Exploration of photoprotective and antibiotic activity of wild Polypodiaceae ferns from Costa Rica.

Skin disorders affect millions of people all over the world. There are limited options to treat dermal illnesses such as vitiligo, psoriasis, and atopic dermatitis (eczema). Central American ferns are a potential source of bioactive metabolites against those diseases. Currently, Polypodium leucotomos Poir. is the only one being commercially utilized for this purpose. In this work, we evaluated the concentration of the skin bioactive compounds: quinic and chlorogenic acid, in the extract of 20 wild ferns from Costa Rica. We also evaluated the antimicrobial capabilities of the crude extracts of wild ferns and the sun protection factor (SPF) of the extracts. We found 19 out of 20 have either an important concentration of the compounds mentioned above or antimicrobial properties. Also, most samples result in higher SPF than P. aureum's rhizome. We also have studied the fern acclimatization, at different shading conditions, finding a significant influence of the culturing conditions on metabolite production. After acclimatization. So far, we demonstrate that various ferns included in this study are a potential source of treatments for skin conditions.

Chemistry and Bioactivity of Microsorum scolopendria (Polypodiaceae): Antioxidant Effects on an Epithelial Damage Model.

Microsorum scolopendia (MS), which grows on the Chilean island of Rapa Nui, is a medicinal fern used to treat several diseases. Despite being widely used, this fern has not been deeply investigated. The aim of this study was to perform a characterization of the polyphenolic and flavonoid identity, radical scavenging, antimicrobial, and anti-inflammatory properties of MS rhizome and leaf extracts (RAE and HAE). The compound identity was analyzed through the reversed-phase high-performance liquid chromatography (RP-HPLC) method coupled with mass spectrometry. The radical scavenging and anti-inflammatory activities were evaluated for DPPH, ORAC, ROS formation, and COX inhibition activity assay. The antimicrobial properties were evaluated using an infection model on Human Dermal Fibroblast adult (HDFa) cell lines incubated with Staphylococcus aureus and Staphylococcus epidermidis. The most abundant compounds were phenolic acids between 46% to 57% in rhizome and leaf extracts, respectively; followed by flavonoids such as protocatechic acid 4-O-glucoside, cirsimaritin, and isoxanthohumol, among others. MS extract inhibited and disaggregated the biofilm bacterial formed and showed an anti-inflammatory selective property against COX-2 enzyme. RAE generated a 64% reduction of ROS formation in the presence of S. aureus and 87.35% less ROS in the presence of S. epidermidis on HDFa cells. MS has great therapeutic potential and possesses several biological properties that should be evaluated.

Floristic and functional diversity of ferns and lycophytes at three elevational zones in the eastern slopes of the northern Andes, Ecuador / Diversidad florística y funcional de helechos y licófitos en tres zonas de elevación en la ladera oriental del noreste de los Andes, Ecuador

The northeastern slope of the Andes is an area of high diversity of ferns and lycophytes. In this study we assessed the diversity patterns of ferns and lycophytes in ten 250 m x 2 m plots installed at three elevational zones, from 418 to 3447 m.a.s.l., in the Napo province of Ecuador. Floristic diversity was measured using Hill numbers and by partitioning species diversity. Three functional diversity indices were calculated from a set of five leaf traits and weighted by species abundance: functional richness (FRic), evenness (FEve) and divergence (FDiv). To disentangle functional diversity from species richness, the standardized effect size (SES) of each index was also calculated. We recorded 148 species, Polypodiaceae and Dryopteridaceae being the most representative families. Species richness continuously decreased with elevation. Floristic composition was highly heterogeneous among elevation zones, with total species turnover found between 1500 and 2000 m.a.s.l. SES-FDiv and SES-FEve were higher than the null expectation for plots at mid-elevations, suggesting that deterministic mechanisms, such as biotic or abiotic filters, could explain assemblage composition at these sites. However, SES-FEve, SES-FRic and SES-FDiv did not differ from the null expectations at low and high elevation sites, suggesting the predominance of a stochastic process. By combining floristic and functional diversity, we were able to infer the observed community patterns with the resource-use strategies of fern and lycophytes in a mountain area, an approach that can be used to understand how assemblages might react to changing environmental conditions.(AU)

La vertiente noreste de los Andes del Ecuador tiene alta diversidad de helechos y licófitos. En este estudio describimos patrones de diversidad de estos grupos en diez parcelas de 250 m x 2 m instaladas en tres zonas altitudinales, de 418 a 3447 m.s.n.m., en la provincia de Napo, Ecuador. La diversidad se calculó utilizando los números de Hill y particionando la diversidad de especies. Tres índices de diversidad funcional fueron estimados a partir de cinco atributos de las hojas ponderados por la abundancia de las especies: riqueza (FRic), uniformidad (FEve) y divergencia (FDiv). Para separar la diversidad funcional de la riqueza, se calculó el tamaño del efecto estandarizado (SES) de cada índice. Registramos 148 especies, siendo Polypodiaceae y Dryopteridaceae las familias más representativas. La riqueza disminuyó continuamente en el gradiente altitudinal. La composición de la comunidad tuvo un cambio total de especies entre 1500 y 2000 m.s.n.m. En parcelas intermedias, SES-FDiv y SES-FEve registraron valores más altos de lo esperado por el modelo nulo, lo que sugiere que los mecanismos deterministas, como los filtros bióticos y abióticos, podrían explicar el ensamble en estas comunidades. En parcelas de altitudes bajas y altas los tres índices no difirieron del valor nulo, lo que sugiere que predominan procesos estocásticos. Al combinar composición florística y diversidad funcional, pudimos identificar patrones de diversidad y estrategias de uso de recursos de helechos y licófitos. Este enfoque se puede utilizar para comprender cómo el ensamble de las comunidades es afectado por la variación en las condiciones ambientales.(AU)

Toxicity assessment of saline extract and lectin-rich fraction from Microgramma vacciniifolia rhizome.

