university centre obergurgl/tyrol, austria 21 – 25 july...

Trends in natural products research: a young scientists meeting of PSE and ÖPhG

University Centre Obergurgl/Tyrol, Austria

21 – 25 July 2013

FINAL PROGRAMME &

BOOK OF ABSTRACTS

ISBN-13-978-0-9565472-3-1

Contents of the printed abstract book and the online edition are copyright of the Phytochemical Society of Europe. You are free to quote passages according to scholarly fair-use, but required cite as your source:

RRJ Arroo (ed.) (2013) Trends in Natural Products Research Abstracts of the Phytochemical Societey of Europe Phytochemical Society of Europe, Leicester ISBN-13-978-0-9565472-3-1

Cover design and artwork by Michael Noisternig and Elisabeth Gstrein ISBN-13-978-0-9565472-3-1 (URL: http://www.phytochemicalsociety.org/leicester/index.html) Copyright © Phytochemical Society of Europe, 2013 PUBLISHER: Phytochemical Society of Europe Leicester School of Pharmacy De Montfort University The Gateway Leicester LE1 9BH UNITED KINGDOM

Chairmen

Prof. Dr. H. Stuppner, Innsbruck

Prof. Dr. Sabine Glasl-Tazreiter, Vienna

Organizing Committee

Prof. Dr. S. Glasl-Tazreiter, Vienna

Prof. Dr. H. Stuppner, Innsbruck

Prof. Dr. F. Bucar, Graz

Prof. S. Gibbons, London

Prof. W. Oleszek, Pulawy

Ing. E. Gstrein, Innsbruck

Ass.-Prof. Dr. S. Sturm, Innsbruck

Scientific Committee

Prof. P. Avato, Bari

Prof. Dr. R. Bauer, Graz

Prof. L. Bohlin, Uppsala

Prof. Dr. V. Dirsch, Vienna

Prof. Dr. G. Ecker, Vienna

Dr. E. Gille, Piatra-Neamt

Prof. V. Lanzotti, Naples

Prof. F. Macías, Cadiz

Prof. Dr. G. Reznicek, Vienna

Prof. Dr. J.M. Rollinger, Innsbruck

Prof. S. Sarker, Wolverhampton

Dr. A. Stafford, Sheffield

Prof. D. Tasdemir, Galway

Congress Secretariate

Ing. E. Gstrein, Innsbruck

REGISTRATION:

Sunday, July 21 2013, 12:30 to 15:30


I

Welcome

Dear Participants

As representatives of the Austrian Pharmaceutical Society (OPhG) it is our pleasure to welcome you to the Young Scientists Meeting at the University Center of Obergurgl. We are very pleased that so many of you did follow our invitation to join the symposium. This conference is the second joint meeting between the two societies, Phytochemical Society of Europe (PSE) and OPhG. The big success of the first conference in April 2009 led to the decision to come back to the University Center of Obergurgl, which provides the perfect infrastructure for such a meeting. In this surrounding, the participants and lecturers have the possibility for intensive scientific exchange and discussion, not only during the lectures, but also afterwards at lunch or dinner in a more familiar atmosphere.

The scientific program comprises 8 plenary lectures, 2 key lectures, 38 short lectures and 33 posters. In addition to the lectures, we scheduled two time slots for workshops, which offer the possibility to participate actively by e. g. working on the computer or interpreting spectra.

In order not to forget to enjoy the magnificent surrounding of the Tyrolean Alps, we planned a one day hike, provided that the weather will be fine. We are very pleased that so many of you decided to join this excursion, which will lead us through the Rotmoostal up to 2720 m altitude.

We are grateful to all people who were involved in organisation and preparation of this congress. We are also grateful to the University of Innsbruck for providing the meeting centre, and the sponsors for their financial support. We hope that the program of our symposium meets your expectation and are looking forward to a prosperous meeting with many fruitful discussions.

Sabine Glasl-Tazreiter

Chairman, General Secretary of the Austrian Pharmaceutical Society PSE Representative of Middle Europe

Hermann Stuppner

Chairman, President of the Austrian Pharmaceutical Society

II

Welcome

Dear Colleagues

On behalf of the Phytochemical Society of Europe (PSE) it is a pleasure and an honour to welcome you to the “Trends in Natural Product Research” meeting held here in beautiful Obergurgl. This Young Scientists PSE meeting builds on the enormous success of the previous Obergurgl meeting organized in 2009 by Professor Stuppner (University of Innsbruck) and Professor Bucar (University of Graz).

The current meeting is supported by the Austrian Pharmaceutical Society (ÖPhG) and the PSE and is excellently organized by Professor Stuppner and Professor Glasl-Tazreiter (University of Vienna). We are very fortunate to be supported by the University of Innsbruck through the use of their superb Alpine conference facility, and by the generous support from our sponsors, Shimadzu, Bruker, Thieme Madaus, VWR and TEVA Czech Industries.

The PSE organizes and specializes in annual Young Scientist Meetings and these are often the first opportunity that young researchers have to present their data as either a poster or an oral presentation. The meetings are less formal than other symposia, and offer the chance for young scientists to present their research in a friendly and collegial environment. These meetings also give junior and senior researchers in natural product research opportunities to share ideas, expertise and enthusiasm.

On behalf of the PSE I thank the organizing and scientific committees for arranging a thoroughly engaging program in one of the most beautiful parts of Europe. I wish all participants a stimulating, instructive and a highly enjoyable meeting!

Yours ever

Simon Gibbons

President of the Phytochemical Society of Europe

III

General Informations

Conference Venue: University Centre Obergurgl Gaisbergweg 5, 6456 Obergurgl/Tyrol, Austria Phone: +43 (512) 507 37201 Fax: +43 (512) 507 37400 Email: [email protected] Website: http://www.uibk.ac.at/obergurgl/

Congress Secretariate: Ing. E. Gstrein University of Innsbruck, Institute of Pharmacy, Pharmacognosy/Austria Tel.:++43/512/507-58403 Email: [email protected]

Office hours: Monday, July 22 and Wednesday, July 24: 08:30 to 12:30, 14:00 to 20:00 Thursday, July 25, 08:30 to 12:30

Posters: Posters will be on display in the lecture hall during the whole conference. Please mount the posters until Monday, July 22, 9:00, the necessary material to fix the posters will be provided by the organizers. The Poster numbers are indicated in the list of poster presentations in the final programme. Presenting authors are asked to be present at their posters during the Poster Session on Monday, July 22, 17:40 – 18:30.

Oral presentations: PC, beamer and overhead projector are available. Please contact the information desk to deliver your PowerPoint presentations at latest in the morning the day your presentation is scheduled. The duration of Plenary lectures is 40 min (35 + 5 min discussion) Key lectures 20 min (18 + 2 min discussion) Short lectures 12 min (10 + 2 min discussion).

Congress Dinner: The congress dinner will take place on Wednesday evening, July 24 2013 at 19:00 in the restaurant at the University Centre Obergurgl. Every participant is invited to join the congress dinner without extra charge.

Language: The congress language is English; no simultaneous translation will be provided

Badges: Badges will be issued to all registered participants and enable access to all scientific sessions.

Liability: The Organizers of the Symposium cannot be held responsible for any loss, theft, damage or injury to any person or property during the symposium, 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 tours at his/her own risk.

IV

Preliminary Time Schedule (Overview)

V

Sunday, July 21 2013

University Centre Obergurgl, Gaisbergweg 5, 6456 Obergurgl/Tyrol, Austria Lecture hall

12:30 – 15:30 Registration

15:30 – 15:40 Opening of the symposium by the chairmen

Prof. Dr. H. Stuppner

President of the Austrian Pharmaceutical Society (ÖPhG) Institute of Pharmacy, University of Innsbruck

Prof. Dr. S. Glasl-Tazreiter

General Secretary ÖPhG; PSE Representative of Middle Europe Department of Pharmacognosy, University of Vienna

Chair: S. Glasl-Tazreiter, H. Stuppner

15:40 - 16:20 Plenary Lecture PL1

Rudolf Bauer

Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Universitätsplatz 4, A-8010 Graz, Austria

QUALITY CONTROL OF HERBAL MEDICINES

16:20 – 16:40 Key Lecture KL1

Mark O’Neil-Johnson, G. Eldridge, W. Maas Sequoia Sciences, Inc, 1912 Innerbelt Business Center Dr. Saint Louis, MO USA; Bruker Biospin, Maning Park, Billerica, MA USA

WHO WILL GET YOU THERE QUICKER, FRED FLINTSTONE OR GEORGE JETSON?


Christoph Carlen, J. F. Vouillamoz, X. Simonnet

Agroscope Changins-Wädenswil ACW, Research Centre Conthey; Mediplant, Swiss Research Centre in Medicinal and Aromatic Plants, 1964 Conthey, Switzerland

BREEDING AND CULTIVATION OF MEDICINAL PLANTS FROM THE ALPS AND THE MEDITERRANEAN

17:30 – 18:30 Welcome Reception

18:30 – 20:30 DINNER

VI

Monday, July 22 2013

07:30 – 9:00 BREAKFAST

Chair: J-L Wolfender, M. O´Neil-Johnson


Pascal Richomme

EA 921 SONAS/SFR 4207 QUASAV, Université d’Angers,16 Bd Daviers, 49045 Angers Cedex 01, France

NEW USES FOR OLD NATURAL PRODUCTS

09:40 – 09:52 Short lecture SL1

F. Antognoni, M. Mandrone, C. Iannello, B. Lorenzi, F. Poli Department of Life Quality Sciences, University of Bologna, Corso Augusto 237, Rimini, Italy PHYTOCHEMICAL ANALYSIS AND BIOLOGICAL ACTIVITIES OF LEAF AND CELL CULTURE EXTRACTS OF TEUCRIUM CHAMAEDRYS

09:52 - 10:04 Short lecture SL2

Ch. Haas, S. Schulz, B. Ludwig, K. Muffler, R. Ulber, T. Bley, J. Steingroewer Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Bergstr. 120, 01062 Dresden, Germany SALVIA SP. IN VITRO CULTURES AS SOURCE OF OLEANOLIC AND URSOLIC ACID


A. Marchev, V. Georgiev, I. Ivanov, N. Kostova, V. Bankova, A. Pavlov Laboratory of Applied Biotechnologies Plovdiv, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Science, 139 Ruski Blvd, 4000 Plovdiv, Bulgaria ANTIOXIDANT ACTIVITY AND GC/MS PROFILES OF SALVIA TOMENTOSA MILL. CELL SUSPENSION CULTURE


S. Schulz, C. Haas, T. Bley, J. Steingroewer Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Bergstr. 120, 01062 Dresden, Germany CHARACTERIZATION OF BIOACTIVE COMPOUNDS IN SAGE CELL CULTURES


Y. Dai, J. van Spronsen, G-J. Witkamp, R. Verpoorte, Y. Hae Choi Natural Products Laboratory, Institute of Biology, Leiden University, 2300 RA Leiden, The Netherlands NEW MEDIUM, NEW PROFILE: NATURAL DEEP EUTECTIC SOLVENTS FOR DRUG DEVELOPMENT

10:40 - 11:10 COFFEE BREAK

VII

Chair: S. Sarker, F. Macias


N. Heffernan, T. Smyth, A. Soler-Villa, R. J. Fitzgerald, N.P. Brunton Department of Biosciences, Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15, Ireland COMPARISON OF SUPERCRITICAL FLUID EXTRACTION AND SOLID-LIQUID EXTRACTION FOR THE DETERMINATION OF PIGMENTS AND CAROTENOIDS FROM TWO IRISH ORIGIN MACROALGAE


D. Kaya, F. N. Yalçın, T. Ersöz Department of Pharmacognosy, Faculty of Pharmacy, University of Hacettepe, TR-06100 Sıhhiye Ankara, Turkey PHARMACOGNOSTIC INVESTIGATIONS ON SOME GENTIANA SPECIES


A. Bisio, G. Mele, G. Romussi, N. De Tommasi Department of Pharmacy, University of Genova, Via Brigata Salerno, 16147 Genoa, Italy EXUDATE COMPOSITION OF SALVIA ELEGANS VAHL. (LAMIACEAE)


F. Merck, X. Fernandez Institut de Chimie de Nice, UMR CNRS 7272, Université Nice Sophia Antipolis, 28 avenue Valrose, F-06108 Nice Cedex 2, France MEDITERRANEAN BIODIVERSITY AS A SOURCE OF NEW NATURAL PRESERVATIVES


O. Roza, N. Lovász, I. Zupkó, J. Hohmann, D. Csupor Department of Pharmacognosy, University of Szeged, Eötvös straße 6, H-6720 Szeged, Hungary HOODIA GORDONII: FACTS BEYOND BELIEFS


M. Engström, M. P. Suber, J-P Salminen, A. E. Hagerman Laboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of Turku, Finland COVALENT STABILIZATION OF ELLAGITANNIN-PROTEIN COMPLEXES

12:30 - 14:00 LUNCH, DISCUSSIONS

Chair: R. Bauer, Ch. Carlen

14:00 – 14:20 Key Lecture KL2

Satyajit D. Sarker, L. Nahar, Md. M. Billah, J. A. Shilpi, F. Sabrin, K. Md. D. Islam, Md. E. Islam, Md. N. Miah and N. Basar Department of Pharmacy, School of Applied Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1LY, United Kingdom

STUDIES ON BANGLADESHI MEDICINAL PLANTS: CONNECTING TRADITIONAL KNOWLEDGE TO SCIENCE

VIII

14:20 - 15:50 WORKSHOP 1:

Satyajit D. Sarker

PUBLISHING IN PEER-REVIEWED JOURNALS: AN INSIGHT FROM THE EDITORS


Chair: R. Bauer, Ch. Carlen


N. A. Muhammad, N. Basar, S. Jamil, S. Binti Shahar Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 Skudai, Johor ANTIBACTERIAL ACTIVITY OF GARCINIA PARVIFOLIA MIQ. AND GARCINIA HOMBRONIANA PIERRE


J. N. Eloff, Lita Pauw Phytomedicine Programme, Department of Paraclinical Sciences, University of Pretoria, Private Bag X04, Onderstepoort, South Africa 0110 IS THERE A CORRELATION BETWEEN THE TAXONOMY AND ANTIMICROBIAL ACTIVITY OF PLANT EXTRACTS?


S. Prasch, K. Schabus, B. Gröblacher, F. Bucar Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Universitätsplatz 4, A-8010 Graz, Austria XANTHORRHIZOL AS ANTIMYCOBACTERIAL OF CURCUMA ZANTHORRHIZA AND A CURCUMA CULTIVAR


O. Bíba, L. Cvak, A. Jegorov, M. Strnad, J. Grúz Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University, Olomouc, Czech Republic SEPARATION AND ISOLATION OF UNKNOWN COMPOUNDS FROM SILYBUM MARIANUM


V. M. Kutluay, U. S. Harput, Y. Genc, S. R. Jensen, I.Saracoglu Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, 06100, Sihhiye, Ankara, Turkey BIOLOGICAL AND PHYTOCHEMICAL STUDIES ON THE GENUS DIGITALIS


W. Waratchareeyakul, K. Chantrapromma, S. Chantrapromma, M. K. Langat, D. A. Mulholland Natural Products Research Group, Department of Chemistry, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK TETRANORTRITERPENOIDS FROM THE HEARTWOOD OF XYLOCARPUS RUMPHII

IX

17:40 - 18:30 POSTER SESSIONS with the presenting authors

18:30 - 19:50 DINNER

Chair: P. Richomme

19:50 - 20:30 Plenary lecture PL4

Jean-Luc Wolfender

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

NEW ADVANCES IN NATURAL PRODUCT RESEARCH FOR COMPREHENSIVE METABOLITE PROFILING AND METABOLOMICS

X

Tuesday, July 23 2013


Botanical Excursion/Mountain hike Flexible date, depending on weather

18:30 - 20:30 DINNER

XI

Wednesday, July 24 2013


Chair: A. Backlund, P. Richomme


Gerhard F. Ecker

Department of Medicinal Chemistry, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria

COMPUTATIONAL DRUG DESIGN


L. O. Demirezer, M. Bodur, P. Gurbuz, N. Ozenver, A. Kuruuzum Uz, Z. Guvenalp, O. Ugur Sezerman Hacettepe University Faculty of Pharmacy Department of Pharmacognosy, 06100, Ankara, Turkey COMPARATIVE DOCKING STUDIES OF ROSMARINIC ACID ON XANTHINE OXIDOREDUCTASE AND ACETYLCHOLINESTERASE


M. K. Langat, D. A. Mulholland Natural Products Research Group, Department of Chemistry, University of Surrey, Guildford, Surrey, GU2 7XH, UK DETERMINATION OF ABSOLUTE STEREOSTRUCTURES OF UNUSUAL NATURAL COMPOUNDS BY QUANTUM CHEMICAL ELECTRONIC CIRCULAR DICHROISM CALCULATIONS


D. Schuster, M. Edtbauer, J. M. Kratz, Ch. E. Mair, S. Hering, J. M. Rollinger Institute of Pharmacy/Pharmaceutical Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria FINDING hERG-BLOCKERS AMONG NATURAL PRODUCTS: VIRTUAL SCREENING WORKFLOW AND EXPERIMENTAL VALIDATION OF HITS


V. Temml, D. Winekenstädde, C. Voss, J. M. Rollinger, H. Stuppner, V. Dirsch, D. Schuster Institute of Pharmacy/Pharmaceutical Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria IDENTIFICATION OF NEW LXR-β MODULATORS BY IN SILICO SCREENING AND BIOLOGICAL EVALUATION


T. Kaserer, M. Lazic, V. Temml, S. Schwaiger, H. Stuppner, D. Schuster Institute of Pharmacy / Pharmaceutical Chemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, Innsbruck, Austria

XII

APPLICATION OF IN SILICO PROFILING TOOLS FOR THE PREDICTION AND RATIONALIZATION OF NATURAL PRODUCT BIOLOGICAL ACTIVITIES


Chair: JN. Eloff, E. Gille


C. Botha, S. Uhlig, T. Vrålstad, E. Rolén, C. Miles Department of Paraclinical Sciences, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa INDOLE-DITERPENES AND ERGOT ALKALOIDS IN CLAVICEPS-INFECTED CYNODON DACTYLON AND PASPALUM SPECIES


W. Hidalgo, M. Reichelt, B. Schneider Biosynthesis/NMR group, Max Planck Institute for Chemical Ecology, Beutenberg Campus, Jena, Germany PHENYLPHENALENONE-TYPE COMPOUNDS AND ITS ROLE AS PHYTOALEXINS IN THE PATHOSYSTEM MUSA- M. FIJIENSIS


C. Gény, V. Dumontet, N. Birlirakis, P. Retailleau, K. Awang, F. Guéritte, M. Litaudon Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, Labex LERMIT, CNRS, Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France FISSISTIGMA LATIFOLIUM: A SOURCE OF NEW MODULATORS OF Bcl-xL/BAK AND Mcl-1/Bid INTERACTIONS


Ch. E. Mair, U. Grienke, S. von Grafenstein, J. Kirchmair, K. R. Liedl, M. Schmidtke, J. M. Rollinger Institute of Pharmacy, Department of Pharmacognosy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria BIOPROSPECTING DIVERSE PLANT SPECIES FOR ANTIVIRAL AGENTS AGAINST UPPER RESPIRATORY TRACT INFECTIONS


U. Grienke, M. Richter, H. Braun, J. Kirchmair, S. von Grafenstein, K. R. Liedl, M. Schmidtke, J. M. Rollinger Institute of Pharmacy/Pharmacognosy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria NEW INSIGHTS INTO THE ANTI-INFLUENZA ACTIVITY OF LICORICE CONSTITUENTS


L.–F. Nothias, V. Dumontet, P. Leyssen, J. Paolini, P. Retailleau, F. Guéritte, Jean Costa, M. Litaudon Laboratoire de Chimie de Produits Naturels, UMR CNRS SPE 6134, University of Corsica, 20250, Corte, France ANTIVIRAL DITERPENES FROM CORSICAN EUPHORBIA SPECIES

XIII

12:30 - 14:00 LUNCH, DISCUSSIONS

14:00 - 15:30 WORKSHOP 2:

G.F. Ecker: COMPUTATIONAL DRUG DESIGN

J-L. Wolfender: STRUCTURE ELUCIDATION OF NATURAL COMPOUNDS


Chair: A. Miron, L-M. Pena-Rodrigues


J. H. Ahn, E. S. Kim, C. Lee, S. Kim, S-H. Cho, B. Y. Hwang, Mi K. Lee College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea CHEMICAL CONSTITUENTS OF LOTUS, THE LEAVES OF NELUMBO NUCIFERA AND THEIR ANTI-OBESITY EFFECTS


S. K. Oettl, J. Gerstmeier, K. Wiechmann, J. Bauer, A. G. Atanasov, C. Malainer, E. H. Heiss, B. Waltenberger, D. Remias, J. Boustie, V. M. Dirsch, H. Stuppner, O. Werz, J. M. Rollinger Institute of Pharmacy/Pharmacognosy, Center for Molecular Biosciences Innsbruck, Leopold-Franzens University of Innsbruck, Austria IN VITRO SCREENING OF ALPINE LICHEN SPECIES FOR ANTI-INFLAMMATORY LEAD STRUCTURES


J. Kovač, A. Klančnik, A. Gornik, Z. Wu, S. Piskernik, F. Bucar, Q. Zhang, S. S. Možina Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jaminkarjeva 101, SI-1000 Ljubljana, Slovenia ACTIVITY OF PINOCEMBRIN ON CAMPYLOBACTER JEJUNI


V. Mathieu, F. Lefranc, R. Kiss Laboratoire de Toxicologie, Faculté de Pharmacie, Université Libre de Bruxelles (ULB), Brussels, Belgium THE ANTICANCER PHARMACOLOGICAL AND TOXICOLOGICAL PROFILES OF NARCICLASINE


L. Rárová, S. Zahler, M. Strnad Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Growth Regulators, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic ANTIANGIOGENIC PROPERTIES OF NATURAL BRASSINOSTEROIDS


