Detection of Airborne Allegen Phl p5 and its Correlation with Poaceae Pollen Counts in Evora, South Portugal

Introduction: Airborne pollen of grasses (Poaceae family) is the main aeroallergen in many european countries, namely in Portugal. Their pollen can be found in the air all over the year but higher concentrations are reached in springtime (Mars to June). Exposure to its allergens is deduced from pollen counts of environmental air samples. The aim of this study is to analyse the relationship between these pollen counts and the concentration of allergen Phl p5, through a new sampling technology, developed under an European LIFE/Environment program. Methods: Airborne pollen were monitored with a Hirst-type sampler (Burkard 7-day pollen trap). Simultaneously, daily air samples were collected with a high volumetric cyclone-type sampler (Coriolis ® d by Bertin Technologies, France) and Phl p5 concentration was measured from liquid samples through a “sandwich” ELISA with a kit from Indoor Biotecnologies®. Both samplers were placed side by side on a meteorological platform at the town center of Evora, 17 m above ground level and 320 m above sea level. ELISA analysis were performed on samples collected between the 2th of Mars and 12 th of June 2007, which includes the main pollen season for grasses and Phleum pratense. Results: Pollen counts from both samplers were not correlated, with absolute values higher in Burkard sampler. Phl p5 concentration in air samples followed the same features of cyclone-type sampler but it was quantified even when pollen counts were low or null, particularly in the beginning of pollen season. Several peaks of allergen were detected at the end of March and on the 1st - 2nd of April. Conclusions: Coriolis ® d cyclone sampler allowed the quantification of Poaceae either by pollen counts and by an ELISA assay but further analysis on the efficiency of sampling and its relationship with biophysical parameters are needed. These results suggest that pollen counts may not reflect exposure to Poaceae pollen. Immuno-sampling could provide a better understanding of exposure to airborne pollen allergens, namely out of the pollen season

Projectos e linhas de investigação no âmbito da Bioquímica aplicada às ciências da saúde

Projectos e linhas de investigação no âmbito da Bioquímica aplicada às ciências da saúde CM Antunes Departamento de Química, Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), Universidade de Évora, Largo dos Colegiais 2, 7000 Évora; Centro de Neurociências e Biologia Celular (CNC), Universidade de Coimbra, 3004-517 Coimbra;[email protected] A área da Bioquímica Clínica tem vindo a ser promovida na Universidade de Évora nos últimos anos. Actualmente são três as principais linhas de investigação em curso: i) uma linha de investigação com um âmbito fundamental na área da fisiopatologia da diabetes tipo 2, onde o principal objectivo consiste no estudo das bases bioquímicas subjacentes ao desenvolvimento desta patologia bem como da função das células secretoras de insulina como elemento central desta síndrome; e duas linhas com um âmbito mais aplicado ii) Selecção e caracterização de produtos provenientes de plantas com efeitos hipoglicemiantes; iii) A pesquisa de marcadores bioquímicos de fácil monitorização e/ou que permitam um diagnóstico precoce da doença, onde o estudo da composição da saliva está actualmente a ser iniciado. Neste âmbito está actualmente aprovado um Projecto intitulado “Plantas mediterrânicas como fontes naturais de agentes anti-diabéticos: efeitos e mecanismos de acção no pâncreas e nas glândulas salivares” apoiado pelo ICAAM. Tem vindo a trabalhar nestas linhas de investigação vários estudantes de Licenciatura, Mestrado e Doutoramento, que têm contribuído para a publicação de diversos trabalhos em congressos, bem como alguns Relatórios de Estágio em Bioquímica. Temos ainda procurado estabelecer colaborações nacionais e internacionais. Neste âmbito está actualmente em curso um projecto europeu – HIALINE (EAHC, Ag nº 2008 11 07) – que visa o estabelecimento e certificação de métodos que permitam quantificar alergénios polínicos no ar exterior que possam vir a contribuir para a previsão mais apurada do risco alérgico para a população. Estão actualmente em curso duas teses de Mestrado de estudantes de Bioquímica no âmbito deste projecto

