghii Rbr. root has been used traditionally in Bangladesh as tonic to brain and in the treatment of nervous system disorders including AD. Therefore, we aimed to investigate the cholinesterase inhibitory activities and antioxidant properties of the extracts from V. roxburghii. METHODS: The crude methanol extract from the roots of plant was sequentially fractionated with petroleum ether, chloroform, ethylacetate and water to yield their corresponding extracts. The extracts were assessed for acetylcholinesterase and butyrylcholinesterase inhibitory activity by modified Ellman method and antioxidant property by several assays including ferric reducing antioxidant power, scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and hydroxyl radical, and inhibition of lipid peroxidation. Endogenous substances in the extracts were analyzed by the standard phytochemical methods and active compound was isolated by the chromatographic methods. RESULTS: Chloroform extract was shown to demonstrate strong ferric-reducing antioxidant power and scavenging activity against DPPH and hydroxyl free radicals when compared with the other extracts and the reference standard catechin. The antioxidant effect was further verified by inhibition of lipid peroxidation in rat brain homogenates. Likewise, the chloroform extract exhibited the highest inhibition against both the acetylcholinesterase and butyrylcholinesterase enzymes with IC50 values of 221.13 and 82.51 µg/ml, respectively. Phytochemical screening revealed a large amount of phenolics and flavonoids in the chloroform extract. Bioactivity guided separation techniques led to the isolation of a strong antioxidant from the chloroform extract and its structure was determined as gigantol on the basis of spectral studies. CONCLUSION: These results suggest that the chloroform extract of V. roxburghii, possibly due to its phenolic compounds, exert potential antioxidant and cholinesterase inhibitory activities, which may be useful in the treatment of AD.
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Antioxidantes , Orchidaceae/química , Extractos Vegetales , Polifenoles , Animales , Antioxidantes/química , Antioxidantes/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Cloroformo , Inhibidores de la Colinesterasa , Extractos Vegetales/química , Extractos Vegetales/farmacología , Polifenoles/química , Polifenoles/farmacología , RatasRESUMEN
BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder clinically characterized by loss of memory and cognition. The effective therapeutic options for AD are limited and thus there is a demand for new drugs. Aegle marmelos (Linn.) (A. marmelos) leaves have been used in traditional medicine to promote intellect and enhance memory. In this study, we evaluated A. marmelos for its acetylcholinesterase (AChE) inhibitory activity and antioxidant property in vitro in the treatment of AD. METHODS: A crude methanol extract and four fractions (petroleum ether, chloroform, ethyl acetate and aqueous) were prepared from the leaves of A. marmelos. The preparations were assessed for AChE inhibitory activity by the Ellman method, and their antioxidant properties were assessed by several assays: reducing power, scavenging of 1,1-diphenyl-2-picrylhydrazyl free radical and hydroxyl radical, and inhibition of lipid peroxidation. Qualitative and quantitative analyses of endogenous substances in A. marmelos were performed by the standard phytochemical methods. RESULTS: Among the different extracts tested, the ethyl acetate fraction exhibited the highest inhibition of AChE activity. In the same way, ethyl acetate fraction showed the highest reducing activity and radical scavenging ability towards the 1,1-diphenyl-2-picrylhydrazyl (half maximal inhibitory concentration = 3.84 µg/mL) and hydroxyl free radicals (half maximal inhibitory concentration = 5.68 µg/mL). The antiradical activity of the ethyl acetate fraction appeared to be similar to that of the reference standard butylated hydroxytoluene and catechin used in this study. In addition, the ethyl acetate fraction displayed higher inhibition of brain lipid peroxidation. Phytochemical screening of different extractives of A. marmelos showed the presence of phenols and flavonoids, alkaloid, saponin, glycoside, tannin and steroids. Quantitative analysis revealed higher contents of phenolics (58.79-mg gallic acid equivalent/g dried extract) and flavonoids (375.73-mg gallic acid equivalent/g dried extract) in the ethyl acetate fraction. CONCLUSION: The results suggest that the ethyl acetate fraction of A. marmelos is a significant source of polyphenolic compounds with potential AChE inhibitory property and antioxidant activity and, thus, may be useful in the treatment of AD.
