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.

Larvicidal effect from different Annonaceae species on Culex quinquefasciatus.

The recent outbreaks of mosquito-borne diseases highlighted the pivotal importance of mosquito vector control in tropical areas worldwide. Several strategies have been developed to control vector populations and disease transmission in endemic areas. The steps to obtain natural active compounds involve the pre-selection in a biological model and subsequently evaluation on specific models. The present study reports the evaluation of 35 extracts, fractions, and essential oils obtained from five species from the Annonaceae family on Artemia salina and Culex quinquefasciatus. The A. salina results were used as a pre-screening for larvicidal test about mosquitoes. A correlation of biological activity in both bioassays was observed for the hydroethanolic extracts and their respective hexane and chloroform fractions of the leaves of Annona species, except A. nutans. The same correlation was also observed for all tested essential oils and petroleum ether extracts from Duguetia species. It was possible to limit an interval of lethality about A. salina, which has a corresponding range to the larvicidal test against the mosquito. The main components present in D. lanceolata essential oil or enriched fraction were α-selinene, aristolochene, (E)-caryophyllene, and (E)-calamenene. For D. furfuracea, the main components present of the underground parts were (E)-asarone, 2,4,5-trimethoxystyrene, spathulenol, and bicyclogermacrene for aerial parts. The A. salina test could be used as a model for the pre-screening of larvicidal activity.

Larvicidal activity of essential oil of Peumus boldus Molina and its ascaridole-enriched fraction against Culex quinquefasciatus.

Culex quinquefasciatus (Say, 1823), known as the domestic mosquito, is a common and abundant species throughout the world, and a cosmopolitan species. The adults of this mosquito are important in terms of public and animal health since they display adaptability to different hosts. In humans, they are responsible for the transmission of various diseases. One manner of control of this vector is the use of insecticidal or larvicidal products, which may have the drawback of toxicity to mammals and can be harmful to the environment. The present work evaluated the larvicidal potential of the essential oil (EO) and ascaridole-enriched fraction (EF4-5) obtained from the leaves of Peumus boldus Molina (boldo). The EO, obtained by steam distillation, was analyzed by GC/MS and fractionated on silica gel. EO and EF4-5, containing 31.4% and 89.5% ascaridole, respectively, were evaluated against C. quinquefasciatus at concentrations of 100, 50 and 25 µg/ml on the third and fourth instars. They showed lethal concentrations (LC50) of 82.14 and 41.85 µg/ml, respectively. Larvae treated with the EF4-5 showed morphological changes in the midgut, with cells possessing a cytoplasm that contained small vacuole-like structures, as well as a nucleus with decondensed chromatin and a cell apex with a short brush border. The cells of the fat body showed larger protein granules, which were acidophilic relative to the larvae of the control group. Moreover, the enriched fraction at a dose of 50 µg/ml showed a residual larvicidal effect according to exposure time on C. quinquefasciatus. This residual effect deserves consideration, since a long-term larvicidal product may be a useful tool for vector control.

Screening of Brazilian medicinal plants for antiviral activity against rotavirus.

ETHNOPHARMACOLOGICAL RELEVANCE: Brazilian medicinal plants traditionally used for the treatment of diarrhoea were investigated for their in vitro antiviral activity against the simian rotavirus SA11. MATERIALS AND METHODS: The ethanolic crude extracts of plants collected in the cerrado of Minas Gerais, Brazil were submitted to phytochemical screening. The cytotoxicity of the extracts was inferred by cellular morphologic alterations. Antiviral activity was assessed by the ability of the extracts to inhibit the cytopathic effect (CPE) of rotavirus on the treated cells. RT-PCR was performed to confirm and/or confront antiviral assay data. RESULTS: The maximum non-toxic concentration ranged from 50 to 500 µg/mL. All extracts were toxic at a concentration of 5000 µg/mL but no extract showed cytotoxicity at 50 µg/mL. The species Byrsonima verbascifolia, Myracrodruon urundeuva, Eugenia dysenterica and Hymenaea courbaril exhibited the strongest in vitro activity against rotavirus. Their extracts prevented the formation of CPE, and RT-PCR analysis detected no amplification of genetic material from rotavirus. Tannins, flavonoids, saponins, coumarins and terpenes were the major classes of natural products found in the leaf extracts that showed antiviral activity. CONCLUSION: Among the species studied, Byrsonima verbascifolia, Eugenia dysenterica, Hymenaea courbaril and Myracrodruon urundeuva showed potential activity against rotavirus and are worthy of further study. The present study corroborates ethnopharmacological data as a valuable source in the selection of plants with antiviral activity and to some extent validates their traditional uses.

