Submitted by Rafael Santana (rafael.silvasantana@ufpe.br) on 2018-02-20T19:15:38Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Tese Isla 08 02 17.pdf: 1341172 bytes, checksum: b9215289b9e41f4bc9bfda4d3ddbaaff (MD5) / Made available in DSpace on 2018-02-20T19:15:38Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Tese Isla 08 02 17.pdf: 1341172 bytes, checksum: b9215289b9e41f4bc9bfda4d3ddbaaff (MD5) Previous issue date: 2015-02-27 / CAPES / O gênero Phoradendron Nutt. compreende um grande número de espécies hemiparasitas conhecidas popularmente como ervas-de-passarinho. A medicina popular atribui a este gênero algumas propriedades farmacológicas como: cicatrizante, analgésica e anticancerígena. Assim, o objetivo desse trabalho foi realizar um estudo fitoquimico e biológico dos extratos das folhas de Phoradendron microphyllum e Phoradendron mucronatum. Os constituintes químicos dos extratos obtidos por maceração em hexano, diclorometano, clorofórmio e acetato de etila foram analisados por CG/EM. A atividade citotóxica desses extratos foi avaliada utilizando as linhagens celulares HEp-2, NCI-H292, MCF-7. Além desses, foram realizados estudos com os extratos metanólicos das folhas de ambas as espécies. O perfil fitoquímico foi analisado por cromatografia em camada delgada, com diferentes fases móveis e reveladores. O conteúdo de fenóis totais foi determinado pelo método de Folin-Ciocalteu; o teor de flavonóides totais pelo complexo formado entre o flavonóide e o alumínio reagente; o teor de taninos pela precipitação com caseína e a atividade antioxidante pelo método de sequestro do radical DPPH. Para avaliação do potencial hemolítico foram usados eritrócitos de camundongos Swiss (Mus musculus) e nos ensaios de citotoxicidade foram utilizadas as linhagens celulares HEp-2, NCI-H292, MCF-7. A atividade antimicrobiana foi realizada pelo método de difusão em Ágar. Para determinação da toxicidade aguda foram utilizados camundongos Swiss, aos quais foram administrados uma dose de 2.000 mg/kg por via oral. A atividade anti-inflamatória foi analisada pelos métodos de bolsão de ar subcutâneo e permeabilidade vascular induzida por ácido acético. O potencial cicatrizante dos extratos em feridas cutâneas foi realizado em ratos Wistar (Rattus norvegicus) por meio de avaliações histológicas. A análise por CG/EM dos extratos das folhas de P. microphyllum e P. mucronatum, possibilitou detectar 51 constituintes de diferentes classes de compostos e apresentaram atividade citotóxica frente as três linhagens testadas. No extrato metanólico das folhas, o perfil fitoquímico evidenciou: açucares redutores, flavonóides, alcalóides, triterpenos/esteróides, monoterpenos, sesquiterpenos e diterpenos, proantocianidinas e leucoantocianidina. O conteúdo fenólico total dos extratos metanólicos das folhas de P. microphyllum e P. mucronatum foi 479,64 ± 44,07 e 139,42 ± 10,78 mg equivalente de ácido tânico/g dos extratos, respectivamente. O extrato metanólico de P. mucronatum apresentou atividade antioxidante. Os extratos metanólicos se mostraram inativos frente à hemólise das hemácias e apresentaram atividade citotóxica frente à linhagem HEp-2. Não foi evidenciada atividade bacteriana frente às linhagens testadas, exceto para o fungo Candida albicans. No ensaio de toxicidade aguda, os animais apresentaram algumas reações tóxicas, porém não letais na dose administrada. Os extratos metanólicos reduziram a migração celular assim como inibiram o aumento da permeabilidade vascular em camundongos com o tratamento por via oral. O creme contendo os extratos metanólicos das folhas de P. mucronatum e P. microphyllum apresentou efeito cicatrizante, reduzindo a área de contração da ferida. Os resultados obtidos mostram