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Acetilcolinesterasa/efectos de los fármacos , Aegle/química , Enfermedad de Alzheimer , Antioxidantes/farmacología , Extractos Vegetales/farmacología , Antioxidantes/química , Flavonoides/química , Flavonoides/farmacología , Humanos , Peroxidación de Lípido/efectos de los fármacos , Fenoles/química , Fenoles/farmacología , Extractos Vegetales/química , Hojas de la Planta/químicaRESUMEN
BACKGROUND: The use of plants and their derived substances increases day by day for the discovery of therapeutic agents owing to their versatile applications. Current research is directed towards finding naturally-occurring antioxidants having anticancer properties from plant origin since oxidants play a crucial role in developing various human diseases. The present study was designed to investigate the antioxidant and anticancer properties of Sygygium fruticosum (Roxb.) (abbreviated as SF). METHODS: The dried coarse powder of seeds of SF was exhaustively extracted with methanol and the resulting crude methanolic extract (CME) was successively fractionated with petroleum ether, chloroform and ethyl acetate to get petroleum ether (PEF), chloroform (CHF), ethyl acetate (EAF) and lastly aqueous (AQF) fraction. The antioxidant activities were determined by several assays: total antioxidant capacity assay, DPPH free radical scavenging assay, hydroxyl radical scavenging assay, ferrous reducing antioxidant capacity and lipid peroxidation inhibition assay. The in vivo anticancer activity of SF was determined on Ehrlich's Ascite cell (EAC) induced Swiss albino mice. RESULTS: All the extractives showed strong antioxidant activities related to the standard. The total antioxidant capacity (TAC) of the fractions was in the following order: EAF>AQF>CME>PEF>CHF. The TAC of EAF at 320 µg/mL was 2.60±0.005 which was significantly higher (p < 0.01) than that of standard catechin (1.37 ± 0.005). The ferrous reducing antioxidant capacity of the extracts was in the following order: EAF>AQF>CME>AA>CHF>PEF. In DPPH free radical scavenging assay, the IC50 value of EAF was 4.85 µg/mL, whereas that of BHT was 9.85 µg/mL. In hydroxyl radical scavenging assay and lipid peroxidation inhibition assay, the EAF showed the most potent inhibitory activity with IC50 of 43.3 and 68.11 µg/mL, respectively. The lipid peroxidation inhibition assay was positively correlated (p < 0 .001) with both DPPH free radical scavenging and hydroxyl radical scavenging assay. The total phenolic contents of SF were also positively correlated (p < 0 .001) with DPPH free radical scavenging, hydroxyl radical scavenging and lipid peroxidation inhibition assay. Based on antioxidant activity, EAF was selected for cytotoxic assay and it was found that EAF inhibited 67.36% (p < 0.01) cell growth at a dose of 50 mg/kg (ip) on day six of EAC cell incubation. CONCLUSIONS: Our results suggest that EAF of seeds of SF possess significant antioxidant and moderate anticancer properties. Seeds of SF may therefore be a good source for natural antioxidants and a possible pharmaceutical supplement.
