Annona muricata L. (soursop) is traditionally used in the treatment of inflammatory diseases, cancer, and infections caused by fungi. The therapeutic activity explored by its medicinal use is ...generally associated with its phytoconstituents, such as acetogenins and alkaloids. However, its potential antifungal bioactivity as well as its mechanism of action remains to be established.
Aim of the study: To evaluate the antifungal activity of the ethanolic extract of A. muricata leaves against multidrug-resistant Candida albicans (ATCC® 10231).
Phytoconstituents were detected by UFLC-QTOF-MS. The minimum inhibitory concentration was determined, followed by the determination of the minimum fungicidal concentration. For planktonic cells, the growth curve and cell density were evaluated. Studies to understand the mechanism of action on the cell envelope involved crystal violet permeability, membrane extravasation, sorbitol protection, exogenous ergosterol binding assay, metabolic activity, and cell viability. Furthermore, mitochondrial membrane potential was assessed.
Our analyses demonstrated a significant inhibitory effect of A. muricata, with the ability to reduce fungal growth by 58% and cell density by 65%. The extract affected both the fungal plasma membrane and cell wall integrity, with significant reduction of the cell viability. Depolarization of the fungal mitochondrial membrane was observed after treatment with A. muricata. Rutin, xi-anomuricine, kaempferol-3O-rutinoside, nornuciferine, xylopine, atherosperminine, caffeic acid, asimilobine, s-norcorydine, loliolide, annohexocin, annomuricin, annopentocin, and sucrose were identified as extract bioactive components.
Our findings show that the A. muricata extract is a source of chemical diversity, which acts as a potential antifungal agent with promising application to the therapy of infections caused by C. albicans.
Display omitted
•Antifungal Annona muricata extract targets the cell envelope of resistant Candida albicans.•Annona muricata extract alters mitochondrial membrane polarization.•Annona muricata extract is a promising antifungal agent.
Candida tropicalis
is one the most relevant biofilm-forming fungal species increasingly associated with invasive mucosal candidiasis worldwide. The amplified antifungal resistance supports the ...necessity for more effective and less toxic treatment, including the use of plant-derived natural products. Scopoletin, a natural coumarin, has shown antifungal properties against plant yeast pathogens. However, the antifungal activity of this coumarin against clinically relevant fungal species such as
C. tropicalis
remains to be established. Here, we investigated the potential antifungal properties and mechanisms of action of scopoletin against a multidrug-resistant
C. tropicalis
strain (ATCC 28707). First, scopoletin was isolated by high-performance liquid chromatography from
Mitracarpus frigidus
, a plant species (family
Rubiaceae
) distributed throughout South America. Next, scopoletin was tested on
C. tropicalis
cultivated for 48h in both planktonic and biofilm forms. Fungal planktonic growth inhibition was analyzed by evaluating minimal inhibitory concentration (MIC), time-kill kinetics and cell density whereas the mechanisms of action were investigated with nucleotide leakage, efflux pumps and sorbitol and ergosterol bioassays. Finally, the scopoletin ability to affect
C. tropicalis
biofilms was evaluated through spectrophotometric and whole slide imaging approaches. In all procedures, fluconazole was used as a positive control. MIC values for scopoletin and fluconazole were 50 and 250 μg/L respectively, thus demonstrating a fungistatic activity for scopoletin. Scopoletin induced a significant decrease of
C. tropicalis
growth curves and cell density (91.7% reduction) compared to the growth control. Its action was related to the fungal cell wall, affecting plasma membrane sterols. When associated with fluconazole, scopoletin led to inhibition of efflux pumps at the plasma membrane. Moreover, scopoletin not only inhibited the growth rate of preformed biofilms (68.2% inhibition at MIC value) but also significantly decreased the extent of biofilms growing on the surface of coverslips, preventing the formation of elongated fungal forms. Our data demonstrate, for the first time, that scopoletin act as an effective antifungal phytocompound against a multidrug-resistant strain of
C. tropicalis
with properties that affect both planktonic and biofilm forms of this pathogen. Thus, the present findings support additional studies for antifungal drug development based on plant isolated-scopoletin to treat candidiasis caused by
C. tropicalis.
The antileishmanial and antifungal activity of 24 methanol extracts from 20 plants, all of them used in the Brazilian traditional medicine for the treatment of several infectious and inflammatory ...disorders, were evaluated against promastigotes forms of two species of
Leishmania (
L. amazonensis and
L. chagasi) and two yeasts (
Candida albicans and
Cryptococcus neoformans). Among the 20 tested methanolic extracts, those of
Vernonia polyanthes was the most active against
L. amazonensis (IC
50 of 4
μg/ml), those of
Ocimum gratissimum exhibited the best activity against
L. chagasi (IC
50 of 71
μg/ml). Concerning antifungical activity,
Schinus terebintifolius,
O. gratissimum,
Cajanus cajan, and
Piper aduncum extracts were the most active against
C. albicans (MIC of 1.25
mg/ml) whereas
Bixa orellana,
O. gratissimum and
Syzygium cumini exhibited the best activity against
C. neoformans (MIC of 0.078
mg/ml).