Leishmanicidal, trypanocidal, and cytotoxic activities of endophytic fungi associated with bioactive plants in Brazil

One hundred and twenty-one isolates of endophytic fungi were recovered from leaves of the bioactive Brazilian plant species Ageratum myriadenia, Palicourea tetraphylla, Piptadenia adiantoides, and Trixis vauthieri. All fungal isolates were cultivated in liquid media and crude extracts were obtained with ethyl acetate. The crude extracts were tested in bioassay panels using Leishmania amazonensis, Trypanosoma cruzi, the enzyme trypanothione reductase (TryR) from Trypanosoma cruzi, and three human cancer cell lines. Thirty-three extracts (27.2 percent) exhibited at least one biological activity. Seventeen extracts (14 percent) were cytotoxic against one or more human cancer cell line with the IC50 values ranged of >0.2 to 25 µg/mL. Twenty-four extracts (19.8 percent) inhibited the activity of TryR, and three showed ability to inhibit the growth of T. cruzi above 60 percent and their IC50 values ranged among 1 to 10 µg/mL. Eleven extracts (9 percent) were able to inhibit the growth of L. amazonensis and showed with IC50 values ranged among 4.6 to 24.4 µg/mL. The endophytic fungi were identified as belonging to the genera Alternaria, Arthrinium, Cochliobolus, Colletotrichum, Penicillium, Fusarium, and Gibberella. An interesting result was obtained for the bioactive isolates UFMGCB 508, 537, 899 and 903, which were related to fungi associated with medicinal plants native to Asia, Australia, Africa, and Polynesia. These results indicate that bioactive plants living in Brazilian ecosystems are a potential host of endophytic fungi able to produce bioactive prototype molecules for drug development against neglected tropical diseases.

Leishmanicidal, trypanocidal, and cytotoxic activities of endophytic fungi associated with bioactive plants in Brazil.

One hundred and twenty-one isolates of endophytic fungi were recovered from leaves of the bioactive Brazilian plant species Ageratum myriadenia , Palicourea tetraphylla , Piptadenia adiantoides, and Trixis vauthieri. All fungal isolates were cultivated in liquid media and crude extracts were obtained with ethyl acetate. The crude extracts were tested in bioassay panels using Leishmania amazonensis , Trypanosoma cruzi, the enzyme trypanothione reductase (TryR) from Trypanosoma cruzi, and three human cancer cell lines. Thirty-three extracts (27.2%) exhibited at least one biological activity. Seventeen extracts (14%) were cytotoxic against one or more human cancer cell line with the IC50 values ranged of >0.2 to 25 µg/mL. Twenty-four extracts (19.8%) inhibited the activity of TryR, and three showed ability to inhibit the growth of T. cruzi above 60% and their IC50 values ranged among 1 to 10 µg/mL. Eleven extracts (9%) were able to inhibit the growth of L. amazonensis and showed with IC50 values ranged among 4.6 to 24.4 µg/mL. The endophytic fungi were identified as belonging to the genera Alternaria , Arthrinium , Cochliobolus , Colletotrichum , Penicillium , Fusarium, and Gibberella. An interesting result was obtained for the bioactive isolates UFMGCB 508, 537, 899 and 903, which were related to fungi associated with medicinal plants native to Asia, Australia, Africa, and Polynesia. These results indicate that bioactive plants living in Brazilian ecosystems are a potential host of endophytic fungi able to produce bioactive prototype molecules for drug development against neglected tropical diseases.

