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FFacolta’ di Farmacia

INTERNATIONAL PSE SYMPOSIUMINTERNATIONAL PSE SYMPOSIUM

CHEMICALSCHEMICALS

PH

YT

PH

YT in Nutrition

and Health

in Nutrition

and Health

27-30 27-30 September September 20112011

Giovinazzo Giovinazzo (BARI), (BARI), ItalyItaly

Programme & AbstractsProgramme & Abstracts

INTERNATIONAL PSE SYMPOSIUM

Phytochemicals in Nutrition and Health

Programme & Abstracts

27-30 September 2011 Giovinazzo (BARI), Italy

FACOLTÀ DI FARMACIA

Organizing Committee

Pinarosa Avato, Chairman (Bari, Italy) Maria Pia Argentieri, Treasurer (Bari, Italy)

Francesco Racaniello, Secretariat (Bari, Italy)

Scientific Committee

Giovanni Appendino (Novara, Italy) Randolph Arroo (Leicester, UK)

Pinarosa Avato (Bari, Italy) Lars Bohlin (Uppsala, Sweden)

Veronique Cheynier (Montpellier, France) Massimo Curini (Perugia, Italy) Virginia Lanzotti (Naples, Italy)

Wieslaw Oleszek (Pulawy, Poland) Vassilios Roussis (Athens, Greece) Satya Sarker (Wolverhampton, UK)

Angela Stafford (Sheffield,UK)

http://www.phytochemicalsociety.org/bari

Under the High Patronage and with the financial support of

Università degli Studi di Bari Aldo Moro Facoltà di Farmacia, Università degli Studi di Bari Aldo Moro

Senato della Repubblica Italiana Ministero della Salute

Regione Puglia Città di Bari

Comune di Bitonto – Assessorato al Turismo Fondazione Cassa di Risparmio di Puglia

Federazione Ordini Farmacisti Italiani Ordine dei Farmacisti Provincia di Bari

Ordine dei Farmacisti Provincia di Brindisi Società Italiana di Fitochimica

Aboca S.p.A Indena S.p.A

Apivita Europe Thermo Scientific

Waters Extrasynthese

Farmalabor – Farmacisti Associati Amarelli

Confetteria Museo Mario Mucci F. Divella S.p.A

Battista Luigi Caffè & Bristol S.r.l Biscottificio Panificio Farinella

Programme

Salone delle Stelle Grand Hotel Riva del Sole

Giovinazzo (Bari)

Programme Tuesday –27 September 12.00-15.00 Registration 15.00 Opening and welcome address

Pinarosa Avato, Chairperson Corrado Petrocelli, Rector of “Universitá degli Studi di Bari Aldo Moro” Roberto Perrone, Dean of “Facoltá di Farmacia, Universitá degli Studi di Bari Aldo Moro” Luigi d’Ambrosio Lettieri, Senator of the Italian Republic, Standing Committee for Health; Vice - President of the Italian Federation of Pharmacists Pier Giorgio Zambonin, President of “Societá Chimica Italiana – Sezione Puglia” Luisa Pistelli, Executive Board of “Societá Italiana di Fitochimica” Wieslaw Oleszek, President of “Phytochemical Society of Europe” Chair: Anna-Marja Aura 15.30-16.10 PL1 Giovanni Appendino (University of Piemonte Orientale, Italy) Food-based drug discovery: overcoming the nutrition-medicine divide 16.10-16.30 SL1 Paul Reddell (EcoBiotics Ltd, Australia) Discovery of new nutraceuticals from Australia’s tropical rainforests 16.30-16.50 SL2 Jafar M.I. Alqudah (Dublin Institute of Technology, Ireland) Screening of irish fruit and vegetable germplasm for novel anti-tumour and pesticidal compounds 16.50-17.30 Coffee Break Chair: Giovanni Appendino 17.30-18.10 PL2 Mercedes Muzquiz (SGIT-INIA, Spain) Bioactive compounds in legumes: pronutritive and antinutritive actions. Implications for nutrition and health 18.10-18.30 SL3 Miriana Durante (ISPA-CNR, Italy) Durum wheat by-products as natural sources of valuable nutrients 18.30-18.50 SL4 Lindsay Brown (University of Southern Queensland, Australia) Anthocyanin-enriched purple carrot juice with low carotenoids attenuates cardiovascular and hepatic symptoms induced by high carbohydrate, high fat-feeding in rats 21.00 “Italian Dragees”: After dinner by “Confetteria & Museo Mario Mucci”

Wednesday –28 September Chair: Paul J. Russell

09.00-09.40 PL3 Wolfgang Gelbmann (European Food Safety Authority – EFSA) EFSA’s Role in Novel Foods and Health Claims 09.40-10.00 SL5 Marcello Salvatore Lenucci (University of Salento, Italy) Tomato organic lycopene: the way to obtain a unique healthy product 10.00-10.20 SL6 Franz Bucar (University of Graz, Austria) Isoflavone composition of soy and red clover dietary supplements 10.20-10.40 SL7 Juan Parrado (University of Sevilla, Spain) Antioxidant and anti-inflammatory properties of a new soluble extract from grape pomace 10.40-11.10 Coffee Break Chair: Jean-Luc Wolfender 11.10-11.50 PL4 Veronique Cheynier (INRA, France) Phenolic compounds: from plants to foods 11.50-12.10 SL8 Ana A. Matias (University of Lisboa, Portugal)

Polyphenols-rich extracts obtained from Prunus avium and Opuntia ficus indica as natural anti-inflammatory modulators for Inflammatory Bowel Diseases.

12.10-12.30 SL9 Emilio Jirillo (University of Bari, Italy) Polyphenols from red wine exert potential beneficial effects to human health 12.30-12.50 SL10 Magdalini A. Papandreou (University of Athens, Greece) Differential antioxidant & anti-amyloid effects of a wild bluberry polyphenol-rich extract: in vitro & in vivo studies 13.00-14.00 Lunch Break 14.00-15.00 Free Poster Session Chair: Franz Bucar 15.00-15.40 PL5 Jean-Luc Wolfender (University of Geneva, Switzerland)

New trends in the analysis of natural products for the detailed metabolite profiling of crude extracts

15.40-16.00 SL11 Vera Schulzova (University of Prague, Czech Republic)

Metabolomic profiling and fingerprinting: new effective approach for plant characterisation and authentication

16.00-16.20 SL12 Margherita Zotti (University of Foggia, Italy) Estrous cycle in female rats influences the neurochemical and neurobehavioural effects of carvacrol 16.20-17.00 Coffee Break Chair: Mercedes Muzquiz 17.00-17.40 PL6 Arnaud Bovy (Plant Research International, The Netherlands) Breeding for improved flavour and health 17.40-18.00 SL13 Jiri Gruz (Palacký University, Czech Republic)

Application of purine derivative LGR-1814 improves functional properties of field-grown lettuce (Lactuca sativa)

18.00-18.20 SL14 Anna Szakiel (University of Warsaw, Poland) Comparison of triterpenoid content of Vaccinium vitis-idaea berries and leaves collected from natural forest habitats in Finland and Poland

18.30-20.00 Poster Session 1 Authors presenting posters with odd numbers should be present at their posters 21.00 Visit to the medieval town of Bitonto

Thursday –29 September Chair: Virginia Lanzotti 09.00-09.40 PL7 Søren Balling Engelsen (University of Copenhagen, Denmark)

NMR metabolomics: advantages and disadvantages for studying bioactive components in food

09.40-10.00 SL15 Marlene Monschein (University of Graz, Austria)

Formation of astragaloside IV from acylated astragalosides during extraction of Astragali radix

10.00-10.20 SL16 Wanda Kisiel (Polish Academy of Sciences, Poland) Sesquiterpenoids from aerial parts of Lactuca aculeata 10.20-10.40 SL17 Christine Tschiggerl (University of Graz, Austria) The volatile fraction of herbal teas 10.40-11.10 Coffee Break Chair: Veronique Cheynier 11.10-11.50 PL8 Anna-Marja Aura (VTT, Finland) Phenolic nutritional phytochemicals as biomarkers of plant food intake 11.50-12.10 SL18 Annalisa Tassoni (University of Bologna, Italy) Elicitation, production and release of polyphenols in fed-batch fermentation of grape cells

12.10-12.30 SL19 Susana M. Cardoso (CERNAS, Portugal) Insights into olive mill wastewaters phenolics 12.30-12.50 SL20 Antonella Leone (ISPA-CNR, Italy)

Molecular mechanisms underlie the biological activities of phytochemicals: searching the gap

13.00-14.00 Lunch Break 14.00-15.00 Free Poster Session Chair: Giuseppe Dalessandro 15.00-15.40 PL9 Barbara Ann Halkier (University of Copenhagen, Denmark) Bioengineering of glucosinolates in yeast 15.40-16.00 SL21 Gemma Farrè (University of Lleida, Spain) Corn metabolic engineering by creating a metabolic sink by expressing the Arabidopsis homologue of the Orange (Or) gene 16.00-16.20 SL22 Mrinal K. Maiti (Indian Institute of Technology, India)

Production of nutritionally desirable fatty acids in seed oil of Indian mustard (Brassica juncea L.) by metabolic engineering

16.20-17.00 Coffee Break Chair: Wieslaw Oleszek 17.00-17.40 PSE-APIVITA Award Ceremony with the lecture by Award Winner 17.40-18.00 SL23 Hemant Poudyal (University of Queensland, Australia)

�-Linolenic acid-rich oil from chia seed (Salvia hispanica L.) induces lipid redistribution, heart and liver protection in diet-induced obese rats

18.00-18.20 SL24 Marie-Hélène Teiten (Hôpital Kirchberg, Grand Duchy of Luxembourg)

Proteomic identification of differentially expressed proteins in curcumin- treated prostate cancer cells

18.30-20.00 Poster Session 2 Authors presenting posters with even numbers should be present at their posters 20.30 Symposium Dinner

Friday –30 September Chair: Arnaud Bovy 09.00-09.40 PL10 Virginia Lanzotti (University of Naples, Italy) Bioactive natural compounds from food plants 09.40-10.00 SL25 Olaf-Georg Issinger (University of Southern Denmark, Denmark)

Natural compounds as selective inhibitors for protein kinases and their potential application in cancer research

10.00-10.20 SL26 Mariangela Miele (University of Genova, Italy) Hazel and other angiosperm plants as new sources of compounds with taxane-like activity 10.20-10.50 Coffee Break Chair: Lindsay Brown 10.50-11.30 PL11 Paul J. Russel (Unilever, UK) Naturals for masses - Chemistry approaches for safe commercialisations 11.30-11.50 SL27 Caroline Mathon (Official Food Control Authority, Switzerland)

Targeted screening of botanicals in herbal dietary supplements by LC- MS/MS with an Information Independent Acquisition approach

11.50-12.10 SL28 Angela Mari (University of Salerno, Italy)

LC-MS/MS determination of marker metabolites in the ethanolic extract and in mother tinctures of Vitex agnus castus fruits

12.10-12.30 Closing and Farewell 14.00 Post Symposium guided sightseeing tour to the UNESCO World Heritage town of Alberobello. Buffet with wine tasting.

Oral Presentations

Oral Presentations – International PSE Symposium on ”Phytochemicals in Nutrition and Health”, 27-30 September 2011

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Food-based drug discovery: overcoming the nutrition-medicine divide Giovanni Appendino

Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Università del Piemonte Orientale, Via Bovio 6, 28100 Novara, Italy

Statins, the commercially most successful drugs ever, are derivatives of biological analogues of lovastatin, a constituent of the red yeast of rice, an ingredient of the Oriental cuisine, and food-plant derived constituents have been developed as mainstream drugs (L-DOPA, theophylline). Yet, edible plants have not yet systematically considered as a source of drugs-leads and drugs. The growing erosion of the drug-dietary supplement interface provides now new opportunities to explore the role of food-derived compounds for the complementary management of various pathologies, especially in the realm of chronic-degenerative diseases, either as a stand-alone agent, or in combination with mainstream drugs.

After an overview of the chemical diversity of food plants-derived phytochemicals and their biological targets, recent studies on the identification of the elusive pharmacophore of curcumin and the clinical development of a phospholipid formulation of curcuminoids will be summarized as exemplificative of how modern science can contribute to the advancement of our knowledge in this field.


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Discovery of new nutraceuticals from Australia’s tropical rainforests Paul Reddella, Victoria Gordona, Peter Parsonsb, Gerald Muenchc, Craig Williamsd a EcoBiotics Ltd, PO Box 1, Yungaburra, Queensland, 4884, Australia b Queensland Institute of Medical Research, Royal Brisbane Hospital, Herston, Queensland, 4029, Australia c School of Medicine, University of Western Sydney, Locked Bag 1797 Penrith South DC, NSW 1797, Australia d Department of Chemistry, University of Queensland, St Lucia, 4072, Australia [email protected]

Australia’s tropical rainforests are amongst the most ancient and diverse in the world. They have a flora characterised by high endemism at both genus and species level and comprise a wide range of forest types from humid lowland rainforests through to palm forests, seasonally deciduous rainforests and tropical mountain cloud forests. The combination of a long geographic isolation, high phylogenetic diversity and a wide range of physical environments creates ideal conditions to underpin high and novel chemical diversity as plants evolve new ‘chemical solutions’ to survive and flourish in these complex and dynamic biological systems.

To date, very few of Australia’s rainforest flora have been examined for their potential applications in the food, cosmetics or pharmaceutical industries. For example, of the more than 200 species (from more than 40 plant families) that are known to produce edible fruits, nuts, tubers, flowers and/or leaves, only the macadamia nut (Macadamia integrifolia; family Proteaceae) and lemon myrtle (Backhousia citrodora; family Myrtaceae) have been commercialised as mainstream ingredients in food or cosmetics, while a small number of other species are used as local food products.

This presentation outlines our approach to discovery and early development of new nutraceutical products from this unique flora. We will (i) highlight how an understanding of rainforest ecology can be used to successfully target and predict potential sources of new nutraceuticals with specific bioactivities and (ii) discuss a specific example of assays, biomarkers and veterinary models that we are using to evaluate potential new anti-inflammatory and neuroprotectant nutraceuticals.


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Screening of irish fruit and vegetable germplasm for novel anti-tumor and pesticidal compounds

Alqudah J.M.I.1, 2, Reilly K.2, Gaffney M.T.2, Martin-Diana A.B.3 & Barry-Ryan C.1

1Dublin Institute of Technology, School of Food Science and Environmental Health, Cathal Brugha St, Dublin 1, Ireland.

2Teagasc R&D Centre, Horticulture Development Department, Kinsealy, Dublin 17, Ireland. 3Instituto Technológico Agrario, Junta de Castilla y León, Consejería de Agricultura y Ganadería,

España.

Plant secondary metabolites are a source of potential novel therapeutic and insecticidal agents. This project is focused on screening Irish grown fruit and vegetables for novel bioactive compounds, for use in food production and biopesticides. Bioassays used include lethality to brine shrimp (Artemia salina) larva, a general bioassay for insecticidal and anti-tumour compounds and the inhibition of Agrobacterium tumefaciens crown gall tumours on potato disc slices. To date aqueous extracts of 173 plant samples have been screened using the brine shrimp bioassay (BSB). Six crops showed positive lethality to brine shrimp at a dilution <0.02 v/v, beetroot (root), cranberry (fruit), garlic (bulb), ginger (rhizome), red onion (bulb) and rhubarb (petiole). These samples also showed significant inhibition of crown gall tumours in the potato disc bioassay (PDB) (>20% inhibition). Methanolic extracts of cranberry (fruit), ginger (rhizome) and rhubarb (petiole) showed positive lethality to brine shrimp in the BSB at concentration <1000 ppm. Methanolic extracts of cranberry (fruit) and rhubarb (petiole) showed significant inhibition of crown gall tumours in the PDB (>20%). There was a significant effect shown between aqueous and methanolic extracts in the BSB and PDB. Phenolic compounds in aqueous extracts of the six positive crop tissues were separated and analysed using thin layer chromatography plates sprayed with Folin Denis reagent.

Brine shrimp bioassays were carried out for both aqueous and methanolic extracts from different tissues of known varieties of beetroot ( ‘Pablo’ and ‘Rocket’), red onion (‘Red Spark’, ‘Red Baron’ and ‘Lilia’) and rhubarb (‘Red Early Superb’, ‘Sutton’ and ‘Victoria’). Beetroot and red onion varieties showed negative results in the BSB for both aqueous and methanolic extracts. Aqueous and methanolic extracts of all rhubarb varieties (petiole and leaf) gave positive results in the BSB. The aqueous extracts of petiole and leaf of rhubarb varieties showed significant inhibition of crown gall tumours in the PDB (>20%) apart from ‘Sutton’ (petiole) which gave 11.5% inhibition. Further investigations will allow identification of useful bio-active compounds of plant origin with potential application in the food, pharmaceutical and biopesticide industry.


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PL2 Bioactive compounds in legumes: pronutritive and antinutritive actions. Implications for nutrition and health

Mercedes Muzquiz

Dpto. de Tecnología de Alimentos. SGIT-INIA. 28080 Madrid, Spain [email protected]

Legumes seeds are employed as a protein source for animal and human nutrition not only for

their nutritional value (high in protein, lipids and dietary fibre), but also their adaptability to marginal soils and climates. Human consumption of legumes has been increased in recent years, being regarded as beneficial food ingredients. In fact, they are recommended as staple food by health organisations and dieticians and they can be considered as functional foods [1].

Legume seeds contain a great number of compounds which qualify as phytochemicals with significant potentials benefits to human health (for example, as anticarcinogenic, hypocholesterolemic or hypoglycemic agents). The term phytochemical has also been used and emphasizes the plant source of the majority of these disease-preventing non-nutritive compounds [2].

Antinutritional compounds vary considerably in their biochemistry. They can be proteins (protease inhibitors, α-amylases, lectins), glycosides (α-galactosides, vicine and convicine), tannins, saponins, alkaloids. Hence, methods for their extraction, determination and quantification are specific of each compound. They do not appear equally distributed in all legumes, and their physiological effects are diverse. Some of these compounds are important in plant defence mechanisms against predators or environmental conditions. Others are reserve compounds, accumulated in seeds as energy stores in readiness for germination [3].

Processing generally improves the nutrient profile of legume seed by increasing in vitro digestibility of proteins and carbohydrates and at the same time there are reductions in some antinutritional compounds [4]. Most antinutritional factors are heat-labile, such as protease inhibitors and lectins, so thermal treatment would remove any potential negative effects from consumption. On the other hand tannins, saponins and phytic acid are heat stable but can be reduced by dehulling, soaking, germination and/or fermentation.

However, in order to exert an effect, either local or systemic, these substances have to survive at least to some extent the digestive process within the gastrointestinal tract. New directions in no-nutritional factors research in the last decade have led to major developments in our understanding of their role in nutrition. The scientific interest in no-nutritional factors is now also turning to studies of their possible useful and beneficial applications as gut, metabolic and hormonal regulators and as probiotic/prebiotic agents [5].

�1� de Roos, N. M. (2004) In: Arnoldi, A. (Ed.), Functional Foods: Cardiovascular Disease and Diabetes.Woodhead

Publishing Ltd., Cambridge, pp. 1-9. [2] Hasler CM, Bloch AS, Thomson CA, Enrione E, Manning C (2004). Journal of American Dietetic Association

104: 814-826. �3� Roberts M (1998). In: M.F. Roberts, M. Wink (eds). Alkaloids. Biochemistry, Ecology, and Medicinal

Applications. Plenum Press. New York and London p. 109-146. �4� Hajos Gy, Osagie AU (2004). In: Muzquiz M., Hill G.D., Cuadrado C., Pedrosa M.M., Burbano, C. (eds.)

Proceedings of the fourth international workshop on antinutritional factors in legume seeds and oilseeds, EAAP publication No. 110, Toledo, Spain, pp. 293-305.

�5� Pusztai A.,Bardocz S., Martin-Cabrejas M.A. (2004) In: Muzquiz M., Hill G.D., Cuadrado C., Pedrosa M.M., Burbano, C. (eds.) Proceedings of the fourth international workshop on antinutritional factors in legume seeds and oilseeds, EAAP publication No. 110, Toledo, Spain, pp.87-100.


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Durum wheat by-products as natural sources of valuable nutrients Miriana Durante1, Marcello Lenucci2, Leonardo Rescio3, Giovanni Mita1, Sofia Caretto1

1 Istituto di Scienze delle Produzioni Alimentari (ISPA), CNR , Via Prov. le Lecce-Monteroni, 73100 Lecce, Italy 2 Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di.S.Te.B.A.), Università del Salento, Via Prov. le Lecce-Monteroni, I-73100 Lecce, Italy 3 PIERRE S.r.l., s.s. 476 Km 17, 650 Zona Industriale, I-73013 Galatina (LE), Italy [email protected]

Based on epidemiological studies, the human consumption of whole grains is associated with a

reduced risk of health disorders, such as cancer and diabetes [1]. However, the outer layers of the wheat kernel (bran plus aleurone layer) together with the embryo (germ), which contain several bioactive compounds, are usually removed during the early stages of milling to produce flour or semolina. As a consequence, mostly of the wheat valuable healthy molecules end up in the by-products. Due to the increasing research interest in establishing new production systems of natural vitamin E [2], attention has been focused on wheat milling by-products, which represent one of the richest known natural sources of vitamin E [3]. Being Apulia the leading Italian region in durum wheat (Triticum durum, ) production, this study was carried out to evaluate the possibility of extracting oil from an enriched germ containing by-product of durum wheat milling by supercritical carbon dioxide (SC-CO2). The SC-CO2 technology gives totally solvent free extracts improving product quality, safety and healthiness. Different operative parameters were tested to optimize oil and vitamin E extraction yields. When 300 bar pressure, 50 °C temperature, 4 L/min CO2 flow and 1 hour extraction time were used, an oil yield of ~6 wt %, containing ~4 mg total tocopherols/g, was obtained. A slight improvement (~7 wt % oil; ~5 mg total tocopherols/g oil) was reached when the starting material was oven-dried for 72 hours at 60°C (residual humidity ~3 % ) and reduced to a particle size of 30 mesh. The oil characterization was carried out by HPLC and GC-MS. The -form was the most abundant tocopherol (2.33 mg/g oil), followed by β/γ–tocopherols (1.99 mg/g oil). Among the identified fatty acids, linoleic was definitely the most abundant (~50%), followed by moderate amounts of oleic (~27%) and palmitic (~19%) acids. Finally, carotenoids were present in the SC-CO2 extracted oil as lutein, zeaxanthin and β-carotene. Oil stability was also evaluated under different combinations of storage conditions (25 °C, 4 °C, day-light, darkness) measuring changes in vitamin E content and hydroperoxide production. Altogether these results highlight the importance of wheat milling industry by-products as rich sources of valuable natural nutrients.

[1] Meyer K.A., Kushi L.H., Jacobs D.R. Jr, Slavin J., Sellers T.A., Folsom A.R. (2000). Carbohydrates, dietary fiber,

incident type 2 diabetes mellitus in older women. American Journal of Clinical Nutrition, 71: 921-930. [2] Caretto S., Nisi R., Paradiso A., De Gara L. (2010). Tocopherol production in plant cell cultures.. Molecular

Nutrition and Food Research, 54 (5): 726-730. [3] Atwell W.A. (2001). Wheat Flour. Eagan Press, St. Paul, Minnesota.


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SL4 Anthocyanin-enriched purple carrot juice with low carotenoids attenuates cardiovascular and hepatic symptoms induced by high carbohydrate, high fat feeding in rats Lindsay Browna and Hemant Poudyalb

aDepartment of Biological and Physical Sciences, University of Southern Queensland, Toowoomba 4350, AUSTRALIA; bSchool of Biomedical Sciences, The University of Queensland, Brisbane, 4072, AUSTRALIA [email protected]

Purple carrots (Dacus carota L.) are rich in anthocyanins such as cyanidin-3-(2ʹʹ-xylose-6ʹʹ-

sinapoyl-glucose-galactoside) imparting the distinctive colour [1, 2]. In the current study, we have investigated whether administration of purple carrot juice (5% of the diet; 15.2 mg/kg/d) for the last 8 weeks of a 16 weeks protocol reverses the structural and functional changes in the heart and liver in rats fed a high carbohydrate, high fat diet (HCHF) (*P<0.05; n=8-10/group). In this model of diet-induced metabolic syndrome, HCHF-fed rats developed hypertension, infiltration of inflammatory cells, cardiac fibrosis, increased cardiac stiffness, endothelial dysfunction, glucose intolerance, increased abdominal fat deposition, altered plasma lipid profile, increased hepatic fat deposition and fibrosis and increased plasma liver enzymes. Purple carrot juice (PC) decreased percent body weight gain (HCHF 14.3±0.9, HCHF+PC 10.3±0.6*) and normalised glucose tolerance, systolic blood pressure (HCHF 145±4.5, HCHF+PC 127.3±1.1* mmHg), left ventricle+septum weight (HCHF 22.3±1.3, HCHF+PC 17.3±0.7* mg/mm tibial length), abdominal circumference (HCHF 22.9±0.6, PC 20.2±0.4* cm), visceral fat pad deposition (HCHF 751±48, HCHF+PC 452±31* mg/mm tibial length), infiltration of inflammatory cells into the left ventricle, collagen deposition in the left ventricle (HCHF 7.8±0.6, HCHF+PC 4.2±0.7* %surface area), diastolic stiffness (HCHF 28.7±0.8, HCHF+PC 22.4±1.3*), acetylcholine-induced vasodilatation, plasma CRP concentrations (HCHF 102.3±4.6, HCHF+PC 23.5±9.2 μmol/L), plasma lipid profile and liver enzymes. Additionally, histological examination of the liver showed reduced inflammatory cell infiltration, fibrosis and lipids deposition. Our data suggests that purple carrot improves cardiovascular and hepatic structure and function through anti-inflammatory effects mediated by its anthocyanin content.

[1] Sun, T., Simon, P.W.,Tanumihardjo, S.A. (2009) Antioxidant phytochemicals and antioxidant capacity of

biofortified carrots ( Daucus carota L.) of various colors. J Agric Food Chem, 57: 4142-4147. [2] Charron, C.S., Kurilich, A.C., Clevidence, B.A., Simon, P.W., Harrison, D.J., Britz, S.J., Baer, D.J.,Novotny, J.A.

(2009) Bioavailability of anthocyanins from purple carrot juice: effects of acylation and plant matrix. J Agric Food Chem, 57: 1226-30.


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EFSA’s role in novel foods and claims

Wolfgang Gelbmann

Nutrition Unit European Food Safety Authority – EFSA Largo N. Palli 5/A, I-43121 Parma [email protected]

Set up in 2002, the European food Safety Authority (EFSA) provide independent scientific advice, opinions, information, and technical support for Community legislation and policies in all fields which have a direct or indirect impact on food and feed safety - including animal health and welfare and plant protection, and nutrition in relation to Community legislation.

EFSA’s risk assessments provide a sound scientific basis for defining policy-driven legislative or regulatory measures required to ensure a high level of consumer protection with regards to food safety. EFSA’s risk assessments are carried out by its Scientific Committee and Scientific Panels, each composed of external experts and specialised in different aspects of food and feed safety.

The EFSA Scientific Panel on Dietetic Products, Nutrition and Allergies (NDA) is the Panel dealing with questions on dietetic products, nutrition and allergies, and other associated subjects such as Novel Food (NF) and Claims.

At the request of the European Commission, the NDA Panel carries out risk assessment of NF (ingredients), defined as food (ingredients) that have not been used for human consumption to a significant degree within the Community, further specified by the NF Regulation (EC) Nº 258/9. Based on an Opinion of the former Scientific Committee for Food, the European Commission has issued Recommendation 97/618/EC of 29 July 1997 concerning the scientific aspects and the presentation of information necessary to support applications for the placing on the market of novel foods and novel food ingredients and the preparation of initial assessment.

Information is required on the specification of the NF, effect of the production process, history of the organism used as the source of the NF, anticipated intake and extent of use, previous human exposure to the NF or its source, the nutritional information, microbiological and toxicological information. The requirements for individual applicants however might differ depending on the composition and the history of the NF. The assessment of NF concerns only risk that might be associated with consumption and is not an assessment of the efficacy of the NF with regard to any claimed benefit.

On the basis of the Novel Food Regulation and the European Commissions Recommendation, the NDA Panel has so far delivered 42 scientific opinions on the safety assessment of a variety of NF such as foodstuffs with added phytosterols, substances with antioxidative properties like synthetic zeaxanthin and lycopene from various sources (Blakeslea trispora, tomato oleoresin, synthetic), NF from third countries (Chia seeds, Allanblackia seed oil, Noni Juice, Noni leaves), Enova oil and vegetable oils high in unsaponifiable matter, NF ingredients with a new or intentionally modified molecular structure (α-cyclodextrin), extracts from Alfalfa, licorice, fermented black beans, yeast and Lentinus edodes, and such to which a production process has been applied (Ice structuring proteins).

Regulation (EC) No 1924/2006 harmonises the provisions that relate to nutrition and health claims and establishes rules governing the Community authorisation of health claims made on foods. This Regulation requires that scientific substantiation should be the main aspect to be taken into account for the use of nutrition and health claims and the food business operators using claims should justify them. A claim should be scientifically substantiated by taking into account the totality of the available scientific data, and by weighing the evidence. In addition, in order to bear claims, foods will have to have appropriate nutrient profiles which will be set. This will enhance the consumers' ability to make informed and meaningful choice. In order to ensure


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PL3 harmonised scientific assessment of these claims, the European Food Safety Authority (EFSA) should carry out such assessments.

The substantiation of health claims includes the assessment of whether or not the food/constituent is sufficiently defined and characterised, to assure EFSA that the food/constituent for which the claim is made is the same as that for which the evidence on efficacy is provided. Also the claimed effect should be defined and comprise a beneficial physiological effect (‘beneficial to human health’). Finally, an assessment will be made of whether or not a cause and effect relationship exists between the consumption of the food/constituent and the claimed effect. In case the Panel considers that a cause-effect relationship is established, it needs to be assessed whether the quantity of food/pattern of consumption required to obtain the claimed effect can be consumed within a balanced diet, whether the proposed wording reflects the scientific evidence (consumer understanding is not addressed by EFSA), and whether the proposed conditions of use are appropriate.

In every case, in assessing each food/health relationship that forms the basis of a claim, all the evidence from the pertinent studies is weighed with respect to its overall strength, consistency and biological plausibility, taking into account the quality of individual studies and with particular regard to the target population group and the conditions of use proposed for the claimed effect.

In this presentation an update on the EFSA’s work on claims will be given.


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Tomato organic lycopene: the way to obtain a unique healthy product

Marcello Salvatore Lenucci1, Leonardo Rescio2, Gabriella Piro1, Giuseppe Dalessandro1

1 Di.S.Te.B.A., Università del Salento, Via prov. Lecce-Monteroni, 73100 Lecce, Italy 2 Pierre S.r.l., S.P. 362 Km 17,650, 73013 Galatina (LE), Italy [email protected]

Lycopene is a carotenoid found in a wide variety of vegetables and fruits. There are more than six hundred natural carotenoids, but only twenty of those have been found in human blood and tissues. Lycopene is the most abundant carotenoid in human blood, followed by β-carotene, lutein and zeaxanthin [1]. The purpose of this work was to obtain a natural, solvent-free lycopene extract from red-ripe tomato fruits. This project was developed in three different steps finalised to control and optimise the entire productive cycle, from fresh tomatoes to the production of organic lycopene. The first step of this work was the accurate selection, cultivation and production of organic grade certified tomato cultivars characterised by high-lycopene content, followed by the biochemical analyses of their antioxidant characteristics [2]. The second step was the set-up of a process to obtain a freeze-dried tomato matrix suitable for the extraction by supercritical carbon dioxide (SC-CO2) from the new selected high-lycopene cultivars [3]. The last step was the optimization of SC-CO2 lycopene extraction from a defined blend of the tomato matrix with a lipid co-matrix obtained from roughly crushed hazelnut seeds. This procedure allowed the simultaneous extraction of lycopene from the tomato matrix and oil from hazelnut co-matrix which improved the solubility of the pigment and increased the yield of the recovered end-product, according to the Pierre S.r.l. patented co-extraction technology [3-5]. The extract was an over-saturated solution of lycopene in hazelnut oil (oleoresin). This innovative procedure showed high lycopene extraction yields (>80%). The extracted oleoresin represents a healthy and high added-value source of lycopene, suitable for nutraceutical, cosmetic and pharmaceutical purposes. It was registered as the first organic grade lycopene. This innovative product is characterized by: 1) the absence of toxic impurities such as solvents, pesticides, heavy metals and dioxins; 2) high natural bioavailability, increased by the presence of the co-extracted natural components from hazelnuts and tomatoes; 3) high antioxidant activity, due to the presence of tomato carotenoids and hazelnut tocopherols and phytosterols.

[1] Rao A.V., Tomatoes, Lycopene & Human Health. Preventing Chronic Diseases. Caledonian Science Press,

Badalona (2006). [2] Lenucci M.S., Cadinu D., Taurino M., Piro G., Dalessandro G. (2006) Antioxidant composition in cherry and

high-pigment tomato cultivars. Journal of Agricultural and Food Chemistry 54: 2606-2613 (2006). [3] Lenucci M.S., Caccioppola A., Durante M., Serrone L., Leonardo R., Piro G., Dalessandro G. (2010) Optimisation

of biological and physical parameters for lycopene supercritical CO2 extraction from ordinary and high-pigment tomato cultivars. Journal of the Science of Food and Agriculture, 90: 1709-1718.

[4] Vasapollo G., Longo L., Rescio L., Ciurlia L. (2004). Innovative supercritical CO2 extraction of lycopene from tomato in the presence of vegetable oil as co-solvent. Journal of Supercritical Fluids, 29: 87-96.

[5] Ciurlia L., Bleve M., Rescio L. (2009) Supercritical carbon dioxide co-extraction of tomatoes (Lycopersicum esculentum L.) and hazelnuts (Corylus avellana L.): a new procedure in obtaining a source of natural lycopene. Journal of Supercritical Fluids, 49: 338-344.


20

SL6

Isoflavone composition of soy and red clover dietary supplements Franz Bucar, Elvira Knauder Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Universitätsplatz 4, A-8010 Graz, Austria [email protected]

Isoflavones from soybean and red clover are supposed to have beneficial effects by alleviating

postmenopausal symptoms or by reducing cancer risk of hormone dependent cancers. However, their efficiency and risk-benefit balance is still discussed controversially [1,2].

Isoflavone preparations are usually marketed as nutraceuticals (dietary supplements). Previous investigations have shown that the proportion of aglycones and glycosides can reveal a wide range [3] but usually this information is not included in the declaration of the products. However, in order to estimate the phytoestrogenic activity the amount of isoflavone aglycones (in free and glycosidated form) in the applied dose should be known.

During the last years by our group a number of isoflavone dietary supplements have been analysed for their isoflavonoid pattern using HPLC-PDA and LC-PDA-ESI-MS analysis.

Sixteen products contained soy extracts, 4 contained red clover isoflavone extracts and 4 were manufactured from mixtures of soy and red clover extracts. Only one half of the products contained the declared amounts ± 15 %. Red clover extracts generally showed a high proportion of aglycones, i.e. 99.1 – 86.8 % of total amount were present as free aglycones, calculated in molar concentrations. Biochanin A and formononetin were the major isoflavones in all investigated red clover supplements. On the other hand, soy products varied to a much greater extent (5.0 – 98.1 % of total amount as glycosides, mean ± S.D. 78.7 ± 29.8 %, calculated in molar concentrations) with daidzin, glycitin and genistin as main glycosides whereas acetylated glycosides usually were present only in minor quantities.

Due to the different absolute amounts and varying ratios of isoflavone aglycones and glycosides in the investigated products, a declaration of the dose as “isoflavone aglycone equivalents” seems to be advantageous. [1] Dixon RA. (2004) Phytoestrogens. Annu. Rev. Plant Biol.,. 55: 225-261 [2] Zhao E., Mu Q. (2011) Phytoestrogen biological actions on mammalian reproductive system and cancer growth.

Sci. Pharm. 79: 1-20 [3] Schwartz H., Sontag G. (2008) Determination of isoflavones in nutritional supplements by HPLC with

coulometric electrode array detection. Monatsh. Chem., 139: 865-872

Compound R1 R2 R3 R4

Daidzein OH H H OH

Genistein OH OH H OH

Glycitein OH H OCH3 OH

Biochanin A OCH3 OH H OH

Formononetin OCH3 H H OH

O

R1

OR2

R3

R4


SL7

21

Antioxidant and anti-inflammatory properties of a new soluble extract from grape pomace

Candiracci M1., Castaño A1., SantaMaría C1., Revilla E1., Dominguez-Barragán M1., Rodríguez-Morgado B1., García-Antras D1., Carballo M3., Bautista J1. Herrera , M.D2., and Parrado J1.

1Departamento de Bioquímica, Biología Molecular. Facultad de Farmacia Universidad de Sevilla. 2Departamento de Farmacología. Facultad de Farmacia Universidad de Sevilla. C/Prof. García Gonzalez 2 , Sevilla 41012 3CITIUS. Universidad de Sevilla

Grape pomace (GP) is a byproduct from wine industry consisting of skins, seeds, and stems. In order to valorize the GP, we have developed an enzymatic process that give rise to a new product consisting in a soluble extract enriched in polyphenols (GPE).

Moderate and regular consumption of red wine has been reported to reduce the risk of cardiovascular diseases [1]. This beneficial effect is attributed, in part, to their high content in polyphenolic compounds that prevent oxidation of low-density lipoproteins [2]. Thus, we have tested antioxidative effect of GPE by three different techniques: Phospho molybdate, ORAC and DDPH. Our results clearly show a dose-dependent antioxidative property of GPE.

On the other hand, it’s widely accepted that inflammation plays a pivotal role in neurodegenerative process as Parkinson´s or Alzheimer disease. The hallmark of neuroinflammation is the activation of microglia, but although microglial activation is the brain´s major defense against immune challenge, activated microglia may also contribute to neurodegeneration through the release of proinflammatory and/or cytotoxic factors as IL-1�, TNF-��, NO and reactive oxygen intermediates among others. Thus it can be assumed that a degree of brain inflammation is required for repair of damaged tissue but excesive inflammation causes neuronal cells death [3,4]. In this sense, it will be interesting to search for molecules that could help to control inflammation in the CNS.

Given that flavonoids and tocopherols inhibit release of proinflammatory cytokines after LPS-activation of microglia [5,6] we search whether the GPE exerts an antiinflammtory effect on N13 microglia cells stimulated with LPS. Interestingly our results show that GPE decreases not only the level of mRNAs of IL-1�, TNF-� and iNOS, but also iNOS protein.

In conclusion our results deeply support that GPE exerts beneficial effect on pathological conditions related to aged as cardiovascular and neurodegenerative processes.

[1] Renaud S. et al. Lancet 339 : 1523–6, 1992, [2] Serafini M., et al. J. Nutr. Biochem. 11: 585–90, 2000. [3] Block M.L. et al. Nature Review Neuroscience 8: 57- 69, 2007. [4] Park K.W. et al. Exp Mol Med 39 (6) : 812-19, 2007. [5] Lu X. et al. J. Neuroinflammation 7: 46-60, 2010, [6] Ha S. K., et al. Neuroscience letters 485(3): 143-7, 2010.


22

PL4

Phenolic compounds : from plants to foods

Véronique Cheynier

INRA, UMR1083 Sciences pour l’Oenologie, place Viala, 34060 Montpellier, France. Phenolic compounds are a large class of plant secondary metabolites, including phenolic acids,

stilbenes, a variety of flavonoids (e.g. flavonols, flavan-3-ols, anthocyanins), and polymeric compounds, based on these three groups of compounds. They play a role in plant defence against biotic and abiotic stress and in attraction processes involved in seed dissemination and pollination. For example, anthocyanins are the pigments of most red and blue plant organs, while flavonols protect plant tissues against UV irradiation and proanthocyanidins (PA, i.e. flavan-3-ol oligomers and polymers, also called condensed tannins) are involved in defence against herbivores, fungi and viruses. Phenolic compounds are equally important for the quality of plant based foods: they are responsible for the colour of red fruits, juices and wines and substrates for enzymatic browning, and are also involved in flavour and taste properties. In particular, astringency is ascribed to precipitation of salivary proteins by tannins. In addition, phenolic compounds may contribute to the health benefit associated to dietary consumption of fruits and vegetables.

