Betulinic Acid-Brosimine B Hybrid Compound Has a Synergistic Effect with Imatinib in Chronic Myeloid Leukemia Cell Line, Modulating Apoptosis and Autophagy.

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the formation of the BCR-ABL (breakpoint cluster region-Abelson) oncoprotein. As many patients display therapeutic resistance, the development of new drugs based on semisynthetic products represents a new potential therapeutic approach for treating the disease. In this study, we investigated the cytotoxic activity, possible mechanism of action of a hybrid compound of betulinic acid (BA) and brosimine B in CML cell lines that are sensitive (K-562) and resistant (K-562R) to imatinib, in addition to evaluating lower doses of imatinib in combination with the hybrid compound. The effects of the compound, and its combination with imatinib, on apoptosis, cell cycle, autophagy and oxidative stress were determined. The compound was cytotoxic in K-562 (23.57 ± 2.87 µM) and K-562R (25.80 ± 3.21 µM) cells, and a synergistic effect was observed when it was associated with imatinib. Apoptosis was mediated by the caspase 3 and 9 intrinsic pathway, and cell cycle evaluation showed arrest at G0/G1. In addition, the hybrid compound increased the production of reactive oxygen species and induced autophagy by increasing LC3II and Beclin-1 mRNA levels. Results suggest that this hybrid compound causes the death of both imatinib-sensitive and -resistant cell lines and may hold potential as a new anticancer treatment against CML.

Ex vivo potential of a quinoline-derivative nail lacquer as a new alternative for dermatophytic onychomycosis treatment.

Introduction. Onychomycosis infections currently show a significant increase, affecting about 10 % of the world population. Trichophyton rubrum is the main agent responsible for about 80 % of the reported infections. The clinical cure for onychomycosis is extremely difficult and effective new antifungal therapy is needed.Hypothesis/Gap Statement. Ex vivo onychomycosis models using porcine hooves can be an excellent alternative for evaluating the efficacy of new anti-dermatophytic agents in a nail lacquer.Aim. Evaluation of the effectiveness of a nail lacquer containing a quinoline derivative on an ex vivo onychomycosis model using porcine hooves, as well as the proposal of a plausible antifungal mechanism of this derivative against dermatophytic strains.Methodology. The action mechanism of a quinoline derivative was evaluated through the sorbitol protection assay, exogenous ergosterol binding, and the determination of the dose-response curves by time-kill assay. Scanning electron microscopy evaluated the effect of the derivative in the fungal cells. The efficacy of a quinoline-derivative nail lacquer on an ex vivo onychomycosis model using porcine hooves was evaluated as well.Results. The quinoline derivative showed a time-dependent fungicidal effect, demonstrating reduction and damage in the morphology of dermatophytic hyphae. In addition, the ex vivo onychomycosis model was effective in the establishment of infection by T. rubrum.Conclusion. Treatment with the quinoline-derivative lacquer showed a significant inhibitory effect on T. rubrum strain in this infection model. Finally, the compound presents high potential for application in a formulation such as nail lacquer as a possible treatment for dermatophytic onychomycosis.

Remarkable capacity of brosimine b to disrupt methicillin-resistant Staphylococcus aureus (MRSA) preformed biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health concern representing about 60% of S. aureus isolated from hospitalized patients in countries such as USA and Brazil in the last years. Additionally, the ability to adhere to surfaces and the development of biofilms are important properties of pathogenic bacteria involved in medical device-associated infections, and staphylococci are recognized as the major etiologic agents in these situations. The aim of this study is to evaluate three Brosimum acutifolium flavonoids, 4'-hydroxy-7,8(2″,2″-dimethylpyran)flavan (1), brosimine b (2) and 4-hydroxy-lonchocarpin (3), regarding their antibiofilm, antibacterial and antioxidant activities. Flavonoids 1 and 2 were able to reduce S. aureus viability within preformed biofilms in 73% at 50 µM while 2 also reduced biofilm biomass in 48% at 100 µM. Flavonoid 3 was not able to reduce biofilm biomass at assessed concentrations. When tested against methicillin-resistant S. aureus (MRSA) strains, 2 (100 µM) reduced 70%-98% of viable bacteria within 24h-old biofilms. The minimum inhibitory concentration against the methicillin-sensitive Staphylococcus aureus ATCC 25904 was 50 µM for the three compounds. In preliminary assays to evaluate cytotoxicity, 1 was highly hemolytic at concentrations above 50 µM while 2 and 3 did not cause significant hemolysis at 100 µM. The antioxidant activity was observed only in the ethanolic extract and 2. In vivo toxicity evaluations using Galleria mellonella larvae as alternative host model resulted in 83.3% survival for treatment with 1, 76.7% for 2, and 100% for 3 at 500 mg/kg. This study highlights the potential of these flavonoids, especially 2, as antibiofilm agent to control preformed S. aureus biofilms.