Microgramma vacciniifolia is broadly used in folk medicine but safety information is unavailable. Therefore, we evaluated the toxicity of a saline extract and a lectin-rich fraction of M. vacciniifolia rhizome. The extract showed hemolytic activity on mice erythrocytes at 1000 µg/mL, whereas the fraction promoted hemolysis (8.57-26.15%) at all tested concentrations (10-1000 µg/mL). Acute toxicity test in mice indicated an LD50 of >5000 mg/kg. Hematological alterations and increased serum alkaline phosphatase level were observed in the treated animals. Transaminases and urea levels increased in the groups treated with the extract or fraction at 5000 mg/kg. Leukocyte infiltration was observed in the liver of extract-treated animals and in the liver and lungs of mice treated with the fraction. The kidneys of animals treated with the fraction at 5000 mg/kg presented hydropic degeneration. The extract and fraction did not induce oxidative stress in the liver and did not show genotoxicity, as examined by micronucleus and comet assays. In conclusion, the preparations were not lethal to mice but caused some signs of toxicity, mainly the fraction. The results indicated the need to evaluate the toxicity of M. vacciniifolia rhizome in other models and in chronic assays.

Ontogenia de los esporangios y esporogénesis del helecho Phymatosorus scolopendria (Polypodiaceae) / Ontogeny of sporangia and sporogenesis of the fern Phymatosorus scolopendria (Polypodiaceae)

Introducción: Las investigaciones sobre ontogenia de los soros, esporangios, paráfisis receptaculares y esporogénesis de los helechos leptosporangiados son escasas en la literatura científica. Objectivos: Describiry analizar la ontogenia de los soros, esporangios, paráfisis receptaculares y esporogénesis de Phymatosorus scolopendria. Métodos: Entre marzo y mayo 2017 (época lluviosa del año) se recolectaron frondas fértiles de P. scolopendria en el campus de la Universidad de Antioquia, Medellín-Colombia.Las frondas fértiles, en diferentes etapas del desarrollo se fijaron y procesaron de acuerdo a protocolos estándar para la inclusión y corte en parafina y resina. Las secciones de 0.5 µm obtenidas en resina se tiñeron con azul de Toluidina que tiñe diferencialmente paredes primarias y secundarias, resalta núcleos celulares, y esporopolenina y de manera secundaria tiñe polifenoles. Para descripciones detalladas, otros cortes se tiñeron con Safranina-azul de alciano que discrimina entre componentes de pared primaria, secundaria, núcleos, cutícula y polifenoles; Hematoxilina-azul de alciano para resaltar núcleos y paredes primarias y Fluoroglucinol ácido para detectar lignina. Las observaciones y registro fotográfico se efectuaron con microscopio fotónico. Para la observación y descripción con microscopía electrónica de barrido (MEB), los soros se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Resultados: Los soros son exindusiados, superficiales, vascularizados y de desarrollo mixto, se encuentran asociados a paráfisis receptaculares multicelulares uniseriadas. Durante el desarrollo del soro primero se diferencian las células epidérmicas receptaculares que darán origen a los esporangios y posteriormente las células que originarán a las paráfisis receptaculares. El esporangio es de tipo leptosporangio de pedicelos largos de una o dos filas de células. Los anillos de los esporangios muestran paredes secundarias con engrosamientos en forma de "U" ricos en lignina. La meiosis es simultánea y las tétradas de esporas se disponen de forma decusada o tetragonal. El tapete celular es inicialmente uniestratificado pero por una división mitótica de tipo periclinal, se torna biestratificado. Las células del estrato interno del tapete pierden la integridad estructural dando origen a un tapete plasmodial que invade los esporocitos en meiosis, el estrato externo persiste hasta la etapa de esporas maduras. En las diferentes etapas de desarrollo del esporodermo, primero se forma el exosporio, compuesto por esporopolenina, seguida del endosporio, conformado por celulosa, pectina y polisacáridos carboxilados y finalmente el perisporio. Los polifenoles fueron detectados, principalmente, en las vacuolas de las células de los esporangios, paráfisis y células receptaculares. Para el momento de la liberación de las esporas, tanto la capa externa del tapete celular como el plasmodial han degenerado por completo. En la cavidad esporangial se aprecian orbículas adyacentes a las esporas. Conclusiones: la ontogenia de los esporangios y esporogénesis de P. scolopendria es similar al descrito previamente para helechos leptosporangiados. Adicionalmente, se indica que las paráfisis receptaculares presentes en los soros de P. scolopendria tienen la función de protección de los esporangios durante las primeras etapas del desarrollo.