que P. microphyllum e P. mucronatum podem ser uma fonte de metabólitos secundários além de apresentar atividades biológicas significativas. No entanto, estudos posteriores serão necessários para isolar e identificar os principais constituintes químicos assim como os possíveis mecanismos de ação envolvidos nas atividades. / The genus Phoradendron Nutt. comprises a large number of hemiparasite species popularly known as herbs-to-bird. Popular medicine attributes to this genus some pharmacological properties such: healing, analgesic and anti-cancer. Thus, the objective of this work was to carry out a phytochemical and biological study of leaves extracts of Phoradendron microphyllum and Phoradendron mucronatum. The chemical constituents of extracts obtained by maceration in hexane, dichloromethane, chloroform and ethyl acetate were analyzed by GC/MS. The cytotoxic activity of these extracts was evaluated using the Hep-2, NCI-H292, MCF-7 cell lines. In addition, studies were carried out with the methanolic extracts of leaves of both species. The phytochemical profile was analyzed by thin layer chromatography, with different mobile phases and revelators. The content of total phenols was determined by the Folin-Ciocalteu method; the total flavonoid content by the complex formed between the flavonoid and the aluminum reagent; the tannin content by casein precipitation and the antioxidant activity by the DPPH radical sequestration method. In order to evaluate the hemolytic potential, erythrocytes of Swiss mice (Mus musculus) were used and the cell lines HEp-2, NCI-H292, MCF-7 were used in the cytotoxicity assays. The antimicrobial activity was performed by the diffusion method in Agar. To determine the acute toxicity, Swiss mice were given a dose of 2,000 mg/kg orally. The anti-inflammatory activity was analyzed by the methods of pockets of subcutaneous air and vascular permeability induced by acetic acid. The wound healing potential of extracts from cutaneous wounds was performed in Wistar rats (Rattus norvegicus) by means of histological evaluations. GC/MS analysis of leaves extracts of P. microphyllum and P. mucronatum allowed to detect 51 constituents of different classes of compounds and presented cytotoxic activity against the three strains tested. In the methanolic extract of the leaves, the phytochemical profile evidenced: reducing sugars, flavonoids, alkaloids, triterpenes/steroids, monoterpenes, sesquiterpenes and diterpenes, proanthocyanidins and leucoantocianidina. The total phenolics contente of the methanolic extracts of the P. microphyllum and P. mucronatum leaves was 479.64 ± 44.07 and 139.42 ± 10.78 mg tannic acid equivalent/g of the extracts, respectively. The methanolic extract of P mucronatum showed antioxidant activity. The methanolic extracts proved inactive against the erythrocyte hemolysis and showed cytotoxic activity against the HEp-2 lineage. There was no evidence of bacterial activity against the tested strains, except for the fungus Candida albicans. In the acute toxicity test, the animals had some toxic but non-lethal reactions at the dose administered. The methanolic extracts reduced cell migration as well as inhibited increased vascular permeability in mice with oral treatment. The cream containing the methanolic extracts of leaves of P. microphyllum and P. mucronatum showed wound healing effects, reducing wound contraction area. The results obtained show that P. mucronatum and P. microphyllum can be a source of secondary metabolic compounds besides showing significant biological activities. However, further studies will be needed to isolate and identify the main chemical constituents as well as the possible mechanisms of action involved in the activities.