Red pepper peptide coatings control Staphylococcus epidermidis adhesion and biofilm formation.

Medical devices (indwelling) have greatly improved healthcare. Nevertheless, infections related to the use of these apparatuses continue to be a major clinical concern. Biofilms form on surfaces after bacterial adhesion, and they function as bacterial reservoirs and as resistance and tolerance factors against antibiotics and the host immune response. Technological strategies to control biofilms and bacterial adhesion, such as the use of surface coatings, are being explored more frequently, and natural peptides may promote their development. In this study, we purified and identified antibiofilm peptides from Capsicum baccatum (red pepper) using chromatography-tandem mass spectrometry, MALDI-MS, MS/MS and bioinformatics. These peptides strongly controlled biofilm formation by Staphylococcus epidermidis, the most prevalent pathogen in device-related infections, without any antibiotic activity. Furthermore, natural peptide-coated surfaces dislayed effective antiadhesive proprieties and showed no cytotoxic effects against different representative human cell lines. Finally, we determined the lead peptide predicted by Mascot and identified CSP37, which may be useful as a prime structure for the design of new antibiofilm agents. Together, these results shed light on natural Capsicum peptides as a possible antiadhesive coat to prevent medical device colonization.

Triterpene Derivatives as Relevant Scaffold for New Antibiofilm Drugs.

New medicines for the treatment of bacterial biofilm formation are required. For thisreason, this study shows the in vitro activity of betulinic acid (BA), ursolic acid (UA) and their twentyderivatives against planktonic and biofilm cells (gram-positive bacterial pathogens: Enterococcusfaecalis, Staphylococcus aureus and Staphylococcus epidermidis). We evaluated the antibiofilm activity(through the crystal violet method), as well as the antibacterial activity via absorbance (OD600) atconcentrations of 5, 25 and 100 µM. Likewise, the cytotoxicity of all compounds was evaluated on akidney African green monkey (VERO) cell line at the same concentration, by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) methodology. We verified for the first timewhether different groups at carbon 3 (C-3) of triterpenes may interfere in the antibiofilm activity withminimal or no antibacterial effect. After the screening of 22 compounds at three distinctconcentrations, we found antibiofilm activity for eight distinct derivatives without antibiotic effect.In particular, the derivative 2f, with an isopentanoyl ester at position C-3, was an antibiofilm activityagainst S. aureus without any effect upon mammalian cells.

In vitro and in silico Activity of Iridoids Against Leishmania amazonensis.

BACKGROUND: Leishmaniasis reaches millions of people around the world. The control of the disease is difficult due to the restricted access to the diagnosis and medication, and low adherence to the treatment. Thus, more efficient drugs are needed and natural products are good alternatives. Iridoids, natural products with reported leishmanicidal activity, can be exploited for the development of anti- Leishmania drugs. The aim of this study was to isolate and to investigate the in vitro activity of iridoids against Leishmania amazonensis and to compare the activity in silico of these compounds with those reported as active against this parasite. METHODS: Iridoids were isolated by chromatographic methods. The in vitro activity of asperuloside (1) and geniposide (2) from Escalonia bifida, galiridoside (3) from Angelonia integerrima and theveridoside (4) and ipolamiide (5) from Amphilophium crucigerum was investigated against promastigote forms of Leishmania amazonensis. Molecular modeling studies of 1-5 and iridoids cited as active against Leishmania spp. were performed. RESULTS: Compounds 1-5 (5-100 µM) did not inhibit the parasite survival. Physicochemical parameters predicted for 1-5 did not show differences compared to those described in literature. The SAR and the pharmacophoric model confirmed the importance of maintaining the cyclopentane[C]pyran ring of the iridoid, of oxygen-linked substituents at the C1 and C6 positions and of bulky substituents attached to the iridoid ring to present leishmanicidal activity. CONCLUSION: The results obtained in this study indicate that iridoids are a promising group of secondary metabolites and should be further investigated in the search for new anti-Leishmania drugs.