During food processing and storage, plant phenolics are converted to a variety of derived compounds, which also affect quality. Understanding the structure of the resulting compounds and their particular properties, as well as the impact of processes on composition changes is essential for controlling food quality. Extraction of phenolics into beverages such as juices, wines and teas, depending on their structure but also on the characteristics of the plant organ, is another important issue for plant transformation industry which has devoted much effort to the development of new extraction processes.

While lower molecular weight compounds are quite easily analysed by HPLC hyphenated with UV-visible spectrophotometry and mass spectrometry, analysis of polymeric compounds remains a challenge. Indeed, strong interactions of polymeric phenolics with plant cell wall material limit their extraction. Besides, their polydispersity results in poor resolution and detection, especially in the case of derived structures. However, approaches combining depolymerisation and small angle X-ray scattering (SAXS) provided new insights on the structure of PA oxidation products (Poncet-Legrand et al., 2010).

Current knowledge on phenolic reactions and resulting structures and their impact on colour, taste, and health properties will be presented. Changes taking place during transformation processes will be illustrated in the case of wine-making, with special emphasis on extraction technologies and oxidation reactions. Poncet-Legrand C, Cabane B, Bautista-Ortın AB, Carrillo S, Fulcrand H, Perez J, Vernhet A (2010), Biomacromolecules, 11, 2376–2386.


SL8

23

Polyphenols-rich extracts obtained from Prunus avium and opuntia ficus indica as natural anti-inflammatory modulators for inflammatory bowel diseases. S. Nunes1, A.T. Serra1,2, M. R. Bronze1,2,3, Catarina M.M. Duarte1,2, A.A. Matias 1,2,3*

1Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Estação Agronómica Nacional, 2780-157 Oeiras, Portugal; 2Instituto de Biologia Experimental e Tecnológica, Avenida da República, Quinta-do-Marquês, Estação Agronómica Nacional, Apartado 12, 2781-901 Oeiras, Portugal; 3iMED, Faculdade de Farmácia da Universidade de Lisboa, Av. das Forças Armadas, 1649-019, Lisboa, Portugal. [email protected]

There are evidences that the regular consumption of polyphenols in early life could reduce or

delay Inflammatory Bowel Diseases (IBD) development in humans. However, IBD are usually treated with pharmacological agents, which could produce secondary effects. It is therefore, urgent to find new and safe compounds able to contribute to prevention or even to treatment of these inflammatory diseases.

In the present study we investigated the preventive effectiveness of polyphenolic-rich extracts obtained from Sweet cherries (Prunus avium) and cactus pear (Opuntia ficus indica) on inflammatory expression in intestinal epithelial human cell lines (caco-2) induced with specific stimuli (LPS and TNF-α combination).

The selected fruits were processed in order to prepare the polyphenols-rich extracts (PRE) and further characterized in terms of polyphenolic compounds, namely anthocyanins and betalains using colorimetric assays and chromatographic techniques.

The inflammatory response of the PRE’s were assessed using intestinal epithelial cells (seeded in transwell™ plates) after pro-inflammatory stimulus (LPS and TNF-α cocktail). The secretion of pro-inflammatory markers (NO excretion, IL-8 and IL-1β secretion) was assayed in the media from both apical and basolateral compartments for inflamed cells as compared to the control.

The monolayer integrity and permeability were assessed by the determination of transepithelial electrical resistance values.

When Caco-2 cells were simultaneously incubated with PREs and the inflammatory stimuli, no significant change in cytotoxicity and TEER values was observed.

In inflamed cells, the incubation with Cherry extract promotes the maintenance of epithelium barrier integrity of the inflamed cells and also reduced significantly the IL- 8 secretion and NO excretion.

Cactus pear extract provoked a slightly decrease in the IL-8 over-secretion. In conclusion, this study provides evidence that cherry and cactus pear extracts could

downregulate the inflammatory response in inflamed intestinal epithelial cells. Further investigation on this subject are in progress.

[1] Kelley DS, Rasooly R, Jacob RA, Kader AA, Mackey BE. Consumption of Bing sweet cherries lowers circulating concentrations of inflammation markers in healthy men and women. J Nutr. 2006, 136:981–6.

[2] Ferretti G, Bacchetti T, Belleggia A, Neri D, Cherry Antioxidants: From Farm to Table, Molecules 2010, 15, 6993-7005.


24

SL9 Polyphenols from red wine exert potential beneficial effects to human health Thea Magronea, Sergio Fontanab, Emilio Jirilloa

aDepartment of Basic Medical Sciences, University of Bari, P.zza G. Cesare 11, 70125 Bari, Italy bFarmalabor Srl, via Oberdan 52, 70053 Canosa di Puglia, Italy [email protected]

Polyphenols (flavonoids and resveratrol) are highly distributed in the plant kingdom and their

biological properties have been extensively studied [1]. Over the past few years, we have investigated the in vitro and in vivo effects of polyphenols

from red grapes. In particular, we have provided evidence that polyphenols isolated from Vitis Vinifera Negroamaro are able to induce release of nitric oxide from human monocytes and to modulate either innate or adaptive human immune response [2]. In this respect, these polyphenols are able to maintain the immunological homeostasis, thus balancing the inflammatory/antinflammatory pathway [3]. In addition, polyphenols interfere with the activation of the NF-�B pathway induced by endotoxins, thus attenuating the proinflammatory wave of cytokines in clinical settings, such as sepsis [4].

Similar effects have been obtained using fermented grape mark (FGM) from Vitis Vinifera Negroamaro and Koshu. Quite interestingly, Koshu-FGM has the ability to attenuate murine experimental colitis in vivo [5]. This finding is also supported by the evidence that Koshu-FGM reduces the secretion of interleukin 1� and tumor necrosis factor � from colitis mice homogenised colon [6]. These data are also supported by in vitro results which clearly demonstrated the ability of both Koshu-FGM and Negroamaro-FGM to induce T regulatory cells (CD4+CD25+FoxP3+) in the human system. Finally, both FGM are able to mitigate allergic reactions inhibiting mast cell and basophil degranulation, release of IgE, infiltration of eosinophils in the respiratory system and passive cutaneous anaphylaxis [7,8].

Clinical trials in patients with allergic disease and inflammatory bowel disease are in progress. [1] Magrone T., Jirillo E. (2010) Polyphenols from red wine are potent modulators of innate and adaptive immune

responsiveness. Proc Nutr Soc, 69: 279-285 [2] Magrone T., Tafaro A., Jirillo F., Panaro MA., Cuzzuol P., Cuzzuol AC., Pugliese V., Amati L., Jirillo E., Covelli

V. (2007) Red wine consumption and prevention of atherosclerosis: an in vitro model using human peripheral blood mononuclear cells. Curr Pharm Des, 13: 3718-3725

[3] Magrone T., Tafaro A., Jirillo F., Amati L., Jirillo E., Covelli V. (2008) Elicitation of immune responsiveness against antigenic challenge in age-related diseases: effects of red wine polyphenols. Curr Pharm Des, 14: 2749-2757

[4] Magrone T., Panaro MA., Jirillo E., Covelli V. (2008) Molecular effects elicited in vitro by red wine on human healthy peripheral blood mononuclear cells: potential therapeutical application of polyphenols to diet-related chronic diseases. Curr Pharm Des, 14: 2758-2766

[5] Magrone T., Kumazawa Y., Jirillo E. (2011) Natural Antioxidants and their Derivatives: Biology and Clinical Application. Curr Top Med Chem, [Epub ahead of print]

[6] Magrone T., Jirillo E. (2011) Potential Application of Dietary Polyphenols from Red Wine to Attaining Healthy Ageing. Curr Top Med Chem, [Epub ahead of print]

[7] Tominaga T., Kawaguchi K., Kanesaka M., Kawauchi H., Jirillo E., Kumazawa Y. (2010) Suppression of type-I allergic responses by oral administration of grape marc fermented with Lactobacillus plantarum. Immunopharmacol Immunotoxicol, 32: 593-599

[8] Kaneko M., Kanesaka M., Yoneyama M., Tominaga T., Jirillo E., Kumazawa Y. (2010) Inhibitory effects of fermented grape marc from Vitis vinifera Negroamaro on antigen-induced degranulation. Immunopharmacol Immunotoxicol, 32: 454-461.


SL10

25

Differential antioxidant & anti-amyloid effects of a wild blueberry polyphenol-rich extract: in vitro & in vivo studies

Magdalini A. Papandreou1, Maria Tsachaki2, Spiros Efthimiopoulos2, Dorothy Klimis-Zacas3, Marigoula Margarity1, Fotini N. Lamari4

1Dept. Biol., Univ. Patras, 2Dept. Biol., Univ. Athens, 3Dept. Food Science & Human Nutrition, Univ. Maine, Orono, ME 04469, USA, 4Lab. Pharmacogn. & Chem. Natural Products, Dept. Pharmacy, Univ. Patras, Greece. [email protected] Blueberries, a rich source of natural anthocyanin antioxidants, have demonstrated a broad spectrum of beneficial biomedical functions. In this study, the effects of a daily (7-day) intraperitoneal (i.p.) administration of a polyphenol-rich extract (PrB) of Vaccinium angustifolium (wild blueberries), on cognitive functions were examined in healthy adult (4 months old), male Balb-c mice (n=8/group) by passive avoidance test. Brain oxidative markers, caspase-3 and acetylcholinesterase (AChE) activity were determined in whole brain homogenates (minus cerebellum). Results showed that PrB-treated mice exhibited a significant improvement in learning and memory, accompanied by reduced lipid peroxidation products, higher total brain antioxidant activity and reduced caspase-3 activity. Furthermore, salt- and detergent-soluble AChE activity was significantly decreased. Thus, the significant cognitive enhancement observed in adult mice after short-term i.p. supplementation with the blueberry extract, is closely related to higher brain antioxidant properties and inhibition of AChE activity. To further delineate the neuroprotective mechanisms, we investigated the antioxidant effects of PrB in three different cell lines against H2O2-induced oxidative damage. PrB suppresses H2O2-initiated SH-SY5Y intracellular cell death (MTT assay) and oxidation (DCF assay). Moderate effects were observed on CHOAPP770 cells, while further damage on HEK293 cells was shown after co-treatment with 250 μΜ H2O2 and PrB. Since Amyloid Precursor Protein (APP) altered metabolism, Aβ-overproduction/aggregation are key pathological hallmarks in AD, we further studied the effects of PrB on Aβ-fibrillogenesis, in vitro, utilizing the thioflavine T assay, and on APP-metabolism in CHOAPP770 cells. Blueberry polyphenols inhibited Aβ-aggregation in a time-dependent manner, while in CHOAPP770 cells, no alterations in APP metabolism were observed as assessed by western blot. Taken together, our results suggest that blueberry polyphenols exhibit antioxidant and/or pro-oxidant properties according to the cellular environment inhibit Aβ-fibrillogenesis in vitro but have no effect on APP metabolism.


26

PL5

New trends in the analysis of natural products for the detailed metabolite profiling of crude extracts

Jean-Luc Wolfender

School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, 30, quai Ernest-Ansermet, CH-1211 Geneva, Switzerland

Plant natural products (NPs) have provided the inspiration for a large number of the active

ingredients in medicine. Their success is probably due to the effects of evolutionary pressure to create biologically active molecules. The large chemical space occupied by NPs is directly linked to a high variability of their intrinsic physicochemical properties that render their separation, detection and characterization challenging. In order to identify all these metabolites, crude plant extract profiling is essential. This is a challenging task that requires methods providing high chromatographic resolution for detailed profiling or high throughput for rapid quantification or fingerprinting analysis. Furthermore these methods should give on-line spectroscopic information for the identification of each individual metabolites for dereplication purposes. In this respect hyphenated techniques such as LC-MS and LC-NMR have played a key role over the last three decades. In phytochemical analysis, the recent introduction of Ultra High Pressure Liquid Chromatography (UHPLC) systems operating at very high pressures and using sub-2 μm packing columns have allowed a remarkable decrease in analysis time and increase in peak capacity, sensitivity and reproducibility compared to conventional HPLC. In complement to this powerful chromatographic method, the introduction of benchtop time–of-flight (TOF)-MS instruments provide sensitive detection and high MS resolution. For de-novo identification of NPs on-line LC-NMR, introduced in the early 90’s, has evolved towards sensitive at-line microflow NMR approaches combining pre-concentration of the LC peaks of interest prior to NMR measurement (LC-SPE-NMR, capillary NMR: CapNMR). With such methods structure determination of targeted compounds at the low microgram level is possible and complement the search in MS and chemotaxonomic data bases for dereplication. The potential and limitations as well as some new trends in the development of UHPLC-MS and micro NMR will be discussed. In particular examples related to metabolomics and de novo biomarker identification will be presented.

Oral Presentations – International PSE Symposium on ”Phytochemicals in Nutrition and Health”, 27-30 September

SL11

27

Metabolomic profiling and fingerprinting: new effective approach for plant characterisation and authentication Vera Schulzova, Hana Novotna, Lukas Vaclavik, Anna Hurajova, Jana Hajslova. Institute of Chemical Technology, Department of Food Chemistry and Analysis, Technická 3, 16628, Prague 6, Czech Republic [email protected]

Food and medicinal plants are a good source of different phytonutrients and nutraceuticals. Content of biological active compounds such as vitamins, carotenoids, phenols, flavonoids, terpenoids etc. differs in individual varieties. The geographic origin and climatic conditions also influence the content of phytonutriets significantly. Until now, a wide range of analytical approaches has been tested for control of quality and authenticity. In our most recent research projects, we have implemented a novel approach based on ambient mass spectrometry [1]. Using the Direct Analysis in Real Time (DART) ion source hyphenated with a high resolution Time of Flight Mass Spectrometer (TOFMS), metabolomic fingerprint (mass spectrum) can be obtained in a few seconds. Metabolomics may be used either for ´fingerprinting´ of samples to perform comparative analyses aimed at detection of differences or for ´profiling´ in which individual sample components (both primary and secondary metabolites) are identified for further chemometric assessment. Using relevant statistical tools, locality, crop years, varieties, and way of farming could be separated. In any case, relevant databases of metabolomic profiles of well characterised samples should be established to have a larger data set for statistical processing thus obtaining recognition and prediction models.

In this study, DART-TOFMS analysis was used for characterisation and authentication of different plant materials. The results of several case studies concerned with authentication of food and medicinal plants (flax seeds, potato tubers, tomatoes, apples, peppers, rose hip, lemon balm, peppermint, stinging nettle etc.) will be presented. This study was carried out within the project MSM 6046137305 and within specific university research MSMT no. 21/2010 supported by Ministry of Education, Youth and Sports of the Czech Republic. [1] Hajslova J., Cajka T., Vaclavik L. (2011) Challenging applications offered by direct analysis in real time (DART) in food-quality and safety analysis. Trend Anal. Chem. 30, 204-218


28

SL12 Estrous cycle in female rats influences the neurochemical and neurobehavioural effects of carvacrol Margherita Zottia, Marilena Colaiannaa, Maria Grazia Morgesea, Lucia Ciuffredaa, Paolo Tuccia, Pinarosa Avatob and Luigia Trabacea

aDipartimento di Scienze Biomediche, Università, V.le Pinto 1, 71121 Foggia, Italy bDipartimento Farmaco-Chimico, Università, Via Orabona 4, 70125 Bari, Italy [email protected]

Carvacrol, the major natural terpenoid constituent of oregano [1] and thyme essential oils is a small and lipophilic compound likely to readily cross the blood-brain barrier. It has been approved for food use and included in the chemical flavorings list, although little is known on its safety.

The use of plant extracts is relatively high among women, especially middle-aged, and even more common among women with depression [2,3]. Some authors have reported antimicrobial, anticancer and antioxidant properties for this terpene, hovewer its possible action in the mammalian central nervous system has not been extensively investigated.

The aim of this study was to evaluate the neurochemical and behavioural effects of carvacrol on physiological and endocrine profile of female rats in proestrus and diestrus phases, characterized by high and low estrogen levels, respectively. In particular, serotonin and its metabolite tissue content in prefrontal cortex and nucleus accumbens, 2h after carvacrol administration (0.15 and 0.45 g/Kg p.o.), were measured. Effects were examined in behavioural tests for alterations in motor activity and depression-related behaviours.

While in proestrus carvacrol reduced serotonin and related metabolite levels in both brain areas, no effects were observed in diestrus phase. Only in proestrus phase, carvacrol induced a depressive-like behaviour in the forced swimming test (FST), without changes in ambulation. The improvement of performance in FST after subchronic treatment with fluoxetine (s.c., 20 mg/kg), suggested the specific involvement of serotonergic system in the depressive-like effects of carvacrol. Regarding endocrine profile, we found that in proestrus phase, carvacrol reduced estradiol levels.

Taken together our data suggest that carvacrol has an estrous-stage specific effect on depressive-behaviours and endocrine parameters involving serotonergic system. [1] Morone-Fortunato, I., Avato P. (2008) Plant Cell Tiss Organ Cult 93, 139-149 [2] Brett, K. M., and Keenan, N. L. (2007). Menopause, New York [3] Barnes, P. M., Powell-Griner, E., McFann, K., and Nahin, R. L. (2004). Advance data, United States


PL6

29

Breeding for improved flavour and health

Yury Tikunova,b, Sjaak van Heusdena,b, Jos Molthoffa, Henri van de Geestb, Ana-Rosa Ballesterc, Irene Romeroa,b, Antonio Granelld, Ric de Vosa,b, Arnaud Bovya,b

aPlant Research International, P.O. Box 16, 6700 AA, Wageningen, Netherlands bCentre for Biosystems Genomics, P.O. Box 98, 6700PB, Wageningen, Netherlands cInstituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Científicas (IATA-CSIC), Valencia, Spain dInstituto de Biología Molecular y Celular de Plantas (IBMCP-CSIC), Universidad Politécnica de Valencia (UPV), Valencia, Spain

Traditionally the most important targets in fruit and vegetable breeding are producer traits, such as yield, disease resistance and shelf life. In the past decade, however, the importance of consumer-driven quality traits, such as flavour, nutritional value and appearance have been increasingly recognised. This is not only due to growing consumer demands for more healthy food with an excellent quality, but also to the availability of various omics tools, which make it possible to study complex, multifactorial traits, such as flavour and health. These quality traits are to a large extent determined by the phytochemical composition of our food products. In order to improve the consumer-quality of food crops, we aim to explore and utilise the natural diversity for flavour- and health-related metabolites. Our general strategy involves the elucidation of biochemical pathways underlying important quality traits using a genetical genomics approach and the isolation of the responsible genes underlying quality-related mQTLs. Tomato is one of the most important vegetable crops worldwide and an important model for fleshy fruits. Cultivated tomato has a very narrow genetic base, due to severe genetic bottlenecks experienced during domestication. In contrast, a lot of genetic diversity can be found in wild relatives of tomato, which are therefore used as novel sources of important agronomic and quality traits. In particular the use of introgression line (IL) populations, in which defined chromosomal segments of wild species have been introgressed into an elite tomato background has been very successful. We analysed a diverse collection of both cultivated and wild tomato germplasm for variation in colour-, health- and flavour-related metabolites, in order to elucidate the biochemical and molecular mechanisms underlying these quality traits. In this lecture, several examples of this approach will be discussed:

1. By screening a Solanum chmielewskii IL population we elucidated the biochemical and molecular basis underlying pink-coloured tomatoes, the predominant tomato type in Asia.

2. This population also revealed a large variation in health-related metabolites, such as carotenoids, flavonoids and alkaloids. We are currently using a candidate gene approach to clone the underlying genes.

3. A set 94 commercial tomato cultivars was evaluated for taste using trained sensory panels and metabolite profiling. We observed a close correlation between the presence of phenylpropanoid-derived volatiles and a “smoky” flavour. The release of these volatiles is determined by their storage form as glycoside conjugate. By a combined genetic, metabolomic and transcriptomic approach the underlying glycosyltransferase gene was isolated.

This research was funded by the Dutch NWO genomics initiative Centre for Biosystems Genomics and the EU project EUSOL (PL-016214).


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SL13 Application of purine derivative LGR-1814 improves functional properties of field-grown lettuce (Lactuca sativa)

Jiri Gruza,b and Lukas Spichala,b

aLaboratory of Growth Regulators, Institute of Experimental Botany AS CR, Slechtitelu 11, CZ 783 71 Olomouc, Czech Republic bCentre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University, Slechtitelu 11, CZ 783 71 Olomouc, Czech Republic

Lettuce is one of the most consumed vegetables in the world. It is particularly popular in Argentina, Australia, Canada, Chile, New Zealand, United States of America and Uruguay, where the mean consumption is 30.6 g person-1 day-1 according to International Estimated Daily Intake published by WHO in 2006. Considering the phytochemical potential of lettuce, the manipulation of endogenous metabolite levels is an advisable strategy to improve its functional properties. The increase in specific bioactive components is also a competitive factor which may increase sales and performance of food companies. In our study, we have found that single application of purine derivative LGR-1814 improved functional properties of field-grown lettuce (Lactuca sativa). ORAC antioxidant capacities of the lettuce plants treated with 10 and 50 μM LGR-1814 were significantly increased by 21 and 32%, respectively. A similar trend was observed when TPC was determined. The increase in antioxidant capacity was correlated with the UPLC-MS/MS determined content of endogenous hydroxycinnamic acids (HCA), including ferulic and 4-coumaric acids, while the content of hydroxybenzoic acids (HBA) was not altered. The fact that levels of only HCA but not HBA were increased in the treated plants is in accordance with the observed induction of PAL activity. The application of LGR-1814 did not result into any significant reduction in yield which is a common side effect of stress and stress-related accumulation of phenolic compounds. In accordance, the levels of malondialdehyde remained unaffected, confirming that lettuce plants were not stressed by the treatment. Our data indicate that LGR-1814 could find interesting applications in improving functional properties, shelf life, and stress protection of edible plants.


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31

Comparison of triterpenoid content of Vaccinium vitis-idaea berries and leaves collected from natural forest habitats in Finland and Poland

Anna Szakiela, Cezary Pączkowskia, Heini Koivuniemib, Satu Huttunenb

a Department of Plant Biochemistry, Faculty of Biology, University of Warsaw, ul. Miecznikowa 1, 02-096 Warszawa, Poland b Botany Division, Department of Biology, University of Oulu, P.O. Box 3000, 90014 Oulu, Finland [email protected]

Vaccinium vitis-idaea (cowberry, lingonberry), known for its edible berries and leaves used in herbal medicine, is a rich source of dietary nutraceutics, phenolics and triterpenoids.

Plant material (berries and two types of foliage: young, immature, current-year leaves and old, previous-year leaves) was collected in natural forest habitats in Finland (65°066 N; 25°458 E) and Poland (52°455 N, 21°332 E) in late August and December 2010. Samples were dried, powdered and extracted in Soxhlet apparatus with diethyl ether. Extracts were fractionated by CC and TLC chromatography on silica gel, and analysed by GC-MS/FID.

Main triterpenoid profile of V. vitis-idaea of both origin consisted of 12 compounds, namely: α- and β-amyrins, betulinol, erythrodiol, fernenol, friedelin, lupeol, svertenol, taraxasterol, uvaol, oleanolic and ursolic acids. Other notable constituents were β–sitosterol, cycloartanol and stigmasta-3,5-dien-7-one. Within each class of triterpenes, compounds based on the ursan skeleton predominated, with α–amyrin being more abundant than β–amyrin, and ursolic acid and uvaol found at higher levels than oleanolic acid and erythrodiol. However, some remarkable qualitative and quantitative differences between berries and leaves obtained from the two locations were found.

The content of triterpene acids was higher in Finnish (approx. 9 mg·g-1 d.w.) than in Polish berries (8 mg·g-1 d.w.) whereas triterpenols were more than twice more abundant in Polish than in Finnish fruits (1 and 0.4 mg·g-1 d.w., respectively). The principal triterpenol in Finnish berries was fernenol whereas in Polish berries α–amyrin was predominating within this class of compounds. Additionally, betulinol and taraxasterol were found in small amounts in Polish berries, whilst they were not detected in Finnish fruits. Berries of both origin contained comparable amounts of free β-sitosterol (1.3 mg·g-1 d.w.) but its esterified form was twice more abundant in Finnish berries. In turn, the amounts of cycloartanol and stigmastadienone were markedly higher in Polish fruits.

In leaves, the total content and profile of triterpenoids varied significantly not only depending on the place of plant origin, but also on leaf age. Generally, in August young leaves contained less triterpenes (4.6 and 3 mg in Finnish and Polish leaves, respectively) than old leaves (6.2 and 6.1 mg). These differences were less significant in December. Triterpene acids were again the most abundant constituents (2.4 and 4.4 mg in young and old Finnish leaves, 3 and 6.1 mg in Polish leaves). The most remarkable difference in triterpene profile concerned the occurrence of fernenol and taraxasterol, the first occurring as the main triterpenol in Finnish, the latter in Polish leaves.

Acknowledgement. This research was supported by POIG.02.02.00-14-024/08-00.


32

PL7

NMR metabolomics: advantages and disadvantages for studying bioactive components in food

Søren Balling Engelsen and Francesco Savorani

Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark [email protected]

Metabolomics is crucial for understanding human health and nutrition. Many lifestyle related

problems are in reality metabolic issues: a mismatch between what is ingested and the needs of the organism. Food health and safety are areas reaping the benefits of metabolomics. Food is eaten as a whole and therefore the combined effect of components has to be taken into account when assessing the health and safety aspect of food and food processes.

In humans and animals, biologically relevant samples can routinely be obtained from blood, small tissue biopsies, saliva, urine and faeces. Metabolomic profiling seeks to determine as many chemical (and physical) features as possible and to identify metabolites or patterns of metabolites that discriminate samples from treated individuals and controls [1].

The two dominant analytical platforms in metabolomics are Liquid Chromatography-Mass Spectrometry (LC-MS) and NMR spectroscopy, which have quite complementary properties. While LC-MS has much higher sensitivity NMR is inherently quantitative, un-biased and non-destructive and has unique properties for measuring fx lipoproteins and chylomicrons in blood [2].

The bottleneck for exploiting the powerful new analytical technologies has become the bioinformatics analysis of the overwhelming amount of data. The combination of NMR spectroscopy with chemometrics has proven to have a tremendous potential for metabonomics [3]. In this approach the multivariate analytical results are compared across large cohorts, in for example intervention studies, for metabolites and/or patterns of metabolites that are able to isolate the effect of the intervention. Chemometrics have also been useful in finding solutions to imperfect analytical methodology that are prone to alignment, normalization and other standardization problems [4, 5] as well as for advanced analysis aimed at exploiting the experimental design and the data structure [6, 7].

This paper tries to sum up the current status of NMR metabonomics for studying bioactive substances in food and their effect in the human body.

[1] Fiehn O. (2009), Metabolomics - the link between genotypes and phenotypes, Plant Molecular Biology, 48: 155-

171. [2] Savorani F., Kristensen M., Larsen F.H., Astrup A., Engelsen S.B. (2010), High throughput prediction of

chylomicron triglycerides in human plasma by nuclear magnetic resonance and chemometrics, Metabolism & Nutrition, 7:43.

[3] Nicholson J.K., Lindon J.C., Holmes E. (1999), 'Metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data, Xenobiotica, 29: 1181-1189.

[4] Savorani F., Tomasi G., Engelsen S.B. (2010), icoshift: a versatile tool for the rapid alignment of 1D NMR spectra, Journal of Magnetic Resonance, 202:190-202.

[5] Rasmussen L.G., Savorani F., Larsen T.M., Dragsted L.O., Astrup A., Engelsen S.B. (2011), Standardization of factors that influence human urine metabolomics, Metabolomics (2011), 7:71-83.

[6] van Velzen E.J.J., Westerhuis J.A., van Duynhoven J.P.M., van Dorsten F.A., Hoefsloot H.C.J., Jacobs D.M., Smit S., Draijer R., Kroner C.I. , Smilde A.K. (2008), Multilevel data analysis of a crossover designed human nutritional intervention study, Journal of Proteome Research, 7:4483-4491.

[7] Bro R., Viereck N., Toft M., Toft H., Hansen P.I., Engelsen S.B. (2010), Mathematical chromatography solves the cocktail party effect in mixtures using 2D spectra and PARAFAC, Trends in Analytical Chemistry, 29: 281-284.


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Formation of astragaloside IV from acylated astragalosides during extraction of Astragali radix Monschein Ma, Ardjomand-Woelkart Ka, Wolf Ia, Rieder Jb, Bauer Ra

a Institute of Pharmaceutical Sciences, Department of Pharmacognosy, Karl-Franzens-University Graz, Universitaetsplatz 4, 8010 Graz, Austria b Bavarian State Research Center for Agriculture, Dept. AQU2, Lange Point 6, 85354 Freising, Germany [email protected]

Astragaloside IV (AGS-IV), a cycloartane-type triterpene glycoside, is used as an active marker for the quality control of Astragali Radix (Huangqi; Astragalus mongholicus Bunge, Leguminosae) in Chinese Medicine [1]. Recently, when astragaloside malonates have been identified, it has already been argued, that qualitative and quantitative analysis of astragaloside IV may not give the correct information about the saponin profile of Astragalus roots [2]. We now have confirmed by HPLC-ELSD (HPLC-Evaporative light scattering detection) analysis that AGS-IV is formed in Astragalus extracts like astragaloside I and II by hydrolysis during extraction. Therefore, for consistent assay results fully implemented hydrolysis with Ammonia R is essential, and the extraction methods of the pharmacopoeias [3] have to be re-evaluated and optimized. [1] The Chinese Pharmacopoeia (Beijing, China) Vol. 1 (2005) 249. [2] Chu C, et al., J Sep Sci. 33(4-5): 570-81 (2010). [3] European Pharmacopoeia Ph.Eur. 7.0

Astragaloside IV


34

SL16 Sesquiterpenoids from aerial parts of Lactuca aculeata Klaudia Michalska, Wanda Kisiel Department of Phytochemistry, Institute of Pharmacology, Polish Academy of Sciences, 31-343 Krakow, Poland [email protected]

The wild lettuce Lactuca aculeata Boiss. (Asteraceae, tribe Cichorieae) is distributed in the Near East and the Anatolian plateau. This species can be easily hybridized with Lactuca sativa L. and thus occupy the primary gene pool for cultivated lettuce improvement. Sesquiterpene lactones, including the guaianolide lactucin and its derivatives as well as the germacranolide lactuside A, are particularly characteristic constituents of Lactuca species and their metabolic function has been strongly implicated in defense against diseases and herbivorous insects. We have recently confirmed the reputed analgesic and sedative activities of lactucin and its derivatives in mice [1]. Moreover, biological studies have shown that 8-deoxylactucin down-regulated cyclooxygenase-2 protein expression and nitric oxide production, and inhibited DNA binding of the central transcription factor NFκB, promoting the expression of multiple genes in response to inflammatory stimulators [2,3]. These data support some Lactuca species as sources of functional food ingredients with anti-inflammatory properties.

Our previous phytochemical study (by HPLC) on variation of seven sesquiterpene lactones in L. aculeata natural populations has shown that this plant is rich in 8-deoxylactucin (up to 1% of dry wt.) [4].

The present study deals with the isolation of ten known sesquiterpene lactones from aerial parts of L. aculeata. These compounds were isolated by silica gel column chromatography followed by preparative TLC and/or semipreparative HPLC, and were characterized by spectroscopic methods as 8-deoxylactucin, jacquinelin, 9α-hydroxyzaluzanin C and its 11β, 13-dihydroderivative, vernoflexuoside and its 11β,13-dihydroderivative, ixerin F, macrocliniside A, crepidiaside B, and lactuside A. In addition, a new lactucin-type guaianolide was isolated and its structure was determined as 11β-hydroxycrepidiaside B on the basis of HR ESIMS, 1H and 13C NMR spectra and 2D NMR techniques. [1] Wesolowska A, Nikiforuk A, Michalska K, Kisiel W, Chojnacka-Wojcik E (2006) Analgesic and sedative

activities of lactucin and some lactucin-like guaianolides in mice. J. Ethnopharmacol. 107: 254-258 [2] Cavin C, Delannoy M, Malnoe A, Debefve E, Touche A, Courtois D, Schilter B (2005) Inhibition of the

expression and activity of cyclooxygenase-2 by chicory extract. Biochem. Biophys. Res. Commun. 327: 742- 749 [3] Ripoll C, Schmidt BM, Ilic N, Poulev A, Dey M, Kurmukov AG, Raskin I (2007) Anti-inflammatory effects

of a sesquiterpene lactone extract from chicory (Cichorium intybus L.) roots. Nat. Prod. Commun. 2:717-722 [4] Beharav A, Ben-David R, Malarz J, Stojakowska A, Michalska K, Dolezalowa I, Lebeda A, Kisiel W (2010)

Variation of sesquiterpene lactones in Lactuca aculeata natural populations from Israel, Jordan and Turkey. Biochem. Syst. Ecol. 38: 602-611.


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The volatile fraction of herbal teas

Christine Tschiggerl and Franz Bucar

Institute of Pharmaceutical Sciences, Department of Pharmacognosy, Karl-Franzens University Graz, Universitätsplatz 4/1, 8010 Graz, Austria [email protected], [email protected]

Herbal teas or tisanes are among the most commonly consumed beverages all over the world.

Often the beneficial health effects of these teas are mainly attributed to their volatiles. Results on the quantitative and qualitative changes of the volatiles during the tea brewing process are scarce [1-3]. Therefore the volatile fraction of rosemary- (Rosmarinus officinalis L., Lamiaceae), lavender- (Lavendula angustifolia Mill., Lamiaceae), fennel- (Foeniculum vulgare Mill. subsp. vulgare var. vulgare (Mill.) Thellung, Apiaceae), chamomile- (Matricaria recutita L., Asteraceae) and thyme infusion (Thymus vulgaris L., Lamiaceae) has been investigated [4-6]. Additionally, the influence of saponin plants (Primula veris L./P. elatior L. and Glycyrrhiza glabra L.) on the volatile fraction in thyme herbal teas has been analyzed [6]. The genuine essential oils of the relevant plant material were compared with the volatile fraction of their infusion extract. The volatiles were obtained by two different methods, hydrodistillation and solid phase extraction. Qualitative and semiquantitative analyses were performed by GC-MS. Some individual compounds of the extracts obtained by SPE were quantified by internal standard method. Volumetric quantification of the essential oil yield of the plant materials was performed by hydrodistillation according to the European Pharmacopoeia. Analogically, the amount of volatile fraction of the infusions was determined.

The results of our investigations can be summarized as follows: 1. Comparison of the chemical classes (hydrocarbons, oxides, alcohols/ethers,

aldehydes/ketones, esters) of the essential oils and of the volatile fraction of the infusion extracts resulted in a different profile of chemical classes, which could be explained by the octanol-water partition coefficient and the boiling point of the individual compounds. In all tea samples significant losses of hydrocarbons could be recorded.

2. Physicochemical interactions between saponins and volatiles (thymol) exist. An influence on the percentage distribution of chemical classes in the volatile fraction of the combined infusion extracts (thyme and cowslip/liquorice) was observed.

3. About 15% to almost 40% of the initial essential oil could be extracted by infusions, depending on the physicochemical nature of the constituents of the essential oil.

4. Hydrodistillation and solid phase extraction are in most cases comparable methods for determining volatiles in the infusion extracts. Exceptions are herbs where reactions of the volatile compounds occur during hydrodistillation processes as in case of chamomile.

5. Semiquantitative peak area% method is in most cases adequate to make conclusions about transfer rates of individual compounds and the composition of the volatile fraction of tisanes. [1] Carnat A., Carnat AP., Fraisse D., Lamaison JL. (1999) The aromatic and polyphenolic composition of lemon verbena tea.

Fitoterapia, 70: 44-49 [2] Carnat A., Carnat AP., Fraisse D., Ricoux L., Lamaison JL. (2004) The aromatic and polyphenolic composition of roman

camomile tea. Fitoterapia, 75: 32-38 [3] Bilia AR., Flamini G., Taglioli V., Morelli I., Vincieri FF. (2002) GC-MS analysis of essential oil of some commercial fennel

teas. Food Chem, 76: 307-310 [4] Tschiggerl C., Bucar F. (2010) Investigation of the volatile fraction of rosemary infusion extracts. Sci Pharm, 78: 483-492 [5] Tschiggerl C., Bucar F. (2010) Volatile fraction of lavender and bitter fennel infusion extracts. Nat Prod Commun, 9: 1431-

1436.


36

PL8

Phenolic nutritional phytochemicals as biomarkers of plant food intake

Anna-Marja Aura

VTT Technical Research Centre of Finland, P.O.Box 1000, Tietotie 2, Espoo, FI-02044 VTT, Finland.

Dietary recommendations define a healthy diet as a one containing substantial amounts of plant foods. Plant foods are rich in dietary fibre and its co-passengers, polyphenols. Dietary fibre is by definition non-digestible fraction of plant foods and is transported by intestinal peristalsis through ileum to the colon, in which the fibre matrix is degraded and the polyphenols are released to undergo larger structural changes by the microbiota than those apparent by the liver and upper intestine. Polyphenols can be divided to six major groups: flavonoids, isoflavonoids, stillbenes, lignans, phenolic acids and tannins: ellagitannins and proanthocyanins, which can be connected with their colonic microbial metabolite profiles. Even more challenges in diversity are apparent as the metabolical processes of plant foods are described by metabolomic profiling techniques. The identified novel metabolites can be found also from the body fluids (plasma and urine) of the volunteers consuming the same plant foods.

Dietary phenolic compounds undergo intestinal and hepatic metabolism, the products of which are also shown in the plasma and urine after consumption of plant foods. Most of the complex colonic metabolites and some phenolic acid metabolites return to the liver and undergo in the enterohepatic-circulation. For this reason the residence time of colonic metabolites has been shown to be over 35 hours before returning to baseline after a single dose. If a person eats or drinks regularly polyphenol-rich foodstuffs, a prediction can be made that the phenolic metabolite level can be fairly high and constant and a connection with systemic health benefits can be made. The mechanisms, however, are under investigation. At the moment we can confirm that the consumption of dietary polyphenols and the circulating metabolites correlate with the plant food intake and serve as biomarkers of intake. The work presented here is supported by project ETHERPATHS (EC Contract no: FP7-KBBE-222639). References Aura, A-M. 2008. Microbial metabolism of dietary phenolic compounds in the colon. Phytochem Rev. 7 (2008): 407-429.


SL18

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Elicitation, production and release of polyphenols in fed-batch fermentation of grape cells

Tassoni Annalisa and Ferri Maura Department of Experimental Evolutionary Biology, University of Bologna, Via Irnerio 42, 40126, Bologna, Italy; [email protected]

Flavonoids and stilbenes are naturally occurring polyphenols, found in several fruits, vegetables and beverages. In the last years, the potential protective effects of many of these compounds against cardiovascular and neurodegenerative diseases, as well as the chemopreventive properties against cancer, have been largely investigated. Cell suspension cultures have been obtained from Vitis vinifera cv. Barbera petioles and berries and treated with several different elicitors in order to increase the in vitro production of stilbenes and flavonoids [1, 2, 3].

Recently, the optimal conditions of grape cell cultures in batch and fed batch fermentations [4], have been studied with the aim to specifically improve the production of mono-glucosylated stilbenes, which are resveratrol derivatives. These compounds are physiologically as active as free resveratrol in cardio- and chemoprotection but more stable and bioavailable after ingestion through diet. Polyphenols were extracted from biomass and culture broths and stilbenes and catechins were analyzed by reverse-phase HPLC-DAD separation [2]. In fed batch conditions the production of mono-glucosides was largely increased together with that of free resveratrol (which was 32-fold higher in bioreactor with respect to jar cultures). The elicitor chitosan [2] was tested in a bioreactor system, demonstrating its efficacy in inducing stilbenes production (23-fold and 103-fold increase of mono-glucosides and free resveratrol respectively compared to untreated fed batch culture). The bioreactor culture conditions also allowed the accumulation of other polyphenols, such as catechins and in particular the most abundant was epigallocatechin-gallate (EGCG, 90% of total catechins). The vast majority of the produced compounds was released in the culture media, a result that represents a relevant advantage for the employment of grape cell cultures in fed batch fermentation for the industrial production of plant polyphenols, also considering the necessity of developing sustainable processes.