IN VITRO AND IN VIVO REMOVAL OF ORAL ANTIDIABETIC AGENTS (METFORMIN) USING ACTIVATED CARBONS

Diabetes is the most worldwide common chronic disease, according the International Diabetes Federation [1], more than 32 million citizens living in the European Union have diabetes, representing nearly 10% of the population, to which we should add equal number of people suffering from impaired glucose tolerance. Diabetes prevalence is growing at alarming rate worldwide, being of particular relevance the type 2 diabetes. Nowadays 285 million people worldwide live with diabetes and it is expected that this numbers will increase by 20% until 2030 due to obesity and the ageing of the population [1]. This growth leads to an increasing consumption of drugs such as oral antidiabetics. Metformin is one of the active principles most commonly used for this purpose being among the pharmaceuticals with the highest production numbers worldwide to treat type 2 diabetes because is cheap, has high level of tolerance and when used in the prescribed dosage is very secure with minimal side effects. However, in case of overdose of metformin upon a ingestion of more than 10 times the prescribed dosage, accidentally or on propose, lactic acidosis and low blood pressure can occur. Overdoses with metformin are relatively uncommon, but may have serious consequences, if medical attention is not given on time, it may lead to coma and ultimately death Because of its spread use another problem must be taken into consideration, which needs to be addressed, the occurrence of metformin residues in sewage and surface waters due to improper discharge of the non-used tablets to regular garbage [2]. This situation is becoming a serious problem of environmental pollution and public health. This paper reports the use of activated carbon produced from biomass for the removal of metformin in 2 different settings. On one hand, from aqueous solutions and, in another hand, from simulated biological fluids (gastric and intestinal) conjugated with in vivo testing

Chemical composition and anti-diabetic properties of Cytisus multiflorus

Bakground and aims: The interest on plants with potential medicinal properties has been increasing worldwide. In the Iberian Peninsula there are some endemic species known by the population for their pharmacologic activity with valorization potential that have not been yet characterized. The white Spanish broom (Cytisus multiflorus) is described as having anti-diabetic effect [1] and in a preliminary the hypoglycemic and hyper-insulinemic effect of an aqueous extract has been shown [2]. The aim of this work was to fractionate and analyse the composition of the aqueous extract of C. multiflorus flowering parts and evaluate its potential as an anti-diabetic agent. Materials and methods: The aqueous extract was primarily fractionated by SPE using water:methanol (W:Me) eluent (a 10% step-wise gradient W:Me from 100:0 to 0:100) followed by high performance liquid chromatography with diode array detector (HPLC-DAD). The most relevant fraction were analysed by LC-MS to determine the chemical composition. Total fenol content was determined by a modified Folin-Ciocalteau method and the anti-oxidant activity was evaluated by the DPPH mehod. Finally, the hipoglicemic potential was evaluated in vivo using glucose intolerant rats (GIR). Results: Eleven fractions of the bulk extract were obtained. Seven of these fractions (10, 30, 40, 50, 60, 70 e 80% Me) were found to have a relevant compounds, mostly flavonoid compounds, namely, rutin (50, 60 and 70% Me fractions), ferrulic acid (30% Me), referred as having hypoglicemic effect. The fractions obtained with 50 and 70% Me showed the highest content in phenol equivalents and the highest anti-oxidant effect were found in the 50 and 60% Me fractions. The 30 and 60% Me fraction had no effect on the post-prandial glicemia. Conclusions: The 30, 50, 60 and 70% Me fractions, due to their chemical composition and anti-oxidant effects were the most promising to have anti-diabetic effect. However, the 30 and 60% Me were found to be ineffective. The 50% Me fraction showed both a high content of flavonoid compounds and the highest anti-oxidant power which suggest that it may constitute the most promising one. The anti-diabetic properties of this fraction should be investigated. [1] Camejo-Rodrigues J. et al. (2003). J. Ethnopharmacol, 89, 199-209 [2] Célia M. Antunes, Laurinda R. Areias, Inês P. Vieira, Ana C. Costa, M. Teresa Tinoco, & Júlio Cruz-Morais (2009). Rev. Fitoterapia 9 (Supl.1): 91