Antileishmanial in vitro activity of essential oil from Myrciaria plinioides, a native species from Southern Brazil

In South American folk medicine members of the genus Myrciaria are used for the treatment of malaria, diarrhoea, asthma, inflammation and post-partum uterine cleansing. The aim of this work was to evaluate its antileishmanial properties (in vitro) of essential oil derived from leaves of Myrciaria plinioides D. Legrand, a plant species that is native in South of Brazil. The essential oil was obtained by hydro-distillation using fresh leaves of M. plinioides. The chemical composition of this essential oil (MPEO, M. plinioides essential oil) was determined by gas chromatography coupled to mass spectrometry (GC-MS). MPEO was assayed in vitro for antileishmanial properties against promastigotes of Leishmania amazonensis and Leishmania infantum, and for cytotoxicity against murine peritoneal macrophages. The MPEO comprised 66 components and was rich in oxygenated sesquiterpenes (82.66%) containing spathulenol (21.12%) as its major constituent. The MPEO was effective against L. amazonensis with IC50 value of 14.16 ± 7.40 µg/mL, while against L. infantum the IC50 value was higher with 101.50 ± 5.78 µg/mL. The MPEO showed significant activity against L. amazonensis, and presented a selectivity index (SI) of 6.60. The results suggest that the essential oil from leaves of M. plinioides is a promising source for new antileishmanial agents against L. amazonensis.

Promising Antibiofilm Activity of Peptidomimetics.

Pathogenic biofilms are a global health care concern, as they can cause extensive antibiotic resistance, morbidity, mortality, and thereby substantial economic loss. Scientific efforts have been made over the past few decades, but so far there is no effective treatment targeting the bacteria in biofilms. Antimicrobial peptidomimetics have been proposed as promising potential anti-biofilm agents. Indeed, these structurally enhanced molecules can mimic the action of peptides but are not susceptible to proteolysis or immunogenicity, the characteristic limitations of natural peptides. Here, we provide insights into antibiofilm peptidomimetic strategies and molecular targets, and discuss the design of two major peptidomimetics classes: AApeptides (N-acylated-N-aminoethyl-substituted peptides) and peptoids (N-substituted glycine units). In particular, we present details of their structural diversity and discuss the possible improvements that can be implemented in order to develop antibiofilm drug alternatives.

Anti-Trichomonas vaginalis activity of ursolic acid derivative: a promising alternative.

Trichomonas vaginalis is an extracellular parasite that binds to the epithelium of the human urogenital tract and causes the sexually transmitted infection, trichomoniasis. In view of increased resistance to drugs belonging to the 5-nitroimidazole class, new treatment alternatives are urgently needed. In this study, eight semisynthetized triterpene derivatives were evaluated for in vitro anti-T. vaginalis activity. Ursolic acid and its derivative, 3-oxime-urs-12-en-28-oic-ursolic acid (9), presented the best anti-T. vaginalis activity when compared to other derivatives, with minimum inhibitory concentration (MIC) at 25 µM. Moreover, 9 was active against several T. vaginalis fresh clinical isolates. Hemolysis assay demonstrated that 9 presented a low hemolytic effect. Importantly, 25 µM 9 was not cytotoxic against the Vero cell lineage. Finally, we demonstrated that compound 9 acts synergistically with metronidazole against a T. vaginalis metronidazole-resistant isolate. This report reveals the high potential of the triterpenoid derivative 9 as trichomonicidal agent.

Peptides as a strategy against biofilm-forming microorganisms: Structure-activity relationship perspectives.

Biofilm forming microorganisms substantially enhance their virulence and drug resistance causing and alternatives are need to combat this health problem. In this context, peptides are an exceptional strategy in drug design and pharmaceutical innovation due to their diverse chemical features, biological activity and biotechnological relevance. Therefore, this study proposes a comprehensive assessment of a wide range of peptides, targeting biofilms. It provides chemical and molecular information and a Structural Activity Relationship perspective in order to delineate minimal requirements for antibiofilm activity and contributing to the development of new antibiofilm agents. In light of this, it was possible to propose a peptide design model (X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20) to be tested in the war against resistant microorganisms.

Adenosine reduces reactive oxygen species and interleukin-8 production by Trichomonas vaginalis-stimulated neutrophils.