J. Csábi, A. Martins, A. Simon, G. Tóth, A. Hunyadi Institute of Pharmacognosy, Faculty of Pharmacy, University of Szeged, 6720 Szeged, Hungary ECDYSTEROID DIOXOLANE DERIVATIVES AS NOVEL MDR-MODULATORS

XIV

17:15 - 17:55 Plenary lecture PL6

Francisco A. Macías, N. Chinchilla, M. Macias, J. L. G. Galindo, G. Guerrero-Vásquez, A. Cala, R. M. Varela, J. M. Igartuburu, J. M. G. Molinillo

Grupo de Alelopatía, Departamento de Química Orgánica, Instituto de Biomoléculas (INBIO), Facultad de Ciencias, Universidad de Cádiz, C/ República Saharaui, s/n, 11510-Puerto Real (Cádiz) Spain

ALLELOPATHY IN THE SEARCH FOR BIOACTIVE COMPOUNDS

19:00 CONGRESS DINNER

XV

Thursday, July 25 2013


Chair: M. Strnad, F. Bucar


Anders Backlund

Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, BMC, Box 574, S-751 23 Uppsala, Sweden

PRACTICAL APPLICATIONS OF CHEMGPS-NP IN (SEMI-)NATURAL PRODUCTS RESEARCH


A. Yam-Puc, F. Escalante-Erosa, K. García-Sosa, F. G. Ramírez-Torres, M. J. Chan-Bacab, W. Einsenreich, C. Huber, N. Knispel, G. Godoy-Hernández and L. M. Peña-Rodríguez Laboratorio 2 de Química Orgánica, Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, C.43 No. 130, Col. Chuburná de Hidalgo, Mérida, Yucatán, México BIOSYNTHESIS OF LUPEOL-3-HYDROXY STEARATE IN THE TROPICAL PLANT, PENTALINON ANDRIEUXII. A 13CO2 STUDY


A.-Ch. Warskulat, E. C. Tatsis, B. Schneider Biosynthesis/NMR group, Max Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, D-07745 Jena BIOSYNTHESIS OF NUDICAULINS IN PETALS OF PAPAVER NUDICAULE


T. J. Smyth, M. Tierney, A. Soler-Villa Department of Biosciences, Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15, Ireland UPLC-MS PROFILING OF LOW MOLECULAR WEIGHT PHLOROTANNIN POLYMERS


L. C. Langat, W. Wetschnig, M. K. Langat, D. A. Mulholland Department of Chemistry, FEPS, University of Surrey, Guildford, Surrey, GU2 7XH, UK CHEMICAL CONSTITUENTS OF URGINAVIA ALTISSIMA (HYACINTHACEAE)


XVI


Giovanni Appendino

Dipartimento di Scienze del Farmaco, Via Bovio 6, 28100 Novara, Italy

SENSE AND DRUGABILITY: CHEMOSENSORY RECEPTORS AS DRUG TARGETS

11:40 – 12:00 AWARDS AND GRANTS

Jeffrey Harborne Award (best oral contribution)

TEVA Awards (best posters)

PSE Travel Grants

ÖPhG Travel Grants

12:00 CLOSING

XVII

Posterpresentations

P 01 Areej H.S. Aldhaher, Moses K. Langat, Daniel J. Driscoll, Dulcie A. Mulholland TIRUCALLANE AND CYCLOARTANE TRITERPENOIDS FROM THE STEM BARK OF TOONA SINENSIS ROEM (MELIACEAE)

P 02 Alaa Alqahtani, Moses K. Langat, Wolfgang Wetschnig, Dulcie A. Mulholland A BUFADIENOLIDE GLYCOSIDE AND A HOMOISOFLAVONOID FROM RHODOCODON CAMPANULATUS (ASPARAGACEAE)

P 03 Sylvin Benjamin Ateba, Dieudonné Njamen, Martin Zehl, Katrin Ukowitz, Hanspeter Kaehlig, Liselotte Krenn PHYTOCHEMICAL INVESTIGATION OF ERIOSEMA LAURENTII DE WILD

P 04 Vessela Balabanova, Reneta Gevrenova, Dimitrina Zheleva-Dimitrova HPLC DETERMINATION OF PHENOLIC ACIDS IN ARNICAE FLOS

P 05 Natacha Bonneau, Isabelle Schmitz Afonso, David Touboul, Alain Brunelle, Pierre Champy

METHODS FOR QUANTITATION OF ANNONACIN IN RAT PLASMA AND BRAIN

P 06 Irina Boz, Elvira Gille, Irina Mihalache, Maria-Magdalena Zamfirache, Rodica Efrose INFLUENCE OF ALTITUDE ON THE CHEMICAL COMPOSITION OF ESSENTIAL OILS OF THYMUS BALCANUS BORBÁS

P 07 Claude-Alain Carron, José F. Vouillamoz, Catherine A. Baroffio, Christoph Carlen ACHILLEA COLLINA 'SPAK': OPTIMAL HARVESTING STAGE

P 08 José Cheel, Pavel Hrouzek, Kateřina Voráčová, Petra Kučerová, Aleksandra Kapuscik, Jiří Kopecký ISOLATION AND PURIFICATION OF A MINOR CONSTITUENT WITH APOPTOSIS-INDUCING ACTIVITY FROM SOIL CYANOBACTERIUM NOSTOC SP.

P 09 Derya Civelek, Ipek Suntar Cigdem Kahraman, I.Irem Tatli, Esra Kupeli Akkol, Zeliha S. Akdemir ESTIMATION OF ANTI-INFLAMMATORY AND ANTINOCICEPTIVE ACTIVITIES OF VERBASCUM PYRAMIDATUM BIEB.

P 10 Sutsawat Duangsrisai, Madalena Pinto, Anake Kijjoa

HYDROXYLATED XANTHONES FROM GARCINIA SUCCIFOLIA KRUZ

P 11 Reneta Gevrenova, Maya M. Zaharieva, Laurence Voutquenne-Nazabadioko, Max Henry, Spiro Konstantinov CYTOTOXICITY ASSESSMENT OF TRITERPENE SAPONINS FROM GYPSOPHILA TRICHOTOMA WEND. TOWARD HUMAN LEUKEMIA CELLS IN CULTURE

P 12 Elvira Gille, Dana Bobit, Georgiana Gavril, Irina Boz, Monica Hancianu ECOLOGICAL CULTURES OF MEDICINAL AND AROMATIC PLANTS COMMERCIALIZED IN FOOD SUPPLEMENTS

P 13 Ruxandra Cretu, Elena Ionescu, Gabriela Mitroi, Elena Iacob, Carmen Tebrencu, Catrinel Giurescu

EXTRACTION PROCESSES TO OBTAIN SOME EXTRACTS ENRICHED IN HYPERICIN FROM HYPERICUM PERFORATUM SPECIES FROM NATURAL POPULATIONS

P 14 Çiğdem Kahraman, Didem Öztürk, Melike Ekizoğlu, Zeliha Ş. Akdemir EVALUATION OF ANTIOXIDANT AND ANTIMICROBIAL POTENTIAL OF AERIAL PARTS AND ROOTS OF FERULA CASPICA BIEB.

XVIII

P 15 O. Kenny, T.J. Smyth, C.M. Hewage, N.P. Brunton, P. McLoughlin

USE OF LC-SPE-NMR AND LC-MS TO CHARACTERISE CAFFEOYLQUINIC DERIVATIVES OF INOSITOL FROM DANDELION ROOT (TARAXACUM OFFICINALE)

P 16 Séverine Boisard, Anne-Marie Le Ray, Marie-Christine Aumond, Patricia Planchenault, Séverine Derbré, Isabelle Péruches, Catherine Flurin and Pascal Richomme CHEMICAL COMPOSITION AND ANTIGLYCOXIDANT ACTIVITIES OF FRENCH ORGANIC PROPOLIS EXTRACTS

P 17 Irina Mihalache, Georgiana Gavril, Adrian Spac, Catalina Drutu, Radu Necula, Elvira Gille THE STUDY OF VOLATILE OIL IN CHEMOVARIETIES OF ORIGANUM VULGARE CULTIVATED IN ROMANIA

P 18 Anca Miron, Cristina Lungu Apetrei, Cosmin Teodor Mihai, Pincu Rotinberg, Adriana Trifan, Ana Clara Aprotosoaie IN VITRO EVALUATION OF ANTIOXIDANT AND ANTIPROLIFERATIVE EFFECTS OF PINUS CEMBRA L. EXTRACTS

P 19 Tamar Muzashvili, Milena Masullo, Mariusz Kowalczyk, Ether Kemertelidze, Wiesław Oleszek, Sonia Piacente, Anna Stochmal NEW FLAVONOIDES FROM HELLEBORUS CAUCASICUS A.BRAUN.

P 20 Doina Danila, Camelia P. Stefanache, Radu Necula, Valentin Grigoras, Anca Miron CONTRIBUTION TO THE PHYTOCHEMICAL STUDY OF VACCINIUM VITIS-IDAEA L. WILD POPULATIONS FROM EASTERN ROMANIAN CARPATHIANS

P 21 Alice W. Njue, Peter K. Cheplogoi, Josiah O. Omolo, Moses K. Langat, Dulcie A. Mulholland ERGOSTANE DERIVATIVES FROM TERMITOMYCES MICROCARPUS (LYOPHYLACEAE)

P 22 P.O. Osarumwense, M.O. Edema, O, Usifoh SYNTHESIS OF 2,3-DISUBSTITUTED QUINAZOLIN-4-ONE BY CONDENSATION OF A NUCLEOPHILE WITH 2-METHYL-6,7 DISUBSTITUTED-1, 3-BENZO-OXAZINE-4-ONE

P 23 Andy J. Pérez, Mariusz Kowalczyk, Ana M. Simonet, Francisco A. Macias, Wiesław Oleszek, Anna Stochmal NEW TRITERPENOID GLYCOSIDES FROM THE AERIAL PARTS OF ALSIKE CLOVER (TRIFOLIUM HYBRIDUM L.)

P 24 Chadaporn Prompanya, Sindhchai Keokitichai, Madalena Pinto, Anake Kijjoa BROMOINDOLES FROM IOTROCHOTA BACULIFERA

P 25 Steliana Rodino, Alina Butu, Marian Butu, Petruta Calina Cornea MAJOR COMPOUNDS AND ANTIMICROBIAL ACTIVITY OF CYNARA SCOLYMUS AND ROSMARINUS OFFICINALIS AGAINST BACILLUS SUBTILIS ATCC 6633 STRAIN

P 26 Jan Šimura, Ondřej Novák, Ladislav Nedbal, Miroslav Strnad DETERMINATION OF TRNA-BOUND CYTOKININS IN MICROALGAE AND CYANOBACTERIA USING UHPLC-MS/MS

P 27 Camelia Stefanache, Doina Danila, Radu Necula, Valentin Grigoras, Elvira Gille COMPARATIVE PHYTOCHEMICAL ANALYSIS FOR AGASTACHE RUGOSA KUNTZE EXPERIMENTAL VARIANTS IN CONVENTIONAL CULTURES

P 28 Jana Steigerová, Lucie Rárová, Kateřina Křížová, Jana Oklešťková, Michaela Šváchová, Zdeněk Kolář and Miroslav Strnad MOLECULAR MECHANISMS OF BRASSIONOSTEROID ACTION AND RECEPTOR INTERACTIONS IN HUMAN CARCINOMA CELLS

XIX

P 29 Carmen Tebrencu, Ruxandra Cretu, Gabriela Mitroi, Elena Iacob, Maria Chiriac, Valentin Grigoras PHYTOCHEMICAL SCREENING OF URTICA DIOICA EXTRACTS: PRELIMINARY STUDY FOR THEIR UTILIZATION AS ANTIMICROBIAL AND ANTIFUNGAL AGENTS

P 30 Tran Thi Van Anh, Stefan Schwaiger, Clemens Malainer, Atanas Georgiev Atanasov, Elke H. Heiss, Verena M. Dirsch, Hermann Stuppner NF-ΚB INHIBITORS FROM EURYCOMA LONGIFOLIA

P 31 Marion Wiggermann, Ute Wittstock POLYACETYLENES FROM DAUCUS CAROTA - BIOSYNTHESIS AND FUNCTION

P 32 Merve Yuzbasioglu, Yasin Genc, U. Sebnem Harput, Zuhal Guvenalp, Iclal Saracoğlu, L. Omur Demirezer, Ayse Kuruuzum-Uz CYTOTOXIC EFFECT OF ARNEBIA PURPUREA ROOT EXTRACT AND ITS NAPHTHOQUINONE CONTENT AGAINST L20B CELL LINE

P 33 Reneta Gevrenova, Nikolay Denkov, Dimitrina Zheleva-Dimitrova FLAVONOID PROFILES AND ANTIOXIDANT ACTIVITY OF BUPLEURUM SPECIES

TRENDS IN NATURAL PRODUCT RESEARCH: A YOUNG SCIENTISTS MEETING OF PSE AND ÖPHG

ABSTRACTS OF

LECTURES & POSTERS


July 21 – 25 2013

2

Plenary Lecture PL1

Quality control of herbal medicines

Rudolf Bauer


For drug approval of herbal medicines in Europe, efficacy, safety and quality have to be

demonstrated. Since pharmaceutical quality is the basis of both, efficacy and safety, it needs to be standardized in order to guarantee reproducible effects of all batches. Therefore, documentation of quality is a major condition of all drug regulatory systems.

Within the European Union, detailed instructions for quality assessment are specified in the Guideline on Good Agricultural and Collection Practice (GACP) for starting materials of herbal origin (EMEA/HMPC/246816/2005; 20.2.2006), the Guideline on Quality of Herbal Medicinal Products/Traditional Herbal Medicinal Products (CPMP/ QWP/2819/00 Rev. 1; 30.3.2006), the Guideline on Specifications: Test procedures and Acceptance Criteria for Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal Products / Traditional Herbal Medicinal Products (CPMP/QWP/2820/00 Rev. 1; 30.3.2006), and the Guideline on Stability Testing of Existing Active Substances and Related Finished Products’ (CPMP/QWP/122/02) [1].

The quality standards of herbal drugs are usually defined in pharmacopoeia monographs. Therefore, the European Pharmacopoeia Commission has established three expert groups (13A, 13B and TCM), which are elaborating such monographs on a high scientific level [2]. Identity, purity, and the content of relevant constituents are the major criteria which need to be specified. Tests for contaminations with heavy metals, pesticides, fungicides, microorganisms and mycotoxins are obligatory as well in order to guarantee a safe use [3].

Since adulterations and substitutions are still common practice, identity and purity tests are very important. Besides microscopy, fingerprint analysis by TLC or HPLC is mainly used for this purpose [4]. However, genetic fingerprinting and DNA sequencing may be the most appropriate tool for the future, but also spectroscopic techniques like NIR and NMR have been evaluated as rapid testing methods [5].

In the assay, regularly only one or two relevant marker compounds are determined. In the future, a more holistic approach should be used, and the effect of mixtures has to be respected and studied. A metabolomics based approach may be used and methods like LC-MS and NMR may be the methods of choice to determine the metabolic profile and correlate it with pharmacological activity [6,7,8].

1. Vlietinck A., Pieters L., Apers S. (2009) Planta Med. 75(7): 683-688. 2. Bauer R., Franz G. (2010) Planta Med. 76(17): 2004-2011. 3. Bauer R. Drug Inf. J. (1998) 32: 101-110. 4. Wagner H., Bauer R., Melchart D., Xiao P.-G., Staudinger A. (2011). Chromatographic Fingerprint Analysis

of Herbal Medicines - Thin-layer and High Performance Liquid Chromatography of Chinese Drugs, Springer-Verlag, Wien.

5. Zhao Z, Hu Y, Liang Z, Yuen JP, Jiang Z, Leung KS. (2006) Planta Med. 72(10): 865-874. 6. Bauer R., Guo D., Hylands P., Fan T.-P., Xu Q. (2013) pp 377-385, in: Fei Fang, Tzi Bun Ng (Eds.)

Antitumor Potential and other Emerging Medicinal Properties of Natural Compounds, Springer Science+Business Media, Dordrecht.

7. van der Greef J., van Wietmarschen H., Schroën J., Wang M., Hankemeier T., Xu G. (2010) Planta Med. 76(17): 2036-2047.

8. Tilton R., Paiva A.A., Guan J.Q., Marathe R., Jiang Z., van Eyndhoven W., Bjoraker J., Prusoff Z., Wang H., Liu S.H., Cheng Y.C. (2010) Chin Med. 5:30.

3

Plenary Lecture PL2

Breeding and cultivation of medicinal plants

Christoph Carlenab, José F. Vouillamoza, Xavier Simonnetb

aAgroscope Changins-Wädenswil ACW, Research Centre Conthey, 1964 Conthey, Switzerland bMediplant, Swiss Research Centre in Medicinal and Aromatic Plants, 1964 Conthey, Switzerland

Plants are the source of many important pharmaceuticals. Especially plants rich in

secondary metabolites are of interest. Collection from wild and agricultural production of medical plants still remain the most important supply for plant–derived pharmaceuticals and natural products. However, harvesting from wild, especially for species with a high demand, can cause loss of genetic diversity and habitat destruction due to overharvesting. The agricultural cultivation of medicinal plants is an interesting alternative and offers several advantages: reliable botanical identification, less genetic, phenotypic and phytochemical diversity, availability of well-defined cultivars adapted to the requirements of the stakeholders, better guarantee for appropriate conservation, less extract variability and instability and a steadier source of raw material.

Agronomic research and development play an essential role to improve cultivation of medicinal plants by increasing their quality, profitability and sustainability. In this context, breeding of new cultivars is a key factor allowing to adapt genotypes to the requirements of the stakeholders. Breeding for increased yield of valuable compounds, for elimination of unwanted compounds, for tolerance against abiotic and biotic stresses and for better homogeneity of the cultivars are important issues. Breeding a new cultivar needs 5 to 15 years according the species and the selection criteria. To react more quickly to the requirements of the stakeholders, methods to accelerate the breeding procedures must be taken into account such as the use of morphological, phytochemical and genetic markers at a very early stage in the reproduction cycle, increasing the number of generations per year, as well as rapid and cheap measurement methods of target traits.

To optimize yield and quality potential of the selected cultivars, research on best cultivation practices are also essential to get information on optimal conditions for seed germination, plant growing (planting, fertilization, irrigation), harvest, drying and conservation. These research results are integrated to the guidelines for GAP (Good Agricultural Practice (GAP) for medicinal plants recommending cultivation procedures optimizing the quality of medicinal plants.

The importance of breeding programs and of improving the cultivation procedures to increase the quality, the homogeneity and the security of supply of plants are discussed.

4

Plenary Lecture PL3

New Uses for Old Natural Products

Pascal Richomme EA 921 SONAS/SFR 4207 QUASAV, Université d’Angers,16 Bd Daviers ,49045 Angers Cedex 01, France The chemistry of the Maillard reaction is known as a complex series of reactions involving reducing sugars and proteins (or amino acids) and leading to a multitude of by-products so-called Advanced Glycation End-products (AGEs). Indeed, a non-enzymatic reaction between the carbonyl group of a reducing sugar and the primary amino groups of a protein produces the corresponding Schiff base, which undergoes an Amadori rearrangement. Then, complex reactions of oxidation, dehydrogenation and condensation lead, via intra- and intermolecular crosslinkage of the proteins, to AGEs, some of which exhibiting fluorescent properties (Figure 1). Accumulation of AGEs in many different cell types affect both extracellular and intracellular structure and function. As an example, there are nowadays many evidence about the adverse effects of AGEs on the vasculature of patients suffering from diabetes, so a number of different therapies to inhibit AGEs are under investigation.

Figure 1. Hodge scheme of the Maillard reaction.

However, besides vitamins (e. g. pyridoxamine), most promising anti-AGEs agents today under investigation -such as guanidine derivatives- are of synthetic origin. In fact, a wide variety of synthetic molecules have already been tested but the chemodiversity of natural products, especially when dealing with secondary metabolites of vegetal origin, still needs to be explored more thoroughly. In this aim we have developed an affordable anti-AGEs screening which could be easily and quickly performed with sufficient quality, repeatability and reproducibility [1]. This automated test allows the bioguided fractionation of crude extracts, uncovering new interest for natural products, even for already known ones [2, 3], as well as the development of cosmetic formulations [4]. 1. S. Derbré, J. Gatto, A. Pelleray, L. Coulon , D. Séraphin, P. Richomme (2010) Anal. Bioanal. Chem. 398: 1747

– 1758. 2. L. Ferchichi, S. Derbré, K. Mahmood, K. Touré, D. Guilet, Marc Litaudon, K. Awang, A. Hamid A. Hadi, A. M. Le Ray, P. Richomme (2012) Phytochem.78: 98-106. 3. A. Lavaud, R. Soleti, A.-E. Hay, P. Richomme, D. Guilet, R. Andriantsitohaina (2012) Biochem. Pharmacol. 83: 514-523 4. S. Derbré, S. Morel, P. Richomme, K. Touré (2012) FR/JPN Patent Deposit.

5

Plenary Lecture PL4

New advances in natural product research for comprehensive metabolite profiling and metabolomics

Jean-Luc Wolfendera

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

The comprehensive analysis of a plant metabolome is very complex. The large chemical space occupied by natural products (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 [1]. This is a 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 metabolite 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 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 [2]. 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) [3]. 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 as well as some application for bioactivity-guided isolation studies at the microgram scale. The impact of these technologies in NP research studies and perspective of use of related state-of-the-art methods in terms of evolution or revolution in the field will be discussed.

Example of a high resolution UHPLC-TOF-MS profiling of the crude leaf extract of A. thaliana.

1. Wolfender JL, Rudaz S, Choi Y, Kim HK (2013) Curr. Med. Chem. 20: 1056-1090. 2. Eugster P, Guillarme D, Rudaz S, Veuthey JL, Carrupt PA, Wolfender JL (2011) J. AOAC Int. 94: 51-70. 3. Wolfender JL, Marti G, Queiroz EF (2010) Curr. Org. Chem. 14: 1808-1832.