Comparison between Poaceae Airborne Pollen Counts and Phl p5 Aeroallergen Quantification in South Europe

The European project HIALINE: Comparison between Poaceae Airborne Pollen Counts and Phl p5 Aeroallergen Quantification in South Europe C. Antunes1,2, R. Ferro2, R. Ribeiro2, Torres M.C.4, M.J. Velasco4H. García-Mozo4, Galán, C4 , R.Brandao1,3, M.Thibaudon5, R. Albertini6 Ugolotti. M.6, Usberti I.6, Dall’Aglio P.6 and the HIALINE team7 1DInstitute of Mediterranean Agricultural and Environmental Sciences –ICAAM, University of Évora, Portugal 2Department of Chemistry, University of Évora, Portugal 3Department of Biology, University of Évora, Portugal 4Department of Botany, Ecology and Plant Physiology, University of Córdoba 5Réseau National de l Surveillance Aerobiologique, Saint-Genis-l’Argentière, France 6Department of Clinical Medicine, Nephrology and Health Sciences, University of Parma, Italy 7J.T.M. Buters, Germany, M. Thibaudon, France, M. Smith, Great Britain, C. Galan, Spain, R. Brandao and C.M. Antunes, Portugal, G. Reese, Germany, R. Albertini, Italy, L. Grewling, Poland, A. Rantio-Lehtimäki, Finland, S. Jäger and U. Berger, Austria, I. Sauliene, Lithuania, L. Cecchi, Italy Introduction: Nowadays, pollinosis is affecting a large percentage of population in countries with a western life style. The existence of allergenic activity in the atmosphere is not only associated to pollen grains and fungal spores, but also to submicronic and paucimicronic biological particles. The origin of these allergens can be due to the rupture of pollen transported in the atmosphere or to the presence of allergens from other parts of the plant making amorphous material with an allergen load. Poaceae pollen is recognized as one of the main causes of allergic disease in all Europe. In this study we have tried to compare Poaceae pollen counts in the air and Phl p 5, one of the major allergens of this family, through the use of a high-volume cascade impactor (Chemvol). This study was done in the frame of the European project HIALINE and it compares the results obtained in 2009 by 4 different partners participating in this project: in Córdoba (Spain), Évora (Portugal), Lyon (France) and Parma (Italy). Methodology: Pollen grains were sampled using a 7-day volumetric Hirst type spore trap. Chemvol high-volume cascade impactor equipped with stages PM>10µm, 10 µm>PM>2.5µm was used for detecting aeroallergens. In each stage polyurethane filters were use as an impacting substrate. Phl p 5 allergen was determined using an allergen specific ELISA. Antibodies for analysis were delivered by Allergopharma Joachim Ganzer KG, the industrial partner in this project. At each location both samplers were placed side-by-side. Results: Most of the allergen was collected in the PM>10µm fraction. Similar profiles between airborne pollen and the total allergenic load was observed during the pollen season. A good correlation was obtained between pollen count and allergen content of the air and a value of 2.5 pg/pollen grain of Poaceae was estimated. Discussion: This is the first year of this project. Nevertheless, results suggest that the allergenic load in outdoor air might be mainly due to pollen bursts. It supports the hypothesis that monitoring the allergens itself in ambient air might be an improvement in allergen exposure assessment. This work was supported in part by the European Agency for Health and Consumers EAHC, Luxembourg, under the grant agreement 2008110

The European project HIALINE: Comparison between Poaceae Airborne Pollen Counts and Phl p5 Aeroallergen Quantification in SW Europe