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Antineoplásicos Fitogénicos/administración & dosificación , Antioxidantes/administración & dosificación , Extractos Vegetales/administración & dosificación , Semillas/química , Syzygium/química , Animales , Antineoplásicos Fitogénicos/química , Antioxidantes/química , Bangladesh , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Extractos Vegetales/química , Semillas/crecimiento & desarrollo , Syzygium/crecimiento & desarrolloRESUMEN
BACKGROUND: Antioxidants play an important role to protect damage caused by oxidative stress (OS). Plants having phenolic contents are reported to possess antioxidant properties. The present study was designed to investigate the antioxidant properties and phenolic contents (total phenols, flavonoids, flavonols and proanthrocyanidins) of methanolic extracts from Morus alba (locally named as Tut and commonly known as white mulberry) stem barks (TSB), root bark (TRB), leaves (TL) and fruits (TF) to make a statistical correlation between phenolic contents and antioxidant potential. METHODS: The antioxidant activities and phenolic contents of methanolic extractives were evaluated by in vitro standard method using spectrophotometer. The antioxidant activities were determined by total antioxidant capacity, DPPH (1,1-diphenyl-2-picrylhydrazine) radical scavenging assay, hydroxyl radical scavenging assay, ferrous reducing antioxidant capacity and lipid peroxidation inhibition assay methods. RESULTS: Among the extracts, TSB showed the highest antioxidant activity followed by TRB, TF and TL. Based on DPPH and hydroxyl radical scavenging activity, the TSB extract was the most effective one with IC50 37.75 and 58.90 µg/mL, followed by TRB, TF and TL with IC50 40.20 and 102.03; 175.01 and 114.63 and 220.23 and 234.63 µg/mL, respectively. The TSB extract had the most potent inhibitory activity against lipid peroxidation with IC50 145.31 µg/mL. In addition, the reducing capacity on ferrous ion was in the following order: TSB > TRB > TL > TF. The content of phenolics, flavonoids, flavonols and proanthocyanidins of TSB was found to be higher than other extractives. CONCLUSION: The results indicate high correlation and regression (p-value <0 .001) between phenolic contents and antioxidant potentials of the extracts, hence the Tut plant could serve as effective free radical inhibitor or scavenger which may be a good candidate for pharmaceutical plant-based products. However, further exploration is necessary for effective use in both modern and traditional system of medicines.
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Antioxidantes/farmacología , Metanol/química , Morus/química , Extractos Vegetales/farmacología , Depuradores de Radicales Libres/farmacología , Radical Hidroxilo/química , Peroxidación de Lípido/efectos de los fármacosRESUMEN
BACKGROUND: Multiple protein kinases have been shown to be involved in the apoptotic neuronal loss of Alzheimer's disease (AD). Although some studies support the role of protein kinase C (PKC) in amyloid precursor protein processing as well as in tau phosphorylation, a direct role for PKC in apoptotic neuronal death remains to be clarified. In the present study, we report on the possible role of PKC in cell survival during conditions of stress through phosphorylation of the X-linked inhibitor of apoptosis protein (XIAP). METHODS: Phosphorylation of XIAP at Ser87 was confirmed by western blot analysis employing phosphorylation dependent anti-XIAP antibody after incubation of recombinant XIAP with active PKC in vitro. And increased phosphorylation of XIAP at the site was also confirmed in SH-SY5Y cells treated with PKC activator, phorbol 12-myristate 13-acetate (PMA). A mutant XIAP construct in which Ser87 was substituted by Ala, was prepared, and transfected to cells. After the transfection of wild or mutant XIAP, cells viability was evaluated by counting living and dead cells treated with PMA during etoposide-induced apoptosis. RESULTS: Recombinant XIAP was phosphorylated at Ser(87) by PKC in vitro and treatment of XIAP-transfected SH-SY5Y cells with a PKC activator, phorbol 12-myristate 13-acetate (PMA) induced phosphorylation of XIAP at Ser(87) . Pulse chase experiments revealed that, when phosphorylated at Ser(87) , wild-type XIAP is more stable than XIAP with a Ser87Ala substitution, which is degraded faster. Importantly, the phosphorylation of XIAP at the site by PKC significantly increased cell survival up to approximately 2.5 times under the condition of apoptosis induced by 25 µg/ml etoposide. CONCLUSION: The findings of the present study indicate a role for PKC, through phosphorylation of XIAP at Ser(87) and its stabilization, in cell survival under conditions of stress and lend strength to the idea that PKC is crucial in regulating neuronal homeostasis, which may be impaired in AD.