6

Plenary Lecture PL5

Computational Drug Design

Gerhard F. Ecker

Department of Medicinal Chemistry, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria

Nowadays computational methods are routinely used in the drug discovery and development process. However, within the past decade the way how computational drug design is conducted changed dramatically. The availability of high performance computing on the desktop, the establishment of cloud and grid services, and, last but not least, the gradual move of cheminformatics to open access, pushed the whole discipline forward. However, highest impact might come from the availability of enormous amounts of data through public data bases such as ChEMBL, PubChem, and ChemSpider, as well as from semantic integration of public data sources, such as utilized in the Open PHACTS Discovery Platform [1]. This not only allows conducting pharmacoinformatics on large scale data sets, but also opens unique possibilities for model generation and -validation.

Within this talk selected case studies for validating docking poses by ligand-based structure-activity relationships [2], for creating predictive classification models for transporters by mining public data bases [3], as well as for predicting side effects based on ligand-receptor interaction profiles[4], will be presented.

We acknowledge financial support provided by the Austrian Science Fund (F3502) and

the Innovative Medicines Initiative (Open PHACTS, 115191)

1. Williams AJ, Harland L, Groth P, Pettifer S, Chichester C, Willighagen EL, Evelo CT, Blomberg N, Ecker GF, Goble C, Mons B (2012) Drug Discovery Today 17: 1188-1198.

2. Richter L, de Graaf C, Sieghart W, Varagic Z, Mörzinger M, de Esch IJP, Ecker GF, Ernst M (2012) Nature Chem Biol 8: 455-464.

3. Pinto M, Trauner M, Ecker GF (2012) Mol Inform 31: 547-553. 4. Michl J, Scharinger C, Zauner M, Freissmuth M, Sitte HH, Pezawas L, Ecker GF, submitted

7

Plenary Lecture PL6

Allelopathy in the search for bioactive compounds

Francisco A. Macíasa, Nuria Chinchillaa, Mariola Maciasb, José L. G. Galindoa, Guillermo Guerrero-Vásqueza, Antonio Calaa, Rosa M. Varelaa, José M. Igartuburua, José M. G. Molinilloa

aGrupo de Alelopatía, Departamento de Química Orgánica, Instituto de Biomoléculas (INBIO), Facultad de

Ciencias, Universidad de Cádiz, C/ República Saharaui, s/n, 11510-Puerto Real (Cádiz) Spain; bDepartamento de Biomedicina, Biotecnología y Salud Pública (Inmunología) y Unidad de Investigación del Hospital

Universitario de Puerto Real, Servicio Central de Investigación en Ciencias de la Salud, Edificio Andrés Segovia, C/ Dr. Marañón 3, 11002–Cádiz (Cádiz) Spain.

Plants have their own communication and defence mechanisms and allelochemicals are,

in fact, natural herbicides within others. Allelopathic studies develop new compounds that may lead to obtain growth inhibitors with different target sites than traditional herbicides. Following the ‘economy of resources’ principle one defence metabolite is cheaper in terms of resource investment (energy, NADPH, carbon) if it can defend the plant from more than one organism.

Here, we illustrate this principle with several examples where allelochemicals, in addition to phytotoxic activities, have shown other interesting biological properties. Thus, epoxycurcuphenols from Helianthus annuus, that were identified as precursors of heliannuols, (a family of allelochemicals isolated from sunflower), showed potential use as immunosuppressants. Similarly, a secoguaianolide isolated from Artemisia gorgonum showed high activity on the wheat coleoptile bioassay, as well as on STS phytotoxicity bioassay. Additionally, this compound was tested on human cell culture showing apoptosis induction on ovarian cancer cells.

Benzoxazinoids are well-known allelochemicals for taking part in the defence strategies of Gramineae, Ranunculaceae, and Scrophulariceae plants. Their phytotoxicity have been widely studied and large QSAR studies have been reported. When a collection of these compounds were tested for potential effects in cultured HeLa cells. The cellular effects observed were the appearance of micronucleus as a genotoxic effect as early as 24 h after incubation with the benzoxazinoids. Other examples are quinones.

Naphthotectone has been proposed as one of the responsible of the allelopathic activity of teak. Preliminary assays have shown that this compound presents citotoxic activity, using HeLa cells, as well.

8

Plenary Lecture PL7

Practical applications of ChemGPS-NP in (semi-)natural products research

Anders Backlund Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, BMC, Box 574, S-751 23

Uppsala, Sweden

In this presentation three recent examples of the application of ChemGPS-NP1,2 in natural products research will be presented, and related to previous studies and scientific challenges. ChemGPS-NP is essentially an eight dimensional (8D) map of physic-chemical property space, onto which the compounds under investigation can be plotted after PCA predicted positioning. This is an efficient approach to rapidly compare or screen very large datasets, and can be done with several aims. In two of these cases, the application has primarily been to interpret and enhance semi-synthetic work in lead optimization3 and mode-of-action determination.4 In the third example,5 a more traditional approach of cross-dataset comparisons is employed. Curious attendees are welcome to submit their own data/structures for discussion – please contact the speaker at [email protected] for more details.

1. Larsson, J. Gottfries, J., Muresan, S., Backlund, A. (2007) ChemGPS-NP: Tuned for navigation in biologically relevant chemical space. J. Nat. Prod. 70: 789-794. 2. Backlund, A. (2010) Topical chemical space in relation to biological space. in: Comprehensive Natural Products II Chemistry and Biology, eds. Mander & Lui. Elsevier, Oxford vol. 3: 47-79. 3. Frédérick, R., Bruyère, C., Vancraeynest, C., Reniers, J., Meinguet, C., Backlund, A., Masereel, B., Kiss, R., Wouters, J. (2012) Novel trisubstituted harmine derivatives with original in vitro anticancer activity. J. Med. Chem. 55: 6489-6501. 4. Lee, C.-L., Lin, Y.-T., Chen, G.-Y., Backlund, A., Yang, J.-C., Wu, C.-C., Hwang, T.-L., Chen, S.-L., Chang, F.-R., Wu, Y.-C. (2012) Synthesis and biological evaluation of phenanthrene derivatives as cytotoxic, antiplatelet aggregation and anti-inflammatory agents with pharmacophore modeling in the human breast cancer cell line MCF-7 and ChemGPS-NP prediction as topoisomerase II inhibitors. PLoS ONE 7:e37897. 5. Muigg, P., Rosén, J., Bohlin, L., Backlund, A. (2012) In silico comparison of marine, terrestrial and synthetic compounds using ChemGPS-NP for navigating chemical space. Phytochem. Rev. DOI 10.1007/s11101-012-9256-2

9

Plenary Lecture PL8

Sense and Drugability: Chemosensory Receptors as Drug Targets

Giovanni Appendino

Dipartimento di Scienze del Farmaco, Via Bovio 6, 28100 Novara, Italy

Over the past decades, there has been a growing awareness that food is a major driver of genetic and epigenetic diversity in humans, and that food, far from being neutral from a pharmacological standpoint, actually contains specific health-promoting (but also potentially damaging) compounds that can modulate transcription factors, receptors, and enzymes involved in homeostatic functions (nutraceuticals). Less attention has been given to the sensory properties of these agents and, in particular, to the relationship between their flavour (taste, odour, trigeminality) and the associated systemic, non-gustatory responses [1]. Interest in this area was fuelled by the discovery that metabotropic- (taste and olfactory receptors) and ionotropic (thermo-TRPs) chemosensory receptors are functionally expressed outside the nasal and orofaringeal areas, where they are critically involved in primary physiological processes (respiration, digestion, reproduction) as well as pain and the defence against microbial-, oxidative- and electrophilic stress [2].

Current research in this area will be summarized, highlighting the pharmacological role of chemosensory responses induced by natural products in hot areas of biomedical research like chronic pain, asthma, and weight control. 1. Nilius B, Appendino G (2013) Rev Physiol Biochem Pharmacol (in press). 2. Behrens M, Meyerhof W. (2011) Physiol Behav 105:4-13.

10

Key Lecture KL1

Who Will Get You There Quicker, Fred Flintstone or George Jetson?

Mark O’Neil-Johnsona, Gary Eldridgea, Werner Maasb

aSequoia Sciences, Inc, 1912 Innerbelt Business Center Dr. Saint Louis, MO USA; bBruker Biospin, Maning

Park, Billerica, MA USA

Sequoia Sciences identifies novel chemistry from its library of structurally diverse small molecules isolated from plants. The proprietary design of this library allows for the biological screening of these compounds at optimal HTS concentrations, without non-drug-like interferences. Sequoia built this analytical process such that rapid isolation and structure elucidation of active compounds could be accomplished. Sequoia’s efforts to date have been to create a high-throughput natural products chemistry program.

The fictitious cartoon characters of the 1960’s network television in the United States, “The Flintstones” and “The Jetsons” will be used to illustrate the ability to create a high-throughput natural products chemistry program without the bottleneck of structure elucidation being the rate limiting step. The improvement of NMR probe technology has created a tremendous sensitivity advantage for today’s natural products chemist. From the early days of 5mm NMR tubes which demanded milligrams of material, to the CapNMR Probe where one could start to realize the sensitivity gains of recording full data sets on 10’s of micrograms of material, to the state of the art 1.7mm MicroCryoProbe.

The scientific strategy that Sequoia employs in order to rapidly uncover the chemical diversity contained in plant natural products for its internal cancer program will be outlined. This presentation will expand upon the ground breaking NMR tube transitions to the CapNMR probe and now the TCI 1.7mm MicroCryoProbe. An example that was realized from this NMR probe advantage will be presented from Sequoia’s cancer program. For the structure elucidation process, this advanced capillary scale NMR cryoprobe acquires complete NMR data sets on micrograms (10ugrams) of material. The MicroCryoProbe technology compliments its Sequoia’s current platform technologies for high-throughput natural products research for its drug discovery program.

11

Key Lecture KL2

Studies on Bangladeshi Medicinal Plants: Connecting Traditional Knowledge to Science

Satyajit D. Sarkera, Lutfun Naharb, Md. Morsaline Billahc, Jamil A. Shilpid, Farah Sabrine, K. Md. Didarul Islamc, Md. Emdadul Islamc, Md. Nesawar Miaha and Norazah Basara

aDepartment of Pharmacy, School of Applied Sciences, University of Wolverhampton, Wulfruna Street,

Wolverhampton WV1 1LY, United Kingdom; bLeicester School of Pharmacy, De Montfort University, The Gateway, Leicester LE1 9BH, United Kingdom; cBiotechnology and Genetic Engineering Discipline, Khulna University, Khulna 9208, Bangladesh; dPharmacy Discipline, Khulna University, Khulna 9208, Bangladesh;

eDepartment of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh

Bangladesh, a South-East Asian country from the low-lying Ganges delta with sub-

tropical climate, enjoys a rich diversity of plants, many of which can be classified as medicinal plants. For thousands of years, people of Bangladesh have explored these medicinal plants for the treatment of various ailments, and thus formed an extensive body of traditional knowledge relating to medicinal uses of plants from the Bangladeshi flora. Traditionally, a significant portion of this knowledge has been passed from generations to generations, and is still in circulation among Bangladeshi population. However, there are not many systematic surveys and reliable documentations available to date. As a result, some of this traditional knowledge-base is disappearing fast. Having felt the need for major long-term initiatives for marrying this traditional medicinal knowledge with modern scientific studies to validate the claims for medicinal values of various plants from the Bangladeshi flora, and to establish the basis of bioprospecting, we have been utilising modern scientific techniques and performing systematic phytochemical and bioactivity studies on Bangladeshi medicinal plants for more than two decades. One of such initiatives is our recent programme on bioprospecting of the plants from the Sundarbans [1], the largest mangrove forest in the world. In the course of our on-going studies, several surveys based on interviews with the users and practitioners of plant-based traditional medicine have been performed [1], and relevant bioactivity assays have been carried out to validate claims. At the same time, several bioactive compounds, mainly with antibacterial, antioxidant, antidiabetic and anticancer properties, have been isolated and identified [2-8]. The talk will provide, using specific examples, an overview on the highlights of our continuing efforts to combine traditional knowledge and modern science in the studies of medicinal plants from the Bangladeshi flora. 1. Shilpi JA, Islam ME, Billah M, Islam KMD, Sabrin F, Uddin JS, Nahar L, Sarker SD (2012) Advances in

Pharmacol. Sci. 1-7, DOI:10.1155/2012/576086. 2. Sarker SD, Muniruzzaman S, Khan SI, Chowdhury AKA (1991) Bangladesh J. Botany 20: 179-182. 3. Chowdhury AKA, Farooque A, Sarker SD (1991) Bangladesh Pharma. J. 10: 5-7. 4. Chowdhury AKA, Sarker SD, Khan IS, Islam SN (1990) Bangladesh Pharma. J. 9: 4-8. 5. Alam MA, Subhan N, Chowdhury SA, Awal MA, Mostofa M, Rashid MA, Hasan CM, Nahar L, Sarker S D

(2011) Braz. J. Pharmacog. 21: 154-164. 6. Uddin SJ, Nahar L, Shilpi JA, Shoeb M, Borkowski T, Gibbons S, Middleton M, Byres M, Sarker SD (2007)

Phytotherapy Res. 21: 757-761. 7. Uddin SJ, Shilpi JA, Alam SMS, Alamgir M, Rahman MT, Sarker SD (2005) J. Ethnopharmacology 101:

139-143. 8. Datta BK, Datta SK, Chowdhury MM, Khan TH, Kundu JK, Rashid MA, Nahar L, Sarker SD (2004) Die

Pharmazie 59: 222-225.

*Corresponding author. E-mail: [email protected]

12

Short Lecture SL1

Phytochemical analysis and biological activities of leaf and cell culture extracts of Teucrium chamaedrys

Fabiana Antognonia, Manuela Mandroneb, Carmelina Iannellob, Beatrice Lorenzib, Ferruccio Polib

aDepartment of Life Quality Sciences, University of Bologna, Corso Augusto 237, Rimini, Italy

bDepartment of Pharmacy and Biotechnology, University of Bologna, Via Irnerio, 42, 40126, Bologna, Italy

Teucrium chamaedrys, commonly called germander, is one of the most common and investigated species of the genus Teucrium, and it has been used for centuries in traditional medicine for many purposes [1]. Phytochemical constituents comprise diterpenes (in particular neo-clerodane diterpenoids), monoterpenes and other classes of compounds including saponins, glycosides (iridoids and phenylethanoids), and flavonoids [2]. Phenylethanoid glycosides (PGs) are the main phenolic components in Teucrium species, and several reports have demonstrated the wide range of biological and pharmacological activities of these compounds [3]. Conversely, for the neo-clerodane diterpenoids, a certain hepatotoxicity has been reported [4].

Plant cell cultures represent a valid alternative to whole plants for the production of bioactive secondary metabolites. In a previous work, cell cultures established from leaf explants of Teucrium chamaedrys were demonstrated to maintain the capacity of producing PGs [5], while did not produce neo-clerodane diterpenes. In this work, leaf and cell cultures extracts were prepared and tested for their in vitro antioxidant and anti-tyrosinase activities.

Ethanolic extracts were obtained from leaf and cell cultures. The extractive yield, calculated after three sequential extractions, was higher in cell culture compared to leaf extracts (77% vs 40%, respectively). Total polyphenol content was also significantly higher in suspension cells than in leaves (21.6% and 8.13%, respectively), and teucrioside, the main PG produced by this species, amounts to 50.48% and 10.24% of total polyphenol content, respectively. In vitro antioxidant activity of cultured cells and leaf extracts was assayed with different tests (DPPH, ABTS, β-carotene bleaching test, FRAP-Ferrozine, and 2-deoxyribose assay) and compared to that of pure teucrioside and Trolox, used as positive control. In all tests, cultured cell extracts revealed a higher Total Antioxidant Capacity (TAC, expressed as mmol TR eq/g extract) than leaf extracts. Using an in vitro assay, T. chamaedrys cell extracts were shown to inhibit the enzyme tyrosinase, a polyphenol oxidase involved in several processes, such as melanogenesis, browning processes of plant-derived foods, and insect moulting process, and the kinetic inhibition parameters, analyzed by a Lineweaver-Burk plot, showed an uncompetitive mechanism. Taken together, these results suggest that T. chamaedrys cell cultures, by virtue of their biological activities, can be exploited in various field of application, such as pharmaceutical, cosmetics, agronomic and food.

1. Stankovic MS, Topuzovic M, Solujic S, Mihailovic V (2010) J Med Plant Res 4: 2092-2098. 2. Bedir E, Manyam R, Khan IA (2003) Phytochem 63: 977–983. 3. Korkina LG, Mikhal’chik EV, Suprun MV, Pastore S, Dal Toso R (2007) Cell Mol Biol 53: 78-83. 4. Gori L, Galluzzi P, Mascherini V, Gallo E, Lapi F, Menniti-Ippolito F, Raschetti R, Mugelli A, Vannacci A,

Firenzuoli F (2011) Basic Clin Pharmacol Toxicol 109: 521-526. 5. Antognoni F, Iannello C, Mandrone M, Scognamiglio M, Fiorentino A, Giovannini P, Poli F (2012)

Phytochem 81:50-59.

13

Short Lecture SL2

Salvia sp. in vitro cultures as source of oleanolic and ursolic acid

Christiane Haasa, Sibylle Schulza, Benjamin Ludwigb, Kai Mufflerb, Roland Ulberb, Thomas Bleya, Juliane Steingroewera

aInstitute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Bergstr. 120,

01062 Dresden, Germany; bInstitute of Bioprocess Engineering, Department of Mechanical & Process Engineering, University of Kaiserslautern, Gottlieb-Daimler-Str. 44, 67663 Kaiserslautern, Germany;

Increasing interest is spent to the triterpenes oleanolic acid (OA) and ursolic acid (UA)

and their pharmacological potential as they possess hepatoprotective, anti-cancer properties, anti-inflammatory and antimicrobial activity for example [1, 2, 3]. Among other plant species especially Salvia species are known as source of OA and UA. Plant in vitro cultures can serve as a production system for OA and UA which fulfil requirements, such as a continuous supply of plant material with constant quality. This enables a GMP-compliant production completely independent from environment which strongly influences the product content and the quantitative phytochemical composition of herbs [4].

The aim of this study was to establish cell suspension cultures of Salvia for the production of OA and UA, to scale up and optimise the production in bioreactors, and to investigate the possibility of a subsequent biotransformation of the triterpenes. The biotransformation could provide derivatives of OA and UA which exhibit further pharmacological potential as reported e.g. for ursonic acid [5].

Cell suspension cultures of the sage species S. officinalis, S. virgata and S. fruticosa were established and finally six cultures were screened for their productivity of OA and UA determined using HPLC. Concerning product content and growth behaviour a suspension culture of S. fruticosa was most appropriate and used for further investigation. An optimisation of the medium resulted in a more than 7-fold higher triterpene concentration compared to the previously used medium. The scale-up procedure with plant suspension culture is a critical step and several cultivation systems were tested. Using a bubble column revealed the best biomass and triterpene production compared to the other systems. The subsequent biotransformation of OA and UA with the bacterium Nocardia iowensis mainly led to their methyl esters, which was qualitatively confirmed by HPLC-MS/MS and HPLC-NMR. It is reported that ursolic acid methyl ester possess higher anti-viral activity against HIV-1 reverse transcriptase than ursolic acid [6].

It can be concluded that a suspension culture of S. fruticosa has high potential as production system for the pharmaceutical interesting oleanolic and ursolic acid, offering independence from environmental influences and pollutions. Optimisations of media and the cultivation system led to an enhanced productivity which could be further increased by different strategies like elicitation. The isolation of OA and UA from the biomass was achieved by ethanolic extraction and the purification of the raw extract is the focus of current studies. This work was supported by Deutsche Forschungsgemeinschaft (BL345/10-1, BL345/10-2). 1. Liu J (2005) J Ethnoparmacol 100: 92 – 94. 2. Sultana N, Ata A (2008) J Enzyme Inhib Med Chem 23: 739 – 756 3. Huang C-Y, Lin C-Y, Tsai C-W, Yin M-C (2011) Toxicol in Vitro 25: 1274 – 1280 4. Smetanska I (2008) Adv Biochem Engin/Biotechnol 111: 187 – 228 5. Poehland BL, Carté BK, Francis TA, Hyland LJ, Allaudeen HS, Troupe N (1987) J Nat Prod 50: 706–713 6. Akihisa T, Ogihara J, Kato J, Yasukawa K, Ukiya M, Yamanouchi S, Oishi K (2001) Lipids 36: 507 – 512

14

Short Lecture SL3

Antioxidant activity and GC/MS profiles of Salvia tomentosa Mill. cell suspension culture

Andrey Marcheva, Vasil Georgieva,b, Ivan Ivanovc, Nadezhda Kostovad, Vasya Bankovad, Atanas Pavlova,c

aLaboratory of Applied Biotechnologies Plovdiv, The Stephan Angeloff Institute of Microbiology, Bulgarian

Academy of Science, 139 Ruski Blvd, 4000 Plovdiv, Bulgaria; bCenter for Viticulture and Small Fruit Research, College of Agriculture and Food Sciences, Florida A & M University, 6505 Mahan Drive, Tallahassee, Fl 32317, USA; cUniversity of Food Technologies, Department of Organic Chemistry, 26 Maritza Blvd, 4002 Plovdiv, Bulgaria; dInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of

Sciences, 9 Acad. Georgi Bonchev str, Sofia 1113, Bulgaria

Aim. The aim of this study was to investigate the antioxidant activity of polyphenolic extracts of S. tomentosa Mill. cell suspension culture grown in shake flasks and mechanically agitated bioreactor. GC/MS profile of the suspension and HPLC analysis of the main phenolic and triterpene compounds were performed as well.

Methods. Phenolic compounds and triterpenoids were quantified by Waters HPLC with UV detector using a RP C18 Supelco column. GC/MS profile was established by Agilent gas chromatograph 7890 MSD 5975C inert (EI 70 eV), HP-5 ms column. The total phenolics and antioxidant activities of the extracts were evaluated by using four complementary methods, based on different reaction mechanisms (DPPH, ABTS, FRAP and CUPRAC).

