A RP-HPLC-DAD method for analysis of Brazilian southeast brown propolis and its leishmanicidal properties.

Propolis is a natural product of great economic and pharmacological importance. The flora surrounding the bee communities is a determining factor in the composition of propolis and therefore in its biological and medicinal properties. Brown propolis is one of the most important types of propolis in Brazil, produced in the southeastern region. The ethanolic extract of a brown propolis sample from Minas Gerais state was chemically characterized for the subsequent development of a RP-HPLC method, validated according to the standards of regulatory agencies. The leishmanicidal activity of this extract was assessed. The brown propolis was characterized by the presence of chemical markers reported on green propolis such as ferulic acid, coumaric acid, caffeic acid, cinnamic acid, baccharin, artepillin and drupanin, indicating a probable origin on Baccharis dracunculifolia. The developed method agreed with the parameters established by the validation guidelines, then proved to be reliable for the analysis of this type of propolis. The brown propolis displayed significant activity against Leishmania amazonensis with IC50 values of 1.8 and 2.4 µg/ml against the promastigote and amastigote forms, respectively. The studied propolis exhibited promising evidence for use as a natural source against L. amazonensis.

Brazilian southeast brown propolis: gas chromatography method development for its volatile oil analysis, its antimicrobial and leishmanicidal activities evaluation.

INTRODUCTION: Propolis is widely used in folk medicine, and many factors can affect its chemical composition, including abiotic factors that can influence plants and bees. Therefore, analytical methods are powerful techniques in the quality control of such products. OBJECTIVE: Develop and validate an analytical method for quantifying volatile compounds in Brazilian brown propolis, and evaluate its biological activities. METHODS: A gas chromatography flame ionisation detector (GC-FID) analytical method was validated, attending the parameters of international validation guidelines as ANVISA 2017 and ICH 2005, for quantification of compounds present in volatile oils from propolis. Evaluation of cytotoxic, antimicrobial, and leishmanicidal activities of the oil. RESULTS: The compounds 1,8-cineole, terpinen-4-ol, α-copaene, ß-caryophyllene, γ-muurolene, nerolidol, spathulenol, and γ-palmitolactone were isolated from the volatile fraction of a Brazilian brown propolis and used in the method validation. All the validation parameters of the method were satisfactory. The volatile fraction displayed a significant leishmanicidal activity, with half maximal inhibition concentration (IC50 ) = 21.3 µg/mL against amastigote forms and IC50 = 25.1 µg/mL against promastigote forms of Leishmania amazonensis. The oil also displayed an antibacterial effect by inhibiting the growth of Streptococcus mutans and Staphylococcus aureus at 25 µg/mL and 50 µg/mL, respectively, but it was not cytotoxic against AGP-01, He-La and CHO-K1cell lines, with IC50 > 100 µg/mL. CONCLUSION: The GC-FID method can be a useful tool in the quality control of propolis material. The southeast brown propolis showed a high chemical complexity in its volatile fraction, which displayed leishmanicidal activity and bactericidal activity.

Inhibition of 19S proteasome deubiquitinating activity in Schistosoma mansoni affects viability, oviposition, and structural changes.

The proteasome is the key player in the cellular protein degradation machinery and is pivotal for protein homeostasis and Schistosoma mansoni (S. mansoni) survival. Our group study provides insights into proteasome inhibitors and reveals that selective schistosomiasis agents represent an interesting branch of proteasome research linked to the development of new drugs for this neglected disease. Here, we explored the phenotypic response of S. mansoni to b-AP15, a bis-benzylidine piperidone that inhibits 26S proteasome deubiquitinases (DUBs), ubiquitin-specific protease 14 (USP14), and ubiquitin carboxyl-terminal hydrolase 5 (UCHL5). b-AP15 induces a modest decrease in egg production in vitro and reduces viability, leading to the death of parasite couples. This inhibitor also induces a twofold increase in the accumulation of polyubiquitinated proteins in S. mansoni adult worms and causes tegument changes such as disintegration, wrinkling, and bubble formation, both throughout the length of the parasite and in the oral sucker. b-AP15 alters the cell organelles of adult S. mansoni worms, and we specifically observed mitochondrial alterations, which are suggestive of proteotoxic stress leading to autophagy. Taken together, these results indicate that the deubiquitinase function of the proteasome is essential for the parasite and support the hypothesis that the proteasome constitutes an interesting drug target for the treatment of schistosomiasis.