The European project HIALINE: Comparison between Poaceae Airborne Pollen Counts and Phl p5 Aeroallergen Quantification in SW Europe C. Antunes1*, R. Ferro1, R. Ribeiro1, Torres M.C.3, M.J. Velasco3H. García-Mozo3, Galán, C3 , R.Brandao2, M.Thibaudon4 and the HIALINE team5 1Department of Chemistry, University of Évora, Portugal 2Department of Biology, University of Évora, Portugal 3Department of Botany, Ecology and Plant Physiology, University of Córdoba 4Réseau National de l Surveillance Aerobiologique, Saint-Genis-l’Argentière, France 5J.Buters, Germanny, M. Thibaudon, France, M. Smith, Great Britain, C. Galan, Spain, R. Brandao and C.M. Antunes, Portugal, R. Albertini, Italy, L. Grewling, Poland, A. Rantio-Lehtimäki, Finland, S. Jäger and U. Berger, Austria, I. Sauliene, Lithuania, L. Cecchi, Italy * Presenting Author: Antunes, C. Department of Chemistry, University of Évora, Portugal. Phone +351 266745319 ; email: [email protected] Introduction: Nowadays, pollinosis is affecting in a large percentage of population in the developed countries. The existence of allergenic activity in the atmosphere is not only associated to pollen grains and fungal spores, but also to submicronic and paucimicronic biological particles. The origin of these allergens can be due to the rupture of pollen transported in the atmosphere or to the presence of allergens from other parts of the plant making amorphous material with allergenic load. Poaceae pollen is recognized as one of the main causes of allergic disease in all Europe. In this study we have tried to compare Poaceae pollen counts in the air and Phl p 5, one of the major allergens of this family, through the use of a high-volume cascade impactor (Chemvol). This study was done in the frame of the European project HIALINE and it compares the results obtained in 2009 by 3 different partners participating in this project: in Córdoba (Spain), Évora (Portugal) and Lyon (France). Methodology: Pollen grains have been taken out using a 7-day volumetric Hirst type spore trap. Chemvol high-volume cascade impactor equipped with stages PM>10µm, 10 µm>PM>2.5µm were used for detecting aeroallergens. In each stage polyurethane filters were applied. Phl p5 allergen was determined using allergen specific ELISA´s. Antibodies for analysis are delivered by Allergopharma Joachim Ganzer KG, the industrial partner in this project. Both samplers were placed side-by-side. Results: Most of the allergen was collected in the PM>10µm fraction. Similar profiles between airborne pollen and the total allergenic load was observed during the pollen season. A good correlation was obtained and a value of 2.5 pg/pollen grain of Poaceae was estimated. Discussion: These results suggest that the allergenic load in outdoor air might be mainly due to pollen bursts. It supports the hypothesis that monitoring the allergens itself in ambient air might be an improvement in allergen exposure assessment. This work was supported in part by the European Agency for Health and Consumers EAHC, Luxembourg, under the grant agreement 2008110

BENEFÍCIOS DA ILUMINAÇÃO NATURAL EM AMBIENTES ESCOLARES

A iluminação natural, além de ser uma fonte renovável, gratuita e sustentável, possui benefícios que vão além do que é conhecido comumente. Ela tem grande influência na vida do ser humano, tanto fisiologicamente quanto psicologicamente. O Brasil está localizado geograficamente em uma posição privilegiada pela iluminação do sol, porém pouco se aproveita quando o assunto é arquitetura, por diversos fatores sociais e econômicos. O estudo da iluminação dentro das edificações se torna importante, pois além dos benefícios citados para o bem do usuário, a eficiência energética se torna uma consequência. Juntando essas duas vertentes, o presente trabalho estuda a iluminação em ambientes escolares, a influência da mesma sobre a vida dos alunos em seu desempenho escolar e soluções arquitetônicas para que a luz entre no ambiente e seja controlada para que não haja ofuscamento

