RESUMO
Antifungal resistance poses a significant challenge to disease management, necessitating the development of novel drugs. Antimicrobial peptides offer potential solutions. This study focused on extraction and characterization of peptides from Adenanthera pavonina seeds with activity against Candida species, Mycobacterium tuberculosis, proteases, and α-amylases. Peptides were extracted in phosphate buffer and heated at 90°C for 10 min to create a peptide rich heated fraction (PRHF). After confirming antimicrobial activity and the presence of peptides, the PRHF underwent ion exchange chromatography, yielding retained and non-retained fractions. These fractions were evaluated for antimicrobial activity and cytotoxicity against murine macrophages. The least toxic and most active fraction underwent reversed-phase chromatography, resulting in ten fractions. These fractions were tested for peptides and antimicrobial activity. The most active fraction was rechromatographed on a reversed-phase column, resulting in two fractions that were assessed for antimicrobial activity. The most active fraction revealed a single band of approximately 6 kDa and was tested for inhibitory effects on proteases and α-amylases. Thermal stability experiments were conducted on the 6 kDa peptide at different temperatures followed by reassessment of antifungal activity and circular dichroism. The 6 kDa peptide inhibited yeasts, M. tuberculosis, human salivary and Tenebrio molitor larvae intestine α-amylases, and proteolytic activity from fungal extracts, and thus named ApPI. Remarkably, ApPI retained antifungal activity and conformation after heating and is primarily composed of α-helices. ApPI is a thermally stable serine protease/α-amylase inhibitor from A. pavonina seeds, offering promise as a foundational molecule for innovative therapeutic agents against fungal infections and tuberculosis.
RESUMO
Six previously undescribed intact limonoids together with four known compounds were isolated from the seeds of Trichilia lepidota subsp. schumanniana (Harms) T.D.Penn. Their structures were characterized based on one- and two-dimensional nuclear magnetic resonance spectra, infrared, ultraviolet, mass spectroscopy results, and optical rotation. All compounds were evaluated for their ability to inhibit nitric oxide production in cultures of RAW 264.7 macrophages stimulated by lipopolysaccharide, cytotoxicity and growth of Mycobacterium tuberculosis strains H37Rv and M299. The compounds 7-deacetyl-11ß,12α-diacetoxy-14,15-epoxyazadirone (5) and walsurin E (9) were the most potent in inhibiting nitric oxide production, although the compounds 1-deshydroxy-12α-acetoxymunronin N (1) and 6α,12α-dihydroxyazadirone (6) also showed controlled potential of this mediator, in addition to being potent growth inhibitors of Mycobacterium tuberculosis H37RV and M299, without cytotoxicity interference. Ring intact limonoids isolated from Trichilia lepidota subsp. schumanniana seeds are a new source of bioactive substances that may be used in the future against diseases such as tuberculosis and other processes related to inflammation.
Assuntos
Limoninas , Meliaceae , Limoninas/química , Óxido Nítrico , Meliaceae/química , Espectroscopia de Ressonância Magnética , MacrófagosRESUMO
Tuberculosis remains a major health problem worldwide. Drug-resistant and hypervirulent Mycobacterium tuberculosis (Mtb) strains can lead to a hyperinflammatory response and necrotic pathology in hyper-reactive individuals that require adjunctive treatment. Plant-derived substances have been investigated for TB treatment, among which flavonoids stand out. We evaluate the anti-Mtb, anti-inflammatory and cytotoxicity activities of fractions and substances 1, 2 and 3 isolated from Erythrina verna through a bioassay guided fractionation. Seven fractions (1, 3-5 and 7-9) obtained from dichloromethane E. verna extract inhibited NO production (IC50 ≤ 15 µg/mL) with none or poor cytotoxic effect, while the fractions 4 and 5 notably reduced TNF-a production. Fractions 4, 6 and 9 suppressed Mycobacterium growth with MIC50 ≤ 20 µg/mL. Fraction 4 was the most potent due to dual biological activities. Erythratidinone and alpinumisoflavone inhibited the growth of Mtb H37Rv and hypervirulent strain in bacterial cultures (MIC50 ≤ 20 µg/mL), with erythratidinone standing out in reducing intracellular growth of Mtb H37Rv (5.8 ± 1.1 µg/mL). Alpinumisoflavone and erythratidinone were capable of inhibiting NO and TNF-α production besides showing significant inhibitory effects against Mycobacterium tuberculosis strains with low toxicity in macrophages. Both substances are promising for further studies focusing on an anti-TB dual treatment approach.