18-Des-hydroxy Cytochalasin: an antiparasitic compound of Diaporthe phaseolorum-92C, an endophytic fungus isolated from Combretum lanceolatum Pohl ex Eichler.

Chemical investigation of the ethyl acetate extract from the endophytic fungus Diaporthe phaseolorum-92C (92C) isolated from the roots of Combretum lanceolatum led to the isolation of 18-des-hydroxy Cytochalasin H (compound 1). The trypanocidal and schistosomicidal activity and cytotoxicity of the extract from 92C were evaluated. The schistosomicidal, leishmanicidal, antimicrobial, and antioxidant actions, as well as the antitumor activity against the breast cancer cells MDA-MB-231 and MCF-7, and the cytotoxicity towards normal human lung fibroblasts GM07492A of compound 1 was tested. The extract from 92C (20 µg/mL) exerted potent trypanocidal activity, reducing 82% of the number of amastigotes and trypomastigotes of Trypanosoma cruzi. Compound 1 at 50 µg/mL killed 50% of Schistosoma mansoni adult worms. Compound 1 reduced the viability of Leishmania amazonenses promastigotes (IC50 = 9.2 µg/mL) and of the cancer cells MDA-MB-231 and MCF-7 (IC50 = 17.5 and 8.88 µg/mL, respectively), presented moderate antioxidant activity, and gave IC50 of 2049.7 ± 39.9 µg/mL for the cytotoxicity towards normal cells GM07492A. This knowledge is highly relevant to the search for new promising compounds for therapeutic purposes.

Bioactive compounds of Aspergillus terreus-F7, an endophytic fungus from Hyptis suaveolens (L.) Poit.

The compounds terrein (1), butyrolactone I (2), and butyrolactone V (3) were isolated from the ethyl acetate extract (EtOAc) of the endophytic fungus Aspergillus terreus-F7 obtained from Hyptis suaveolens (L.) Poit. The extract and the compounds presented schistosomicidal activity against Schistosoma mansoni; at 100 µg/mL for EtOAc extract, 1297.3 µM for compound 1, 235.6 µM for compound 2, and 454.1 µM for compound 3, they killed 100% of the parasites after 72 h of treatment. Compounds 1, 2, and 3 exerted moderate leishmanicidal activity against Leishmania amazonensis (IC50 ranged from 23.7 to 78.6 µM). At 235.6 and 227.0 µM, compounds 2 and 3, respectively, scavenged 95.92 and 95.12% of the DPPH radical (2,2-diphenyl-1-picryl-hydrazyl), respectively. Regarding the cytotoxicity against the breast tumor cell lines MDA-MB-231 and MCF-7, compound 2 gave IC50 of 34.4 and 17.4 µM, respectively, while compound 3 afforded IC50 of 22.2 and 31.9 µM, respectively. At 117.6 µM, compound 2 inhibited the growth of and killed the pathogen Escherichia coli (ATCC 25922). Compounds 1, 2, and 3 displayed low toxicity against the normal line of human lung fibroblasts (GM07492A cells), with IC50 of 15.3 × 103, 3.4 × 103, and 5.8 × 103 µM, respectively. This is the first report on (i) the in vitro schistosomicidal and leishmanicidal activities of the EtOAc extract of A. terreus-F7 and compounds 1, 2, and 3; and (ii) the antitumor activity of compounds 2 and 3 against MDA-MB-231 and MCF-7 cells.

Proteasome stress responses in Schistosoma mansoni.

