RESUMO
BACKGROUND: The chemistry of Costa Rican propolis from Apis mellifera remains underexplored despite its potential applications. This study identified its chemical composition, linking chemotypes to antioxidant potential. METHODS: Proton nuclear magnetic resonance (1H NMR) spectra were obtained for 119 propolis extracts and analyzed using multivariate analyses. In parallel, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was used to assess antioxidant activity. A generalized linear regression model (GLM) correlated this with its chemical profiles and geographical origin. Chromatographic methods were used to isolate active and inactive compounds, which were identified using nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). RESULTS: Principal component analysis (PCA) revealed three chemical profile groups for the 119 propolis extracts, explaining 73% of the total variance with two components. Radical scavenging activity was found to correlate with chemical composition. Isolation yielded n-coniferyl benzoate in type I (EC50 = 190 µg/mL, ORAC = 0.60 µmol TE/µmol) and nemorosone in type II (EC50 = 300 µg/mL, ORAC = 0.7 µmol TE/µmol). Type III was represented in terpene-like components, which exhibited lower antioxidant activity. CONCLUSIONS: This study categorizes Costa Rican propolis into three chemical types and identifies two key components linked to antioxidant activity. Notably, nemorosone, a valuable natural product, was found to be highly concentrated in a particular region of Costa Rica.
Assuntos
Própole , Animais , Própole/química , Antioxidantes/química , Costa Rica , Benzofenonas/químicaRESUMO
Fungi exhibit a wide range of ecological guilds, but those that live within the inner tissues of plants (also known as endophytes) are particularly relevant due to the benefits they sometimes provide to their hosts, such as herbivory deterrence, disease protection, and growth promotion. Recently, endophytes have gained interest as potential biocontrol agents against crop pathogens, for example, coffee plants (Coffea arabica). Published results from research performed in our laboratory showed that endophytic fungi isolated from wild Rubiaceae plants were effective in reducing the effects of the American leaf spot of coffee (Mycena citricolor). One of these isolates (GU11N) from the plant Randia grandifolia was identified as Daldinia eschscholtzii (Xylariales). Its antagonism mechanisms, effects, and chemistry against M. citricolor were investigated by analyzing its volatile profile alone and in the presence of the pathogen in contactless and dual culture assays. The experimental design involved direct sampling of agar plugs in vials for headspace (HS) and headspace solid-phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS) analysis. Additionally, we used ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS) to identify nonvolatile compounds from organic extracts of the mycelia involved in the interaction. Results showed that more volatile compounds were identified using HS-SPME (39 components) than those by the HS technique (13 components), sharing only 12 compounds. Statistical tests suggest that D. eschscholtzii inhibited the growth of M. citricolor through the release of VOCs containing a combination of 1,8-dimethoxynapththalene and terpene compounds affecting M. citricolor pseudopilei. The damaging effects of 1,8-dimethoxynaphthalene were corroborated in an in vitro test against M. citricolor pseudopilei; scanning electron microscopy (SEM) photographs confirmed structural damage. After analyzing the UHPLC-HRMS/MS data, a predominance of fatty acid derivatives was found among the putatively identified compounds. However, a considerable proportion of features (37.3%) remained unannotated. In conclusion, our study suggests that D. eschscholtzii has potential as a biocontrol agent against M. citricolor and that 1,8-dimethoxynaphthalene contributes to the observed damage to the pathogen's reproductive structures.
