Mitracarpus frigidus reduces lipid metabolism and PGE2 levels in inflammatory cells.

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-ß), 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-ß 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.

Antifungal Annona muricata L. (soursop) extract targets the cell envelope of multi-drug resistant Candida albicans.

ETNOPHARMACOLOGICAL RELEVANCE: 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). MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Isolation and Chemical Characterization of Antifungal, Antioxidant, and Anti-Inflammatory Compounds from Centrosema coriaceum using GC/MS, UFLC-QTOF-MS, and FACE.

In recent years, natural products with biological activities have been increasingly researched. The elucidation of phytoconstituents is necessary for the development of drugs as a natural alternative for the treatment of various diseases. The work aimed to evaluate in vitro and in silico bioactivities of hexane (CCHE) and methanol (CCME) fractions of ethanolic extract from Centrosema coriaceum Benth (Fabaceae) leaves and elucidate their phytoconstituents. CCHE and CCME showed antifungal activity for Candida glabrata (MIC of 1000 µg/mL) with fungistatic effect and action in cell envelope by sorbitol and ergosterol assays. CCHE and CCME presented promising antioxidant activity against the DPPH radical with IC50 of 13.61±0.50 and 6.31±0.40 µg/mL, respectively, and relative antioxidant activity (RAA%) of 45.77±3.61/ 28.53±2.25 % for CCHE and 82.18±2.25/51.99±3.23 % for CCME when compared to rutin and quercetin, respectively. Moreover, these fractions demonstrated promising results for the inhibition of lipid peroxidation by ß-carotene/linoleic acid assay. For anti-inflammatory and cytotoxicity activities, CCHE and CCME significantly inhibited the production of nitric oxide and TNF-α, without toxicity on murine intraperitoneal macrophages, respectively. Esters, alkanes, steroids, tocopherols, and terpenes were identified in CCHE by GC/MS. Flavonoids, phenolic acids, and disaccharides were detected in CCME by UFLC-QTOF-MS and FACE. Furthermore, rutin was purified from CCME. In silico predictions evidenced that compounds present in both fractions have high affinity to the fungal membrane besides antioxidant and anti-inflammatory activities. Based on these observations, CCHE and CCME have a noteworthy potential for the design of novel antifungal and anti-inflammatory agents that should be explored in future studies.

Pharmacological investigation of antioxidant and anti-inflammatory activities of aqueous extract from Mitracarpus frigidus (Rubiaceae).

OBJECTIVES: This study aimed to evaluate the potential of aqueous extract from Mitracarpus frigidus aerial parts (MFAq) in the treatment of inflammation and oxidative stress, as well as to characterize its chemical constituents. METHODS: Total phenolic and flavonoid contents were determined, and phytoconstituents were detected by ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UFLC-QTOF-MS). The antioxidant activity was evaluated by DPPH, TAC and ß-carotene/linoleic acid assays. In-vitro anti-inflammatory activity, cell viability and cell cycle were performed in J774A.1 cell line. In-vivo anti-inflammatory activity was investigated by two ear oedema assays (croton oil and phenol). KEY FINDINGS: Chlorogenic acid, clarinoside, quercetin-hexosylpentoside, rutin, kaempferol-3-O-rutinoside, kaempferol-rhamnosylhexoside, quercetin-pentosylrhamnosylhexoside, harounoside, 2-azaanthraquinone and sucrose were identified by UFLC-QTOF-MS. MFAq showed antioxidant activity, which was positively correlated to the content of phenolic compounds. MFAq significantly inhibited the production of nitric oxide, did not decrease viability in MTT assay (all concentrations) and showed no changes in membrane permeability and cell cycle of J774A.1 cell line. Furthermore, MFAq showed a reduction in ear oedema in all tested doses. CONCLUSION: MFAq was effective in some antioxidant and inflammatory parameters, in the experimental conditions that were used in the study. This is the first report of chemical composition and bioactivities from this extract.

Combining UFLC-QTOF-MS analysis with biological evaluation of Centrosema coriaceum (Fabaceae) leaves.

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.

Spilanthol as a promising antifungal alkylamide for the treatment of vulvovaginal candidiasis.