The proteasome proteolytic system is the major ATP-dependent protease in eukaryotic cells responsible for intracellular protein turnover. Schistosoma mansoni has been reported to contain an ubiquitin-proteasome proteolytic pathway, and many studies have suggested a biological role of proteasomes in the development of this parasite. Additionally, evidence has suggested diversity in proteasome composition under several cellular conditions, and this might contribute to the regulation of its function in this parasite. The proteasomal system has been considered important to support the protein homeostasis during cellular stress. In this study, we described in vitro effects of oxidative stress, heat shock, and chemical stress on S. mansoni adults. Our findings showed that chemical stress induced with curcumin, IBMX, and MG132 modified the gene expression of the proteasomal enzymes SmHul5 and SmUbp6. Likewise, the expression of these genes was upregulated during oxidative stress and heat shock. Analyses of the S. mansoni life cycle showed differential gene expression in sporocysts, schistosomulae, and miracidia. These results suggested that proteasome accessory proteins participate in stress response during the parasite development. The expression level of SmHul5 and SmUbp6 was decreased by 16-fold and 9-fold, respectively, by the chemical stress induced with IBMX, which suggests proteasome disassembly. On the other hand, curcumin, MG132, oxidative stress, and heat shock increased the expression of these genes. Furthermore, the gene expression of maturation proteasome protein (SmPOMP) was increased in stress conditions induced by curcumin, MG132, and H2O2, which could be related to the synthesis of new proteasomes. S. mansoni adult worms were found to utilize similar mechanisms to respond to different conditions of stress. Our results demonstrated that oxidative stress, heat shock, and chemical stress modified the expression profile of genes related to the ubiquitin-proteasome system and suggested that the proteasome might be important in the cellular stress response in this parasite.

Structurally modified natural sesquiterpene lactones constitute effective and less toxic schistosomicidal compounds.

Sesquiterpene lactones are known to be active, but are also known to present high cytotoxicity. In the present work an evaluation of how slight structural alterations affect the cytotoxicity and the schistosomicidal activity of sesquiterpene lactones was undertaken. More specifically, we assessed the activity of budlein-A, a furanoheliangolide sesquiterpene lactone, and four of its derivatives. The structural modifications of budlein-A, presented in this work, diminished the cytotoxicity and changed the antiparasitary behavior of the molecule. They also provided data for a better understanding of the sesquiterpene lactone cytotoxicity. The establishment of the structures of three synthesized sesquiterpene lactones on the basis of NMR and HRESIMS data is also presented here. Complete and detailed (1)H and (13)C 1D and 2D NMR data, with measurements of all J values and all multiplicities clarified, are presented for five sesquiterpene lactones for the first time.

Biochemical characterization and role of the proteasome in the oxidative stress response of adult Schistosoma mansoni worms.

The trematode Schistosoma mansoni, an important parasite of humans, is the principle agent of the disease schistosomiasis. In the human host, one of the most important stress factors of this parasite is the oxidative stress generated by both the metabolism of the worm and the immune system of the host. The proteasomal system is responsible for protein homeostasis during oxidative stress. The 26S proteasome is a multicatalytic protease formed by two compartments, a 20S core and regulatory particle 19S, and controls the degradation of intracellular proteins, hence regulating many cellular processes. In the present report, we describe the biochemical characterization and role of the 20S proteasome in the response of adult S. mansoni worms exposed to hydrogen peroxide. Characterization of the response to the oxidative stress included the evaluation of viability, egg production, mortality, tegument integrity, and both expression and activity of proteasome. We observed decreases in viability, egg production as well as 100% mortality at the higher concentrations of hydrogen peroxide tested. The main changes observed in the tegument of adult worms were peeling as well as the appearance of bubbles and a decrease of spines on the tubercles. Furthermore, there were increases in 26S activity to the same extent as 20S proteasome activity, although there was increase of 20S proteasome content, suggesting that degradation of protein oxidized in adult worms is due to the 20S proteasome. It was demonstrated that adult S. mansoni worms are sensitive to oxidative stress, and that a variety of processes in this parasite are altered under this condition. The work contributes to a better understanding of the mechanisms employed by S. mansoni to survive under oxidative stress.