Trichomonas vaginalis is a flagellated protozoan that affects the human urogenital tract causing 276.4 million new infections a year. The parasite elicits a vaginal mucosal infiltration of immune cells, especially neutrophils which are considered to be primarily responsible for cytological change observed at the infection site as well as the major contributor in the inflammatory response against the parasite. Extracellular nucleotides and their nucleosides are signaling compounds involved in several biological processes, including inflammation and immune responses. Once in the extracellular space, the nucleotides and nucleosides can directly activate the purinergic receptors. Herein, we investigated the involvement of purinergic signaling on the production of reactive oxygen species (ROS) and cytokines by T. vaginalis-stimulated neutrophils. Parasites were able to induce an increase in ROS and IL-8 levels while they did not promote IL-6 secretion or neutrophil elastase activity. Adenine and guanine nucleotides or nucleosides were not able to modulate ROS and cytokine production; however, when T. vaginalis-stimulated neutrophils were incubated with adenosine and adenosine deaminase inhibitor, the levels of ROS and IL-8 were significantly reduced. These immunosuppressive effects were probably a response to the higher bioavailability of adenosine found in the supernatant as result of inhibition of enzyme activity. The involvement of P1 receptors was investigated by immunofluorescence and A1 receptor was the most abundant. Our data show that the influence of purinergic signaling, specifically those effects associated with adenosine accumulation, on the modulation of production of proinflammatory mediators by T. vaginalis-stimulated neutrophils contribute to the understanding of immunological aspects of trichomoniasis.

Standardized Passiflora incarnata L. Extract Reverts the Analgesia Induced by Alcohol Withdrawal in Rats.

Passiflora incarnata L. (Passifloraceae) has been traditionally used for treatment of anxiety, insomnia, drug addiction, mild infections, and pain. The aim of this study was to investigate the effect of a commercial extract of P. incarnata in the analgesia induced by alcohol withdrawal syndrome in rats. In addition, brain-derived neurotrophic factor and interleukin-10 levels were evaluated in prefrontal cortex, brainstem, and hippocampus. Male adult rats received by oral gavage: (1: water group) water for 19 days, 1 day interval and water (8 days); (2: P. incarnata group) water for 19 days, 1 day interval and P. incarnata 200 mg/kg (8 days); (3: alcohol withdrawal group) alcohol for 19 days, 1 day interval and water (8 days); and (4: P. incarnata in alcohol withdrawal) alcohol for 19 days, 1 day interval and P. incarnata 200 mg/kg (8 days). The tail-flick and hot plate tests were used as nociceptive response measures. Confirming previous study of our group, it was showed that alcohol-treated groups presented an increase in the nociceptive thresholds after alcohol withdrawal, which was reverted by P. incarnata, measured by the hot plate test. Besides, alcohol treatment increased brain-derived neurotrophic factor and interleukin-10 levels in prefrontal cortex, which was not reverted by P. incarnata. Considering these results, the P. incarnata treatment might be a potential therapy in the alcohol withdrawal syndrome. Copyright © 2017 John Wiley & Sons, Ltd.

Caatinga plants: Natural and semi-synthetic compounds potentially active against Trichomonas vaginalis.

Trichomonas vaginalis causes trichomoniasis; the most common but overlooked non-viral sexually transmitted disease worldwide. The treatment is based at 5'-nitroimidazoles, however, failure are related to resistance of T. vaginalis to chemotherapy. Caatinga is a uniquely Brazilian region representing a biome with type desert vegetation and plants present diverse biological activity, however, with few studies. The aim of this study was to investigate the activity against T. vaginalis of different plants from Caatinga and identify the compounds responsible by the activity. A bioguided fractionation of Manilkara rufula was performed and four major compounds were identified: caproate of α-amyrin (1b), acetate of ß-amyrin (2a), caproate of ß-amyrin (2b), and acetate of lupeol (3a). In addition, six derivatives of α-amyrin (1), ß-amyrin (2) and lupeol (3) were synthesized and tested against the parasite. Ursolic acid (5) reduced about 98% of parasite viability after 2h of incubation and drastic ultrastructural alterations were observed by scanning electron microscopy. Moreover, 5 presented high cytotoxicity to HMVII and HeLa cell line and low cytotoxicity against Vero line at 50 µM (MIC against the parasite). Metronidazole effect against T. vaginalis resistant isolate was improved when in association with 5.

Anti-Trichomonas vaginalis activity from triterpenoid derivatives.