Spilanthol is a bioactive alkylamide from the native Amazon plant species, Acmella oleracea. However, antifungal activities of spilanthol and its application to the therapeutic treatment of candidiasis remain to be explored. This study sought to evaluate the in vitro and in vivo antifungal activity of spilanthol previously isolated from A. oleracea (spilanthol(AcO)) against Candida albicans ATCC® 10231™, a multidrug-resistant fungal strain. Microdilution methods were used to determine inhibitory and fungicidal concentrations of spilanthol(AcO). In planktonic cultures, the fungal growth kinetics, yeast cell metabolic activity, cell membrane permeability and cell wall integrity were investigated. The effect of spilanthol(AcO) on the proliferation and adhesion of fungal biofilms was evaluated by whole slide imaging and scanning electron microscopy. The biochemical composition of the biofilm matrix was also analyzed. In parallel, spilanthol(AcO) was tested in vivo in an experimental vulvovaginal candidiasis model. Our in vitro analyses in C. albicans planktonic cultures detected a significant inhibitory effect of spilanthol(AcO), which affects both yeast cell membrane and cell wall integrity, interfering with the fungus growth. C. albicans biofilm proliferation and adhesion, as well as, carbohydrates and DNA in biofilm matrix were reduced after spilanthol(AcO) treatment. Moreover, infected rats treated with spilanthol(AcO) showed consistent reduction of both fungal burden and inflammatory processes compared to the untreated animals. Altogether, our findings demonstrated that spilanthol(AcO) is an bioactive compound against planktonic and biofilm forms of a multidrug resistant C. albicans strain. Furthermore, spilanthol(AcO) can be potentially considered for therapeutical treatment of vulvovaginal candidiasis caused by C. albicans. LAY SUMMARY: This study sought to evaluate the antifungal activity of spilanthol against Candida albicans ATCC® 10 231™, a multidrug-resistant fungal strain. Our findings demonstrated that spilanthol(AcO) can be potentially considered for therapeutical treatment of vulvovaginal candidiasis caused by C. albicans.

Whole slide imaging is a high-throughput method to assess Candida biofilm formation.

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.

Antifungal Activity of the Natural Coumarin Scopoletin Against Planktonic Cells and Biofilms From a Multidrug-Resistant Candida tropicalis Strain.

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.

Antibacterial and Antibiofilm Activities of Psychorubrin, a Pyranonaphthoquinone Isolated From Mitracarpus frigidus (Rubiaceae).

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) Staphylococcus aureus 33591 and 33592 and Staphylococcus pyogenes 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 S. aureus 33592 and S. pyogenes 10096 while an additive effect was detected for S. aureus 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.

Tibolone induces serotonin, estrogen, and progesterone receptor expression but not contractile response to serotonin in the rat uterus.

Most studies on the effect of tibolone on the uterus have focused on the endometrium dismissing the importance of the myometrium. The aim of the present study was to investigate some estrogen-like actions of tibolone in the uterus assessed by: 1) the expression of estrogen, progesterone, and serotonin receptors, and 2) the myometrial contraction induced by serotonin. Estradiol (250 µg), progesterone (50 mg), or testosterone (25 mg) pellets were implanted to ovariectomized rats. Tibolone (0.5 mg/day) was orally administered. An implanted pellet containing vehicle or an equivalent volume of water p.o., were used as controls. Sixty days after beginning the treatments, rats were killed and uterus removed. One horn was processed to evaluate estrogen-alpha, progesterone A and B, and serotonin-2A receptors expression, and the other one was used for studying contraction to serotonin and 60 mM potassium solution. The present data showed that tibolone-induced expression of estrogen, progesterone, and serotonin receptors, but did not induce uterine contractile response to either serotonin or potassium solution. These findings suggest that, in the uterus, tibolone may exert molecular estrogenic actions such as the induction of receptor expression, but not a physiological response as the estrogen-dependent contraction to serotonin.

Hemodynamic and oxidative stress profile after exercise in type 2 diabetes.

This study investigated the effect of an acute bout of exercise (>85% VO2Max) on biochemical, hemodynamic and oxidative stress variables in sedentary and physically active subjects with type 2 diabetes (T2D). Blood measurements were taken before and after a treadmill test on 12 sedentary non-diabetes subjects (ND), 12 sedentary type 2 diabetes (T2S) and 9 physically active T2D subjects (T2DA). T2DS subjects before and after the treadmill test showed a higher plasma glucose (123.2 +/- 19.0 mg/dL versus 108.9 +/- 16.8 mg/dL, p < 0.001), HbA1C (8.7 +/- 2.4% versus 7.3 +/- 1.2%, p < 0.001) and body fat% (21.3 +/- 5.7% versus 34.6 +/- 4.5%, p < 0.001) than T2DA subjects. T2DA had higher VO2Max (37.7 +/- 3.5 versus 29.5 +/- 3.2, p < 0.05), time on treadmill (22.3 +/- 2.1 min versus 16.1 +/- 2.1 min, p < 0.05), hemoglobin (17.9 +/- 0.9 g/dL, p < 0.05) and lower blood pressure levels in comparison to ND and T2DS subjects. Thiobarbituric acid substances (TBARS) in T2DS were higher than in T2DA subjects (0.27 +/- 0.1 nmol/mL versus 0.21 +/- 0.1 nmol/mL, p < 0.05). Glutathione (GSH) levels were similar among the groups. Physically active type 2 diabetes subjects had a more favorable biochemical, hemodynamic and oxidative stress profile than sedentary subjects. The coexistence of a poor cardiopulmonary performance and high oxidative stress environment can determine a profile of high risk for serious cardiovascular events in patients with diabetes.