Searching for plant-derived antivirals against dengue virus and Zika virus.

BACKGROUND: The worldwide epidemics of diseases as dengue and Zika have triggered an intense effort to repurpose drugs and search for novel antivirals to treat patients as no approved drugs for these diseases are currently available. Our aim was to screen plant-derived extracts to identify and isolate compounds with antiviral properties against dengue virus (DENV) and Zika virus (ZIKV). METHODS: Seven thousand plant extracts were screened in vitro for their antiviral properties against DENV-2 and ZIKV by their viral cytopathic effect reduction followed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, previously validated for this purpose. Selected extracts were submitted to bioactivity-guided fractionation using high- and ultrahigh-pressure liquid chromatography. In parallel, high-resolution mass spectrometric data (MSn) were collected from each fraction, allowing compounds into the active fractions to be tracked in subsequent fractionation procedures. The virucidal activity of extracts and compounds was assessed by using the plaque reduction assay. EC50 and CC50 were determined by dose response experiments, and the ratio (EC50/CC50) was used as a selectivity index (SI) to measure the antiviral vs. cytotoxic activity. Purified compounds were used in nuclear magnetic resonance spectroscopy to identify their chemical structures. Two compounds were associated in different proportions and submitted to bioassays against both viruses to investigate possible synergy. In silico prediction of the pharmacokinetic and toxicity (ADMET) properties of the antiviral compounds were calculated using the pkCSM platform. RESULTS: We detected antiviral activity against DENV-2 and ZIKV in 21 extracts obtained from 15 plant species. Hippeastrum (Amaryllidaceae) was the most represented genus, affording seven active extracts. Bioactivity-guided fractionation of several extracts led to the purification of lycorine, pretazettine, narciclasine, and narciclasine-4-O-ß-D-xylopyranoside (NXP). Another 16 compounds were identified in active fractions. Association of lycorine and pretazettine did not improve their antiviral activity against DENV-2 and neither to ZIKV. ADMET prediction suggested that these four compounds may have a good metabolism and no mutagenic toxicity. Predicted oral absorption, distribution, and excretion parameters of lycorine and pretazettine indicate them as candidates to be tested in animal models. CONCLUSIONS: Our results showed that plant extracts, especially those from the Hippeastrum genus, can be a valuable source of antiviral compounds against ZIKV and DENV-2. The majority of compounds identified have never been previously described for their activity against ZIKV and other viruses.

Trypanocidal Trixikingolides From Trixis Vauthieri.

Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi. Only two drugs are available, with the drawback of low rate of cure in the chronic phase of the disease and undesirable side effects. These facts highlight the need to find new compounds for Chagas disease chemotherapy. We describe the isolation and identification of an inseparable mixture of two new trixikingolides from Trixis vauthieri, a plant from family Asteraceae, which present outstanding in vitro trypanocidal activity, with IC50 value of 0.053 µM against the intracellular trypomastigotes and amastigotes forms of T. cruzi infecting L929 cells. The IC50 of the mixture against the host cells is 68 times higher and about 70 times more potent than benznidazole, the reference drug used as control at the experiments. The next step, which depends on obtaining larger quantities of the mixture, is to test it on mice infected with T. cruzi.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth) used as a remedy to treat fever and malaria in the Amazon

Infusions of Aspidosperma nitidum (Apocynaceae) wood bark are used to treat fever and malaria in the Amazon Region. Several species of this family are known to possess indole alkaloids and other classes of secondary metabolites, whereas terpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitidumine have been described in A. nitidum . In the present study, extracts from the wood bark, leaves and branches of this species were prepared for assays against malaria parasites and cytotoxicity testing using human hepatoma and normal monkey kidney cells. The wood bark extracts were active against Plasmodium falciparum and showed a low cytotoxicity in vitro, whereas the leaf and branch extracts and the pure alkaloid braznitidumine were inactive. A crude methanol extract was subjected to acid-base fractionation aimed at obtaining alkaloid-rich fractions, which were active at low concentrations against P. falciparum and in mice infected with and sensitive Plasmodium berghei parasites. Our data validate the antimalarial usefulness of A. nitidum wood bark, a remedy that can most likely help to control malaria. However, the molecules responsible for this antimalarial activity have not yet been identified. Considering their high selectivity index, the alkaloid-rich fractions from the plant bark might be useful in the development of new antimalarials.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth) used as a remedy to treat fever and malaria in the Amazon.

Infusions of Aspidosperma nitidum (Apocynaceae) wood bark are used to treat fever and malaria in the Amazon Region. Several species of this family are known to possess indole alkaloids and other classes of secondary metabolites, whereas terpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitidumine have been described in A. nitidum . In the present study, extracts from the wood bark, leaves and branches of this species were prepared for assays against malaria parasites and cytotoxicity testing using human hepatoma and normal monkey kidney cells. The wood bark extracts were active against Plasmodium falciparum and showed a low cytotoxicity in vitro, whereas the leaf and branch extracts and the pure alkaloid braznitidumine were inactive. A crude methanol extract was subjected to acid-base fractionation aimed at obtaining alkaloid-rich fractions, which were active at low concentrations against P. falciparum and in mice infected with and sensitive Plasmodium berghei parasites. Our data validate the antimalarial usefulness of A. nitidum wood bark, a remedy that can most likely help to control malaria. However, the molecules responsible for this antimalarial activity have not yet been identified. Considering their high selectivity index, the alkaloid-rich fractions from the plant bark might be useful in the development of new antimalarials.