RESUMO
Streptomyces symbionts in insects have shown to be a valuable source of new antibiotics. Here, we report the genome sequence and the potential for antibiotic production of "Streptomyces sp. M54", an Actinobacteria associated with the eusocial wasp, Polybia plebeja. The Streptomyces sp. M54 genome is composed of a chromosome (7.96 Mb), and a plasmid (1.91 Kb) and harbors 30 biosynthetic gene clusters for secondary metabolites, of which only one third has been previously characterized. Growth inhibition bioassays show that this bacterium produces antimicrobial compounds that are active against Hirsutella citriformis, a natural fungal enemy of its host, and the human pathogens Staphylococcus aureus and Candida albicans. Analyses through TLC-bioautography, LC-MS/MS and NMR allowed the identification of five macrocyclic ionophore antibiotics, with previously reported antibacterial, antitumor and antiviral properties. Phylogenetic analyses placed Streptomyces sp. M54 in a clade of other host-associated strains taxonomically related to Streptomyces griseus. Pangenomic and ANI analyses confirm the identity of one of its closest relatives as Streptomyces sp. LaPpAH-199, a strain isolated from an ant-plant symbiosis in Africa. In summary, our results suggest an insect-microbe association in distant geographic areas and showcase the potential of Streptomyces sp. M54 and related strains for the discovery of novel antibiotics.
Assuntos
Actinobacteria , Streptomyces , Vespas , Actinobacteria/genética , Animais , Antibacterianos/farmacologia , Cromatografia Líquida , Humanos , Hypocreales , Filogenia , Streptomyces/genética , Espectrometria de Massas em TandemRESUMO
INTRODUCTION: Comparative analysis of metabolic features of plants has a high potential for determination of quality control of active ingredients, ecological or chemotaxonomic purposes. Specifically, the development of efficient and rapid analytical tools that allow the differentiation among species, subspecies and varieties of plants is a relevant issue. Here we describe a multivariate model based on LC-MS/MS fingerprinting capable of discriminating between subspecies and varieties of the medicinal plant Chamaecrista nictitans, a rare distributed species in Costa Rica. METHODS: Determination of the chemical fingerprint was carried out on a LC-MS (ESI-QTOF) in negative ionization mode, main detected and putatively identified compounds included proanthocyanidin oligomers, several flavonoid C- and O-glycosides, and flavonoid acetates. Principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA) and cluster analysis of chemical profiles were performed. RESULTS: Our method showed a clear discrimination between the subspecies and varieties of Chamaecrista nictitans, separating the samples into four fair differentiated groups: M1 = C. nictitans ssp. patellaria; M2 = C. nictitans ssp. disadena; M3 = C. nictitans ssp. nictitans var. jaliscensis and M4 = C. nictitans ssp. disadena var. pilosa. LC-MS/MS fingerprint data was validated using both morphological characters and DNA barcoding with ITS2 region. The comparison of the morphological characters against the chemical profiles and DNA barcoding shows a 63% coincidence, evidencing the morphological similarity in C. nictitans. On the other hand, genetic data and chemical profiles grouped all samples in a similar pattern, validating the functionality of our metabolomic approach. CONCLUSION: The metabolomic method described in this study allows a reliably differentiation between subspecies and varieties of C. nictitans using a straightforward protocol that lacks extensive purification steps.