Psychorubrin, a natural pyranonaphthoquinone found in different plants, has become an interesting compound in the search for new antimicrobial therapeutic agents. Here, we investigated the potential ...antagonistic activity of psychorubrin against planktonic and biofilm bacteria. First, psychorubrin was tested against several Gram-positive and Gram-negative bacteria strains by a broth microdilution susceptibility method. Second, bacterial killing assay, bacterial abundance, and membrane viability were evaluated. The nucleotide leakage assay was used to verify membrane destabilization while antibiofilm activities were analyzed by the effect on established biofilm, static biofilm formation, isolation of biofilm matrix assay and scanning electron microscopy. In parallel, the combinatorial effect of psychorubrin and chloramphenicol was evaluated by the checkerboard method. Psychorubrin was active against Gram-positive bacteria, showing rapid time-dependent kinetics of bacterial killing, amplified nucleotide leakage, and greater activity against the methicillin-resistant species (MRSA)
33591 and 33592 and
10096. Psychorubrin also interfered with the composition of the biofilm matrix by reducing the total content of carbohydrates and proteins. A synergic effect between psychorubrin and chloramphenicol was observed for
33592 and
10096 while an additive effect was detected for
33591. Our findings demonstrate, for the first time, an antagonistic activity of psychorubrin against bacteria not only in their planktonic forms but also in biofilms, and identify bacterial membranes as primary targets for this compound. Based on these observations, psychorubrin has a good potential for the design of novel antimicrobial agents.
Bryophyllum pinnatum (Lam.) Oken (Crassulaceae), popularly known in Brazil as “folha-da-fortuna”, is a plant species used in folk medicine for the external and internal treatment of inflammation, ...infection, wound, burn, boil, ulcers and gastritis, and several other diseases. The present study aimed to perform the chemical characterization and the evaluation of the topical anti-inflammatory effect of the ethanol extract of Bryophyllum pinnatum leaves (EEBP) in acute and chronic mice ear edema models induced by different irritant agents.
The EEBP chemical characterization was performed by HPLC–UV DAD. Ear edema on Swiss mice was induced by the topical application of Croton oil (single and multiple applications), arachidonic acid, phenol, capsaicin and ethyl phenylpropiolate (EPP). The topical anti-inflammatory effect of EEBP was evaluated by measuring the ear weight (acute inflammation models) and thickness (chronic inflammation model). Histopathological analyses of ear tissue samples sensitized with Croton oil (single and multiple applications) were also performed.
The flavonoids rutin, quercetin, luteolin and luteolin7-O-β-d-glucoside were detected in EEBP. Topical application of EEBP significantly (P<0.001) inhibited the ear edema induced by Croton oil single application (inhibition of 57%), arachidonic acid (inhibition of 67%), phenol (inhibition of 80%), capsaicin (inhibition of 72%), EPP (inhibition of 75%) and Croton oil multiple application (55% after 9 days). Histopathological analyses confirmed the topical anti-inflammatory effect of EEBP since it was observed reduction of edema, epidermal hyperplasia, inflammatory cells infiltration and vasodilation.
The results suggest that EEBP is effective as a topical anti-inflammatory agent in acute and chronic inflammatory processes possibly due to inhibition of arachidonic acid pathway, which justify the traditional use of Bryophyllum pinnatum as a remedy for skin disorders.
Display omitted
Centrosema coriaceum Benth belongs to Fabaceae family and have few studies of biological activity and chemical composition. Thus, the aims of this work were to determine chemical profile of the ...ethanolic extract of C. coriaceum leaves (CCE) by UFLC-QTOF-MS and to evaluate its in vitro biological potential. CCE showed MIC value of 1000 µg/mL against Candida glabrata (fungistatic effect) and high affinity in cell envelope by increasing cell permeability in nucleotide leakage, sorbitol and ergosterol assays. CCE showed antioxidant activity in all assays performed. For the anti-inflammatory and cytotoxicity activities, CCE, at all tested concentrations, significantly inhibited the production of nitric oxide and did not decrease J774A.1 cell viability below 70%. Finally, rutin, kaempferol-3O-rutinoside, caffeic acid, and sucrose were identified in CCE by UFLC-QTOF-MS. These results suggest, for the first time, that C. coriaceum has interesting antifungal, antioxidant, and anti-inflammatory activities.
Abstract
Objectives
To evaluate the ability of the aqueous extract of Mitracarpus frigidus (MFAq) to inhibit lipid body formation and inflammatory mediator production in macrophages stimulated with ...lipopolysaccharide (LPS) and interferon gamma (IFN-γ).
Methods
MFAq was chemically characterized by ultrafast liquid chromatography/quadruple time-of-flight tandem mass spectrometry. The macrophages obtained from mice were incubated with MFAq. Cell viability and membrane integrity were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and propidium iodide assays, respectively. Moreover, NO, reactive oxygen species (ROS), transforming growth factor beta (TGF-β), prostaglandin E2 (PGE2) levels and lipid bodies (LBs) were examined in macrophages that were stimulated with LPS and IFN-γ and treated with MFAq. Finally, molecular docking analysis was conducted to investigate the interaction of MFAq with the cyclooxygenase 2 (COX-2) enzyme.