This project arose because of the desire by the resident Ateles research observers, at Santa Rosa, to attempt to ascertain a function for what they deemed ‘uncharacteristic’ behaviours. The following were important considerations meriting further investigation. • Phoradendron (mistletoe) consumption by Ateles spp. appeared to be present only at Santa Rosa; • Mistletoe selection was considered deliberate, as mature host leaf (which was available) was not selected; • More than one mistletoe was available in the normal home range of the monkey, but selection was dominated by one mistletoe/host combination; • Consumption was of relatively small volumes; • There was an apparent seasonality to the use of mistletoe; • Consumption of mature, mistletoe leaf occurred at times when there was no shortage of suitable fruit; • Limitations of the Ateles spp. digestive system made exploitation of mature leaf potentially problematic; • The monkeys were also seen consuming geophageous material; on occasion, this closely followed mistletoe consumption; • There were no published reports of Ateles spp. geophagy in Central America and/or tropical dry forest habitat. The aim of the subsequent investigation was to determine if there was any beneficial function that could be attributed to the materials and so provide a link to a self-mediation hypothesis for mistletoe and/or geophagy or to relate the determined geophagy properties to the other published functional hypotheses for geophagy. The novel aspect of this project was the development of a specific ‘gastric model’ reflecting the differences between Ateles and human digestive systems. This modified model was used to investigate geophagic and Phoradendron samples. It was hoped that this approach would lead to the identification or constituents in the samples, which may have physiological significance. Samples of the two species of mistletoe identified were collected from three Phoradendron/host tree combinations. Samples were extracted using the simulated gastric conditions and the extracts analysed. Analytical ‘fingerprints’ of the gastric extracts of the two species were obtained together with the antimicrobial activities of the extracts. Species variation in Phoradendron constituents and antimicrobial activity was detected. The principal difference between the eaten and non-eaten Phoradendron species was identified using HPLC and LC-MS, as chlorogenic acid. Chlorogenic acid has antibacterial and antioxidant properties, stimulates the immune system and has activities related to regulation of blood sugar levels. Phoradendron consumption wass during the wet season, which may be a period of increased bacterial and parasite infection. It also coincides with a change in dietary fruits. The geophagic samples were taken from sites previously used by Ateles, together with ‘control’ sites found in the home range of the study group. Analysis of the physical properties and characteristics was undertaken to attempt to identify the mineral content material. Further analyses then investigated the behaviour of the material in relation to the commonly accepted hypotheses for geophagy. Where possible these were investigated using the simulated gastric conditions. The physical characteristics of the samples did not resemble the previously published reports for geophagic material used by humans or non-human primates. The results failed to detect the presence of montmorillonite and only a suggestion of the presence of kaolinite. The results do not suggest that it functions as an antacid, an anti-diarrhoeal or mineral supplement. An increase in antibacterial activity was seen when geophagic material and Phoradendron samples were incubated together. A putative hypothesis for the mechanism of Fe limitation was suggested by the physical properties of the geophagic material and the Fe chelating potential of the chemical constituents of the Phoradendron leaf.

Made available in DSpace on 2014-06-12T15:04:43Z (GMT). No. of bitstreams: 2 arquivo4493_1.pdf: 788690 bytes, checksum: 9083fdd72e97b7ffb548d159cd7df2ba (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2002 / Foram analisados os números cromossômicos, a estrutura do núcleo interfásico, o padrão de condensação cromossômica profásica e o comportamento meiótico em 14 espécies brasileiras das famílias Loranthaceae e Viscaceae. Todas as espécies apresentaram núcleos interfásicos reticulados e padrão de condensação profásico uniforme. Os três gêneros estudados de Loranthaceae apresentaram 2n=16 e as três espécies do único gênero analisado de Viscaceae, Phoradendron, apresentaram 2n=28. Apenas três espécies possuíam registro cariológico anterior, sendo as demais contagens, inéditas. A análise do complemento cromossômico mitótico de duas espécies de Phoradendron revelou uma maior assimetria cariotípica, enquanto as espécies dos outros gêneros foram mais similares entre si e mais simétricas. O comportamento meiótico, analisado em 11 espécies, foi geralmente regular, embora tenham sido observadas algumas irregularidades meióticas em muitas amostras. Em uma das amostras de Struthanthus syringifolius foi encontrado um tetravalente em anel na maioria dos meiócitos. Estas irregularidades meióticas parecem não influir na estabilidade dos números cromossômicos nas duas famílias e sugerem que haja uma variabilidade estrutural maior do que a observada por técnicas citológicas convencionais. Os resultados obtidos indicam que as populações brasileiras possuem a mesma constância do número cromossômico por gênero observada em representantes dessas famílias em outras regiões. É sugerido que essa estabilidade cromossômica esteja relacionada à quantidade elevada de DNA nuclear que caracteriza a maioria dessas espécies

The mistletoe Phoradendron californicum is a common parasite on the mesquite plant Prosopis juliflora. Seeds of the parasite are deposited upon the host plant by birds and perhaps other agents. Normally seedlings of the parasite become established when the elongating radicle of the embryo comes into contact with a host branch and forms a holdfast. From the underside of the holdfast the primary haustorium enters the host tissue and establishes the parasitic union. Later the aerial portion of the parasite develops. Some seeds of P. californicum follow a different sequence; they stimulate the host to produce gum in the tissue beneath the seed by apparent dissolution of cells. The exuded gum elevates the developing seed from the host branch preventing establishment of the host-parasite union. The possible significance of this apparent defense mechanism is discussed.