Trichomonas vaginalis is a flagellated parasite that causes trichomonosis, the most common non-viral sexually transmitted disease (STD) in the world. Worryingly, trichomonosis is associated to increased transmission of HIV. Due to high frequency of the infection during pregnancy and the development of metronidazole-resistant isolates, therapeutic alternatives to 5-nitroimidazole are being searched. Triterpenes are natural products presenting several biological activities such as anti-protozoal activity. The aim of this study was to evaluate the in vitro anti-T. vaginalis activity from betulinic and ursolic acids, as well as semisynthetic derivatives obtained. Compounds obtained from betulinic acid presented better activity than those from ursolic acid. Piperazine derivatived from betulinic acid presented minimum inhibitory concentration (MIC) value of 91.2 µM, and the kinetic growth curve performed with parasites treated with this most active compound revealed complete inhibition of trophozoite proliferation at 2 h of incubation and total abolition of trophozoite growth in 24 h, revealing that the piperazine derivative is an efficient trichomonacidal molecule. The same compound promoted total erythrocyte lysis and lactate dehydrogenase (LDH) liberation of 83 and 100% (at 45.6 and 91.2 µM, respectively), indicating parasite membrane damage. The piperazine derivative demonstrated cytotoxic effect against the HMVII and HeLa cell lineages at the MIC value. This is the first report of semisynthetic triterpenoid derivatives with anti-T. vaginalis activity, revealing the high potential of these compounds as trichomonacidal agents.

Antithrombotic effect of chikusetsusaponin IVa isolated from Ilex paraguariensis (Maté).

The triterpene chikusetsusaponin IVa was isolated from the fruit of Ilex paraguariensis. Using biochemical and pharmacological methods, we demonstrated that chikusetsusaponin IVa (1) prolongs the recalcification time, prothrombin time, activated partial thromboplastin time, and thrombin time of normal human plasma in a dose-dependent manner, (2) inhibits the amidolytic activity of thrombin and factor Xa upon synthetic substrates S2238 and S2222, (3) inhibits thrombin-induced fibrinogen clotting (50% inhibition concentration, 199.4 ± 9.1 µM), and (4) inhibits thrombin- and collagen-induced platelet aggregation. The results also indicate that chikusetsusaponin IVa preferentially inhibits thrombin in a competitive manner (K(i)=219.6 µM). Furthermore, when administered intravenously to rats, chikusetsusaponin IVa inhibited thrombus formation in a stasis model of venous thrombosis, although it did not induce a significant bleeding effect. Chikusetsusaponin IVa also prolonged the ex vivo activated partial thromboplastin time. Altogether, these data suggest that chikusetsusaponin IVa exerts antithrombotic effects, including minor hemorrhagic events. This appears to be important for the development of new therapeutic agents.

Phenolic compounds from maté (Ilex paraguariensis) inhibit adipogenesis in 3T3-L1 preadipocytes.

Leaves of Ilex paraguariensis are used to prepare a tea known as maté which is a common beverage in several South American countries. The ethanol extract was fractionated to identify the compounds responsible for the anti-adipogenic activity in 3T3-L1 cells. Extracts of both fresh and dried maté leaves were subjected to column chromatography using molecular permeation to obtain the saponin (20 % yields) and the polyphenol extracts (40 % yields) from the fresh and dried leaves. The phenolic content was determined using high-performance liquid chromatography analysis and the Folin-Ciocalteau method. Also, maté extracts (50 µg/ml to 1,000 µg/ml) did not display citotoxicity using MTT. The polyphenol extract from the dried leaves was the most effective (50 µg/ml) in the inhibition of triglyceride accumulation in 3T3-L1 adipocytes, and rutin (100 µg/ml) likely accounted for a large portion of this activity. Additionally, maté extracts had a modulatory effect on the expression of genes related to the adipogenesis as PPARγ2, leptin, TNF-α and C/EBPα.

Anti-Trichomonas vaginalis activity of saponins from Quillaja, Passiflora, and Ilex species.

Trichomonas vaginalis is a flagellated protozoan that causes trichomonosis, the most prevalent non-viral STD worldwide. The pathogen has been associated with serious health consequences including predisposition to cervical cancer and adverse pregnancy outcomes and infertility. It also acts as a co-factor in HIV transmission and acquisition. The 5-nitroimidazole drugs are used in the treatment, however, treatment noncompliance is observed, and a growing number of T. vaginalis isolates resistant to the drugs have been related. Saponins are natural products possessing many biological activities such as antiprotozoan activity. The aim of this study was to evaluate the anti-T. vaginalis activity of saponins from Quillaja, Passiflora, and Ilex species. Saponins from Passiflora alata and Quillaja saponaria presented the best anti-T. vaginalis activity (MIC = 0.025%). In addition, all samples induced erythrocyte lysis and LDH release. As far as we know, this is the first report demonstrating the potential anti-T. vaginalis activity of these saponins.