Evaluation of the in vitro schistosomicidal, leishmanicidal, and trypanocidal activities of the capsaicin metabolite, Capsicum frutescens, and Capsicum baccatum extracts and of their analysis of the main constituents by HPLC/UV and CG/MS.

Neglected tropical diseases are significant causes of death and temporary or permanent disability for millions living in developing countries. Unfortunately, there is no effective treatment for these diseases. Thus, this work aimed to conduct a chemical analysis using HPLC/UV and GC/MS to identify the major constituents of the hydroalcoholic extracts of Capsicum frutescens and Capsicum baccatum fruits, evaluating these extracts and their constituents' schistosomicidal, leishmanicidal and trypanocidal activities. The results obtained for the extracts of C. frutescens are better when compared to those obtained for C. baccatum, which can be related to the different concentrations of capsaicin (1) present in the extracts. The lysis of trypomastigote forms results for capsaicin (1) led to a significant value of IC50 = 6.23 µM. Thus, the results point to capsaicin (1) as a possible active constituent in these extracts.

The lignan (-)-hinokinin displays modulatory effects on human monoamine and GABA transporter activities.

The neurotransmitter transporters of the SLC6 family play critical roles in the regulation of neurotransmission and are the primary targets of therapeutic agents used to treat clinical disorders involving compromised neurotransmitter signaling. The dopamine and norepinephrine transporters have been implicated in clinical disorders such as attention deficit hyperactivity disorder (ADHD) and substance abuse. The GABA transporters (GATs) serve as a target for anxiolytic, antidepressant, and antiepileptic therapies. In this work, the interaction with neurotransmitter transporters was characterized for a derivative of the lignan (-)-cubebin (1), namely, (-)-hinokinin (2). Using in vitro pharmacological assays, 2 selectively inhibited the human dopamine and norepinephrine transporters, in a noncompetitive manner possibly mediated by binding to a novel site within the transporters, and displayed low affinity for the serotonin transporter. Compound 2 also specifically inhibited the GAT-1 GABA transporter subtype. Compound 2 is not a substrate of the carriers as it had no effect on the efflux of either of the neurotransmitters investigated. This compound is inactive toward glutamate and glycine transporters. These results suggest that 2 may serve as a tool to develop new therapeutic drugs for ADHD and anxiety that target the DAT, NET, and GAT-1 transporters.

In vitro antiparasitic activity and chemical composition of the essential oil obtained from the fruits of Piper cubeba.

Protozoans of the trypanosomatid family cause the neglected tropical diseases leishmaniasis and trypanosomiasis, for which few drugs are available. In this context our group has recently reported that the essential oil obtained by steam distillation of the fruits of Piper cubeba is active against Schistosoma mansoni. Therefore, we have investigated the in vitro effects of the essential oil against the trypomastigote and amastigote forms of Trypanosoma cruzi isolated from an LLCMK2 cell line culture and the promastigote forms of Leishmania amazonensis. The in vitro activity of the essential oil against trypomastigotes of T. cruzi increased upon rising concentrations, giving IC50 values of 45.5 and 87.9 µg ·â€ŠmL⁻¹ against trypomastigotes and amastigotes, respectively. The essential oil was not active against L. amazonensis, since it displayed lyses of only 24 % at 400 µg ·â€ŠmL⁻¹, and an IC50 of 326.5 µg ·â€ŠmL⁻¹. Therefore, the essential oil should be further investigated to determine the compounds responsible for the observed activities, as well as its mechanism of action.

Immunomodulatory effect of the alkaloidic extract of Solanum lycocarpum fruits in mice infected with Schistosoma mansoni.