Antinociceptive and anti-inflammatory effects of the essential oil of Eugenia candolleana DC., Myrtaceae, on mice / Efeito antinociceptivo e antiinflamatório do óleo essencial de Eugenia candolleana DC. (Myrtaceae) em roedores

Eugenia candolleana DC. (Myrtaceae), commonly known as "murta" or "murtinha", is a plant species without any chemical or pharmacological study described in the literature. It has been popularly used for the treatment of pain and fever. This report aimed to investigate the possible antinociceptive and anti-inflammatory effects of the essential oil extracted from fresh leaves of Eugenia candolleana DC. (EOEc) in rodents. Following intraperitoneal injection, EOEc (25, 50 and 100 mg/kg) reduced the number of writhes significantly in a writhing test and the number of paw licks during phase two of formalin test (p < 0.001). However, administration of EOEc did not alter the time of reaction in hot plate test. Furthermore, EOEc inhibited (p < 0.01) the carrageenan-induced leukocyte migration to the peritoneal cavity. These results indicate antinociceptive and anti-inflammatory properties of EOEc probably mediated via inhibition of prostaglandin synthesis or other peripherally pathway.

Eugenia candolleana DC. (Myrtaceae), conhecida popularmente como "murta" ou "murtinha", é uma espécie vegetal sem estudos químicos e farmacológicos descritos na literatura, distribuída no Nordeste brasileiro, principalmente, na Zona da Mata. Na medicina popular do Estado de Sergipe é utilizada no tratamento de distúrbios febris e da dor. O presente estudo buscou avaliar as possíveis atividades antinociceptiva e antiinflamatória do óleo essencial extraído das folhas de E. candolleana DC (OEEc) em roedores. A administração intraperitoneal (i.p.) do OEEc (25, 50 e 100 mg/kg) reduziu significativamente o número de contorções no teste das contorções abdominais e a duração da lambida da pata na segunda fase do teste da formalina (p < 0,001). Entretanto, a administração do OEEc não alterou o tempo de reação no teste da placa quente. No experimento de peritonite induzido por carragenina, o OEEc reduziu de forma significativa (p < 0,01) a migração de leucócitos para a cavidade peritoneal. Os resultados obtidos sugerem que o OEEc possui ação antinociceptiva, provavelmente mediado por mecanismos periféricos, e ação antiinflamatória.

Differential acetyl cholinesterase inhibition by volatile oils from two specimens of Marlierea racemosa (Myrtaceae) collected from different areas of the Atlantic Rain Forest.

The volatile oil composition and anti-acetyl cholinesterase activity were analyzed in two specimens of Marlierea racemosa growing in different areas of the Atlantic Rain Forest (Cananéia and Caraguatatuba, SP, Brazil). Component identifications were performed by GC/MS and their acetyl cholinesterase inhibitory activity was measured through colorimetric analysis. The major constituent in both specimens was spathulenol (25.1% in Cananéia and 31.9% in Caraguatatuba). However, the first one also presented monoterpenes (41.2%), while in the Carguatatuba plants, this class was not detected. The oils from the plants collected in Cananéia were able to inhibit the acetyl cholinesterase activity by up to 75%, but for oils from the other locality the maximal inhibition achieved was 35%. These results suggested that the monoterpenes are more effective in the inhibition of acetyl cholinesterase activity than sesquiterpenes as these compounds are present in higher amounts in the M. racemosa plants collected in Cananéia.

Essential oils from Calyptranthes concinna, C. lucida and C. rubella (Myrtaceae)

Essential oils from Calyptranthes concinna, C. lucida and C. rubella, collected in Southern Brazil, were analyzed by GC and GC/MS. Sixty-two compounds were identified representing about 98 por cento of the oil contents. All samples were rich in cyclic sesquiterpenes (more then 90 por cento), mainly those from cadinane, bisabolane and germacrane cyclization pathway. The mainly components characterized were byclogermacrene (22.1 por cento in C. concinna; 11.7 por cento in C. rubella), cis-calamenene (10.3 por cento in C.concinna), beta-caryophyllene (16.5 por cento in C. rubella; 9.4 por cento in C. lucida), beta-bisabolene (25.5 por cento in C. lucida), spathulenol (15.4 por cento in C. rubella) and caryophyllene oxide (7.6 por cento in C. concinna)

Screening of Brazilian plants for the presence of peroxides

Chloroform or dichloromethane extracts of 357 southern Brazilian plant species were tested for the presence of peroxides by thin-layer chromatography, using the spray reagent from Huber & Fröhlke. >From the species tested, 71 (20 por cento) showed positive results and most of them (56 por cento) are Asteraceae species. The species tested were mainly from Asteraceae, but 55 more families were screened, in a total of 77 genera surveyed...