[1] Tassoni A. et al. (2005). Jasmonates and Na-orthovanadate promote resveratrol production in Vitis vinifera L. cv.

Barbera cell cultures. New Phytologist, 166: 895-905 [2] Ferri M. et al. (2009) Chitosan treatment induces changes of protein expression profile and stilbene distribution in

Vitis vinifera cell suspensions. Proteomics, 9: 610-624 [3] Ferri M. et al. (2011). Increasing sucrose concentrations promote phenylpropanoid biosynthesis in grapevine cell

cultures. Journal of Plant Physiology, 168: 189-195 [4] Ferri M. et al. (2011). Chitosan elicits mono-glucosylated stilbene production and release in fed-batch bioreactor

cultures of grape cells. Food Chemistry, 124: 1473-1479


38

SL19 Insights into olive mill wastewaters phenolics Susana M. Cardosoa,b*, Soraia I. Falcãoa, António M. Peresb,c, Olivia R. Pereirad, Maria R.M. Dominguese

aCERNAS - Escola Superior Agrária, Instituto Politécnico de Coimbra, Bencanta, 3040-316 Coimbra, Portugal; bCIMO, LSRE - Escola Superior Agrária, Instituto Politécnico de Bragança, Campus de Sta. Apolónia, 5301-855 Bragança, Portugal; cEscola Superior de Saúde, Instituto Politécnico de Bragança, Av. D. Afonso V, 5300-121 Bragança, Portugal; dCentro de Espectrometria de Massa, Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro, Portugal [email protected]

Olive mill wastewaters (OMW) have high contents of phenolic compounds, which possess a wide range of biological activities. Therefore, OMW are regarded as a potent source of biophenols for food and pharmaceutical industries. Important biophenols occurring in OMW include tyrosol, oleuropein and hydroxytyrosol, with the latter being commercially exploited as a novel ingredient with a broad use in dietary supplements, functional foods and natural cosmetics [1,2]. Nevertheless, the phenolic profile of OMW is extremely complex and many compounds remain unknown. The elucidation of new phenolic compounds in this matrix is an important task to encourage the search of more bioactive compounds. Indeed, hydroxytyrosyl acyclodihydroelenolate and p-coumaroyl-6’- secologanoside were recently identified in OMW and proved to have greater antioxidant scavenging activity than hydroxytyrosol and oleuropein [3].

The present study evaluates the recovery of some biophenols from three Portuguese OMW. For that an ethyl acetate liquid-liquid extraction procedure, which has been previously optimized for hydroxytyrosol extraction [4], has been used. On the other hand, this work also contributes to the knowledge of OMW phenolic composition, through the elucidation of major phenolic constituents in purified methanol (PME) or aqueous acetone extracts (PAAE).

Data showed that the ethyl acetate extracts mostly contained hydroxytyrosl, although its content was much variable for the three OMW samples (0.25 – 1.91 g L-1). Oleuropein, tyrosol and luteolin-7-O-glucoside were found in the range of 0.02 – 0.23 g L-1, 0.08 – 0.35 g L-1 and 0.3 – 0.75 g L-1, respectively. New phenolic compounds identified in PME comprised oleuropein and ligstroside isomers which contain the glucose unit linked to the aromatic moiety, as well as some di-glucoside derivatives of those two compounds. In addition, polymeric compounds derived from ligstroside glucoside isomer or composed of oleuropein monomers were identified as major components of the PAAE. Future studies focusing the abundance of these new phenolic compounds in OMW, as well as their bioactivities, are now needed for determining their possible industrial exploitation. [1] Takaç S., Karakaya A. (2009). Recovery of Phenolic Antioxidants from Olive Mill Wastewater. Rec Pat Chem

Eng, 2: 230-237 [2] Soni MG., Burdock GA., Christian MS., Bitler CM., Crea R. (2006) Safety assessment of aqueous olive pulp

extract as an antioxidant or antimicrobial agent in foods. Food Chem Toxicol, 44: 903-915 [3] Obied H K., Karuso P., Prenzler PD., Robards K. (2007) Novel Secoiridoids with Antioxidant Activity from

Australian Olive Mill Waste. JAFC, 55: 2848-2853 [4] De Marco E., Savarese M., Paduano A., Sacchi R. (2007). Characterization and fractionation of phenolic

compounds extracted from olive oil mill wastewaters. Food Chem, 104: 858-867


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Molecular mechanisms underlie the biological activities of phytochemicals: searching the gap

Cristiano Longoa, Roberto Zefferinob, Antonella Leonea

a Istituto di Scienze delle Produzioni Alimentari, Consglio Nazionale delle Ricerche (ISPA-CNR), via Prov.le Lecce Monteroni, 73100, Lecce, Italy b Dipartimento di Scienze Mediche e del Lavoro, Università di Foggia, Foggia, Italy [email protected]

The health benefit of plant-derived foods is established and new researches are identifying

active plant derived molecules among the thousands phytochemicals. Throughout life, the exposure to specific phytochemicals can affect gene expression via reversible epigenetic mechanisms. This has recently launched re-exploration of nutritional, botanical or phytopharmaceutical compounds for their epigenetic effects in order to identify promising nutraceuticals or cosmeceuticals [1].

Polyphenols from grape seed extracts (GSE) possess a broad spectrum of chemo-protective properties, and lycopene, found in tomatoes and other vegetables, have been proposed to modulate hormonal and immune systems, metabolic pathways, and gap junction intercellular communication (GJIC). In animal cells, GJIC has been implicated in the cell growth control via adaptive responses: differentiation, proliferation and apoptosis. GJIC is deficient in many human tumors and its restoration or upregulation is associated with decreased cell proliferation. In the carcinogenesis process, the reversible inhibition of GJIC has been hypothesized to be involved in the tumor promotion phase.

Some innovative, safe and environmentally friendly processes such as supercritical carbon dioxide (S-CO2) extraction are developing for the antioxidant production from plant matrices. GSE and new S-CO2-extracted oleoresins obtained from tomato and tomato added with grape seeds were analyzed for antioxidant activities and biological properties on human keratinocytes [2] and human breast cancer cell (MCF-7) cultures. Plant extracts showed the ability to enhance the GJIC and connexin 43 expression in human cell cultures, and were able to completely overcome the GJIC inhibition induced by the tumor promoter HgCl2.

The knowledge of molecular mechanisms underlies the phytochemical effects and the availability of environmentally friendly methods to produce plant-derived molecules with potentially healthy properties, from natural sources (e.g., tomato and waste winery by-products), could lay new bases for preventive nutrition. [1] vel Szic KS., Ndlovu MN., Haegeman G., Berghe WV. (2010) Nature or nurture: Let food be your epigenetic

medicine in chronic inflammatory disorders. Biochemical Pharmacology, 80: 1816–183. [2] Leone A., Zefferino R., Longo C., Leo L., Zacheo G. (2010) Supercritical CO2-extracted tomato oleoresins

enhance gap junction intercellular communications and recover from mercury chloride inhibition in keratinocytes. Journal of Agricultural Food Chemistry, 58: 4769-4778.


40

PL9

Bioengineering of glucosinolates production in yeast B.A. Halkier1*, U.H. Mortensen2, L. Albertsen2, B. Salomonsen1, M. Mikkelsen1, J.K. Vester, F. Geu-Flores1

1VKR Centre for Pro-Active Plants, LIFE-KU, 1871 Frederiksberg C 2Centre for Microbial Technology, DTU, 2800 Lyngby [email protected] Epidemiological studies have demonstrated reduced risk of developing cancer upon consumption of diets rich in cruciferous vegetables. Key players in this chemoprevention are the natural products glucosinolates, in particular the methionine-derived glucoraphanin which is highly abundant in broccoli. Improved nutrition by functional foods or health-promoting dietary supplements is an attractive means for prevention of lifestyle-based diseases. Towards this goal, we have engineered the production of both simple and complex glucosinolates into tobacco (1,2) transferred the entire glucoraphanin biosynthetic pathway consisting of thirteen genes from Arabidopsis into the non-cruciferous tobacco by transient expression. The engineering involves the chloroplast-localized chain elongation machinery (5 genes) that converts methionine to dihomomethionine, and the cytosolic, ER-anchored core structure pathway (8 genes) that converts dihomomethionine to the glucoraphanin. More recently, we have embarked on development of a technology platform for engineering plant pathways exemplified by glucosinolates into yeast. Our progress in this respect will be discussed. [1] Geu-Flores et al. (2009) Engineering the production of benzylglucosinolate identifies an uncharacterized

biosynthetic enzyme. Nature Chem. Biol., 5, 575. [2] Mikkelsen et al. (2010) Reconstitution of the glucoraphanin biosynthetic pathway. Mol.Plant. 3, 751.


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The Arabidopsis Orange (Or) gene generates a metabolic sink and enhances accumulation of carotenoids in cereal cells and plants Gemma Farre1, Sol Rivera1, Chao Bai1, Ramon Canela1, Gerhard Sandmann2, Changfu Zhu1, Teresa Capell1, Paul Christou1,3 1 Departament de Producció Vegetal i Ciencia Forestal, Universitat de Lleida, Lleida, Spain 2Molecular Biosciences, J. W Goethe Universitaet, Frankfurt, Germany 3Institucio Catalana de Recerca I Estudis Avancats, Barcelona, Spain

Carotenoids accumulate in chromoplasts, which arise directly from proplastids in dividing tissues and from other non-photosynthetic plastids, such as leucoplasts and amyloplasts [1]. In all cases, chromoplasts accumulate large amounts of carotenoids in specialized lipoprotein-sequestering structures [2]. A spontaneous mutation in cauliflower lead to the discovery of the Orange (Or) gene, which showed a profound effect on patterns of carotenoid accumulation in cauliflower plants [3]. In plants cells, chromoplasts contain specialized structures that function as a metabolic sink to sequester large amounts of carotenoids [4]. The orange (or) gene is responsible for the accumulation of high levels of β-carotene in various tissues normally devoid of carotenoids. A functional role for or involves the differentiation of non-colored plastids into chromoplasts, thus generating a sink for carotenoid accumulation [5]. Lopez et al. [4] were able to increase carotenoid content in potato tubers by expressing a cauliflower or gene under the control of a potato granule-bound starch synthase (GBSS) promoter. The total carotenoid levels in the Or-expresing transgenic lines were up to 6-fold higher than in the wild-type tubers. This demonstrates that creating a metabolic sink has a positive effect on carotenoid accumulation.

We have introduced the Arabidopsis or gene into white corn, which normally accumulates only trace amounts of carotenoids (lutein and zeaxanthin). Expression of or was controlled by the endosperm specific wheat LMW glutenin promoter. We report molecular and expression analyses of T1 seeds from independent transgenic plants as a prelude to a more in depth analyses at the metabolite level. Transgenic T1 seeds exhibited a color phenotype and even though carotenoid levels have not been measured yet in these plants, our preliminary data provide exciting opportunities to enhance carotenoid levels in corn. In order to investigate the mechanisms of carotenoid accumulation and enhance carotenoid content in rice endosperm, we genetically engineered rice (Oryza sativa) with the Arabidopsis thaliana orange gene (Ator), Z. mays phytoene synthase 1 (Zmpsy1), and Pantoea ananatis phytoene desaturase (PacrtI). Here we report preliminary results in rice callus tissue which demonstrate that the Ator gene enhances carotenoid accumulation in rice dedifferentiated tissues. We are in the process of regenerating plants from colored rice tissue to ascertain whether carotenoid levels will also be increased in rice seeds. We acknowledge financial support from Ministry of Science and Innovation-MICINN, Spain (Grant BFU2007-61413) and European Research Council Advanced Grant BIOFORCE. [1] Marano et al. (1993); [2] Bartley and Scolnik (1995); [3] Crisp et al. (1975); [4] Lopez et al. (2008); [5] Lu et al. (2006)


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SL22 Production of nutritionally desirable fatty acids in seed oil of Indian mustard (Brassica juncea L.) by metabolic engineering

Surajit Bhattacharyaa, Saheli Sinhab, Prabuddha Deyb, Natasha Dasa, Mrinal K. Maitia,b

aDepartment of Biotechnology, bAdv. Lab. for Plant Genetic Engineering, Advanced Technology Development Center,Indian Institute of Technology Kharagpur, Kharagpur 721302, India [email protected]

Development of a designer oilseed crop with the improved yield attributes and enhanced

nutritional quality for the benefits of mankind and animal husbandry is now achievable with the combination of genetic engineering and plant breeding. In spite of their immense importance, the fatty acid profiles of most oilseed crops are imbalanced that necessitate the use metabolic engineering strategies to overcome the various shortfalls in order to improve the nutritional quality of these edible oils.

Indian mustard (Brassica juncea L.), being one of the important oilseed crops in Indian subcontinent naturally contains ~50% nutritionally undesirable very long chain unsaturated fatty acids (VLCUFAs), e.g. erucic acid (C22:1). In order to divert the carbon flux from the VLCUFAs to other health beneficial fatty acids, a hairpin RNA-mediated gene silencing construct under the seed specific napin promoter was used to down-regulate the endogenous fatty acid elongase [1]. The mature seeds of the transgenic B. juncea plant lines developed by Agrobacterium-mediated transformation showed significant decrease (80-82%) in the VLCUFAs along with noteworthy increase of oleic acid (C18:1), and displayed a few potentially yield attributing traits.

Further attempts have been initiated for seed-specific down-regulation of the endogenous stearoyl-acyl carrier protein (ACP) desaturase by similar RNAi approach and over-expression of a heterologous stearoyl-ACP thioesterase of FatB type [2]. For these purposes, two different genetic constructs have been prepared for B. juncea transformation using Agrobacterium-based binary plasmid. Transgenic lines are expected to have increased amount of stearic acid (C18:0), a nutritionally desirable saturated fatty acid in B. juncea seed oil. [1] Sinha S., Jha JK., Maiti MK., Basu A., Mukhopadhyay UK., Sen SK. (2007) Metabolic engineering of fatty acid

biosynthesis in Indian mustard (Brassica juncea) improves nutritional quality of seed oil. Plant Biotechnol Rep, 1: 185-197.

[2] Ghosh SK., Bhattacharjee A., Jha JK., Mondal AK., Maiti MK., Basu A., Ghosh D., Ghosh S., Sen SK. (2007) Characterization and cloning of a stearoyl/oleoyl specific fatty acyl-acyl carrier protein thioesterase from the seeds of Madhuca longifolia (latifolia). Plant Physiol. Biochem., 45: 887-897.


SL23

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α-Linolenic acid-rich oil from chia seed (Salvia hispanica L.) induces lipid redistribution, heart and liver protection in diet-induced obese rats

Hemant Poudyala and Lindsay Brownb

aSchool of Biomedical Sciences, The University of Queensland, Brisbane, 4072, AUSTRALIA; bDepartment of Biological and Physical Sciences, University of Southern Queensland, Toowoomba 4350, AUSTRALIA. [email protected]

Chia oil contains the essential n-3 fatty acid, �-linolenic acid (~58% ALA) [1, 2]. This study

has assessed whether chia oil attenuates the metabolic, cardiovascular and hepatic signs in rats fed a high carbohydrate, high fat (H) diet (carbohydrates 52% (w/w), fat 24% (w/w) with 25% (w/v) fructose in drinking water). Diets of the treatment groups were supplemented with 3% chia oil after 8 weeks on H diet for a further 8 weeks. Compared to the H rats, chia oil-supplemented H-rats had improved insulin sensitivity and glucose tolerance, reduced visceral adiposity, decreased hepatic steatosis, reduced cardiac and hepatic inflammation and fibrosis without changes in plasma lipids. Chia oil induced lipid redistribution with lipid trafficking away from the visceral fat and liver with increased accumulation in the heart. The stearoyl-CoA desaturase-1 substrate (saturated fatty acids) concentrations were increased in the heart, liver and the adipose tissue of chia oil supplemented rats with a decrease in the products (monounsaturated fatty acids). Trans-vaccenic acid was preferentially stored in the adipose tissue; the relatively inert oleic acid was stored in sensitive organs such as liver and heart and linoleic acid, the precursor of arachidonic acid, the pro-inflammatory fatty acid, was preferentially metabolised. These results strongly suggest ALA-induced lipid redistribution underlies the heart and liver protection in diet-induced obese rats. [1]Alvarez-Chavez, L.M., Valdivia-Lopez, M.D., Aburto-Juarez, M.D.,Tecante, A. (2008) Chemical characterization

of the lipid fraction of Mexican chia seed (Salvia hispanica L.). International Journal of Food Properties, 11: 687-697.

[2] Ayerza, R. (2009) The seed's protein and oil content, fatty acid composition, and growing cycle length of a single genotype of chia (Salvia hispanica L.) as affected by environmental factors. Journal of Oleo Science, 58: 347-354.


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SL24 Proteomic identification of differentially expressed proteins in curcumin-treated prostate cancer cells Marie-Hélène Teiten a, Anthoula Gaigneaux a, Sébastien Chateauvieuxa, Billing AMb, Renaut Jc, Mario Dicato a, Marc Diederich a a Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, 9 rue Edward Steichen, L-2540 Luxembourg, Grand Duchy of Luxembourg b Laboratoire National de la santé, Institute of Immunology, CRP-Sante, L-1950 Luxembourg. cCRP-Gabriel Lippmann, Department Environment and Agro-biotechnologies, L-4422 Belvaux, Luxembourg

Prostate cancer is the most common cancer in men in the western world. Lifestyle, diet and environmental as well as genetic factors (e.g. deregulations of the Wingless (Wnt) signaling pathway) promote the malignant transformation of healthy prostatic epithelium. Due to the high prevalence and the slow progressive development of prostate cancer, primary prevention appears as an attractive strategy to eradicate prostate cancer. During the last decades, curcumin (diferuloylmethane), a natural compound issued from the roots of turmeric plant Curcuma Longa, was described as a potent chemopreventive agent as it exhibits anti-inflammatory, anti-carcinogenic, anti-proliferative, anti-angiogenic and anti-oxidant properties in various cancer cells. The present study was designed to identify proteins involved in curcumin anti-cancer activity in androgen dependent and independent human prostate cancer cells using a two-dimensional electrophoresis–based proteomic analysis (2D-DIGE). Our approach permitted to revealed 425 proteins differentially expressed after curcumin treatment. Identification of selected proteins by MALDI-TOF-MS led to the conclusion that curcumin modulated proteins implicated in protein folding (Hsps family members, PP2R1A), RNA splicing (RBM17, DDX39), cell death (HMGB1, NPM1), androgen receptor signaling (NPM1, FKBP4/FKBP52) and that this natural compound has an impact on miR183 and miR141 expression. Taken altogether these data reinforced the chemopreventive potential of curcumin by showing that curcumin modulates the expression of proteins that potentially contribute to prostate carcinogenesis.


PL10

45

Bioactive natural compounds from food plants

Lanzotti V. Dipartimento di Scienza degli Alimenti, Università di Napoli Federico II, via Università 100, 80055 Portici, Napoli, Italy. [email protected]

Bioactive natural compounds from food plants have been the focus of researches for decades, firstly due to their pharmacological effects, and secondly due to their defence properties against plant diseases. Recent years have seen a renewed interest of such a research because of the development of analytical techniques allowing the structure elucidation of compounds isolated in low amounts and the application of genetic engineering and combinatorial chemistry to phytochemistry.

Our research in this field started twenty years ago and was mainly addressed to the phytochemical investigation of food plants belonging to Allium genus.1-3

In particular, we isolated and characterized of a large number of bioactive plant metabolites including apolar compounds such as tiosulfinate and sulphur compounds but also polar compounds such as flavonoid glycosides and saponins1-3 from A. porrum, A. ascalonicum, A. triquetrum, A. cepa var. Tropea, A. elburzense, A. hirthifolium, A. atroviolaceum, and very recently from A. cepa4 and A. sativum. 5

The isolated compounds subjected to a biological screening showed cytotoxicity, and antiproliferative, antispasmodic and antifungal properties.

In addition, the isolation of a high number of structurally-related analogues allowed to perform Structure Activity Relationship studies without any chemical modification which gave information on the key pharmacophoric elements of these new class of potential drugs. [1] Lanzotti, V. (2005) Bioactive Saponins from Allium and Aster plants, Phytochemistry Rev., 4: 95-110. [2] Lanzotti, V. (2006) The analysis of onion and garlic, J. Chromat. A, 1112: 3-22. [3] Benkemblia, N. and Lanzotti, V. (2007) Allium Thiosulfinates: Chemistry, Biological Properties and their

Potential Utilization in Food Preservation, Food, 1, 193-201. [4] Lanzotti, V, Romano, A., Lanzuise S., Bonanomi G., Scala F. (2011) Antifungal saponins from white onion,

Allium cepa L., in preparation. [5]Lanzotti, V., Barile E., Antignani E., Bonanomi G., Scala F. (2011) Bioactive Compounds from Allium sativum

L., in preparation


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SL25 Natural compounds as selective inhibitors for protein kinases and their potential application in cancer research Johanne Hadsbjerga, Sarah Willkomma, Katharina Nöskea, Tine D Rasmussena, Elena Brunsteinb, Karsten Niefindb, Olaf-Georg Issingera

aInstitut for Biokemi og Molekylærbiologi, Syddansk Universitet, Campusvej 55, 5230 Odense Denmark bInstitut für Biochemie, Universität zu Köln, Zülpicher St. 47, 50674 Köln, Germany [email protected]

Protein kinases have become the pharmaceutical industry’s most studied class of targets and

some 10 protein kinase inhibitors have so far been approved for the treatment of cancer. Recently, protein kinase CK2 has been considered a druggable kinase, and increased efforts are undertaken to find potent and selective inhibitors also for this kinase [1].

A Natural Compound Library from the "The NCI/DTP Open Chemical Repository" was screened for inhibitors of protein kinase CK2. The screening was performed using the catalytic subunit and the heterotetrameric holoenzyme using a luminometric test platform as described [2]. Several potential compounds were identified as CK2 inhibitors. We have focused on the characterization of resorufin and hypericin by measuring IC50, Ki, Vmax and Km. Moreover, we have determined the selectivity of the compounds towards a panel of 60 protein kinases and investigated their effect on selected human prostate and kidney tumor cell lines.

When WiT49 and CAL-54 cell lines were treated with 50 μM hypericin 55% and 40% inhibition of protein kinase CK2 activity was observed, respectively as compared to untreated controls.

Special focus was on the activity and expression status of selected signaling molecules of key signaling pathways, e.g. PI3K, AKT/PKB; p38, ERK, JNK, AMPK.

Treatment with 50 uM hypericin caused a reduction in S473 phosphorylation in AKT1/PKB and an increase in the phosphorylation of T180/Y182 in p38.

How these alterations relate to the concomitant inhibition of CK2 activity is a matter of discussion [1] Guerra, B., & Issinger, O. (2008). Protein kinase CK2 in human diseases. Current medicinal chemistry, 15(19),

1870-1886. [2] Boldyreff, B., Rasmussen, T. L., Jensen, H. H., Cloutier, A., Beaudet, L., Roby, P., & Issinger, O. (2008).

Expression and purification of PI3 kinase alpha and development of an ATP depletion and an alphascreen PI3 kinase activity assay. Journal of biomolecular screening:13(10),1035-1040.


SL26

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Hazel and other angiosperm plants as new sources of compounds with taxane-like activity

Mariangela Mielea,b, Anna Maria Mumota, Achille Zappab,c, Paolo Romanob, Laura Ottaggiob

aDepartment of Pharmaceutical Sciences, University of Genova, V.le Benedetto XV, 3, 16132, Genova, Italy bDepartment of Advanced Diagnostic Technologies, National Institute for Cancer Research, Largo R. Benzi, 10, 16132, Genova, Italy cDepartment of Informatics, Systems and Telematics, University of Genoa, Via Opera Pia 13, 16132, Genova, Italy [email protected]

Taxol is an effective antineoplastic drug, originally extracted from the bark of Taxus brevifolia,

acting on microtubule dynamics and affecting cell cycle progression[1]. The yield of Taxol and its precursors from Taxus species is very low and it is not sufficient to satisfy the commercial requirements. For this reason, most of the drug for clinical use is currently produced by semi-synthesis, starting from a natural precursor, the taxane 10-deacetylbaccatin III [2]. The finding of taxanes in differentiated and undifferentiated tissue of Corylus avellana [3,4], suggested that the production of these compounds is not a peculiarity of Taxus genera, and that, developing appropriate methodologies, other species could be used for the future production of Taxol and taxanes.

The aim of the present work is to identify species other than the slow growing Taxus to be employed as commercial source of Taxol and its precursors.

With this purpose, extracts from differentiated tissues of several plant species were analysed by ELISA and HPLC. Methanolic extracts obtained from hazel and other angiosperms have been shown to contain Taxol and other taxanes. Some of these extracts have also been shown to possess antimitotic activity on cultured cancer cell lines. Our finding suggested that the biosynthesis of taxanes occur in hazel as well as in other Angiosperms. Thus, other species could be employed for the commercial production of Taxol and other antineoplastic compounds. Studies are in progress to verify whether the enzyme involved in taxane biosynthesis, only reported for Taxus species, are also present in different species producing taxanes. With this purpose a comparison of involved genes is being carried out through specialized databanks and software. This work was partially supported by grant of Compagnia di San Paolo. [1] Wani M et al. (1971) Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and

antitumor agent from Taxus brevifolia J Am Chem Soc, 93: 2325-2327. [2] Holton RA et al. (1995) Taxol Science and Applications, Suffness M. CRC Press, Boca Raton 97-12. [3] Bestoso F et al. (2006) In vitro cell cultures obtained from different explants of Corylus avellana produce Taxol

and taxanes BMC Biotechnology, 6: 45. [4] Ottaggio L et al. (2008) Taxanes from Shells and Leaves of Corylus avellana JNP, 71: 58-60.


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PL11

Naturals for the masses – Chemistry approaches for safecommercialisation

Russell, P.J. Safety & Environmental Assurance Centre, Unilever, Colworth Science Park, Sharnbrook, Bedford, UK [email protected]

There is a growing consumer interest in food and cosmetic products which contain botanicals as an ingredient with an established or perceived functional benefit which is often intrinsically linked to traditional medicine cultures throughout the world. The challenges in safely commercialising truly efficacious plant derived materials which are substantiated by strong functional claims are great due to their inherent chemical complexity.

Traditional medicine usage is well documented for many natural products but can only be used to support development programmes where both the indication and extract composition are the same as historically used. This should be confirmed through raw material authentication and comprehensive holistic fingerprinting approaches, which we define as a ‘unique visual pattern representing the presence of known and/or unknown characteristic chemical components’. The reductive nature of pharmacopeia marker approaches do not provide sufficient insight into the composition of the complex material as a whole and the concept of a holistic analytical representation also retains respect for the synergistic philosophy of completeness advocated in many traditional therapies.

Partially or heavily refined extracts and those which are no longer being used for the traditional indication are often desired for commercialisation to increase efficacy and provide protectable intellectual property. The approach to risk assessment therefore requires a combination of fingerprinting techniques and comprehensive individual chemical component investigation which can be resource consuming. In addition to these challenges there appears to be a lack of definition from international regulators as to the requirements for the safety assessment of natural products, placing the emphasis on industry to design and provide robust supporting data packages.

Here we present a framework approach to define the analytical requirements to support the development of natural products through a defined safety programme using risk based approaches.


SL27

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Targeted screening of botanicals in herbal dietary supplements by LC-MS/MS with an information dependent acquisition approach

Mathon C.a,b, Duret M.a, Edder P. a, Bieri S.a, Christen P.b

a Official Food Control Authority of Geneva, Quai Ernest-Ansermet 22, 1211 Geneva 4, Switzerland b School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, Quai Ernest-Ansermet 30, CH-1211 Geneva 4, Switzerland [email protected] Herbal Dietary Supplements (HDS) are more popular now than ever before. The commercial success of botanical ingredients is largely due to a growing interest for products supporting a healthy lifestyle. HDS are widely marketed with various health claims. These phytopreparations are easily available to consumers through several distribution channels: OTC in pharmacies, in supermarkets, herbalist’s shops or via the Internet. In this context, problems related on undeclared, unauthorized [1] or toxic botanicals in HDS is of growing importance, because HDS have generally not been through a rigorous drug testing process. Furthermore, there is a need to conduct anti-fraud analyses by confirming the presence of the declared botanicals. Chemical methods already exist for the detection of plants but they are usually specific for a few plants only. In this study, a generic method was developed for the multi-target screening of biomarkers in HDS. This method uses high performance liquid chromatography coupled to hybrid mass spectrometry (Q-Trap) with an Information Dependent Acquisition (IDA) approach generating MS/MS spectra which can be compared with an in-house library. Botanical preparations were unselectively extracted by sonication in methanol. Each plant was characterized with at least one biomarker, which in turn was identified with its retention time, two specific transitions and their corresponding ratio as well as three Enhanced Product Ion (EPI) scans. With this method, almost hundred biomarkers were targeted by two successive analyses in the positive and negative ElectroSpray Ionisation (ESI) modes which finally allowed to screen more than 60 plants. [1] Swiss guidance on the categorization of botanicals intended for use as therapeutic preparations or food

supplements (2010). List of botanicals, version 1.3, Federal Office of Public Health, Switzerland.


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SL28 LC-MS/MS determination of marker metabolites in the ethanolic extract and in mother tinctures of Vitex agnus castus fruits Angela Mari, Paola Montoro, Sonia Piacente Dipartimento di Scienze Farmaceutiche e Biomediche, Università di Salerno, via ponte don Melillo, 84084 Fisciano (SA), Italy [email protected]

Vitex agnus-castus L. (Verbenaceae), chasteberry, is one of the most popular and effective herbs used for the prevention and treatment of pre-menstrual syndrome (PMS) and hyperprolactinemia because of its hormone-like effect [1]. Although its mechanism of action has only partially been determined, dopaminergic activities were found in polar and apolar fractions of the fruits of V. agnus-castus [2]. The chemical composition of polar extracts is characterized by the presence of iridoids and flavonoids [3]. Among these latter, casticin, a methoxylated flavonol, is considered by European Pharmacopoeia (Ph. Eur.) the reference compound for standardization of dry extracts of the species. A large number of V. agnus-castus products routinely used by women against PMS are commercially available, most of them being mother tinctures whose standardization is required.

Many analytical studies are reported about the constituents of the essential oil of chasteberry fruits but very few reports occur about LC/MS studies of the polar fraction of these fruits [4]. Targeted mass spectrometry approach is considered one of the most powerful way to obtain metabolite profiling of biological samples so it represents an ambitious tool in metabolomic quantitative analysis field [5]. While MS itself provides sensitive detection and the ability to identify metabolites based on MS or MS/MS spectra, LC-MS techniques avoiding signal suppression phenomena, provide identification of isobaric ions based on differences in their retention times. According to this, HPLC/ESI-triple quadrupole (QqQ)-MS using a very sensitive and selective mass tandem experiment such as Multiple Reaction Minitoring is considered one of the most suitable techniques for quantification of plant metabolites.

Our investigation deals with the characterization of the ethanolic extract obtained from the fruits of chasteberry. An analytical method for the quantitative determination of seven compounds, previously reported in literature [3], was developed by using HPLC/ESI-QqQMSMS in order to obtain a full metabolite profile of the extract. Five commercially available mother tinctures of V. agnus-castus fruits were analysed under the same analytical conditions and their chromatographic profiles were compared. In spite of the small contents in casticin, all the extracts exhibited higher amounts of the iridois aucubin and agnuside. That is the reason why, according to our results, these two compounds could be considered more suitable than casticin, suggested by the Ph. Eur, for standardization procedures. [1] Liu J., Burdette J.E., Xu H., Gu C., van Breemen R.B., Bhat K.P.L., Booth N., Constantinou A.I., Pezzuto J.M., Fong H.H.S.,

Farnsworth N.R., and Bolton J. L. (2001). Evaluation of Estrogenic Activity of Plant Extracts for the Potential Treatment of Menopausal Symptoms. J. Agric. Food Chem, 49: 2472-2479.

[2] Lucks B.C., Sorensen J., Veal L. (2002). Vitex agnus-castus essential oil and menopausal balance: a self-care survey. Compl. Ther. in Nursing and Midwifery, 8: 148–154.

[3] Chen S.N., Friesen B.J., Webster D., Nikolic D., van Breemen R.B., Wang Z.J. Fong H.H.S., Farnsworth N.R., Pauli G.F. (2010). Phytocostituents from Vitex angus-castus fruits. Fitoter, 82(4): 528-33.

[4] Hoberg E.; Meier B.; Sticher O. (2001). Quantitative high performance liquid chromatographic analysis of casticin in the fruits of Vitex agnus castus. Pharm. Biol, 39(1): 57-61.

[5] Wolfender J.L., Glauser G., Boccard J. and Rudaz S. (2009). MS-based Plant Metabolomic Approaches for Biomarker Discovery. Nat. Prod. Com, 4 (10): 1417-1430.

International PSE Symposium on ”Phytochemicals in Nutrition and Health”, 27-30 September 2011

51

PSE-APIVITA Award Lecture


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Contribution to Phytochemistry in recent years Sonia Piacente Dipartimento di Scienze Farmaceutiche e Biomediche, Università di Salerno, via Ponte Don Melillo, 84084 Fisciano (SA), Italy [email protected] This presentation will be focused on some of the results of phytochemical investigations performed in recent years. -Phenolic constituents of Yucca spp. Yucca schidigera (Agavaceae) is one of the major commercial source of steroidal saponins. It possesses the GRAS label which allows its use as foaming agent in soft drink (root beer), pharmaceutical, cosmetic, food, and feeding-stuffs industries. The main application of yucca products is in animal nutrition, in particolar as a feed additive to reduce ammonia and fecal odors in animal excreta. The positive effects of dietary supplementation with yucca products on the growth rates, feed efficiency, and health of livestock seem to be due not only to the saponin constituents but also to other constituents. These observations prompted us to investigate the phenolic constituents of Y. schidigera and of another species of the same genus, Y. gloriosa, largely cultivated in Eastern Georgia. Yuccaols and gloriosaols, showing very unusual spiro-structures made up of one (yuccaols from Y. schidigera) or two (gloriosaols from Y. gloriosa) C15 units linked via a �-lactone ring to a stilbenic moiety related to resveratrol, were isolated [1-2]. Yuccaols showed potent radical scavenging activity, were found to reduce the expression of the inducible isoform of nitrogen oxide synthase and to inhibit in a dose-dependent manner the Vascular Endothelial Growth Factor (VEGF)-induced proliferation, migration, and Platelet Activating Factor (PAF) synthesis in Kaposi’s sarcoma cells. Antiproliferative pregnane and secopregnane glycosides from Solenostemma argel -In the frame of a bilateral Italy-Egypt project, Solenostemma argel Hayne (Asclepiadaceae), an Egyptian wild perennial erect shrub growing in the eastern desert and along the Nile in South Egypt, used in traditional medicine as a purgative, has been investigated. From the pericarps, hairy seeds and leaves new 14,15-secopregnane glycosides, namely argelosides A – J, and new 15-keto pregnane glycosides, namely stemmosides C – K, have been isolated [3-4]. On the basis of the anticancer and cytotoxic activities reported for pregnane derivatives and considering the very unusual structural features of argelosides and stemmosides, the anti-proliferative activity of these compounds in Kaposi’s sarcoma (KS) cells has been evaluated. A dose dependence study was performed to test the effects of argelosides and stemmosides on the VEGF-induced KS cell proliferation in a concentration range 0.1 – 20 M. Results indicated that 15-keto-pregnane glycosides and 14,15-secopregnane glycosides reduced the VEGF-induced KS cell proliferation in a dose-dependent manner. -Cardenolides as cytotoxic agents

Over the past few years, there has been a marked increase in the number of reports of cardiotonic steroids-induced anticancer effects. Cardiac glycosides-mediated effects are linked to their Na+/K+-ATPase binding properties. In particular calactin-related cardenolides showing trans fused A/B rings and a single sugar in a unique “dioxanoid” attachment (doubly linked) seem to markedly bind Na+/K+-ATPase α1 subunit, overexpressed in some cancer cell lines. In this frame doubly-linked cardiac glycosides isolated from the roots of Pergularia tomentosa were tested in an vitro growth inhibitory assay including six different human cancer cell lines and for their ability to inhibit Na+/K+-ATPase activity [5]. The data revealed for these cardenolides a marked


54

cytotoxic activity (IC50 in the range 0.02- 4.00 M) and a strong correlation between their citotoxicity and their ability to inhibit sodium pump. Polyisoprenylated benzophenones from Garcinia cambogia fruits and their effects on cytokine signalling Garcinia cambogia L. (Guttiferae) is a small or medium-sized tree whose fruits are used in traditional Indian medicine as anti-inflammatory remedy. Garcinol, a polyisoprenylated benzophenone derivative occurring in G. cambogia, has been reported to reduce COX-2 and iNOS expression as well as NO production, in LPS-stimulated macrophages. In order to provide deeper insight into the biological effects of garcinol, we carried out its isolation from the fruits of G. cambogia. This phytochemical investigation afforded garcinol, guttiferones I, J and K, the new guttiferones M and N along with oxy-guttiferones K, K2, I and M which are the first example of tetracyclic xanthones derived from the oxidation of polyisoprenilated benzophenones, from natural source [6]. On the basis of the reported inhibition of STAT-1 activation by garcinol, the interaction between immobilized STAT-1 and some guttiferones was evaluated by Surface Plasmon Resonance (SPR). All the tested compounds were able to form complexes with STAT-1, exhibiting KD in the range 3-20 μM. Concomitantly, we tested the isolated compounds for their ability to modulate cytokine signalling in different cell lines. The results showed that cytokine-induced STAT-1 and NF-κB activations, as assessed by EMSA, were dose-dependently inhibited by garcinol, guttiferone M and, to minor extent, by guttiferone K (range 10-20 μM) in most cell lines. [1] Balestrieri C., Felice F., Piacente S., Pizza C., Montoro P., Oleszek W.,Visciano V., Balestrieri M. L. (2006)

Relative effects of phenolic constituents from Yucca schidigera Roezl. bark on Kaposi's sarcoma cell proliferation, migration, and PAF synthesis. Biochemical Pharmacology , 7: 1479-1487.

[2] Bassarello C., Bifulco G., Montoro P., Skhirtladze A., Benidze M., Kemertelidze E., Pizza, C, Piacente, S. (2007) Yucca gloriosa: A Source of Phenolic Derivatives with Strong Antioxidant Activity. Journal of Agricultural and Food Chemistry, 55: 6636-6642.

[3] Plaza A., Perrone A., Balestrieri C., Balestrieri M. L., Bifulco G., Carbone V., Hamed A., Pizza C., Piacente S. (2005) New antiproliferative 14,15-secopregnane glycosides from Solenostemma argel. Tetrahedron , 61: 7470-7480.

[4] Plaza A., Perrone A., Balestrieri M. L., Felice F., Balestrieri C., Hamed A., Pizza C., Piacente S. (2005) New unusual pregnane glycosides with antiproliferative activity from Solenostemma argel. Steroids , 70: 594-603.

[5] Piacente S., Masullo M., De Neve N., Dewelle J., Hamed A., Kiss R., Mijatovic T. (2009) Cardenolides from Pergularia tomentosa display cytotoxic activity resulting from their potent inhibition of Na+/K+-ATPase. Journal of Natural Products, 72: 1087-1091.

[6] Masullo,M., Bassarello C., Suzuki H., Pizza C, Piacente S. Polyisoprenylated benzophenones and an unusual polyisoprenylated tetracyclic xanthone from the fruits of Garcinia cambogia. (2008) Journal of Agricultural and Food Chemistry, 56: 5205-5210.