Introduction: Research about the ontogeny of sori, sporangia, receptacular paraphyses and sporogenesis of leptosporangiate ferns are scarce in the scientific literature. Objectives: To describe and analyze the ontogeny of sori, sporangia, receptacular paraphyses and sporogenesis of Phymatosorus scolopendria. Methods: Fertile fronds of P. scolopendria were collected in the campus of the Universidad de Antioquia, Medellín, Colombia, during the months March and May (annual rain season) of 2017. The fertile fronds of the samples at different developmental stages were fixed and processed according to the standard protocols for embedding and sectioning in paraffin and resin. Sections of 0.5 µm obtained in resin were stained with Toluidine blue, which differentially stains primary and secondary walls, highlights the cell nucleus and sporopolenin and secondarily stains polyphenols. For detailed descriptions, additional sections were processed with Safranin-Alcian blue, allowing the distinction of components of primary and secondary walls, nuclei, cuticle and polyphenols; Hematoxylin-Alcian blue to enhance nuclei and primary walls and Phloroglucinol-HCl for lignin. Observations and photographic records were done with a photonic microscope. For the observations and descriptions with scanning electron microscopy (SEM), the sori were dehydrated with 2,2-dimethoxypropane, critical point dried and coated with gold. Results: The sori are exindusiate, superficial, vascularized and have mixed development; they are associated with uniseriate and multicellular receptacle paraphyses. During the development of the sori, the epidermal cells of the receptacle that will form the sporangia are the first differentiated followed by those forming the receptacle paraphyses. The sporangium is leptosporangiate, with long stalks formed by one or two cell rows. The annulus of the sporangia displays secondary walls with U-shaped thickenings rich in lignin. The meiosis is simultaneous and the spore tetrads are arranged in a decussate or tetragonal shape. The cellular tapetum is initially unistratified but becomes bistratified after a periclinal division. The cells of the internal strata of the cellular tapetum loose structural integrity giving rise to a plasmodial tapetum that invades the meiotic sporocytes. During the sporoderm development, the sporopollenin-composed exospore is the first formed followed by the endospore, composed by cellulose, pectin and carboxylated polysaccharides; the process ends with the perispore. Polyphenols were mainly detected on vacuoles in cells of the sporangium, paraphysis and receptacle. When the time comes for the spore maturation, the remnants of cellular and the plasmodial tapeta have fully degenerated. Abundant orbicles are seen near the spores in the sporangial cavity. Conclusions: The ontogeny of the sporangia and sporogenesis of P. scolopendria are similar to the previously described for leptosporangiate ferns. Furthermore, in P. scolopendria, the receptacle paraphyses of the sori have a role protecting the sporangium during the early development stages.

Samambaias epífitas em trilhas do Parque Guairacá, município de Paula Freitas, Paraná / Epiphytic ferns in trails of the Guairacá Park, municipality of Paula Freitas, Paraná state

Os epífitos desempenham importantes funções ecológicas, e em particular as samambaias, mostram uma diversidade significativa em ambientes florestais, ocupando microambientes específicos e diversos. A diversidade de samambaias epifíticas no estado do Paraná ainda é relativamente pouco conhecida. A região de Paula Freitas, em particular, não apresenta estudos específicos para este grupo de plantas. Neste sentido, o presente estudo teve por finalidade avaliar a riqueza de espécies de samambaias epífitas em regiões de trilhas no Parque Municipal Guairacá em Paula Freitas, Paraná. Foram registradas 10 espécies distribuídas em oito gêneros e quatro famílias. A família Polypodiaceae foi a mais rica na área avaliada (oito espécies), reflexo das características morfoanatômicas comuns às espécies da família. Os resultados encontrados contribuem com o reconhecimento das características florísticas e ecológicas de samambaias epífitas ocorrentes no estado do Paraná.(AU)

Epiphytes play important ecological role, and the ferns shows a significant diversity in the forest environments, due to the numerous specific and diverse microenvironments. The diversity of epiphytic ferns in Paraná state is still relatively unknown. The Paula Freitas region, in particular, does not present specific studies for this group of plants. In this sense, the present study aimed to evaluate the diversity of epiphytic ferns in regions of trails in the Guairacá Municipal Park in Paula Freitas, Paraná. Ten species in eight genera and four families were recorded. The family Polypodiaceae (eight species) was the richest in the evaluated area, reflecting the morphoanatomic characteristics common to the family species. The results contribute to the recognition of the floristic and ecological characteristics of epiphytic ferns occurring in the state of Paraná.(AU)

Ontogenia e histoquímica de los esporangios y escamas receptaculares del helecho epífito Pleopeltis macrocarpa (Polypodiaceae) / Ontogeny and histochemistry of sporangia and receptacle scales of the epiphytic fern Pleopeltis macrocarpa (Polypodiaceae)