Stachytarpheta gesnerioides Cham: chemical composition of hexane fraction and essential oil, antioxidant and antimicrobial activities / Stachytarpheta gesnerioides Cham: composición química de la fracción hexánica y aceite esencial, actividades antioxidante y antimicrobiana

In the present study we investigated the chemical composition of hexane fraction and essential oil of Stachytarpheta gesnerioides (Verbenaceae) by GC-MS, total phenol and flavonoid contents. The antioxidant capacity and antimicrobial activity were investigated in five extracts of leaves of S. gesnerioides. Aqueous and 100 percent ethanol extracts were prepared by dynamic maceration. Hexane, ethyl acetate and methanol extracts were prepared by Soxhlet extraction. The essential oil (EO) and hexane fraction (HF) are mainly composed by guaiol. Moreover, the HF is also rich in the monoterpene alpha-pinene. The total phenol content ranged from 0.85 to 22.74 mg gallic acid equivalent /100mg dry extract at Folin–Ciocalteu’s reagent method. The total flavonoid concentration ranged from 0.68 to 13.65 mg rutin equivalent /100mg dry extract, detected using 8 percent aluminium chloride. The ethyl acetate extract (IC50=9.41 ug/ml) showed the highest antioxidant activity. The extracts were found to be effective to inhibit the microorganisms tested.

Se han investigado la composición química de la fracción hexánica (FH) y aceite esencial (AE) de Stachytarpheta gesnerioides (Verbenaceae) por GC-MS, el contenido de fenoles totales y flavonoides. La capacidad antioxidante y actividad antimicrobiana fueron investigadas en cinco extractos de hojas de S gesnerioides. Extractos acuosos y etanolico fueron preparados por la maceración dinámica y extracción continua en Soxhlet con hexano, acetato de etilo y metanol. Las fracciones AE y FH están compuestas principalmente por guaiol. La fracción FH es también rica en alfa-pineno. El contenido de fenoles totales varió desde 0,85 hasta 22,74 mg de ácido gálico/100 mg de extracto seco (Folin-Ciocalteu). La concentración total de flavonoides varió desde 0,68 hasta 13,65 mg en equivalentes de rutina/100 mg de extracto seco, que se detectó mediante reacción con cloruro de aluminio al 8 por ciento. El extracto de acetato de etilo (CI50=9,41 ug/ml) enseño la más grande actividad antioxidante. Los extractos se encontraron eficaces para inhibir los microorganismos ensayados.

Antifungal activity of trichothecenes from Fusarium sp. against clinical isolates of Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM), a human mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis, is a serious public health problem in several countries of Latin America. In our search we found that the crude extract of the endophytic fungus UFMGCB 551 was able to inhibit several clinical strains of P. brasiliensis, and was also active in the bioautographic assay against Cladosporium sphaerospermum. The endophytic fungus UFMGCB 551 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae). The fungus was identified as Fusarium sp. based on its macro- and micro-morphology, and on the sequence of the internally transcribed spacer regions (ITS) of its rRNA gene. The chromatographic fractionation of the fungal extract was guided by the bioautographic assay to afford three known trichothecene mycotoxins: T2-toxin (1) and a mixture of 8-n-butyrylneosolaniol (2) and 8-isobutyrylsolaniol (3). The minimal inhibitory concentrations (MIC) of the these compounds against eleven clinical strains of P. brasiliensis were evaluated and found to be in the range between 75 and 640 nmol l(-1) for 1 and 160-640 nmol l(-1) for the mixture of 2 and 3.

Leishmanicidal metabolites from Cochliobolus sp., an endophytic fungus isolated from Piptadenia adiantoides (Fabaceae).

Protozoan parasites belonging to genera Leishmania and Trypanosoma are the etiological agents of severe neglected tropical diseases (NTDs) that cause enormous social and economic impact in many countries of tropical and sub-tropical areas of the world. In our screening program for new drug leads from natural sources, we found that the crude extract of the endophytic fungus Cochliobolus sp. (UFMGCB-555) could kill 90% of the amastigote-like forms of Leishmania amazonensis and inhibit by 100% Ellman's reagent reduction in the trypanothione reductase (TryR) assay, when tested at 20 microg mL(-1). UFMGCB-555 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae) and identified based on the sequence of the internally transcribed spacer (ITS) regions of its ribosomal DNA. The chromatographic fractionation of the extract was guided by the TryR assay and resulted in the isolation of cochlioquinone A and isocochlioquinone A. Both compounds were active in the assay with L. amazonensis, disclosing EC(50) values (effective concentrations required to kill 50% of the parasite) of 1.7 microM (95% confidence interval = 1.6 to 1.9 microM) and 4.1 microM (95% confidence interval = 3.6 to 4.7 microM), respectively. These compounds were not active against three human cancer cell lines (MCF-7, TK-10, and UACC-62), indicating some degree of selectivity towards the parasites. These results suggest that cochlioquinones are attractive lead compounds that deserve further investigation aiming at developing new drugs to treat leishmaniasis. The findings also reinforce the role of endophytic fungi as an important source of compounds with potential to enter the pipeline for drug development against NTDs.