Poster Presentations - International PSE Symposium on ”Phytochemicals in Nutrition and Health”, 27-30 September 2011

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Poster Presentations

Poster Presentations – International PSE Symposium on ”Phytochemicals in Nutrition and Health”, 27-30 September 2011

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Poster Presentations - International PSE Symposium on ”Phytochemicals in Nutrition and Health”, 27-30 September 2011

P1 Nutritional and nutraceutical potential of saprotrophic and mycorrhizal wild edible mushrooms from Northeast Portugal

Lillian Barrosa,b, Cátia Grangeiaa,b, Anabela Martinsb, Isabel C.F.R. Ferreiraa,b

aCIMO-ESA, Instituto Politécnico de Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-855 Bragança, Portugal. bEscola Superior Agrária, Instituto Politécnico de Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-855 Bragança, Portugal. [email protected]

Mushrooms are appreciated all over the world not only by their texture and flavour, but also by their chemical, nutritional [1] and functional properties [2,3]. The consumption of wild growing mushrooms has been preferred to eating of cultivated fungi in many countries of central and Eastern Europe. Nevertheless, the knowledge of the nutritional value of wild growing mushrooms is limited. The present study reports the nutritional and nutraceutical potential of five saprotrophic (Calvatia utriformis, Clitopilus prunulus, Lycoperdon echinatum, Lyophyllum decastes, and Macrolepiota excoriata) and five mycorrhizal (Boletus erythropus, Boletus fragrans, Hygrophorus pustulatus, Russula cyanoxantha, and Russula olivacea) wild edible mushrooms. The nutritional composition was determined on the bases of moisture, proteins, fat, carbohydrate and ash. For the nutraceutical potential the antioxidant activity was evaluated by different in vitro chemical and biochemical assays: scavenging effects on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals, reducing power, inhibition of �-carotene bleaching and inhibition of lipid peroxidation in brain cells homogenates by TBARS (thiobarbituric acid reactive substances) assay. The results were compared to bioactive compounds: phenolics and ascorbic acid (determined by spectrophotometric techniques); tocopherols (HPLC/fluorescence), sugars (HPLC/RI) and fatty acids (GC/FID). Mycorrhizal species revealed higher sugars concentration (16–42 g/100 g dw) than the saprotrophic mushrooms (0.4–15 g/100 g). Furthermore, fructose was found only in mycorrhizal species (0.2–2 g/100 g). The saprotrophic L. decastes, and the mycorrhizal species B. erythropus and B. fragrans gave the highest antioxidant potential, mainly due to the contribution of polar antioxidants such as phenolics and sugars [4]. The bioactive compounds found in wild mushrooms give scientific evidence to traditional edible and medicinal uses of these species. FCT (Portugal) and COMPETE/QREN/UE- research project PTDC/AGR-ALI/110062/2009. L. Barros also thanks to FCT, POPH-QREN and FSE for her grant (SFRH/BPD/4609/2008). [1] Kalač P. (2009). Chemical composition and nutritional value of European species of wild growing mushrooms: A

review. Food Chemistry, 113: 9–16. [2] Ferreira ICFR., Vaz JA., Vasconcelos MH., Martins A. (2010). Compounds from Wild Mushrooms with

Antitumor Potential. Anti-cancer Agents in Medicinal Chemistry 10: 424-436. [3] Ferreira ICFR., Barros L., Abreu RMV. (2009). Antioxidants in Wild 320 Mushrooms. Current Medicinal

Chemistry, 16:1543-1560. [4] Grangeia C., Heleno SA., Barros L., Martins A., Ferreira ICFR. (2011). Effects of trophism on nutritional and

nutraceutical potential of wild edible mushrooms. Food Research International, 44: 1029–1035.


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P2 In vitro inhibition of nitric oxide production and free radical scavenging activity of non-cultivated Mediterranean vegetables Filomena Conforti, Mariangela Marrelli, Giancarlo A. Statti, Francesco Menichini Department of Pharmaceutical Sciences, Faculty of Pharmacy and Nutritional and Health Science, University of Calabria, I-87036 Rende (CS), Italy [email protected]

The consumption of non-cultivated botanicals may play a central role in the diet of some population groups but very few ethnopharmacological and phytopharmacological studies have exhaustively dealt. Epidemiological studies have indicated that dietary intake of antioxidant substances from plants is inversely associated with mortality from coronary heart disease and inflammation [1].

Inflammation is critical in recruiting immune cells and molecules to the site of infection for defence. Among participating cells, macrophages play a central role in organizing the release of inflammation mediators, including prostaglandin E2 (PGE2), nitric oxide (NO) and cytokines that promote host protection, as well as cause pathological consequences such as tissue oedema and abnormal histological change [2]. The RAW 264.7 mouse macrophage cell line is widely used for studies of inflammation, due to its reproducible response to lipopolysaccharide (LPS), mediated by toll-like receptor 4 (TLR4) [3].

Hydroalcoholic extracts from three non-cultivated vegetables traditionally consumed in Southern Italy, belonging to the Lamiaceae family, were tested for their free radical scavenging activity (FRSA) in the DPPH (1,1- diphenyl-2-picrylhydrazil radical) screening assay [4] and for their in vitro inhibition of niric oxide (NO) production [5]. The strongest antiradical activity was shown for Mentha spicata subsp. glabrata (leaves) with IC50 value 6 g/ml. The same extract showed high in vitro inhibition of nitric oxide production with an activity comparable to a reference drug, indomethacin. M. spicata subsp. glabrata had relatively high polyphenols content 334 mg per g of extract.

Dietary plants are being recognized as a source of numerous bioactive compounds. Many of them were shown to possess antioxidant properties and inhibit redox-sensitive intracellular signaling pathways leading to cell activation. However, the search for new plants exerting anti-inflammatory properties continues, especially among the plants included in the diets regarded as "health beneficial" ones. Clearly, this research has shown that such local food plants are a treasure trove for developing leads for new nutraceuticals and pharmaceuticals, especially for inflammatory conditions. [1] Giugliano D. (2000). Dietary antioxidants for cardiovascular prevention. Nutrition, metabolism, and cardiovascular diseases,

10: 38-44. [2] Wang L., Tu Y.C., Lian T.W., Hung J.T., Yen J.H., Wu M.J. (2006). Distinctive antioxidant and antiinflammatory effects of

flavonols. Journal of Agricultural and Food Chemistry, 54: 9798– 9804. [3] Cao X.Y., Dong M., Shen J.Z., Wu B.B., Wu C.M., Du X.D., Wang Z., Qi Y.T., Li B.Y. (2006). Tilmicosin and tylosin have

anti-inflammatory properties via modulation of COX-2 and iNOS gene expression and production of cytokines in LPS-induced macrophages and monocytes. International journal of antimicrobial agents, 27: 431– 438.

[4] Parejo I., Viladomat F., Bastida J., Rosas-Romero A., Flerlage N., Burillo J., Codina C. (2002). Comparison between the radical scavenging activity and antioxidant activity of six distilled and nondistilled Mediterranean herbs and aromatic plants. Journal of Agricultural and Food Chemistry, 50: 6882-6890.

[5] Prakash H., Ali A., Bala M., Goel H.C. (2005). Anti-inflammatory effects of Podophyllum hexandrum (RP-1) against lipopolysaccharides induced inflammation in mice. Journal of pharmacy & pharmaceutical sciences, 8: 107-114.


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A new clerodane diterpenoid from the aerial part exudate of Salvia chamaedryoides Cav. Angela Bisioa, Emanuela Giacomellia, Gianluca Damonteb, Annalisa Salisb, Daniele Fraternalec, Giovanni Romussia, Sergio Cafaggia, Donata Riccic, Nunziatina De Tommasid

aDipartimento di Chimica e Tecnologie Farmaceutiche e Alimentari, Università di Genova, Via Brigata Salerno 16147 Genova, Italy bDipartimento di Medicina Sperimentale e Centro di Eccellenza per la Ricerca Biomedica, Università di Genova, Viale Benedetto XV 7, 16132, Genova, Italy cDipartimento di Scienze dell’Uomo, dell’Ambiente e della Natura, Università di Urbino, Via Bramante 28, Urbino, Italy dDipartimento di Scienze Farmaceutiche,Università di Salerno, Via Ponte Don Melillo, 84084 Salerno, Italy [email protected]

Salvia chamaedryoides Cav. [1] is a Mexican species whose aerial part exudate showed herbicide activity against Papaver rhoeas L. and Avena sativa L. in a preliminary test [2]. The surface exudate, obtained by rinsing the plant material with CH2Cl2, was subjected to repeated column chromatography on Sephadex LH-20 , silica gel and HPLC-MS and MS2 experiments followed by semi-preparative RP-HPLC, yielding a new clerodane diterpenoid (1).

1 was identified by IR and NMR analysis, including TOCSY, COSY, HSQC and HMBC experiments, and ESI-TRAP-MS and HR-MS analysis.

(1)

[1] Epling C. (1940) A Revision of Salvia, subgenus Calosphace. In: Repertorium Specierum Novarum Regni

Vegetabilis. Vol.110. Fedde F., University of California Press: Berkley, California. [2] Bisio A., Fraternale D., Giacomini M., Giacomelli E., Pivetti S., Russo E., Caviglioli G., Romussi G., Ricci D.,

De Tommasi N. (2010) Phytotoxicity of Salvia spp. exudates. Crop Protection, 29: 1434-1446.


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P4 Chemical study on Pavetta corymbosa Augustine A Ahmadu1and Abdulkarim Agunu2

1Department of Pharmaceutical and Medicinal Chemistry Faculty of Pharmacy, Niger-Delta University,Wilberforce island, Bayelsa State-NIGERIA. 2Department of Pharmacognosy and drug development,Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria-NIGERIA. [email protected]; The genus Pavetta have long being used in ethnomedicine as anti-malaria, remedy for tuberculosis and for relieve of stomach pain[1].The literature does not report any phytochemical studies on Pavetta corymbosa. The dichloromethane and the ethyl acetate extracts were investigated for phytochemical constituents. Fractionation of the dichloromethane extract by Flash column chromatography, sephadex LH-20 and Preparative TLC afforded the trietrpenes: α-amyrin, Lupenol, ursolic acid and a mixture (1:1) of β-sitosterol and Stigmasterol, while the ethylacetate extract was fractionated over sephadex LH-20 eluted with methanol to give the known flavonoids: Quercetin, Quercetin 7-O-rhamnoside and Kaempferol. The structures were elucidated by NMR spectroscopy and compared with literature [2-5] and are reported here for the first time. [1] Dalziel J.M. and Hutchinson J. (1955). Useful plants of West Africa. Crown Agents for oversea publication,

London [2] Bishi D.S., Padalia R.C., Joshi S.C., Tewari A. and Mathela S.C. (2010). Constituents of Nepeta clarkei; Hook F.

Indian Journ. Of Chem. Vol.49B:807-811 [3] Ahmed Z., Zahra D.N. and Malik A. (2006). Phytochemical studies on Abuliton kara. Journ.of Chem. Soc.Pak.

28:3,295-297. [4] Zhang Tao, YE Qi, FENG Chun and Chen Yaoming (2007). Chemical study on Gladiolus gandavensis. Chin. J.

Appl Environ Biol. 13:5,635-640. [5] Mabry T. J et al (1970). The Systematic Identification of Favonoids. Spring- Verlag Publication New York.


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Physicochemical characterization and bioactivity of Portuguese Propolis towards its standardization Soraia I. Falcãoa, b, Cristina Freirea, Miguel Vilas-Boasb

a REQUIMTE/Departamento de Química e Bioquímica, Faculdade de Ciências do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal bMountain Research Centre (CIMO), Polytechnic Institute of Bragança, Campus de Sta. Apolónia, Apartado 1172, 5301-855 Bragança, Portugal [email protected]

Propolis is a sticky dark colored material collected by honeybees, from leaf buds and other

secretions of numerous tree species (alder, birch, palm, pine, poplar and willow) around the hive, mix with beeswax and used to reinforce the combs and to keep the hive environment aseptic.[1] This hive product is widely used in folk medicine since ancient times and recently gained popularity all over the world as an important ingredient of health foods and cosmetics. Propolis is thought to improve human health and to prevent diseases such as inflammation, heart disease, diabetes and even cancer.[2]

Phenolic compounds are the major constituents of propolis representing approximately half of the resin, while beeswax, volatiles and pollen represent 30%, 10% and 5%, respectively. The chemical composition of this bee product is highly variable and complex, depending strongly on the plant sources available to the bees at the site of collection as well as with the geographic and climatic conditions of the site.

This research outlines the extensive physicochemical characterization of propolis from different Portuguese regions, a country with botanical diversity, in order to establish quality criteria parameters for commercial purposes and thus for its valorization. For that, forty samples from continental Portugal and islands, were characterized and subjected to hydro-ethanol extraction according to our previous work.[3] Several physicochemical parameters like water, ash and waxes content, color index, spectrophotometric determination of phenolic compounds, and antioxidant capacity were determined in either crude propolis or ethanol extracts. The results for crude propolis show that water content ranged from 2% to 14%, ashes values ranged between 0.5-16.1% and the wax from 2% to 35%. According to the CIELAB color system, samples showing an orange to dark orange color like in interior north, coast and Azores archipelago presented higher values of a* and b* coordinates, comparing to the samples from interior center, south and Madeira island with darker colors. The phenolic composition of the extracts was achieved measuring the content in total phenols (10-850 mg eq/100g extract), flavones/flavonols (3-73 mg eq/100g) and flavanones/ dihydroflavonols (2-73 mg eq/100g). The bioactivity of the extracts was evaluated by the DPPH free radical scavenging activity with the EC50 ranging from 0.008 mg/mL to 0.093 mg/mL and by the reducing power, with values in the interval of 110 to 757 mg eq/100g extract. The analyzed samples presented a wide variability for the different physicochemical parameters which reflect the phyto-geography differences at the site of collection. [1] Bankova VS., De Castro SL., Marcucci MC. (2000) Propolis: recent advances in chemistry and plant origin.

Apidol., 31: 3-15. [2] Banskota A H., Tezuka Y., Kadota SH. (2001) Recent progress in pharmacological research of propolis.

Phytother. Res., 15: 561-571. [3] Falcão S., Vilas-Boas M., Estevinho LM., Barros C., Domingues MRM., Cardoso SM. (2010) Phenolic

characterization of Northeast Portuguese Propolis: usual and unusual compounds. Anal. Bioanal. Chem., 396: 887-897.


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P6 A new cytotoxic dihydrophenantrene against human cancer cells from Dioscorea membranacea roots

Arunporn Itharat a, Srisopa Ruangnoob, Pagakrong Thongdeeyingc

a Department of Applied Thai Traditional Medicine , b Ph.D program on Nutraceutical, Graduate School, cDivision of Herbal and Food Research, Faculty of Medicine, Thammasart University, Rungsit, Klongloung, Pathumthanee, 12121 Thailand [email protected];

The roots of Dioscorea membrancea has been used as anticancer drug in Thai traditional

medicine (1). Bioassay-guided isolation was used to separate the cytotoxic ingredients from the ethanolic extract of Dioscorea membranacea by testing against five human cancer cell lines, i.e. large cell lung carcinoma COR-L23, liver cancer cell HepG2, prostate cancer cell PC3, breast cancer cell line MCF-7, cervical cancer cell line Hela, one normal human lung cell MRC5 using the SRB assay(2).. Two known dihydrophenantrene compounds [2,4 dimethoxy-5,6-dihydroxy-9,10-dihydrophenanthrene (1) and 5-hydroxy-2,4,6-trimethoxy-9,10-dihydrophenanthrene (2)] and a new dihydrophenantrene compound as 5,6,2 -trihydroxy 3,4-methoxy, 9,10- dihydrophenanthrene (3) were isolated. 1 showed the highest cytotoxic activity against lung, breast and prostate cancer cell lines (IC50= 14.89 , 17.49 and 19.04 µM respectively), and 2 showed selective cytotoxic activity against prostate cancer (IC50= 23.54µM). 3 showed selective cytotoxic against only breast cancer cells (IC50= 31.41 µM).

[1] Itharat, A.,.Houghton,P.J., E.Eno-Ammguaye, Burke, P.J.,..Sampson, J.H and Raman, A. (2004) In vitro cytotoxic activity of Thai medicinal plants used traditionally to treat cancer. J Ethnopharmacology, 90 :33-38.

[2] Skehan, P., Storeng, R., Scudier., D., Monks, A., Mc Mahon, J., Vistica, D., Warren, J.T., Bokesch, H., Kenney, S. and Boyd, M.R. (1990). New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82: 1107-1112.


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Effects of daily consumption of Rosmarinus officinalis and Hypericum vesiculosum infusions on rodent cognitive function and cerebral acetylcholinesterase activity Ferlemi A.V.1, Lamari F.N.1, Iatrou G.2, Margariti M.2

1Department of Pharmacy, University of Patras, 2Department of Biology, University of Patras [email protected]

Cholinergic system is involved in the regulation of several central nervous system functions, like learning/memory. Acetylcholinesterase (AChE) at the synaptic gap plays a key role in cholinergic neurotransmission since it hydrolyses the available acetylcholine. Rosmarinus officinalis (rosemary) is a perennial shrub, native to the Mediterranean region, with many culinary and other traditional uses. Hypericum vesiculosum is an endemic species of Greek flora, which has not been yet studied, although other Hypericum have anti-depressant and wound healing properties. Aim of our project was to study the effects of daily consumption of rosemary and H. vesiculosum infusions on cognitive function and AChE activity [salt-soluble (SS) and detergent-soluble (DS) fraction] in whole brain and cerebellum of adult mice. Animals were separated in 3 groups: control-group, Ro-treated group (2% w/v of R. officinalis tea for 4 weeks) and Hv-treated group (2% w/v of H. vesiculosum tea for 4 weeks). Mice body weight was measured at regular time intervals. Effects on learning/memory were assessed using the step-through latency test and on AChE activity by the colorimetric method of Ellman. Our results demonstrate that tea drinking affect neither mouse and tissue weight nor learning/memory indices. There is a tendency of improvement in the cognitive function of Ro-mice, although not statistically significant. Moreover, in Ro-mice, AChE activity was lower (p<0,05) [19,29% (SS), 43,76% (DS) and 25,74% (SS), 19,83% (DS) in whole brain and cerebellum] than the control group. H. vesiculosum did not affect effectively learning and memory but decreased AChE activity [(13,73% (SS), 8,79% (DS) and 18,53% (SS), 15,43% (DS) in whole brain and cerebellum respectively] compared to the controls. Conclusively, our data suggest that daily consumption of both infusions for 4 weeks induced significant decrease of cerebral AChE activity, although the complex function of learning/memory was not affected.


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P8 Cytotoxic activity against lung and prostate cancer cells of the ethanolic extract from Antidesma thwaitesianum Müll. Arg. leaves

Bhanuz Dechayonta, Pakakrong Thondeeying b, Arunporn Itharatc

aGraduated School, b Division of Herbal and Food Research cDepartment of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University, Klongluang , patumtanee, Thailand [email protected]

Antidesma thwaitesianum Müll.Arg. is belongs to the family Euphorbiaceae[1]. It is mostly

found in northeast of Thailand. This plant is traditionally used to treat a various diseases such as anemia, leucorrhoea, stimulate blood circulation, diuretic and abscess. It was reported that the fruits of A. thwaitesianum contain high antioxidant activity [2] but leaves not were reported. Thus, the objectives of this research were studied cytotoxicity of crude extract and fractions from leaves of A. thwaitesianum against two types of human cancer cell lines ; large cell lung carcinoma (CORL-23) and prostate cancer cell lines (PC3), by using SRB assay [3].

The ethanolic extract of leaves and the fractions were separated by using vacuum liquid chromatography (VLC) as hexane (1,000 ml as F1), hexane – chloroform (500:500 ml as F2), chloroform(500 ml 3 times as F3, F4, F5), chloroform- methanol (750:750 ml as F7) and then methanol (2000 ml as F8). After running VLC, the different fractions were collected to test cytotoxic activity.

The results indicate that crude extract showed the specific cytotoxicity against COR-L23 (IC50= 27.91 μg/ml) but it exhibited low cytotoxicity against PC3 (IC50= 75.41 μg/ml). The activity of fractions shows that the F5 showed the highest cytotoxicity against COR-L23 (IC50= 6.52 µg/ml). We concluded that the principle cytotoxic compound should be isolated from fraction F5.

[1] Hoffmann P., (1999). The genus Antidesma (Euphorbiaceae) in Madagascar and the Comoro Islands. KewBull, 54:877–85.

[2] Puangpronpitag, D., Areejitranusorn, P., Boonsiri, P., Suttajit, M., Yongvanit, P., (2008) Antioxidant Activities of Polyphenolic Compounds Isolated from Antidesma thwaitesianum Müll. Arg. Seeds and Marcs. Journal of Food Science, 73: 648-653.

[3] Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, I., Vistica,D., Waren, J.T., Bokesch, H., Kenney, S., Boyd, M.R., (1990). New colorimetric cytotoxicity assay for anticancer drug screening. Journalof National Cancer Institute, 82: 1107–1112.


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Comparison of antimicrobial, antioxidant, anti-inflammatory activities and total phenolic contents of female and male trees from Carica papaya roots. Jitpisute Chunthorng-Orna , Arunporn Itharat b

aGraduate School, bDepartment of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University, Pathumthani, 12120, Thailand [email protected]

The female (CPF) and male tree (CPM)of Carica papaya roots are used to treat HIV patients by Thai folk doctors of Thailand. In this research, the water and ethanolic extracts of Carica papaya roots (female and male trees) were tested in vitro biological effects such as the antimicrobial activity using disc diffusion assay and minimal inhibitory concentration (MIC) by using the modified reasazurin assay (1,2) against four species of microorganisms: Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Candida albicans. The antioxidant activity also tested by DPPH assay(3). Their anti-inflammatory activity were tested by nitric oxide (NO) inhibitory activity using RAW264.7 cells (4). Total phenolic content also test by Folin-Ciocalteau colorimetric method (5). The results found that the ethanolic extract of CPF showed antibacterial activity against only one of Bacillus subtilis (clear zone= 7 mm , MIC value = 2.5 mg/ml), contrast with the ethanolic extract of CPM showed antibacterial activity against only one of Staphylococcus aureus (clear zone= 7.3 mm, MIC value = 2.5 mg/ml). The both water extract of CPF and CPM had no antibacterial activity. The ethanolic extract and the water extract of CPM showed antioxidant activity (EC50 of 8.48 and 41.66µg/ml respectively), while the ethanolic extract and the water extract of CPF had no antioxidant activity (EC50 of > 100µg/ml). The ethanolic extract of CPM showed highest total phenolic content (83.78 mg/g), as for the water extract of CPM, CPF and the ethanolic extract of CPF showed low total phenolic content (32.53, 16.71 and 6.86 mg/g respectively). For the water extract of CPF exhibited the highest activity against the NO inhibitory effect, followed by the ethanolic extract of CPF, the water extract of CPM and the ethanolic extract of CPM respectively, but the water of CPM showed only one of toxicity with RAW264.7 cells. These results can supports the traditional used as combination of female and male trees from Carica papaya roots for treatment of HIV patients because the ethanolic of CPM showed high antioxidant and total phenolic content , and the both extract of CPF showed high antibacterial and anti-inflammatory . [1] Lorian, V. 1996. Antibiotics in Laboratory Medicine. 3rd ed.Williams&Wilkins, Baltimore. McNicholl, B.P.,

McGrath, J.W. and Quinn, J.P. 2006. Water Res, 41 : 127–133. [2] Sarker, S.D., Nahar, L. and Kumarasamy, Y. 2007. Microtitre plate-based antibacterial assay incorporating

resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods, 42:321–324.

[3] Yamazaki, K. Hashimoto, A. Kokusenya, Y. Miyamoto, T. and Sato, T. 1994. Electrochemical method for estimating the antioxidative effect of methanol extracts of crude drugs. Chem. Pharm. Bull., 42: 1663-1665.

[4] Folin, O. and Ciocalteu, V. 1927. On tyrosine and tryptophan determination in proteins. J. Biol Chem., 27: 627–650.

[5] Tewtrakul, S. & Subhadhirasakul, S. (2008) Effects of compounds from Kaempferia parviflora on nitric oxide, prostaglandin E2 and tumor necrosis factor-alpha productions in RAW264.7 macrophage cells. Journal of Ethnopharmacology, 120, 81–84.


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P10 Alkaloids of Hippeastrum papilio

Jean Paulo de Andrade1,2, Strahil Berkov1, Kelly B. da Silva2, Liana G. Sachet2, Francesc Viladomat1, Carles Codina1, José Angelo S. Zuanazzi2, Jaume Bastida1

1Departament de Products Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII s/n, E-08028 Barcelona, SPAIN.

2Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, 90610-000, Porto Alegre, RS, BRAZIL.

[email protected];

The Amaryllidaceae alkaloids constitute a large group of isoquinoline alkaloids with a wide range of chemical structures and interesting biological properties [1]. The use of galanthamine in palliative therapy for mild-to-moderate Alzheimer’s Disease (Reminyl®) has prompted a search for analogous compounds bearing the galanthamine-type skeleton [2] as well as new Amaryllidaceae species for a sustainable production of this compound [3].

The genus Hippeastrum is endemic to South America, principally Brazil. Earlier studies have reported the isolation of galanthamine from Hippeastrum species and cultivars, but mainly as a minor compound.

Hippeastrum papilio, found in the South of Brazil, has been chosen for phytochemical study., We have identified six known alkaloids from bulbs and leaves, namely galanthamine (2), which was found in relatively high quantities, narwedine (3), haemanthamine (4), 11-hydroxyvittatine (5), 8-O-demethylmaritidine (6) and vittatine (7). We also report the isolation of the new compound, 11β-hydroxygalanthamine (1).

Alkaloid 1 is an epimer of the alkaloid previously described as habranthine, which was isolated from Pancratium maritimum with an uncertain stereochemistry of the hydroxyl substituent at position 11. Using modern 2D NMR, we provide the correct assignment of 1, confirming that the compound hitherto known as habranthine is in fact 11α-hydroxygalanthamine. Interestingly, 11β-hydroxygalathamine (1) has demonstrated a significant ability to inhibit the acetylcholinesterase enzyme.

[1] Bastida, J.; Lavilla, R.; Viladomat, F. Chemical and biological aspects of Narcissus alkaloids. In: The alkaloids:

Chemistry and Biology. 2006. v.63. p.99-179. [2]Berkov, S.; Codina, C.; Viladomat, F.; Bastida, J. N-alkylated galanthamine derivatives: potent

acetylcholinesterase inhibitors from Leucojum aestivum. Bioorg Med Chem Lett 2008, 18, 2263-2266. [3] Berkov, S.; Bastida, J.; Viladomat, F.; Codina, C. Development and validation of GC-MS method for rapid

determination of galanthamine in Leucojum aestivum and Narcissus ssp.: a metabolomic approach. Talanta, 2010, 83, 1455.


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Activity of compounds extracted from Salvia aurea against methicillin- resistant Staphylococci. Schito AM.a, Piatti G.a, Bisio A.b, Giacomelli E.b, Romussi G.b, Pruzzo C.c

aDept. of Integr. Surgical and Diagnostic Sciences, University of Genoa, Largo R. Benzi 10, 16132 Genoa, Italy bDept of Chemistry and Pharmaceutical and Food Technologies, University of Genoa, Via Brigata Salerno 16147 Genova, Italy cDept of Biology, University of Genoa, Viale Benedetto XV, 5 16132 Genoa, Italy - [email protected]

Methicillin-resistant Staphylococcus aureus (MRSA) and S.epidermidis (MRSE) are Gram positive pathogens that represent a leading cause of serious human diseases worldwide and are responsible for substantial morbidity and mortality, both within the healthcare system and in the community [1,2]. We describe here the activity of three compounds, 12 methoxy-carnosic acid (MCA), 7-methoxy-rosmanol (MR) and rosmanol (R), derived from S.aurea, on 5 clinical S.aureus strains (2 MRSA and 3 MSSA) and 5 S.epidermidis strains (3 MRSE and 2 MSSE). The three molecules, identified by IR and NMR analysis, were obtained from the surface exudates by rinsing the fresh aerial part plant material with CH2Cl2 and repeated column chromatography on Sephadex LH-20 and silica gel. Table 1 shows the minimal inhibitory concentrations (MICs) of the compounds, determined by standard methods [3]. Killing curve assays, at 4 x MIC concentrations, were performed on S.aureus and S.epidermidis according to recommended procedures [3]. A description of the activity expressed on a representative MRSA and MRSE strain is shown in Fig 1 and Fig 2. MC and R displayed a clear bacteriostatic effect while MR possessed a bactericidal activity on the same pathogen. On S.epidermidis all tree compounds manifested a bacteriostatic effect. [31]Our results indicate that MCA, MR and R isolated from S.aurea represent molecules capable of inhibiting or killing methicillin-resistant Staphylococci. Investigation on their mechanisms of action are highly warranted.

Tab. 1: MIC values for the indicated compounds are expressed in μg/ml

Fig 1: S. aureus Oxa-R - time kill curves of a rapresentative MRSA strain. Compounds were tested at 4 X their respective MIC (log UFC/ml vs. hours).

Fig 2: S. epidermidis Oxa-R - time kill curves of a rapresentative MRSE strain. Compounds were tested at 4 X their respective MIC(log UFC/ml vs. hours).

[1] Mandell, Douglas and Bennett (2005) Principles and Practice of Infectious Diseases, 5th ed.; Mandell, G.,Bennett, J.; Dolin, R., Eds.; Churchill Livingstone: Philadelphia; pp 2147− 2152 and 2069− 2089

[2] Witte W, Cuny C, Klare I, Nübel U, Strommenger B., Werner G (2008) Emergence and spread of antibiotic-resistant Gram-positive bacterial pathogens. Int J Med Microbiol. Jul;298(5-6):365-77.

[3] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; sixteenth informational supplement. Document M100-S16. Wayne, PA: CLSI; 2006.


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P12 Influence of benzylaminopurine on antioxidant activity and phenolic content on in vitro propagated Lamium album L. Milena Dimitrova, Zhenya Yordanova, Veneta Kapchina-Toteva Department of Plant Physiology, Faculty of Biology, Sofia University “St. Kliment Ohridski”, 8 Dragan Tsankov Blvd., 1164 Sofia, Bulgaria

Lamium album L., commonly known as “white dead nettle” is perennial herb from Lamiaceae family widely used in folk and official medicine. Lamium album L. contains variety of compounds: phenolics, iridoids, triterpenes, saponines, fatty acids, phytoecdysteroids, essential oils, tannins, amines. The effect of different concentration (0,1-3,0 mg.l-1) of cytokinin BA (6-benzylaminopurine) on in vitro propagation and the amount of secondary metabolites and total antioxidant activity in Lamium album L. was examined. All concentrations of BA suppressed root formation and stimulated callusogenesis. Stimulation of shoot number was observed on MS medium supplemented with 1.6 and 2,6 mg.l-1 BA compared with control plants propagated on MS medium without plant growth regulators. Shoots cultivated on MS medium supplemented with 1.4 and 2.0 mg.l-1 BA showed increased content of total phenols. The highest concentrations of flavonoids were obtained in MS medium supplemented with BA from 1.6 up to 2.0 mg.l-1. The highest content of antioxidant activity was achieved on MS medium supplemented with BA 1.4 and 1.6 mg.l-1 compared with control plants. In addition, the amount of secondary metabolites and antioxidant capacity was established in formed callus in underground part of in vitro propagated Lamium album plants. Significant stimulation of total phenols in callus was observed on MS medium supplemented with BA in concentrations - 0.5, 0.8 mg.l-1 and 2.2, 2.6 mg.l-1. The highest content of antioxidant activity was achieved on MS medium supplemented with BA - 0.2, 0.5 and 0.8 mg.l-1. The amount of flavonoids in callus was approximately threefold lower than that established in shoots. Therefore the higher concentrations of BA enhanced content of phenolic compounds with high antioxidant activity in shoots, but lower concentrations of BA (0.2, 0.5 and 0.8 mg.l-1) stimulated accumulation of secondary metabolites in formed callus. This work was financially supported by the grand № DTK-02-29/2009 of Ministry of Education, Bulgaria.


P13

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Currant seed extracts showing protective effect on human lymphocytes DNA

Dejan Gođevaca, Vele Teševićb, Vlatka Vajsa, Slobodan Milosavljević, b Gordana Zdunić c, Miroslava Stankovićd

aInstitute of Chemistry, Technology and Metallurgy, Njegoševa 12, University of Belgrade, Serbia bFaculty of Chemistry, Studentski trg 12-16, University of Belgrade, Serbia cInstitute of Nuclear Science ″Vinča″, University of Belgrade, Serbia dInstitute for Medicinal Plants Research, Tadeuša Košćuška 1, 11000 Belgrade, Serbia [email protected]

On the basis of LC/UV/MS analysis, 35 compounds from the extracts of seeds of six cultivars of currants (two cv. of black, red and white currant) were identified. Black currants cultivars contained protocatechuic acid and p-hydroxibenzoic acid, and traces of nitril containing phenolic acids. Synapoyl glucoside is characteristic for cv. Malling Jewel. Dihydrophaseic acid glycosides and carboxymethylindol glycosides were present mainly in white and red currant cultivars. Blackcurrant seeds contained higher amount of flavonoids, especially rutin, isoquercetin and taxifolin.

The currant seed extracts were tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using cytochalasin-B blocked micronucleus (CBMN) assay. [1] The frequency of MN was scored in binucleated cells, and the best antioxidant potential showed the cultivars of black currants. At a concentration of only 0.17 mg/ml, extract of the seed cv. Malling Jewel effected a decrease in the frequency of MN for 60% compared with control cell cultures.

These findings could support the potential benefits of currant seed extracts and their constituents in a multitude of disease states, including cancer, cardiovascular disease, and neurodegenerative disorders.

[1] Fenech M., Morley AA. (1993) The cytokinesis-block micronucleus technique: a detailed description of the

method and its application to genotoxicity studies in human populations. Mut. Res. 285: 35-44


70

P14 Inhibitory effect of Kaempferia parviflora on NFкB, Rel A, NOS, COX-2 and TNF-�� mRNA expression

Chutha Sae-wong, Supinya Tewtrakul, Pimpimon Tansakul

Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla 90112, Thailand [email protected]

Nuclear factor-kappa B (NFкB) is dimeric complexes of p50/RelA (p50/p65) which plays an important role in controlling inflammatory gene activation[1] such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2)[2] which are involve in inflammation process. To clarify the anti-inflammatory mechanism of the active compounds from Kaempferia parviflora, the effects of 5,7-dimethoxyflavone, trimethylapigenin and tetramethylluteolin on the expression of mRNA were examined using semi-quantitative analysis of RT-PCR technique. The result revealed that 5,7-dimethoxyflavone decreased cellular iNOS, COX-2 and Rel A mRNA level and this compound also affected the β-actin mRNA expression. It was indicated that inhibitory effect of this compound cause of fault positive effect. Trimethylapigenin dose-dependently suppressed iNOS, COX-2 and Rel A mRNA level, while inhibitory effect on NFкB was partly affected at the highest concentration. Tetramethylluteolin selective inhibited Rel A, but slightly inhibited iNOS mRNA, while the expression of COX-2, NFкB and TNF-� was not affected [Figure 1]. In conclusion, the mechanism of anti-inflammation of K. parviflara is due to the suppressive effect of trimethylapigenin mainly through the inhibition of COX-2 enzyme and the selective inhibitory effect of tetramethylluteolin through Rel A mRNA expression. Conc N C 3 10 30 100 N C 3 10 30 100 N C 3 10 30 100 µg/ml

iNOS

COX-2

NFкB

Rel A

TNF-�

β-actin (A) (B) (C)

5, 7-Dimethoxyflavone Trimethylapigenin Tetramethylluteolin

Figure 1 Effect of 5, 7-dimethoxyflavone (A), trimethylapigenin (B) and tetramethylluteolin (C) on the expression of mRNA

[1] Lima T.M., Gorjao R., Hatanaka E., Cury-Boaventula M.F., Silva E.P.P., Procopio J., et al. (2007). Mechanisms

by which fatty acids regulate leucocyte function. Clinical Science 113: 65-77. [2] Haridas, V., Arntzenm, C.J., Gutterman, J.U. (2001). Avicins, a family of triterpenoid saponins from Acacia

Victoriae (Bentham), inhibit activation of nuclear localization and ability to bind DNA. Proceedings of the National Academy of Sciences of the United States of America 98: 557-562.


P15

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Antioxidant activity of Uva di Troia Canosina ad acino piccolo: comparison of different extraction methods Filomena Corboa, Angelo Lovecea, Claudio Brunoa, Flavia la Forgiab, Sergio Fontanab, Mariagrazia Rosellia, Antonio Laghezzaa, Giovanni Lentinia, Carlo Franchinia, Solomon Habtemariamc

aDipartimento Farmaco Chimico, Università degli Studi di Bari, via Orabona 4, 70125 Bari, Italy bFarmalabor S.R.L. Ricerca e Sviluppo via Pozzillo, Z.I. 70053 Canosa di Puglia (Bari), Italy cPharmacognosy Research Laboratories, Medway School of Science, University of Greenwich Central Avenue Chatham Maritime Kent ME4 4T

Over the past few years a big effort has been done for the valorisation of food, agricultural residues, and wastes by creating added-value using bio-products, bio-fuels, or food ingredients. Grape is one of the world’s largest fruit crops with a global production of around 55 million tonnes in 2008 and 26 million tonnes of wine.1 This also generates an enormous amount of waste, including grape skins and seeds.

Polyphenols, contained in grapes, exhibit a wide range of biological effects as antioxidants, antimicrobials, and modulators of various enzyme systems. Beneficial effects for human health have been demonstrated in experimental studies on LDL-cholesterol oxidation, platelet aggregation, cardiovascular diseases,2–4 and type 2 diabetes.5

Skins and seeds of Uva di Troia Canosina cultivar from red-vinification were used in this work. The dried marc was extracted by both traditional and microwave-assisted extraction (MAE) techniques, the extracts obtained were analyzed by capillary zone electrophoresis (CZE) and evaluated as antioxidant agents by the DPPH assay. [1] OIV (2009). Organisation internationale de la vigne et du vin. <http://www.oiv.int> [2] Leifert, W.R., Abeywardena, M.Y., (2008) Cardioprotective actions of grape polyphenols, Nutrition Research, 28,

729–737 [3] Anastasiadi, M., Pratsinis, H., Kletsas, D., Skaltsounis, A.L., Haroutounian, S.A., (2009) Bioactive non coloured

polyphenols content of grapes, wines and vinification by-products. Evaluation of the antioxidant activities of their extracts. Food Research International, doi:10.1016/j.foodres.2009.11.017

[4] Iriti, M., Faoro, F., (2010) Bioactive chemicals and health benefits of grapevine products. Bioactive Foods in Promoting Health, 581, 620

[5] A. M. Hodge, D. R. English, K. O’Dea and G. G. Giles, (2006) Diabetic Med., 23, 690–697


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P16 Nitric oxide inhibitory effect and antioxidant activity of the extracts from Musa sapientum bracts and flowers

Pathompong Phuakleea, Arunporn Itharatb aGraduated School, Faculty of Medicine, Thammasat University. bDepartment of Applied Thai traditional Medicine, Faculty of Medicine, Thammasat University, Thailand [email protected]

Musa sapientum Linn. (Musaceae) or banana has been commonly used as health food and

drugs in Thai traditional medicine. For the example, bract and flower parts of Musa sapientum Linn. were used as drugs and health foods for postpartum care. Thus, the objective of this research was investigated anti-inflammatory activity by determination on inhibitory effect on Nitric oxide production on RAW264.7 cell which were stimulated by lipopolysaccharide (LPS)[1] and tested antioxidant activity by using DPPH radical scavenging assay[2] of bract and flower parts of banana extracts. The dried bracts and flowers of Musa sapientum were extracted by macerated with 95% ethanol, 50%ethanol, soaking in water and boiling in water or decoction. The results were found that the water extract by soaking of flower showed the highest anti-inflammatory activity, with an IC50 of 9.75 µg/ml. The decoction extract of flower showed the highest antioxidant activity, with an EC50 of 12.35 μg/ml, followed by the 95% ethanolic and soaking extract of flowers, with EC50 values of 15.57 and 22.86 μg/ml, respectively. It was concluded that the flower of Musa sapientum Linn. may be considered potentially useful as a source of natural anti-inflammatory and antioxidant ingredients and it was support using in Thai traditional medicine as food and anti-inflammation for postpartum care. [1] Tewtrakul, S., and Subhadhirasakul, S. Effects of compounds from Kaempferia parviflora on

nitric oxide, prostaglandin E2 and tumor necrosis factor-alpha productions in RAW 264.7 macrophage cells. Journal of Ethanopharmacology, 2008; 120:81-84.