Results. Total phenolics, antioxidant activity and HPLC analysis were performed at different growth stages of the cell suspension in shake flasks. The amount of total phenolics increased with the cultivation time and was correlated with the increasing amount rosmarinic acid (RA). Total phenolics (220.58 mg/L) and RA (10.93 mg/L) had maximal values at 11th day of the cultivation which corresponded with the maximal level of the accumulated dry biomass (ADB=6.61 g/L). The biosynthesis of ursolic (UA) and oleanolic (OA) acids (45.19 mg/L and 15.52 mg/L) had maximum at the same day of cultivation. The polyphenolic extract had the strongest effect on reducing Cu in CUPRAC method (283.12 mM TE/g DW) and the highest radical scavenging activity in DPPH (73.17 mM TE/g DW and radical inhibition of 38.08%). The cultivation in stirred tank bioreactor was characterized with lower ADB=4.68 g/L, which resulted in lower but comparable metabolite concentrations: RA=9.86 mg/L, UA=44.02 mg/L and OA=12.29 mg/L. The antioxidant activity according to CUPARC and DPPH was 156.23 and 30.15 mM TE/g DW. The antioxidant activity was higher in comparison with BHT (7.83 and 195.45 mM TE/g for CUPRAC and DPPH). The presence of the phenolic compounds and triterpenoids were proven by GC/MS analysis. Maslinic and corosolic acids were detected for the first time in in vitro cultures of S. tomentosa Mill.

Conclusions. This is the first report that reveals the powerful antioxidant activity of S. tomentosa Mill. cell suspension culture. The stable growth and metabolite characteristics confirm that this suspension is a promising alternative for production of phenolic acids and triterpenoids as well. To our knowledge this is the first report for the presence of maslinic and corosolic acids in S. tomentosa suspensions.

Acknowledgements: This research was supported by the Bulgarian Science Foundation, Bulgarian Ministry of Education and Science (Project DMU – 02/9, 2009).

15

Short Lecture SL4

Characterization of bioactive compounds in sage cell cultures

S. Schulza, C. Haasa, T. Bleya, J. Steingroewera

aInstitute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Bergstr. 120,

01062 Dresden, Germany

Plant in vitro cultures are a prospective alternative for biochemicals production e. g. oleanolic (OA) and ursolic acid (UA) present in plants and cell cultures of Salvia sp. These triterpenoids are of pharmaceutical importance and known to provide anti-inflammatory, anti-tumour as well as anti-HIV effects [1]. Although a diversity of chromatographic methods is already reported for the determination of these triterpenes, a complete separation of OA and UA by HPLC remains still difficult [2]. Especially in the first part of in vitro culture establishment, in many cases low production rates as well as low availability of biomass are problematic. Thus, the purpose of our research was to develop a suitable analysis protocol for evaluation of triterpenic acid yield in plant raw material and in vitro cultures supporting selection processes. Moreover, detailed studies of valuable bioactive compounds produced by sage cell suspensions are lacking and had to be revealed for an evaluation of the potentialities of these in vitro cultures.

Different strategies enhancing the separation for a sensitive and effective HPLC-UV method were investigated and the method developed was validated for linearity, precision, accuracy, limits of detection and quantification. Further metabolites were determined by GC-MS analysis.

A baseline separation of the isomers OA and UA by HPLC enabled detection limits of below 0.4 µg/ml and quantification limits of about 1.2 µg/ml. Over the tested concentration range a good linearity was observed (R² > 0.9999). The variations of the method were below 6% for intra- and inter-day assays of concentration. Plant material and cell suspension cultures recoveries were between 85-98% for both compounds using eco-friendly ethanol as extraction solvent. Thus, a simple, sensitive and accurate RP-HPLC method has been successfully applied to the determination of OA and UA in different sample types of Salvia sp.

Additionally, metabolite profiling of cell suspension culture extracts by GC-MS has shown the production variability of different plant metabolites and especially the presence of further pharmaceutical compounds as plant phenols and sterols. These studies provide a method suitable for screening plant and plant cell culture productivity of valuable triterpenic acids and highlighted interesting co-products of plant cell cultures. This work has been supported by European Social Funds and the Free State of Saxony, project number 080938406, Deutsche Forschungsgemeinschaft, project ID: BL 345/10-1 and Zentrales Innovationsprogramm Mittelstand des BMWi (ZIM) KF2049810SA2. 1. Muffler K, Leipold D, Scheller M-C, et al., (2011) Process Biochem., 46: 1 – 15. 2. Martelanc M, Vovk I, Simonovska B (2009) J. Chromatogr. A., 1216: 6662 – 6670.

16

Short Lecture SL5

New medium, new profile: natural deep eutectic solvents for drug development

Yuntao Daia, Jaap van Spronsenb, Geert-Jan Witkampb, Robert Verpoortea, Young Hae Choia*

aNatural Products Laboratory, Institute of Biology, Leiden University, 2300 RA Leiden, The Netherlands;

bDepartment of Biotechnology, Delft University of Technology, Delft, The Netherlands

Developing green solvents with low toxicity, low cost and high solubilisation ability is an important issue for drug development. A new type of green solvents, natural deep eutectic solvents (NADES) have been proposed by our group to extend the range of ionic liquids (ILs) and deep eutectic solvents (DES) and to explore their applications in health related areas [1,2]. Natural deep eutectic solvents are composed of natural primary metabolites from organisms such as sugars, sugar alcohols, organic acids, amino acids, and amines. A series of NADES have been developed and they were characterized by extensive intermolecular interactions [1,2]. They possess excellent solvent properties such as negligible volatility at room temperature, liquid state even below 0 oC, polarity of a broad range. As solvents, in view of the benefits of environment and economy, NADES present many striking advantages including biodegradability, sustainability, low costs, simple preparation, and low toxicity. All those properties make them of interest for applications in health related areas such as pharmaceuticals, food, and cosmetics. They have already been used in enzyme reactions, biotransformations and dissolving DNA [3,4].

Solubility tests with some representative NADES [2] show that NADES can dissolve a wide range of metabolites of low to medium polarity such as taxol and carthamin, and macromolecules such as starch and protein. It is noteworthy that the solubility of poorly water-soluble compounds such as quercetin, ginkgolide B increased in NADES by 18 to 400,000 times if compared in water. Their high solubilizing capacity is related to their supramolecular structure with strong hydrogen bonding capacity.

Experiments with different NADES [5] in extracting phenolic compounds demonstrate that the extractability of both polar and less polar metabolites was greater with NADES than conventional solvents such as water and ethanol. The water content in NADES proved to have the biggest effect on the yield of phenolic compounds, whereas no obvious effects from extraction time and the ratio of material weight to NADES volume were observed. Most major phenolic compounds were recovered from NADES with a ratio between 75%-97%. These simple, low-cost, green solvents can be applied to the dissolution of poorly water-soluble compounds, and extraction and isolation of natural products from biomaterials. This holds promise for further applications of NADES in pharmaceutical, cosmetics, and food industry. 1. Choi, Y.H., Van Spronsen, J., Dai, Y. (2011). Plant Physiol. 156:1701-05. 2. Dai, Y., Spronsen, J. V.,Witkam, G. J., Verpoorte, R., Choi, Y. H. (2013) Anal. Chim. Acta 766: 61-68. 3. Gorke, J. T., Srienc, F., Kazlauskas, R. J. (2008) Chem. Commun. 1235-1237. 4. Zhao, H., Baker, G. A., Holmes, S. (2011) Org. Biomol. Chem. 9: 1908-1916. 5. Dai, Y., Verpoorte, R., Choi, Y. H. (2013) Anal. Chem. Submitted.

17

Short Lecture SL6

Comparison of Supercritical Fluid Extraction and Solid-liquid extraction for the determination of pigments and carotenoids

from two Irish origin macroalgae

N.Heffernana,c, T. Smytha, A. Soler-Villab, R. J. Fitzgeraldc, N.P. Bruntond

aDepartment of Biosciences, Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15, Ireland; bIrish

Seaweed research Group, Ryan Institute (Environmental marine and Energy Research),National University of Ireland, Galway, Ireland; cDepartment of Life Science, University of Limerick, Castletroy, Limerick, Ireland;

dSchool of Agriculture and Food Science,University College Dublin, Belfield, Dublin 4, Ireland.

Marine invertebrates are rich sources of molecules with applications in pharmaceuticals, cosmetics, nutraceuticals and agrochemicals. These molecules include antioxidant carotenoids such as fucoxanthin which could have applications in prevention of free radical mediated conditions such as cardiovascular disease and as food ingredients to prevent oxidative deterioration. To date, little work has been undertaken to determine the carotenoid and pigment content of the brown macroalgae species Laminaria digitata and Fucus serratus. However, although there are only few reports of its use, supercritical-carbon dioxide (SC-CO2) is well known as an efficient method to extract hydrophobic molecules such as carotenoids from natural matrices. In the present study the efficiency of SC-CO2 for the extraction of carotenoids from macroalgae was compared to two other techniques: Supercritical fluid extraction (SFE) with the use of a modifier (10% ethanol) and Solid-liquid extraction (SLE, hexane and acetone at a ratio of 7:3). To make the comparison, an optimisation of the extraction conditions was performed and all extractions were carried out at the best selected conditions for each technique. A high performance liquid chromatography method based on a reverse phase C8 column was developed for the simultaneous separation of chlorophylls and carotenoids. Carotenoids were resolved within 30 min by using a mobile phase consisting of water (A) and methanol (B) at 1mL/min and detection at 450nm. Internal standards fucoxanthin and xanthophyll were used to quantify the carotenoids. Results indicate that SC-CO2 extraction resulted in the highest purity of carotenoids but the lowest yield while SLE showed the highest yield but a lower purity of carotenoids.

18

Short Lecture SL7

Pharmacognostic Investigations on some Gentiana species

Duygu Kayaa, Funda Nuray Yalçına, Tayfun Ersöza

aDepartment of Pharmacognosy, Faculty of Pharmacy, University of Hacettepe, TR-06100 Sıhhiye Ankara, Turkey

The genus Gentiana (Gentianaceae) consists of species with important phytochemical

properties and has been widely used in traditional folk medicine. Iridoids, secoiridoids, C-glycosylflavones and xanthones are considered as the most promising groups of compounds responsible for the pharmacological activities of these species. Moreover, they are also evaluated as the important key compounds in the aspect of chemotaxonomy of the genus Gentiana [1].

The aim of this work, which was planned as a continuation of our previous studies on Gentiana species in our department, was to identify the chemical composition as well as MAO-A enzyme inhibition activities of the above ground parts of Gentiana pyrenaica, G. verna subsp. balcanica and G. verna subsp. pontica growing on the mountains Uludağ (Bursa, 3200 m) and Zigana Mts. (Trabzon, 1795 m) in Turkey.

n-BuOH extracts of the plants were prepared and the MAO-A inhibitory activity of these extracts were tested. By means of chromatographic studies on the H2O extracts, a new secoirioid glucoside (1), three new benzophenone glucosides (2-4) in addition to two known xanthone glucosides (5,6) were isolated from G. verna subsp. pontica; a C-glycosylflavone (7) and two phenolic glucosides (8,9) were isolated from G. pyrenaica. The chemical composition of G. verna subsp. balcanica was analyzed by using an HPLC method with the comparison of standard compounds. All three mentioned Gentiana species and two benzophenone glucosides (2,3) were found to possess MAO-A inhibitory activity.

1 5

6

R1 R2 2 H CH3 3 OH H 4 H H

1. Jensen, S, Schripsema, J (2002). Chemotaxonomy and pharmacology of Gentianaceae. L. Struwe & V. A. Albert (Ed.). in Gentianaceae: Systematics and Natural History, Cambridge University Press.

R 8 H 9 OCH3

7

19

Short Lecture SL8

Exudate composition of Salvia elegans Vahl. (Lamiaceae)

Angela Bisioa, Giacomo Melea, Giovanni Romussia, Nunziatina De Tommasib aDepartment of Pharmacy, University of Genova, Via Brigata Salerno, 16147 Genoa, Italy; bDipartimento per lo

Studio del Territorio e sue Risorse, University of Genova, Corso Europa 26 16132 Genova, Italy; b Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Via

Ponte Don Melillo, 84084 Salerno, Italy

Salvia elegans Vahl is a Mexican species belonging to subgenus Calosphace, section Incarnatae [1], widely used in Mexican traditional medicine for alleviate central nervous system ailments, stomach aches wounds, and post-partum pain [2]. The leaves of S. elegans exude a sweet pineapple scent [2], and its essential oil has been described [3].The hydroalcoholic extract of this species has shown anxiolytic and antidepressant effects [4]; this extract has been fractionated revealing the presence of ursolic, oleanolic and 3β,23-dihydroxyolean-12-en-28-oic acids, together with 3-acetoxy-7-methoxyflavone [5]. As a secoisopimarane diterpenoid previously isolated by us from the exudate material of Salvia cinnabarina M. Martens et Galeotti exerted antispasmodic [6] hypotensive [7], anxiolytic [8] and antimutagenic [9] activity, in this work we investigated the exudate of S. elegans as this species is closely related to S. cinnabarina in order to verify if also this species produced this compound.

The surface exudate, obtained by rinsing the aerial parts of S. elegans with CH2Cl2, was extracted with n-hexane. The "hexane-soluble" fraction was then subjected to repeated column chromatography on Sephadex LH-20 eluting with CHCl3/CH3OH 7:3 and silica gel, eluting with n-hexane/CHCl3/CH3OH from 100:0:0 to 0:0:100, and to HPLC-MS followed by semi-preparative RP-HPLC (Waters, uBondapack RP18, 5 um, 10 × 250 mm, isocratic conditions: eluent CH3OH/H2O 70:30). 3,4-secoisopimara-4(18),7,15-triene-3-oic acid previously isolated by S. cinnabarina was obtained [6] along with trans-communic acid [10], ursolic acid and spathulenol [3], and identified by IR, 1D and 2D-NMR experiments, HR-MS analysis and by comparison with literature data. 1. Epling C (1940) A Revision of Salvia, Subgenus Calosphace. Vol. 110. University of California Press,

Berkley, California. 2. Jenks A A, Kim S-C (2013) J. Ethnopharmacol. 146: 214-224. 3. Mathew J, Toppil JE (2011) Pharm. Biol. 49: 456-463. 4. Herrera-Ruiz M, Garcıa-Beltran Y, Mora S, Dıaz-Veliz G, Viana GSB, Tortoriello J, Ramirez G (2006) J.

Ethnopharmacol.107: 53–58. 5. Marquina S, García Y, Alvarez L, Tortoriello J (2008) Nat. Prod. Commun. 3: 185-188. 6. Romussi G, Ciarallo G, Bisio A, Fontana N, De Simone F, De Tommasi N, Mascolo N, Pinto L (2001)

Planta Med. 67: 153–155. 7. Alfieri A, Maione F, Bisio A, Romussi G, Mascolo N, Cicala C (2007) Phytother. Res. 21, 690–692. 8. Maione F, Bonito MC, Colucci MA, Cozzolino V, Bisio A, Romussi G, Cicala C, Pieretti S, Mascolo N

(2009) Nat. Prod. Commun. 4: 469 – 472. 9. Di Sotto A, Mastrangelo, Romussi G, Bisio A, Mazzanti G (2009) Food Chem. Toxicol. 47: 2092–2096. 10. Barrero AF, Herrador MM, Arteaga P, Arteaga JF, Arteaga AF (2012) Molecules 17: 1448-1467.

20

Short Lecture SL9

Mediterranean biodiversity as a source of new natural preservatives

Florence Mercka, Xavier Fernandeza

aInstitut de Chimie de Nice, UMR CNRS 7272, Université Nice Sophia Antipolis, 28 avenue Valrose, F-06108 Nice Cedex 2, France

Preserving biodiversity is a priority since the concept of hotspot was introduced for the

first time [1]. The Mediterranean area, for example, appears to be one of the 34 terrestrial biodiversity hotspots, and deserves to be explored for its notable vegetal richness [2]. Biodiversity actually plays an important role in several domains like biology, ecology and economics but also in chemistry. Natural products, essentially secondary metabolites, are widely studied and employed in pharmaceutical, cosmetic and food industry. Besides these environmental considerations, our research focuses on one major challenge cosmetic industrials are currently facing, namely cosmetics preservation. Since synthetic preservatives such as parabens are no longer welcome in our society due to their possible effect on cancer cells development [3], a recent trend is to develop natural alternatives to these preservatives. Since natural products such as essential oils, which are already used by industrials, are often responsible for skin allergies, there is currently a need to develop both innovative and safe ingredients.

Our project named NATUBAVAL aims at finding new natural preservatives issued from local biodiversity. A careful examination of the literature combined with ancient recordings of folk medicine and traditional use permitted us to select several plant species. Plants were extracted by solid-liquid extraction and a phytochemical fingerprint of each extract was established by means of HPLC-DAD-ELSD, HPTLC, GC-MS and GC-FID. All extracts were subsequently investigated for antimicrobial and antioxidant activities by microbiological and chemical assays. Among approximately thirty species, ten appeared to be interesting, and we finally focused on two plants that exhibited valuable activities. Bioguided fractionation and isolation of the major constituents followed by their structural elucidation by UPLC-HRMS, 1D- and 2D-NMR led us to identify new bioactive natural compounds. Moreover, a relationship between phytochemical fingerprints and biological activities has been determined by HPTLC-EDA analysis and permitted us to develop a new bioanalytical method for the rapid screening of plant extracts.

This original work allowed us to identify new natural extracts that are currently subjected to development as inventive preservatives for cosmetics. Thanks to our industrial and associative partnerships, we are also evaluating cosmetic formulas containing our extracts. 1. N. Myers, R.A. Mittermeier, C.G. Mittermeier, G.A.B. da Fonseca, J. Kent (2000) Nature 403: 853 – 860. 2. R.A. Mittermeier, P. Robles Gil (2004) Hotspots revisited, Cemex, Mexico. 3. P.D. Darbre, A. Aljarrah, W.R. Miller, N.G. Coldham, M.J. Sauer, G.S. Pope (2004) J. Appl. Toxicol. 24(1):

5 – 13.

21

Short Lecture SL10

Hoodia gordonii: facts beyond beliefs

Orsolya Rozaa, Norbert Lovászb, István Zupkób, Judit Hohmanna, Dezső Csupora

aDepartment of Pharmacognosy, University of Szeged, Eötvös straße 6,H-6720 Szeged, Hungary; bDepartment of Pharmacdynamics and Biopharmacy, University of Szeged, Eötvös straße 6, 6720 Szeged, Hungary

Hoodia gordonii (Masson) Sweet ex Decne is a succulent plant consumed by Bushmen

in South-Africa in order to reduce appetite [1]. Its proposed active compound for weight-loss, P57, an oxypregnane glycoside was shown to cause increased ATP production in the hypothalamus after intracerebroventricular administration [2]. However, the exclusive importance of this compound is debated, as there was no detectable P57 in the brain after oral administration in CD1 female mice according to Madgula et. al. [3]. The only available human clinical study reported no change in body weight or energy expenditure, but administration of a Hoodia gordonii purified extract was associated with significant increases in blood pressure and pulse rate [4].

The aim of the study was to elucidate the underlying mechanism of these symptoms. Our present work has focused on the effect of a Hoodia gordonii product on beta adrenergic receptors in rat uterus to explore the potential role of beta adrenergic receptor agonist activity in the possible cardiovascular adverse effect of the plant. The uterus-relaxant effect of Hoodia spray was investigated on spontaneous and 25 mM KCl-induced contractions alone and in the presence of 10 µM propranolol. The extract was administered cumulatively into the organ bath and the effect of the solvent system was excluded in separate experiments. The product elicited a marked and concentration dependent relaxation against both spontaneous and stimulated contractions. The inhibition of spontaneous contractility was significantly decreased in the presence of propranolol. Based on the propranolol-sensitive component of the uterine action of the product a sympathomimetic effect with substantial β-receptor-mediated contribution is proposed, which may explains the reported cardiovascular side effects in the human clinical trial.

The adulteration among diet and weight management supplements occurs frequently; usually active pharmaceuticals are used as adulterants to intensify the anticipated effect. Thus chromatographic comparison of the analysed product and authentic plant material confirmed that the herbal product was based on Hoodia extract and its bioactivity is linked to the compounds of the plant. We also tested the Hoodia spray for the presence of some frequent adulterants having sympathomimetic property as well. Amphetamine, methamphetamine, sibutramine and ephedrine were not detected in the product. 1. van Heerden FR (2008) Hoodia gordonii: a natural appetite suppressant, J Ethnopharmacol, 119: 434-437 2. MacLean DB, Luo LG (2004) Increased ATP content/production in the hypothalamus may be a signal for

energy-sensing of satiety:studies of the anorectic mechanism of a plant steroidal glycoside, Brain Res,1020: 1-11

3. Madgula VL, Ashfaq MK, Wang YH, Avula B, Khan IA, Walker LA, Khan SI (2010) Bioavailability, pharmacokinetics, and tissue distribution of the oxypregnane steroidal glycoside P57AS3 (P57) from Hoodia gordonii in mouse model, Planta Med 76: 1582-1586

4. Blom WA, Abrahamse SL, Bradford R, Duchateau GS, Theis W, Orsi A, Ward CL, Mela DJ (2011) Effects of 15-d repeated consumption of Hoodia gordonii purified extract on safety, ad libitum energy intake, and body weight in healthy, overweight women: a randomized controlled trial, Am J Clin Nutr, 94: 1171-1181

22

Short Lecture SL11

Covalent stabilization of ellagitannin-protein complexes

Marica Engströma, Matthew P. Suberb, Juha-Pekka Salminena, Ann E. Hagermanb

aLaboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of Turku, Finland, Turku, FI; bDepartment of Chemistry & Biochemistry Miami University, Oxford Ohio USA

The nature of the interaction between tannins and protein can be described as covalent

or non-covalent based on whether the molecules are irreversibly bound to each other or not. In present study the interaction between bovine serum albumin (BSA) and eight different hydrolysable tannins were examined. The goal was to define conditions that promoted formation of covalently stabilized tannin-protein adducts. We postulated that covalent interactions would be favored by more basic pH values, and that the tendency of the tannin to be oxidized under basic conditions would be correlated with tendency to bind covalently to BSA. SDS-PAGE was used to qualitatively assess the formation of covalent complexes, and MALDI-TOF MS was used to evaluate stoichiometry of binding. The protein precipitation capacities (PPC) of the compounds were also examined.

