Activity of Combined Antifungal Agents Against Multidrug-Resistant <Emphasis Type="Italic">Candida glabrata</Emphasis> Strains

In this study, we evaluated the in vitro activity of echinocandins, azoles, and amphotericin B alone and in combination against echinocandin/azole-sensitive and echinocandin/azole-resistant Candida glabrata isolates. Susceptibility tests were performed using the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute document M27-A3. The checkerboard method was used to evaluate the fractional inhibitory concentration index of the interactions. Cross-resistance was observed among echinocandins; 15% of the isolates resistant to caspofungin were also resistant to anidulafungin and micafungin. Synergistic activity was observed in 70% of resistant C. glabrata when anidulafungin was combined with voriconazole or posaconazole. Higher (85%) synergism was found in the combination of caspofungin and voriconazole. The combinations of caspofungin with fluconazole, posaconazole and amphotericin B, micafungin with fluconazole, posaconazole and voriconazole, and anidulafungin with amphotericin B showed indifferent activities for the majority of the isolates. Anidulafungin combined with fluconazole showed the same percentage of synergism and indifference (45%). Antagonism was detected in 50% of isolates when micafungin was combined with amphotericin B. Combinations of echinocandins and antifungal azoles have great potential for in vivo assays which are required to evaluate the efficacy of these combinations against multidrug-resistant C. glabrata strains.     

In Vitro Activity of Berberine Alone and in Combination with Antifungal Drugs Against Planktonic Forms and Biofilms of <Emphasis Type="Italic">Trichosporon Asahii</Emphasis>

Trichosporon asahii (T. asahii) is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients, with high mortality rates up to 80% despite treated with antifungal drugs. The biofilms-forming ability of T. asahii on indwelling medical devices may account for the resistance to antifungal drugs. Berberine (BBR) has been demonstrated to have antifungal activity and synergistic effects in combination with antifungal drugs against pathogenic fungi. In the present study, the in vitro activities of BBR alone or combined with fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), caspofungin (CAS) and amphotericin B (AMB) against planktonic forms and biofilms of 21 clinical T. asahii isolates were evaluated using checkerboard microdilution method and XTT reduction assay, respectively. The fractional inhibitory concentration index (FICI) was used to interpret drug interactions. BBR alone did not exhibit significant antifungal activities against both T. asahii planktonic cells (MICs, 32 → 128 μg/ml) and T. asahii biofilms (SMICs, >128 μg/ml). However, BBR exhibited synergistic effects against T. asahii planktonic cells in combination with AMB, FLC and CAS (FICI ≤ 0.5) and exhibited synergistic effects against T. asahii biofilms in combination with AMB and CAS (FICI ≤ 0.5). BBR/ITC and BBR/VRC combinations yielded mainly indifferent interactions against T. asahii planktonic cells. BBR/FLC, BBR/ITC and BBR/VRC combinations also yielded indifferent interactions against T. asahii biofilms. Our study highlights the therapeutic potential of BBR to be used as an antifungal synergist in combination with antifungal drugs against T. asahii infections, especially BBR/AMB combination. Further in vivo studies are needed to validate our findings.     

In Vitro Effect of Amphotericin B on Candida albicans,Candida glabrata and Candida parapsilosis Biofilm Formation

Candida spp. biofilm is considered highly resistant to conventional antifungals. The aim of this study was to investigate the in vitro effect of amphotericin B on Candida spp. biofilms at different stages of maturation. We investigated the activity of amphotericin B against 78 clinical isolates of Candida spp., representing three species, growing as planktonic and sessile cells, by a widely accepted broth microdilution method. The in vitro effect on sessile cell viability was evaluated by MTT reduction assay. All examined strains were susceptible to amphotericin B when grown as free-living cells. At the early stages of biofilm maturation 96.7–100.0 % strains, depending on species, displayed amphotericin B sessile minimal inhibitory concentration (SMIC) ≤1 μg/mL. Mature Candida spp. biofilm of 32.1–90.0 % strains displayed amphotericin B SMIC ≤1 μg/mL. Based on these results, amphotericin B displays species- and strain-depending activity against Candida spp. biofilms.     