Schistosomiasis is a chronic disease caused by trematode flatworms of the genus Schistosoma; it accounts for more than 280,000 deaths annually. In this work we investigated the effect of the alkaloidic extract obtained by acid-base extraction of the dried fruits of Solanum lycocarpum on schistosomiasis. We used this extract at concentrations of 10, 20, and 40 mg/kg to treat mice infected with Schistosoma mansoni in different phases of the parasite cycle, and we compared its effect with that of the positive control praziquantel (60 mg/kg). We evaluated the results on the basis of the number of macrophages, eggs, and granulomas; we also assessed nitric oxide (NO) and interferon-gamma (IFN-γ) production. Animals treated with a daily dose of 10 or 20 mg/kg alkaloidic extract between the 37th and 41st day of infection showed increased number of macrophages, elevated NO and IFN-γ concentrations, and reduced number of eggs and granulomas in the liver. The alkaloidic extract of S. lycocarpum fruits displayed an immunomodulatory effect on mice infected with S. mansoni, so its potential to treat schistosomiasis deserves further studies.

Leishmanial activity of Brazilian brown propolis and its diterpenes.

Propolis is a natural product widely used in folk medicine. Among its various applications, its antiparasitic properties stand out. Due to its great biodiversity, Brazil is a major producer of several types of propolis. This study proposes to evaluate the leishmanicidal properties of the hydroalcoholic extract of propolis collected in the southern region of Brazil (Brown propolis - HEBP) and its main isolated compounds: abietic acid (1), 13-epi-cupressic acid (2), 13-epi-torulosol (3), dehydroabietic acid (4), cis-communic acid (5) and ent-agatic acid (6). In general, the diterpenes did not show activity against the promastigotes of Leishmania (Leishmania) amazonensis at the evaluated concentrations. However, the HEBP was very active with an inhibition concentration of 50% at 8.32 µg/mL. Moreover, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) assays showed morphological and structural alterations in promastigote forms of L. (L.) amazonensis when incubated with HEBP.

HPLC method for quantifying verbascoside in Stizophyllum perforatum and assessment of verbascoside acute toxicity and antileishmanial activity.

We report the chemical composition of the crude leaf extracts obtained from Stizophyllum perforatum (Cham.) Miers (Bignoniaceae), a simple high-performance liquid chromatography-diode array detection (HPLC-DAD) method based on mangiferin as an internal standard to quantify verbascoside, and the verbascoside acute oral toxicity and antileishmanial activity. HPLC-high-resolution mass spectrometry-DAD (HPLC-HRMS-DAD) analyses of the crude ethanol S. perforatum leaf extracts (CE-1 and CE-2) revealed that verbascoside was the major constituent in both extracts. CE-1 was purified, and verbascoside and casticin, among other compounds, were isolated. The developed HPLC-DAD method was validated and met the required standards. Investigation of the CE-2 acute toxicity indicated a lethal dose (LD50) greater than 2,000 mg/kg of body weight. Both CE-1 and CE-2 exhibited antileishmanial activity. The isolated compounds, verbascoside and casticin, also displayed antileishmanial activity with effective concentrations (IC50) of 6.23 and 24.20 µM against promastigote forms and 3.71 and 18.97 µM against amastigote forms of Leishmania amazonensis, respectively, but they were not cytotoxic to J774A.1 macrophages. Scanning electron microscopy of the L. amazonensis promastigotes showed that the parasites became more rounded and that their plasma membrane was altered in the presence of verbascoside. Additionally, transmission electron microscopy demonstrated that vacuoles emerged, lipids accumulated, kinetoplast size increased, and interstitial extravasation occurred in L. amazonensis promastigotes exposed to verbascoside. These findings suggest that S. perforatum is a promising candidate for further in vivo investigations against L. amazonensis.

In vitro schistosomicidal activity of some brazilian cerrado species and their isolated compounds.

