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1.
J Biol Chem ; 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32414842

RESUMO

Cyclotides are plant-derived peptides characterized by a ~30-amino-acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and one known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, M, and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.

2.
ACS Chem Biol ; 12(9): 2324-2334, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28741926

RESUMO

Gomesin, a disulfide-rich antimicrobial peptide produced by the Brazilian spider Acanthoscurria gomesiana, has been shown to be potent against Gram-negative bacteria and to possess selective anticancer properties against melanoma cells. In a recent study, a backbone cyclized analogue of gomesin was shown to be as active but more stable than its native form. In the current study, we were interested in improving the antimicrobial properties of the cyclic gomesin, understanding its selectivity toward melanoma cells and elucidating its antimicrobial and anticancer mode of action. Rationally designed analogues of cyclic gomesin were examined for their antimicrobial potency, selectivity toward cancer cells, membrane-binding affinity, and ability to disrupt cell and model membranes. We improved the activity of cyclic gomesin by ∼10-fold against tested Gram-negative and Gram-positive bacteria without increasing toxicity to human red blood cells. In addition, we showed that gomesin and its analogues are more toxic toward melanoma and leukemia cells than toward red blood cells and act by selectively targeting and disrupting cancer cell membranes. Preference toward some cancer types is likely dependent on their different cell membrane properties. Our findings highlight the potential of peptides as antimicrobial and anticancer leads and the importance of selectively targeting cancer cell membranes for drug development.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Aranhas/química , Animais , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fungos/efeitos dos fármacos , Humanos , Leucemia/tratamento farmacológico , Bicamadas Lipídicas/metabolismo , Melanoma/tratamento farmacológico , Micoses/tratamento farmacológico
3.
J Nat Prod ; 80(5): 1522-1530, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28471681

RESUMO

Cyclotides are a large family of naturally occurring plant-derived macrocyclic cystine-knot peptides, with more than 400 having been identified in species from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families. Nevertheless, their specialized distribution within the plant kingdom remains poorly understood. In this study, the diversity of cyclotides was explored through the screening of 197 plants belonging to 43 different families. In total, 28 cyclotides were sequenced from 15 plant species, one of which belonged to the Rubiaceae and 14 to the Violaceae. Every Violaceae species screened contained cyclotides, but they were only sparsely represented in Rubiaceae and nonexistent in other families. The study thus supports the hypothesis that cyclotides are ubiquitous in the Violaceae, and it adds to the list of plants found to express kalata S and cycloviolacin O12. Finally, previous studies suggested the existence of cyclotide isoforms with either an Asn or an Asp at the C-terminal processing site of the cyclotide domain within the precursor proteins. Here we found that despite the discovery of a few cyclotides genuinely containing an Asp in loop 6 as evidenced by gene sequencing, deamidation of Asn during enzymatic digestion resulted in the artifactual presence of Asp isoforms. This result is consistent with studies suggesting that peptides can undergo deamidation after being subjected to external factors, including pH, temperature, and enzymatic digestion.


Assuntos
Ciclotídeos/isolamento & purificação , Cistina/isolamento & purificação , Fabaceae/química , Proteínas de Plantas/isolamento & purificação , Rubiaceae/química , Solanaceae/química , Violaceae/química , Sequência de Aminoácidos , Ciclotídeos/química , Cistina/química , Estrutura Molecular , Proteínas de Plantas/química
4.
ACS Chem Biol ; 10(11): 2491-500, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26322745

RESUMO

Cyclotides are macrocyclic proteins produced by plants for host defense. Although they occur sparsely in other plant families, cyclotides have been detected in every Violaceae plant species so far screened. Many of the Violaceae species examined until now have been from closely related geographical regions or habitats. To test the hypothesis that cyclotides are ubiquitous in this family, two geographically isolated (and critically endangered) species of Australasian Violaceae, namely Melicytus chathamicus and M. latifolius, were examined. Surprisingly, we discovered a suite of cyclotides possessing novel sequence features, including a lysine-rich nature, distinguishing them from "conventional" cyclotides and suggesting that they might have different physiological activities in plants to those reported to date. The newly discovered cyclotides were found to bind to lipid membranes and were cytotoxic against cancer cell lines but had low toxicity against red blood cells, which is advantageous for potential therapeutic applications. This suite of novel Lys-rich cyclotides emphasizes the broad diversity of cyclotides in Violaceae species.