Antifungal Activity of Chitosan-Coated Poly(lactic-co-glycolic) Acid Nanoparticles Containing Amphotericin B

Amphotericin B (AmB) is one of the most used drugs for the treatment of systemic fungal infections; however, the treatment causes several toxic manifestations, including nephrotoxicity and hemolytic anemia. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. In this work, the antifungal efficacy of chitosan-coated PLGA nanoparticles containing AmB was evaluated in 20 strains of fungus isolates from patients with vulvovaginal candidiasis (01 Candida glabrata and 03 Candida albicans), bloodstream infections (04 C. albicans and 01 C. tropicalis) and patients with urinary tract infection (04 Candida albicans, 02 Trichosporon asahii, 01 C. guilhermondii, 03 C. glabrata) and 01 Candida albicans ATCC 90028. Moreover, the cytotoxicity over erythrocytes was evaluated. The single-emulsion solvent evaporation method was suitable for obtaining chitosan-coated PGLA nanoparticles containing AmB. Nanoparticles were spherical in shape, presented mean particle size about 460 nm, positive zeta potential and encapsulation efficiency of 42%. Moreover, nanoparticles prolonged the AmB release. All the strains were susceptible to plain AmB and nanostructured AmB, according to EUCAST breakpoint version 8.1 (resistant > 1 μg/mL), using broth microdilution method. In C. albicans (urine, blood, and vulvovaginal secretion isolates, and 1 ATCC), the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.5 μg/mL. In urine and vulvovaginal secretion isolates of C. glabrata, the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.015 μg/mL. In urine isolates of C. guilhermondii, the MIC value of AmB-loaded nanoparticles was 0.12 μg/mL and EUCAST was 0.06 μg/mL. In blood isolates of C. tropicalis, the MIC value of AmB-loaded nanoparticles was 0.5 μg/mL and EUCAST was 0.25 μg/mL. Finally, in urine isolates of T asahii, the MIC value of AmB-loaded nanoparticles was 1 μg/mL and EUCAST varied from 0.5 to 1 μg/mL. In the cytotoxicity assay, plain AmB was highly hemolytic (100% in 24 h) while AmB-loaded chitosan/PLGA nanoparticles presented negligible hemolysis.     

Non-Healing Ulcer Due to Trichosporon loubieri in an Immunocompetent Host and Review of Published Reports

We report here a case of non-healing ulcer due to Trichosporon loubieri in an apparently immunocompetent female. The identity of isolate was confirmed by DNA sequencing of D1/D2 region of 26S rDNA. The minimum inhibitory concentrations of the isolate were amphotericin B—0.5 μg/ml; fluconazole—4 μg/ml; posaconazole—0.25 μg/ml; voriconazole—0.06 μg/ml. The patient was managed by extensive debridement and oral fluconazole 150 mg daily for 6 weeks. She responded to therapy. To the best of our knowledge, till date, this is the fourth report of human infection due to T. loubieri and the first of its kind in an immunocompetent host. A review of published literature on infections due to T. loubieri is also included.     

Antifungal Resistance and Virulence Among <Emphasis Type="Italic">Candida</Emphasis> spp. from Captive <Emphasis Type="Italic">Amazonian manatees</Emphasis> and West Indian Manatees: Potential Impacts on Animal and Environmental Health

This work aimed at evaluating the antifungal susceptibility and production of virulence factors by Candida spp. isolated from sirenians in Brazil. The isolates (n = 105) were recovered from the natural cavities of Amazonian and West Indian manatees and were tested for the susceptibility to amphotericin B, itraconazole, and fluconazole and for the production of phospholipases, proteases, and biofilm. The minimum inhibitory concentrations (MICs) for amphotericin B ranged from 0.03 to 1 µg/mL, and no resistant isolates were detected. Itraconazole and fluconazole MICs ranged from 0.03 to 16 µg/mL and from 0.125 to 64 µg/mL, respectively, and 35.2% (37/105) of the isolates were resistant to at least one of these azole drugs. Concerning the production of virulence factors, phospholipase activity was observed in 67.6% (71/105) of the isolates, while protease activity and biofilm production were detected in 50.5% (53/105) and 32.4% (34/105) of the isolates, respectively. Since the natural cavities of manatees are colonized by resistant and virulent strains of Candida spp., these animals can act as sources of resistance and virulence genes for the environment, conspecifics and other animal species, demonstrating the potential environmental impacts associated with their release back into their natural habitat.     