Efeito Hipoglicemiante de um Extracto Aquoso de Cytisus multiflorus

Efeito Hipoglicemiante de um Extracto Aquoso de Cytisus multiflorus I. P. Vieira1, A. C. Costa1,2, D. M. Teixeira1,2,3, C. M. Antunes1,4 & J. Cruz-Morais1,2 1Departamento de Química; 2Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), 3Laboratório Hércules, Universidade de Évora, Largo dos Colegiais 2, 7000 Évora; 4Centro de Neurociências e Biologia Celular (CNC), Universidade de Coimbra, 3004-517 Coimbra; [email protected] Actualmente o interesse pelas plantas medicinais tem vindo a aumentar pois estas apresentam diversas actividades farmacológicas, entre as quais se inclui o efeito anti-diabético. A flora nativa portuguesa inclui várias plantas às quais se atribuem propriedades hipoglicemiantes, sem que estas tenham, no entanto, sido ainda cientificamente demonstradas. O objectivo deste trabalho foi estudar a possível acção hipoglicemiante de Cytisus multiflorus, uma planta da flora portuguesa tradicionalmente usada como agente etnofarmacológico no tratamento da diabetes. O efeito do extracto aquoso de C. multiflorus foi avaliado em roedores que apresentaram anomalias das curvas de tolerância à glicose oral, seleccionados duma colónia de ratos Wistar. Com este ensaio, determinaram-se as insulinemias pós-prandiais e foram avaliados alguns indicadores serológicos e histológicos de toxicidade [1]. Em resposta a este tratamento com o extracto de C. multiflorus, observou-se uma diminuição significativa das glicemias pós-prandiais dependente da dose. Observou-se também, um aumento dependente da dose das insulinemias pós-prandiais. Deste modo, o extracto aquoso teve um efeito hipoglicemiante, provavelmente devido à estimulação da secreção de insulina, comprovando-se a sua validade como agente etnofarmacológico para o controlo da diabetes tipo 2 [1]. Perante estas evidências, procedeu-se ao fraccionamento do extracto de C. multiflorus, de forma a caracterizá-lo e a identificar as principais famílias de compostos nele presentes. Além disso, pretende-se também avaliar in vitro a potencial acção das fracções, identificando assim a(s) fracção(ões) activa(s) no controlo da diabetes tipo 2. Futuramente, estas fracções irão ser testadas em linhas celulares secretoras de insulina (BRIN-BD11) e/ou estudos in vivo, para avaliar o possível efeito insulinotrópico bem como os mecanismos de acção do extracto. Uma vez identificadas as fracções activas, proceder-se-á à identificação dos principais princípios activos que possam ser responsáveis pela actividade hipoglicemiante e/ou insulinotrópica, utilizando técnicas analíticas como Cromatografia Líquida de Elevada Eficiência (HPLC) com detecção de Diode Array (HPLC-DAD) e de Espectrometria de Massa (LC-MS). [1] C.M. Antunes, L.R. Areias, I.P. Vieira, A.C. Costa, M.T. Tinoco, & J. Cruz-Morais (2009). Rev. Fitoterapia 9 (Supl.1): 91 (Abstract)