SDS-PAGE experiments showed that higher pH, a higher tannin: protein ratio, and longer incubation times favor the formation of covalently stabilized complexes. The MALDI results suggested that the less easily oxidized compounds behave more similarly at both pH 5 and pH 7 whereas for the more easily oxidized compounds formation of the complexes was very pH-dependent. For example, vescavaloninic acid, which has high oxidative activity, exhibits strong pH dependence (Fig. 1). At pH 5 up to two moles of vescavaloninic acid bound per mole BSA, but all the bound tannin could be removed by ultrafiltration, indicating noncovalent complexes. At pH 7 complexes that were stable to ultrafiltration were formed, comprising one vescavaloninic acid per BSA as a covalent adduct.

Our results indicate all the tannins examined formed covalent complexes. The compounds that are less oxidatively active covalently react with protein at both lower and higher pH but the more oxidatively active tannins only formed covalent bonds at higher pH values. We also noted that the less oxidatively active tannins had higher protein precipitating capacity. We speculate that ability to cross link and precipitate protein is an absolute pre-requisite for formation of covalent bonds, and that oxidative activity is a less critical parameter that dictates pH-dependence.

Figure 1. MALDI-TOF spectra of BSA after reaction with vescavaloninic acid at pH 5 (A) or pH 7 (B), or unreacted BSA (C). Samples were incubated for 90 min at 37°C, ultrafiltered through a 30kD MWCO filter, and mixed with the matrix for analysis. The molecular weight of BSA is 66 kD, vescavaloninic acid is 1.1 kD.

23

Short Lecture SL12

Antibacterial Activity of Garcinia parvifolia Miq. and Garcinia hombroniana Pierre

Noor Amira Muhammada, Norazah Basara, Shajarahtunnur Jamila, Saleha Binti Shaharb

aDepartment of Chemistry, Faculty of Science, bDepartment of Industrial Biotechnology, Faculty of Bioscience and Medical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor

The genus Garcinia, which belongs to family Clusiaceae, is well-known as a source of natural phenolic-containing compounds such as xanthones, biflavonoids, benzophenones and triterpenoids [1,2,3]. G. hombroniana has been known as ‘‘seashore mangosteen’’ or commonly known as ‘‘Beruas’’ or ‘‘Manggis Hutan’’ in Malay. The roots and leaves were traditionally used to treat itchiness and treatment of skin diseases in Malaysia [4]. G. parvifolia which known as “kandis” was also documented medicinally used to treat fever [5]. Phytochemical studies on the stem bark of Garcinia parvifolia and the pericarp of Garcinia hombroniana have been performed. The extracts were purified using vacuum liquid chromatography (VLC) and column chromatography (CC) to obtain pure compounds named as rubraxanthone (1), parvixanthone G (2) cowanin (3), garcihombronane B (4), garcihombronane C (5) and stigmasterol (6). The structures of all compounds were characterized by spectroscopic methods including 1D and 2D NMR, IR and MS. The extracts and pure compounds isolated were tested for their antibacterial activity using the disc diffusion method, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Among all the extracts tested, the acetone extract of the pericarp of G. hombroniana showed the most significant antibacterial activity with both MIC and MBC values of 225 µg/mL. The pure compound, cowanin (3), isolated from the bark of G. parvifolia, exhibited the strongest antibacterial activity compared to other xanthones with a MBC of 225 µg/mL.

1. Niu S, Li Z, Ji F, Liu G, Zhao N, Liu X, Jing Y, Hua H (2012) Xanthones from the stem bark of Garcinia

bracteata with growth inhibitory effects against HL-60 cells Phytochemistry 77 280-286. 2. Rukachaisirikul V, Saelim S, Karnsomchoke P, and Phongpaichit S (2005) Friedolanostanes and Lanostanes

from the Leaves of Garcinia hombroniana J. Nat. Prod 68 1222-1225. 3. Rukachaisirikul V, Saelim S, Karnsomchoke P, and Phongpaichit S (2005) Friedolanostanes and Lanostanes

from the Leaves of Garcinia hombroniana J. Nat. Prod 68 1222-1225. 4. John, K J, Senthil Kumar, R, Suresh, C P, George, J K, Abraham, Z (2008) Occurrence, distribution and

economic potential of seashore mangosteen (Garcinia hombroniana Pierre) in India Genet Resour Crop Evol. 55 183–186.

5. Kardono, L B S, Hanafi, M, Sherley, G, Kosela, S, and Harrison, L J (2006) Bioactive Constituents of Garcinia porrecta and G. parvifolia Grown in Indonesia Pakistan Journal of Biological Sciences 9(3) 483-486.

O

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OH O1

2

34 5

6

7

89

1112

13

14

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18

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4a

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10a

(1)

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OHHO

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(2)

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OOH

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(3)

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OH

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(4)

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OH

(5)

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OH

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(6)

24

Short Lecture SL13

Is there a correlation between the taxonomy and antimicrobial activity of plant extracts?

Jacobus N Eloff, Lita Pauw

Phytomedicine Programme, Department of Paraclinical Sciences, University of Pretoria, Private Bag X04, Onderstepoort, South Africa 0110

There have been thousands of publications on investigating plant extracts for

antimicrobial activity to address the problem of development of resistance against current antibiotics. Many authors have focused on plants used traditionally to treat infections. Traditional healers have mainly water available as an extractant and we have proven many times that aqueous extracts nearly always had very low antimicrobial activity. We have also found that antimicrobial compounds isolated from plants usually have a much lower activity than expected. We decided to screen leaf extracts of trees randomly collected against 8 important bacterial and fungal pathogens. We investigated more than 700 different tree species with a serial dilution microplate method using tetrazolium violet as growth indicator [1]. We only used acetone as extractant [2].

In hardly any case did the acetone leaf extracts have a minimum inhibitory concentration (MIC) higher than 2.5 mg/ml. In many cases extracts had activities of 20 µg/ml. There were large differences in the sensitivity of different pathogens to extracts. Mycobacterium smegmatis a non-pathogenic form of the tuberculosis pathogen was the most sensitive and methicillin resistant Staphylococcus aureus was the most resistant. The controversial aspect of what MIC of a plant extract should be considered as interesting will be discussed based on these results.

There were large differences in activity between different tree families. Only results obtained with the two nosocomial Gram positive bacteria Staphylococcus aureus and Enterococcus faecalis of plant families with at least nine species investigated will be discussed. The four families with the highest activities (average MIC in mg/ml) were Anacardiaceae (0.20), Moraceae (0.24), Combretaceae (0.27) and Celastraceae (0.30). The four families with the lowest activities were Apocynaceae (0.73), Asteraceae (0.80) and Rutaceae (0.83). In some cases the differences were statistically significant. Some promising genera and species to investigate further have also been identified. Several patents have already been registered after investigating potentised extracts of these species in animal and field experiments [3].

It appears that there is a potential value in investigating plant extracts for treating microbial infections.

1. Eloff JN (1998a) Planta Med. 64: 64-711. 2. Eloff JN (1998b) J Ethnopharm. 60: 1-8. 3. Suleiman MM, Duncan JN, Eloff JN, Naidoo V (2012) BMC Veterinary Research 8: 210

25

Short Lecture SL14

Xanthorrhizol as antimycobacterial of Curcuma zanthorrhiza and a Curcuma cultivar

Sandra Prasch, Kerstin Schabus, Barbara Gröblacher, Franz Bucar


The treatment against TB requires a combination therapy over several months

which bears the risk of emerging multi-resistant or even total-drug-resistant strains. Therefore, new antimycobacterials are in need to overcome this threat. There are only a few new compounds in the pipeline for approval and only one compound (bedaquiline®) approved recently for combination therapy of MDR TB [1] with the drawback of not changing the resistance problem on a long term.

Efflux pumps are involved in the development of intrinsic antibiotic resistance due to their ability to transport cytotoxic compounds out securing the survival of the cell. Therefore administered antibiotics are not able to reach the effective concentration level. In Mycobacterium tuberculosis (MTB) there is evidence for several dozens of such efflux pumps, such as MmpL7 which is involved in isoniazid resistance [2], currently the most important antituberculotic drug. Effective efflux pump inhibitors (EPI) could be administered together with the common antibiotics returning their full effectiveness or prolongate the period until resistance develops.

Previous studies lead to identification of a number of EPIs from extracts of species of Zingiberaceae plant family. [3,4] As a consequence, the aim of this study was the screening of further Zingiberaceae plants for their potential antimycobacterial effects as well as their resistance modifying activities. In course of this screening, the hexane extract of the roots of two Curcuma spp., Curcuma zanthorrhiza Roxb. and a Curcuma cultivar (Siam® Curcuma ´Spring´) proved to show low minimal inhibitory concentrations (MIC) of 16 mg/L and modulation factors (MF) of up to 4 (in combination with ethidium bromide, EtBr) in Mycobacterium smegmatis mc² 155. The antimycobacterial effect in both cases could be attributed to the presence of the sesquiterpenoid xanthorrhizol which has not been identified in the cultivar until now.

H3C

HO

Xanthorrhizol 1. FDA (2012) http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm333695.htm 2. Li XZ, Nikaido H (2009) Drugs 69: 1555 – 1623. 3. Groeblacher B, Maier V, Kunert O, Bucar F (2012) J Nat Prod 75: 1393 – 1399. 4. Groeblacher B, Kunert O, Bucar F (2012) Bioorg Med Chem 20: 2701 – 2706.

26

Short Lecture SL15

Separation and isolation of unknown compounds from Silybum marianum

Ondřej Bíbaa,b, Ladislav Cvakc, Alexandr Jegorovc, Miroslav Strnada,b, Jiří Grúza,b

aCentre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University, Olomouc, Czech Republic; bLaboratory of Growth Regulators, Institute of Experimental Botany AS

CR, Olomouc, Czech Republic; cTeva Czech Industries s.r.o., Opava, Czech Republic

Silybum marianum, a plant nowadays spread worldwide, has been used in traditional medicine for treatment of liver diseases since ancient times. Extract from seeds, silymarin, was proven to be hepato-protective with strong antioxidant activity and anticancer potential. In our preliminary screening we found compounds other than currently known flavonolignans silybin A, B, isosilybin A, B, silydianin, silychristin, silychristin B or the flavanonol taxifolin. The aim of our work was therefore to develop an HPLC method for separation of all components and later on to isolate and identify the unknown compounds.

A new method based on high-performance liquid chromatography coupled with UV detection was developed to determine compounds present in silymarin. Their separation was enabled by fine adjustment of both mobile phase pH and elution gradient steps. Two unknown compounds were isolated from silymarin by subsequent application of normal phase column chromatography followed by low pressure reversed phase chromatography. NMR spectroscopy confirmed one of them as currently unknown silybin isomer and the other one as diconiferyl alcohol, constituent already known from other species.

27

Short Lecture SL16

Biological and Phytochemical Studies On The Genus Digitalis

V. Murat Kutluaya, U. Sebnem Harputa, Yasin Genca, Soren R.Jensenb, Iclal Saracoglua

aDepartment of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, 06100, Sihhiye, Ankara, Turkey; bDepartment of Chemistry, The Technical University of Denmark, DK-2800, Lyngby, Denmark In the Flora of Turkey, the genus Digitalis is represented by nine species [1]. Digitalis

species contain biologically active compounds such as cardenolides and phenylethanoid glycosides [2]. Leaves of Digitalis species are still the major source for the isolation of cardenolides used to treat cardiac insufficiency in humans. As the cardenolides are major compounds of Digitalis genus, most of the researches are focused on cardenolides so other active compounds such as phenolics haven’t been investigated for their role in biological activity of Digitalis extracts. In the present study, comparative biological investigations were performed on the aqueous extracts of D. ferruginea L. subsp. schischkinii (Ivan)Werner and D.lamarckii Ivan on the basis of their cytotoxic and antioxidative activities. Cytotoxic activity was determined through the Hep-2 cancer cell line by MTT method and antioxidative activity through the exhibition of scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), superoxide (SO) and 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals [3,4]. It was found that both extracts showed concentration dependent activity. Since the extract of D. ferruginea subsp. schischkinii was determined to be more active, it was applied to the polyamide column chromatography for the fractionation. For the purpose of bioactivity guided isolation, main fractions from the polyamide column of D. ferruginea subsp. schischkinii were also investigated for their cytotoxic and antioxidative activities. A fraction, rich in phenylethanoid glycosides, was found as the most active. Phytochemical studies on the active fractions resulted in the isolation of seven compounds which also showed cytotoxic activities against Hep-2 cell line. Among them forsythiaside, calceorioside A, and methyl caffeoate were identified by using 1D- and 2D-NMR techniques. Structural determination studies on the other bioactive compounds are in progress. This study was supported by The Scientific and Technological Research Council of Turkey TUBITAK Project No:108T518. 1. Davis PH (1978) Flora of Turkey and East Eagean Islands, University Press, Edinburg, Vol 6. 2. Calıs I, et al. (1999) Pharmazie, 12, 1926-1930 3. Saracoglu I, et al. (1995) Biol Pharm Bull, 18, 1396-1400 4. Harput, US, et al. (2011) Rec. Nat. Prod., 5, 100-107

28

Short Lecture SL17

Tetranortriterpenoids from the Heartwood of Xylocarpus rumphii

Watcharee Waratchareeyakula,b, Kan Chantraprommac, Suchada Chantraprommad, Moses K. Langata, Dulcie A. Mulhollanda

aNatural Products Research Group, Department of Chemistry, Faculty of Engineering and Physical Sciences,

University of Surrey, Guildford, Surrey GU2 7XH, UK; bDepartment of Chemistry, Faculty of Science and Technology, Rambhai Barni Rajabhat University, Chantaburi 22000, Thailand; cInstitute of Research and

Development, Walailak University, Nakhon Si Thammarat 80160, Thailand; dDepartment of Chemistry, Faculty of Science, Prince of Songkla University, Songkhla 90110, Thailand

Three novel C-23 epimeric xylorumpholides A-C (1-3) and four novel C-21 epimeric

xylorumpholides D-G (4-7) were isolated from the heartwood of X. rumphii. Compounds 1-7 have a hemiacetal group which opens and closes in solution making it impossible to purify. Acetylation enabled separation of the and forms. Column chromatographic separation of the acetylated fraction of impure 1-7 led to the isolation of one acetylated derivative of 1 (1a), four acetylated derivatives of 2 (2a-d), two acetylated derivatives of 3 (3a and 3b), one acetylated derivative of 5 (5a), two acetylated derivatives of 6 (6a and 6b) and one acetylated derivative of 7 (7a). Three compounds, 1-3 were tested at one concentration, 1 x 10-5 M, against several leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast cancer cell lines. The compounds did not meet activity criteria in the one-dose NCI59 cell test for further testing.

1 : R1 = R2 = 2-methylbutyryl, R3 = OH, R4 = H 1a : R1 = R2 = 2-methylbutyryl, R3 = OAc, R4 = H 2 : R1 = 2-methylbutyryl, R2 = isobutyryl, R3 = OH, R4 = H 2a : R1 = 2-methylbutyryl, R2 = isobutyryl, R3 = -OAc, R4 = H 2b : R1 = 2-methylbutyryl, R2 = isobutyryl, R3 = -OAc, R4 = H 2c : R1 = 2-methylbutyryl, R2 = isobutyryl, R3 = -OAc, R4 = Ac 2d : R1 = 2-methylbutyryl, R2 = isobutyryl, R3 = -OAc, R4 = Ac 3 : R1 = isobutyryl, R2 = 2-methylbutyryl, R3 = OH, R4 = H 3a : R1 = isobutyryl, R2 = 2-methylbutyryl, R3 = -OAc, R4 = H 3b : R1 = isobutyryl, R2 = 2-methylbutyryl, R3 = -OAc, R4 = H

4 : R1 = R2 = 2-methylbutyryl, R3 = OH, R4 = H 5 : R1 = CH3, R2 = isobutyryl, R3 = OH, R4 = H 5a : R1 = CH3, R2 = isobutyryl, R3 = OAc, R4 = H 6 : R1 = R2 = isobutyryl, R3 = OH, R4 = H 6a : R1 = R2 = isobutyryl, R3 = OAc, R4 = H 6b : R1 = R2 = isobutyryl, R3 = OAc, R4 = Ac 7 : R1 = H, R2 = 2-methylbutyryl, R3 = OH, R4 = H 7a : R1 = H, R2 = 2-methylbutyryl, R3 = OAc, R4 = H

Figure 1 Compounds were isolated from the heartwood of X. rumphii and their derivatives

O

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OR1

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29

Short Lecture SL18

Comparative Docking Studies of Rosmarinic Acid on Xanthine Oxidoreductase and Acetylcholinesterase

L. Omur Demirezera, Mine Bodura, Perihan Gurbuza, Nadire Ozenvera, Ayse Kuruuzum Uza, Zuhal Guvenalpb, O. Ugur Sezermanc

aHacettepe University Faculty of Pharmacy Department of Pharmacognosy, 06100, Ankara-Turkey, bAtaturk University Faculty of Pharmacy Department of Pharmacognosy, Erzurum-Turkey, cSabancı University Faculty

of Engineering and Natural Sciences, Istanbul-Turkey In our previous study acetylcholinesterase inhibitory effects of different extracts

prepared from three Salvia species (S. trichoclada, S. fruticosa, S. verticillata) were tested in isolated guinea pig ileum by acetylcholine stimulated contractions together with Ellman method and their free radical scavenging effect were measured by DPPH in vitro showing bioactivities in the tested extracts [1]. In recent times, computational methods including database search algorithms, determining quantitative structure-activity relationships, similarity search methods, computational modeling and docking have been developed and widely applied to pharmacological hypothesis development and testing. These methods have been widely used to investigate potential drug targets [2].

Rosmarinic acid is the main compound of Salvia sp. which has diverse biological effects in the cell through interactions with proteins like receptors, enzymes or transporter proteins. Xanthine oxidoreductase (XOR) catalyses the oxidation of hypoxanthine to xanthine or xanthine to uric acid in the metabolic pathway of purine degradation and generates the superoxide radical. Superoxide dismutase (SOD) catalyze the dismutation of superoxide into oxygen and hydrogen peroxide [3]. Acetylcholinesterase (ACE) is a serine protease that hydrolyzes the neurotransmitter acetylcholine, and acetylcholinesterase inhibitors are used against Alzheimer’s disease. Alzheimer has mitochondrial anomalies affecting cytochrome-c oxidase, and these anomalies may contribute to the abnormal production of free radicals. Oxidative stress has been implicated in the development of several neurodegenerative diseases including Alzheimer [4]. Galantamine, physostigmine and huperzin A are clinically used against Alzheimer’s disease [5,6,7].

In this study, acetylcholinesterase (ACE), xanthin oxidoreductase (XOR) and superoxide dismutase (SOD) were selected as targets and then computationally determined and their relative binding energies to rosmarinic acid, physostigmine, galantamine, huperzin A, gallic acid and ascorbic acid and their docking results were compared. According to the docking results for the focused targets, -ACE, XOR and SOD- , rosmarinic acid showed the lowest binding energy. This result give an impression rosmarinic acid may become novel therapeutic candidate for the treatment of Alzheimer’s disease. 1. Gurbuz P (2007) Salvia trichoclada Benth. üzerinde farmakognozik araştırmalar. 2. Yong UC (2008) In-silico approaches in the study of traditional Chinese herbal medicine. 3. Marcus DL, Thomas C, Rodriguez C, Simberkoff K, Tsai JS, Strafaci JA, Freedman ML (1998) Experimental

Neurology 150(1):40-44. 4. Christen Y (2000) The American Journal of Clinical Nutrition 71(2):621-629. 5. Prvulovic D, Hampel H, Pantel J (2010) Expert Opinion on Drug Metabolizm & Toxicology 6(3):345-354. 6. Stern Y, Sano M, Mayeux R (1987) Annals of Neurology 22(3):306-310. 7. Rafii MS, Walsh S, Little JT, Behan K, Reynolds B, Ward C, Jin S, Thomas R, Aisen PS (2011) Neurology

76(16):1389-1394.

30

Short Lecture SL19

Determination of Absolute Stereostructures of Unusual Natural Compounds by Quantum Chemical Electronic Circular

Dichroism Calculations

Moses K. Langata, Dulcie A. Mulhollanda

aNatural Products Research Group, Department of Chemistry, University of Surrey, Guildford, Surrey, GU2

7XH, UK

Phytochemical investigation of the Madagascan Tachiadenus longiflorus Griseb. (Gentianaceae) and Russian Pinus pumila (Pall.) Regel (Pinaceae) gave unusual natural products, langaside (1) and pumilol (2) respectively whose carbon skeletons have not been reported previously. The absolute configurations of 1 and 2 were determined by comparing their experimental and simulated ECD curves. Conformational searches were done on possible isomers that were consistent to correlations in their NOESY spectra using the MMFF basis set built into Spartan08 software. Conformers which were under 2 kcal/mol were subjected to TDDFT calculations using a B3LYP method at 6-31G (d, f) level built into Gaussian09 software. The resulting ECD curves of the selected conformers were Boltzmann weighted [1] and compared to the experimental ECD curves for 1 and 2.

1. Pescitelli G, Di Bari L, Berova N (2011) Chem. Soc. Rev. 40: 4603-4625

H

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2

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31

Short Lecture SL20

Finding hERG-Blockers among Natural Products: Virtual Screening Workflow and Experimental Validation of Hits

Daniela Schustera, Michael Edtbauera, Jadel M. Kratzb, Christina E. Mairc, Steffen Heringd, Judith M. Rollingerc

aInstitute of Pharmacy / Pharmaceutical Chemistry and Center for Molecular Biosciences Innsbruck (CMBI),

University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria; bPrograma de Pós-Graduação em Farmácia, Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina, Campus

Universitário Trindade, 88040-900, Florianópolis, SC, Brazil; c Institute of Pharmacy / Pharmacognosy and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria ; dDepartment of Pharmacology and Toxicology, University of Vienna, Althanstraße 14, A-1090 Vienna,

Austria.