Fungicidal activity of the human peptide hepcidin 20 alone or in combination with other antifungals against Candida glabrata isolates

Candida glabrata infections are often difficult to eradicate due to the intrinsically low susceptibility to azoles of this species. In addition, C. glabrata has also been shown to be insensitive to several cationic peptides, which have been shown to be promising novel therapeutic candidates for the treatment of fungal infection. In this study, the in vitro fungicidal activity of the human cationic peptide hepcidin 20 (Hep-20) was evaluated against clinical isolates of C. glabrata with different levels of fluconazole susceptibility. Interestingly, all isolates were susceptible to Hep-20 (100–200 μg/ml) at pH 7.4, whereas the fungicidal effect of the peptide was higher (50 μg/ml) at acidic pH values. In addition, an increased antifungal activity was observed for Hep-20 with amphotericin B and a synergistic effect was demonstrated for the Hep-20/fluconazole and Hep-20/caspofungin combinations.     

Veronaea botryosa: Molecular Identification with Amplified Fragment Length Polymorphism (AFLP) and In vitro Antifungal Susceptibility

Inter- and intraspecific genomic variability of 18 isolates of Veronaea botryosa originating from clinical and environmental sources was studied using amplified fragment length polymorphism (AFLP). The species was originally described from the environment, but several severe cases of disseminated infection in apparently healthy individuals have been reported worldwide. All tested strains of V. botryosa, identified on the basis of sequencing and phenotypic and physiological criteria prior to our study, were confirmed by AFLP analysis, yielding a clear separation of V. botryosa as a rather homogeneous group from related species. In vitro antifungal susceptibility testing resulted in MIC₉₀s across all strains in increasing order posaconazole (0.25 μg/ml), itraconazole (1 μg/ml), voriconazole (4 μg/ml), terbinafine (4 μg/ml), caspofungin (8 μg/ml), anidulafungin (8 μg/ml), isavuconazole (16 μg/ml), amphotericin B (16 μg/ml), and fluconazole (32 μg/ml). Overall, the isolates showed a uniform pattern of low MICs of itraconazole and posaconazole, but high MICs for remaining agents. The echinocandins (caspofungin and anidulafungin) had no activity against V. botryosa. There was no statistically significant difference between susceptibilities of environmental (n = 11) and clinical (n = 7) isolates of V. botryosa (P > 0.05).     

Chromoblastomycosis due to Fonsecaea pedrosoi and F. monophora in Cuba

We report two cases of chromoblastomycosis due to Fonsecaea pedrosoi and F. monophora in otherwise healthy Cuban males. Direct microscopic examination of biopsies revealed muriform cells, the hallmark of chromoblastomycosis. The suspected agents were recovered in culture, identified on the basis of morphological criteria and confirmed by sequencing of the internal transcribed spacer regions of rDNA. Final treatment consisted of surgical excision. The patients were successfully cured since there was no relapse after a follow-up of more than a year. In vitro antifungal susceptibility testing of both isolates showed that itraconazole and posaconazole had potent activity. High MICs of amphotericin B (2 μg/ml), fluconazole (>64 μg/ml), anidulafungin (8 μg/ml) and caspofungin (8 μg/ml) were found.     