<p>This thesis examines the isolation and characterisation (biological and chemical) of polypeptides from plants. A fractionation protocol was developed and applied on 100 plant materials with the aim of isolating highly purified polypeptide fractions from small amounts of plant materials. The polypeptide fractions were analysed and evaluated for peptide content and biological activities. A multitarget functional bioassay was optimised as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes. In this assay, the neutrophil was challenged with an inflammatory mediator, <i>N</i>-formyl methionyl-leucyl-phenylalanine (fMLP), or with platelet activating factor (PAF), to induce exocytotic release of the enzyme elastase, which then was quantified by photometric determination of the product p-nitroanilide (pNA) formed from a chromogenic substrate for elastase. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation.</p><p>Phoratoxin B and four new peptides, phoratoxins C-F, were isolated from <i>Phoradendron tomentosum</i>. In addition, the cardiac glycoside digitoxin was isolated from <i>Digitalis purpurea</i>. All these substances expressed cytotoxicity and a neutrophil challenging activity.</p><p>Phoratoxins C-F were similar to earlier described phoratoxins A and B, which belong to the group of thionins. All the peptides were evaluated for cytotoxicity in a human cell line panel. Phoratoxin C was the most potent towards the cell lines (mean IC₅₀: 160 nM), and was therefore investigated further on tumour cells from patients. Correlation analysis of the log IC₅₀ values indicated a mechanism of action different from clinically used archetypal cytotoxic drugs. Phoratoxin C also showed selective toxicity to the solid tumours when compared to the haematological cancer types. The phoratoxin C was 18 times more potent towards the solid tumour samples from breast cancer cells (87 nM) compared to the tested haematological malignancies.</p><p>The structure-activity relationship concerning cytotoxicity was evaluated for digitoxin and related cardiac glycosides. Digitoxin was shown to be potent, with the average IC₅₀ 37 nM being within the therapeutic concentration used for cardiac congestion (13-45 nM). Digitoxin expressed selective toxicity towards solid tumours from patients compared to haematological malignancies.</p>

This thesis examines the isolation and characterisation (biological and chemical) of polypeptides from plants. A fractionation protocol was developed and applied on 100 plant materials with the aim of isolating highly purified polypeptide fractions from small amounts of plant materials. The polypeptide fractions were analysed and evaluated for peptide content and biological activities. A multitarget functional bioassay was optimised as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes. In this assay, the neutrophil was challenged with an inflammatory mediator, N-formyl methionyl-leucyl-phenylalanine (fMLP), or with platelet activating factor (PAF), to induce exocytotic release of the enzyme elastase, which then was quantified by photometric determination of the product p-nitroanilide (pNA) formed from a chromogenic substrate for elastase. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation. Phoratoxin B and four new peptides, phoratoxins C-F, were isolated from Phoradendron tomentosum. In addition, the cardiac glycoside digitoxin was isolated from Digitalis purpurea. All these substances expressed cytotoxicity and a neutrophil challenging activity. Phoratoxins C-F were similar to earlier described phoratoxins A and B, which belong to the group of thionins. All the peptides were evaluated for cytotoxicity in a human cell line panel. Phoratoxin C was the most potent towards the cell lines (mean IC50: 160 nM), and was therefore investigated further on tumour cells from patients. Correlation analysis of the log IC50 values indicated a mechanism of action different from clinically used archetypal cytotoxic drugs. Phoratoxin C also showed selective toxicity to the solid tumours when compared to the haematological cancer types. The phoratoxin C was 18 times more potent towards the solid tumour samples from breast cancer cells (87 nM) compared to the tested haematological malignancies. The structure-activity relationship concerning cytotoxicity was evaluated for digitoxin and related cardiac glycosides. Digitoxin was shown to be potent, with the average IC50 37 nM being within the therapeutic concentration used for cardiac congestion (13-45 nM). Digitoxin expressed selective toxicity towards solid tumours from patients compared to haematological malignancies.