The human ether-a-go-go-related gene (hERG) channel plays a critical role in cardiac action potential repolarization. Block of this potassium channel can lead to arrhythmia and increased incidence of sudden death [1]. Although several drugs have been removed from the market for this reason, e.g. astemizole, and hERG block testing is an established part of preclinical drug candidate testing, it has just recently been of interest to assess the potential cardiotoxic risks of botanicals [2].

The goal of this study was to design, experimentally validate, and apply a virtual screening workflow to identify novel hERG channel blockers, with a special focus on the investigation of natural products.

A ligand-based pharmacophore model collection was developed and theoretically evaluated against databases of known hERG blockers from the literature and drug-like decoys. The seven most complementary, high-quality models were then used for virtual screening of our Innhouse database and commercially available compound libraries. Fifty chemically diverse compounds, including natural products, were selected from the hitlists for bioactivity testing on Xenopus laevis oocytes, using a voltage clamp technique [3]. Cells were treated with 30 µM solutions of the compounds, and 30% reduction of the peak tail hERG current was established as the cut-off for positive blockade. This campaign identified 20 so far unknown hERG blockers inducing at 30 µM an inhibition between 32 and 79%.

In summary, we have demonstrated that our virtual screening approach was successful in identifying novel hERG blockers. The experimental evaluation of virtual hits identified the most reliable pharmacophore models which represent a valuable predictive tool in the assessment of potentially cardiotoxic natural compounds.

Acknowledgement: This work was supported by a Marie Curie International Research Staff Exchange Scheme Fellowship within the 7th European Community Framework Program (hERGscreen, 295174) and by the Austrian Science Fund (P22395 to SH). DS thanks the University of Innsbruck for her position in the Erika Cremer Habilitation Program. 1. Sanguinetti MC, Tristani-Firouzi M (2006) Nature 440: 463-69. 2. Schramm A, Baburin I, Hering S, Hamburger M (2011) Planta Med. 77: 692-97. 3. Baburin I, Beyl S, Hering S (2006) Pflugers. Arch. 453: 117-23.

32

Short Lecture SL21

Identification of new LXR-β modulators by in silico screening and biological evaluation

Veronika Temmla, Daniel Winekenstäddeb, Constance Vossc, Judith M. Rollingerb, Hermann Stuppnerb, Verena Dirschc, Daniela Schustera

aInstitute of Pharmacy/Pharmaceutical Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria; bInstitute of Pharmacy/Pharmacognosy and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, A-6020 Innsbruck,

Austria; cUniversity of Vienna, Department of Pharmacognosy, Althanstrasse 14, A-1090 Vienna, Austria

In the search for new cholesterol-lowering drugs the liver X receptors (LXRα and LXRβ) have been identified as key regulators of cholesterol metabolism. The LXRs induce cellular cholesterol efflux and up-regulate high-density lipoprotein (HDL) [1]. Many synthetic compounds targeting LXR in vivo induce lipogenesis, hypertriglyceridemia, and hepatic steatosis, believed to be caused by LXRα agonism [2]. Thus, selective LXRβ modulation with small molecules emerges as a desirable goal in drug development.

In a first approach to identify LXR ligands, previously developed 3-D pharmacophore models [3] were used to screen natural product databases. Among several biologically evaluated virtual hits, bernumidine and some structural analogues showed activity and a 2-3-fold higher activation of LXRβ versus LXRα. Additionally a shape based ROCS screening [4] was performed. A β-selective ligand discovered by Molteni et al [5, 22r, Figure] was set as a shape query for the commercial Specs compound database. In this approach, a synthetic triazole compound (S9) was identified as active, but not as LXRβ-selective.

Both natural and synthetic actives were tested in human macrophages and liver cells for

LXR target gene expression, such as ATP-binding cassette transporter (ABCA1/ ABCG1), FAS, and SREBP-1c. All compounds substantially enhanced the ABCA1 protein expression. However, the LXRβ-selective compounds from the cell assay only slightly promoted cholesterol efflux in macrophages. Remarkably, the synthetic hit S9 induced ABCA1 and ABCG1 expression and increased cholesterol efflux to a similar extent as the control agonist GW3965. While the agonist GW3965 significantly induced lipid accumulation in HepG2 cells, S9 did not show these hepatotoxic effects and is therefore an interesting compound for further investigation and in vivo studies. 1. Miao B, Zondlo S, Gibbs S, Cromley D, Hosagrahara V, Kirchgessner T, Billheimer J, Mukherjee R (2004)

Journal of Lipid Research 45: 1410-1418. 2. Viennois E, Mouzat K, Dufour J, Morel L, Lobaccaro J, Baron S (2012) Molecular and cellular

endocrinology, 351: 129–41. 3. von Grafenstein S, Mihaly-Bison J, Wolber G, Bochkov V, Liedl K, Schuster D. (2012) J. Chem. Inf.,

52:1391-1400. 4. OMEGA version 2.2.1; OpenEye Scientific Software, Santa Fe, NM, USA; www.eyesopen.com (2007) 5. Molteni V, Li X, Nabakka J, Liang F, Wityak J, Koder A, Vargas L, Romeo R, Mitro N, Mak P,Seidel M,

Haslam J, Chow D, Tuntland T, Spalding T, Brock A, Bradley M, Castrillo A, Tontonoz P, Saez E (2007), J. Med. Chem. 50:4255-4259.

Acknowledgement: FWF, NFN Austrian Science Fund S10711, S10703, S10704; Erika Cremer habilitation program and Nachwuchsförderung of the University of Innsbruck.

33

Short Lecture SL22

Application of in silico profiling tools for the prediction and rationalization of natural product biological activities

Teresa Kaserera, Milos Lazica, Veronika Temmla, Stefan Schwaigerb, Hermann Stuppnerb, Daniela Schustera

aInstitute of Pharmacy / Pharmaceutical Chemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, Innsbruck, Austria; bInstitute of Pharmacy / Pharmacognosy and Center for

Molecular Biosciences, University of Innsbruck, Innrain 80/82, Innsbruck, Austria Parallel profiling of one compound against multiple targets appears to be an emerging trend in computer-aided drug development and is of special interest for natural drug research. Natural products often represent complex mixtures of diverse compounds, which complicate the identification of the bioactive components. Even if the biological effects are well-documented, the underlying modes of action often remain elusive. The prediction of ligand-target interactions can aid in the identification of targets for compounds of biological origin (so-called target-fishing) and thereby elucidate the mechanisms responsible for the observed activity. Several different in silico profiling tools, many of them publicly accessible, including pharmacophore- and 2D-similarity-based methods, will be listed and an example of natural product profiling will be presented.

34

Short Lecture SL23

Indole-Diterpenes and Ergot Alkaloids in Claviceps-infected Cynodon dactylon and Paspalum species

Christo Bothaa, Silvio Uhligb, Trude Vrålstadb, Elin Rolénb, Christopher Milesb

aDepartment of Paraclinical Sciences, University of Pretoria, Private Bag X04, Onderstepoort 0110, South

Africa; bNational Veterinary Institute, P.O. Box 750 Sentrum, 0106 Oslo, Norway

Paspalum grass, such as P. distichum or P. dilatatum, may be parasitized by the ergot fungus Claviceps paspali which has for several decades been known to synthesize indole-diterpenoid alkaloids. These compounds induce tremors and locomotory disturbances (“staggers”) when cattle ingest the infected seedheads[1]. Cynodon dactylon (Bermuda grass, kweek) is another species of grass that may be parasitized with a similar fungus, namely Claviceps cynodontis [2]. Bermuda grass samples infected with a clavicipitaceaous fungus and ergotized P. dilatatum and P. distichum seedheads from South Africa and Claviceps paspali sclerotia from New Zealand were extracted with acetone, filtered and subjected to HPLC-MS analysis.

The ergotized Bermuda grass contained a complex mixture of indole-diterpenes, of which the tremorgens paspalitrem A and B, as well as paspaline and paspalinine, represented major constituents. The highest concentration (about 150 μg/g) was found for paspalitrem B. The ergot alkaloids ergonovine and ergine (lysergic acid amide) were found to co-occur with the indole-diterpenes at concentrations of about 10 ng/g.

The indole-diterpene profile of the extract from the ergotized Bermuda grass was similar to that of Claviceps paspali sclerotia. However, the C. paspali sclerotia contained in addition the ergot alkaloids agroclavine and elymoclavine.

It was concluded that the tremorgenic syndrome that is sometimes observed with the grazing of Bermuda grass is likely caused by indole-diterpenoid mycotoxins.

1. Kellerman TS, Coetzer JAW, Naudé TW, Botha CJ (2005) Plant Poisoning and Mycotoxicoses of Livestock

in Southern Africa Second Edition, Oxford University Press, Cape Town, South Africa. 2. Langdon RF (1954) New species of Claviceps. Univ. Queensl. Pap., Dep.Bot. 3: 37–40.

35

Short Lecture SL24

Phenylphenalenone-type compounds and its role as phytoalexins in the pathosystem Musa- M. fijiensis

William Hidalgoa, Michael Reicheltb, Bernd Schneidera

aBiosynthesis/NMR group, Max Planck Institute for Chemical Ecology, Beutenberg Campus, Jena, Germany; bBiochemistry Department, Max Planck Institute for Chemical Ecology, Beutenberg Campus, Jena, Germany

Bananas are among the most important crops worldwide since they not only represent a

staple food but also the major economical income for many producing countries. However, the production of commercial cultivars of Musa (banana) belonging to the Cavendish subgroup is enormously affected by insects, nematodes and microbial infections. The Black Sigatoka Disease (BSD), caused by the ascomycete fungus Mycosphaerella fijiensis, is considered the most detrimental disease of this crop [1, 2]. Despite the attempts of rearing genetically modified Musa plants resistant to this pathogen, the use of fungicides still remains the only available method for the control of this disease. Understanding how the pathogen recognizes its host and how the plant responds to the pathogen attack constitute the major challenge in order to address this problem in a more ecological friendly way. According to previous results the plant’s defense mechanism involves the expression of pathogenesis related proteins (PR), morphological modifications and the production of a class of secondary metabolites named phenylphenalenones (PP’s) that due to their antimicrobial properties have been considered as the main phytoalexins occurring in the Musa genus [3, 4]. However, little is known about whether the resistance in Musa against phytopathogens is correlated in some extent with the chemical topology, content and distribution of these metabolites around the infection site. In this study, two varieties of Musa plants: Khai Thong Ruang (resistant) and Williams (susceptible) were used to explore the spatial and temporal resolution of triggered phenylphenalenones during the BSD development. NMR and HPLC analysis were used for the identification and quantification of the main phenylphenalenone-type compounds and related natural products. The results show that the resistant variety is able to recognize the pathogen at early stage of the interaction and produces a large pool of PP’s, unlike the susceptible variety which displays a late response and a weak chemical profile. Bioassays to determine the antifungal properties of each compound are still in progress. 1. Stover R. H. (1979). Trans. Br. Mycol. Soc. 69: 500-502 2. Mourichon X., Carlier J. and Fouré E. (1997). Enfermedades de Musa: Hoja Divulgativa No. 8, CIRAD

/INIBAP, Montpellier, 1-4 3. Torres J., Calderón H., Rodríguez-Arango E., Morales J., Arango R. (2012). Eur. J. Plant Pathol. 133: 887-

898 4. Otálvaro F., Nanclares J., Vásquez L.E., Quiñonez W., Echeverri F., Arango R. and Schneider B. (2007). J.

Nat. Prod. 70: 887-890

36

Short Lecture SL25

Fissistigma latifolium: a source of new modulators of Bcl-xL/Bak and Mcl-1/Bid interactions

Charlotte Génya, Vincent Dumonteta, Nicolas Birlirakisa, Pascal Retailleaua, Khalijah Awangb, Françoise Guérittea, Marc Litaudona

aCentre de Recherche de Gif, Institut de Chimie des Substances Naturelles, Labex LERMIT, CNRS, Avenue de la

Terrasse, 91198 Gif sur Yvette Cedex, France. bDepartment of chemistry, University of Malaya, 59100 Kuala Lumpur, Malaysia.

It is now well established that the over-expression of anti-apoptotic proteins such as Bcl-xL and Mcl-1 plays a role in cancer development and can be correlated with resistance to cancer therapeutics. These proteins are thus considered to be challenging targets for the development of novel anti-cancer treatment, but a strict inhibition of Bcl-xL may result in apoptosis "escape" through Mcl-1 pathway [1]. The search for dual inhibitors is therefore essential to restore the apoptotic process. Thus, similarly to the methodology used for Bcl-xL/Bak interaction [2], an affinity displacement assay using fluorescent polarization, based on the binding of a fluorescein-labeled peptide (BH3-domain of Bid) to Mcl-1, has been developed. A biological screening on the modulation of Bcl-xL/Bak interaction was initially conducted on 9 000 plant extracts, leading to the isolation of new active compounds such as meiogynin A [2] or kingianin G [3]. The most active extracts were also screened on Mcl-1/Bid interaction. The bio-guided fractionation of the EtOAc bark extract from Fissistigma latifolium led to the isolation of one new prenylated chalcone (1) with potent binding affinity for Bcl-xL and Mcl-1, and three new substituted monoterpenoids (2, 3, 4, 5). The isolated compounds possess a carbon skeleton that we suggest to be biologically formed via an enzymatic catalysis of a Diels-Alder reaction.

In this communication, we will report (i) the development of the bioassay on Mcl-1/Bid interaction used for the bio-guided fractionation of Fissistigma latifolium and (ii) the chemical investigation of this Malaysian plant using spectroscopic and crystallographic methods.

1. Tse, C. et al., (2008) Cancer Res. 68: 3421-3428. 2. Litaudon, M., et al., (2009) J. Nat. Prod. 72: 480–483. 3. Leverrier, A., et al., (2011) Phytochem. 72: 1443-1452.

(±)

1. R1= quinone ; R2=H ; R3=(CH2)2CH(CH3)2 2. R1=OH ; R2=H ; R3=(CH2)2CH(CH3)2

3. R1=CH3 ; R2=H ; R3=(CH2)2CH(CH3)2

4. R1=CH3 ; R2=(CH2)2CH(CH3)2 ; R3=H 5. R1=heterocycle ; R2=H ; R3=(CH2)2CH(CH3)2

37

Short Lecture SL26

Bioprospecting diverse plant species for antiviral agents against upper respiratory tract infections

Christina E. Maira, Ulrike Grienkea, Susanne von Grafensteinb, Johannes Kirchmairc, Klaus R. Liedlb, Michaela Schmidtked, Judith M. Rollingera

aInstitute of Pharmacy, Department of Pharmacognosy, Center for Molecular Biosciences Innsbruck, University

of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria; bInstitute of General, Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80-82, 6020

Innsbruck, Austria; cUnilever Centre for Molecular Sciences Informatics, Department of Chemistry Lensfield Road, Cambridge, CB2 1EW, UK; dDepartment of Virology and Antiviral Therapy, Jena University Hospital,

Hans-Knoell-Straße 2, 07745 Jena, Germany

Upper respiratory tract infections (URIs) are caused by various bacteria and viruses. In this work, we focused on the identification of natural constituents with antiviral activity on human rhinovirus (HRV2), a predominant cause of the common cold, influenza virus A and coxsackie virus (CVB3), both of them being responsible for more severe forms of URIs. Starting from a literature survey to explore knowledge from folk medicine, 66 plant species of interest were identified. The selection focuses on the flora of the Alpine region with Lamiaceae, Apiaceae and Asteraceae representing the most prominent families.

Following the acquisition and collection of the selected plants, the obtained extracts were tested for their antiviral potential against the three viruses in cytopathic effect (CPE) inhibition assays. About 12 % of the extracts had an IC50 lower than 50 µg/mL for influenza virus A/HK/68 in MDCK cells. A comparable ratio of active extracts was found for HRV2, and 8 % of all extracts showed activity for CVB3, the latter two being tested in HeLa cells. Of all active extracts, 31 % inhibited two and 8 % all three distinct viruses.

In addition to these experimental investigations, a structure-based computational approach was followed, aiming to support the interpretation of ethno-pharmacological data and guide isolation. As a preliminary result, five out of the 66 plant species were found to contain constituents that were predicted in silico to be active on influenza virus neuraminidase and nucleoprotein, as well as the HRV capsid protein 1. In the course of the experimental evaluation of these five extracts, all of them were confirmed to exhibit significant antiviral activity, affirming the potential of this bioprospecting approach in finding valuable starting points for target-based drug discovery. This work is supported by the Austrian Science Fund (FWF P24587 & P23051) and the European Social Fund (ESF and TMWAT Project 2011 FGR 0137).

38

Short Lecture SL27

New insights into the anti-influenza activity of licorice constituents

Ulrike Grienkea, Martina Richterb, Heike Braunb, Johannes Kirchmairc, Susanne von Grafensteind, Klaus R. Liedld, Michaela Schmidtkeb, Judith M. Rollingera

aInstitute of Pharmacy/Pharmacognosy, Center for Molecular Biosciences Innsbruck, University of Innsbruck,

Innrain 80-82, 6020 Innsbruck, Austria; bDepartment of Virology and Antiviral Therapy, Jena University Hospital, Hans-Knoell-Straße 2, 07745 Jena, Germany; cUnilever Centre for Molecular Informatics,

Department of Chemistry, Lensfield Road, Cambridge, CB2 1EW, UK; dInstitute of General, Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80-82,

6020 Innsbruck, Austria

Neuraminidase (NA), a key enzyme in viral replication and spread, is the first-line drug target to combat influenza. Based on a shape-focused virtual screening approach, the roots of Glycyrrhiza glabra L. (licorice) were identified as plant material with an accumulation of molecules that show 3D similarities to previously discovered influenza NA inhibitors [1]. Moreover, licorice is the most frequently cited herb in TCM for treating respiratory tract infections [2].

Phytochemical investigations revealed twelve constituents identified as (E)-1-[2,4-dihydroxy-3-(3-methyl-2-butenyl)phenyl]-3-(8-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl)-2-propen-1-one (1), 3,4-dihydro-8,8-di-methyl-2H,8H-benzo[1,2-b:3,4-b']dipyran-3-ol (2), biochanin B (3), glabrol (4), glabrone (5), hispaglabridin B (6), licoflavone B (7), licorice-glycoside B (8) & E (9), liquiritigenin (10), liquiritin (11), and prunin (12). A distinct inhibition of the cytopathic effect in MDCK cells was observed for compounds 3 and 5 (IC50s 40.3; 37.4 µM). On the target level, chemiluminescence (CL)-based NA inhibition assays were performed using the NA of different influenza virus strains including A/342/09 (H1N1), an oseltamivir-resistant virus isolate. Strikingly, 11 compounds showed IC50s in the low micromolar to even nanomolar range. For most constituents 2- to 10-fold higher concentrations were necessary to inhibit the NA of the oseltamivir-resistant virus. In addition, the NA of Clostridium perfringens was shown to be susceptible in CL- as well as fluorescence-based assays.

In this work, we report novel insights into the anti-influenza potential of licorice constituents which includes also a critical discussion of possible assay interference problems [3].

1. Grienke U, et al. (2010) J. Med. Chem. 53: 778 – 786. 2. Ge H, et al. (2010) Nat. Prod. Rep. 27: 1758 – 1780. 3. Chamni S, De-Eknamkul W (2013) Expert Opin. Ther. Patents23: 409 – 423. This work is supported by the Austrian Science Fund (FWF: P24587 & P23051) and the European Social Fund (ESF & TMWAT Project 2011 FGR 0137).

39

Short Lecture SL28

Antiviral Diterpenes from Corsican Euphorbia Species

Louis-Félix Nothiasab, Vincent Dumontetb, Pieter Leyssenc, Julien Paolinia, Pascal Retailleaub, Françoise Guéritteb, Jean Costaa and Marc Litaudonb

aLaboratoire de Chimie de Produits Naturels, UMR CNRS SPE 6134, University of Corsica, 20250, Corte,

France; bCentre de recherche de Gif, LabEx LERMIT, Institut de Chimie des Produits Naturels ICSN, CNRS UPR 2301, 1 avenue de la terrasse, 91198, Gif-sur-Yvette, France; cLaboratory for Virology and Experimental

Chemotherapy, Rega Institute for Medical Research, KULeuven, Minderbroedersstraat, B3000 Leuven, Belgium.

Aim of the work The invasive tiger mosquito species, Aedes albopictus, is a serious health concern since

it is one of the main vector for the transmission of viral pathogens, such as dengue and chikungunya virus. In human, the chikungunya virus (CHIKV) is responsible for an acute disease, characterized by a triad of fever, arthralgia and maculopapular rash. Currently, no specific antiviral therapy or vaccine is available for the treatment or prevention of this disease. [1]

In the course of identifying selective inhibitors of chikungunya replication, a previous phytochemical investigation of two Trigonostemon species (Euphorbiaceae) led to the characterization of new chlorinated daphnane orthoesters and tigliane endowed with a potent antiviral activity. [2] [3]

Within the remarkable flora of Corsica, the Euphorbiaceae familly is well represented by the genus Euphorbia including 31 species, from which 17 are endemic of Mediterranean areas. [4]. This study aims at investigating the antiviral activities of Corsican Euphorbia species.

Methodology and results Fifty-two EtOAc and MeOH extracts have been prepared from 9 Euphorbia species,

and were evaluated for selective antiviral activity in a virus-cell-based assay for CHIKV. Several extracts exhibited potent inhibitory activity on CHIKV replication. A bioactivity-guided purification of the EtOAc extract of Euphorbia amygdaloides subsp semiperfoliata (Viv.) A.R.Sm, an endemic plant of Corsica and Sardinia, afforded 13 jatrophanes esters, from which 9 are new, along with 2 deoxyphorbols and one abietane diterpene. Their planar structures and relative configurations were determined by HRMS, 1D and 2D NMR spectroscopic analyses, and their absolute configurations by X-ray diffraction. One compound exhibited strong and selective inhibitory activity on CHIKV replication with an EC50 of 1.3 μM and a selectivity index approximating 200. In this communication, we will report identification and biological activities of the purified compounds and will highlight some structure-activity relationships.