Comparison of Commercial Methods and the CLSI Broth Microdilution to Determine the Antifungal Susceptibility of Candida parapsilosis Complex Bloodstream Isolates from Three Health Institutions in Rio de Janeiro,Brazil

Two commercial methods, the Etest and Vitek 2, were compared with the Clinical and Laboratory Standards Institute broth microdilution method to determine the susceptibility of Candida parapsilosis complex to amphotericin B, caspofungin, fluconazole, voriconazole, and itraconazole. One-hundred bloodstream isolates of C. parapsilosis complex from three hospitals in Rio de Janeiro city, Brazil, between 1998 and 2006 were analyzed. C. parapsilosis sensu stricto (61 %) was the predominant species, followed by C. orthopsilosis (37 %) and C. metapsilosis (2 %). Most isolates were susceptible to the tested drugs. However, one C. parapsilosis sensu stricto isolate was considered resistant for amphotericin B. The essential agreement was 100 % between the methods, except for itraconazole (96.3 %). The categorical agreement varied for fluconazole and itraconazole by Etest and for amphotericin B and fluconazole by Vitek 2. This study reinforces the suitability of the commercial methods in routine clinical microbiology laboratories for antifungal susceptibility testing.     

Diphenyl diselenide modulates nucleotidases,reducing inflammatory responses in the liver of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis>-infected mice

The aim of this study was to verify the effect of diphenyl diselenide (PhSe)₂ on hepatic nucleotidases and on the concentration of purines in mice infected by Toxoplasma gondii. The animals were divided into four groups: Group A (uninfected), Group B (uninfected and treated with (PhSe)₂), Group C (infected), and Group D (infected and treated with (PhSe)₂). The inoculation (groups C and D) was performed with 50 cysts of T. gondii (ME-49 strain). Mice from groups B and D were treated with 5 μmol kg^?1 of (PhSe)₂. Liver tissue from infected mice showed less severe inflammation, elevated ATP/ADO ratio, elevated NTPDase, 5′nucleotidase, and ADA activities compared to the uninfected group (Group A; P < 0.05). However, infected and treated mice showed decreased ATP levels and elevated ADO levels, as well as higher NTPDase and 5′nucleotidase activities and decreased ADA activity in the hepatic tissue compared to the infected group (P < 0.05). Moreover, the (PhSe)₂ treatment of infected mice reduced the hepatic inflammation and showed an immunomodulatory effect on ectonucleotidases of hepatic lymphocytes, which it returned to basal levels. Therefore, chronic infection by T. gondii induces hepatic inflammation in mice, and it is possible that purine levels and nucleotidase activities in hepatic tissue are related to the pathogenesis of the infection in this tissue. The treatment with (PhSe)₂ was able to reverse the hepatic inflammation in mice chronically infected, possibly due to the modulation of purinergic enzymes that produce an anti-inflammatory profile through the purinergic system in the liver tissue.     

Diphenyl Diselenide Reduces Mechanical and Thermal Nociceptive Behavioral Responses After Unilateral Intrastriatal Administration of 6-Hydroxydopamine in Rats

Parkinson’s disease (PD) patients, in addition to motor dysfunction, also present alterations in pain sensation. The present study characterized the antinociceptive effects of diphenyl diselenide ((PhSe)₂) in a model of nociception induced by unilateral, intrastriatal 6-hydroxydopamine (6-OHDA) injection in rats. Male adult Wistar rats received 20 μg/3 μl of 6-OHDA (in saline solution containing 0.02 % of ascorbic acid) or 3 μl of vehicle into the right striatum (1.0 mm anterior, 3.0 mm lateral, and 5.0 mm ventral—with respect to the bregma). Thirty days after injection, rats received (PhSe)₂ intragastrically at a dose of 10 mg/kg 1 h before behavioral tests (von Frey hairs, hot plate, tail immersion, formalin, and open field). Our results demonstrated that 6-OHDA injection to rats augmented the response frequency of von Frey hairs (VHF) stimulation, besides reducing the thermal withdrawal latency in the hot plate test. Importantly, the (PhSe)₂ treatment decreased the mechanical allodynia measured by the response frequency of VHF stimulation and diminished the thermal nociception in the hot plate test in 6-OHDA-injected rats. In conclusion, these results revealed that a single oral administration of (PhSe)₂ 1 h prior to the accomplishment of the behavioral tests was effective to attenuate the increased mechanical and thermal nociception caused by a single intrastriatal 6-OHDA injection to rats. Furthermore, other clarifying studies are warranted to improve the evidence bases for future clinical use of (PhSe)₂ as a new alternative therapy for the treatment of painful symptoms associated to PD.     

