In vitro evaluation against Leishmania amazonensis and Leishmania chagasi of medicinal plant species of interest to the Unified Health System.

Leishmaniasis is a disease of public health relevance that demands new therapeutic alternatives due to the toxicity of conventional treatments. In this study, 27 plants of interest to the Unified Health System (SUS) were evaluated for cytotoxicity in macrophages, leishmanicidal activity and production of nitric oxide (NO). None of the species demonstrated cytotoxicity to macrophages (CC50 >100 µg/mL). Extracts from Chenopodium ambrosioides, Equisetum arvense, Maytenus ilicifolia showed greater efficacy in inducing the death of Leishmania amazonensis amastigotes with IC50 of 68.4, 82.3, 75.7 µg/mL, respectively. The species Cynara scolymus, Punica granatum and Passiflora alata were the most effective in inducing an increase in the indirect concentration of NO (41.31, 29.30 and 28.86 µM, respectively) in cultures of macrophages infected with L. amazonensis. Furthermore, Punica granatum was also the most effective species in inducing an increase in NO in macrophages infected by Leishmania chagasi (19.90 µM). The results obtained so far support the continuation of studies, with the possibility of developing safer and more effective treatments for leishmaniasis, using natural products. The identification of plants that stimulate the production of NO in macrophages infected by Leishmania opens doors for more detailed investigations of the mechanism of action of these natural products.

Antarctic organisms as a source of antimicrobial compounds: a patent review.

Currently, antimicrobial resistance has become a global public health problem, which has made the need for new antimicrobial compounds to deal with resistant infections an emergency. However, environments that once offered so many innovative molecules, now already exhaustively exploited, do not meet this need. In this context, a geographically isolated, under-explored and extreme environment, such as Antarctica, which holds organisms with unique physiological and biochemical characteristics, assumes great importance as a potential source of new compounds with antimicrobial activity. In this patent review, we investigate the state of technological development in the field of antimicrobial compounds obtained from Antarctic organisms, highlighting the main countries and researchers active in the field, the species utilized, the compounds obtained, and their possible therapeutic applications. As results, few patent documents were found, however they encompass a wide diversity of compounds and species, indicating a great antimicrobial potential present in Antarctic biota, including compounds active against the most important human pathogenic microorganisms, such as including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus spp. and multi-resistant Mycobacterium tuberculosis. Furthermore, due to the increasing trend in patent applications, a significant rise in the number of patents in this area is expected in the coming years.

Extracellular hydrolytic enzymes produced by yeasts from Antarctic lichens.

In the Antarctic environment, yeasts are versatile eukaryotes that have shown wide dispersion in different substrates, producing active enzymes in extreme conditions, but their relevance in biotechnological applications is largely unknown. The aim of this study was to evaluate the production of extracellular hydrolases by yeasts isolated from Antarctic lichens and molecularly identify these isolates. From a total of 144 isolates on the screening, 109 (76%) produced at least one of the hydrolases tested, with most activities for proteases 59 (41%), cellulases 58 (40%), esterases 57 (39%), lipases 29 (20%), amylases 23 (16%) and pectinases 20 (14%). Among these isolates, 76 were identified, most belonged to the phylum Basidiomycota (n=73) with the dominance of Vishniacozyma victoriae (n=27), Cystobasidium alpinum (n=3), Mrakia niccombsii (n=3), Cystobasidium laryngis (n=2), Bannozyma yamatoana (n=2), Holtermanniella nyarrowii (n=2), and Glaciozyma martinii (n=2). This study is the first one reporting extracellular enzyme production by yeasts isolated from thallus of the species of Antarctic lichens Lecania brialmontii, Polycauliona candelaria, Usnea capillacea, Cladonia metacorallifera, and Polycauliona regalis. With these data, it's possible to confirm lichens as a source of hydrolase-producing yeasts, reinforcing the potential of these microorganisms in bioprospecting studies of catalytic molecules from polar regions that may be useful in promising biotechnological applications.

Antarctic environments as a source of bacterial and fungal therapeutic enzymes.

Microbial therapeutic enzymes are the protagonists in the pharmacological treatment of different human diseases. The intrinsic enzymatic characteristics, such as high affinity and specificity to the corresponding substrate, enable effective therapies, with minimal adverse effects and complete remission. However, immunogenicity, short half-life, low enzymatic yield, and low selectivity regarding available enzyme drugs are currently the main obstacles to their development and the broad adherence to therapeutic protocols. By harboring adapted and still unexplored microbial life, environments of extreme conditions, such as Antarctica, become especially important in the prospecting and development of new enzymatic compounds that present higher yields and the possibility of genetic improvement. Antarctic microorganisms have adaptation mechanisms, such as more fluid cell membranes, production of antifreeze proteins and enzymes with more malleable structures, more robust, stable, selective catalytic sites for their respective substrates, and high antioxidant capacity. In this context, this review aims to explore enzymes synthesized by bacteria and fungi from Antarctica as potential drug producers, capable of providing therapeutic efficacy, less adverse effects, and lower production costs with highlight to L-Asparaginase, collagenase, superoxide dismutase and ribonucleases. In addition, this review highlights the unique biotechnological profile of these Antarctic extremophile microorganisms.