Introducción: Las investigaciones sobre la ontogenia de los esporangios y más aún, de la estructura y función de las escamas receptaculares presentes en los soros de algunas especies de helechos, son escasos en la literatura científica. Objetivos: Describir y analizar la ontogenia de los esporangios y las escamas receptaculares de Pleopeltis macrocarpa. Metodología: Durante marzo y mayo de 2017 se recolectaron frondas fértiles de esta especie en los troncos de árboles en el vivero El Edén de las Flores en el municipio de Rionegro, Antioquia-Colombia. Las muestras se fijaron y procesaron de acuerdo a protocolos estándar para la inclusión y corte en parafina y resina. Las secciones obtenidas en resina (0.5 µm) se tiñeron con azul de Toluidina. Para descripciones adicionales sobre la anatomía e histoquímica se aplicaron reactivos específicos para determinar paredes primarias, secundarias, núcleos, lignina, polifenoles, polisacáridos, sustancias pécticas y celulosa. Las observaciones y registro fotográfico se efectuaron con microscopio fotónico y microscopía de epifluorescencia. Para observaciones con microscopía electrónica de barrido (MEB), los soros se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Resultados: Los soros son superficiales, vascularizados y de desarrollo mixto y están cubiertos por escamas receptaculares que se desprenden con la maduración de los esporangios. El esporangio de tipo leptosporangio tiene pedicelos largos de paredes primarias, anillos de los esporangios muestran paredes secundarias con engrosamientos en forma de "U" ricos en lignina. Las células epidérmicas de los receptáculos originan a los esporangios y las escamas receptaculares. Los eventos de división mitótica de estas dos estructuras son inicialmente similares, pero luego divergen para la diferenciación reproductiva y vegetativa de estos dos órganos. La meiosis es simultánea y las tétradas de esporas se disponen de forma decusada o tetragonal. El tapete celular es inicialmente uniestratificado pero por una división mitótica se torna biestratificado. Las células del estrato interno del tapete se rompen dando origen a un tapete plasmodial. En el desarrollo del esporodermo, primero se forma el exosporio, compuesto por esporopolenina, luego el endosporio compuesto de celulosa, pectina y polisacáridos carboxilados y finalmente el perisporio. Los resultados histoquímicos y de epifluorescencia indican que las paredes celulares tanto de los esporangios como las escamas receptaculares inmaduras son de naturaleza celulósica. Al madurar, estas estructuras, así como las células de la pared del esporangio mantienen esta composición. En tanto que las células epidérmicas de los escudos de las escamas receptaculares maduras se caracterizan por mostrar cutícula engrosada. Los polifenoles están presentes durante todas las etapas de desarrollo de los esporangios y escamas receptaculares. Los almidones son abundantes en etapas tempranas del desarrollo en las células del receptáculo y primordios de los esporangios. Conclusiones: La ontogenia de los esporangios de P. macrocarpa es similar al descrito para helechos leptosporangidos. Las escamas receptaculares son estructuras principalmente de protección, su morfología y composición de las paredes celulares evitan la desecación o perdida de humedad en los esporangios durante las etapas lábiles de su desarrollo. Estos resultados concuerdan con la función de protección atribuida a las escamas peltadas pluricelulares presentes en las estructuras vegetativas de algunas especies de helechos y angiospermas tolerantes a la sequía.

Introduction: The ontogeny of sporangia and furthermore the structure and function of the receptacle scales showed by the sori of some fern species are topics scarcely represented in the scientific literature. Objectives: To describe and analyze the ontogeny of sporangia and receptacle scales of Pleopeltis macrocarpa. Methods: During March and April of 2017, fertile fronds of P. macrocarpa were collected from tree stems located in the plant nursery "El Edén de las flores", municipality of Rionegro, Antioquia, Colombia. The samples were fixed and processed according to the standard protocols for embedding and sectioning in paraffin and resin. Sections obtained in resin (0.5 µm) were stained with Toluidine blue. The additional descriptions of the anatomy and histochemistry required specific reagents, applied for the determination of primary walls, secondary walls, nuclei, lignin, polyphenols, polysaccharides, pectic substances and cellulose. The observations and photographic records were performed by photonic and epifluorescence microscopy. For the scanning electron microscopy (SEM) technique, the sori were dehydrated with 2,2- Dimethoxypropane, dried to critical point and coated with gold. Results: The sori are superficial, vascularized and have mixed development, covered by receptacle scales that detach as the sporangia reaches maturity. The leptosporangiate type sporangium have long stalks of primary walls, the annulus of the sporangia shows secondary walls with "U" shaped thickenings rich in lignin. The epidermal cells of the receptacle originate the sporangia and receptacle scales. The mitotic division events of these two structures are initially similar, but then diverge for the reproductive and vegetative differentiation of these two organs. Meiosis is simultaneous and the spore tetrads are arranged in a decussate or tetragonal shape. The cellular tapetum is initially unstratified but becomes bistratified by mitotic division. The inner layer of the tapetum cells break originating a plasmodial tapetum. During the sporoderm development, the first structure formed is the exospore, composed of sporopolenin, followed by the endospore composed of cellulose, pectin and carboxilated polysaccharides, and finally the perispore. The histochemistry and epifluorescence results indicate that both the sporangia and immature receptacle scales have cell walls of cellulosic. These structures as well as those of the sporangium wall cells maintain its composition during maturation. Whereas, the epidermal wall cells of the shields from the mature receptacle scales are characterized by thickened cuticle. The polyphenols are present during all the development stages of the sporangia and receptacle scales. Starch is abundant in the early stages of development of the receptacle cells and sporangial primordia. Conclusions: The ontogeny of the sporangia of P. macrocarpa is similar to the described for leptosporangiate ferns. The receptacle scales are mainly protective structures, its morphology and cell wall composition prevent desiccation or humidity loss of the sporangia during the labile stages of development. These results agree with the protective function attributed to the peltated pluricellular scales present in the vegetative structures of drought tolerant species of ferns and angiosperms.