[2] Yamasaki, K., Hashimoto, A. Kokusenya, Y., Miyamoto, T. and Sato, T. Electrochemical method for estimating the antioxidative effects of methanol extract of crude drugs, Chem. Pharm. Bull.1994; 42:1663-1665.


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Correlation between phenolic content and antioxidant activity of infusions from eleven Mediterranean plant species

Sandra Gonçalves, Diogo Gomes, Patrícia Costa, Anabela Romano

Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology (IBB-CGB), Faculty of Sciences and Technology, University of Algarve, Campus de Gambelas, Ed. 8, 8005-139 Faro, Portugal. [email protected]

Antioxidants scavenge free radicals by initiating and propagating oxidative chain reactions, thus delaying or preventing intracellular oxidative damage [1]. Natural antioxidants such as phenolics and flavonoids have been shown to possess multiple pharmacological activities through their antioxidant properties, including neuroprotective and anti-ageing activities. Highly heterogeneous soil and climatic conditions of the Mediterranean region have resulted in an increased diversity of plant species, most of them with medicinal properties that deserve further investigation. In this study, the total phenolic content of extracts from eleven Mediterranean plants and their related antioxidant activities were determined. The extracts were prepared as infusions (cold and hot), as they are traditionally used in popular medicine. The infusions were investigated for their effect on deoxyribose degradation, reducing properties and Fe2+-chelating abilities. The free radical scavenging activity and the total phenolic contents were also determined by using DPPH and Folin-Ciocalteu assays, respectively. The capacity of hot infusions to prevent Fe2+-induced lipid peroxidation in mouse´s brain (in vitro) was also investigated. The correlation between phenolic content and the antioxidant activity, obtained with each method, was calculated according to Pearson’s test. The phenolic content was significantly higher in hot infusions than in cold infusions of all the species. Among the species studied, the highest phenolic contents were observed in Arbutus unedo (1693.1 118.2 and 2256.8 15.6 µmolGAEgps

-1 in cold and hot infusions, respectively), Myrtus communis (1006.1 54.4 and 1244.6 30.6 µmolGAEgps

-1) and Pistacia lentiscus (759.5 74.4 and 1359.1 33.2 µmolGAEgps

-1). The infusions from these species also showed the highest Fe2+ chelating ability, reducing power, as well as DPPH free-radical scavenging and inhibitory activity toward lipid peroxidation. A positive correlation was found between total phenolic content and antioxidant activity obtained with all the methods excepting hydroxyl radical-scavenging (deoxyribose degradation). The results of this research represent a good basis for further analysis of the selected plants to discovery new antioxidant compounds. S. Gonçalves and P. Costa acknowledge a grant from Portuguese Science and Technology Foundation (FCT, SFRH/BPD/31534/2006 and SFRH/BD/63505/2009). [1] Valko M., Leibfritz D., Moncola J., Cronin M.T.D., Mazura M., Telser J. (2007). Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39: 44–84.


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P18 New natural compounds of Dionaea muscipula as anticancerous agents François Gaaschta, Monika Jain a, Marie-Hélène Teiten a, Marc Schumacher a, Gilbert Kirsch b, Denyse Bagrel c, Mario Dicato a, Marc Diederich a aLaboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, 9 rue Edward Steichen, L-2540 Luxembourg, Grand Duchy of Luxembourg bLaboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, Université Paul Verlaine de Metz, Technopôle, 1 Boulevard Arago, F-57070, Metz, France cLaboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, Université Paul Verlaine de Metz, Campus Bridoux, Rue du Général Delestraint, F-57070, Metz, France

Since centuries, natural compounds are used in traditional medicines. Recent studies showed that such substances are able to block the appearance and the development of many diseases such as cancers. The discovery and characterization of new molecules will permit to fill out the drug library of new therapeutic molecules that needs to become greater due to the emergence of new diseases and the appearance of resistance towards conventional treatments.

The Venus Flytrap (Dionaea muscipula Solander Ex Ellis) is the most well known carnivorous plant. Inherent from the United States, this plant catches and digests small preys with an active trap. It was already described that this plant contains many therapeutic molecules previously described in other plants like myricetin, quercetin, plumbagin and ellagic acid but any specific natural compound has been discovered till today in Dionaea muscipula. The aim of this project is to discover and to characterize new and/or specific therapeutic molecules issued from Dionaea muscipula.

We prepared methanolic leaves extracts of Dionaea muscipula. These extracts were shown to be uptaken by K562 cells and to be cytotoxic toward five different leukaemia cell lines (HL-60, Jurkat, K562, Raji, U937) and four adherent cancer cell lines: A549 (lung), MDAMB-231 (breast), PC-3 (prostate), SAOS-2 (bone) but have no effect on HT-29 cells (colon). Purification of the extract by preparative HPLC-MS has shown that the extract contains 7 major compounds and that among these different substances, at least one is unknown and exerts cytotoxicity towards K562 cells. Analysis of cell death by fluorescence microscopy after Hoechst/propidium iodide staining showed that this cytotoxic fraction induces cell death mainly by necrosis. Preliminary results showed that this fraction was till now unknown as therapeutic substance and will be identified by NMR.


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Effects of a rice bran enzymatic extract on cytokines implicated in the inflammatory state of adipose tissue in a rat model of obesity.

Candiracci Ma., Parrado Ja., Justo M.L. b, Rodríguez-Rodríguez R. b, Castaño A.a and Herrera M.D.b

a Departamento de Bioquímica y Biología molecular. b Departamento de Farmacología. Facultad de Farmacia. Universidad de Sevilla. C/ Profesor García González, nº 2, 41012- Sevilla. Spain.

Obesity is characterized by a chronic low-grade inflammation in adipose tissue that is believed to contribute to the genesis of insulin resistance, leads to type 2 diabetes, a risk factor for cardiovascular diseases. The inflammation of the adipose tissue is mainly due an influx of macrophages that secrete proinflammatory cytokines as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin 1 beta (IL-1 β) (1, 2).

At the same time, it is well-known that soluble factors released by adipocytes have roles at the interface between the immune and the metabolic systems. In this sense, adiponectin is an anti-inflammatory factor known to be decreased in individuals with visceral obesity and states of insulin-resistance (3).

Attenuating inflammation in adipose tissue beneficially modifies metabolic disturbances and reduces disease risk, even in the absence of obesity reduction (4, 5). We have previously described that obese Zucker rats fed on Rice Bran Enzymatic Extract enriched-standard diet (RBEE) ameliorated metabolic parameters related to insulin resistance and cardiovascular disease. Thus, the main goal of present work was to investigate the ability of RBEE to prevent the inflammatory state in the adipose tissue.

Our results show that RBEE-diets (1% or 5%), significantly decrease the upregulation of mRNAs of inflammatory cytokines (TNF-�, IL-6) found in obese Zucker rats. However, RBEE-diets did not reverse the downregulation of adiponectin found in obese Zucker rats. In conclusion we hypothesize that RBEE has a beneficial effect on inflammatory state in the adipose tissue that is mainly carried out on macrophages-derived cytokines. [1] Hotamisligil GS, Shargill NS, Spiegelman BM. (1993) Science, 259:87-91. [2] Bastard JP, Maachi M, Van Nhieu JT, Jardel C, Bruckert E, Grimaldi A, et al. (2002) J Clin Endocrinol Metab.

87: 2084-9. [3] Tilg H, Moschen A.R. (2006) Nature Reviews. 6: 772-83. [4] Kanda H, Tateya S, Tamori Y, Kotani K, Hiasa K, Kitazawa R, Kitazawa S, Miyachi H, Maeda S, Egashira K,

Kasuga M. (2006) J Clin Invest. 2006; 116: 1494–1505. [5] Todoric J, Loffler M, Huber J, Bilban M, Reimers M, Kadl A, Zeyda M, Waldhausl W, Stulnig TM. (2006)

Diabetologia 49: 2109-19.


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P20 Metabolic engineering of carotenoid biosynthesis in riceendosperm

Chao Bai1, Sol M Rivera2, Vicente Medina1, Gerhard Sandmann3, Ramon Canela2, Changfu Zhu1, Teresa Capell1 and Paul Christou1, 4

1Departament de Producció Vegetal i Ciència Forestal, Universitat de Lleida-CRA, Spain 2Departament de Química, UdL, Av. Alcalde Rovira Roure, 191, Lleida, 25198, Spain 3Molecular Biosciences, J.W. Goethe Universitaet, Frankfurt am Main, Germany 4Institucio Catalana de Recerca i Estudis Avancats, Barcelona, Spain [email protected]

Vitamin A is an essential nutrient in the human diet. Although many crops are rich sources of pro-vitamin A, millions of people rely on staple crops that contain minimal levels of this nutrient, for example rice. Several strategies have been used to enhance carotenoid accumulation in plants. The amounts of carotenoids in plant tissues and organs do not appear to depend solely on carotenogenic enzyme activities responsible for their synthesis. The upstream precursor (MEP derived IPP and GGPP) pathway may also positively influence their accumulation [1], while downstream degradation pathways may deplete the carotenoid pool [2]. The identification and characterization of a novel gene mutation in cauliflower (or) which appears to mediate the differentiation of proplastids and/or non-colored plastids into chromoplasts accompanied by a concomitant accumulation of carotenoids indicated that creating a metabolic sink to sequester carotenoids is another important mechanism for their accumulation in plants [3]. In order to investigate the mechanisms of carotenoid accumulation and enhance carotenoid content in rice endosperm, we genetically engineered rice (Oryza sativa) with the Arabidopsis thaliana orange gene (Ator)/A. thaliana 1-deoxy-D-xylulose 5-phosphate synthase (Atdxs); Zea mays phytoene synthase 1 (Zmpsy1), and Pantoea ananatis phytoene desaturase (PacrtI). Here we report preliminary results from early experiments which demonstrate that co-transformation is a useful method for engineering such complex traits in rice plants. [1] Enfissi EMA., Fraser PD., Lois LM., Boronat A., Schuch W., Bramley PM. (2005) Metabolic engineering of the

mevalonate and non-mevalonate isopentenyl diphosphate-forming pathways for the production of health-promoting isoprenoids in tomato. Plant Biotechnol J, 3: 17–27

[2] Auldridge ME., Block A., Vogel JT., Dabney-Smith C., Mila I., Bouzayen M., Magallanes-Lundback M., DellaPenna D., McCarty DR., Klee HJ. (2006) Characterisation of three members of the Arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family. Plant J, 45: 982–993

[3] Li L., Van Eck J. (2007) Perspectives: metabolic engineering of carotenoid accumulation by creating a metabolic sink . Transgenic Research, 16: 581-585


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Effect of quercetin on hepatic steatosis in ob/ob mice

Jung-In Kim, Jihye Kim, Haneul Choi, Min-Jung Kang, Soomi Jeong, Minju Kim School of Food and Life Science, Inje University, Gimhae, South Korea [email protected]

Obesity and hypertriglycerinemia are risk factors associated with non-alcoholic fatty liver disease (1). Quercetin (QE) exerts anti-hypertriglyecmic effect in mice fed high cholesterol diet (2). The aim of this study is to investigate effect of QE on obesity, hyperlipidemia, and hepatic steatosis in ob/ob mice. Four-week-old male ob/ob mice (n=16) and their lean littermates (Lean-control, n=8) were purchased. After 1 week of adaptation, ob/ob mice were fed either AIN-93G diet (ob/ob-control) or a diet containing QE at 0.08% for 10 weeks. Lean-control animals were fed AIN-93G diet. Animals were sacrificed after an overnight fast. Serum levels of triglycerides, cholesterol and adiponectin and activity of alanine amininotransferase (ALT) were measured. Liver lipids were extracted and triglyceride levels were measured. The data were analyzed by one-way ANOVA and Tukey’s test (p<0.05). Consumption of QE did not influence body weight of ob/ob mice significantly. Serum triglyceride, cholesterol, and ALT and hepatic triglyceride levels of ob/ob-control group were significantly higher than lean-control group. Consumption of QE significantly decreased serum triglyceride, cholesterol, and ALT and hepatic triglyceride levels compared with ob/ob control group. Serum adiponectin of ob/ob mice was lower than lean-control group. Reduced serum ALT of ob/ob mice was significantly increased by consumption of QE. These results indicate that QE could alleviate hyperlipidmia and hepatic steatosis in ob/ob mice. Table 1. Serum lipid profile, adiponectin, and ALT and hepatic triglycerides

mean ± SD. P<0.05 [1] Angulo P. (2002) Nonalcoholic fatty liver disease. N Engl J Med, 16: 1221-1231 [2] Azuma K, Ippoushi K, Terao J. (2010) Evaluation of tolerable levels of dietary quercetin for exerting its

antioxidative effect in high cholesterol-fed rats. Food Chem Toxicol, 48: 1117–1122

Group Lean-control ob/ob-control ob/ob-QE

Serum triglycerides (mg/dL) 64.0 ± 10.6 a 134.3 ± 17.4 c 107.0 ± 14.7 b

Serum cholesterol (mg/dL) 90.4 ± 14.2 a 171.7 ± 25.6 c 142.0 ± 19.8 b

Serum adiponectin ( g/mL) 13.8 ± 2.4 b 7.6 ± 1.3 a 11.0 ± 1.6 b

Serum ALT (IU/L) 49.4 ± 10.4 a 197.6 ± 35.0 c 139.5 ± 24.0 b

Hepatic triglycerides (mg/g liver) 13.0 ± 2.9 a 65.5 ± 1.6 c 42.1 ± 5.9 b


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P22 Olive oil phenols improve the altered human intestinal permeability. Study in vitro P. Trisolini1, A. Ignazzi1, F. Pezzolla1, D. Lorusso1, S. Fiume2, L. Debellis2 and M.A. Maselli1 1Scientific Institute of Gastroenterology “S de Bellis”, Castellana Grotte, Bari; 2 Dept. of General and Environmental Physiology, University of Bari.

In ex vivo and in vitro models olive oil phenols Hydroxytyrosol (HT) has shown to have role of "radical scavenger" and to be also involved in the inhibition of oxidation processes related to alteration cellular aging and degenerative diseases[1, 2]. However nothing is known about HT participation on human colonic mucosa transport process. Therefore, we tested the effects of HT on electrophysiological parameters of non stimulated and stimulated human colon.

Segments of sigmoid colon were obtained from 6 patients (78 mean age yrs, range 75-81 yrs), undergoing left hemicolectomy for non-obstructive sigmoid cancer. Colonic mucosa were taken from macroscopically normal areas. Colonic mucosa deprived of the muscle layer was mounted in Ussing chambers and bathed in oxygenated Krebs solution at 37°C. Functional parameters: transepithelial voltage (Vt), short-circuit current (Icc) and transepithelial resistance (Rt), were recorded in resting condition and under stimulation with CARB (100M), forskolin and IBMX in the absence or in the presence of HT (1M, 10M e 100M). Statistical analysis was performed by paired t-Student test. P<0.05 was considered to be statistically significant.

The exposure to HT in non stimulated tissues produced not significant variations of the Vt, Icc and transepithelial resistance.The exposure to HT in CARB, forskolin and IBMX stimulated tissues produced a progressive dose dependent reduction of the Vt significant at 100M (p<0.05) and 10M (p<0.01); a not significant reduction of the Icc at the same concentrations and a significant change in transepithelial resistance at 100M (p<0.05).

HT could help improve the altered intestinal permeability through a reduction of the transport activity and paracellular permeation in stimulated and non stimulated human colon respectively. [1] Dowd Y et al. (2004) Bioc. Pharm. 68:2003-8 [2] Fabiani R et al. (2002) Eur. J. Cancer Prev.11:351-8


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Phenolic composition of Trifolium scabrum L. aerial parts.

Iwona Kowalska1, Dariusz Jędrejek 1 , Łukasz Cieśla 1 , Sonia Piacente2, Milena Mosullo2, Wieslaw Oleszek1 and Anna Stochmal 1

1 Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, ul. Czartoryskich 8, 24-100 Pulawy, Poland 2Department of Pharmaceutical Sciences, University of Salerno, via Ponte Don Melillo-84084, Fisciano, Salerno, Italy [email protected]

Flavonoid-derived plant natural products have long been known to function as floral pigments for the attraction of insect pollinators, as signal molecules for beneficial microorganism in the rhizosphere, and as antimicrobial defense compounds. Isoflavones are naturally occurring diphenolic compounds found mainly in the legume family of plants and represent the main class of phytoestrogens of current interest in clinical nutrition.

Trifolium pratense L. (red clover) is (besides soybean) one of the plant sources of these compounds. Extracts of Trifolium pratense L. are commercially available as isoflavone enriched dietary supplements meant for long-term use as a phytoestrogen source and ’’natural’’ form of hormone replacement therapy. Preliminary analysis of 57 Trifolium species for isoflavone concentration showed that Trifolium scabrum L. (rough clover) has higher total isoflavone contents than Trifolium pratense L. 72.76 and 39.51 mg/g dry matter, respectively [1]. Thus, we suggest that Trifolium scabrum L. can be considered as natural and very good commercial source of these compounds.

The aim of the present research was to isolate and identify phenolic constituents of T. scabrum L. aerial parts. It was cultivated in an experimental field of the Institute of Soil Science and Plant Cultivation in Pulawy, Poland. Plants were harvested at the beginning of flowering. The plant material was lyophilized, finely powdered, and used for the successive extraction according to previously developed techniques [2]. Phenolic fractions were separated by C18 preparative column chromatography into individual isoflavonoids. Purity of isolated compounds was identified by spectra analysis (UV, ESI-MS/MS, 1H NMR) and by comparison with the literature data. Among fifteen isolated and characterized substances from the extract of Trifolium scabrum L., thirteen were isoflavones or isoflavone glucosides and two were flavonol glucosides. [1] Oleszek W., Stochmal A., Janda B. Concentration of isoflavones and other phenolics in the aerial parts of

Trifolium species. 55, 8095-8100, 2007. [2] Stochmal, A., Piacente, S., Pizza, C., Riccardis, F., Leitz, R., Oleszek, W. Alfalfa (Medicago sativa L.)

flavonoids. 1. Apigenin and luteolin glycosides from aerial parts. J. Agric. Food Chem., 49, 753-758, 2001.


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P24 Evaluation of antioxidant and free radical scavenging potential of Lamium album L. growing in Bulgaria Miglena Spasimirova Valyovaa, Milena Angelova Dimitrovab, Yordanka Angelova Ganevaa, Veneta Mihova Kapchina-Totevab, Zhenya Petkova Yordanovab

aDepartment of Organic Chemistry, Faculty of Chemistry, Sofia University “St. Kliment Ohridski”, 1 James Bourchier Blvd., 1164 Sofia, Bulgaria. bDepartment of Plant Physiology, Faculty of Biology, Sofia University “St. Kliment Ohridski”, 8 Dragan Tsankov Blvd., 1164 Sofia, Bulgaria.

In vitro antioxidant capacity and total phenolic and flavonoid contents of different extracts,

obtained from in vivo and in vitro cultivated plants of Lamium album L. were tested. Lamium album L. is valuable medicinal plant which possesses astringent, antispasmodic, anti-inflammatory, antibiotic and bacteriostatic properties. Three different assays (DPPH-free radical scavenging activity, ABTS-free radical scavenging activity and Phosphomolybdenum redusing power) for screening of antioxidant activity were applied. The highest phenolic content was detected in in vivo methanol extracts, obtained by thermostat and Soxhlet extractions (MET and MES). The highest concentrations of flavonoids were obtained in in vivo MET and MES (62.05 ± 2.25 and 82.11 ± 4.3, respectively). In vivo MET and MES demonstrated the highest DPPH scavenging potentials than other extracts with IC50 values of 20.62 ± 0.49 and 29.86 ± 0.15 μg.ml-1, respectively. The highest ABTS scavenging potential in in vivo and in vitro L. album L. extracts were found for MES and MET (0.65 ± 0.009 and 0.60 ± 0.036 TEAC). The results indicated that the highest reducing potential towards transition metals was found in in vivo ethanol extract (237.67 ± 1.66 mM.g-1) obtained by thermostat extractions. The total phenolic content correlated with the radical scavenging activity. The results revealed that L. album L. could be an interesting source of natural antioxidants with potential use in food supplements. To the best of our knowledge this is the first report which indicates total phenolic and flavonoid contents, antioxidant capacity of in vitro propagated plants of L. album L. by comparison with in vivo grown plants. This work was financially supported by the grand No. DTK-02-29/2009 of Ministry of Education Youth and Science, Bulgaria.


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Evaluation of the saponin content in lines of common beans (Phaseolus vulgaris L.) selected in Italy Aldo Tavaa, Barbara Pintusa, Agata Ursinoa, Bruno Campionb

aCRA-FLC Centro di Ricerca per le Produzioni Foraggere e Lattiero Casearie, v.le Piacenza 29, 26900 Lodi, Italy bCRA-ORL Unità di ricerca per l’Orticoltura, via Paullese 28, 26836 Montanaso Lombardo (LO), Italy [email protected]

The common bean (Phaseolus vulgaris L.) is one of the most important staple food worldwide,

mainly thanks to its relatively high content of protein and other valuable nutrients such as complex carbohydrates, minerals, together with some vitamins that are of particular relevance for human health. Beans also contain other secondary metabolites such as phytates, protease inhibitors, polyphenols, lectins, raffinosaccharides and saponins that can affect the nutritional value of this legume [1].

In a series of breeding studies aimed at obtaining bean grains with improved nutritional properties, several lines of P. vulgaris were selected and characterized by a low content of antinutritional factors such as lectines and phytates [2,3].

From a nutritional point of view, among all reported metabolites, saponins might represent a positive trait due to their ability to lower plasma LDL cholesterol concentration by interfering with cholesterol absorption [4].

The present paper reports an evaluation of saponin/sapogenin content of Italian lines of common beans selected for their improved nutritional properties.

[1]Norton G., Bliston FA., Bressani R. (1985) Biochemical and nutritional attributes of grain legumes. In: Summerfield RJ., Roberts EH. ed. Grain legume crops, Collins, London.

[2] Campion B., Perrone D., Galasso I., Bollini R. (2009) Common bean (Phaseolus vulgaris L.) lines devoid of major lectin proteins. Plant Breeding, 128: 199-204

[3] Campion B., Sparvoli F., Doria E., Tagliabue G., Galasso I., Fileppi M., Bollini R., Nielsen E. (2009) Isolation and characterization of an lpa (low phytic acid) mutant in common bean (Phaseolus vulgaris L.). Teor Appl Genet, 118: 1211-1221

[4] Carr TP., Jesch ED. (2006) Food components that reduce cholesterol absorption. In: Taylor SL. ed. Advances in food nutrition research. Vol 51. Academic Press, London.


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P26 Glucosinolates from “Mugnolo”, a variety of Brassica oleracea Argentieri Maria Pia., Pascale Rossana, Avato Pinarosa Dipartimento Farmaco-Chimico,Università degli Studi “Aldo Moro”di Bari- Via Orabona 4,I- 70125 Bari – ITALY [email protected]

Plants of the Brassicaceae family are widespread in the Mediterranean region where they are

widely consumed cooked or as salad vegetables (cabbage, Brussel sprouts, cauliflower, broccoli, turnips, radishes, cress) or condiments (horseradish, mustard, capers). These plants are a good source of bioactive compounds of nutraceutical relevance including flavonoids, minerals, vitamins, and glucosinolates (GLSs). These are responsible for the plant pungent aroma and bitter taste and are the most extensively studied bioactive compounds in the Brassicaceae family. GLSs consist of a large group of sulphur-containing secondary metabolites with cancer protection properties [1]. In recent years, GLSs, and particularly their hydrolysis products isothiocyanates, have attracted great attention in the scientific world due to their potential anticarcinogenic effects [2]. Sulforaphane, the isothiocyanate resulting from the hydrolysis of glucoraphanin, the main GLS in broccoli (B. oleracea var italica) has been shown to reduce tumor formation induced by chemical carcinogens in several animal models [3].

Within the Brassica genus, B. oleracea includes a number of varieties which, due their importance as edible crops, have been studied for their content of GLSs. These include B. oleracea var gemmifera (Brussels sprouts), var capitata (cabbage), var acephala (kale) and particularly B. oleracea var italica (broccoli). In this study we have analyzed the metabolic profile of a poorly studied variety of B. oleracea locally called “mugnolo”, traditionally cultivated in Southern Italy (Puglia region) and largely consumed in the diet. Results obtained by HPLC and ESI-MS/MS demonstrate that “ mugnolo” inflorescences are characterized by the presence of the aliphatic GLSs glucoraphanin (1.79 μmol/g), glucoiberin, glucoerucin, and sinigrin, of the aromatic GLSs glucobarberin (0.56 μmol/g) and gluconasturtin, and of indole GLSs glucobrassicin (3.51 μmol/g), neoglucobrassicin, 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin.

We also analyzed the GLS profile of “mugnolo” after cooking in water. The results showed variations in the GLSs content with a 50% fall of their concentration [4].

[1] Moreno DA, Carvajal M, Lopez-Berenguer C, Garcìa-Viguera C. (2006) Chemical and biological characterization of nutraceutical compounds of broccoli. J Pharm Biomed; 41: 1508–1522.

[2] Cartea ME, Velasco P. (2008) Glucosinolates in Brassica food: bioavailability in food and significance for human health. Phytochem Rev; 7: 213–229.

[3] Jeffery EH, ArayaM. (2009) Physiological effects of broccoli consumption. Phytochem Rev; 8: 283–298. [4] Argentieri M.P., Accogli R., Fanizzi F.P., Avato P. (2011) Glucosinolates profile of “mugnolo”, a variety of

Brassica oleracea L. native to Southern Italy (Salento). Planta Med; 77: 287-292.


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Antioxidant properties of Saffron against H2O2- & selenite-induced oxidative stress: in vitro & in vivo studies Fotini N. Lamari1, Magdalini A. Papandreou2, Andriana P. Dimakopoulou1, Evangelos Drimtzias3, Anastasia-Barbara Ferlemi1, Olga Makri3, Paul Cordopatis1, Costas Georgakopoulos3, Marigoula Margarity2

1Department of Pharmacy, 2Department of Biology, 3School of Medicine, University of Patras, 26504 Patras, Greece [email protected]

Imbalance between oxidative challenge and antioxidant protection is implicated in normal (ageing) and pathological processes. Crocus sativus has been used for medicinal purposes for millennia. Its phytochemical composition is unique due to the presence of water-soluble carotenoids, crocins, which are glycosidic esters of crocetin. The antioxidant properties of saffron were studied in human neuroblastoma SH-SY5Y cells treated with H2O2, in neonatal rats treated with sodium selenite -a well known cataractogenic model-, and in aged mice. Results in vitro showed that both saffron and crocetin provided strong protection in rescuing cell viability, repressing ROS production and decreasing caspase-3 activation against H2O2-induced toxicity in SH-SY5Y cells. Experiments in aged mice showed that mice treated intraperitoneously with saffron (60 mg/kg body weight (BW)) exhibited significant improvement in learning and memory (passive avoidance test), accompanied by significantly lower brain lipid peroxidation and higher reduced glutathione. Salt- and detergent-soluble AChE activity remained unchanged. Thus, the significant cognitive enhancement conferred by saffron administration in aged mice, is more closely related to the antioxidant reinforcement. Moreover, saffron administration to 9 days old Wistar rat pups (60 mg/kg BW, 10th and 11th postnatal days) significantly protected them against selenite-induced (20 μmol/kg BW on 9th day) cataract formation. Saffron treatment protected lens from selenite-induced lipid peroxidation and protein proteolytic degradation. Region-specific cerebral protective effects were also afforded to saffron-treated rats against selenite-induced toxicity; lipid peroxidation levels were significantly lower and catalase levels being significantly higher in cerebral cortex, while total antioxidant power was increased in all tested brain regions. Our data suggest that saffron and its constituents could be effective against various oxidative stressors.


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P28 Surface sterilization and in vitro shoot multiplication of Dioscorea membranacea Pierre ex Prain&Burkill

Yaowapha Jirakiattikula and Arunporn Itharatb

aDepartment of Agricultural Technology, Faculty of Science and Technology, Thammasat University, Rangsit Campus, Thailand bApplied Thai Traditional Medicine Centre, Faculty of Medicine, Thammasat University, Rangsit Campus, Thailand [email protected]

Dioscorea membranacea Pierre ex Prain & Burkill is a Thai medicinal plant which its rhizome

is used for treatments of inflammation, lymphopath, cancers, veneral diseases, leprosy and dermopathy [1]. The rhizome requirement for medicine ingredient preparations has been considerably increased therefore mass multiplication through plant tissue culture technique needs to be applied. In order to establish an efficient in vitro regeneration system for D. membranacea, explant surface sterilization and culture medium for multiplication were examined. Single nodes were sonicated for 0, 15, 30, 45 and 60 min followed by surface sterilization with 10% (v/v) and 5% (v/v) sodium hypochlorite for 15 and 10 min, respectively. The results showed that the highest survival percentages were 28.4% and 22.0% after sonication for 30 and 45 min, respectively which were not significantly different between the treatments. On the other hand, contamination totally occurred when sterilized without or with sonication for 15 min. For shoot multiplication, the in vitro nodes were cultured on MS medium supplemented with 2 or 3 mg/l BA with or without 0.2 or 0.5 mg/l NAA for 4 weeks. It was found that MS medium supplemented with 2 or 3 mg/l BA gave the greatest generated shoot number, shoot length, and number of shoots and nodes which were significantly different among the treatments. [1] Tewtrakul, S. and A. Itharat. (2007) Nitric oxide inhibitory substances from the rhizome of Dioscorea

membranacea. Journal of Ethnopharmacology, 109: 412-416


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Activity of Plectranthus barbatus extract against inflammatory response in human neutrophils

Pedro Luís Faléa, Mariana Alves Filipea, Lia Ascensãob, Maria Luísa Serralheiroa, Lurdes Miraa

a Centro de Química e Bioquímica, FCUL, Campo Grande, 1749-016 Lisboa, Portugal b Centro de Biotecnologia Vegetal, (IBB), FCUL, Campo Grande, 1749-016 Lisboa, Portugal [email protected]

Plectranthus barbatus Andrews (Lamiaceae), known as “falso boldo”, is used in South Africa

and South America for a wide range of therapeutical purposes. Previous in vitro studies showed that a P. barbatus extract, prepared as decoction, has promising antioxidant and anticholinesterase properties, and the extract composition is almost unaltered during a in vitro simulated digestion, especially in the content of its main component, rosmarinic acid [1]. In addition, after administration of the extract to rats its components were found circulating in plasma [2].

Inflammation is a complex biological response to harmful stimuli involving the vascular system, the immune system and the injured cells. Although it has primarily a protective function, the destructive effects can largely surpass the gravity of the stimulus. Therefore, excessive inflammatory responses are often associated with diseases, as atherosclerosis, allergies and myopathies. The high production of reactive oxygen species, such as hypochlorous acid, by activated neutrophils, for the purpose of killing invading microorganisms, is responsible for the tissue damage associated with the inflammation process.

In the present work, neutrophils were isolated from blood of human donors and were activated by phorbol 12-myristate 13-acetate, to stimulate the production of reactive oxygen species. The hypochlorous acid was measured by the taurine chlorination method, in the presence and in the absence of P. barbatus extract and rosmarinic acid.

P. barbatus extract was able to scavenge hypochlorous acid produced by the activated neutrophils in a dose-dependent manner. The IC50 for the hypochlorous acid scavenging activity was determined as 10.7±3.1 µg.mL-1 for the P. barbatus extract, and 3.62 ± 0.42 µM for the isolated rosmarinic acid. The content of rosmarinic acid in the plant extract seems to be the main responsible for the hypochlorous scavenging activity.

The low IC50 values shown by the plant extract and its main component for the scavenging of hypochlorous acid, produced by human neutrophils, together with the fact that rosmarinic acid can be found circulating in plasma after intragastric administration [2], allow to consider that P. barbatus herbal tea and rosmarinic acid may be useful in the treatment of inflammation related health problems. [1]Porfírio S., Falé PL., Madeira PJA., Florêncio MH, Ascensão L., Serralheiro M.L.M. (2010)

Antiacetylcholinesterase and antioxidant activities of Plectranthus barbatus tea, after in vitro gastrointestinal metabolism. Food Chem., 122: 179-187.

[2]Falé PL., Madeira PJA., Florêncio MH., Ascensão L., Serralheiro MLM. (2011) Function of Plectranthus barbatus herbal tea as neuronal acetylcholinesterase inhibitor. Food Funct., 2: 130-136.


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P30 Elucidation of rate limiting steps of ascorbate biosynthesis in maize Georgina Sanahujaa*,Gemma Farrea, Ludovic Bassiéa, Changfu Zhua,Teresa Capella, Paul Christoua,b

aPVCF, Universitat de Lleida, Av. Alcalde Rovira Roure, 191, Lleida, 25198, Spain; bICREA Institucio Catalana de Recerca I Estudis Avançats, Passeig Lluis Companys, 23, Barcelona 08010, Spain [email protected]

L-Ascorbate (AA), also known as vitamin C, plays an important role as an antioxidant and it also has various physiological roles. In plants it is implicated in photoprotection of the photosynthetic apparatus, regulation in cell division and conferring tolerance to environmental stresses. Plants and many animals can synthesize AA, but humans lack the final enzyme in AA biosynthesis, L-gulonolactone oxidase. As a result, they depend on dietary sources of AA. Regulation of AA synthesis varies among plant species and different tissues. As a prelude to a more refined engineering of the AA pathway in plants we investigated rate-limiting steps in the maize AA biosynthetic pathway. We carried out a targeted transcriptome profiling of critical endogenous AA pathway genes in four distinct maize varieties, representing diverse germplasm. We will present data from quantitative Real Time PCR (qRT-PCR) indicating a broad variation in the levels of mRNA accumulation in the different varieties of maize we analyzed. We have thus identified rate limiting steps in the AA in different maize varieties. This information will aid in the design of more targeted genetic engineering strategies to enhance vitamin C in maize and other plants. We acknowledge financial support from Ministry of Science and Innovation-MICINN and European Research Council Advanced Grant BIOFORCE.


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Antioxidant activity and antiproliferative properties of Ficus carica cv. Dottato fruits.

Mariangela Marrelli, Filomena Conforti, Giancarlo A. Statti, Francesco Menichini

Department of Pharmaceutical Sciences, Faculty of Nutrition and Health Sciences, University of Calabria, I-87036 Rende (CS), Italy [email protected]

The fig tree is intensively cultivated in the Mediterranean region, from Turkey to Spain and

Portugal; it is also grown commercially in parts of USA and, to a smaller extent, in India and Japan. Ficus carica Linn. may be important not only as an edible plant: in traditional medicine the fruits were utilised for their antipyretic, purgative and anti-inflammatory properties [1].

Epidemiological studies have shown that there is a clear significant positive association between intake of natural products and reduced rate of heart disease mortalities, common cancers and other degenerative diseases. Free radicals present in human organism cause oxidative damage to various molecules, such as lipids, proteins and nucleic acids, being involved in the initiation of those diseases [2]. Antioxidant compounds, such as phenolics, scavenge free radicals, inhibit the oxidative mechanism that lead to degenerative illnesses. Important findings also support the free radical hypothesis in skin carcinogenesis [3].

In the present study we describe the evaluation of antioxidant and antiproliferative activities of various phytocomplexes obtained from fruits of Ficus carica cv. Dottato from Calabria, Cosenza district (Italy), collected in three different months. The work aimed to assess how the stage of ripeness influences the biological activity of examined plant, since the phonologic stage is an important factor that influences the compositional quality of fruits and vegetables.

The antioxidant activity of extracts from Ficus carica cv. Dottato was investigated by the �-carotene bleaching test [4]. The first harvest (June) of the total extracts exhibited the best inhibition of peroxidation with an IC50 of 0.004 mg/ml after 30 minutes of incubation. Among single fractions obtained from total extracts the dichloromethane fraction from II harvest (July) showed the best activity with an IC50 value of 0.020 mg/ml. The phototoxicity of F. carica samples was investigated for the first time on the human tumor cell line C32 (amelanotic melanoma) [5]. Dichloromethane fractions showed the best antiproliferative activity with an IC50 <5 μg/ml independently of the harvest.

The present study suggests that all the fruits extracts have moderate to potent antioxidant and antiproliferative activity. June is the best month to collect F. carica cv. Dottato fruits in order to have the best phytocomplex that showed the higher antioxidant activity.

[1] Patil VV., Patil VR. (2011) Ficus carica Linn.-An overview. Res J Med Plant, 5: 246-253 [2] Garcia-Alonso M., de Pascual-Teresa S., Santos-Buelga C., Rivas-Gonzalo JC. (2004) Evaluation of the

antioxidant properties of fruits. Food Chem, 84: 13-18. [3] Sander CS., Chang H., Hamm F., Elsner P., Thiele JJ. (2004) Role of oxidative stress and the antioxidant network

in cutaneous carcinogenesis. Int J Dermatol, 43: 326-335. [4] Ismail A., Marjan ZM., Foong CW. (2004) Total antioxidant activity and phenolic content in selected vegetables.

Food Chem, 87:581-586. [5] Barraja P., Diana P., Montalbano A., Dattolo G., Cirrincione G., Viola G., Vedaldi D., Dall'Acqua F. (2006)

Pyrrolo[2,3-h]quinolinones: A new ring system with potent photoantiproliferative activity. Bioorg Med Chem, 14: 8712-8728


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P32 Some morphology and genetic relationship of Dioscorea membranacea using molecular technique

Nantana Jaengsuwan

Department of Applied Biology, Thepsatri Rajabhat University, Lop-Buri, Thailand, 15000 [email protected]

Huakhaoyen in Thai name is commomly used in Thai Traditional Medicine as cancer drug [1],

it showed the best seller in Thai Pharmacy drugstore. However, it is often collected difference types [2]. Thus, the objective of this research is to investigate some morphology and genetic relationship of Dioscorea membranacea using molecular technique of 22 Huakhaoyen (Dioscorea membranacea) samples collected from different provinces in Thailand including Nakhon Ratchasima, Lop Buri, Suphan Buri, Uthai Thani and Chumphon. The morphology in this study was studied by shape, leaves, fruit, stem color, stipule, epidermal cell and stomata size. Most characteristics were not distinctively classified. There were two types of stem color, blank and white. The amplified fragment length polymorphism (AFLP) and the software programs computer were used to construct the genetic relationship[3,4,5]. The results of DNA fingerprint from AFLP technique could be classified into a phylogenetic tree. The tree was divided into two groups. The first group consisted of 1 sample from Huakhaoyen (be identified as Smilax corbularia Kunth) which was a marker in this study. The second group consisted of 22 samples from all of Huakhaoyen (Dioscorea membranacea) in the research. The mean Polymorphic Information Content (PIC) score of AFLP was 0.22. There was genetic diversity of Huakhaoyen (Dioscorea membranacea) in Thailand [1] Itharat, A., Houghton, P.J., Ammguaye, E.E., Burke, P.J., Sampson, J.H. and Raman, A. (2004). In vitro cytotoxic

activity of Thai medicinal plants used traditionally to treat cancer. J. Ethnopharmacology, 90 :33-38. [2] Boonyaratanakornkit, L. and Chantarateptawan, V. (1993). Identification and Specification of Khaao-Yen-Neua,

Khaao-Yen-Tai. Thai J. Pharm. Sci., 17: 79-90. [3] Rohlf, F.J. 1998. NTSYS-pc: numerical taxonomy and multivariate analysis system. Version 2.0. New York:

Setauket. [4] Worapa S., Norihiko T., Aree W., Piyada T., Paisan L., Akito K. and Duncan A. V. 2006. Genetic Diversity of the

Vigna Germplasm from Thailand and Neighboring Regions Revealed by AFLP Analysis. Genet. Res. Crop Evol. 53: 1043-1059

[5] Xiaowei N., Yelin H., Lin W., Renchao Z., Shulin D., Darong W., Bosun W., Guohua S., Tian T. and Suhua S. 2006. Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP). Genetica. 127: 177-183.