Synthesis, Antileishmanial Activity and in silico Studies of Aminoguanidine Hydrazones (AGH) and Thiosemicarbazones (TSC) Against Leishmania chagasi Amastigotes.

BACKGROUND: Leishmaniasis is a worldwide health problem, highly endemic in developing countries. Among the four main clinical forms of the disease, visceral leishmaniasis is the most severe, fatal in 95% of cases. The undesired side-effects from first-line chemotherapy and the reported drug resistance search for effective drugs that can replace or supplement those currently used in an urgent need. Aminoguanidine hydrazones (AGH's) have been explored for exhibiting a diverse spectrum of biological activities, in particular the antileishmanial activity of MGBG. The bioisosteres thiosemicarbazones (TSC's) offer a similar biological activity diversity, including antiprotozoal effects against Leishmania species and Trypanosoma cruzi. OBJECTIVES: Considering the impact of leishmaniasis worldwide, this work aimed to design, synthesize, and perform a screening upon L. chagasi amastigotes and for the cytotoxicity of the small "inhouse" library of both AGH and TSC derivatives and their structurally-related compounds. METHODS: A set of AGH's (3-7), TSC's (9, 10), and semicarbazones (11) were initially synthesized. Subsequently, different semi-constrained analogs were designed and also prepared, including thiazolidines (12), dihydrothiazines (13), imidazolines (15), pyrimidines (16, 18) azines (19, 20), and benzotriazepinones (23-25). All intermediates and target compounds were obtained with satisfactory yields and exhibited spectral data consistent with their structures. All final compounds were evaluated against L. chagasi amastigotes and J774.A1 cell line. Molecular docking was performed towards trypanothione reductase using GOLD® software. RESULTS: The AGH's 3i, 4a, and 5d, and the TSC's 9i, 9k, and 9o were selected as valuable hits. These compounds presented antileishmanial activity compared with pentamidine, showing IC50 values ranged from 0.6 to 7.27 µM, maximal effects up to 55.3%, and satisfactory SI values (ranged from 11 to 87). On the other hand, most of the resulting semi-constrained analogs were found cytotoxic or presented reduced antileishmanial activity. In general, TSC class is more promising than its isosteric AGH analogs, and the beneficial aromatic substituent effects are not similar in both series. In silico studies have suggested that these hits are capable of inhibiting the trypanothione reductase from the amastigote forms. CONCLUSION: The promising antileishmanial activity of three AGH's and three TSC's was characterized. These compounds presented antileishmanial activity compared with PTD, showing IC50 values ranged from 0.6 to 7.27 µM, and satisfactory SI values. Further pharmacological assays involving other Leishmania strains are in progress, which will help choose the best hits for in vivo experiments.

Carbamoyl-N-aryl-imine-urea: a new framework to obtain a putative leishmanicidal drug-candidate.

Leishmaniasis is a neglected parasitic disease, and current treatment includes limitations of toxicity, variable efficacy, high costs and inconvenient doses and treatment schedules. Therefore, new leishmanicidal drugs are still an unquestionable medical need. In this paper we described the design conception of a new framework, the carbamoyl-N-aryl-imine-urea, to obtain putative leishmanicidal drug-candidates. Compounds 9a-e and 10a-e were designed and synthesized and their leishmanicidal activity was studied in comparison to pentamidine, miltefosine and meglumine antimoniate. The conformational profile of the new carbamoyl-N-aryl-imine-urea framework was investigated by X-ray diffraction studies, using compound 9a as a model. The plasma stability of this putative peptide mimetic subunit was studied for compound 10e (LASSBio-1736). Among the congeneric series, LASSBio-1736 was identified as a new antileishmanial drug-candidate, displaying plasma stability, cytotoxicity against amastigote forms of L. amazonensis and L. braziliensis, and leishmanicidal activity in a cutaneous leishmaniasis murine model, without preliminary evidence of hepatic or renal toxicity.

Flavonoids induce cell death in Leishmania amazonensis: in vitro characterization by flow cytometry and Raman spectroscopy.