Morfogénesis de la fase sexual de los helechos epífitos Microgramma mortoniana y Pleopeltis macrocarpa (Polypodiaceae) de la Reserva Natural Punta Lara, Buenos Aires, Argentina / Sexual morphogenesis phase of the epiphytic ferns Microgramma mortoniana and Pleopeltis macrocarpa (Polypodiaceae) from Punta Lara Natural Reserve, Buenos Aires, Argentina

Resumen Pleopeltis macrocarpa y Microgramma mortoniana son dos helechos epífitos de la familia Polypodiaceae hallados con baja frecuencia en la Reserva Natural Punta Lara (Buenos Aires, Argentina). El objetivo de este trabajo fue estudiar la morfogénesis de gametófitos epífitos para fortalecer poblaciones vulnerables y contribuir a su conservación. Se recolectaron hojas fértiles. Las esporas se sembraron en placas de Petri con medio de cultivo Dyer. Las esporas se caracterizaron por ser monoletes, elipsoidales, amarillentas y con ornamentación verrucosa. La germinación fue del tipo Vittaria y el desarrollo del gametofito es del tipo Drynaria. El esporofito de P. macrocarpa surgió a los 500 días y el de M. mortoniana a los 120. El patrón de germinación, el desarrollo del gametofito, el glóbulo lipídico en la célula protálica y los pelos unicelulares capitados en el margen podrían considerarse en la sistemática del grupo. La demora en la formación de esporofitos a través de la reproducción sexual, permite inferir que el éxito de su propagación estaría sujeto a la reproducción vegetativa.

Abstract The Punta Lara Natural Reserve is located on the riverside of the La Plata River in the province of Buenos Aires, Argentina. It is the Southernmost relict in the world of subtropical riparian forest. The epiphytic ferns studied in this work belong to the Polypodiaceae family: Microgramma mortoniana and Pleopeltis macrocarpa. Plant communities are subject to high levels of anthropization and introduction of exotic species. The goals of this work are to provide information on the morphogenesis of epiphytic gametophytes and to extend knowledge of their life cycles, contributing to their conservation. Sowing was carried out in Dyer medium. In both species the spores are monolete, ellipsoidal, yellowish and with verrucate sculpture. The equatorial diameter is 60-61 μm, the polar diameter is 39-42 μm. The germination is the Vittaria type; in M. mortoniana occurs at 20 days, while in P. macrocarpa occurs at six days. The filaments are uniseriate of 3-6 cells in length. The gametophyte development is Drynaria type. The cordated form is given after 40 days. In M. mortoniana, buds originated after 40 days. In P. macrocarpa, after 120 days, clathrate trichomes scale-like appear mainly on the margins of the gametophyte. The gametangia are typical of leptosporangiate ferns. The sporophyte of M. mortoniana emerged after 120 days and that of P. macrocarpa arose after 500 days, its blades are simple, spatulate and unicellular and multicellular branching hairs were observed. The germination pattern, gametophyte development, the presence of a lipid globule in the prothalic cell and the formation of unicellular capitated hairs are relevant characters that could be considered for systematic group. The delay in the formation of sporophytes through sexual reproduction, allows us to infer that the success of their establishment in situ would be given by the vegetative reproduction through creeping rhizomes and buds of gametophytes. Rev. Biol. Trop. 66(3): 1078-1089. Epub 2018 September 01.

Microgramma vacciniifolia (Polypodiaceae) fronds contain a multifunctional lectin with immunomodulatory properties on human cells.

In this study, we report the purification and characterization of a multifunctional lectin (MvFL) from Microgramma vacciniifolia fronds as well as its immunomodulatory properties on human peripheral blood mononuclear cells (PBMCs). MvFL (pI 4.51; 54kDa) is a glycoprotein able to inhibit trypsin activity and that has sequence similarities (32% coverage) with a plant RNA-binding protein. Hemagglutinating activity of MvFL was not altered by heating at 100°C for 30min, but was reduced in alkaline pH (8.0 and 9.0). Fluorimetric analyses showed that this lectin did not undergo marked conformational changes when heated. However, the MvFL conformation changed depending on the pH. MvFL at 6.25-25µg/mL was not cytotoxic to lymphocytes present among PBMCs. The PBMCs incubated for 24h with the lectin (12.5µg/mL) showed increased TNF-α, IFN-γ, IL-6, IL-10, and nitric oxide production. MvFL also stimulated T lymphocytes from PBMCs to differentiate into CD8+ cells. The activation (indicated by CD28 expression) of these cells was also stimulated. In conclusion, MvFL is a heat-stable and multifunctional protein, with both lectin and trypsin inhibitor activities, and capable of inducing predominantly a Th1 response in human PBMCs as well as activation and differentiation of T lymphocytes.

Antimicrobial, cytotoxic, and anti-inflammatory activities of Pleopeltis polylepis.