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Metabolic engineering of terpenoid biosynthesis in transgenic tobacco plants Bruna Miralpeix1, René Hoefer2, Danièle Werck2, Lemeng Dong3, Harro J. Bouwmeester3, Teresa Capell1, Paul Christou1,4

1Departament de Producció Vegetal I Ciència Forestal (PVCF), Universitat de Lleida, Av. Alcalde Rovira Roure, 177, Lleida, E-25198, Spain. 2 Institut de Biologie Moléculaire des Plantes, CNRS-UPR2357, Université de Strasbourg, 28 Rue Goethe, 67083 Strasbourg Cedex, France. 3Laboratory of Plant Physiology, Wageningen University, P.O. Box 658, 6700 AR Wageningen, the Netherlands. 4 Institució Catalana de Recerca I estudis avançats (ICREA Barcelona, Spain) [email protected]

Catharanthus roseus is a rich source of alkaloids; more than 130 different compounds have so far been reported, of which vincristine is highly important. Vincristine is mainly used to treat acute leukaemia, Hodgkin’s disease, rhabdomyosarcomas, neuroblastoma, breast carcinoma, among other lymphomas. C. roseus leaves accumulate about 5.8 g/g (fresh weight) vinblastine and 0.9 g/g vincristine. These amounts represent the lowest level of any medicinally useful alkaloid produced on a commercial basis. Most of the effort to-date to enhance the accumulation of these and other molecules have concentrated on non-engineered cell lines under elicitation, precursor feeding, and culture media optimization. Although these classical methods have resulted in a thorough characterization of the pathway, metabolic engineering offers a promising approach for improved product composition and increased yields in transgenic plants and cultured cells.

We engineered into tobacco plants the early part of terpenoid biosynthesis aiming to create plants accumulating 10-hydroxygeraniol. A number of transgenic plants were recovered after transformation with a geraniol synthase (GES) from Valeriana officialis and a geraniol 10-hydroxylase (CYP76B6) from C. roseus; or GES with G10H from Arabidopsis. Molecular, expression and metabolite analysis is currently ongoing to identify transgenic plants accumulating 10-hydroxygeraniol and other metabolites. Subsequent experiments will involve introduction of additional heterologous transgenes into these plants to recreate the biosynthetic pathway to the important metabolites secologanin and strictosidine.

The research leading to these results has received funding from the European Community's Seventh Framework Programme FP7/2007-2013 under grant agreement n° 222716


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P34 Quali-quantitative profiling of the polar extracts of the flowers of Ammi visnaga plants grown in different conditions Assunta Napolitanoa, Hela Kallel Sellamib, Milena Masulloa, Samira Smitib, Sonia Piacentea, Cosimo Pizzaa

aDipartimento di Scienze Farmaceutiche, Facoltà di Farmacia, Università degli Studi di Salerno, via ponte Don Melillo, 84084 Fisciano, SA, Italy

bUniversity of El-Manar II, Faculty of Sciences, Unity of Research of Vegetal Ecology, Campus Academia, 2092 Tunis, Tunisia.

Ammi visnaga (L.) Lam. is an annual and biennial herb, belonging to the Umbelliferae family. It is widely spread in the Mediterranean region and in particular in the north of Tunisia, where it is known as “guabeba” and used as medicinal plant. Egyptians referred to this plant as “khella”, on the basis of its major active metabolite, the furanochromone khellin. Khellin products are commercially available in European market for oral and parenteral administration in the treatment of bronchial asthma and angina pectoris as vasodilator agent [1]. Along with furanochromones like khellin, visnagin, and khellol-glucoside, Ammi visnaga is a valuable natural source of pyranocoumarins such as visnadin, a coronary vasodilator 5–10 times stronger than khellin [2].

Considering that the level of these compounds is greatly influenced by genetic factors and environmental conditions [3], the study of the metabolite expression of A. visnaga plants originating from so far unexplored Mediterranean areas, such as Tunisia, prompted us to carry out the first phytochemical investigation of Tunisian species grown both in spontaneous way and cultivated in restrained and in abiotic stress conditions, to highlight qualitative and quantitative differences of metabolite composition in comparison with literature data for non endemic species and to assess the response of A. visnaga plant to stressed environmental conditions.

To define and to promptly compare the qualitative metabolite profiles obtained for the methanol extract of the flowers of both spontaneous and cultivated plants, stressed and not stressed, an analytical approach based on HPLC-ESIMS technique was applied. Subsequently, the flower methanol extract of spontaneous A. visnaga was purified by different chromatographic steps. The structures of isolated compounds were elucidated by extensive spectroscopic methods such as 1D- and 2D-NMR experiments as well as ESIMSn analysis. Twenty-two compounds belonging to the furanocoumarin, pyranocoumarin, furanochromone, and chromone classes, including two novel furanochromones, were identified. Finally, a quantitative comparison by LC-MS between the metabolite content of the flowers of A. visnaga grown in spontaneous and in restrained and stressed environmental conditions was carried out. [1] Gunaydin K., Beyazit N. (2004) The chemical investigations on the ripe fruits of Ammi visnaga (Lam.) Lamarck

growing in Turkey. Nat. Prod. Res., 18: 169-175 [2] Kursinszki L., Troilina J., Szõke E. (1998) Determination of visnagin in Ammi visnaga hairy root cultures using

solid-phase extraction and high-performance liquid chromatography. Microchem. J., 59: 392-398 [3] Gunaydin K., Erimb F.B. (2002) Determination of khellin and visnagin in Ammi visnaga fruits by capillary

electrophoresis. J. Chromatogr. A, 954: 291–294


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Harnessing valuable botanicals from Areca catechu pericarps Adinpunya Mitra, Mahesh Kumar Natural Product Biotechnology Group, Agricultural and Food Engineering Department Indian Institute of Technology Kharagpur, Kharagpur – 721 302, India [email protected]

Areca catechu Linn. is a tropical palm belongs to Arecaceae family. Areca fruit is an ovoid drupe, which becomes orange/reddish when ripened. A. catechu plant is being used as herbal medicine in many countries because of the presence of different natural products including phenolics, alkaloids and carotenes. While exploring the possibility of utilizing pericarp wastes of A. catechu as a promising source for harnessing natural products of economic importance, a wide range of secondary metabolites were detected upon fruit maturity.

Young pericarp when extracted with methanol and subjected to column chromatography using Sephadex LH-20 as matrix, yielded 4-hydroxybenzaldehyde as the major soluble phenolics. The second major soluble phenolics found in the young pericarp extract was 4-hydroxybenzoic acid. Interestingly, 4-hydroxybenzoic acid was detected earlier from A. catechu as one of major cell wall-bound phenolic acids, and the content appeared to be the highest (~4 mg/g cell wall dry mass) among 20 Arecaceae species studied. By contrast, a number of carotenes were detected and isolated from the soluble pericarp extracts of ripened Areca fruit. Among them, β-carotene was the major one. The second major carotene in the pericarp tissue was found to be ζ-carotene, which was identified on the basis of spectral information available in the literature. TLC coupled with UV-VIS spectroscopy formed the basis for separation, identification and quantification of the pericarp carotenoids. A reverse phase thin layer chromatography (RP-TLC)-based separation was standardized for the first time for pericarp carotenoids of A. catechu, and the method was validated subsequently. Solid phase extraction was also performed for rapid separation of β-carotene, using Sep-Pak® cartridge. The total carotenoid content of pericarp tissue was 0.04 mg/g dry mass of pericarp tissue using β-carotene as standard. The β-carotene content found in TLC-separated and Sep-Pak® cartridge-separated fractions was 0.11 mg/g and 0.12 mg/g dry mass, respectively. Further it was found that β-carotene only constitutes around 30% of the total carotenoid content in ripened A. catechu pericarp as estimated through quantitation of TLC separated and Sep-Pak® cartridge-separated fractions. Qualitative determination of free-radical scavenging activity showed scavenging capacity maximum at 30 min, when 1,1-diphenyl-2-picryl hydrazyl (DPPH) was used as a free-radical. ζ-carotene, also showed antioxidant activity, and even stronger than that of β-carotene.


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P36 Exact mass and QToF fragmentation database of bioactive Amaryllidaceae alkaloids

Laura Torras-Claveriaa, Strahil Berkova, Francesc Viladomata, Carles Codinaa, Jaume Bastidaa

aDepartament de Productes Naturals, Facultat de Farmàcia, Universitat de Barcelona, Avda. Joan XXIII s/n, 08028, Barcelona, Catalonia, Spain

A particular feature of the Amaryllidaceae family is the consistent presence of an exclusive group of alkaloids, which are known as Amaryllidacae alkaloids. These compounds are responsible for many pharmacological activities, including antiviral, antitumour and cholinesterase inhibition, and show considerable structural diversity. The nine main skeleton types of Amaryllidaceae alkaloids are Norbelladine, Lycorine, Homolycorine, Crinine, Haemanthamine, Narciclasine, Tazettine, Montanine and Galanthamine. Amaryllidaceae plants have been used for thousands of years as herbal remedies, and in the last 50 years alkaloids from their extracts have been the object of active chemical investigation [1].

Classical phytochemical structural elucidation is time- and solvent-consuming, and often results in the isolation and identification of very few compounds. Although gas chromatography coupled to mass fragmentation technique (GC-MS) has been usually used for identification of Amaryllidaceae alkaloids, sometimes new compounds are present in concentrations too low for NMR analysis and identification. In these cases, GC-MS combined with exact mass analysis and fragmentation obtained from quadrupole time-of-flight with electrospray ionisation (ESI-QToF) can be an effective method for the structural elucidation of new Amaryllidaceae alkaloids [2].

This work presents the exact mass and QToF fragmentation data base of several Amaryllidaceae alkaloids. Five groups of alkaloids were analysed (Homolycorine, Lycorine, Haemanthamine, Galanthamine and Other), with between 3 and 19 compounds per group. The exact mass of the [M+H]+ ion as well as its Product Ion Scan (PIS) fragmentation were obtained for each alkaloid. This method could also distinguish between some stereoisomers of Haemanthamine-type alkaloids, which is not possible by GC-MS.

The valuable structural information of Amaryllidaceae alkaloids obtained by ESI-QToF can contribute to the phytochemical investigation of Amaryllidaceae plants and allows a faster identification of unknown bioactive alkaloids without prior isolation, requiring smaller amounts of plant material and reducing the chemical and time consumption. [1] Bastida J., Lavilla R., Viladomat F. (2006) The Alkaloids, Elsevier, Chicago. [2] Torras-Claveria L., Berkov S., Jáuregui O., Caujapé J., Viladomat F., Codina C., Bastida J. (2010) Metabolic

profiling of bioactive Pancratium canariense extracts by GC-MS. Phytochemical Analysis, 21: 80-88


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Suitability of ancient and modern cereals to produce novel functional fermented foods with a high content of antioxidant compounds Maura Ferri1, Andrea Gianotti2 and Annalisa Tassoni1

1Dept. Experimental Evolutionary Biology, University of Bologna,Via Irnerio 42, 40126, Bologna, Italy: [email protected] 2Dept. Food Sciences, University of Bologna, Viale Fanin 44, 40100, Bologna, Italy

Epidemiological studies have strongly suggested that consumption through the diet of fruits, vegetables and grains, plays a crucial role in the prevention of heart diseases, cancer, diabetes and Alzheimer pathologies. This effect seems to be related to the high levels of phytochemicals present in these foods, that combat oxidative stress helping to maintain a balance between oxidants and antioxidants. Therefore, increased consumption of foods containing high levels of antioxidants has been recommended.

The traditional sourdough fermentation process of cereals performed by Lactic Acid Bacteria (LAB), not only improves flavour and texture of the products, but also enables consumption of whole meal cereals, which are well known for their high nutritional quality and health-promoting properties including antioxidant protection [1]. In particular Lactobacillus plantarum during fermentation of vegetal matrices showed an increase of phenolic compounds.

This work is aimed to obtain functional cereal based fermented foods with high content of antioxidant compounds starting from two different cereals, durum wheat and Kamut khorasan. The fermentation process was performed by selected L. plantarum strains and their ability to increase the quali/quantitative profile of antioxidant compounds was considered. Total polyphenols, total flavonoids, antioxidant activities and detailed polyphenolic profile, were analysed. Preliminary results indicated, in most of the fermented flour samples, an increase of total polyphenols levels and antioxidant activities as consequence of the L. plantarum action.

After fermentation a wider range of polyphenols were synthesized by the L. plantarum both in durum wheat and Kamut matrices. In particular, a large spectrum of catechins (such as epicatechin, epigallocatechin and catechin), were detected. These findings open the way to future applications of L. plantarum strains with the aim of formulating new functional foods with improved healthy properties. [1] Gianotti A. et al. (2011) Role of cereal type and processing in whole grain in vivo protection from oxidative

stress. Frontiers in Bioscience, 16: 1609-1618


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Antioxidant and cytotoxic activities against lung cancer cells of Thai Medicinal plants for cancer treatment in Kumpramong Temple

Onmanee Prajuabjindaa, Arunporn Itharatb

aStudent of Master Degree of Medical Science Program (Appiled Thai Traditional Medicine) Faculty of Medicine, Thammasat University. bDepartment of Appiled Thai Traditional Medicine, Faculty of Medicine, Thammasat University, Thailand [email protected]

Thai medicinal preparation of Kumpramong Temple has been used for cancer treatment more

than ten years ago. It composed with eleven plants. This research were investigated on antioxidant and cytotoxic activities against lung cancer cell of Thai medicinal plants used to treat cancer of Kumpramong Temple. The extract procedures was maceration method by 95% ethanol and dry by evaporator. The antioxidant activity determined by using DPPH radical scavenging assay [1]. The cytotoxic activity against large cell lung carcinoma (CORL-23) determined by SRB assay [2]. The results showed that the ethanolic extract of Hydnophytum formicarum showed the highest antioxidant activity (EC50 = 3.47±0.23 µg/ml) than Thai medicinal preparation of Kumpramong Temple (EC50 = 4.98+0.06 µg/ml). The ethanolic extract of Tectona grandis stems showed good cytotoxic activity against CORL-23 by IC50 values 7.78 ± 0.75 µg/ml than Thai medicinal preparation of Kumpramong Temple.

This results should be supported using of those plants in Thai traditional medicine to treated cancer patients although the cytotoxic activity of Kumpramong preparation showed less activity but it showed good evidence as antioxidant activity which is good for treatment cancer patients. [1].Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, I., Vistica,D., Waren, J.T., Bokesch, H., Kenney, S.,

Boyd, M.R., 1990. New colorimetric cytotoxicity assay for anticancer drug screening. Journal of National Cancer Institute 82, 1107–1112.

[2].Yamazaki, K. Hashimoto, A. Kokusenya, Y. Miyamoto,T. and Sato, T. 1994. Electrochemical method for estimating the antioxidative effect of methanol extracts of crude drugs, Chem. Pharm. Bull., 42: 1663-1665


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Phytochemical investigation of phenolic compounds of elderflowers growing at different altitudes

Marlene Monschein, Franz Bucar

Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Universitätsplatz 4/1, A-8010 Graz, Austria [email protected]

Elderflowers, the flowers of Sambucus nigra L. (Common elder, Caprifoliaceae) are used as

herbal tea to treat colds and fever and to prepare elderflower cordial for food purposes. They are known for their diaphoretic and secretolytic effects. Sambucus nigra is distributed almost all over Europe, west and central Asia as well as in northern Africa. The aim of this study was to investigate to which extent the different environmental conditions at various altitudes influence the formation of phenolic substances in elderflowers. The changes of the levels of flavonoids and phenolic acids in samples collected at two altitudinal levels (800 m and 1000 m) have been studied over two growing periods (year 1: 2007 and year 2: 2008, respectively) in order to identify individual compounds as markers. The dried, powdered plant material (10 to 14 samples per altitude and year) was extracted with methanol 80 % (v/v) using ASE (Accelerated Solvent Extraction) and used for further qualitative and quantitative HPLC-PDA-ESI-MS- analysis [1].

Qualitative analysis resulted in the identification of 5 hydroxycinnamic acids including 3- and 5-O-caffeoylquinic acid, 3,5- and 4,5-O-dicaffeoylquinic acid, and 7 flavonol glycosides of isorhamnetin, kaempferol and quercetin. Quantitative analysis of elderflowers showed statistically significant to highly significant elevated levels of rutin, narcissin, 3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid in samples collected at higher altitude. Increased levels but no statistical significance of chlorogenic acid and astragalin could be determined. The assumption that the content of flavonol 3-O-glycosides with adjacent hydroxyl groups in ring B is increased in plants growing at higher altitude could not be confirmed [1, 2]. Furthermore, the hypothesis that plants at higher altitude produce more free radical scavengers due to the harsher climatic conditions and increased ultraviolet radiation could not be verified in general [2]. The radical scavenging capacity, measured by the DPPH assay, showed IC50 values ranging from 9.38 to 14.37µg/ml. A highly significant rise of radical scavenging capacity linked to increasing altitude could only be recorded in year 2 (p = <0,001). Acknowledgements: The Dr.-Heinrich-Jörg foundation and the Office of Research Management and Service, University of Graz are gratefully acknowledged for financial support. [1] Rieger, G., Muller, M., Guttenberger, H. und Bucar, F. (2008) Influence of altitudinal variation on the content of

phenolic compounds in wild populations of Calluna vulgaris, Sambucus nigra, and Vaccinium myrtillus. J. Agric. Food Chem. 56(19): 9080-9086.

[2] Ganzera, M., Guggenberger, M., Stuppner, H. und Zidorn, C. (2008) Altitudinal variation of secondary metabolite profiles in flowering heads of Matricaria chamomilla cv. BONA. Planta Med. 74(4): 453-457.


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P40 Growth of Dioscorea membranacea Pierre ex Prain & Burkill and dioscorealide B accumulation at different harvest times

Panumart Rithichaia, Arunporn Itharatb

aDepartment of Agricultural Technology, Faculty of Science and Technology, Thammasat University, Klongluang, Pathumthani 12121, Thailand bApplied Thai Traditional Medicine Center, Faculty of Medicine, Thammasat University, Klongluang, Pathumthani 12121, Thailand [email protected]

The rhizome of Dioscorea membranacea Pierre ex Prain & Burkill, popularly knows as Khao-

Yen-Tai, has been used to treat cancer in Thai traditional medicine. Dioscorealide B (DB), a naphthofuranoxepin is the bioactive compound isolated from its rhizome which reduces inflammation and possesses cytotoxic effect against human breast cancer cells [1, 2]. In order to produce rhizomes for medicinal used, the appropriate harvest time must be investigated. Therefore, the aim of the present study was to examine growth of D. membranacea and DB accumulation at different harvest times. The 10-16 cm in length of mother rhizomes (MR) was planted individually in the pot. Growth of aboveground shoot, rhizome development and DB accumulated in rhizomes were determined at 3 months interval until 15 months after planting (MAP). Results revealed that dry weight and the number of aboveground shoots dramatically increased throughout the experiment. MR accumulated 4.0 %w/w DB at the beginning of experiment. The degradation of MR started at 9 MAP and completely degraded at 12 MAP. The daughter rhizomes (DR) were formed at 3 MAP and gradually developed until the final of experiment. At 15 MAP, DR had 41.8 g dry weight/plant, 14.8 cm in length and 2.7 %w/w DB accumulation. [1] Itharat A., Houghton PJ., Ammgusye EE., Burke PJ., Sampson JH., Raman A. (2004) In vitro cytotoxic activity

of Thai medicinal plants used traditionally to treat cancer. Journal of Ethnopharmacology, 90: 33-38. [2] Hiransai P., Ratanachaiyavong S., Itharat A., Graidist P., Ruengrairatanaroj P., Purintrapiban J. (2010)

Dioscorealide B suppresses LPS-induced nitric oxide production and inflammatory cytokine expression in RAW 264.7 macrophages: The inhibition of NF-ĸB and ERK ½ activation. Journal of Cellular Biochemistry, 109: 1057-1063.


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Strong free radical scavenging phenolics from Acacia nilotica pods

Mariateresa Maldinia, Paola Montoroa, Arafa I. Hamedb, Usama A. Mahalelb, Wieslaw Oleszekc , Anna Stochmalc, and Sonia Piacentea.

aDipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy, bFaculty of Science, South Valley University, Aswan 81528, Egypt cDepartment of Biochemistry, Institute of Soil Science and Plant Cultivation, ul. Czartoryskich 8, 24-100 Pulawy, Poland. [email protected]

Acacia nilotica Lamarack (Mimosa nilotica, Mimosaceae) is a medium size tree growing in tropical and subtropical countries and native from Egypt to Mozambique. It is extensively used for the treatment of various inflammatory diseases and it is reported to be rich in polyphenolic constituents [1], in particular in catechins which are hypothesized to possess antioxidant properties and to play a role in the anti-inflammatory activity of several plants [2]. Antinflammatory activity is probably related to their antioxidant activity [3] and to their inhibition of arachidonic acid metabolism via cyclooxygenase and lypoxygenase pathways [4]. Due to the complexity of catechin derivatives, the investigation of this class of natural compounds has been limited by difficulties in their separation. In the present work, rationalization of the phenolics occurring in the EtOH extract of Acacia nilotica pods, on the basis of ESI-MS and ESI-MS/MS profiles, has been proposed, together with an LC–ESI-MS qualitative study performed by using a C18 polar endcapped stationary phase. The fragmentation pattern obtained evidenced the presence in A. nilotica pods of galloylated- catechins and gallocatechins along with galloylated glucose derivatives. The structures were confirmed by NMR, after isolation of the pure compounds.

On the basis of the fragmentation patterns, quantitative analyses were performed by LC–ESI-MS/MS.

In addition, the radical scavenging activities of the EtOH extract, enriched fractions and pure compounds were investigated, by using the TEAC assay. The correlation between quantitative analysis and free radical scavenging activity confirmed the interest of this species as a rich source of very strong antioxidant principles. [1] Kaur K., Michael H., Arora S., Härkönen P., Kumar S. (2005) In vitro bioactivity-guided fractionation and

characterization of polyphenolic inhibitory fractions from Acacia nilotica (L.) Willd. ex Del. J Ethnopharmacol., 99 (3): 353-60.

[2] Benavides A., Montoro P., Bassarello C., Piacente S., Pizza C. (2006) Catechin derivatives in Jatropha macrantha stems: characterisation and LC/ESI/MS/MS quali-quantitative analysis.Journal of Pharmaceutical and Biomedical Analysis, 40: 639-647.

[3] Goncalves C., Dinis T., Batist MT. (2005) Antioxidant properties of proanthocyanidins of Uncaria tomentosa bark decoction: a mechanism for anti-inflammatory activity. Phytochemistry, 66: 89-98.

[4] Zhang W., Liu H., Xie K., Yin L., Li Y., Kwik-Uribe C.L., Zhu X. (2005) Procyanidin dimer B2 [epicatechin-(4β-8)-epicatechin] suppresses the expression of cyclooxygenase-2 in endotoxin-treated monocytic cells. Biochemical and Biophysical Research Communications, 345, 508-515.


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P42 Neuropharmacological study of Eryngium planum L. extract Thiem B.a, Ożarowski M.a,b, Szulc M.c , Kujawski R.b, Mrozikiewicz P.M.b,d, Mikołajczak P.L.b,c aDepartment of Pharmaceutical Botany and Plant Biotechnology, University of Medical Sciences, 14 Sw. Marii Magdaleny , 61-861 Poznań, Poland, bInstitute of Natural Fibres and Medicinal Plants, 27 Libelta Str., 61-707 Poznań , cDepartment of Pharmacology, University of Medical Sciences, Rokietnicka 5a, 60-806 Poznań, dLaboratory of Experimental Pharmacogenetics, Department of Clinical Pharmacy and Biopharmacy, University of Medical Sciences, Święcickiego 6, 61-781 Poznań, Poland; [email protected]

Eryngium planum L.(Apiaceae), used in folk medicine in Europe contains phenolic acids,

triterpene saponins, flavonoids, essential oil, acetylenes and coumarins [1]. The presence of the active compounds determine their therapeutic activity: diuretic, antidiabetic, expectorant, spasmolytic, antiinflammatory, antinociceptive, haemolytic, apoptotic and antimycotic. The activities of Eryngium depend mainly on its high saponin content, but presence of phenolic acids could play an important role. Triterpene saponins as well as phenolics: chlorogenic, caffeic and rosmarinic acids and their glucosid derivatives have a wide range of biological and health promoting properties. Neurobehavioral and neuroprotective effect on CNS have been also describe for these compounds [2-3]. In traditional herbal medicine numerous plants from Apiaceae family have been used to treat age related cognitive disorders and neurodegenerative diseases [4].

The aim of the present studies were to investigate neuroactive properties of methanol extracts from E.planum roots.

The influence of extract of E. planum radix (ER) on chosen aspects of its pharmacological profile using rat’s scopolamine-induced amnesia model and spontaneous activity, motor coordination, anxiety-related reactions and long term memory were assessed [5]. The effect of ER extract on acetylcholinesterase, butyrylcholinesterase and secretase mRNA expression level in hippocampus, striatum and frontal cortex of rats were studied on the molecular level. The possible role of ER extract in the prevention or treatment of neurodegenerative disorders is discussed.

[1] Duke JA et. al (2002) Handbook of medicinal herbs. London CRC Press 277-278 [2] Sparg et al. (2004) Biological activities and distribution of plant saponins. J Ethnopharmacol 94:219-243 [3] Pereira et al. (2005) Neurobehavioral and genotoxic aspects of rosmarinic acid. Pharmacol Research 52:199-203 [4] Adams M et.al. (2007) Plants traditionally used in age related brain disorders – A survey of ethnobotanical

literature. J Ethnopharmacol 113:363-381. [5] Mikołajczak P et al. (2002) Effects of acamprosate and some polyamine site ligands of NMDA receptor on short-

term memory in rats. Eur. J. Pharmacol. 444:83-96.


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Using DART-TOFMS for quality assessment of rose hip (Rosa Canina) fruits Hana Novotna, Vera Schulzova, Ivana Sosnovcova, Jana Hajslova Department of Food Chemistry and Analysis, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague 6, Czech Republic [email protected]

Rose hip fruits have been traditionally used for the preparation of tea, infusions and jams and are known as a rich source of many nutritionally valuable substances. The most important of these is ascorbic acid (vitamin C), with content around 1%. Besides the high content of ascorbic acid, rose hips have a significant content of other antioxidants, e.g., carotenoids (β-carotene and lycopene), phenolic compounds, folates, tocopherols, and many others. The high antioxidant potential of rose hip is used in food supplements, where the addition of rose hips enhances the effect of synthetic vitamin C. Triterpenic acids also contribute to the antioxidative potential. Furthermore, these acids show antitumor activity. Recently, a new active compound, a galactolipid named GOPO®, was discovered in rose hip. This substance shows strong anti-inflammatory and antioxidant effects, which can be helpful for the treatment of osteoarthritis.

For metabolomic profiling of rose hips we used a unique ionization source Direct Analysis in Real Time (DART) coupled with a High Resolution Time of Flight Mass Spectrometer (HR-TOFMS). This technique enabled complex quality assessment of rose hip based nutraceuticals and products. High Resolution TOFMS also enables the identification of many compounds occurring in rose hip extracts. For quantification of nutritionally important compounds modern analytical techniques were used. Acknowledgement: This study was carried out with support from the Ministry of Education, Youth and Sports, Czech Republic, the project MSM 6046137305 and specific university research (MSMT no. 21/2010)


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P44 In Vitro cytotoxic activity against human lung cancer cells of the ethanolic extract from Benjakul preparation and its isolated compounds

Ruchilak Rattarom 1, Intouch Sakpakdeejaroen 2, Arunporn Itharat 2 1 Student of Doctor of Philosophy, Faculty of Medicine, Thammasat University, Thailand 2Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University, Thailand [email protected]

Benjakul is widely used in Thai traditional medicine as balance health and and have also been used for cancer treatment by folk doctors(1). It composed with five plants such as Piper chaba fruit, Piper samentosum root, Piper interuptum stem, Plumbago indica root and Zingiber officinale rhizome in equal proportions(2). In this study, the ethanolic extract from Benjakul preparation and its five cytotoxic compounds were isolated by bioassay guide isolation. Cytotoxic activity was used to test against three types of human lung cancer cell lines (COR-L23, A549 and NCI-H226) and one normal human lung myofibroblasts cell line (MRC-5) by SRB assay (3). The results found that the ethanolic extract of Benjakul showed stronger cytotoxic activity against NCI-H226 than COR-L23 and A549 (IC50 values as 11.62±1.55, 24.26±0.50 and 34.40±0.37 μg/ml, respectively) but less active with normal cell line (MRC-5) (IC50 values 69.92±3.46 μg/ml). The chloroform fraction which was separated by vacumn liquid chromatography, showed the highest cytotoxic activity against all types of lung cancer cells, so five compounds were isolated from this fraction (piperine, plumbgin, 6-gingerol, 6-shogaol and myristicin). Plumbagin exhibited the highest cytotoxic activity against COR-L23, A549 and NCI-H226 (IC50 values 0.42±0.09, 0.53±0.01 and 0.78±0.15 μg/ml, respectively) and 6-shogaol shown the second most effective cytotoxicity (IC50 values 4.59±0.52, 5.15±0.15 and 4.99±0.03 μg/ml, respectively). Piperine exhibited specific cytotoxicity against NCI-H226 and COR-L23 more than A549. On the other hand, 6-gingerol and myristicin exhibited specific cytotoxic activity against A549 higher than NCI-H226 and COR-L23. These results can support using Benjakul preparation for lung cancer treatment.

[1] Itharat, A. Singchangchai, P. and Ratanasuwan, P. (1998). Wisdom of Southern Thai traditional doctors. Research Report of Prince Songkhla University, Songkhla, Thailand.

[2] Sakpakdeejaroen, I and Itharat, A. Cytotoxic compounds against breast adenocarcinoma cells (MCF-7) from Pikutbenjakul. J Health Res 2009, 23(2), 71-76.

[3] Skehan, P., Storeng, R., Scudier., D., Monks, A., Mc Mahon, J., Vistica, D., Warren, J.T., Bokesch, H., Kenney, S. and Boyd, M.R. (1990). New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82: 1107-1112.


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Watermelon as a source of bioactive molecules Marcello Salvatore Lenucci, Stefania Grassi, Anna Eleonora Caprifico, Maria Chiara Contino, Anna Montefusco, Gabriella Piro, Giuseppe Dalessandro Di.S.Te.B.A., University of Salento, Via Prov. Lecce-Monteroni, 73100 Lecce, Italy [email protected]

Watermelon (Cucumis citrullus (L.) Ser.) is a typical summer fruit, known for its refreshing and thirst-quenching power. From the nutritional point of view it has attracted considerable interest for the presence of bioactive molecules with undisputed beneficial properties for human health, such as lycopene, citrulline and fibers. Many epidemiological studies highlight lycopene involvement in the prevention of degenerative pathologies such as cardiovascular diseases, diabetes and certain types of cancer due to its high antioxidant activity, beside other still poorly understood mechanisms of action [1]. Similarly, the water-soluble, non-essential, non-proteic amino acid citrulline is showing a huge potential as therapeutic agent in the prevention and treatment of various metabolic diseases playing a central role in cell metabolism and organ functionality [2]. Eventually, the health benefits offered by a high-fiber diet in reducing risk for developing coronary heart disease, stroke, hypertension, diabetes, obesity, and certain gastrointestinal disorders are widely recognized [3]. The nutritional importance of these bioactive molecules makes the preparation of natural extracts and their use in the formulation of high quality supplements, and/or pharmaceuticals, a valuable alternative to the classical production and distribution chain of watermelon, opening new opportunities for agri-food processing industry.

The pellet and the supernatant obtained by centrifugation of the homogenized watermelon flesh (cultivar Dumara) were freeze-dried giving a lycopene rich matrix (~3.3 mg/g f-d.m.), constituted mainly of clumps of parenchyma cells, and a slightly pink powder containing soluble sugars (principally fructose) and high amount of citrulline (>10 mg/g f-d.m.), respectively. Both fractions are considerably enriched in dietary fibers.

Under the right conditions (absence of light and air, and temperature of -20 °C), the matrix can be stored for a period longer than six months, providing a readily extractable watermelon product available over time. The citrulline rich powder was stable for several months at room temperature and could be used as such or after further purification.

Recently, the interest in safe and clean extraction technologies has increased. Supercritical CO2 has been successfully used to extract lycopene from freeze-dried tomato matrices [4]. The chemical and physical parameters (pressure, temperature, CO2 flux and particle size) applied on tomato, were used as a starting point to identify the optimal conditions for lycopene extraction from the freeze-dried watermelon matrix. The presence of lipids from the watermelon seed endosperm had a positive effect on the amount and yield of extracted oleoresin. The addition of a lipid co-matrix, consisting of roughly crushed hazelnuts, did not further increase the extraction yield, whereas the use of ethanol as co-solvent resulted in a significant increase in lycopene extraction. In the optimal operative conditions, lycopene yield exceeded 75% and an oleoresin containing ~20 mg/g lycopene was obtained. [1] Kong K.-W., Khoo H.-E., Prasad K.N., Ismail A., Tan C.-P., Rajab N.F. (2010) Revealing the power of the natural red pigment

lycopene. Molecules, 15: 859-987. [2] Curis E., Nicolis I., Moinard C., Osowska S., Zerrouk N., Bénazeth S., Cynober L. (2005) Almost all about citrulline in

mammals. Amino Acids, 29: 177-205. [3] Anderson J.W., Baird P., Davis Jr R.H, Ferreri S., Knudtson M., Koraym A., Waters V., Williams C.L. (2009) Health benefits

of dietary fiber. Nutrition Reviews, 67(4): 188-205. [4] Lenucci M.S., Caccioppola A., Durante M., Serrone L., Leonardo R., Piro G., Dalessandro G. (2010) Optimisation of

biological and physical parameters for lycopene supercritical CO2 extraction from ordinary and high-pigment tomato cultivars. Journal of the Science of Food and Agriculture, 90: 1709-1718.


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P46 Adaptation of leaves in Ligularia fischeri (Ledeb.) Turcz to sun, shade and drought: chlorophylls and phenolic compounds Sang Min Kim, Je-Seung Jeon, Yu-Jin Jung, Linna Ly, Byung-Hun Um

Functional Food Center, Korean Institute of Science and Technology Gangneung Institute, Gangneung, Gangwon-do, 210-340, Korea [email protected]

Ligularia fischeri (Ledeb.) Turcz is a commercial leafy vegetable that has been eaten for a long time in Korea. The HPLC chromatograms of leaf extracts collected from different areas in Korea showed a significant variation in caffeoylquinic acid derivatives (CQAs) [1] and two new tri-O-caffeoylquinic acids were purified and structurally identified by NMR and MS from this plant.

As this plant prefer the low-light and wet environment, in the present study, the effect of sun and drought was investigated by applying shade condition for 4 weeks to the plants growing under sun-light. The differences were described on chlorophyll, phenolics levels, containing CQAs, flavonoids, and anthocyanins.

Sun leaves showed higher levels of leaf thickness, % of dry weight than shade leaves. In addition, total phenolic and total flavonoid levels in sun leaves were found in higher levels compared to shade leaves. Especially, four major CQAs (5-CQA, 3,4-, 3,5-, and 4,5-DCQA) were induced according to the temperature increase, while in shade leaves, these compounds were continuously decreased in 4 weeks test period. The shade leaves possessed higher chlorophyll levels on fresh weight basis with bigger leaves in comparison to sun leaves. In a drought condition, sun leaves turned from green to purple and total anthocyanin content was increased, while phenolic and chlorophyll levels were decreased slightly.

The results clearly demonstrate that the leaves of L. fischeri show adaptation response to high irradiance and drought and these characteristics can be useful information in growing this plant for different commercial application. [1] Shang YF., Kim SM., Song DG., Pan CH., Lee WJ., Um BH. (2010) Isolation and identification of

antioxidant compounds from Ligularia fischeri. J Food Sci, 75:C530-C535


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The correlation between the total phenolic and flavonoid contents and their antioxidant properties of Thai rice cultivars in chemical and cell-based assays Pintusorn Hansakula, Umarat Srisawata, Nusiri Lerdvuthisopona, Arunporn Itharatb

aDepartment of Preclinical Science, Faculty of Medicine, Thammasat University, Rangsit Campus, Klong Luang, Pathumthani, 12120 Thailand. b Department of Applied Thai Traditional Medicine Center, Faculty of Medicine, Thammasat University, Rangsit Campus, Klong Luang, Pathumthani, 12120 Thailand. [email protected]

Free radicals induce oxidative stress in various cell components, leading to some certain

diseases. Due to adverse effects of some synthetic antioxidants, plant-derived antioxidants have become a profitable alternative to prevent oxidative stress in cells. In this study, the ethanolic extracts of brown rice and rice bran from Thai rice cultivars: Sangyod red rice and Dawk Mali 105 white rice were evaluated for the correlation between the total phenolic and flavonoid contents and their antioxidant properties through two chemical assays: DPPH radical-scavenging and inhibition of lipid peroxidation assays as well as a cell-based assay: scavenging capacity against intracellular superoxide in cells using dichlo-rofluorescein diacetate (DCF-DA).

All the rice extracts displayed their antioxidant activities in a dose dependent manner through the DPPH scavenging assay, the anti-lipid peroxidation assay, and the cell-based DCF-DA assay with EC50 values in the range of 3.54-230.71 μg/ml, 1.09-6.39 mg/ml, and 0.55-1.19 mg/ml, respectively. The ethanolic extract of Sangyod rice bran and brown rice exhibited the highest and the second highest inhibitory effect as compared to those of Dawk Mali 105. The total phenolic and flavonoid contents of all the rice extracts were in the range of 18.97-189.20 mg gallic acid equivalent per gram rice extract and 18.75-46.13 mg rutin hydrate equivalent per gram rice extract, respectively. The ethanolic extract of Sangyod rice bran contained the highest content of both studied compounds. Positive correlations were observed between the phenolic contents and the three antioxidant assays: the DPPH assay (R = 0.999), the anti-lipid peroxidation assay (R = 0.752), and the cell-based DCF-DA assay (R = 0.990). Furthermore, significant antioxidant effects of flavonoid contents were also observed in the DPPH assay (R = 0.909) and the cell-based DCF-DA assay (R = 0.836), but not in the anti-lipid peroxidation assay (R = 0.435). Importantly, the ethanolic extracts of Sangyod extracts were shown to be more effective than their water extracts (1).

These findings suggest that ethanolic extracts of brown rice and rice bran of Sangyod red rice can be promising sources of potential natural antioxidants. This study is also one of a few reports that perform a correlation analysis between antioxidant activity and total phenolic and flavonoid contents in Thai rice varieties (2).

[1] Srisawat U., Panunto W., Kaendee N., Tanuchit S., Itharat A., Lerdvuthisopon N., Hansakul P. (2010) Determination of phenolic compounds, flavonoids, and antioxidant activities in water extracts of Thai red and white rice cultivars. J Med Assoc Thai. 93 Suppl 7:S83-91.

[2] Butsat S., Siriamornpun S. (2010) Phenolic acids and antioxidant activities in husk of different thai rice varieties. Food Sci Technol Int. 16(4):329-36.