Miconia langsdorffii Cogn. (Melastomataceae), Roupala montana Aubl. (Proteaceae), Struthanthus syringifolius (Mart.) (Loranthaceae), and Schefflera vinosa (Cham. & Schltdl.) Frodin (Araliaceae) are plant species from the Brazilian Cerrado whose schistosomicidal potential has not yet been described. The crude extracts, fractions, the triterpenes betulin, oleanolic acid, ursolic acid and the flavonoids quercetin 3-O-ß-D-rhamnoside, quercetin 3-O-ß-D-glucoside, quercetin 3-O-ß-D-glucopyranosyl-(1-2)-α-L-rhamnopyranoside and isorhamnetin 3-O-ß-D-glucopyranosyl-(1-2)-α-L-rhamnopyranoside were evaluated in vitro against Schistosoma mansoni adult worms and the bioactive n-hexane fractions of the mentioned species were also analyzed by GC-MS. Betulin was able to cause worm death percentage values of 25% after 120 h (at 100 µM), and 25% and 50% after 24 and 120 h (at 200 µM), respectively; besides the flavonoid quercetin 3-O-ß-D-rhamnoside promoted 25% of death of the parasites at 100 µM. Farther the flavonoids quercetin 3-O-ß-D-glucoside and quercetin 3-O-ß-D-rhamnoside at 100 µM exhibited significantly reduction in motor activity, 75% and 87.5%, respectively. Biological results indicated that crude extracts of R. montana, S. vinosa, and M. langsdorffii and some n-hexane and EtOAc fractions of this species were able to induce worm death to some extent. The results suggest that lupane-type triterpenes and flavonoid monoglycosides should be considered for further antiparasites studies.

Evaluation of the schistosomicidal activity of the steroidal alkaloids from Solanum lycocarpum fruits.

Solanum lycocarpum (Solanaceae), a Brazilian medicinal plant known as "wolf fruit," contains about 1.5% of glycoalkaloids in its dried fruits, consisting mainly of solamargine and solasonine. The present work reports the obtainment of the alkaloidic extract of the S. lycocarpum fruit by acid-base extraction and the isolation of the major alkaloid heterosides by chromatographic means, as well as the evaluation of their in vitro schistosomicidal activities. The in vitro schistosomicidal activities of the alkaloidic extract of S. lycocarpum fruits and its isolated steroidal alkaloids were undertaken against adult worms of Schistosoma mansoni. The alkaloidic extract (20, 32, and 50 µg mL(-1)), solasonine (50 µM), solamargine (32 and 50 µM), and equimolar mixture of glycoalkaloids (20, 32, and 50 µM) lead to the separation of all couple worms and extensive disruption on their teguments, such as sloughing, as well as their deaths within 24 h of incubation. In addition, the alkaloidic extract (10 and 15 µg mL(-1)), solasonine (50 µM), solamargine (10, 15, and 20 µM), and equimolar mixtures of glycoalkaloids (10 and 15 µM) reduced the development of eggs produced by the adult worms. Solamargine, containing the sugar chain moiety chacotriose, was more active than the solasonine, which contains solatriose sugar chain moiety. A synergistic effect was also observed for a mixture of solamargine and solasonine. Therefore, the alkaloidic extract of S. lycocarpum, and its major components, solamargine and solasonine, showed promising schistosomicidal activity.

Schistosomicidal evaluation of flavonoids from two species of Styrax against Schistosoma mansoni adult worms.

CONTEXT: Schistosomiasis is a major health problem worldwide. Thus, the search for new schistosomicidal agents from natural sources can provide prototypes for drug discovery. OBJECTIVE: The present study investigated the chemical composition of the EtOAc fractions of Styrax pohlii Pohl (Styracaceae) (EF-SP) aerial parts and S. camporum A. DC. leaves (EF-SC), as well as schistosomicidal activities against Schistosoma mansoni adult worms, which have not yet been studied. MATERIALS AND METHODS: The crude ethanol extracts of S. camporum leaves and S. pohlii aerial parts (EE-SC and EE-SP) were partitioned with n-hexane, EtOAc, and n-BuOH. The EtOAc fractions were purified by preparative HPLC. The crude extracts, EtOAc fractions and pure compounds were tested against S. mansoni adult worms in vitro. RESULTS: The purification procedure resulted in the isolation of kaempferol-3-O-(2'',4''-di-O-(E)-p-coumaroyl)-ß-d-glucopyranoside (1), kaempferol-3-O-(2'',6''-di-O-(E)-p-coumaroyl)-ß-d-glucopyranoside (2), quercetin (3), and kaempferol (4). The bioassay results indicated that EE-SC, EF-SC, EF-SP, and compounds 2 and 4 are able to separate coupled S. mansoni adult worms. Additionally, EE-SC, EF-SP, and compound 4 killed the adult schistosomes in vitro at 100 µg/mL and 100 µM. DISCUSSION AND CONCLUSION: This is the first time that the presence of compounds 1-2 in S. pohlii and 3-4 in S. camporum has been reported. Additionally, biological results indicated that S. pohlii and S. camporum have great potential as a source of active compounds.