Assuntos
Chamaecrista/química , Chamaecrista/metabolismo , Metabolômica/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Análise por Conglomerados , Análise Discriminante , Análise Multivariada , Fenóis/química , Análise de Componente Principal/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Three new diketopiperazines (1-3), cyclo(l-Pro-d-trans-Hyp) (1), cyclo(l-Pro-d-Glu) (2), and cyclo(d-Pro-d-Glu) (3) and five known diketopiperazines (4-8) were isolated from the endolichenic fungus Colpoma sp. CR1465A identified from the Costa Rican plant Henriettea tuberculosa (Melatomataceae). The structures of the new compounds 1-3 were elucidated using a combination of extensive spectroscopic analyses, including 2D NMR and HR-MS, and their absolute configurations were determined by a combination of NOESY analysis and Marfey's method. Cyclo(l-Pro-d-allo-Thr) (4) was recently isolated from a South China Sea marine sponge Callyspongia sp., but its NMR spectroscopic data were not reported, and cyclo(l-Pro-l-Asp) (5) was previously reported but only as a synthetic product. The NMR data assignments of compounds 4 and 5 are reported for the first time. All of the isolated compounds were tested for antifungal and antimicrobial properties.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Callyspongia/química , Dicetopiperazinas/química , Dicetopiperazinas/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Animais , Antibacterianos/química , Antifúngicos/química , Costa Rica , Avaliação Pré-Clínica de Medicamentos/métodos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade MicrobianaRESUMO
Methods to identify the bioactive diversity within natural product extracts (NPEs) continue to evolve. NPEs constitute complex mixtures of chemical substances varying in structure, composition, and abundance. NPEs can therefore be challenging to evaluate efficiently with high-throughput screening approaches designed to test pure substances. Here we facilitate the rapid identification and prioritization of antimalarial NPEs using a pharmacologically driven, quantitative high-throughput-screening (qHTS) paradigm. In qHTS each NPE is tested across a concentration range from which sigmoidal response, efficacy, and apparent EC50s can be used to rank order NPEs for subsequent organism reculture, extraction, and fractionation. Using an NPE library derived from diverse marine microorganisms we observed potent antimalarial activity from two Streptomyces sp. extracts identified from thousands tested using qHTS. Seven compounds were isolated from two phylogenetically related Streptomyces species: Streptomyces ballenaensis collected from Costa Rica and Streptomyces bangulaensis collected from Papua New Guinea. Among them we identified actinoramides A and B, belonging to the unusually elaborated nonproteinogenic amino-acid-containing tetrapeptide series of natural products. In addition, we characterized a series of new compounds, including an artifact, 25-epi-actinoramide A, and actinoramides D, E, and F, which are closely related biosynthetic congeners of the previously reported metabolites.
Assuntos
Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/farmacologia , Streptomyces/química , Antimaláricos/química , Produtos Biológicos/química , Costa Rica , Sedimentos Geológicos/química , Biologia Marinha , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/química , Papua Nova Guiné , Filogenia , Plasmodium falciparum/efeitos dos fármacos , Streptomyces/genéticaRESUMO
Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales (26% of 169 total), while the majority of actinomycetes belonged to the genus Streptomyces (86% of 241 total). Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, α, ß and γ-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (Anthostomella sp.), two Streptomyces and one Bacillus bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.
Assuntos
Actinobacteria/classificação , Bactérias Aeróbias/classificação , Parede Celular/metabolismo , Besouros/microbiologia , Fungos/classificação , Células Vegetais/metabolismo , Actinobacteria/enzimologia , Actinobacteria/isolamento & purificação , Animais , Bactérias Aeróbias/enzimologia , Bactérias Aeróbias/isolamento & purificação , Besouros/anatomia & histologia , Besouros/classificação , Costa Rica , DNA Bacteriano/análise , DNA Fúngico/análise , Fungos/enzimologia , Fungos/isolamento & purificação , Intestinos/microbiologia , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNARESUMO
From the methanol root extract of Godmania aesculifolia, a species selected in a multinational OAS program aimed at discovering antifungal compounds from Latin American plants, a new chavicol diglycoside (1), the known 3,4-dihydroxy-2-(3-methylbut-2-en-1-yl)-3,4-dihydronaphthalen-1(2H)-one (2), and lapachol (3) were isolated and characterized by 1D and 2D NMR and MS techniques. Only 3 exhibited fairly good activity against a panel of clinical isolates of Cryptococcus neoformans (MIC50 between 7.8 and 31.2 µg/mL) and moderate activities against Candida spp. and non-albicans Candida spp.