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P48 Acetylcholinesterase inhibition and antioxidant activity of three Asteraceae species from the Portuguese Aromatic Flora Ana Margarida Rodriguesa, Pedro Luís Faléb, Lia Ascensãoa, Maria Luísa Serralheirob

a Centro de Biotecnologia Vegetal (IBB), FCUL, Campo Grande, 1749-016 Lisboa, Portugal b Centro de Química e Bioquímica, FCUL, Campo Grande, 1749-016 Lisboa, Portugal [email protected]

The Portuguese Flora is very rich in aromatic species used for centuries in traditional medicine in the form of "teas", aqueous extracts obtained by infusion or decoction of different parts of plants, mainly belonging to Lamiaceae and Asteraceae families [1]. In the present work the antioxidant and acetylcholinesterase inhibitory activities were evaluated for the decoctions of stems/leaves and flowers of three Asteraceae species (Helichrysum italicum, Santolina impressa and Solidago virgaurea) collected from natural populations occurring throughout western Portugal sandy soils. The in vitro metabolism by the action of gastric and pancreatic artificial juices, as a simulation of the digestion of the extracts, was also studied.

The antioxidant activity of the aqueous extracts was assessed by the DPPH method. The decoction of H. italicum flowers showed the lowest EC50 value, representing the lowest concentration required to consume 50% of DPPH (10.14±0.02 µg/mL). The highest EC50 was obtained with the decoction of S. impressa flowers (14.70±0.06 µg/mL). As observed for the antioxidant activity, the lowest IC50 for acetylcholinesterase activity was found in H. italicum flowers decoction (302.52±1.14 µg/mL). The highest IC50 corresponded to the decoction of S. impressa leaves (578.98±13.70 µg/mL). All the aqueous extracts from flowers of these three species show a higher acetylcholinesterase inhibitory activity when compared with those found in their leaves extracts. The decoctions from flowers of H. italicum showed the highest tested biological activities.

The results of the in vitro metabolism revealed that decoctions did not suffer appreciable degradation, showing no significant changes both in its chemical composition and biological activities.

The high activities found in the three Asteraceae herbal teas could have a useful effect in the treatment of Alzheimer’s disease (AD) patients, as the inhibition of acetylcholinesterase is the most effective therapy for AD and the antioxidant activity has been connected with the capacity to scavenge the free radicals formed during the inflammation processes [2].

[1] Neves, J. M., Matos, C., Moutinho, C., Queiroz, G. & Gomes, L. R. (2009) Ethnopharmacological

notes about ancient uses of medicinal plants in Trás-os-Montes (northern of Portugal). Journal of Ethnopharmacology, 24: 270-283.

[2] Howes, M. J. R., Perry, N. S. L. and Houghton, P. J. (2003) Plants with traditional uses and activities relevant to the management of Alzheimer’s Disease and other cognitive disorders. Phytotherapy Research, 17: 1-18.


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Rosmarinus officinalis extract: a promising clinical management of Krabbe leukodystrophy

Venera Cardilea, Giovanna Pannuzzoa, Adriana Grazianoa, Juan Garbarinob, Nicolas Troncosoc and Alessandra Russod

aDepartment of Biomedical Sciences, University of Catania, V.le A. Doria 6, 95125 Catania, Italy bDepartment of Chemistry, University T.F. Santa Maria, Casilla 110-V, Valparaiso, Chile cLo Vicuña & Cia., Av. Santa María 5888, Vitacura, Santiago dDepartment of Sciences of Drug, University of Catania, V.le A. Doria 6, 95125, Catania, Italy [email protected]

Krabbe disease or globoid cell leukodystrophy is an autosomal recessively inherited disorder

caused by the deficiency of galactocerebrosidase, the lysosomal enzyme that catalyzes the hydrolysis of galactose from galactosylceramide and galactosylsphingosine (psychosine). Psychosine accumulation results in the loss of myelin and oligodendrocytes in the brain of Krabbe patients as well as twitcher mice (natural model of human Krabbe disease). Several approaches for the treatment of Krabbe disease are targets of research, but current treatment opinions for the disease are very limited. Gene therapy might be effective; however, a stable and strong expression vector is required [1]. Some studies reported that brains from brains from patients with Krabbe disease and twitcher show expression of the inflammatory cytokine TNF-�, and IL-6, and established the relationship of psychosine and cytokine-induced cellular redox alterations by increase in reactive oxygen species (ROS) and the inhibition of peroxisomal functions [1].

Rosmarinus officinalis L. (Labiatae) is used as a folk medicine around the world. In medicine, the extract is receiving increasing attention due to its anti-inflammatory and antioxidative constituents [2]. There are several reports that have established carnosic acid as the major phenolic diterpenoid present in rosemary leaves with antioxidant activity [2]. The aim of the present research was to demonstrate suppression of the apoptotic effects of psychosine in cultured oligodendrocyte progenitor mice cells (OLP-II) with a R. officinalis methanolic extract, containing 39.7 % of carnosic acid. Protection of the OLP-II cells against psychosine was shown using the MTT test (the ability of the tetrazolium salt MTT to form a dark blue formazan product by mitochondrial dehydrogenase in viable cells) and assay of expression of p53 and TNF-related apoptosis-inducing ligand (TRAIL). The results showed a time-dependent and concentration-dependent decrease of OLP-II viability on exposure to psychosine and dose-dependent protection with the examined extract. They also demonstrated that psychosine-induced p53 induction of apoptosis and TNF-related apoptosis-inducing ligand receptors could be decreased by this natural product. These preliminary data suggest that an integration with phytochemicals may constitute a promising clinical management of the late-infantile and juvenile forms of Krabbe leukodystrophy. [1] Pannuzzo G., Cardile V., Costantino-Ceccarini E., Alvares E., Mazzone D., Perciavalle V. (2010). A galactose-

free diet enriched in soy isoflavones and antioxidants results in delayed onset of symptoms of Krabbe disease in twitcher mice. Mol. Genet. Metab. 100: 234-40.

[2] Russo A., Lombardo L., Troncoso N., Garbarino J. and Cardile V. (2009). Rosmarinus officinalis L. extract inhibits human melanoma cell growth. Natural Prod. Commun. 4: 1707-1710.


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P50 Antioxidant properties of Asplenium trichomanes, Polypodiaceae Jelica Srdanova, Veljko Krstonosica, Marija Krickovica, Neda Lakica, Biljana Bozina aDepartment of Pharmacy, Faculty of Medicine, University of Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad, Serbia [email protected]

Presence of natural antioxidants is of a great importance, not only because of the impact on the

sustainability of oil, but also for important functions in human organism [1]. Maidenhair spleenwort (Asplenium trichomanes, Polypodiaceae) has a long tradition of usage in traditional medicine as a cure for respiratory illnesses. Also, it is used for the treatment of psoriasis. Today, when oxidative stress plays an important role in pathophysiology of diseases of modern age, antioxidant potential of maidenhair spleenwort could be of a great interest.

The objective of this study was to assess the oxidative stability of sunflower and corn oil and effectiveness of Asplenium trichomanes to slow down the peroxidation process of oils. Peroxidation was evaluated through measuring of peroxide values during 45 days. Also, total phenolic (gallic acid equivalents-GAE) and flavonoid content (quercetine equivalents-QE) and scavenging activity of 45% ethanol extract on 2,2-diphenyl-1-pycryl hydrazyil radical (DPPH�) were determined [2].

Leaves of wild-growing plants of Asplenium trichomanes were collected in July of 2010 at Fruska gora mountain, Republic of Serbia. Voucher specimen of collected plant material (No. A-75/10) was confirmed and deposited at the Herbarium of the Laboratory for Pharmacognosy, Faculty of Medicine, University of Novi Sad.

The results showed that Asplenium trichomanes exhibited protective acitivity against oxidative deterioration only in sunflower oil (34.21%), comparing to the pure oil, without plant material.

The amount of dry extract (d.e.) in dried plant material was 12.79%. Total amount of phenolics was 1.11 mg of GEA/g d.e. Flavonoids were present in the amount of 22.19 QE/g d.e. In DPPH-test, assayed extract exhibited notable antioxidant activity (IC50=13.38μg/ml).

The obtained results provide confirmation of the strong antioxidant and protective activity of Asplenium trichomanes herb extract, partially explaining some of traditional uses of this fern.

[1] Adnan A. et al. (2009) Analytical investigation of oxidative deterioration of sunflower oil stored under different

conditions. Eletronic Journal of Environmental, Agricultural and Food Chemistry, 8(10), [1043-1051]. [2] Bozin B., Mimica-Dukic N., Samojlik I., Anackov G., Igic R. (2008) Phenolics as antioxidants in garlic (Allium

sativum L., Alliaceae). Food Chemistry, 111(4), [925-929].


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Antioxidant activities, gamma oryzanol, vitamin E and total phenolic content of hom mali 105 rice (Oryza sativa) in different extraction methods Sakuntala Uttama a, Intouch Sakpakdeejaroen b, Rujulak Rattarom c, Arunporn Itharat b a Student of Ph.D in Medical Science (Nutraceutical)program, Faculty of Medicine, Thammasat University. Klongluang, Pathumtanee, Thailand b Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University, Klongluang, Pathumtanee, Thailand c Faculty of Pharmacy, Mahasarakham University, Khamreang, Mahasarakham, Thailand [email protected]

The objectives of this research are to investigate for the antioxidant activities, gamma

oryzanol , vitamin E and total phenolic content of Hom Mali 105 Rice (Oryza sativa) in

different extraction methods of three parts of rice as follow: rice bran, brown rice and white rice. The methods of extraction were maceration by ethanol 95% (ME) supercritical fluid extraction (SFE), boiling in water and dry by freeze dryer (BF),Water from washing rice and freeze dried (WR), expression method (EX) and soxhlet extraction method by using different solvent as hexane , chloroform and methanol (SXH, SXC and SXM respectively). DPPH assay (1) was carried for antioxidant activity and gamma oryzanol and vitamin E content were determined by HPLC (2,3 ). Total phenolic content was determine by using Folin–Ciocalteu reagent (3) The result found that BF showed the highest of percentage of yield (21.73%).white rice exhibited the highest antioxidant activity and it reduced free radical scavenging at concentration 100µg/ml as 48.34%. The SXM method gave the highest total phenolic content follow by SXC and ME are 20.6, 17.01 and 12.37 mg Gallic acid equivalent/ g samples of extracts respectively. ME method of rice bran gave the highest gamma oryzanol and also vitamin E content are 340, and 205 mg per 100 g of extracts respectively. This study is a guide for extraction product form rice the method of extraction and part of rice should be chosen for production of health product as essential bionutrients.

[1] Yamasaki, K. Hashimoto, A. Kokusenya, Y. Miyamoto T. and Sato, T. (1994). Electrochemical method for

estimating the antioxidative effect of methanol extracts of crude drugs, Chem. Pharm. Bull 42 : 1663-1665. [2] Edison B. (2009). Analysis of Tocopherols by High Performance Liquid Chromatography. E-Journal of Chemistry

6(2), 395-398. [3] Chotimakorn, C., Benjakul, S. and Silalai, N. (2008). Antioxidant components and properties of five long-grained

rice bran extracts from commercial available cultivars in Thailand. Food Chemistry, 111, 636-641.


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P52 Anti-inflammatory activities of Smilax corbularia Kunth and its constituents Srisopa Ruangnooa, Arunporn Itharata

aDepartment of Applied Thai Traditional Medicine Centre, Faculty of Medicine, Thammasat University, Klongluang, Pathumthani, 12120 Thailand [email protected]

Smilax corbularia Kunth (Smilacaceae) is a Thai medicinal plant locally known as ‘Hua-Khao-

Yen Neua’ . Its roots were used to treat inflammations such as arthritis [1,2]. Thus, the objective of this study is to investigate on anti-inflammatory activity of two extracts (ethanolic and water extracts) of this plant and its constituentsby determination of inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW 264.7 cell lines. Nitric oxide in the culture supernatant was measured by Griess reaction [3]. The ethanolic extract of Smilax corbularia Kunth exhibited the most inhibitory activity, with an IC50 value of 83.90+3.82 μg/ml. From this extract, three compounds [ engeletin (1) astilbin (2) and quercetin (3),] were isolated and further investigated for their inhibitory properties of NO production. It was found that quercetin (3) possessed the highest activity (IC50 = 9.17 μg/ml or 27.12 μM), whereas engeletin (1) and astilbin (2) exhibited mild activity. The ethanolic , water extracts of Smilax corbularia Kunth and compounds 1–3 were also evaluated for the inhibitory effect on LPS-stimulated PGE2 release from RAW 264.7 cells. The results found that engeletin (1) and astilbin (2) possessed potent activity against PGE2release with IC50 values of 19.67+0.01 μg/ml or 43.81 μM and 19.72+0.02 μg/ml or 45.31 μM, respectively, whereas, quercetin (3) exhibited mild activity. The present study may support using Smilax corbularia Kunth as inflammatory drug by Thai folk doctors . [1] Pongbunrod, S. (1976). Mai Thiet Mueang Thai Kasembanakit. Bangkok, pp.120–122. [2] Tungtrongjit, K. (1978). Pramuan Supphakun Ya Thai. Bangkok, pp. 107–108. [3] Tewtrakul, S. and Itharat, A. (2007). Nitric oxide inhibitory substances from the rhizomes of Dioscorea

membranacea. J.Ethnopharmacol. 109:412-416.


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Bioactive compounds from Capparis spinosa subsp. rupestris

Argentieri Maria Piaa, Macchia Francescob, Papadia Paridec, Fanizzi Francesco Paoloc, Avato Pinarosaa

aDipartimento Farmaco-Chimico, Università, Via Orabona 4, I-70125 Bari, Italy bOrto Botanico, Università, Via Orabona 4, I-70125, Bari, Italy c Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università, I-73100 Lecce, Italy [email protected]

The genus Capparis includes about 250 species distributed in the Mediterranean regions which

can be distinguished mainly based on their morphological traits such as leaf shape, flowers and presence/absence of spines. Some species are used as food and have application in medicine for skin protection [1].

According to the official European food legislation, commercial capers consumed as condiment are the flower buds of C. spinosa L., which represents a high polymorphic species with still an uncertain taxonomic position. It has been separated by some Authors into two different subspecies, namely C. spinosa L. subsp. spinosa and C. spinosa L. subsp. rupestris (Sibth. & Sm.) Nyman. The latter is also reported as C. spinosa subsp. rupestris (Veill. in Duhamel) Jafri and classified as a synonymous species of C. orientalis Veill., from which it might be derived [2].

Caper plants used industrially are generally referred to as C. spinosa, however, the less defined taxonomy and the fact that several cultivars and some species overlap with others may produce economic implications due to the quality variability of the commercial products.

The objective of the present study was to determine the spectrum of phytochemical constituents of C. spinosa subsp. rupestris in order to evaluate the presence of bioactive metabolites and compare the chemical profile of this species with that of other studied Capparis sp. and especially with C. spinosa subsp. spinosa. Chemical composition of oil and glucosinolates from seeds, as well as of glucosinolates and flavonoids from the aerial parts of the plant have been determined and data presented here.

The overall phytochemical data from the analysis of C. spinosa subsp. rupestris (syn. C. orientalis) do not differentiate this subspecies from C. spinosa subsp. spinosa, their compositional profiles resulted in fact largely superimposable. However, obtained results show that C. spinosa subsp. rupestris represents a very rich source of bioactive compounds of nutraceutical relevance, especially of the antioxidant rutin. [1] Rivera, D., Inocencio, C., Obón, M.C., Alcaraz, F. (2003). Review of food and medicinal uses of Capparis L.

subgenus Capparis (Capparidaceae). Econom. Bot. 57: 515-534. [2] Inocencio, C., Rivera, D., Obón, M.C., Alcaraz, F., Barreña, J-A. (2006). A systematic revision of Capparis

section Capparis (Capparaceae). Ann. Missouri Bot. Gard. 93: 122-149.


110

P54 Isolation of bioactive compounds from Saponaria officinalis L. Barbara Szajwaj1, Łukasz Pecio1, Małgorzata Szumacher-Strabel2, Adam Cieślak2, Wiesław Oleszek1 and Anna Stochmal1 1Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, ul. Czartoryskich 8, 24-100 Pulawy, Poland 2 Department of Animal Nutrition and Feed Management, RUMEN PULS Poznan, Wolynska 33, 60-637 Poznan, Poland [email protected]

Saponaria officinalis L. commonly named soapwort or bouncing-bet is a perennial plant from the carnation family (Caryophyllaceae), it’s native range extends throughout Europe, northern Africa and western to central Asia, but currently is also cultivated in many countries. Soapwort has been used in medicine as an expectorant in bronchitis, and in folk medicine it is still used for skin complaints and rheumatic disorders [1-4]. S. officinalis is a rich source of triterpenoid saponins but only part of them have been known. Thus, the aim of the present research was to focus on roots, because they contain about 5% of saponins and about 30% of oligosaccharides, which have not been investigated and until now only two were known.

The methanolic extract (233 g) from 500 g of dry, powdered roots of S. officinalis was fractionated on C18 preparative column with H2O (162.99 g), 30% MeOH (8.51 g) and 90% MeOH (49.90 g). Next, 30% and 90% methanolic fractions were suspended in H2O and extracted with EtOAc. The EtOAc extraction of 30% MeOH fraction yielded 681 mg of phenolic compounds, while from 90% MeOH fraction 45.41 g of saponins and 2.98 g of catechins were obtained. The fraction containing sugars (H2O fraction) was applied to a column chromatography on DEAE-cellulose, and the column was washed with H2O. This afforded 155.06 g of pure sugars. All groups of compounds (phenolics, sugars and saponins) were analyzed for their composition using a Waters ACQUITY UPLCTM system coupled to Waters TQ Detector (Waters Corp.) in full scan mode (m/z = 200-2000) on a HSS C18 (1.0 x 100 mm, 1.8 µm) column.

Future research on these fractions will be focused on the separation of sugars, phenolics, catechins and saponins into individual compounds. The activity of the fraction containing saponins and isolated individual saponins will be tested using ruminants animal models. Saponins demonstrate beneficial effects in defaunation (selective removal of protozoa) of the rumen and manipulation of the end products of fermentation [5]. [1] Jia Z., Koike K., Nikaido T. (1998) Major triterpenoid saponins from Saponaria officinalis. J. Nat. Prod., 61:

1368-1373 [2] Jia Z., Koike K., Nikaido T. (1999) Saponarioside C, the first α-D-galactose containing triterpenoid saponin, and

five related compounds from Saponaria officinalis. J. Nat. Prod., 62: 449-453 [3] Jia Z., Koike K., Nikaido T. (1999) New triterpenoid saponins and sapogenins from Saponaria officinalis. J. Nat.

Prod., 62: 1655-1659 [4] http://www.ars-grin.gov/cgi-bin/npgs/html/genform.pl [5] Wina E., Muetzel S., Becker K. (2005) The impact of saponins or saponin-containing plant materials on ruminant

production – a review. J. Agric. Food Chem., 53: 8093-8105


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The influence of isoflavonoid genistein incorporated in hydroxy-propyl-gamma-cyclodextrin on experimental B164A5 murinic melanoma model on mice

Tiulea Corinaa, Peev Cameliaa, Feflea Stefanaa, Dehelean Cristinab, Soica Codrutac , Andrei Motocd

aUniversity of Medicine and Pharmacy Victor Babes, Timisoara, Romania, Department of Pharmacognosy bUniversity of Medicine and Pharmacy Victor Babes, Timisoara, Romania, Department of Toxicology cUniversity of Medicine and Pharmacy Victor Babes, Timisoara, Romania, Department of Pharmaceutical Chemistry dUniversity of Medicine and Pharmacy Victor Babes, Timisoara, Romania, Department of Anatomy [email protected]

Genistein (4,5,7-trihydroxyisoflavone) is the aglicon of genistin and has been identified as the

predominant isoflavone in soybean, the vegetal product from Glycine max Fam. Fabaceae. This compound is a specific inhibitor of protein tyrosine kinase and was found to exhibit chemopreventive, cardioprotective and antiosteoporotic activities. One of the major problems of this active compound is its poor water solubility. Because genistein has a poor water solubility and the compound was injected in saline solution in order to increase the biodisponibility a complex with hydroxyl-propyl- gama-cyclodextrin (HPGCD) in a molar ratio 1:2 by kneading method was prepared. In order to test the complex formation scanning electron microscopy and differencial scanning calorimetry assay were performed. Results suggest that incorporation took place. Ten C57BL/6J mice, female of eight weeks were s.c. inoculated with 0.2 ml x 10 6 B164A5 melanoma cell suspension prepared after standard protocols using as solvent saline solution. Mice were divided in two groups and one group was injected with genistein complex s.c.at daily doses of genistein of 15 mg/kg b. w. for five days from the first day of experiment. Animals were inspected daily for the development of tumors, which became visible after 10 days from inoculation. On day 14 mice were sacrificed. Macroscopic examination was made. After that tissue samples were fixed in 10% formalin solution and were embedded in paraffin and cut at 4 microns. Finally after deparaffinized the samples were stained with H&E (hematoxylin-eosin) and microscopically analyzed. One can observe a decease in the number and dimension of tumors in mice treated with the complex genistein:HPGCD than in case of untreated mice. HE analyze showed that the diagnosis of malignant melanoma on skin level was present in both groups, but in case of untreated mice liver metastasis were also present.

The conclusion of this study is that genistein incorporated in hydroxyl-propyl- gama-cyclodextrin reduces the number and dimension of subcutaneous tumors and also the distant metastases on a period of study of two weeks.


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P56 SPOTLight: sustainable production of thapsigargin using light

Corinna Weitzel, Andreas Harald Klem, Tom Manczak, Trine Bundgaard Andersen, Joachim Møller Larsen, Henrik Toft Simonsen Department of Plant Biology and Biotechnology, VKR Research Centre Pro-Active Plants, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark [email protected]

The sesquiterpene lactone (SQL) thapsigargin is a promising agent for cancer therapy inducing

apoptosis by inhibiting the sarco/endoplasmatic calcium ATPase (SERCA). Covalently linked to a short peptide, the molecule can be directed to prostate cancer cells where it is activated in the presence of prostate specific antigen (PSA) [1]. This modification facilitates targeted treatment of this slowly growing cancer type.

Nature’s only source of thapsigargin are two species belonging to the genus Thapsia (Apiaceae) that is widely distributed throughout the Mediterranean area. Of the various Thapsia species only T. garganica and T. gymnesica produce thapsigargin whereas numerous sesquiterpenes, also some with related structures, can be found in all Thapsia species [2, 3, 4]. Once thapsigargin is established on the market, future demands cannot be ensured by isolating the drug from T. garganica without endangering wild populations. Since complexity of the chemical structure precludes organic synthesis, a biotechnological production platform will be the method of choice [5]. This platform will be created by our group by transferring thapsigargin’s metabolic pathway into the moss Physcomitrella patens that can be easily genetically transformed, grown in liquid culture and maintained in simple mineral medium thereby facilitating stable and inexpensive supply [5].

Until now, little is known about SQL biosynthesis in Apiaceae plants. In contrary, most investigations have been focused on artemisin or costunolid, both produced by Asteraceae. Even though SQL isolated from Apiaceae plants contain stereochemical conformations different from those found in Asteraceae [3], comparison of the biochemical data with that of related SQL enabled us to propose an enzymatic pathway by which a thapsigargin precursor could be generated [3]. Several steps in this pathway are believed to be carried out by cytochrome P450’s. Systematic investigation of the various steps will be possible by utilization of the unique high throughput sequencing database of T. garganica and T. villosa available in house. So far, this data lead to the cloning of two sesquiterpene synthases and eight CYP450 candidate genes, which are currently under investigation concerning their substrate specificity and their role in thapsigargin biosynthesis.

[1] Søhoel H, Jensen AM, Moller JV, Nissen P, Denmeade SR, Isaacs JT, Olsen CE, Christensen SB (2006) Natural

products as starting materials for development of second-generation SERCA inhibitors targeted towards prostate cancer cells. Bioorg Med Chem 14: 2810-2815

[2] Christensen SB, Andersen A, Smitt UW (1997) Sesquiterpenoids from Thapsia species and medicinal chemistry of the thapsigargins. Fortschr Chem Org Naturst 71: 129-167

[3] Drew DP, Krichau N, Reichwald K, Simonsen HT (2009) Guaianolides in apiaceae: perspectives on pharmacology and biosynthesis. Phytochem Rev 8: 581-599

[4] Drew DP, Rasmussen SK, Avato P, Simonsen HT (2011) Comparison of volatiles and essential oil components from Thapsia garganica L. and Thapsia villosa L. and discussion of guaianolide biosynthesis in Apiaceae. Phytochem. Anal. in press

[5] Drew DP, Krichau N, Reichwald K, Simonsen HT (2009) Guaianolides in apiaceae: perspectives on pharmacology and biosynthesis. Perspec. Med. Chem. 3:1-6


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Determination of phytoestrogens in natural products by liquid chromatography–tandem mass spectrometry Ivana Sosnovcova, Anna Hurajova, Vera Schulzova, Jana Hajslova

Department of Food Chemistry and Analysis, Institute of Chemical Technology Prague, Technická 5, 166 28 Prague 6 – Dejvice, Czech Republic, [email protected]

Phytoestrogens are plant-derived compounds, which commonly occur in a wide range of food- and feedstuff. Isoflavones, the well known group of phytoestrogens, are the most abundant in soybeans but they can be also found in alfalfa sprouts and in other legumes. The main isoflavones presented in plant matrices are daidzein, genistein, glycitein, formononetin and biochanin A. As suggested in recently published studies, these plants may comprise substantial part of dairy cows feed, thus presenting a potential source of isoflavones in bovine milk and milk products. Phytoestrogens represent biologically active compounds showing estrogenic activity similar to that of sex hormones – estrogens [1]. Epidemiologic studies have suggested a protective effect of isoflavones against a number of adult chronic diseases, including coronary heart disease and breast, endometrial, and prostate cancers. Previously, analysis of phytoestrogens in food was performed using either HPLC-UV or GC-MS technique [2].

In the presented study, suitable analytical method for determination of phytoestrogens in different matrices was developed. The performance characteristics achieved using an ultra performance liquid chromatography (UPLC) coupled to a tandem mass spectrometer (MS/MS) were evaluated, low limits of quantification (LOQs) were the key criteria that enable control of compounds and their metabolites even at very low levels. The main aim of this study was to monitor the content of phytoestrogens in different plants, soy-based foodstuff and nutraceuticals. Transfer via feedstuffs into the bovine milk and milk products, as well as the levels of equol (daidzein metabolite) were investigated.

Acknowledgement: This study was carried out within the projects NPVII 2B08073 supported by the Ministry of Education, Youth and Sports of the Czech Republic, partly from the projects MSM 6046137305, and Specific University Research (MSMT No. 21/2011). [1] Krajcova A., Schulzova V., Lojza J., Krizova L., Hajslova J. (2010) Phytoestrogens in bovine plasma and milk –

LC-MS/MS analysis. Czech J. Food Sci. 28, 264-274 [2] Dabrowski W. M., Sikorski Z. E. (2005) Toxins in Food. CRC Press, 85-104


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P58 Anti-inflammatory effects of Thai Traditional medicine called Ridsiduangmahakan Suchada Kittisrisopita, Arunporn Itharata

aDepartment of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University, 12120, Pathumthani, Thailand [email protected]

Ridsiduangmahakan (RSD) preparation was used to treat hemorrhoid patients by Thai

traditional doctors long time ago. Thus, this study was aimed to investigate anti-inflammation of this preparation by three anti-inflammation pathway: inhibitory activity on LPS induced NO production, TNF-� release and LPS-stimulated PGE2 in macrophage cells (RAW264.7) [1]. The preparation was macerated with 95%ethanol and its crude extract characteristic was divided as two part (solid and oil). The percentage of yields of the ethanolic extract were 16.30% (oil 4.68% and solid 11.62%). The results of RSD extract (oil, solid and oil+solid) on inhibitory activity on LPS induced NO production showed IC50 values of 48.1±1.8, 56.9±5.1 and 78.9±0.6 µg/ml, respectively and no cytotoxicity observed. Indomethacin was used as positive control with IC50 value of 20.32 g/ml or 56.78 M. The inhibitory effects on LPS-induced TNF-� release in RAW 264.7 cells were found that RSD (oil) gave IC50 value better than indomethacin (IC50 value of 31.861.86 g/ml and 51.413.58 g/ml or 143.6910.01 M, respectively) whereas RSD (solid) and RSD (oil+solid) did not exhibit inhibitory effect on TNF-� (IC50 values > 100 g/ml). In addition, the inhibitory effects of RSD (oil) on LPS-stimulated PGE2 showed the best IC50 value ( 8.851.60 µg/ml) followed by RSD (solid+oil) 16.804.04 µg/ml and RSD (solid) 21.223.29 µg/ml (IC50 value of indomethacin = 1.00±0.43 µg/ml or 2.801.20 M). These results lead to conclude that RSD preparation is anti-inflammatory drug through COX-2 inhibition pathway rather than through inhibition on TNF-� and NO production. It was suggested that oil of RSD preparation should be selected to prepare as antiinflammation drug and also supported using by folk doctors for treatment hemorrhoid patients.

[1] Tewtrakul, S. & Subhadhirasakul, S. (2008) Effects of compounds from Kaempferia parviflora on nitric oxide,

prostaglandin E2 and tumor necrosis factor-alpha productions in RAW264.7 macrophage cells. Journal of Ethnopharmacology, 120, 81–84.


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Varietal and environmental effects on some nutraceuticals in selected Spanish common beans (Phaseolus vulgaris L.)

Mercedes M. Pedrosa, Mara Sacristán, Carmen Burbano, Alejandro Varela, Blanca Cabellos, Carmen Cuadrado, Mercedes Muzquiz

Instituto Nacional de Investigación y Tecnología y Alimentaria (INIA), Dpto Tecnología de Alimentos, Ctra A Coruña KM 7.5, 28850 Madrid, Spain mmartí[email protected]

Beans are the most important grain legumes for direct human consumption in the world. Their production is almost twice that of chickpea, which is the second most important grain legume. They are consumed as mature grain, as immature seed, as well as a vegetable (both leaves and pods) [1]. Nowadays, the nutritional value of legumes is gaining considerable interest in developed countries because of the demand for healthy foods [2,3]. Several reports claim that inclusion of legumes in the daily diet has many beneficial physiological effects in controlling and preventing various metabolic diseases such as diabetes mellitus, coronary heart disease and colon cancer [4]. Related to nutraceutics foods, there are some preview researches about the antinutrients’s behavior in legumes, most particularly in beans. These substances are plant secondary metabolites, now increasingly recognized for their potential benefits for human health, amongst them are trypsin inhibitors, phytates, phenolic substances and α-galactosides [5]. Recent research has revealed the possible therapeutic properties of phytate in the prevention breast and colon cancers, probably due to its anti-oxidant properties. Phytate has also been implicated in the reduction of cholesterol and other lipids due to its presence in high fiber diets [6]. Moreover, trypsin inhibitors are effective in preventing, or suppressing, carcinogen-induced transformation in vitro and carcinogenesis in animals. Prebiotic effect has been recognized in �-galactosides like as fiber functions and could be used to functional ingredient once it have been extracted and purified. In addition, phenolic compounds are attributed antioxidant properties. The aim of this work is to determine the composition on some nutraceutical compounds (galactosides, inositol phosphates, trypsin inhibitors and phenolic compounds) for some selected Spanish varieties of common beans and to verify the effect of the environment on the content of these compounds. Most of the research on Spanish dry beans has been related to varietal selection. The criteria for selection have always been resistance to disease or high yields but never phytochemical composition. So, the results obtained will show a starting point in the hypothesis of selection of bean varieties with high nutritional value and rich in bioactive components, as well as the development of new functional foods derived from them. Acknowledgements: INIA, Project RTA2008-00007-C02. [1] Sathe S.K. , (2002) Crit. Rev. Biotechnol., 22, 175–223 [2] Shehata N.A., Darwish N., El-Nahry F., Andel-Razek F.A. (1988) Nahrung, 32, 3–8 [3] Simpson H.C., Lousley R.S., Greekie M., Hockaday T.D.R., Carter R.D., Mann J.I. (1981).Lancet, 1, 1-4 [4] Tharanathan R.N., Mahadevamma S. (2003) Trends Food Sci. Technol., 14, 507–518, [5] Kigel J. (19999 Biotech. Agron. Soc. Environ., 3, 205–209 [6] Midorikawa K., Murata M., Oikawa S., Hiraku Y., Kawanishi S. (2001) Biochem. Biophys. Res. Commun., 288,

552–557


116

P60 Antibacterial activity against biofilm formation of usnic acid and hyperforin

A. Rosatoa, B.P.I. Schiavoneb, G. Caggianod, M.Piarullib, L. Verottac, F. Corboa, C. Franchinia aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Bari Aldo Moro, Bari, Italy bDoctorate School of Biomedical Sciences, Hygiene Section, University of Bari Aldo Moro, Bari, Italy cDepartment of Organic and Industrial Chemistry, University of Milano, Milano dDepartment of Biomedical Science, Human Oncology Section,University of Bari Aldo Moro, Bari, Italy [email protected]

In the human body, bacteria are present in biofilms in essentially every niche that they colonize such as skin flora, oropharyngeal and nose flora, intestinal flora, in addition bacteria adhere to endovascular structures. Thus these microorganisms can compromise a wide number of medical devices, exposing patients at risk for local and systemic infectious complications.

Many natural products show antimicrobial activity against a number of bacteria, but only a small number of these are also modulators (inhibitors or disrupters) of the biofilm formation, one of the limits of many antibiotics to be efficacious [1].

In the light of these evidences we decided to test two natural phloroglucinols: usnic acid, a secondary lichen metabolite, and hyperforin [2], a phytochemical compound, and some of their derivatives, against biofilms. The assays were performed on biofilms from Gram+ and Gram- bacterial strains using in vitro diffusion cell bioassay system [3].

Scanning electronic microscopy (S.E.M.) revealed that the presence of these substances modified and disrupted the structure of the tested biofilms. In addition, to test cell viability of bacterial strains, viable counts of the biofilms of Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC 43300 and Enterococcus faecalis ATCC 29212 were performed [4]. Actually we are supporting these antimicrobial activity on pathogenic oropharyngeal flora using Streptococcus mutans ATCC 35668 and Streptococcus sanguinis ATCC 10556 forming biofilms as bacterial strains.

The in vitro model used could represent an useful system to know the interactions that appear to operate on biofilm. The experimental details and results of the study are reported in this communication. [1] Richards, Justin J.; Melander, Christian. (2009) Small molecule approaches toward the non-microbicidal

modulation of bacterial biofilm growth and maintenance. Anti-Infective Agents in Medicinal Chemistry, 8(4), 295-314.

[2] P. Avato , F. Raffo , G. Guglielmi , C. Vitali and A. Rosato. Extracts from St. John’s Worth and their antimicrobial activity. Phytotherapy Research. 18, 230-232. 2004.

[3] Y. H. Samaranayake, J. Ye, J. Y. Y. Yau, B. P. K. Cheung, and L. P. Samaranayake (2005) In Vitro Method To Study Antifungal Perfusion in Candida Biofilms J. Clin. Microbiol. 43: 818 - 825.

[4] Mohammed A. S. Alem and L. Julia Douglas (2004) Effects of Aspirin and Other Nonsteroidal Anti-Inflammatory Drugs on Biofilms and Planktonic Cells of Candida albicans Antimicrob. Agents Chemother. 48: 41 – 47.


P61

117

Antidiabetic effect of extract of Terminalia arjuna in streptozotocin-induced diabetic Wistar rats Tan Benny KH, Yong Edwin WW, Hsu A Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, MD 11, #05-09, 10 Medical Drive, Singapore 117597 [email protected]

Terminalia arjuna is an important medicinal plant widely used against various ailments.

Numerous studies have shown antioxidant, cardiotonic and hypoglycaemic, properties of the herb1, 2. However few studies have been conducted to probe its anti-diabetic potential. Oxidative stress has been implicated in the development of diabetic complications such as hypertension. In view of the close link between diabetes, oxidative stress and cardiovascular disease, we investigated the anti-diabetic capability of a 70% methanolic extract of T. arjuna administered orally for 28 days to streptozotocin (STZ)-induced diabetic rats.

A dose-response study of T. arjuna extract on glucose tolerance in STZ-diabetic rats (results in Figure 1) was followed up by a 28-day treatment with the extract. The extract significantly reduced fasting serum glucose level in STZ-diabetic rats at the end of the 28-day period. Decreased hepatic glucose-6-phosphatase activity and increased muscle glycogen content were found in the extract-treated rats. GLUT4 expression in skeletal muscle was also enhanced in these rats. Serum and pancreatic insulin concentrations were not significantly increased while serum and hepatic triglyceride and total cholesterol levels were significantly decreased. Interestingly, T. arjuna-treated rats had elevated GSH levels and augmented activities of antioxidant enzymes.

Results from this study provide support for the use of T. arjuna in the treatment and possible prevention of complications of diabetes mellitus. Future purification and bioassay-guided studies are planned to identify the bioactive component(s) in the herb plant. Further detailed studies can then be conducted to elucidate the exact mechanism(s) underlying the potential effectiveness of T. arjuna as an anti-diabetic herb.

Figure 1. Dose-response effect of T. arjuna extract on glucose tolerance in STZ-diabetic rats

[1] Sunyana Jain, P. P. (2009). Terminalia arjuna: a sacred medicinal plant: phytochemical and pharmacological

profile. Phytochemistry Reviews, 8 (2): 491-502 [2] B. Ragavan, S. K. (2006). Antidiabetic effect of T arjuna Bark Extract in Alloxan-Induced Diabetic Rats. Indian

Journal of Clinical Biochemistry, 21 (2): 123-128.


P62

118

Simultaneous determination of salicylates and benzoic acid in food

by SPME-LC-UV-DAD

A. Aresta, G. Lioce, C.G.

Zambonin

Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, via Orabona 4, 70126 Bari

[email protected]

Salicylate, acetylsalicylic acid (aspirin) and their derivatives (salicylates) are used as fungicidal

and antimicrobial agents in pharmaceuticals preparations (external use) as well as in the treatment

of inflammatory processes as antipyretic and analgesic drugs (internal use).

Salicylates have been also used in beverages and foods for preservation, but it has been

forbidden since the sixties in several countries due to its toxicity [1].

These chemicals occur also naturally in many plants, including many fruits vegetables, and

herbs. Salicylates in plants act as a natural immune hormone and preservative, protecting the

plants against diseases, insects, fungi, and harmful bacteria.

Salicylates are generally regarded as safe for adults, even if high enough doses are harmful to

everyone. However, most people can handle average amounts of salicylate in food, products and

medications without any adverse affects on their health. Unfortunately, there is a small percent of

the population for which even a small dose of salicylates can be a problem. Some adults and

children may develop symptoms and health problems from salicylates which are dose-related.

This is called ‘Salicylate Sensitivity’ or ‘Salicylate Intolerance’.

Individuals that are sensitive to salicylates may suffer with urticaria, angiooedema, rhinitis,

bronchial asthma and recurrent nasal polyps [2]. The chronic nature of some of these clinical

presentations, without other obvious cause, may suggest an underlying etiology related to dietary

salicylates. A low salicylate diet may be of clinical benefit to such affected individuals. This

cannot be established however, until the salicylate content of different food and drinks is known.

Data on the salicylate content of foods are scarce and contradictory. Our aim was to develop an

accurate analytical method to measure the salicylates content of selected food commodities.

Existing papers on this topic have been essentially based on chromatographic techniques, after

purification of the analytes by means of complicate isolation procedures. A good alternative could

be represented by the use of solid-phase microextraction (SPME), a solventless technique initially

coupled to GC and later interfaced also to LC [3].

Thus, in the present work, a solid-phase microextraction (SPME)–LC–UV-DAD method for

the simultaneous determination of salicylates and benzoic acid in food samples was developed for

the first time using a polydimethylsiloxane/divinylbenzene (PDMS/DVB) coated fiber. The

procedure required very simple sample pretreatments, isocratic elution, and provided highly

selective extractions. The applicability of the method was demonstrated on different food

products, i.e. kiwis, blueberries, lemons, mandarins, oranges, broad beans, and commercial fruit

juices.

[1] T.M. Brown, A.T. Dronsfield, P.M. Ellis and J.S. Parker, Educ. Chem., 35, 1998, 47-51.