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Enfermedad de Alzheimer/fisiopatología , Apoptosis/fisiología , Proteína Quinasa C/fisiología , Serina/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Antineoplásicos Fitogénicos/farmacología , Encéfalo/fisiopatología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Etopósido/farmacología , Humanos , Neuroblastoma , Neuronas/efectos de los fármacos , Fosforilación , TransfecciónRESUMEN
Two alpha-N-acetylgalactosaminidases, alpha-N-acetylgalactosaminidase (alpha-GalNAcase) I and II, were purified from the digestive organ of starfish. Purified alpha-GalNAcase I and II gave nearly single protein bands on SDS-polyacrylamide gel electrophoresis, individually. Even the final preparation of alpha-GalNAcase I contained alpha-galactosidase activity, while alpha-GalNAcase II was almost free from that activity with p-nitrophenyl and 4-methylumbelliferyl alpha-N-acetylgalactosaminides as substrates. alpha-GalNAcase I and II both hydrolyzed terminal alpha-N-acetylgalactosaminyl linkages of the natural compounds investigated: Forssman hapten glycolipid, blood group A active oligosaccharide and GalNAc-alpha1-O-serine. On the other hand, oligosaccharides, and glycolipid containing alpha-galactosyl terminals were hydrolyzed by alpha-GalNAcase I but not by alpha-GalNAcase II. The substrate specificities and other enzymatic properties of alpha-GalNAcase I were similar to those of human placental alpha-GalNAcase, but distinct from alpha-GalNAcase II.
Asunto(s)
Oligosacáridos/aislamiento & purificación , Estrellas de Mar/enzimología , alfa-N-Acetilgalactosaminidasa/aislamiento & purificación , Animales , Electroforesis en Gel de Poliacrilamida , Especificidad por SustratoRESUMEN
Based on the inherent alpha-galactosidase activity, squid liver contains two different alpha-N-acetylgalactosaminidases (alpha-GalNAcases): alpha-N-acetylgalactosaminidase I (alpha-GalNAcase I), which typically exhibits the alpha-galactosidase activity and alpha-N-acetylgalactosaminidase II (alpha-GalNAcase II), which is devoid of such activity. The molecular properties of the alpha-GalNAcases that may account for their enzymological differences are as yet unknown. In this study, we have characterized and compared the chemical and immunological properties of alpha-GalNAcase I and alpha-GalNAcase II. Analysis of the N-terminal sequence of the first twenty amino acids revealed the striking homology between alpha-GalNAcase I and alpha-GalNAcase II. Digestion of alpha-GalNAcase I and alpha-GalNAcase II generated the peptide maps that display similarities in peptide pattern, indicating their close relationship in structure. Polyclonal antibodies were generated in rabbits against the purified alpha-GalNAcase I and alpha-GalNAcase II for comparison of the immunological properties. Both Western blot and surface plasmon resonance (SPR) studies showed that the anti-alpha-GalNAcase II antibody reacted with both alpha-GalNAcase I and alpha-GalNAcase II, whereas the anti-alpha-GalNAcase I antibody reacted only with alpha-GalNAcase I, indicating the presence of common as well as unique antigenic determinants on alpha-GalNAcase I and alpha-GalNAcase II. Taken together, these results suggest that alpha-GalNAcase I and alpha-GalNAcase II are closely related with regard to structure and that their nonhomologous domains are possibly responsible for the differences in enzymatic properties.
Asunto(s)
Decapodiformes/enzimología , Epítopos/inmunología , Hígado/enzimología , alfa-N-Acetilgalactosaminidasa/química , alfa-N-Acetilgalactosaminidasa/inmunología , Secuencia de Aminoácidos , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Mapeo Epitopo , Isoenzimas , Datos de Secuencia Molecular , Mapeo Peptídico , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , alfa-N-Acetilgalactosaminidasa/aislamiento & purificaciónRESUMEN
Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3zeta. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3zeta is approximately 3-folds higher than that between unphosphorylated 4R-tau and 14-3-3zeta. Phosphorylation of tau by protein kinase A (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3zeta to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3zeta. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3zeta exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3zeta suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.