Diphenyl diselenide and diphenyl ditelluride: neurotoxic effect in brain of young rats, in vitro

This study examined whether maturity of rat brain may be relevant for the sensitivity to diphenyl diselenide (PhSe)₂ and diphenyl ditelluride (PhTe)₂ on [³H]glutamate uptake and release, in vitro. Brain synaptosomes were isolated from young (14- and 30-day-old) and adult rats and incubated at different concentrations of (PhSe)₂ or (PhTe)₂. The results demonstrated that the highest concentration (100 μM) of (PhSe)₂ and (PhTe)₂ inhibited the [³H]glutamate uptake by synaptosomes of brain at all ages. In the adult brain, (PhSe)₂ did not inhibit the [³H]glutamate uptake at the lowest concentration (10 μM). The highest concentration of (PhTe)₂ inhibited the [³H]glutamate uptake more in the 14-day-old than in the 30-day-old rats or adult rats. In the 30-day-old animals, the highest concentration of (PhSe)₂, and the lowest concentration of (PhTe)₂, increased the basal [³H]glutamate release. At the highest concentration, (PhTe)₂ increased the basal and K⁺-stimulated glutamate release on all ages evaluated. The results suggest that (PhSe)₂ and (PhTe)₂ caused alterations on the homeostasis of the glutamatergic system at the pre-synaptic level. These alterations were age-, concentration-, and compound-dependent. The maturity of rat brain is relevant for the glutamatergic system sensitivity to (PhSe)₂ and (PhTe)₂ .     

Caulerprenylols A and B,two rare antifungal prenylated para-xylenes from the green alga Caulerpa racemosa

Two new prenylated para-xylenes, named caulerprenylols A (1) and B (2), were isolated from the green alga Caulerpa racemosa, collected from the Zhanjiang coastline, China. The structures of the two metabolites were elucidated on the basis of detailed spectroscopic analysis. This is the first report of prenylated para-xylenes from marine algae and from marine organisms as well. Moreover, caulerprenylol B (2) is also characterized by an uncommon indane ring system. In in vitro bioassays, the new compounds exhibited a broad spectrum of antifungal activity against Candida glabrata (537), Trichophyton rubrum (Cmccftla), and Cryptococcus neoformans (32609) with MIC₈₀ values between 4 and 64 μg/mL when compared to amphotericin B (MIC₈₀ values of 2.0, 1.0, and 4.0 μg/mL, respectively) as a positive control and showed no growth inhibition activity against the tumor cells HL60 and A549.     

Clinical and Microbiological Investigation of Fungemia from Four Hospitals in China

In this study, fungemia cases from four tertiary hospitals located in Shanghai and Anhui province in China from March 2012 to December 2013 were enrolled to investigate clinical features, species distribution, antifungal susceptibility and strain relatedness. During the study period, 137 non-duplicate cases and their corresponding isolates were collected. Six different genera of fungi were identified, of which Candida spp. were the most common (126/137, 91.97 %), with C. albicans predominating (48/137, 35.03 %). The non-Candida fungi rate reached 8.03 % (11/137), and Pichia spp. was the most common (5/137, 3.65 %). Compared with C. albicans, non-C. albicans fungi-associated fungemia was more likely in younger (p = 0.004) and male patients (χ ² = 6.2618, p = 0.0123) and patients from ICUs (χ ² = 6.3783, p = 0.0116). Overall, the susceptible/WT rates of common Candida spp. to fluconazole, itraconazole, voriconazole, flucytosine, amphotericin B and caspofungin were 74.63, 92.31, 93.16, 96.58, 100 and 95.69 %, respectively. C. tropicalis and C. guilliermondii had a low susceptibility to fluconazole: 79.95 and 77.78 %, respectively. No isolates were resistant/WT to caspofungin, but C. parapsilosis and C. guilliermondii had high MIC₉₀ values; 1 and 2 mg/L, respectively. In terms of genotyping, MLST was taken for C. glabrata and C. tropicalis, while microsatellite marker analysis was used for C. albicans and C. parapsilosis. C. glabrata was predominantly clone ST7, accounting for 75 %, while the other isolates showed genetic diversity. Considering the increased proportion of non-C. albicans fungi and the presence of endemic clones of C. glabrata, it is essential to undertake additional surveillance of fungemia.     