Leishmaniasis comprises a group of infectious diseases with worldwide distribution, of which both the visceral and cutaneous forms are caused by Leishmania parasites. In the absence of vaccines, efficacious chemotherapy remains the basis for leishmaniasis control. The available drugs are expensive and associated with several secondary adverse effects. Due to these limitations, the development of new antileishmanial compounds is imperative, and plants offer various perspectives in this regard. The present study evaluated the in vitro leishmanicidal activity of flavonoids isolated from Solanum paludosum Moric. and investigated the mechanisms of cell death induced by them. These compounds were evaluated in vitro for their antileishmanial activity against Leishmania amazonensis promastigotes and they showed prominent leishmanicidal activity. The EtOAc fraction, gossypetin 3,7,8,4'-tetra-O-methyl ether (1), and kaempferol 3,7-di-O-methyl ether (3) were selected to be used in an in vitro assay against L. amazonensis amastigotes and cell death assays. The flavonoids (1) and (3) presented significant activity against L. amazonensis amastigotes, exhibiting the IC50 values of 23.3 ± 4.5 µM, 34.0 ± 9.6 µM, and 10.5 ± 2.5 µM for the EtOAc fraction, (1), and (3), respectively, without toxic effects to the host cells. Moreover, (1) and (3) induced blocked cell cycle progression at the G1/S transition, ultimately leading to G1/G0 arrest. Flavonoid (3) also induced autophagy. Using Raman spectroscopy in conjunction with principal component analysis, the biochemical changes in the cellular components induced by flavonoids (1) and (3) were presented. The obtained results indicated that the mechanisms of action of (1) and (3) occurred through different routes. The results support that the flavonoids derived from S. paludosum can become lead molecules for the design of antileishmanial prototypes.

Chemical constituents from Sidastrum paniculatum and evaluation of their leishmanicidal activity

AbstractSidastrum paniculatum (L.) Fryxell, Malvaceae, is popularly known in Brazil as “malva-roxa” or “malvavisco”. The species is found mainly in Northeast region where it is used by locals to treat spider bites and bee stings. Aiming to identify the chemical compounds from S. paniculatum secondary metabolism and to contribute to the chemotaxonomic knowledge of Malvaceae family, a phytochemical study of S. paniculatum was carried out. Besides that, the isolated compounds were evaluated for antileishmanial activity against promastigotes of Leishmania braziliensis. By using chromatographic techniques the study resulted the isolation of eight compounds: 3-oxo-21β-H-hop-22(29)-ene; sebiferic acid; sitosterol 3-O-β-d-glucopyranoside/stigmasterol 3-O-β-d-glucopyranoside; phaeophytin a; 132(S)-hydroxyphaeophytin a; 132(S)-hydroxy-(173)-ethoxyphaeophorbide a and 7,4′-di-O-methylisoescutellarein. The structure of all isolated compounds was elucidated by spectroscopic analysis, including two-dimensional NMR techniques. In addition, the isolated compounds phaeophytin a; 132(S)-hydroxyphaeophytin a; 132(S)-hydroxy-(173)-ethoxyphaeophorbide a and 7,4′-di-O-methylisoescutellarein exhibited antileishmanial activity against promastigotes of L. braziliensis.

Design, synthesis and in vitro trypanocidal and leishmanicidal activities of novel semicarbazone derivatives.

Trypanosomatids are protozoan parasites that cause various diseases in human, such as leishmaniasis, Chagas disease and sleeping sickness. The highly syntenic genomes of the trypanosomatid species lead the assumption that they can encode similar proteins, indicating the possibility to design new antitrypanosomatid drugs with dual trypanosomicidal and leishmanicidal activities. In this work a series of compounds (6a-h and 7a-h), containing a semicarbazone scaffold as a peptide mimetic framework, was designed and synthesized. From this series compound 7g (LASSBio-1483) highlighted, showing dual in vitro trypanosomicidal and leishmanicidal activities, with potency similar to the standard drugs nifurtimox and pentamidine. This data, taken together with its good in silico druglikeness profile and its great chemical and plasma stability, make LASSBio-1483 (7g) a new antitrypanosomatid lead-candidate.

Synthesis, leishmanicidal activity and theoretical evaluations of a series of substituted bis-2-hydroxy-1,4-naphthoquinones.

A series of eight substituted bis-2-hydroxy-1,4-naphthoquinone derivatives was synthesized through lawsone condensation with various aromatic and aliphatic aldehydes under mild acidic conditions. The title compounds were evaluated for antileishmanial activity in vitro against Leishmania amazonensis and Leishmania braziliensis promastigotes; six compounds showed good activity without significant toxic effects. The compound with the highest activity was used for an in vivo assay with Leishmania amazonensis.