Trypanosoma cruzi: evaluation of (-)-cubebin derivatives activity in the messenger RNAs processing.

No fully effective treatment has been developed since the discovery of Chagas' disease. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effective in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing, which is unusual RNA processing reaction, and it implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. Cubebin derivatives seem to provide treatments with less collateral effects than benznidazole and showed similar or better trypanocidal activities than benznidazole. Therefore, the cubebin derivatives ((-)-6,6'-dinitrohinokinin (DNH) and (-)-hinokinin (HQ)) interference in the mRNA processing was evaluated using T. cruzi permeable cells (Y and BOL (Bolivia) strains) following by RNase protection reaction. These substances seem to intervene in any step of the RNA transcription, promoting alterations in the RNA synthesis, even though the RNA processing mechanism still occurs. Furthermore, HQ presented better activity against the parasites than DNH, meaning that BOL strain seems to be more resistant than Y.

Deubiquitinating enzymes as possible drug targets for schistosomiasis.

Deubiquitinating enzymes (DUBs) are conserved in Schistosoma mansoni and may be linked to the 26S proteasome. Previous results from our group showed that b-AP15, an inhibitor of the 26S proteasome DUBs UCHL5 and USP14 induced structural and gene expression changes in mature S. mansoni pairs. This work suggests the use of the nonselective DUB inhibitor PR-619 to verify whether these enzymes are potential target proteins for new drug development. Our approach is based on previous studies with DUB inhibitors in mammalian cells that have shown that these enzymes are associated with apoptosis, autophagy and the transforming growth factor beta (TGF-ß) signaling pathway. PR-619 inhibited oviposition in parasite pairs in vitro, leading to mitochondrial changes, autophagic body formation, and changes in expression of SmSmad2 and SmUSP9x, which are genes linked to the TGF-ß pathway that are responsible for parasite oviposition and SmUCHL5 and SmRpn11 DUB maintenance. Taken together, these results indicate that DUBs may be used as targets for the development of new drugs against schistosomiasis.

An enquiry into antileishmanial activity and quantitative analysis of polyhydroxylated steroidal saponins from Solanum paniculatum L. leaves.

Solanum paniculatum L. is species whose fruits are widely consumed in Brazil as a tonic beverage with higher content of steroidal saponins. In this work, we developed an analytical method for the quantification of the eight saponins present in the 70 % ethanol extract from the leaves using ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS). Besides, the eight spirostanic saponins were screened for in vitro antileishmanial activity against promastigote and amastigote forms of Leishmania (L.) amazonensis. Substances 1, 2 and 3 were found to be the most active compounds, with inhibitory concentration (IC50) values of 8.51 ± 4.38, 10.75 ± 6.85 and 10.45 ± 4.21 µM, respectively, against promastigote forms and effective concentration (EC50) values of >25, 17.73 ± 0.99 and 19.57 ± 0.84 µM, respectively, against amastigote forms. The cytotoxic test with compounds 1-3 evidenced low toxicity in murine macrophage cells, with values above 50 µM at concentration lower than 25 µM. These findings show that saponins 1-3 should be evaluated in further studies for the treatment of cutaneous leishmaniasis.