AIM OF THE STUDY: Pleopeltis polylepis (Polypodaceae) is a fern used in the traditional Mexican medicine to treat fever, bleeding, typhoid, cough, pertussis, chest pain, and renal and hepatic diseases. The aim of this study was to analyze the bioactivities of different extracts, fractions and isolated compounds from this species to scientifically validate its medicinal applications. MATERIALS AND METHODS: Aerial parts of P. polylepis were macerated and extracted consecutively with hexane, chloroform, and methanol. These extracts were subsequently fractionated and compounds from hexane and methanol extracts were purified. The antimicrobial activity was assessed using a panel of eight Gram-positive and -negative bacterial and four fungal strains. The cytotoxicity of the compounds was assessed by flow cytometry using propidium iodide and the human-derived monocytic cell line THP-1. The anti-inflammatory activity was investigated by measuring the secretion of interleukin-6 and IL-10 using also the cell line THP-1. RESULTS: Various extracts, fractions and compounds obtained from this plant showed antibacterial activity against both Gram-positive and -negative strains. Antifungal activity was confirmed only in Candida albicans and Tricophyton mentagrophytes. Two fractions and two isolated compounds (butyl myristate and ß-sitosterol) showed no significant cytotoxicity and were further evaluated for their anti-inflammatory activity. All four samples tested showed an anti-inflammatory activity similar to prednisone used as a control. CONCLUSIONS: The benefit of P. polylepis as a traditional plant related to its antimicrobial and anti-inflammatory activities was confirmed by in vitro assays. To the best of our knowledge, this is the first study reporting the isolation and bioactivities of extracts, fractions or isolated compounds from P. polylepis.

Vascular epiphytes in a remnant of seasonal semideciduous forest in zona da mata, State of Minas Gerais, Brazil / Epífitas vasculares em um remanescente de floresta estacional semidecidual na zona da mata, Minas Gerais, Brasil

This study was performed in a remnant of Seasonal Semideciduous Forest at Fazenda Fortaleza de Sant'Anna, in the municipality of Chácara, Minas Gerais, Brazil (22.0129S, 43.8628W), in an area of ca. 1 ha, via monthly expeditions throughout the year 2012. We found 91 species of vascular epiphytes, distributed in 44 genera and 12 families. The richest families were Orchidaceae (35 spp.), Bromeliaceae (18 spp.), Polypodiaceae (10 spp.), Piperaceae (nine spp.) and Araceae (seven spp.). Two species were recorded for the first time in Minas Gerais: Rodriguezia sticta and Stelis oligantha (Orchidaceae). Rhipsalis oblonga (Cactaceae) was recollected in the state after about 100 years without records. Four species are cited in the list of endangered plants in Minas Gerais: Nematanthus crassifolius (Gesneriaceae) and Cattleya bicolor (Orchidaceae) are in the category "Vulnerable" (VU), Nidularium azureum (Bromeliaceae) and Pleurothallis pectinata (Orchidaceae) are "Critically Endangered" (CR). Nidularium azureum is also cited as CR in the Red Book of Brazilian Flora. Most species were classified as characteristic holoepiphytes and anemochory was the most frequent dispersion syndrome.

O estudo foi realizado em um remanescente de Floresta Estacional Semidecidual, na Fazenda Fortaleza de Sant'Anna, município de Chácara, Minas Gerais, Brasil (22,0129S, 43,8628W), em uma área aproximada de 1 ha, através de expedições mensais durante o ano de 2012. Foram encontradas 91 espécies de epífitas vasculares, distribuídas em 44 gêneros e 12 famílias. As famílias mais ricas foram Orchidaceae (35 spp.), Bromeliaceae (18 spp.), Polypodiaceae (10 spp.), Piperaceae (nove spp.) e Araceae (sete spp.). Duas espécies foram registradas pela primeira vez para Minas Gerais: Rodriguezia sticta e Stelis oligantha (Orchidaceae). Rhipsalis oblonga (Cactaceae) foi recoletada no estado depois de cerca de 100 anos sem registros. Quatro espécies estão presentes na lista de plantas ameaçadas de extinção em Minas Gerais: Nematanthus crassifolius (Gesneriaceae) e Cattleya bicolor (Orchidaceae) na categoria "Vulnerável" (VU), Nidularium azureum (Bromeliaceae) e Pleurothallis pectinata (Orchidaceae) como "Criticamente em Perigo" (CR). Nidularium azureum também é citado como CR no Livro Vermelho da Flora do Brasil. A maioria das espécies foi classificada como holoepífita característica e a síndrome de dispersão mais frequente foi a anemocoria.

Pesticidal and pest repellency activities of rhizomes of Drynaria quercifolia (J. Smith) against Tribolium castaneum (Herbst).

BACKGROUND: Tribolium castaneum (Herbst) is a harmful pest of stored grain and flour-based products in tropical and subtropical region. In the present study, rhizome of Drynaria quercifolia (J. Smith) was evaluated for pesticidal and pest repellency activities against T. castaneum, using surface film method and filter paper disc method, respectively. In addition, activity of the isolated compound 3,4-dihydroxybenzoic acid was evaluated against the pest. RESULTS: Chloroform soluble fraction of ethanol extract of rhizome of D. quercifolia showed significant pesticidal activity at doses 0.88 to 1.77 mg/cm(2) and significant pest repellency activity at doses 0.94 to 0.23 mg/cm(2). No pesticidal and pest repellency activity was found for petroleum ether, ethyl acetate and methanol soluble fractions of ethanol extract as well as for 3,4-dihydroxybenzoic acid. CONCLUSION: Considering our findings it can be concluded that chloroform soluble fraction of rhizome of D. quercifolia is useful in controlling T. castaneum of stored grain and flour-based products.

Morphometric differences of Microgramma squamulosa (Kaulf.) de la Sota (Polypodiaceae) leaves in environments with distinct atmospheric air quality.