Antileishmanial Phenylpropanoids from the Leaves of Hyptis pectinata (L.) Poit.

Hyptis pectinata, popularly known in Brazil as "sambacaitá" or "canudinho," is an aromatic shrub largely grown in the northeast of Brazil. The leaves and bark are used in an infusion for the treatment of throat and skin inflammations, bacterial infections, pain, and cancer. Analogues of rosmarinic acid and flavonoids were obtained from the leaves of Hyptis pectinata and consisted of two new compounds, sambacaitaric acid (1) and 3-O-methyl-sambacaitaric acid (2), and nine known compounds, rosmarinic acid (3), 3-O-methyl-rosmarinic acid (4), ethyl caffeate (5), nepetoidin A (6), nepetoidin B (7), cirsiliol (8), circimaritin (9), 7-O-methylluteolin (10), and genkwanin (11). The structures of these compounds were determined by spectroscopic methods. Compounds 1-5, and 7 were evaluated in vitro against the promastigote form of L. braziliensis, and the ethanol extract. The hexane, ethyl acetate, and methanol-water fractions were also evaluated. The EtOH extract, the hexane extract, EtOAc, MeOH:H2O fractions; and compounds 1, 2 and 4 exhibited antileishmanial activity, and compound 1 was as potent as pentamidine. In contrast, compounds 3, 5, and 7 did not present activity against the promastigote form of L. braziliensis below 100 µM. To our knowledge, compounds 1 and 2 are being described for the first time.

Antinociceptive activities of crude methanolic extract and phases, n-butanolic, chloroformic and ethyl acetate from Caulerpa racemosa (Caulerpaceae) / Atividade antinociceptiva do extrato metanólico bruto e das fases n-butanólica, clorofórmica e acetato de etila de Caulerpa recemosa (Caulerpaceae)

In this study, we attempted to identify the possible antinociceptive actions of n-butanolic phase, chloroformic phase, ethyl acetate phase and crude methanolic extract obtained from Caulerpa racemosa. This seaweed is cosmopolitan in world, mainly in tropical regions. The n-butanolic, chloroformic, ethyl acetate phases and crude methanolic extract, all administered orally in the concentration of 100 mg/kg, reduced the nociception produced by acetic acid by 47.39 percent, 70.51 percent, 76.11 percent and 72.24 percent, respectively. In the hotplate test the chloroformic and ethyl acetate phase were activite in this models. In the neurogenic phase on formalin test, were observed that crude methanolic extract (51.77 percent), n-butanolic phase (35.12 percent), chloroformic phase (32.70 percent) and indomethacin (32.06 percent) were effective in inhibit the nociceptive response. In the inflammatory phase, only the ethyl acetate phase (75.43 percent) and indomethacin (47.83 percent) inhibited significantly the nociceptive response. Based on these data, we can infer that the ethyl acetate phase shows a significant anti-inflammatory profile, whose power has not yet been determined. However, pharmacological and chemical studies are continuing in order to characterize the mechanism(s) responsible for the antinociceptive action and also to identify other active principles present in Caulerpa racemosa.

Neste estudo, tentamos identificar a atividade antinociceptiva do extrato metanólico bruto e das fases n-butanólica, clorofórmica e acetato de etila provenientes da alga Caulerpa racemosa. Esta alga é cosmopolita no mundo, principalmente em regiões tropicais. O extrato metanólico bruto e as fases n-butanólica, clorofórmica e acetato de etila foram administrados por via oral, na concentração de 100 mg/kg. Estes foram capazes de reduzir a nocicepção produzida pelo ácido acético, sendo 47,39 por cento, 70,51 por cento, 76,11 por cento e 72,24 por cento, respectivamente. No ensaio da placa quente as fases clorofórmica e acetato de etila foram ativas neste modelo. Na fase neurogênica do teste de formalina, foi observado que o extrato metanólico bruto (51,77 por cento), fase n-butanólica (35,12 por cento), fase clorofórmica (32,70 por cento) e indometacina (32,06 por cento) foram eficazes em inibir a resposta nociceptiva. Na fase inflamatória, apenas a fase acetato de etila (75,43 por cento) e indometacina (47,83 por cento) foram capazes de inibir significativamente a resposta nociceptiva. Com base nestes dados, podemos sugerir que o a fase acetato de etila apresenta um significativo efeito anti-inflamatório, cuja potência ainda não foi determinada. No entanto, estudos farmacológicos e químicos serão necessários, a fim de caracterizar o mecanismo responsável pela ação antinociceptiva e também para identificar outros princípios ativos presentes na alga Caulerpa racemosa.