Assuntos
Erythrina , Mycobacterium tuberculosis , Anti-Inflamatórios/farmacologia , Antituberculosos/farmacologia , Flavonoides/farmacologia , Humanos , Cloreto de Metileno , Testes de Sensibilidade Microbiana , Extratos Vegetais/farmacologia , Fator de Necrose Tumoral alfaRESUMO
Microbial resistance to available drugs is a growing health threat imposing the need for the development of new drugs. The scaffold of plant defensins, including their γ-cores, are particularly good candidates for drug design. This work aimed to improve the antifungal activity of a previous design peptide, named A36,42,44γ32-46VuDef (for short DD) against yeasts by altering its biochemical parameters. We explore the correlation of the biological activity and structure of plant defensins and compared their primary structures by superimposition with VuDef1 and DD which indicated us the favorable position and the amino acid to be changed. Three new peptides with modifications in charge, hydrophobicity (RR and WR) and chirality (D-RR) were designed and tested against pathogenic yeasts. Inhibition was determined by absorbance. Viability of mammalian cells was determined by MTT. The three designed peptides had better inhibitory activity against the yeasts with better potency and spectrum of yeast species inhibition, with low toxicity to mammalian cells. WR, the most hydrophobic and cationic, exhibited better antifungal activity and lower toxicity. Our study provides experimental evidence that targeted changes in the primary structure of peptides based on plant defensins γ-core primary structures prove to be a good tool for the synthesis of new compounds that may be useful as alternative antifungal drugs. The method described did not have the drawback of synthesis of several peptides, because alterations are guided. When compared to other methods, the design process described is efficient and viable to those with scarce resources.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Linhagem Celular , Defensinas/química , Defensinas/farmacologia , Desenho de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Leveduras/efeitos dos fármacos , Leveduras/crescimento & desenvolvimentoRESUMO
Scientific advances have not been enough to combat the growing resistance to antimicrobial medicines. Antimicrobial peptides (AMPs) are effector molecules of the innate immune defense system in plants and could provide an important source of new antimicrobial drugs. The aim of this work was to extract, purify, characterize, and evaluate the antifungal activities present in fractions obtained from Capsicum annum fruits through reversed-phase chromatography. The fractions named F2 and F3 presented the highest inhibitory activity against Candida and Mycobacterium tuberculosis species. In addition, we identified two sequences of AMPs in the F2 and F3 fractions through mass spectrometry that showed similarity to an already well-characterized family of plant defensins. A plasma membrane permeabilization assay demonstrated that the peptides present in F2, F3, and F4 fractions induced changes in the membrane of some yeast strains, culminating in permeabilization. The production of reactive oxygen species was induced by the fractions in some yeast strains. Fractions F2, F3, and F4 also did not show toxicity in macrophage or monocyte cultures. In conclusion, the obtained data demonstrate that the AMPs, especially those present in the fractions F2 and F3, are promising antimicrobial agents that may be useful to enhance the development of new therapeutic agents for the treatment of diseases.
Assuntos
Antifúngicos , Capsicum/química , Defensinas , Frutas/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Defensinas/isolamento & purificação , Defensinas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
A phytochemical study of leaves and twigs of Psychotria nuda resulted in 19 compounds, including five indole alkaloids, N,N,N-trimethyltryptamine, lyaloside, strictosamide, strictosidine, and 5α-carboxystrictosidine; two flavonolignans, cinchonain Ia and cinchonain Ib; an iridoid, roseoside; a sugar, lawsofructose; a coumarin, scopoletin; a diterpene, phytol; three triterpenes, pomolic acid, spinosic acid, and rotungenic acid; and five steroids, sitosterol, stigmasterol, campesterol, ß-sitosterol-3-O-ß-d-glucoside, and ß-stigmasterol-3-O-ß-d-glucoside. Some compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis and their ability to inhibit NO production by macrophages stimulated by lipopolysaccharide (LPS). The compounds pomolic acid, spinosic acid, strictosidine, and 5α-carboxystrictosidine displayed antimycobacterial activity with minimum inhibitory concentrations ranging from 7.1 to 19.2 µg/mL. These compounds showed promising inhibitory activity against NO production (IC50 3.22 to 25.5 µg/mL). 5α-carboxystrictosidine did not show cytotoxicity against macrophages RAW264.7 up to a concentration of 100 µg/mL. With the exception of strictosamide, this is the first report of the occurrence of these substances in P. nuda.
Assuntos
Alcaloides/análise , Antibacterianos/análise , Antioxidantes/análise , Psychotria/química , Triterpenos/análise , Alcaloides/farmacologia , Animais , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Lipopolissacarídeos/efeitos adversos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Óxido Nítrico/metabolismo , Extratos Vegetais/química , Folhas de Planta/química , Células RAW 264.7 , Triterpenos/farmacologiaRESUMO
Abstract The chemical study of roots from Azadirachta indica A. Juss., Meliaceae, led to the isolation of two new terpenoids, limonoid morenolide and diterpene 17-hydroxy-sandaracopimar-8,15-dien-11-one, in addition to the four well-known limonoids nimbinene, nimbinal, nimbandiol and salannin, and three diterpenoids nimbidiol, ferruginol, and 6,7-dehydroferruginol. Their structural elucidations were based on one and bidimensional Nuclear Magnetic Resonance and Electrospray ionization mass spectrometry spectra data which was compared to the data found in literature. The anti-inflammatory, cytotoxic and antimycobacterial activities of the identified terpenoids were evaluated.