Plants growing in environments with different atmospheric conditions may present changes in the morphometric parameters of their leaves. Microgramma squamulosa (Kaulf.) de la Sota is a neotropical epiphytic fern found in impacted environments. The aims of this study were to quantitatively compare structural characteristics of leaves in areas with different air quality conditions, and to identify morphometric parameters that are potential indicators of the effects of pollution on these plants. Fertile and sterile leaves growing on isolated trees were collected from an urban (Estância Velha) and a rural (Novo Hamburgo) environment, in Rio Grande do Sul, Brazil. For each leaf type, macroscopic and microscopic analyses were performed on 192 samples collected in each environment. The sterile and fertile leaves showed significantly greater thickness of the midrib and greater vascular bundle and leaf blade areas in the rural environment, which is characterized by less air pollution. The thickness of the hypodermis and the stomatal density of the fertile leaves were greater in the urban area, which is characterized by more air pollution. Based on the fact that significant changes were found in the parameters of both types of leaves, which could possibly be related to air pollutants, M. squamulosa may be a potential bioindicator.

Drynaria total flavonoids decrease cathepsin K expression in ovariectomized rats.

This study investigated the effects of Drynaria total flavonoids on cathepsin K serum concentrations and gene expression, biomechanics and bone mineral density (BMD) of the tibial shaft in ovariectomized rat models of osteoporosis, and mechanism in the prevention and cure of osteoporosis. Seventy-two female Sprague-Dawley rats were divided into six groups. The rats in each group were subjected to gastric lavage after the model was established. The tibial shaft of the right hindlimb was obtained to measure the BMD. Serum cathepsin K concentrations were determined. The cathepsin K mRNA expression was also determined using fluorescent quantitative polymerase chain reaction. The three-point bending method was performed to measure the maximum bending load of the tibial shaft. The total flavonoid and normal groups had significant differences in serum cathepsin K concentrations compared with that in the estrogen group (P<0.05). The total flavonoid and sham-operated groups also showed significant differences in cathepsin K mRNA expression compared with that in the normal group (P<0.01). The maximum bending load of the rats in the total flavonoid group was significantly different from that in the estrogen group (P<0.05) and the sham-operated and normal groups (P<0.01). The high-dose total flavonoid group elicited a better effect on BMD than that by the medium- and low-dose groups (P<0.05). Thus, Drynaria total flavonoids inhibited the serum cathepsin K concentration and increased the maximum bending load of the tibial shaft in ovariectomized rats.

Toxic effects of Microgramma vacciniifolia rhizome lectin on Artemia salina, human cells, and the schistosomiasis vector Biomphalaria glabrata.

The present study evaluated the toxicity of Microgramma vacciniifolia rhizome lectin (MvRL) to Artemia salina, human tumour cell lines (larynx epidermoid carcinoma Hep-2, NCI-H292 lung mucoepidermoid carcinoma, and chronic myelocytic leukaemia K562), and normal peripheral blood mononuclear cells (PBMCs), as well as to Biomphalaria glabrata embryos and adults. MvRL was toxic to A. salina (LC50=159.9 µg/mL), and exerted cytotoxic effects on NCI-H292 cells (IC50=25.23 µg/mL). The lectin (1-100 µg/mL) did not affect the viability of K562 and Hep-2 tumour cells, as well as of PBMCs. MvRL concentration of 1, 10, and 100 µg/mL promoted malformations (mainly exogastrulation) in 7.8%, 22.5%, and 27.7% of embryos, respectively, as well as delayed embryo development in 42.0%, 69.5%, and 54.7% of embryos, respectively. MvRL at a concentration of 100 µg/mL killed B. glabrata embryos (17.7%) and adults (25%). Further, MvRL damaged B. glabrata reproductive processes, which was evidenced by observations that snails exposed to the lectin (100 µg/mL) deposited fewer eggs than those in the control group, and approximately 40% of the deposited eggs exhibited malformations. Comparison of these results with that from A. salina assay indicates that MvRL is adulticidal at the concentration range which is toxic to environment. In conclusion, the cytotoxicity of MvRL on tumour cell and absence of toxicity to normal cell indicate its potential as chemotherapeutic drug. Also, the study revealed that the lectin is able to promote deleterious effects on B. glabrata embryos at environmentally safe concentrations.

Pesticidal and pest repellency activities of rhizomes of Drynaria quercifolia (J. Smith) against Tribolium castaneum (Herbst)

BACKGROUND: Tribolium castaneum (Herbst) is a harmful pest of stored grain and flour-based products in tropical and subtropical region. In the present study, rhizome of Drynaria quercifolia (J. Smith) was evaluated for pesticidal and pest repellency activities against T. castaneum, using surface film method and filter paper disc method, respectively. In addition, activity of the isolated compound 3,4-dihydroxybenzoic acid was evaluated against the pest. RESULTS: Chloroform soluble fraction of ethanol extract of rhizome of D. quercifolia showed significant pesticidal activity at doses 0.88 to 1.77 mg/cm² and significant pest repellency activity at doses 0.94 to 0.23 mg/cm². No pesticidal and pest repellency activity was found for petroleum ether, ethyl acetate and methanol soluble fractions of ethanol extract as well as for 3,4-dihydroxybenzoic acid. CONCLUSION: Considering our findings it can be concluded that chloroform soluble fraction of rhizome of D. quercifoliais useful in controlling T. castaneum of stored grain and flour-based products.