In Vitro Susceptibility of Candida Isolates from Organ Transplant Recipients to Newer Antifungals

Organ transplant recipients (OTR) are at higher risk of developing life-threatening infections. In this study, we tested 527 Candida isolates obtained from the oral and genital mucosa from OTR and healthy controls in order to monitor antifungal susceptibility patterns in this particular risk group. Testing was carried out in parallel for already marketed azoles and anidulafungin. Minimal inhibitory concentrations (MICs) were determined using the E-test^® for azoles and CLSI broth microdilution for anidulafungin. Overall, there was no difference in the distribution of Candida spp. for both groups, C. albicans being the most frequently isolated Candida sp. followed by C. glabrata. Also, there were only minor differences in the susceptibility patterns to all antifungal agents. All C. albicans isolates were fully susceptible to fluconazole and voriconazole. In C. glabrata, 2.2 % (n = 1) were resistant to fluconazole, and 82.6 % (n = 38) to itraconazole, and in C. krusei, 66.7 % (n = 2) were resistant in itraconazole. All strains were susceptible to voriconazole. Only fluconazole showed a higher rate of resistant C. glabrata isolates for OTR (3.7 %), whereas the control group showed only intermediate susceptible and no resistant isolates. As there are no breakpoints established for posaconazole by CLSI, breakpoints determined by EUCAST were used. A total of 87.9 % of C. albicans, 81.3 % of C. parapsilosis and 66.7 % of C. tropicalis were considered susceptible. C. glabrata and C. krusei showed higher MIC values and thus lesser susceptibility than the other Candida species. There were no differences observed between OTR and control groups. For anidulafungin, 99.8 % of C. albicans isolates were susceptible, 0.2 % were intermediate, whereas for C. glabrata, only 95.3 % were susceptible, 0.2 % were resistant and 4.5 % were interpreted as intermediate. Interestingly, the two resistant isolates were found in the control group. Also, the controls showed a marginally higher percentage of intermediate strains compared to the transplant patients. All in all, resistant isolates were only observed for C. glabrata of the control group.     

Extent and Mode of Action of Cationic Legume Proteins against Listeria monocytogenes and Salmonella Enteritidis

The methylated soybean protein and methylated chickpea protein (MSP and MCP) with isoelectric points around pI 8 were prepared by esterifying 83 % of their free carboxyl groups and tested for their interactions with Listeria monocytogenes and Salmonella Enteritidis. The two substances exhibited a concentration-dependent inhibitory action against the two studied bacteria with a minimum inhibitory concentration of about 100 μg/mL. The IC_50 % of the two proteins against L. monocytogenes (17 μg/mL) was comparable to penicillin but comparatively much lower (15 μg/mL) than that of penicillin (85 μg/mL) against S. Enteritidis. The two proteins could inhibit the growth of L. monocytogenes and S. Enteritidis by about 97 and 91 %, respectively, after 6–12 h of incubation at 37 °C. The constituting subunits of MSP (methylated 11S and methylated 7S) were both responsible for its antimicrobial action. Transmission electron microscopy of the protein-treated bacteria showed various signs of cellular deformation. The cationic proteins can electrostatically and hydrophobically interact with cell wall and cell membrane, producing large pores, pore channels and cell wall and cell membrane disintegration, engendering higher cell permeability leading finally to cell emptiness, lysis and death.     