Assuntos
Anisóis/isolamento & purificação , Antifúngicos/isolamento & purificação , Bignoniaceae/química , Glicosídeos/isolamento & purificação , Naftoquinonas/isolamento & purificação , Derivados de Alilbenzenos , Anisóis/química , Anisóis/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Aspergillus/efeitos dos fármacos , Candida/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Glicosídeos/química , Glicosídeos/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Microsporum/efeitos dos fármacos , Estrutura Molecular , Naftoquinonas/química , Naftoquinonas/farmacologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Trichophyton/efeitos dos fármacosRESUMO
Targeting and inhibiting CMG2 (Capillary Morphogenesis Gene protein 2) represents a new strategy for therapeutic agents for cancer and retinal diseases due to CMG2's role in blood vessel growth (angiogenesis). A high throughput FRET (Förster Resonance Energy Transfer) assay was developed for the identification of CMG2 inhibitors as anti-angiogenetic agents. Bioassay-guided separation led to the isolation and identification of two new compounds (1 and 2) from CR252M, an endophytic fungus Coccomyces proteae collected from a Costa Rican rainforest, and one known compound (3) from CR1207B (Aurapex penicillata). Secondary in vitro assays indicated anti-angiogenic activity. Compound 3 inhibited the endothelial cell migration at 52 µM, but did not show any endothelial cell antiproliferative effect at 156 µM. The structure of the two new compounds, A (1) and B (2), were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D NMR experiments.
Assuntos
Ascomicetos/química , Proteínas de Membrana/antagonistas & inibidores , Fenóis/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Costa Rica , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Humanos , Estrutura Molecular , Fenóis/química , Fenóis/isolamento & purificação , Receptores de Peptídeos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
In this study, 137 actinomycetes were isolated from subtidal marine sediments in the North Pacific and Caribbean coasts of Costa Rica. Bioinformatics analysis of the 16S rRNA gene sequences assigned the isolates to 15 families and 21 genera. Streptomyces was the dominant genus while the remaining 20 genera were poorly represented. Nearly 70% of the phylotypes presented a coastal-restricted distribution whereas the other 30% were common inhabitants of both shores. The coastal tropical waters of Costa Rica showed a high diversity of actinomycetes, both in terms of the number of species and phylogenetic composition, although significant differences were observed between and within shores. The observed pattern of species distribution might be the result of several factors including the characteristics of the ecosystems, presence of endemic species and the influence of terrestrial runoff.
Assuntos
Actinobacteria/classificação , Actinobacteria/isolamento & purificação , Biodiversidade , Sedimentos Geológicos/microbiologia , Actinobacteria/genética , Actinobacteria/crescimento & desenvolvimento , Oceano Atlântico , Análise por Conglomerados , Costa Rica , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Oceano Pacífico , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Natural concentration of antimalaric components in Tropical arthropods (in vitro). Alcohol, hexane and dichlorometane extracts of 751 samples of Costa Rican arthropods were studied for the presence of antimalaric components. With Plasmodium berghei we set an in vitro model in which the effect of the extract was determined by staining of the parasites with cresil brilliant blue. Active extracts at concentration of 50 mg or less, were considered positive. Promissory extracts were found in the orders Lepidoptera (24.1%), Coleoptera (32.8%), Hemiptera (38.5%) and Polydesmida (81.3%). Since most of the Lepidoptera samples were in the immature stages, the relation with the host plant was analyzed. Cannaceae, Flacourtiaceae, Crisobalanaceae, Lauraceae, Fagaceae, Ulmaceae, Rosaceae, Asteraceae, Rubiaceae, Lauraceae and Caprifoliaceae were related with the Lepidoptera larvae, and an antimalaric effect has been reported in most of these families. In the orders Polydesmida, Opiliones and Blattodea, the extract from adults also had some important effect, probably because all of them fed on plants. Polydesmida and Opiliones have chemical substances that probably serve as defensive purposes; these chemicals could also have some antiparasitic effect. Therefore, the detection of antimalaric components in arthropod species led to the identification of plants with promissory antimalaric components. Rev. Biol. Trop. 56 (2): 473-485. Epub 2008 June 30.