Assuntos
Ciclotídeos/química , Lisina/química , Violaceae/química , Sequência de Aminoácidos , Células Cultivadas , Ciclotídeos/farmacologia , Motivos Nó de Cisteína , Eritrócitos/efeitos dos fármacos , Humanos , Lisina/farmacologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray
5.
Peptides ; 63: 38-42, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25451333

RESUMO

The main bacterium associated with skin infection is Staphylococcus aureus, occurring especially in infections acquired via surgical wounds, commonly leading to lethal hospital-acquired infections, emphasizing the importance of identifying new antimicrobial compounds. Among them, cyclotides have gained interest due to their high stability and multifunctional properties. Here, cycloviolacin 2 (CyO2) and kalata B2 (KB2) were evaluated to determinate their anti-staphylococcal activities using a subcutaneous infection model. Anti-staphylococcal activities of 50mM for KB2 and 25mM for CyO2 were detected with no cytotoxic activities against RAW 264.7 monocytes. In the in vivo assays, both cyclotides reduced bacterial load and CyO2 demonstrated an increase in the phagocytosis index, suggesting that the CyO2 in vivo anti-staphylococcal activity may be associated with phagocytic activity, additionally to direct anti-pathogenic activity.


Assuntos
Antibacterianos/farmacologia , Peptídeos Cíclicos/farmacologia , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Infecção da Ferida Cirúrgica/tratamento farmacológico , Animais , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Fagocitose/efeitos dos fármacos , Células RAW 264.7 , Infecções Cutâneas Estafilocócicas/microbiologia , Infecção da Ferida Cirúrgica/microbiologia
6.
Asian Pac J Trop Med ; 6(9): 673-81, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23827142

RESUMO

OBJECTIVE: To evaluate in vitro antimicrobial activities of selected 58 ethno-medicinal plant extracts with a view to assess their therapeutic potential. METHODS: A total of 58 traditional Chinese medicinal plants were carefully selected based on the literature review and their traditional use. The antimicrobial activities of ethanol extracts of these medicinal plants were tested against fungi (Aspergillus fumigatus), yeast (Candida albicans), gram-negative (Acinetobacter baumannii and Pseudomonas aeruginosa) and gram-positive bacteria (Staphylococcus aureus). The activities were tested at three different concentrations of 1.00, 0.10 and 0.01 mg/mL. The data was analysed using Gene data Screener program. RESULTS: The measured antimicrobial activities indicated that out of the 58 plant extracts, 15 extracts showed anti-fungal activity and 23 extracts exhibited anti-bacterial activity. Eight plant extracts have exhibited both anti-bacterial and anti-fungal activities. For instance, Eucommia ulmoides, Polygonum cuspidatum, Poria cocos and Uncaria rhyncophylla showed activity against both bacterial and fungal strains, indicating their broad spectrum of activity. CONCLUSIONS: The results revealed that the ethanol extracts of 30 plants out of the selected 58 possess significant antimicrobial activities. It is interesting to note that the findings from the current study are consistent with the traditional use. A clear correlation has also been found between the antimicrobial activity and the flavonoid content of the plant extracts which is in agreement with the literature. Hence, the results presented here can be used to guide the selection of potential plant species for the isolation and structure elucidation of novel antimicrobial compounds in order to establish the structure-activity relationship. This in turn is expected to lead the way to the discovery of novel antimicrobial agents for therapeutic use.


Assuntos
Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Fungos/efeitos dos fármacos , Plantas Medicinais/química , Anti-Infecciosos/química , Bactérias/crescimento & desenvolvimento , Avaliação Pré-Clínica de Medicamentos , Medicamentos de Ervas Chinesas/química , Fungos/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade
7.
BMC Complement Altern Med ; 12: 173, 2012 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-23038995