Morphological, anatomical, macro and micromolecular markers for Solanum cernuum identification

The plants consumed as remedy by the population may have imprecise taxonomical identification. If these plants are used for the production of phytomedicines such misidentification may affect the quality of the product. Hereby, we describe markers for identification of the entire plant or grounded plant material or the crude extract of Solanum cernuum Vell. (Solanaceae). Specimens from four localities were collected, analyzed and compared. Morphological characters were used to identify the plant when it is not grounded or extracted. However, when the plant material is grounded, the set of trichomes may be used as anatomical marker. The region ITS1, 5.8S and ITS2 of the nuclear ribosomal DNA was cloned and sequenced. The sequence, with length of about 600 base pairs, being 48.1 percent AT , was deposited in GenBank under the accession number DQ837371. Once this sequence is specific to S. cernuum, it was used as marker for this species. For the crude extract, chromatographic profiles of the leaves extracts were obtained by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Two flavonoids were isolated and identified as quercitrin and afzelin. So, this study presents morphological, anatomical, macro and micromolecular markers to identify S. cernuum.

Plantas consumidas como remédio nem sempre são identificadas taxonomicamente de maneira correta. Se estas plantas forem utilizadas para obtenção de uma droga vegetal ou um fitoterápico, tal erro pode afetar a qualidade do produto final. Neste trabalho são descritos marcadores para a identificação de Solanum cernuum Vell. (Solanaceae), esteja a planta íntegra, triturada ou como extrato bruto. Indivíduos de quatro localidades de Minas Gerais foram coletados, analisados e comparados. Os caracteres morfológicos foram utilizados para a planta íntegra. Para a planta triturada, o conjunto dos tricomas foi utilizado como marcador anatômico. Um marcador macromolecular também foi determinado. Para tal a região ITS1, 5.8S e ITS2 do DNAr foi clonada e seqüenciada. A seqüência, com cerca de 600 pares de bases dos quais 48,1 por cento são AT, foi depositada no GenBank sob o número de acesso DQ837371. Por ser uma seqüência específica para S. cernuum, ela pode ser usada como marcador desta espécie. Para o extrato bruto foram determinados perfis cromatográficos de extratos das folhas por cromatografia em camada delgada e por cromatografia líquida de alta eficiência. Dois flavonóides foram isolados e identificados como quercitrina e afzelina. Assim, neste trabalho foram determinados marcadores morfológicos, anatômicos, macro e micromoleculares para identificar S. cernuum.

Eleutherinone, a novel fungitoxic naphthoquinone from Eleutherine bulbosa (Iridaceae).

The dichloromethane extract prepared from the underground parts of Eleutherine bulbosa (Miller) Urban (Iridaceae) showed strong activity in the direct bioautography assay with the phytopathogenic fungus Cladosporium sphaerospermum. This assay was used to guide the fractionation of this extract and allowed the isolation of four compounds: the new naphthoquinone eleutherinone[8-methoxy-1-methyl-1,3-dihydro-naphtho(2,3-c)furan-4,9 -dione] and the known compounds, previously isolated from this species, eleutherin [9-methoxy-1(R),3(S)-dimethyl-3,4-dihydro-1H-benzo(g)isochromene-5,10-dione], isoeleutherin [9-methoxy-1(R),3(R)-dimethyl-3,4-dihydro-1H-benzo(g)isochromene-5,10-dione], and eleutherol [4-hydroxy-5-methoxy-3(R)-methyl-3H-naphtho(2,3-c)furan-1 -one]. All quinonoid compounds showed strong antifungal activity in the bioautography assay at 100 g/spot, while eleutherol was inactive.

Eleutherinone, a novel fungitoxic naphthoquinone from Eleutherine bulbosa (Iridaceae)

The dichloromethane extract prepared from the underground parts of Eleutherine bulbosa (Miller) Urban (Iridaceae) showed strong activity in the direct bioautography assay with the phytopathogenic fungus Cladosporium sphaerospermum. This assay was used to guide the fractionation of this extract and allowed the isolation of four compounds: the new naphthoquinone eleutherinone[8-methoxy-1-methyl-1,3-dihydro-naphtho(2,3-c)furan-4,9 -dione] and the known compounds, previously isolated from this species, eleutherin [9-methoxy-1(R),3(S)-dimethyl-3,4-dihydro-1H-benzo(g)isochromene-5,10-dione], isoeleutherin [9-methoxy-1(R),3(R)-dimethyl-3,4-dihydro-1H-benzo(g)isochromene-5,10-dione], and eleutherol [4-hydroxy-5-methoxy-3(R)-methyl-3H-naphtho(2,3-c)furan-1 -one]. All quinonoid compounds showed strong antifungal activity in the bioautography assay at 100 æg/spot, while eleutherol was inactive