Key findings
Chlorogenic acid, clarinoside, harounoside, rutin, kaempferol-3O-rutinoside and 2-azaanthraquinone were identified in MFAq. MFAq significantly inhibited NO, ROS and LBs, and did not affect the membrane integrity of macrophages. MFAq-treated cells showed significantly lower levels of TGF-β and PGE2. Molecular docking demonstrated that the compounds found in MFAq are able to inhibit COX-2 by binding to important residues in the catalytic site.
Conclusions
MFAq interferes with lipid metabolism in stimulated macrophages, leading to the reduction of important inflammatory mediators. Furthermore, MFAq can directly inhibit the COX-2 enzyme or inhibit its expression owing to its ability to reduce NO production.
This study aimed to further investigate the cytotoxicity against tumor cell lines and several bacterial strains of Annona squamosa and its mode of action. Methanol extracts of A. squamosa leaves ...(ASL) and seeds (ASS) were used. ASL showed significant antibacterial activity against S. aureus, K. pneumoniae and E. faecalis with MIC values of 78, 78 and 39 µg/mL respectively. Moreover, ASL exhibited significant biofilm disruption, rapid time dependent kinetics of bacterial killing, increased membrane permeability and significantly reduced the cell numbers and viability. Regarding the cytotoxicity against tumor cell lines, ASS was more active against Jurkat and MCF-7 cells, with CI50 1.1 and 2.1 µg/mL, respectively. ASL showed promising activity against Jurkat and HL60, with CI50 4.2 and 6.4 µg/mL, respectively. Both extracts showed lower activity against VERO cells and reduced the clonogenic survival at higher concentrations (IC90) to MCF-7 and HCT-116 lineages. The alkaloids anonaine, asimilobine, corypalmine, liriodenine nornuciferine and reticuline were identified in extracts by UPLC-ESI-MS/MS analysis. This study reinforced that A. squamosa presents a remarkable phytomedicinal potential and revealed that its antimicrobial mechanism of action is related to bacterial membrane destabilization.
New strategies that enable fast and accurate visualization of Candida biofilms are necessary to better study their structure and response to antifungals agents. Here, we applied whole slide imaging ...(WSI) to study biofilm formation of Candida species. Three relevant biofilm-forming Candida species (C. albicans ATCC 10231, C. glabrata ATCC 2001, and C. tropicalis ATCC 750) were cultivated on glass coverslips both in presence and absence of widely used antifungals. Accumulated biofilms were stained with fluorescent markers and scanned in both bright-field and fluorescence modes using a WSI digital scanner. WSI enabled clear assessment of both size and structural features of Candida biofilms. Quantitative analyses readily detected reductions in biofilm-covered surface area upon antifungal exposure. Furthermore, we show that the overall biofilm growth can be adequately assessed across both bright-field and fluorescence modes. At the single-cell level, WSI proved adequate, as morphometric parameters evaluated with WSI did not differ significantly from those obtained with scanning electron microscopy, considered as golden standard at single-cell resolution. Thus, WSI allows for reliable visualization of Candida biofilms enabling both large-scale growth assessment and morphometric characterization of single-cell features, making it an important addition to the available microscopic toolset to image and analyse fungal biofilm growth.
Display omitted
•Antibacterial Annona muricata extract target the cell membranes of Gram-positive and Gram-negative bacteria.•The alkaloids may be, at least in part, responsible for the bioactivities ...reported by this study.•This plant, commonly used as food, may be useful to treat or prevent bacterial infections.
Annona muricata has become an interesting subject in the search for new therapeutic agents. We investigated the bacterial mode of action of the methanolic extract of A. muricata leaves (AML). AML extract was tested against several bacteria strains by broth microdilution susceptibility method. The bacterial killing assay, bacterial abundance and membrane viability analysis were made using fluorescent probes. The nucleotide leakage and outer membrane (OM) permeability assays were used to verify membrane destabilization. The biochemical reaction profile was carried out on a VITEK®2 system. For UPLC-ESI–MS/MS Analysis an Acquity UPLC system was used. AML was active against both Gram-negative and Gram-positive bacteria, showing greater activity against S. aureus, S. typhimurium and E. faecalis. AML exhibited rapid time dependent kinetics of bacterial killing. DAPI staining revealed that AML inhibited the bacterial growth, while the LIVE/DEAD BacLight analysis showed that AML induced an increase in dead cells. AML increased nucleotide leakage and was also capable of increasing the OM permeability in the tested bacteria. Differences between the stressed clones and controls observed in the biochemical characterization were not enough to modify the strain identity. UPLC-ESI–MS/MS analysis revealed the presence of the alkaloids anonaine, asimilobine, corypalmine, lirioderine, nornuciferine, xylopine and reticuline. Our findings demonstrate, for the first time, a broad spectrum of antibacterial activity for AML and identify that bacterial membranes (both plasma and outer membranes) are primary targets of this extract. Based on these observations, AML has a good potential for the design of novel antimicrobial agents.
PDF