Leaf Dimorphism Of Microgramma Squamulosa (Polypodiaceae): a qualitative and quantitative analysis focusing on adaptations to epiphytism.

The epiphytic fern Microgramma squamulosa occurs in the Neotropics and shows dimorphic sterile and fertile leaves. The present study aimed to describe and compare qualitatively and quantitatively macroscopic and microscopic structural characteristics of the dimorphic leaves of M. squamulosa, to point more precisely those characteristics which may contribute to epiphytic adaptations. In June 2009, six isolated host trees covered by M squamulosa were selected close to the edge of a semi-deciduous seasonal forest fragment in the municipality of Novo Hamburgo, State of Rio Grande do Sul, Brazil. Macroscopic and microscopic analyzes were performed from 192 samples for each leaf type, and permanent and semi-permanent slides were prepared. Sections were observed under light microscopy using image capture software to produce illustrations and scales, as well as to perform quantitative analyses. Fertile and sterile leaves had no qualitative structural differences, being hypostomatous and presenting uniseriate epidermis, homogeneous chlorenchyma, amphicribal vascular bundle, and hypodermis. The presence of hypodermal tissue and the occurrence of stomata at the abaxial face are typical characteristics ofxeromorphic leaves. Sterile leaves showed significantly larger areas (14.80cm2), higher sclerophylly index (0.13g/cm2) and higher stomatal density (27.75stomata/mm2) than fertile leaves. The higher sclerophylly index and the higher stomatal density observed in sterile leaves are features that make these leaves more xeromorphic, enhancing their efficiency to deal with limited water availability in the epiphytic environment, compared to fertile leaves.

Leaf dimorphism of Microgramma squamulosa (Polypodiaceae): a qualitative and quantitative analysis focusing on adaptations to epiphytism

The epiphytic fern Microgramma squamulosa occurs in the Neotropics and shows dimorphic sterile and fertile leaves. The present study aimed to describe and compare qualitatively and quantitatively macroscopic and microscopic structural characteristics of the dimorphic leaves of M. squamulosa, to point more precisely those characteristics which may contribute to epiphytic adaptations. In June 2009, six isolated host trees covered by M. squamulosa were selected close to the edge of a semi-deciduous seasonal forest fragment in the municipality of Novo Hamburgo, State of Rio Grande do Sul, Brazil. Macroscopic and microscopic analyzes were performed from 192 samples for each leaf type, and permanent and semi-permanent slides were prepared. Sections were observed under light microscopy using image capture software to produce illustrations and scales, as well as to perform quantitative analyses. Fertile and sterile leaves had no qualitative structural differences, being hypostomatous and presenting uniseriate epidermis, homogeneous chlorenchyma, amphicribal vascular bundle, and hypodermis. The presence of hypodermal tissue and the occurrence of stomata at the abaxial face are typical characteristics of xeromorphic leaves. Sterile leaves showed significantly larger areas (14.80cm²), higher sclerophylly index (0.13g/cm²) and higher stomatal density (27.75stomata/mm²) than fertile leaves. The higher sclerophylly index and the higher stomatal density observed in sterile leaves are features that make these leaves more xeromorphic, enhancing their efficiency to deal with limited water availability in the epiphytic environment, compared to fertile leaves.

El helecho epífito Microgramma squamulosa se encuentra en el Neotrópico y tiene hojas estériles y fértiles dimorfas. El objetivo de este estudio fue describir y comparar cuantitativa y cualitativamente la organización estructural de las hojas de la M. squamulosa, investigando las características morfológicas y anatómicas, y señalando los factores que contribuyen a la adaptación al ambiente epífito. Los análisis macroscópicos y microscópicos se realizaron a partir de 192 muestras de hojas de cada tipo. Láminas permanentes y semi-permanentes fueron preparadas y las secciones fueron observadas en el microscopio de luz. Hojas fértiles y estériles no mostraron diferencias estructurales cualitativas, son hipostomáticas, presentan epidermis uniseriada, haz vascular anficrival e hipodermis. La presencia de hipodermis y aparatos estomáticos en la nsuperficie abaxial de la epidermis son típicas de hojas xeromórficas. Hojas estériles presentaran áreas significativamente mayores, tasa más alta de esclerofilia y mayor densidad de estomas que hojas fértiles. La mayor tasa de esclerofilia y mayor densidad de estomas son características que hacen las hojas estériles más xeromórficas, aumentando su eficiencia para hacer frente a la disponibilidad de agua limitada en el ambiente epífito, en comparación con las hojas fértiles.

Long-term male aggregations of Euglossa melanotricha Moure (Hymenoptera: Apidae) on fern fronds Serpocaulon triseriale (Pteridophyta: Polypodiaceae).

A communal dormitory of male orchid bees, Euglossa melanotricha Moure, was monitored over a one-year period, when they passed the night in the fronds of a Serpocaulon triseriale (Polypodiaceae) fern. The bees used the same fronds continuously, moving to neighboring fronds as senescence set in. As many as 49 males were observed together on any one night, clinging to the midribs on the abaxial surface of up to five fern blades with their mandibles. A number of males returned to the same site to pass the night continuously over a number of months, and were observed making physical contact with one another without provoking agonistic behavior. Males of E. melanotricha appeared to prefer sleeping at sites close to nests and potential sources of odoriferous essences, such as orchids (Orchidaceae).