Efeito hipoglicemiante de um extracto aquoso de Cytisus multiflorus

Actualmente o interesse pelas plantas medicinais tem vindo a aumentar pois estas apresentam diversas actividades farmacológicas, entre as quais se inclui o efeito anti-diabético. A flora nativa portuguesa inclui várias plantas às quais se atribuem propriedades hipoglicemiantes, sem que estas tenham, no entanto, sido ainda cientificamente demonstradas. O objectivo deste trabalho foi estudar a possível acção hipoglicemiante de Cytisus Multiflorus, uma planta da flora portuguesa tradicionalmente usada como agente etnofarmacológico no tratamento da diabetes (1). O efeito do extracto aquoso de C. multiflorus foi avaliado em roedores que apresentaram anomalias das curvas de tolerância à glicose oral, seleccionados duma colónia de ratos Wistar. O extracto foi preparado a partir das extremidades florais da planta com água por refluxo durante 10 min. O extracto foi filtrado e seco em evaporador rotativo sob pressão e foi armazenado a 4Cº até posterior administração aos animais. Prepararam-se cinco grupos de cinco fêmeas que foram diariamente administrados, com sonda gástrica, com soro fisiológico (controlo negativo), glicazida (controlo positivo) e três doses diferentes de extracto aquoso da planta (40, 110 e 220mg/Kg), durante 28 dias. As glicemias em jejum e pós-prandiais foram medidas durante o teste. No fim do teste, determinaram-se as insulinemias pós-prandiais e foram avaliados alguns indicadores serológicos e histológicos de toxicidade. Em resposta ao tratamento com o extracto de C. multiflorus, observou-se uma diminuição significativa das glicemias pós-prandeais dependente da dose. O extracto induziu um aumento dependente da dose das insulinemias pós-prandiais. O efeito máximo do extracto de C. multiflorus foi semelhante ao observado em resposta à administração de glicazida. As glicemias em jejum não foram significativamente alteradas. Em conclusão, o extracto aquoso teve um efeito hipoglicemiante, provavelmente devido à estimulação da secreção de insulina, comprovando-se a sua validade como agente etnofarmacológico para o controlo da diabetes tipo 2. Agradecimentos: Este trabalho foi apoiado pelo ICAAM e pelo CNC. Referências: 1. Camejo-Rodrigues J. et al. (2003). J. Ethnopharmacol, 89, 199-209

Differential Ole e 1 Release from Olea Airborne Pollen in the Southwest Iberian Peninsula. Results from the HIALINE Study

Background: Ole e 1 is the major allergen of olive pollen (Olea europaea L.), the second largest cause of pollinosis in some areas from the Mediterranean Region. Although it has been assumed that airborne pollen is a representative parameter for allergen exposure, variability of allergen content and/or release from pollen has been demonstrated for other taxa. The aim of this study was to: i) estimate the correlation between daily airborne olive pollen and Ole e 1 in ambient air; ii) evaluate the annual and geographical variation of pollen and allergenic loads in southwest Iberian Peninsula; iii) evaluate the contribution of meteorological parameters to ambient Ole e 1 loads variations. Methods: Airborne Ole e 1 and olive pollen were assessed simultaneously in Cordoba, Spain and Evora, Portugal. Aeroallergens were collected in 2009-2011 using prewashed polyurethane foam as impacting substrate (Rupprecht & Patashnick ChemVol®2400 high-volume cascade impactor, Albany, NY, USA). Flow was adjusted to 800 L/min with a rotameter controlled high-volume pump (Digitel DHM-60, Ludesch, Austria). After extraction, Ole e 1 was quantified by ELISA. Airborne Olea pollen was monitored with a Burkard Hirst type Seven-Day Recording Volumetric SporeTrap®. Both samplers were placed side-by-side with the air input at the same level. Results: The aeroallergen and airborne pollen profiles overlapped during pollen seasons, however, deviations between pollen counts and allergen load were found. Annual pollen index of Olea was 3-4 folds higher in Spain (29,956, 26,274 and 42,223 in Spain versus 12,524, 7,144 and 10,499 in Portugal). A 4-9 fold difference in aeroallergen load was observed (14,375, 18,913 and 20,989 in Portugal and 108,720, 80,972 and 171,248 in Spain). Annual Ole e 1/pollen was 3.1-4.0 in Spain, 0.8-2.6 in Portugal and was positively correlated with precipitation prior to pollen season. Conclusions: These results have shown that Ole e 1 is mostly associated with olive pollen grains but aeroallergen load was not always directly proportional to airborne pollen counts. This suggests that Ole e 1 quantification is a better marker for olive allergen exposure. In conclusion, aeroallergen monitoring may contribute to a better understanding of the Ole e 1 exposure from airborne pollen. Acknowledgments: This study is integrated in the European project HIALINE (Executive Agency for Health and Consumers, grant agreement No 2008 11 07). 1st&2nd author equally contributed to the work