Effects of a Standardized Phenolic-Enriched Maple Syrup Extract on β-Amyloid Aggregation,Neuroinflammation in Microglial and Neuronal Cells,and β-Amyloid Induced Neurotoxicity in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Published data supports the neuroprotective effects of several phenolic-containing natural products, including certain fruit, berries, spices, nuts, green tea, and olive oil. However, limited data are available for phenolic-containing plant-derived natural sweeteners including maple syrup. Herein, we investigated the neuroprotective effects of a chemically standardized phenolic-enriched maple syrup extract (MSX) using a combination of biophysical, in vitro, and in vivo studies. Based on biophysical data (Thioflavin T assay, transmission electron microscopy, circular dichroism, dynamic light scattering, and zeta potential), MSX reduced amyloid β_1?42 peptide (Aβ_1?42) fibrillation in a concentration-dependent manner (50–500 μg/mL) with similar effects as the neuroprotective polyphenol, resveratrol, at its highest test concentration (63.5?% at 500 μg/mL vs. 77.3?% at 50 μg/mL, respectively). MSX (100 μg/mL) decreased H₂O₂-induced oxidative stress (16.1?% decrease in ROS levels compared to control), and down-regulated the production of lipopolysaccharide (LPS)-stimulated inflammatory markers (22.1, 19.9, 74.8, and 87.6?% decrease in NOS, IL-6, PGE₂, and TNFα levels, respectively, compared to control) in murine BV-2 microglial cells. Moreover, in a non-contact co-culture cell model, differentiated human SH-SY5Y neuronal cells were exposed to conditioned media from BV-2 cells treated with MSX (100 μg/mL) and LPS or LPS alone. MSX-BV-2 media increased SH-SY5Y cell viability by 13.8?% compared to media collected from LPS-BV-2 treated cells. Also, MSX (10 μg/mL) showed protective effects against Aβ_1?42 induced neurotoxicity and paralysis in Caenorhabditis elegans in vivo. These data support the potential neuroprotective effects of MSX warranting further studies on this natural product.     

In vitro and in silico characterization of angiogenic inhibitors from <Emphasis Type="Italic">Sophora interrupta</Emphasis>

Sophora interrupta Bedd, (Fabaceae) is used in Indian folk medicine to treat cancer. Angiogenesis is one of the crucial characteristics of cancer metastasis and is regulated by vascular endothelial growth factor (VEGF). In this study, we examined the antiangiogenic properties of the root ethyl acetate extract of Sophora interrupta by various methods. In vitro antioxidant activity (100–600 μg/ml) of S. interrupta ethyl acetate (SEA) extract was evaluated by DPPH and ABTS, anti-inflammatory activity (50, 100 and 150 μg/ml) by estimating nitric oxide (NO) levels, anti-angiogenic activity (200 and 500 μg/ml) was validated by chorio allantoic membrane (CAM) assay and in silico molecular dynamic (MD) simulations analyses (25 ns) were performed to identify the anti-angiogenic compounds extracted from root extract. The antioxidative activity of SEA extract at IC₅₀ (200?±?0.6 μg/mL) is equal to that of ascorbic acid at IC₅₀ (50?±?0.6 μg/mL), and the anti-inflammatory activity of SEA extract at IC₅₀ (150?±?0.2 μg/mL) was inhibited significantly by nitric oxide (NO) production. The SEA extract significantly reduced the sprouting of new blood vessels at ID₅₀ 500?±?0.13 μg/mL in the CAM assay. Gas chromatography–mass spectrometry analysis of the SEA extract detected 34 secondary metabolites, of which 6a,12a-dihydro-6H-(1,3)dioxolo(5,6)benzofuro(3,2-c)chromen-3-ol (maackiain) and funiculosin formed strong hydrogen bond interactions with Lys 920, Thr 916 and Cys 919 (2H), as well as Glu 917 of VEGFR2, and these interactions were similar to those of the anti-angiogenic compound axitinib. Significant findings in all the assays performed indicate that SEA extract has potential anti-angiogenic compounds that may interfere with VEGF-induced cancer malignancy.