[2] S. Jantti-Alanko, E. Holopainen, and H. Malmberg, Rhinology, 8, 1989, 59–64.

[3] C.G. Zambonin, Anal Bioanal. Chem., 375, 2003, 73–80.

mailto:[email protected]


P63

119

Formulation and characterization of new packaging material incorporating chitosan nanoparticles-vitamin E and C for food shelf life improvement

Calvano C.D.a, Trapani G.b, Barnaba M.a, De Giglio E.a, Cafagna D.a, Aresta A.a, Zambonin C.G. a, c a Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, via Orabona 4, 70126 Bari bDipartimento Farmaco Chimico, Università degli Studi di Bari Aldo Moro, via Orabona 4, 70126 Bari cCentro Interdipartimentale SMART, Università degli Studi di Bari Aldo Moro, via Orabona 4, 70126 Bari [email protected]

One of the principal causes of food quality deterioration is the oxidation of unsaturated lipids initiated by free radicals. When lipids are exposed to environmental factors such as air, light and temperature, oxidation reactions start to produce undesirable flavours, rancid odours, discoloration and other forms of spoilage. During oxidation, a series of volatile and non-volatile compounds are originated, altering sensory proprieties, causing rancid flavors and decreasing the nutritional quality and safety, which may play a role in the development of some diseases [1]; in addition lipid oxidation could cause a consumer rejection of the food product.

The rate of autoxidation depends on temperature, pH, the degree of unsaturation of the fatty acids and the number of unsaturated fatty acids in the triacylglycerol or phospholipid molecule, as well as on the availability of oxygen and transition metal ions. The other major cause of food spoilage during storage is the bacterial contamination arising from several sources, such as the animal itself, the external environment and its handling.

Packaging is important to preserve the quality and safety of fruits, vegetables and processed foods for assured shelf life extension. The new generation of edible coatings is especially designed to allow the incorporation and/or controlled release of antioxidants, vitamins, nutraceuticals, and natural antimicrobials [2-4].

Among packaging material, chitosan has been well-known for its excellent film-forming property, antimicrobial activity, and unique coagulating ability with metal and other lipid and protein complexes. It seems to be due to the presence of high density of amino groups and hydroxyl groups in the chitosan polymer structure. Its high binding ability and antimicrobial properties are both beneficial in developing new applications of this natural polymer in food preservation. Herein, we aimed at the formulation of chitosan-cyclodextrin nanoparticles (NPs) obtained by the ionic gelation process in order to load vitamin E and C selected as powerful antioxidant agents. In fact, vitamin E is important in the prevention of lipid peroxidation, whereas vitamin C reacts effectively with superoxide and hydroxyl radicals. Vitamins C and E and vitamin precursors (e.g. carotenoids) should reduce the rate of initiation or prevent the propagation of free radicals as notable non-enzymatic antioxidants.

The NPs will be characterized in terms of light scattering, zeta-potential, vitamins content and by X-Ray Photoelectron Spectroscopy (XPS) measurements. Afterwards, the NPs loading vitamin C and E will be dispersed on polymeric films that are usually found as food packaging materials.

[1] R. M. Adibhatla, J.F. Hatcher Antioxidants & redox signaling, 12, 2010, 125-169 [2] M. Vargas, C.Pastor, A.Chiralt, D. J. McClements, C. Gonz lez-Mart nez Crit Rev Food Sci Nut, 48, 2008, 496–511. [3] P. K Dutta, S. Tripathi, G. K. Mehrotra, J. Dutta, Food Chem 114, 2009,1173-1182. [4] P.E. Chiu, L.S. Lai, Int J Food Microbiol 139, 2010, 23-30.


Complete List of Presentations


PLENARY LECTURES

SHORT LECTURES

SL 1 P. Reddell, V. Gordon, P. Parsons, G. Muench, C. Williams- Discovery of New Nutraceuticals from Australia’s Tropical Rainforests.

SL 2 J.M.I.Alqudah, K..Reilly, M.T.Gaffney, A.B. Martin-Diana & C.Barry-Ryan - Screening of Irish Fruit and Vegetable Germplasm for Novel Anti-Tumor and Pesticidal Compounds.

SL 3 M. Durante, M. Lenucci, L. Rescio, G. Mita, S. Caretto.- Durum Wheat By-Products as Natural Sources of Valuable Nutrients

SL 4 L. Brown, H. Poudyal- Anthocyanin-enriched purple carrot juice with low carotenoids attenuates cardiovascular and hepatic symptoms induced by high carbohydrate, high fat feeding in rats.

SL 5 M. Lenucci, L. Rescio, G. Piro, G. Dalessandro - Tomato organic lycopene: the way to obtain a unique healthy product.

SL 6 F. Bucar, E. Knauder - Isoflavone Composition of Soy and Red Clover Dietary Supplements.

SL 7 Candiracci, A. Castaño, C. SantaMaría, E. Revilla, M. Dominguez-Barragán, B. Rodríguez-Morgado, D. García-Antras, M. Carballo, J. Bautista, M.D. Herrera, J. Parrado - Antioxidant and anti-inflammatory properties of a new soluble extract from grape pomace. M.

SL 8 S. Nunes, A.T. Serra, M. R. Bronze, Catarina M.M. Duarte, A.A. Matias - Polyphenols-rich extracts obtained from Prunus avium and opuntia ficus indica as Natural anti-inflammatory modulators for Inflammatory Bowel Diseases.

SL 9 T. Magrone, S. Fontana, E. Jirillo.Polyphenols from red wine exert potential beneficial effects to human health.

SL10 M.A. Papandreou, M. Tsachaki, S. Efthimiopoulos, D. Klimis-Zacas, M. Margarity, F. N. Lamari - Differential antioxidant and anti-amyloid effect of a wild blueberry polyphenol-rich extract: in vitro and in vivo studies.

SL11 V. Schulzova, H. Novotna, L. Vaclavik, A. Hurajova, J. Hajslova - Metabolomic Profiling and Fingerprinting: New Effective Approach for Plant Characterisation and Authentication.

SL12 M. Zotti, M. Colaianna, M.G. Morgese, L.Ciuffreda, P. Tucci, P. Avato, L. - Estrous cycle in female rats influences the neurochemical and neurobehavioural effects of carvacrol

SL13 J. Gruz, L. Spichal - Application of purine derivative LGR-1814 improves functional properties of field-grown lettuce (Lactuca sativa).

SL14 A. Szakiel, C. Pączkowski, H. Koivuniemi, S.Huttunen - Comparison of triterpenoid content of Vaccinium vitis-idaea berries and leaves collected from natural forest habitats in Finland and Poland.

SL15 M. Monschein, K. Ardjomand-Woelkart, I. Wolf, J. Rieder, R. Bauer - Formation of astragaloside IV from acylated astragalosides during extraction of Astragali Radix

SL16 K. Michalska, W.Kisiel - Sesquiterpenoids from aerial parts of Lactuca aculeate SL17 The volatile fraction of herbal teas.Christine Tschiggerl and Franz Bucar SL18 Tassoni A., Ferri M. - Elicitation, production and release of polyphenols in fed-batch fermentation of grape

PL 1 G. Appendino - Food-based drug discovery: overcoming the nutrition-medicine divide.

PL 2 M. Muzquiz - Bioactive compounds in legumes: pronutritive and antinutritive actions. Implications for nutrition and health.

PL 3 PL 4 V. Cheynier - Phenolic compounds : from plants to foods.

PL 5 J.L.Wolfender - New trends in the analysis of natural products for the detailed metabolite profiling of crude extracts.

PL 6 Y. Tikunov, S. van Heusden, J. Molthoff , H. van de Geest , A. Ballester, I. Romero, A. Granell, R. de Vos, A. Bovy - Breeding for improved flavour and health.

PL 7 S.B. Engelsen, F. Savorani - NMR metabolomics: advantages and disadvantages for studying bioactive components in food.

PL 8 A.M. Aura - Phenolic nutritional phytochemicals as biomarkers of plant food intake.

PL 9 B. A. Halkier,U.H. Mortensen, L. Albertsen, B. Salomonsen, M. Mikkelsen, J. K.Vester, F. Geu-Flores - Bioengineering of glucosinolates production in yeast.

PL10 V. Lanzotti - Bioactive Natural Compounds from Food Plants. PL11 P. J. Russell - Naturals for the masses – Chemistry approaches for safe commercialization.


cells.

SL19 S. M. Cardoso, S.I. Falcão, A.M. Peres, O.R. Pereira, M.R.M. Domingues Insights into olive mill wastewaters phenolics.

SL20 C. Longo, R. Zefferino, A. Leone - Molecular Mechanisms Underlies the Biological Activities of Phytochemicals: searching the gap

SL21 G. Farre, S. Rivera, C. Bai, R. Canela, G. Sandmann, C. Zhu, T. Capell, P. Christou - The Arabidopsis Orange (Or) gene generates a metabolic sink and enhances accumulation of carotenoids in cereal cells and plants.

SL22 S. Bhattacharya, S. Sinha, P. Dey, N. Das, M. K. Maiti - Production of nutritionally desirable fatty acids in seed oil of Indian mustard (Brassica juncea L.) by metabolic engineering.

SL23 H. Poudyal, L. Brown - α-Linolenic acid-rich oil from chia seed (Salvia hispanica L.) induces lipid redistribution, heart and liver protection in diet-induced obese rats.

SL24 M-H. Teiten, A. Gaigneaux, S. Chateauvieux, A.M Billing, J. Renaut, M. Dicato, M. Diederich - Proteomic identification of differentially expressed proteins in curcumin-treated prostate cancer cells.

SL25 J. Hadsbjerg, S. Willkomm, K. Nöske, T.D. Rasmussen, E. Brunstein, K.. Niefind, O.G. Issinger - Natural compounds as selective inhibitors for protein kinases and their potential application in cancer research.

SL26 M. Miele, A.M. Mumot, A. Zappa, P. Romano, L. Ottaggio - Hazel and Other Angiosperm Plants as New Sources of Compounds with Taxane-Like Activity.

SL27 C. Mathon, M.Duret, P. Edder, S. Bieri, P. Christen - Targeted screening of botanicals in herbal dietary supplements by LC-MS/MS with an Information Dependent Acquisition approach.

SL28 A. Mari, P. Montoro, S. Piacente - LC-MS/MS determination of marker metabolites in the ethanolic extract and in mother tinctures of Vitex agnus castus fruits.

POSTERS

P 1 L. Barros, C. Grangeia, A. Martins, I. C.F.R. Ferreira.- Nutritional and nutraceutical potential of saprotrophic

and mycorrhizal wild edible mushrooms from Northeast Portugal.

P 2 F. Conforti, M. Marrelli, G. A. Statti, F. Menichini -In vitro inhibition of nitric oxide production and free radical scavenging activity of non-cultivated Mediterranean vegetables.

P 3 A. Bisio, E. Giacomelli, G. Damonte, A. Salis, D. Fraternale, G. Romussi, S. Cafaggi, D. Ricci, N. - De Tommasi - A new clerodane diterpenoid from the aerial part exudate of Salvia chamaedryoides Cav.

P 4 A. A Ahmadu, A. Agunu - Chemical study on Pavetta corymbosa.

P 5 S. I. Falcão, C. Freire, M. Vilas-Boas - Physicochemical characterization and bioactivity of Portuguese Propolis towards its standardization.

P 6 A. Itharat, S. Ruangnoo, P. Thongdeeying - A new cytotoxic dihydrophenantrene against human cancercCells from Dioscorea membranacea roots.

P 7 A.V. Ferlemi, F.N. Lamari, G. Iatrou, M. Margariti - Effects of daily consumption of Rosmarinus officinalis and Hypericum vesiculosum infusions on rodent cognitive function and cerebral acetylcholinesterase activity.

P 8 B. Dechayont, P. Thondeeying, A. Itharat - Cytotoxic activity against lung and prostate cancer cells of the ethanolic extract from Antidesma thwaitesianum Müll. Arg. Leaves.

P 9 J. Chunthorng-Orn, A. Itharat - Comparison of antimicrobial, antioxidant, anti inflammatory activities and total phenolic contents of female and male trees from Carica papaya Roots.

P10 J. P. de Andrade, S. Berkov, K. B. da Silva, L. G. Sachet, F. Viladomat, C. Codina, J. A. S. Zuanazzi, J. Bastida - Alkaloids of Hippeastrum papilio.

P11 A.M. Schito, G. Piatti, A. Bisio, E. Giacomelli, G. Romussi, C. Pruzzo - Activity of compounds extracted from Salvia aurea against methicillin- resistant Staphylococci.

P12 M. Dimitrova, Z. Yordanova, V. Kapchina-Toteva - Influence of benzylaminopurine on antioxidant activity and phenolic content on in vitro propagated Lamium album L.

P13 D. Gođevac, V. Tešević, V. Vajs, S. Milosavljević, G. Zdunić , M. Stanković - Currant seed extracts showing protective effect on human lymphocytes DNA.

P14 C. Sae-wong, S. Tewtrakul, P. Tansakul - Inhibitory effect of Kaempferia parviflora on NFкB, Rel A, NOS, COX-2 and TNF-� mRNA expression.

P15 F. Corbo, A. Lovece, C. Bruno, F. La Forgia, S. Fontana, M. Roselli, A. Laghezza, G. Lentini, C. Franchini, S. Habtemariam - Antioxidant activity of Uva di Troia Canosina ad acino piccolo: comparison of different extraction methods.

P16 P. Phuaklee, A. Itharat - Nitric oxide inhibitory effect and antioxidant activity of the extracts from Musa sapientum bracts and flowers.

P17 S. Gonçalves, D. Gomes, P. Costa, A. Romano - Correlation between phenolic content and antioxidant activity of infusions from eleven Mediterranean plant species.

P18 F. Gaascht, M. Jain, M-H. Teiten, M. Schumacher, G. Kirsch, D. Bagrel, M. Dicato, M. Diederich -New


natural compounds of Dionaea muscipula as anticancerous agents.

P19 M. Candiracci, J. Parrado,M.L. Justo, R.Rodríguez-Rodríguez, A. Castaño, M.D. Herrera - Effects of a rice bran enzymatic extract on cytokines implicated in the inflammatory state of adipose tissue in a rat model of obesity.

P20 C. Bai, S. M. Rivera, V. Medina, G. Sandmann, R. Canela, C. Zhu, T. Capell, P. Christou - Metabolic engineering of carotenoid biosynthesis in riceendosperm.

P21 J-I. Kim, J. Kim, H. Choi, M-J. Kang, S. Jeong, M. Kim- Effect of quercetin on hepatic steatosis in ob/ob mice.

P22 P. Trisolini, A. Ignazzi, F. Pezzolla, D. Lorusso, S. Fiume, L. Debellis, M.A. Maselli - Olive oil phenols improve the altered human intestinal permeability. Study in vitro.

P23 I. Kowalska, D. Jędrejek, Ł. Cieśla, S. Piacente, M. Mosullo, W. Oleszek, A. Stochmal - Phenolic composition of Trifolium scabrum L. aerial parts.

P24 M. S. Valyova, M. A. Dimitrova, Y. Ganeva, V. M. Kapchina-Toteva, Z. P. Yordanova - Evaluation of antioxidant and free radical scavenging potential of Lamium album L. growing in bulgaria.

P25 A. Tava, B. Pintus, A. Ursino, B. Campion - Evaluation of the saponin content in lines of common beans (Phaseolus vulgaris L.) selected in Italy.

P26 M.P. Argentieri, R. Pascale, P. Avato - Glucosinolates from “Mugnolo”, a variety of Brassica oleracea.

P27 F. N. Lamari, M. A. Papandreou, A. P. Dimakopoulou, E. Drimtzias, A-B. Ferlemi, O. Makri, P. Cordopatis, C. Georgakopoulos, M. Margarity - Antioxidant properties of Saffron against H2O2- & Selenite-induced oxidative stress: in Vitro & in Vivo Studies.

P28 Y. Jirakiattikul, A. Itharat - Surface sterilization and in vitro shoot multiplication of Dioscorea membranacea Pierre ex Prain & Burkill.

P29 P. L. Falé, M. A. Filipe, L. Ascensão, M. L. Serralheiro, L. Mira - Activity of Plectranthus barbatus extract against inflammatory response in human neutrophils.

P30 G. Sanahuja, G. Farre, L. Bassié, C. Zhu,T. Capell, P. Christou - Elucidation of rate limiting steps of ascorbate biosynthesis in maize.

P31 M. Marrelli, F. Conforti, G. A. Statti, F. Menichini - Antioxidant activity and antiproliferative properties of Ficus carica cv. Dottato fruits.

P32 J. Nantana - Some Morphology and Genetic Relationship of Dioscorea membranacea using Molecular Technique.

P33 B. Miralpeix, R. Hoefer, D. Werck, L. Dong, H. J. Bouwmeester, T. Capell, P. Christou - Metabolic engineering of terpenoid biosynthesis in transgenic tobacco plants.

P34 A. Napolitano, H. K. Sellami, M. Masullo, S. Smiti, S. Piacente, C. Pizza - Quali-quantitative profiling of the polar extracts of the flowers of Ammi visnaga plants grown in different conditions.

P35 A. Mitra, M. Kumar - Harnessing valuable botanicals from Areca catechu pericarps.

P36 L. Torras-Claveria, S. Berkov, F. Viladomat, C. Codina, J. Bastida - Exact mass and QToF fragmentation data base of bioactive Amaryllidaceae alkaloids.

P37 M. Ferri, A. Gianotti, A. Tassoni - Suitability of ancient and modern cereals to produce novel functional fermented foods with a high content of antioxidant compounds.

P38 O. Prajuabjinda, A. Itharat - Antioxidant and cytotoxic activities against lung cancer cells of Thai Medicinal Plants for Cancer treatment in Kumpramong Temple.

P39 M. Monschein, F. Bucar - Phytochemical investigation of phenolic compounds of elderflowers growing at different altitudes.

P40 P. Rithichai, A. Itharat - Growth of Dioscorea membranacea Pierre ex Prain & Burkill and dioscorealide B accumulation at different harvest times.

P41 M. Maldini, P. Montoro, A. I. Hamed, U. A. Mahalel, W. Oleszek, A. Stochmal, S. Piacente - Strong free radical scavenging phenolics from Acacia nilotica pods.

P42 B. Thiem, M. Ożarowski, M. Szulc, R. Kujawski, P. M. Mrozikiewicz, P.L. Mikołajczak Neuropharmacological study of Eryngium planum L. extract.

P43 H. Novotna, V. Schulzova, I. Sosnovcova, J. Hajslova - Using DART-TOFMS for quality assessment of rose hip (Rosa Canina) fruits.

P44 R. Rattarom, I. Sakpakdeejaroen, A. Itharat - In Vitro cytotoxic activity against human lung cancer cells of the ethanolic extract from Benjakul preparation and its isolated compounds.

P45 Watermelon as a source of bioactive molecules. Marcello Salvatore Lenucci, Stefania Grassi, Anna Eleonora Caprifico, Maria Chiara Contino, Anna Montefusco, Gabriella Piro, Giuseppe Dalessandro

P46 S. M. Kim, J-S. Jeon, Y-J. Jung, L. Ly, B-H. Um - Adaptation of leaves in Ligularia fischeri (Ledeb.) Turcz to sun, shade and drought: chlorophylls and phenolic compounds.

P47 P. Hansakul, U. Srisawat, N. Lerdvuthisopon, A. Itharat - The correlation between the total phenolic and flavonoid contents and their antioxidant properties of Thai rice cultivars in chemical and cell-based assays.

P48 A. M. Rodrigues, P. L. Falé, L. Ascensão, M. L. Serralheiro - Acetylcholinesterase inhibition and antioxidant activity of three Asteraceae species from the Portuguese Aromatic Flora.


P49 V. Cardile, G. Pannuzzo, A. Graziano, J. Garbarino, N. Troncoso, A. Russo - Rosmarinus officinalis extract: a promising clinical management of Krabbe leukodystrophy.

P50 J. Srdanov, V. Krstonosic, M. Krickovic, N. Lakic, B. Bozin - Antioxidant properties of Asplenium trichomanes, Polypodiaceae.

P51 S. Uttama, I. Sakpakdeejaroen, R. Rattarom, A. Itharat - Antioxidant activities, gamma oryzanol ,vitamin E and total phenolic content of hom Mali 105 Rice (Oryza sativa) in different extraction methods.

P52 S. Ruangnoo, A. Itharat - Anti-inflammatory activities of Smilax corbularia Kunth and its constituents.

P53 M.P. Argentieri, F. Macchia, P. Papadia, F.P. Fanizzi, P. Avato - Bioactive compounds from Capparis spinosa subsp. Rupestris.

P54 B. Szajwaj, Ł. Pecio, M. Szumacher-Strabel, A. Cieślak, W. Oleszek, A. Stochmal - Isolation of bioactive compounds from Saponaria officinalis L.

P55 C. Tiulea, C. Peev, S. Feflea, C. Dehelean, C. Soica, A. Motoc - The influence of isoflavonoid genistein incorporated in hydroxy-propyl-gamma-cyclodextrin on experimental B164A5 murinic melanoma model on mice.

P56 C. Weitzel, A. H. Klem, T. Manczak, T. A. Bundgaard, J. M. Larsen, H. S. Toft - SPOTLight: sustainable production of thapsigargin using light.

P57 I. Sosnovcova, A. Hurajova, V. Schulzova, J. Hajslova - Determination of phytoestrogens in natural products by liquid chromatography–tandem mass spectrometry.

P58 S. Kittisrisopit, A. Itharat - Anti-inflammatory Effects of Thai Traditional Medicine called Ridsiduangmahakan.

P59 M. M. Pedrosa, M. Sacristán, C. Burbano, A. Varela, B. Cabellos, C. Cuadrado, M. Muzquiz - Varietal and environmental effects on some nutraceuticals in selected Spanish common beans (Phaseolus vulgaris L.).

P60 A. Rosato, B.P.I. Schiavone, G. Caggiano, M.Piarulli, L. Verotta, F. Corbo, C. Franchini - Antibacterial activity against biofilm formation of usnic acid and hyperforin.

P61 KH. Tan Benny, W.W. Yong Edwin, A. Hsu - Antidiabetic effect of extract of Terminalia arjuna in streptozotocin-induced diabetic Wistar rats.

P62 A. Aresta, G. Lioce, C.G. Zambonin - Simultaneous determination of salicylates and benzoic acid in food by SPME-LC-UV-DAD

P63 C.D. Calvano, G. Trapani, M. Barnaba, E. De Giglio, D. Cafagna, A. Aresta, C.G. Zambonin - Formulation and characterization of new packaging material incorporating chitosan nanoparticles-vitamin E and C for food shelf life improvement


Authors´ Index


A page Agunu A. 60 Ahmadu A.A. 50

Albertsen L. 40 Alqudah J.M.I. 13

Andersen T.B. 112 Appendino G. 11 Ardjomand-Woelkart K. 33 Aresta A. 118, 119 Argentieri M.P. 82, 109 Ascensão L. 85, 104 Aura A-M. 36 Avato P. 28, 82, 109 B page Bagrel D. 74 Bai C. 41, 76 Ballester A-R. 29 Barnaba M. 120 Barros L. 57 Barry-Ryan C. 13 Bassié L. 86 Bastida J. 66, 92

Bauer R. 33 Bautista J. 21 Berkov S. 66, 92 Bhattacharya S. 42 Bieri S. 49 Billing A.M. 44 Bisio A. 59, 67 Bouwmeester H.J. 89 Bovy A. 29 Bozin B. 106 Bronze M.R. 23 Brown L. 16, 43 Bruno C. 71 Brunstein E. 46 Bucar F. 20, 35, 95 Burbano C. 115 C page Cabellos B. 115 Cafaggi S. 59 Cafagna D. 119 Calvano C.D. 119 Caggiano G. 116

Campion B. 81 Candiracci M. 21, 75 Canela R. 41, 76 Capell T. 41,76,86,89 Caprifico A.E. 19, 101 Carballo M. 21 Cardile V. 105 Cardoso S.M. 38 Caretto S. 15 Castaño A. 21, 75 Chateauvieux S. 44 Cheynier V. 22 Choi H. 77 Christen P. 49 Christou P. 41,76,86,89 Chunthorng-Orn J. 64 Cieśla Ł. 79 Cieślak A. 110 Ciuffreda L. 28 Codina C. 66, 92 Codruta S. 111 Colaianna M. 28 Conforti F. 58, 87 Contino M.C. 19, 101 Corbo F. 71, 116 Cordopatis P. 83 Costa P. 68 Cuadrado C. 115 D page da Silva K.B. 66 Dalessandro G. 19, 101 Damonte G. 59 Das N. 42 de Andrade J.P. 66 De Giglio E. 120 De Tommasi N. 59 Debellis L. 78 Dechayont B. 64 Dehelean C. 111 Dey P. 42 Dicato M. 44, 74 Diederich M. 44, 74 Dimakopoulou A.P. 81 Dimitrova A.M. 68, 80 Domingues M.R.M. 38 Dominguez-Barragán M. 21 Dong L. 89


Drimtzias E. 81 Duarte C.M.M. 23 Durante M. 15 Duret M. 49 E page Edder P. 49 Efthimiopoulos S. 25 Engelsen S-B. 32 F page Falcão S.I. 38, 61 Falé P.L. 85, 104 Fanizzi F.P. 109 Farre G. 41, 86 Feflea S. 111 Ferlemi A.V. 63 Ferlemi A-B. 83 Ferreira C.F.R.I. 57

Ferri M. 37, 93 Filipe M.A. 85 Fiume S. 78 Fontana S. 24, 71 Franchini C. 71, 116 Fraternale D. 59 Freire C. 61 G page Gaascht F. 74 Gaffney M.T. 13

Gaigneaux A. 44 Ganeva A.Y. 80 Garbarino J. 105 García-Antras D. 21 Gelbmann W. 17 Georgakopoulos C. 83 Geu-Flores F. 40 Giacomelli E. 59, 67 Gianotti A. 93 Gođevac D. 69 Gomes D. 73 Gonçalves S. 73 Gordon V. 12 Granell A. 29 Grangeia C. 57 Grassi S. 19, 101

Graziano A. 105 Gruz J. 30 H page Habtemariam S. 71 Hadsbjerg J. 46 Hajslova.J. 27, 99, 113 Halkier B.A. 40 Hamed A.I. 97 Hansakul P. 103 Herrera M.D. 21, 75 Hoefer R. 89 Hurajova A. 27, 113 Hsu A. 117 Huttunen S. 31 I page

Iatrou G. 58 Ignazzi A. 78 Issinger O-G. 46 Itharat A. 62,64,65,72

84,94,96,100, 103,107,108, 114

J page Jaengsuwan N. 88 Jain M. 74 Jędrejek D. 79 Jeon J-S 102 Jeong S. 77 Jirakiattikul Y. 84 Jirillo E. 24 Jung Y-J. 102 Justo M.L. 75 K page Kang M-J. 77 Kim J. 77 Kim J-I. 77 Kim M. 77 Kim S.M. 102 Kirsch G. 74 Kisiel W. 34 Kittisrisopit S. 114


Klem A.H. 112 Klimis-Zacas D. 25 Knauder E. 20 Koivuniemi H. 31 Kowalska I. 79

Krickovic M. 106 Krstonosic V. 106 Kujawski R. 98 Kumar M. 91 L page La Forgia F. 71 Laghezza A. 71 Lakic N. 106 Lamari F.N. 63, 83 Lanzotti V. 45 Larsen J.M. 112 Lentini G. 71 Lenucci M.S. 14, 19, 101 Leone A. 39 Lerdvuthisopon N. 103 Lioce G. 119 Longo C. 39 Lorusso D. 78 Lovece A. 71 Ly L. 102 M page Macchia F. 109 Magrone T. 24 Mahalel U.A. 97 Maiti M.K. 42 Makri O. 83 Maldini M. 97 Manczak T. 112 Margarity M. 25, 63, 83 Mari A. 50 Marrelli M. 58, 87 Martin-Diana A.B. 13

Martins A. 57 Maselli M.A. 78 Masullo M. 90 Mathon C. 49 Matias A.A. 23 Medina V. 76 Menichini F. 58, 87 Michalska K. 34

Miele M. 47 Mikkelsen M. 40 Mikołajczak P.L. 98 Milosavljević S. 69 Mira L. 85 Miralpeix B. 89 Mita G. 15 Mitra A. 91 Molthoff J. 29 Monschein M. 33, 95 Montefusco A. 19, 101 Montoro P. 50, 97 Morgese M.G. 28 Mortensen U.H. 40 Mosullo M. 79 Motoc A. 111

Mrozikiewicz P.M. 98

Muench G. 12 Mumot A.M. 97 Muzquiz M. 14, 115 N page Napolitano A. 90 Niefind K. 46 Nöske K. 46 Novotna H. 27, 99 Nunes S. 23 O page

Oleszek W. 79, 97, 110 Ottaggio L. 47 Ożarowski M. 98 P page Pączkowski C. 31 Pakakrong Thondeeying P 62, 64 Papadia P. 109 Pannuzzo G. 105 Papandreou M.A. 25, 83 Parrado J. 21, 75 Parsons P. 12 Pascale R. 82

Pecio Ł. 110 Pedrosa M.M. 115 Peev C. 111 Pereira O.R. 38


Peres A.M. 38 Pezzolla F. 78 Phuaklee P. 72 Piacente S. 50, 79, 90, 97 Piarulli M. 116 Piatti G. 62 Pintus B. 81 Piro G. 19, 101 Pizza C. 90 Poudyal H. 16, 43 Prajuabjinda O. 94 Pruzzo C. 67 R page Rasmussen T.D. 46 Rattarom R. 100,107 Reddell P. 12 Reilly K. 13 Rescio L. 15 Revilla E. 21 Ric de Vos R. 29 Ricci D. 59 Rieder J. 33 Rithichai P. 96 Rivera S. 41, 76 Rodrigues A.M. 104 Rodríguez-Morgado B. 21 Rodríguez-Rodríguez R. 75 Romano A. 47, 73 Romero I. 29 Romussi G. 59, 67 Rosato A. 116 Roselli M. 71 Ruangnoo S. 61, 108 Russell P.J. 48 Russo A. 105 S page

Sachet L.G. 64 Sacristán M. 115 Sae-wong C. 70 Sakpakdeejaroen I. 100,107 Salis A. 59 Salomonsen B. 40 Samira Smiti S. 90 Sanahuja G. 86 Sandmann G. 41, 76

SantaMaría C. 21 Savorani F. 32 Schiavone B.P.I. 116

Schito AM. 67 Schulzova V. 27,99,113 Schumacher M. 74 Sellami H.K. 90 Serra A.T. 23 Serralheiro M.L. 85, 104 Simonsen H.T. 112 Sinha S. 42 Sosnovcova I. 99, 113 Spichal L. 30 Srdanov J. 106 Srisawat U. 103 Stanković M. 69 Statti G.A. 58, 87 Stochmal A. 79, 97, 110 Szajwaj B. 110 Szakiel A. 31 Szulc M. 98 Szumacher-Strabel M. 110 T page Tan Benny K.H. 117 Tansakul P. 70 Tassoni A. 37, 93 Tava A. 81 Teiten M-H. 44, 74 Tešević V. 69 Tewtrakul S. 70 Thiem B. 98 Tikunov Y. 29 Tiulea C. 111

Torras-Claveria L. 92 Toteva V.K. 68, 80 Trabace L. 28 Trapani G. 120 Trisolini P. 78 Troncoso N. 105 Tsachaki M. 25 Tschiggerl C. 35 Tucci P. 28 U page Um B-H. 102 Ursino A. 81


Uttama S. 107 V page Vaclavik L. 27 Vajs V. 69 Valyova S.M. 80 van de Geest H. 29 van Heusden S. 29

Varela A. 115 Verotta L. 116 Vester J.K. 40 Viladomat F. 92 Vilas-Boas M. 61 W page Weitzel C. 112 Werck D. 89

Williams C. 12 Willkomm S. 46 Wolf I. 33 Wolfender J-L. 26 Y page Yong Edwin W.W. 117 Yordanova P.Z. 68, 80 Z page Zambonin C.G. 118, 119 Zappa A. 47 Zdunić G. 69 Zefferino R. 39 Zhu C. 41, 76, 86 Zotti M. 28 Zuanazzi J.A.S. 66


List of participants


Country Last-name First-name Email AUSTRALIA BROWN Lindsay [email protected] GORDON Victoria [email protected]

POUDYAL Hemant [email protected] REDDELL Paul [email protected]

AUSTRIA BUCAR Franz [email protected] MONCHEIN Marlene [email protected]

TSCHIGGERL Christine [email protected]; BULGARIA DIMITROVA Milena [email protected] GANEVA Yordanka A. [email protected] CZECH REPUBLIC GRUZ Jiri [email protected] NOVOTNA Hana [email protected]

SCHULZOVA Vera [email protected] SOSNOVCOVA Ivana [email protected] SPICHAL Lukas [email protected]

DENMARK BOLDYRETT Brigitte [email protected] ENGELSEN SØren B. [email protected]

HALKIER Barbara A. [email protected] ISSINGER Olaf-Georg [email protected] WEITZEL Corinna [email protected]

ENGLAND RUSSELL Paul J. [email protected] FINLAND AURA Anna-Marja [email protected] FRANCE CHEYNIER Veronique [email protected]

GERMANY BAUER Anne Katrin [email protected]; [email protected]

GREECE FERLEMI Anastasia [email protected] LAMARI Fotini N. [email protected]

PAPANDREOU Magdalini A. [email protected] INDIA MAITI Mrinal K. [email protected] MITRA Adinpunya [email protected] IRELAND ALQUADH Jafar [email protected] ITALY APPENDINO Giovanni [email protected] ARESTA Antonella [email protected]

ARGENTIERI Maria Pia [email protected] AVATO Pinarosa [email protected] BISIO Angela [email protected] CALVANO Cosima D. [email protected] CONFORTI Filomena [email protected] CORBO Filomena [email protected] DALESSANDRO Giuseppe [email protected] DURANTE Miriana [email protected] FERRI Maura [email protected]


GELBMANN Wolfgang [email protected] GIACOMELLI Emanuela [email protected] JIRILLO Emilio [email protected] LANZOTTI Virginia [email protected] LENUCCI Marcello S. [email protected] LEONE Antonella [email protected] LUCCHETTI Vito [email protected] MARELLI Mariangela [email protected] MARI Angela [email protected] MIELE Mariangela [email protected] MONTORO Paola [email protected] NAPOLITANO Assunta [email protected] PIACENTE Sonia [email protected] PISTELLI Luisa [email protected] ROSELLI Mariangela [email protected] RUSSO Alessandra [email protected] SCHIAVONE Brigida [email protected] SOSSAI Dimitri [email protected] TASSONI Annalisa [email protected] TAVA Aldo [email protected] TOMÈ Franca [email protected] TRISOLINI Pietro [email protected] ZOTTI Margherita [email protected]

LUXEMBOURG GAASCHT Francois [email protected] TEITEN Marie-Hèlène [email protected] NIGERIA AHMADU Augustine [email protected] NORWAY BRUSTAD Irena [email protected] POLAND KISIEL Wanda [email protected] KOWALSKA Iwona [email protected]

OLESZEK Wieslaw [email protected] STOCHMAL Anna [email protected] SZAJWAJ Barbara [email protected] SZAKIEL Anna [email protected] THIEM Barbara [email protected]

PORTUGAL BARROS Lilian [email protected] CARDOSO Susana [email protected]

DUARTE Catarina [email protected] FALCAO Soraia I. [email protected] FALÈ Pedro Luis [email protected] GONCALVES Sandra [email protected] MATIAS Ana [email protected]

ROMANIA TIULEA Corina [email protected] SERBIA GODEVAC Dejan [email protected] SRDANOV Jelica [email protected] SINGAPORE TAN BENNY Kwong Huat [email protected] SOUTH KOREA KIM Jung-In [email protected] KIM Sang Min [email protected]


SPAIN BAI Chao [email protected] CANDIRACCI Manila [email protected]

CASTANO NAVARRO

Angelica [email protected]

de ANDRADE Jean Paulo [email protected] FARRÈ Gemma [email protected] HERRERA Gonzalez

M.D. [email protected]

MIRALPEIX Bruna [email protected] MUZQUIZ Mercedes [email protected] PARRADO Rubio Juan [email protected] PEDROSA Mercedes M. mmartí[email protected] RODRIGUEZ MORGADO Bruno [email protected]

SANAHUJA Georgina [email protected] SERRALHEIRO Maria Luisa [email protected] TORRAS-CLAVEIRA

Laura [email protected]

SWITZERLAND CHRISTEN Philippe [email protected] MATHON Caroline [email protected]

WOLFENDER Jean-Luc [email protected] THAILAND ITHARAT Arunporn [email protected] CHUNTHORNG-

ORN Jitpisute [email protected]

DECHAYONT Bhanuz [email protected] HANSAKUL Pinturson [email protected] JAENGSUWAN Nantana [email protected] JIRAKIATTIKUL Yaowapha [email protected] KITTISRISOPIT Suchada [email protected] PHUAKLEE Pathompong [email protected] PRAJUABJINDA Onmanee [email protected] RATTAROM Ruchilak [email protected] RITHICAI Panumart [email protected] SAE-WONG Chutha [email protected] SRISOPA Ruangnoo [email protected] UTTAMA Sakuntala [email protected]

THE NETHERLANDS BOVY Arnaud [email protected]


General Information Information and registration desk The information and registration desk will be available at the Conference Venue. Participants can pick up their personal conference materials at the registration desk, which will be open as follows:

Tuesday-27 September 2011, 12.00-18.00 Wednesday-28 September 2011, 8.45-18.00 Thursday-29 September 2011, 8.45-18.00

Friday-30 September 2011, 8.45-12.00 Language The conference language is English. There will be no simultaneous interpretation. Name badges Badges will be issued to all participants and accompanying persons and allow access to all scientific sessions. Projection Please contact the local organizers to deliver your Powerpoint projections. Posters There will be two poster sessions. Posters with odd numbers (Poster Session 1) will be on display from Tuesday-27 September to Wednesday-28 September. Posters with even numbers (Poster session 2) will be on display from Thursday-29 September to Friday-30 September. Presenting Authors with odd poster numbers are asked to be present to their posters during the Poster session 1 on Wednesday, 18.30-20.00. Presenting Authors with even poster numbers are asked to be present to their posters during the Poster session 2 on Thursday, 18.30-20.00. Refreshments and meals Coffee, tea and other refreshments are included in the registration fee for all registered participants. Lunches and dinners are included in the hotel accomodation fee for participants staying at Grand Hotel Riva del Sole. Registered participants who are not staying at Grand Hotel Riva del Sole and wish to have lunch/dinner at the Congress may buy tickets at the Registration desk. Social Programme Symposium Dinner will take place at Grand Hotel Riva del Sole on Thursday-29 September, 20:30 pm. Gala dinner is included in the registration fee for all registered participants. Please confirm your participation at the Registration Desk. Tuesday-27 September, 9:00 p.m.: “Italian Dragees”: After dinner by the “Confetteria & Museo Mario Mucci”. Wednesday-28 September, 9.00 p.m.: Visit to the medieval town of Bitonto


A post Symposium guided sightseeing tour to the UNESCO World Heritage town of Alberobello will take place on Friday-30 September, 14:00 pm. A buffet with wine tasting will be offered free of charge to all registered participants. Certificate of attendance/Payment receipt All registered participants will receive the Certificate of Attendance and the receipt for the payment of the registration fee together with the conference materials. Liability The Congress organizers cannot be held responsible for any loss, theft, damage or injury to any person or property during the Congress, whatever the cause may be. The liability of persons and enterprises providing means of transportation or other services, remains unaffected. Each Congress participant and accompanying person takes part in all the congress events at his/her own risk.

Regione Puglia

Ordine dei Farmacisti della Provincia di Bari

SOCIETA’ ITALIANA DI FITOCHIMICA

Ordine dei Farmacisti della Provincia di Brindisi

Under the High Patronage and with the financial support of:

Federazione Ordini Farmacisti Italiani

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