Asunto(s)
Proteínas 14-3-3/metabolismo , Secuencias Repetitivas de Aminoácido , Tauopatías/metabolismo , Proteínas tau/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Humanos , Fosforilación , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de Superficie , Proteínas tau/química , Proteínas tau/genéticaRESUMEN
In tauopathies, tau protein is hyperphosphorylated, ubiquitinated, and accumulated in the brain; however, the mechanisms underlying this accumulation remain unclear. To gain an understanding of the role of proteases in the metabolism of tau protein, in the present study we evaluated the effects of protease inhibitors in SH-SY5Y human neuroblastoma cells and COS-7 cells transfected with the tau gene. When cells were treated with 0.1-10 micromol/L of lactacystin and 1.0-20 micromol/L of MG-132 (inhibitors of proteasome), 0.1-10 micromol/L of CA-074Me (a cathepsin inhibitor), and 0.1-2 micromol/L of puromycin (a puromycin-sensitive aminopeptidase (PSA) inhibitor) for up to 24 h, there were no significant changes in tau protein levels. However, pulse-chase experiments demonstrated that the proteolysis of tau protein in SH-SY5Y cells was attenuated following treatment of cells with 200 nmol/L puromycin. Increased tau protein levels were also observed in SH-SY5Y cells treated with short interference (si) RNA to PSA to inhibit the expression of PSA. These data suggest that PSA is a protease that catalyses tau protein predominantly in SH-SY5Y cells. The protein metabolism of tau-containing mutations of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) was also investigated using pulse-chase experiments. The results indicate attenuated proteolysis of tau in cells transfected with mutant tau genes after 48 h. Further immunocytochemical analysis and subcellular fractionation experiments revealed that the mutations did not alter the intracellular distribution of tau and suggested that impaired accessibility of tau to PSA is unlikely to account for the attenuated proteolysis of tau protein. Western blotting with phosphorylation-dependent antibodies revealed that phosphorylation levels of tau at Thr(231), Ser(396), and Ser(409) were increased in cells transfected with V337M, R406W, and R406W mutant tau genes, respectively. Together, the data suggest that attenuated proteolysis of FTDP-17 mutant tau may be explained by increased phosphorylation levels, resulting in resistance to proteolysis.
Asunto(s)
Alelos , Aminopeptidasas/genética , Mapeo Cromosómico , Cromosomas Humanos Par 17/genética , Análisis Mutacional de ADN , Demencia Frontotemporal/genética , Tauopatías/genética , Proteínas tau/genética , Animales , Células COS , Muerte Celular/genética , Línea Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Demencia Frontotemporal/diagnóstico , Humanos , Tauopatías/diagnóstico , TransfecciónRESUMEN
The microtubule associated protein tau is a major component of neurofibrillary tangles in Alzheimer disease brain, however the neuropathological processes behind the formation of neurofibrillary tangles are still unclear. Previously, 14-3-3 proteins were reported to bind with tau. 14-3-3 Proteins usually bind their targets through specific serine/threonine -phosphorylated motifs. Therefore, the interaction of tau with 14-3-3 mediated by phosphorylation was investigated. In this study, we show that the phosphorylation of tau by either protein kinase A (PKA) or protein kinase B (PKB) enhances the binding of tau with 14-3-3 in vitro. The affinity between tau and 14-3-3 is increased 12- to 14-fold by phosphorylation as determined by real time surface plasmon resonance studies. Mutational analyses revealed that Ser214 is critical for the phosphorylation-mediated interaction of tau with 14-3-3. Finally, in vitro aggregation assays demonstrated that phosphorylation by PKA/PKB inhibits the formation of aggregates/filaments of tau induced by 14-3-3. As the phosphorylation at Ser214 is up-regulated in fetal brain, tau's interaction with 14-3-3 may have a significant role in the organization of the microtubule cytoskeleton in development. Also as the phosphorylation at Ser214 is up-regulated in Alzheimer's disease brain, tau's interaction with 14-3-3 might be involved in the pathology of this disease.
Asunto(s)
Proteínas 14-3-3/metabolismo , Fosforilación , Serina/metabolismo , Proteínas tau/metabolismo , Proteínas 14-3-3/inmunología , Animales , Anticuerpos/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Inmunoprecipitación/métodos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Resonancia por Plasmón de Superficie/métodos , Factores de Tiempo , Proteínas tau/farmacologíaRESUMEN
Chloroform extract (CE) of Achyranthes ferruginea and N-trans-feruloyl-4-methyldopamine (1) showed remarkable antimicrobial activities against a wide range of bacteria and fungi. Both crude extract (CE) and compound 1 showed significant cytotoxicity of LC(50) at 16.21 microg/ml and 11.70 microg/ml, respectively.