1. Moro, M.L. et al. (2010) Intl. Health, 2; 3: 223-227 2. Allard, P.-M. et al. (2012) Phytochemistry, 84: 160-168 3. Bourjot, M. et al. (2012) J. Nat. Prod., 75;12: 2183–2187 4. Jeanmonod, D.; Gamisans, J. (2007) Flora Corsica, Edisud, Aix-en-Provence.

Ortep view of jatrophane ester A3

40

Short Lecture SL29

Chemical constituents of Lotus, the leaves of Nelumbo nucifera and their anti-obesity effects

Jong Hoon Ahna, Eun Sil Kimb, Chul Leea, Soonok Kimb, Soo-Hyun Choc, Bang Yeon Hwanga, Mi Kyeong Leea

aCollege of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea;bWildlife Genetic Resources Center, National Institute of Biological Resources, Incheon 404-708, Korea; cNational Institute of

Ecology, Seocheon, Chungnam 325-814, Korea

Obesity is defined as abnormal or excessive fat accumulation that stems from a prolonged imbalance between the levels of energy intake and expenditure and has been steadily increasing and has become a serious health issue worldwide [1]. Nelumbo nucifera Gaertn. (Nymphaeaceae), commonly called lotus, is widely distributed throughout Eastern Asia. It has been used for food and medicine for a long time and Flavonoids and benzylisoquinoline alkaloids have been reported in lotus leaves [2-3]. The beneficial effects of lotus leaves on obesity have been extensively investigated [4]. A phytochemical investigation of N. nucifera leaves led to the isolation of 13 megastigmanes (1 - 13), including a new megastigmane, nelumnucifoside A (1), and a new eudesmane sesquiterpene, nelumnucifoside B (14), eight alkaloids (15 - 22), eleven flavonoids (23 - 33) and three flavolignans (34-36). Their chemical structures were determined based on spectroscopic methods including 1D, 2D NMR and MS spectrometry. The relative and absolute stereochemistry of the compounds was determined by NOESY and CD spectrometry, respectively. Compounds 19 and 22 significantly inhibited pancreatic lipase, whereas compounds 15 and 16 showed a strong inhibitory effect on adipocyte differentiation [5]. Therefore, the leaves of N. nucifera have potential as an anti-obesity agent by inhibiting pancreatic lipase and adipocyte differentiation. 1. Kopelman PG (2000) Nature 404: 635 – 643. 2. Lin HY, Kuo YH, Lin YL, Chiang W (2009) J Agric Food Chem 57:6623-6629 3. Kashiwada Y, Aoshima A, Ikeshiro Y, Chen YP, Furukawa H, Itoigawa N, Fujioka T, Mihashi K (2005)

Bioorg Med Chem 13: 443-448 Wu CH, Yang MY, Chan KC, Chung PJ, Ou TT, Wang CJ (2010) J Agric Food Chem 58: 7075-7081.5. Ahn JH, Kim ES, Lee C, Kim S, Cho SH, Hwang BY, Lee MK (2013) Bioorg Med Chem Lett 23: 3604-3608 Please insert the reference to the published version of this study: Bioorg & Med Chem Lett 2013; 23(12): 3604-3608.

41

Short Lecture SL30

In vitro Screening of Alpine Lichen Species for Anti-Inflammatory Lead Structures

Sarah K. Oettla, Jana Gerstmeierb, Katja Wiechmannb, Julia Bauerc, Atanas G. Atanasovd, Clemens Malainerd, Elke H. Heissd, Birgit Waltenbergera, Daniel Remiasa, Joel Boustiee, Verena M. Dirschd,

Hermann Stuppnera, Oliver Werzb, Judith M. Rollingera

aInstitute of Pharmacy/Pharmacognosy, Center for Molecular Biosciences Innsbruck, Leopold-Franzens University of Innsbruck, Austria; bChair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy,

Friedrich Schiller University of Jena, Germany; cDepartment of Pharmaceutical Analytics, Pharmaceutical Institute, University Tuebingen, Tuebingen, Germany; dDepartment of Pharmacognosy, University of Vienna, Austria; eInstitute of Chemical Sciences of Rennes, UMR 6226, Team PNSCM, University of Rennes 1, France

Although lichens provide a vast diversity of small chemical entities with a variety of

reported bioactivities, they are still underestimated and underexplored with respect to pharmaceutical lead discovery. Based on a previously performed pharmacophore-based virtual screening on mPGES-1, we identified lichen constituents as potent mPGES-1 inhibitors [1]. Here, we focused on further anti-inflammatory properties of lichen compounds. Samples of 17 Alpine lichen species were collected in Tyrol, South Tyrol and Upper Austria and unambiguously identified using morphological and microchemical methods. Ethanolic crude extracts of these lichen species were screened in vitro for their potency to inhibit two targets within eicosanoid biosynthesis, namely microsomal prostaglandin E2 synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO), as well as NF-κB transactivation activity. The extract of Cetrelia monachorum (Zahlbr.) W.L. Culb. & C.F. Culb., a lichen species of the Parmeliaceae family, showed an outstanding pharmacological in vitro profile in the applied assays and thus was identified as promising source for novel anti-inflammatory lead structures.

Phytochemical investigation of the ethanolic crude extract resulted in the isolation and identification of 11 constituents, belonging to depsides and derivatives of orsellinic acid, olivetolic acid and olivetol. The two depsides imbricaric acid and perlatolic acid exhibited inhibition of mPGES-1 (IC50 = 1.9 and 0.4 µM, respectively) and 5-LO tested in a cell-based assay (IC50 = 5.3 and 1.8 µM, respectively) and on the purified enzyme (IC50 = 3.5 and 0.4 µM, respectively). Dual inhibition of 5-LO and mPGES-1 provides safer and more effective anti-inflammatory properties [2]. Furthermore, these two main constituents, imbricaric acid and perlatolic acid, were quantified in the extract with a content of 15.22% and 9.10%, respectively. They showed significant inhibition of TNF-α-induced NF-κB activation in luciferase reporter cells with IC50 values of 2.0 and 7.0 µM, respectively. Our findings attest imbricaric acid and perlatolic acid a pronounced threefold anti-inflammatory profile [3], which warrants further investigation on their pharmacokinetics and in vivo efficacy. Based on these properties, C. monachorum represents a valuable source for novel bioactive ingredients with high potential for pharmacological intervention for inflammatory disorders.

Acknowledgements: Supported by the Austrian Science Fund (NFN-project DNTI: S10703, S10704).

1. Bauer J, Waltenberger B, Noha SM, Schuster D, Rollinger JM, Boustie J, Chollet M, Stuppner H, Werz O (2012) ChemMedChem 7: 2077 - 2081.

2. Radmark O, Samuelsson B (2010) J Intern Med 268: 5 - 14. 3. Oettl S, Gerstmeier J, Wiechmann K, Bauer J, Atanasov AG, Malainer C, Heiss EH, Waltenberger B, Remias

D, Boustie J, Dirsch VM, Stuppner H, Werz O, Rollinger JM (2013) submitted

42

Short Lecture SL31

Activity of pinocembrin on Campylobacter jejuni

Jasna Kovača, Anja Klančnika, Aleksandra Gornika, Zuowei Wub, Saša Piskernika, Franz Bucarc, Qijing Zhangb, Sonja Smole Možinaa

aDepartment of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jaminkarjeva 101,

SI-1000 Ljubljana, Slovenia; bDepartment of Veterinary Microbiology and Preventive Medicine, College of

Veterinary Medicine, Iowa State University, 1600 S 16th St, IA-50011 Ames, USA; cDepartment of

Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Universitätsplatz 2, A-8010 Graz, Austria

Campylobacteriosis is a leading bacterial food-borne disease worldwide facing increasing resistance against several clinical antibiotics [1]. Therefore, new effective natural antimicrobials are needed for application in medicine and/or antimicrobial prevention in food production and supply chains. One of the active natural compounds is pinocembrin – a flavonoid compound with various bioactivities, found in the extracts of some medicinal plants (e.g. Alpinia katsumadai) [2]. We investigated the activity of different concentrations of pinocembrin on Campylobacter jejuni, to evaluate how the applied concentration influences its mode of action. For this purpose the minimal inhibitory concentration of pinocembrin (MIC=64 µg/ml) was determined by broth microdilution. It was further tested also in subinhibitory (16 µg/ml) and suprainhibitory (128 µg/ml) concentrations. The influence of the subinhibitory concentration of pinocembrin on gene expression was evaluated in C. jejuni NCTC 11168 mutant of the cmeR transcriptional repressor by microarray analysis and confirmed with qRT-PCR. Further, the physiological influence of pinocembrin (between subinhibitory and suprainhibitory values) on time-kill kinetics, intracellular oxidation and membrane integrity was evaluated with broth macrodilution, dihydrodichlorofluorescein diacetate and Bacterial Viability Kit LIVE/DEAD BacLight, respectively. The results on molecular level show upregulated expression of several motility-related genes, as well as genes involved in redox signalling and antioxidant defence. Influence of pinocembrin on antioxidant defence was reconfirmed phenotypically, where it induced efficient neutralization of reactive oxygen species and even promoted bacterial growth in subinhibitory concentrations. However, MIC significantly decreased viability of cells, whereas suprainhibitory concentration worked bactericidally, and it severely affected the membrane integrity. Activity of pinocembrin is dose-dependent, therefore, the concentration for its application needs to be carefully chosen.

Figure 1: Chemical structure of pinocembrin

1. EFSA (2013) EFSA J 1: 3129. 2. Klančnik A, Gröblacher B, Kovač J, Bucar F, Smole Možina S (2012) J Appl Micro 113:1249-62.

43

Short Lecture SL32

The Anticancer Pharmacological and Toxicological Profiles of Narciclasine

Véronique Mathieua, Florence Lefrancb, Robert Kissa

aLaboratoire de Toxicologie, Faculté de Pharmacie, bService de Neurochirurgie, Hôpital Erasme; Université Libre de Bruxelles (ULB), Brussels, Belgium.

Narciclasine is a plant growth regulator whose anti-tumor effects have been known for

millennia from folk medicine extracts of Narcissus bulbs [1]. It displays potent in vitro anti-tumor activity below 100 nM in the NCI 60 cancer cell line panel (http://dtp.nci.nih.gov/). It is far less toxic to normal than to cancer cells [2]. The compound is pro-apoptotic at high concentrations (>1µM) to cancer cells of epithelial origin, i.e. carcinoma cells [2], but not to glioma cells [3]. In carcinoma cells, narciclasine pro-apoptotic activity relates to its activation of the initiator caspases of the death receptor pathway [2]. In glioma cells, which are naturally resistant to apoptosis, narciclasine impairs the organization of the actin cytoskeleton at concentrations which are anti-proliferative (IC50 values of 30-90nM) [3, 4]. Same features are observed in melanoma cells [5] that are also naturally resistant to apoptosis. The actin cytoskeleton is implicated in both cell proliferation (cytokinesis) and cell migration (including the metastatic process). Narciclasine-induced effects on the actin cytoskeleton occur through activation of the ROCK/LIMK/cofilin pathway [4]. Narciclasine also impairs protein syntheses in cancer cells through the inhibition of the eEF1A elongation factor, a feature that in turns also impact on the actin cytoskeleton organization [5].

Narciclasine appears more active in an intracranial xenograft model of human non-small-cell lung cancer (NSCLC) than in the same model orthotopically grafted into the lungs of mice [3]. Taxol was ineffective in this intra-cranial model and the differences in activity between the two compounds may reflect the ability of narciclasine to more readily cross the blood-brain barrier [4]. In the same manner, the treatment of human glioblastoma orthotopic xenograft- [4] and melanoma metastasis orthotopic xenograft- [5] bearing immunocompromized mice with non-toxic doses of narciclasine significantly increases the survival of these mice. Narciclasine anti-tumor effects are of the same magnitude than temozolomide, the drug associated with the highest therapeutic benefits in treating glioblastoma patients.

In conclusion, specific and selective targeted delivery approaches should be envisaged to move narciclasine in pre-clinical trials, and hopefully in clinical trials for patients with brain tumors, including gliomas and brain metastases. 1. Kornienko A, Evidente A. (2008) Chem Rev 108:1982-2014. 2. Dumont P, Ingrassia L, Rouzeau S, Ribaucour F, Thomas S, Roland I, Darro F, Lefranc F, Kiss R. (2007) Neoplasia

9:766-776. 3. Ingrassia L, Lefranc F, Dewelle J, Pottier L, Mathieu V, Spiegl-Kreinecker S, Sauvage S, El Yazidi M, Dehoux M,

Berger W, Van Quaquebeke E, Kiss R. (2009) J Med Chem 52:1100-1114. 4. Lefranc F, Sauvage S, Van Goietsenoven G, Mégalizzi V, Lamoral-Theys D, Debeir O, Spiegl-Kreinecker S, Berger

W, Mathieu V, Decaestecker C, Kiss R. (2009) Mol Cancer Ther 8:1739-1750. 5. Van Goietsenoven G, Hutton J, Becker JP, Lallemand B, Robert F, Lefranc F, Pirker C, Vandenbussche G, Van

Antwerpen P, Evidente A, Berger W, Prévost M, Pelletier J, Kiss R, Kinzy TG, Kornienko A, Mathieu V. (2010) FASEB J 24:4575-4584.

44

Short Lecture SL33

Antiangiogenic properties of natural brassinosteroids

Lucie Rárováa, Stefan Zahlerb, Miroslav Strnada aCentre of the Region Haná for Biotechnological and Agricultural Research, Department of Growth Regulators,

Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic; bDepartment of Pharmacy, Centre of Drug Research – Pharmaceutical Biology, Butenandtstrasse 5-13, 81377

Munich, Germany

Brassinosteroids are phytohormones, small-molecule organic compounds occurring in plants. They regulate various types of processes in plants, such as growth, development and resistance to stresses. Brassinosteroids are studied for their influence on human cells, e.g. cell viability, proliferation, apoptosis and their mechanism of action. The antiproliferative effects on human cancer cells were estimated in vitro recently [1, 2].

The cellular and molecular mechanisms of action of these phytohormones in animal and human cells are still largely unknown. Endothelial primary cells HUVEC (Human Umbilical Vein Endothelial Cells) were used for: proliferation assay (staining with Calcein AM), migration assay, tube formation, flow cytometry, Western blotting, reporter assay and fluorescence polarization-based competitive binding assay.

Natural brassinosteroids inhibit proliferation, migration and tube formation of human endothelial cells. Some of the tested compounds interacted with steroid receptors. Our results provide the first evidence that a large group of brassinosteroids can inhibit angiogenesis in vitro. Brassinosteroids constitute a novel group of human steroid receptor activators or inhibitors with capacity to inhibit angiogenesis.

This work was supported by grant No. ED0007/01/01 Centre of the Region Haná for

Biotechnological and Agricultural Research and FP6-2002-Life Sciences & Health, PRO-KINASE Research Project, Project no. LSHB-CT-2004-503467. 1. Malikova J, Swaczynova J, Kolar Z, Strnad M. (2008) Anticancer and antiproliferative activity of natural

brassinosteroids. Phytochemistry 69: 418–426. 2. Steigerová J, Oklešťková J, Levková M, Rárová L, Kolář Z, Strnad M. (2010) Brassinosteroids cause cell

cycle arrest and apoptosis of human breast cancer cells. Chem Biol Interact. 188(3):487-496.

45

Short Lecture SL34

Ecdysteroid dioxolane derivatives as novel MDR-modulators

Joseph Csábia, Ana Martinsb, András Simonc, Gábor Tóthc, Attila Hunyadia

aInstitute of Pharmacognosy, Faculty of Pharmacy, University of Szeged, 6720, Szeged, Hungary; bDepartment of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, 6720, Szeged, Hungary; cDepartment of Inorganic and Analytical Chemistry, Budapest University of Technology and

Economics, 1111, Budapest, Hungary

Multidrug resistance (MDR) is a major cause of failure in cancer chemotherapy. We have previously investigated the effect of ecdysteroids on MDR cancer cells, and from these studies we obtained some promising results [1]. Our goal was to obtain novel dioxolane derivatives of ecdysteroids as potential anti-MDR leads.

Ecdysteroids are hydroxylated steroids with a characteristic 7-en-6-one moiety in their B-ring. Their most abundant representative in nature is 20-hydroxyecdysone (20E), a relatively hydrophilic compound [2]. Some of its more lipofilic derivatives were found to be efficient modulators of MDR, acting in strong synergism with doxorubicin on a murine cancer cell line over-expressing the human ABCB1 transporter. Synthesis of these compounds from 20E is not so complicated. However, despite its simplicity, only a few of such derivatives have been described. In our work we obtained compounds with structural modifications primarily at the 20,22 but also at the 2,3 positions. Interestingly, those compounds that have their added moiety at 20,22 are more stable (also in acidic environment [1]) and can be stereo selective.

In this work we focused our attention on producing novel, apolar ecdysteroid-derivatives disubstituted with various aldehydes and ketones. All reaction products were synthesized by using phosphomolybdic acid as catalyst. Bioactivity testing was carried out on L5178 mouse T-cell lymphoma cells (non MDR) and their sub-cell line transfected with pHa MDR1/A retrovirus, overexpressing the human ABCB1 efflux pump (MDR cell line). Several compounds showed promising activity which revealed novel structure-activity relationships to be discussed in the presentation.

Acknowledgements: The authors acknowledge support of the European Union co-funded by the European Social Fund (TÁMOP 4.2.2/B-10/1-2010-0012 and TÁMOP-4.2.2.A-11/1/KONV-2012-0035), and that of the Fundação para a Ciência e para a Tecnologia, Portugal (PEsT-OE/SAU/UI0074/2011). Martins A was supported by the grant SFRH/BPD/81118/2011, FCT, Portugal.

1. Martins, A. et al.; J. Med. Chem. 2012, 55, 5034-5043. 2. Báthori, M. et al.; Curr. Med Chem. 2008, 15, 75-91.

46

Short Lecture SL35

Biosynthesis of lupeol-3-hydroxy stearate in the tropical plant, Pentalinon andrieuxii. A 13CO2 study

Alejandro Yam-Puc,a Fabiola Escalante-Erosa,a Karlina García-Sosa,a Fabiola G. Ramírez-Torres,a Manuel J. Chan-Bacab,c Wolfgang Einsenreich,d Claudia Huber,d Nihat Knispel,d Gregorio Godoy-

Hernández,b and Luis M. Peña-Rodrígueza*

aLaboratorio 2 de Química Orgánica, Unidad de Biotecnología, bUnidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, C.43 No. 130, Col. Chuburná de Hidalgo, Mérida,

Yucatán, México. cDepartamento de Microbiología Ambiental y Biotecnología, Universidad Autónoma de Campeche, Av. Agustín Melgar s/n, Campeche, México. dLehrstuhl für Biochemie, Technische Universität

München, Lichtenbergstr. 4, D-85747 Garching, Germany

The biosynthesis of the triterpene lupeol-3-hydroxy-stearate (1) was investigated in growing plants of Pentalinon andrieuxii Muell. Arg. (Apocynaceae) by pulse-chase experiments using 13CO2 as a tracer. 13C NMR spectroscopy showed a highly distinct labeling pattern in 1 with 12 pairs of directly adjacent 13C-atoms in the lupeol moiety. The stearate moiety was also characterized by the presence of 13C2-isotopologues. This profile gave clear evidence for the mevalonate origin of lupeol and the fatty acid-type biosynthesis of the stearate moiety. The deoxyxylulose phosphate pathway had not contributed to the pathway since 13C3-isotopologues were not detected. The study documents for the first time the expected mevalonate pathway for the biosynthesis of a triterpene under quasi physiological conditions.

O

OOH

12

12

34

56

7

89

10

11

1213

1415

16

1718

1920 21

22

23 24

25 26

27

28

29

30

1'2'

3'

4'

5'18'

1

47

Short Lecture SL36

Biosynthesis of nudicaulins in petals of Papaver nudicaule

Anne-Christin Warskulata, Evangelos C. Tatsisa, Bernd Schneidera

aBiosynthesis/NMR group, Max Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, D-07745 Jena

Yellow nudicaulins obtained from Papaver nudicaule are a unique group of flower pigments in nature. Their elusive structure was investigated for almost 70 years until a first proposal was published in 2006 [1]. It was shown that the aglycone contains an indole ring and a chalcone-like moiety, which usually occurs in the flavonoid biosynthesis. A retrobiosynthetic study with 13CO2 suggested a mixed origin of the two parts of the molecule but further details on the pathway are still obscure.

To investigate the annelation between the indole and flavonoid part, derivatives generated by hydrolysis, hydrogenation and permethylation were prepared and analyzed by NMR, MS and ECD. The results of these experiments led to a revision of the proposed structure and absolute configuration [2].

For a detailed study on the nudicaulins biosynthesis an experimental system for feeding 13C-labelled precursors was established. Petals from closed buds were sliced and suspended in a buffer containing labelled precursors. From those experiments nudicaulins, which are enriched with 13C were obtained. Analysis of extracts by NMR spectroscopy and MS revealed the 13C incorporation and enabled conclusions on biosynthetic precursor-product relationships.

The results of feeding experiments with [13C6]glucose showed that the de novo biosynthesis of nudicaulins is performed in situ in petals. Further feeding experiments demonstrated that typical precursors of the flavonoid pathway are incorporated into the polyphenol-like part of the molecule and anthranilic acid is incorporated into the indole part of the nudicaulin aglycone.

Syntheses of more advanced precursors and investigations on the nudicaulin production in developing petals are in progress. Analyses of enzymes involved in the synthesis are in preparation. In addition enzymatic studies on the hydrolysis of our target molecules are ongoing. 1.