Extractos alcohólicos, hexánicos y diclorometánicos de 751 muestras de artrópodos fueron estudiados por la presencia de actividad antimalárica. En este trabajo se empleó un modelo murino usando el Plasmodium berghei, modelo que es biológicamente similar a la malaria humana. El estudio fue realizado determinando el efecto del extracto sobre el parásito por la inclusión o no del colorante azul de cresil brillante. Estimando como positivos aquellos extractos cuya actividad antimalárica se mostró en concentraciones no mayores de 50 mg, se encontró que los órdenes más promisorios fueron Lepidoptera (24.1%), Polydesmida (81.3%), Blattodea (25%) y Opiliones, entre otros. Las formas inmaduras de Lepidoptera fueron las más positivas, por lo que se analizaron las plantas hospederos de donde se alimentaban dichos organismos. Las familias de estas plantas eran Malvaceae, Acanthaceae, Rutaceae, Myrtaceae, Solanaceae, Fabaceae, Urticaceae, Anacardiaceae, Rosaceae, Asteraceae, Rubiaceae, Lauraceae y Caprifoliaceae. Especies de casi todas estas familias han sido reportadas con actividad antimalárica. En el caso de los órdenes Polydesmida, Opiliones y Blattodea, cuyas formas adultas presentaron alguna actividad contra P. berghei, encontramos que todos esos grupos se alimentan también de plantas. En el caso de Opiliones sus especies son predadores de lepidópteros, coleópteros, hemípteros fitófagos y otros artrópodos, además de que producen sustancias de defensas tales como alcoholes, cetonas y quinonas, entre otros, todo lo cual podría explicar la actividad encontrada. Algunas especies del Orden Polydesmida, también secretan ciertas sustancias químicas, las cuales podrían tener un efecto antiparasitario. Así, a través de este trabajo en artrópodos hemos llegado a identificar fuentes vegetales potenciales para componentes antimaláricos.
Assuntos
Animais , Camundongos , Antimaláricos/farmacologia , Artrópodes/química , Comportamento Alimentar/fisiologia , Malária/tratamento farmacológico , Extratos Vegetais/farmacologia , Extratos de Tecidos/farmacologia , Antimaláricos/isolamento & purificação , Artrópodes/classificação , Artrópodes/fisiologia , Modelos Animais de Doenças , Extratos de Tecidos/isolamento & purificaçãoRESUMO
Alcohol, hexane and dichlorometane extracts of 751 samples of Costa Rican arthropods were studied for the presence of antimalaric components. With Plasmodium berghei we set an in vitro model in which the effect of the extract was determined by staining of the parasites with cresil brilliant blue. Active extracts at concentration of 50 mg or less, were considered positive. Promissory extracts were found in the orders Lepidoptera (24.1%), Coleoptera (32.8%), Hemiptera (38.5%) and Polydesmida (81.3%). Since most of the Lepidoptera samples were in the immature stages, the relation with the host plant was analyzed. Cannaceae, Flacourtiaceae, Crisobalanaceae, Lauraceae, Fagaceae, Ulmaceae, Rosaceae, Asteraceae, Rubiaceae, Lauraceae and Caprifoliaceae were related with the Lepidoptera larvae, and an antimalaric effect has been reported in most of these families. In the orders Polydesmida, Opiliones and Blattodea, the extract from adults also had some important effect, probably because all of them fed on plants. Polydesmida and Opiliones have chemical substances that probably serve as defensive purposes; these chemicals could also have some antiparasitic effect. Therefore, the detection of antimalaric components in arthropod species led to the identification of plants with promissory antimalaric components.