RESUMO

BACKGROUND: The main aim of this study is to evaluate the antioxidant and anti-inflammatory properties of forty four traditional Chinese medicinal herbal extracts and to examine these activities in relation to their antioxidant content. METHODS: The antioxidant activities were investigated using DPPH radical scavenging method and yeast model. The anti-inflammatory properties of the herbal extracts were evaluated by measuring their ability to inhibit the production of nitric oxide and TNF-α in RAW 264.7 macrophages activated by LPS and IFN- γ, respectively. The cytotoxic effects of the herbal extracts were determined by Alomar Blue assay by measuring cell viability. In order to understand the variation of antioxidant activities of herbal extracts with their antioxidant contents, the total phenolics, total flavonoids and trace metal (Mg, Mn, Cu, Zn, Se and Mo) quantities were estimated and a correlation analysis was carried out. RESULTS: Results of this study show that significant levels of phenolics, flavonoids and trace metal contents were found in Ligustrum lucidum, Paeonia suffuticosa, Salvia miltiorrhiza, Sanguisorba officinalis, Spatholobus suberectus, Tussilago farfara and Uncaria rhyncophylla, which correlated well with their antioxidant and anti-inflammatory activities. Some of the plants displayed high antioxidant and anti-inflammatory activities but contained low levels of phenolics and flavonoids. Interestingly, these plants contained significant levels of trace metals (such as Zn, Mg and Se) which are likely to be responsible for their activities. CONCLUSIONS: The results indicate that the phenolics, flavonoids and trace metals play an important role in the antioxidant activities of medicinal plants. Many of the plants studied here have been identified as potential sources of new antioxidant compounds.


Assuntos
Anti-Inflamatórios/química , Antioxidantes/química , Medicamentos de Ervas Chinesas/química , Plantas Medicinais/química , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Linhagem Celular , Medicamentos de Ervas Chinesas/farmacologia , Flavonoides/química , Flavonoides/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Fenóis/química , Fenóis/farmacologia
8.
Curr Top Med Chem ; 12(14): 1534-45, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22827522

RESUMO

Cyclic peptides typically have much higher stability and improved biopharmaceutical properties over their linear counterparts. Our work focuses on the discovery of naturally occurring disulfide-rich cyclic peptides and their applications in drug design. These peptides provide a design basis for re-engineering natural acyclic peptides to improve their biopharmaceutical properties by chemically linking their termini. Here we describe examples of the discovery of the cyclotide family of peptides, their chemical re-engineering to introduce desired pharmaceutical activities, studies of their biopharmaceutical properties and applications of cyclization technologies to naturally occurring toxins, including conotoxins and scorpion toxins. In the case of the conotoxin Vc1.1, we produced an orally active peptide with potential for the treatment of neuropathic pain by cyclising the native peptide. In the case of the scorpion toxin chlorotoxin, a cyclised derivative had improved biopharmaceutical properties as a tumour imaging agent over the naturally occurring linear chlorotoxin. Ongoing chemical and structural studies of these classes of disulfide-rich peptides promise to increase their value for use in dissecting biological processes in plants and mammals while also providing leads to new classes of biopharmaceuticals.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Dissulfetos/química , Descoberta de Drogas , Glioma/tratamento farmacológico , Neurotoxinas/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/química , Glioma/patologia , Humanos , Modelos Moleculares , Neuralgia/tratamento farmacológico , Neurotoxinas/administração & dosagem , Neurotoxinas/química , Peptídeos Cíclicos/administração & dosagem
9.
J Agric Food Chem ; 59(23): 12361-7, 2011 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-22023309

RESUMO

The antioxidant, anti-inflammatory, and cytotoxic activities of water and ethanol extracts of 14 Chinese medicinal plants were investigated and also their total phenolics and flavonoid contents measured. The antioxidant activity was evaluated in a biological assay using Saccharomyces cerevisiae , whereas the radical scavenging activity was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Total phenolics and flavonoid contents were estimated by Folin-Ciocalteu and aluminum chloride methods, respectively. The anti-inflammatory activities of the plant extracts were determined by measuring the inhibition of production of nitric oxide (NO) and TNF-α in LPS and IFN-γ activated RAW 264.7 macrophages. Their cytotoxic activities against macrophages were determined by Alamar Blue assay. Four plants, namely, Scutellaria baicalensis , Taxillus chinensis , Rheum officinale , and Sophora japonica , showed significant antioxidant activity in both yeast model and also free radical scavenging methods. The ethanol extract of S. japonica showed highest levels of phenolics and flavonoids (91.33 GAE mg/g and 151.86 QE mg/g, respectively). A positive linear correlation between antioxidant activity and the total phenolics and flavonoid contents indicates that these compounds are likely to be the main antioxidants contributing to the observed activities. Five plant extracts (S. baicalensis, T. chinensis, S. japonica, Mahonia fortunei , and Sophora flavescens ) exhibited significant anti-inflammatory activity by in vitro inhibition of the production of NO and TNF-α with low IC(50) values. These findings suggest that some of the medicinal herbs studied in this paper are good sources of antioxidants.


Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Flavonoides/análise , Fenóis/análise , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Animais , Linhagem Celular , Macrófagos/efeitos dos fármacos , Camundongos
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