Assuntos
Antimaláricos/farmacologia , Artrópodes/química , Comportamento Alimentar/fisiologia , Malária/tratamento farmacológico , Extratos Vegetais/farmacologia , Extratos de Tecidos/farmacologia , Animais , Antimaláricos/isolamento & purificação , Artrópodes/classificação , Artrópodes/fisiologia , Modelos Animais de Doenças , Camundongos , Extratos de Tecidos/isolamento & purificaçãoRESUMO
We have previously identified a crude extract of the plant Chamaecrista nictitans (Fabaceae) with antiviral activity against herpes simplex virus. The main objectives of this research were to identify the step of the replication cycle of herpes simplex inhibited by the extract, and to attempt to characterize the chemical characteristics of this extract. The crude extract from--Chamaecrista nictitans (Fabaceae) was extracted with a mixture of diclorometane/methanol, and further fractionated following a bioassay-guided protocol using a combination of preparative thin layer and column chromatography. Toxicity and bioassay experiments were carried out in monolayers of Vero cells. The antiviral activity of the extract was assessed by total inhibition of cytopathic effect after three-day incubation. The highest concentration of the extract which was not toxic to the cells was 200 ptg/ml. Western blot and immunofluorescence techniques were used to elucidate the antiviral mechanism of the extract by infecting Vero cells with the virus at different times and monitoring the synthesis of viral proteins. A 60 kDa protein was detected at 2 hr and 8 hr post-infection but no additional proteins were synthesized at later time intervals, and cytopathic effect was not observed after 24 hr. This result indicates that the extract acts at the intracellular level in order to inhibit late transcription. However, it does not inhibit transcription/translation of early viral proteins. These results were confirmed by immunofluorescence experiments. A strong fluorescent signal was observed in control cell monolayers at 24 hr post infection, accompanied with a clear cytopathic effect. In contrast, in the presence of acyclovir or the extract, cells showed very discrete immunofluorescence, characterized by a punctuated pattern, and no cytopathic effect was observed. Neutralization assays were performed using pre-incubation of virus with either specific herpes simplex-1 antiserum, 200...
Assuntos
Animais , Antivirais/farmacologia , Fabaceae/química , Replicação Viral , Simplexvirus , Aciclovir/farmacologia , Chlorocebus aethiops , Células Vero , Testes de Sensibilidade MicrobianaRESUMO
We have previously identified a crude extract of the plant Chamaecrista nictitans (Fabaceae) with antiviral activity against herpes simplex virus. The main objectives of this research were to identify the step of the replication cycle of herpes simplex inhibited by the extract, and to attempt to characterize the chemical characteristics of this extract. The crude extract from--Chamaecrista nictitans (Fabaceae) was extracted with a mixture of diclorometane/methanol, and further fractionated following a bioassay-guided protocol using a combination of preparative thin layer and column chromatography. Toxicity and bioassay experiments were carried out in monolayers of Vero cells. The antiviral activity of the extract was assessed by total inhibition of cytopathic effect after three-day incubation. The highest concentration of the extract which was not toxic to the cells was 200 ptg/ml. Western blot and immunofluorescence techniques were used to elucidate the antiviral mechanism of the extract by infecting Vero cells with the virus at different times and monitoring the synthesis of viral proteins. A 60 kDa protein was detected at 2 hr and 8 hr post-infection but no additional proteins were synthesized at later time intervals, and cytopathic effect was not observed after 24 hr. This result indicates that the extract acts at the intracellular level in order to inhibit late transcription. However, it does not inhibit transcription/translation of early viral proteins. These results were confirmed by immunofluorescence experiments. A strong fluorescent signal was observed in control cell monolayers at 24 hr post infection, accompanied with a clear cytopathic effect. In contrast, in the presence of acyclovir or the extract, cells showed very discrete immunofluorescence, characterized by a punctuated pattern, and no cytopathic effect was observed. Neutralization assays were performed using pre-incubation of virus with either specific herpes simplex-1 antiserum, 200 microg/ml of the extract or 20 microg/ml of acyclovir. After 1 hr incubation, cells were infected and monitored for cytopathic effect. Only the virus treated with acyclovir showed viral activity, while no cytopathic effect was induced by samples of virus incubated with the extract. It is concluded that the extract inhibits both the attachment of the virus to the cell and the secondary transcription of the virus within the cells. Chemical characterization of the extract showed the presence of tannins.