Schliemann, W., Schneider, B., Wray, V., Schmidt, E., Nimtz, M., Porzel, A., Böhm, H. (2006) Phytochemistry 67: 191-201

2. Tatsis, E., Schaumlöffel, A., Warskulat, A.-C., Massiot, G., Schneider, B., Bringmann, G. (2013) Organic Letters 15: 156-159

48

Short Lecture SL37

UPLC-MS profiling of low molecular weight phlorotannin polymers

T.J. Smytha, M. Tierneya, A. Soler-Villa

aDepartment of Biosciences, Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15, Ireland; bIrish Seaweed research Group, Ryan Institute (Environmental marine and Energy Research),National University of

Ireland, Galway, Ireland.

Phlorotannins are a group of complex polymers, found in particular brown seaweeds,

composed solely of the monomer phloroglucinol (1,3,5-trihydroxybenzene) [1]. Their structural complexity arises from the number of possible linkage positions between each monomer unit [2]. This study aimed to profile the phlorotannin composition and level of isomerisation present in brown Irish macroalgae using UPLC-MS utilising a triple quadrupole mass spectrometer. Phlorotannin-enriched fractions from aqueous ethanol extracts were analysed by UPLC-MS performed in multiple reaction monitoring mode to detect molecular ions consistent with the molecular weights of phlorotannins. Ascophyllum nodosum and Pelvetia canaliculata appeared to contain predominantly larger phlorotannins (degree of polymerisation (DP) of 6-13 monomers) compared to Fucus spiralis (DP of 4-6 PGUs). Extracted ion chromatograms for each species were analysed to profile the level of isomerisation for specific molecular weights of phlorotannins between three to sixteen monomers. The level of phlorotannin isomerisation within the extracts of the individual macroalgal species differed to some degree, resulting in substantially different numbers of phlorotannin isomers for particular molecular weights. A similar UPLC-MS/MS separation procedure, as outlined in this study, may be used in the future as a means of screening the metabolite profile of macroalgal extracts, therefore, allowing extract consistency to be monitored for standardisation purposes.

1. Tierney, M. S., Smyth, T. J., Rai, D. K., Soler-Vila, A., Croft, A. K., Brunton, N. (2013). Food Chemistry, 139, 753–761

2. Steevensz, A. J., MacKinnon, S. L., Hankinson, R., et al. (2012). Phytochemical Analysis, 23 (5): 547-53

49

Short Lecture SL38

Chemical Constituents of Urginavia altissima (Hyacinthaceae)

Linda C. Langata, Wolfgang Wetschnigb, Moses K. Langat,a Dulcie A. Mulhollanda

aDepartment of Chemistry, FEPS, University of Surrey, Guildford, Surrey, GU2 7XH, UK; bInstitute of Plant

Sciences, Karl-Franzens-University Graz, Holteigasse 6, 8010 Graz, Austria

Urginavia altissima (L.F) Speta (syn. Urginea altissima (L.F) Baker, Drimia altissima (L.F) Ker Gawl) is an African species belonging to the Urgineeae tribe of Scilloideae subfamily of the Asparagaceae family (formerly Hyacinthaceae). This species is widespread in sub-Saharan Africa and investigations of the synonymous Urginea altissima and Drimia altissima have yielded bufadienolides [1, 2]. A phytochemical study of Urginavia altissima has yielded seven novel bufadienolides (compounds 1-7). Compounds 4 and 7 were separated after acetylation of a mixture. Other compounds isolated in this study from Urginavia altissima include three homoisoflavonoids and two sesquiterpenoids which are both only the second reported occurrence from the Urgineeae tribe. The isolation of bufadienolides from Urginavia altissima follows the chemotaxonomic trend of the Urgineeae tribe of the Asparagaceae family. The structures of the compounds isolated were determined by mass spectrometry, IR, 1D and 2D NMR spectroscopic experiments. Novel compounds will be submitted to NCI for anticancer screening.

1. Pohl T, Koorbanally C, Crouch NR, Mulholland DA (2001) Phytochem. 58: 557-561. 2. Dagne E, Mammo W, Alemu M, Casser I (1994) Bull. Chem. Soc. Ethiop. 8: 85-89.

O

O

OH

H

H

H

R1O

HO

O

O

R2

OH

H

H

H

R1O

R3

O

O

CHO

OH

H

H

H

R1OH

6. R1 = OH 7. R1= 2’,3’,4’,6’-tetra-O-

acetyl-O-β-D-glucose

1. R1 = α-OH 2. R1 = β-OH 3. R1 = α-O-β-D-

glucose

4. R1 = 2’,3’,4’,6’-tetra-O-acetyl-O-β-D-glucose, R2= CH3, R3= =O

5. R1= O-β-D-glucose, R2=CH2OH, R3= β-OH

50

Poster P 01

Tirucallane and Cycloartane Triterpenoids from the Stem Bark of Toona sinensis Roem (Meliaceae)

Areej H.S. Aldhahera,b, Moses K. Langata, Daniel J. Driscolla and Dulcie A. Mulhollanda

aDepartment of Chemistry, FEPS, University of Surrey, Surrey, GU2 7XH, UK; bDepartment of Biology, College

of Science, University of Basrah, Basrah, IRAQ. Toona sinensis Roem (Meliaceae) (syn. Cedrela sinensis A. Juss) is a woody plant that

is widely distributed in eastern and south eastern Asia from India, Nepal, China, Burma, Thailand, Malaysia, Java to Europe [1,2]. Different parts of T. sinensis are used in traditional medicine, the stem bark is used as an adstringent and depurative, the fruits are used to treat eye infections and the roots are used as a corrective for amenorrhea [1]. The roots of T. sinensis are reported to give limonoids, lignans and phenolic compounds [1] and several parts of C. sinensis, restricted to China and Korea, and widely regarded as synonym of T. sinensis, has yielded limonoids, apotirucallane and tirucallane triterpenoids [3,4,5]. The genus Toona has been reported to yield flavonoids, phenolics, alkaloids, limonoids, apo- and tirucallane-type triterpenoids [1,6].

The stem bark of T. sinensis collected from the grounds of the University of Surrey, Guildford, UK was analysed for its chemical constituents. Three minor tirucallane (1-3) and two minor cycloartane-type (4, 5) triterpenoids alongside four common phytosterols were identified from the dichloromethane extract of the stem bark of T. sinensis. The structures of the compounds were identified by the analysis of their NMR spectroscopic data, HRMS and by comparison against published data. Previous biological studies have demonstrated that extracts of T. ciliata and limonoids and triterpenoids from T. ciliata and T. sinensis have been anticancer properties [6,7,8,9], therefore compounds 1-5 will be submitted to the NCI59 anticancer screening programme.

1. Dong X, Zhu Y, Bao G, Hu F, Qin G (2013) Molecules 18: 2840-2850. 2. Edmonds JM, Staniforth M (1998) Curtis’s Botanical Magazine 15: 186-193. 3. Mitsui K, Saito H, Yamamura R, Fukaya H, Hitotsuyanagi Y, Takeya K (2007) Chem. Pharm. Bull. 55: 1442-

1447. 4. Mitsui K, Maejima M, Fukaya H, Hitotsuyanagi Y, Takeya K ( 2004) Phytochemistry 65: 3075-3081. 5. Mitsui K, Maejima M, Saito H, Fukaya H, Hitotsuyanagi Y, Takeya K (2005) Tetrahedron 61: 10569-10582. 6. Zhang F, Wang J, Gu Y, Kong L, (2012) J. Nat Prod 75: 538- 546. 7. Yang S, Zhao Q, Xiang H, Liu M, Zhang Q, Xue W, Song B, Yang S (2013) Cancer Cell International 13:12. 8. Chowdhary R, Rashid R B, Sohrab M H, Hasan C M (2003) Pharmazie 58:272-273. 9. Chowdhary R, Hasan C M, Rashid M A. (2003) Pharmaceutical Bio 41(4):281-83.

51

Poster P 02

A Bufadienolide Glycoside and a Homoisoflavonoid from Rhodocodon campanulatus (Asparagaceae)

Alaa Alqahtania, Moses K. Langata, Wolfgang Wetschnigb and Dulcie A. Mulhollanda

aDepartment of Chemistry, FEPS, University of Surrey, Surrey, GU2 7XH, U; b Institute of Plant Sciences, University of Graz, Holteigasse 6, 8010 Graz, Austria

Rhodocodon campanulatus is a member of the bulbous Urgineeae tribe of the

Scilloideae subfamily of the expanded Asparagaceae family (formerly Hyacinthaceae). Plants of the Urgineeae tribe are used as traditional remedies for the treatment of several ailments, such as infections, rheumatism, inflammation and disorders associated with the central nervous system [1]. The Urgineeae tribe is distributed from South Africa to the Mediterranean, Saudi Arabia, India and Myanmar [2]. The chemical constituents of plants of the Rhodocodon genus are not documented and hence the plant was investigated for chemotaxonomical reasons. In this study we report the isolation of a novel bufadienolide glycoside and a known homoisoflavonoid from the ethanol extract of the bulbs of Rhodocodon campanulatus.

The major compounds were novel bufadienolide glycoside, 1, 3β-(O-β-D-glucopyranoside)-14β-hydroxybufa-20,22-dienolid-19-al, and the known homoisoflavonoid, 2, 5,7-dihydroxy-3-(3-hydroxy-4-methoxybenzyl)chroman-4-one, previously isolated from the South African Scilla kraussi [3]. The structures of 1 and 2 were determined by the analysis of their NMR and MS spectra. The absolute configuration at C-3 for 2 was determined in this study as S on the basis of its electronic circular dichroism study. A positive Cotton effect at 290 was in agreement to those reported for homoisoflavonoids with H-3 in β position [4].

1. Goel A, Ram VJ, (2009) Tetrahedron 65: 7865-7913. 2. Koorbanally NA, Koorbanally C, Harilal A, Mulholland DA, Crouch NR, (2004) Phytochem 65: 3069-3073. 3. Crouch NR., Mulholland DA (1999) Phytochem 51: 943-946. 4. Adinolfi M, Barone G, Corsaro MM, Mangoni L (1988) Tetrahedron 44: 15, 4981-4888.

O

O

OH

HO

H

H

H

O

12

34

5

10

67

89

1112

13

14 1516

17

20

21

2423

22

19

18

O

H

H

OH

H

HO

H

HOH

OH

1'2'3'

4'5'

6'

1

52

Poster P 03

Phytochemical investigation of Eriosema laurentii De Wild

Sylvin Benjamin Atebaa,b, Dieudonné Njamena, Martin Zehlb, Katrin Ukowitzb, Hanspeter Kaehligc, Liselotte Krennb

aDepartment of Animal Biology and Physiology, University of Yaounde I, Box 812 Yaounde, Cameroon;

bDepartment of Pharmacognosy, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria; cInstitute of Organic Chemistry, University of Vienna,Währingerstr.38, A-1090 Vienna, Austria

Eriosema laurentii De Wild is a medicinal plant widely used in Cameroon for the

treatment of infertility and various gynaecological and menopausal complaints. Despite this widespread use as natural remedy, a phytochemical analysis of this plant has not been carried out, yet. Therefore, based on the traditional claims, a thorough phytochemical investigation of a methanol extract of the aerial parts of E. laurentii from Cameroon was undertaken aiming to identify estrogenic compounds.

The phytochemical analysis was performed by liquid-liquid partition of the extract, by column chromatography on silica gel and Sephadex LH-20 as well as by high performance counter current chromatography (HPCCC) of the fractions. For dereplication, thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) were applied. Mass spectrometry and nuclear magnetic resonance (NMR) techniques were used for the structure elucidation of new compounds. Using these methods, two new compounds, namely 5,7-dihydroxy-2'-methoxy-6",6"-dimethyl-pyrano(2",3": 4',5') isoflavone (I) and the highly prenylated 6,8,3'-triprenyl-dihydromorin (II), were isolated and structurally elucidated. Eleven known flavonoids/isoflavonoids were identified as genistein, genistin, 2’-hydroxygenistein, lupinalbin A, luteolin, luteolin-7-O-glucoside, homoorientin, quercetin, quercetin-3-O-methylether, isovitexin and eriosemaone D for the first time in E. laurentii. These results indicate that the methanol extract of the aerial parts of E. laurentii contains several phytoestrogens and support the traditional use of this plant against infertility and in menopausal problems.

2

3

O

O

OH

HO

OH

HO OH

6

89

10

1'

3'

6a

6b

6c

6d

6e

Chemical Formula: C30H36O7

Molecular Weight: 508,60

2

4

O

O

HO

OHOO

3

56

78

9

10 1'

2'3'

4'

5'6' 4''

5''

6''

Chemical Formula: C21H18O6

Molecular Weight: 366,36

I II

53

Poster P 04

HPLC Determination of Phenolic Acids in Arnicae Flos

Vessela Balabanova, Reneta Gevrenova, Dimitrina Zheleva-Dimitrova

Department of Pharmacognosy, Faculty of Pharmacy, Medical University - Sofia, Dunav str. 2, 1000 Sofia, Bulgaria

Arnica montana L. is an important medicinal plant with great medicinal value. Recently,

the species has been investigated for the content of phenolic acids and flavonoids since they are responsible for many potential health benefits [1, 2].

In this study the quantitative determination of protocatechic, chlorogenic and caffeic acids in Arnicae flos by reverse phase high-performance liquid chromatography (RP-HPLC) is reported. Samples of Arnicae flos from eight different origins were selected for the assay as following: one Bulgarian and one Polish cultivated collection, two cultivars, three collections grown in Botanical Garden in Finland, and one purchased from a Pharmacy Drugstore.

The effect of extracting solvent was investigated and optimal extraction of assayed phenolic acids was achieved by 80% methanol. The subsequent HPLC separation of the analytes was performed on Hypersil ODS C18 column using linear gradient elution with a mobile phase composed of 20 mM phosphate buffer (pH 3.22) and methanol, and with UV detection at 280 and 310 nm. The detection limits were 0.188 μg/ml, 0.073 μg/ml and 0.186 μg/ml for protocatechic, chlorogenic and caffeic acids, respectively. Chlorogenic acid was the predominant phenolic acid in the studied samples being present in amounts between 1.16 ± 0.09 mg/g dry weight in cultivar “ARBO” and 4.22 ± 0.03 in Arnicae flos collected from Botanical Garden at Joensuu University, Finland, where the caffeic acid was accounted for up to 0.22 ± 0.01 mg/g dry weight. The protocatechic acid was detected in much lower quantities ranging from 0.01 ± 0.001 mg/g to 0.14 ± 0.01 mg/g.

The results revealed that the sample from Botanical Garden at Joensuu University, Finland, tended to have the highest amounts of phenolic acids compared to the other samples. The Bulgarian collection is also a promising source of valuable phenolic compounds. 1. Gawlik-Dziki U, Swieca M, Sugier D, Cichocka J (2009) Herba pol 55: 60-71. 2. Craciunescu O, Constantin D, Gaspar A, Toma L, Utoiu E, Moldovan L (2012) Chem Cent J 6: 97 doi:10.1186/1752-153X-6-97.

54

Poster P 05

Methods for quantitation of annonacin in Rat plasma and brain

Natacha Bonneaua, Isabelle Schmitz Afonsob, David Touboulb, Alain Brunelleb, Pierre Champya

aDepartment of Pharmacognosy,UMR CNRS 8076 BioCIS, Faculty of Pharmacy, Univ. Paris-Sud, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry, France ; bInstitut de Chimie des Substances Naturelles,

Department of Mass Spectrometry, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France

Annonaceous acetogenins [1] are neurotoxic molecules distributed in the Annonaceae family. They have been found in several parts of the plants, including worldwide consumed fruits like Soursop (Annona muricata). A link between the consumption of Soursop and a high prevalence of atypical parkinsonisms has been established by epidemiological studies [2]. The most abundant acetogenin in Soursop is annonacin, hence it has become a main concern to evaluate its pharmacokinetics parameters, and to know if it is absorbed and particularly if - and to which extent - it is distributed to the brain, as expected.

We developed quantitation methods for annonacin in rat plasma and brain, using UPLC-ESI-TQ, with annonacinone as internal standard. We used MRM mode with a focus on 112 uma loss which is caracteristic of this type of molecules. We used a gradient water / acetonitrile: 35 / 65 to 15 / 85 in 5 minutes as mobile phase, and an Acquity UPLC BEH C18 column. We prepared calibration standards from 0.25 ng/mL to 100 ng/mL in rat plasma spiking 10 μL of annonacin solution of the appropriate concentration, and 10 μL of IS solution at the concentration of 100 ng/mL, in 100 μL of plasma. Protein precipitation was performed by addition of 1 mL of methanol, centrifugation, evaporation of the supernatant under stream-flow, reconstitution in 100 μL of methanol. 5 μL were injected in 3 replicates. The calibration curve was linear (R2 = 0.998). The lower limit of quantitation was 0.25 ng/mL, with a S/N ratio > 300, a CV of 10% and an accuracy of 10% calculated with the low range of the calibration curve (5 dots). Intraday and interday repetability were calculated at 4 concentrations, 2 consecutive days, 3 injections a day, then again five days later. They were respectively < 10% and < 15% for each concentration (0.25; 1; 10; 100 ng/mL). Extraction recovery was > 80%. We compared matrix effect in different plasma matrices. The method was applied to plasma and brain samples after intravenous and oral administration. Annonacin was administered to rats at the concentration of 0.5 mg/kg by intravenous route, and 100 mg/kg or 10 mg/kg by oral route. Blood samples were collected periodically during 24 or 48 hours. At these doses, the molecule could be detected in plasma until 24 hours, after administration by both routes. We prepared brain homogenates in methanol with a ratio of 20% (m/v). Expecting lower concentration, we prepared a calibration curve from 0.025 ng/mL to 0,75 ng/mL, spiking 10 μL of annonacin solution of the appropriate concentration, and 10 μL of IS solution at the concentration of 100 ng/mL, in 1 mL of brain homogenate. The LLOQ was 0.075 ng/mL with a S/N ratio > 1000, a CV < 10% and an accuracy < 15%. This calibration curve was performed for a first estimation of the amount in brain after administration by both routes. Sample preparation optimization and analytical validation are underway. Methods are presented as well as preliminary results.

1. A. Bermejo, I. Barrachina, E. Estornell, D. Cortes (2005) Nat. Prod. Rep. 22: 269–303. 2. A. Lannuzel, M. Ruberg, P.P. Michel (2008) Mov. Disord. 23: 2122–2128.

55

Poster P 06

Influence of altitude on the chemical composition of essential oils of Thymus balcanus Borbás

Irina Boza,b, Elvira Gillec, Irina Mihalached, Maria-Magdalena Zamfiracheb, Rodica Efrosea

aINCDSB - Institute of Biological Research, Lascar Catargi Street, no. 47, Iasi, Romania; bFaculty of Biology, “Alexandru Ioan Cuza” University, Carol I Bd., 20A, Iasi, Romania; c„Stejarul” Biological Research Centre, no.6 Alexandru cel Bun, Piatra Neamt, Romania; dDepartment of Pharmacognosy-Phytotherapy, Faculty of

Pharmacy, University of Medicine and Pharmacy “Gr.T.Popa”, University Street, No.16, Iasi, Romania

Aim: In this paper the authors investigate the changes that occur in the chemical composition of volatile oil of Th. balcanus, a species that has been collected in the anthesis stage, in areas with different altitudes, in the period of 2011-2012. It is known that the altitude is an important environmental factor that influences the essential oil content and the chemical composition [1]. The essential oil yield and chemical composition varied significantly, too, depending on the locations where the plants grew [2].

Methods applied: The volatile oil has been extracted using a Clevenger hydro-distillation process, according to European Pharmacopoeia. The separation and the identification of the components have been carried out with GC-MS (gas chromatography coupled with mass spectrometry) Agilent 6890 N with a spectrometric mass detector 5973 and an auto sampler. The separated compounds were identified by means of the NIST Mass Spectral Library, and the peak position was confirmed by the Kovats retention index.

Results and conclusions: Regarding the influence of altitude on essential oils, there is some literature on species belonging to the Lamiaceae family, including species of the genus Thymus [3; 4], but in the literature reviewed so far we have found very few data on the influence of altitude on the volatile oil of Th. balcanus. Our results showed that the altitude can affect both the qualitative and quantitative chemical composition of the volatile oil. Thus, at higher altitudes, the volatile oil of Th. balcanus contains a high amount of thymol, amount decreasing with the altitude. Species that grow at lower altitudes contain large amounts of farnesol in the volatile oil. There have also been a number of differences in the essential oil, from one year to another. In conclusion, we can affirm that the altitude is an important environmental factor that can influence the chemical composition of volatile oils. Our results come to complete the limited data existing in the literature on volatile oils of Th. balcanus and on the environmental factors that may influence the production and chemical composition.

1. Vokou D, Kokkini S, Bessiere JM (1993) Geographic variation of Greek oregano (Origanum vulgare ssp.

hirtum) essential oils. Biochem. Syst. Ecol., 21: 287–295. 2. Uribe-Hernandez CJ, Hurtado-Ramos JB, Olmedo-Arcega ER, Martinez-Sosa MA (1992) The essential oil of

Lippia graveolens H.B.K. from Jalisco, Mexico, J. Essent. Oil Res. 4: 647-649. 3. Kisgyörgy Z, Csedö K, Hörster H, Gergely J, Racz G (1983) The volatile oil of the more important

indigenous Thymus species occurring in the composition of Serpylli herba, Rev. Med. (Tirgu-Mures, Rom.), 124-130.

4. Kulevanova S, Ristic M, Stafilov T (1998) The essential oils of Thymus balcanus, T. ciliatopubescens and T. pseudo-atticus from Macedonia, Acta Pharm., 48:119-126.

56

Poster P 07

Achillea collina 'Spak': optimal harvesting stage

Claude-Alain Carron, José F. Vouillamoz, Catherine A. Baroffio, Christoph Carlen

Agroscope Changins-Wädenswil ACW, Research Centre Conthey, 1964 Conthey, Switzerland

Achillea collina Becker (ex Rchb.) is a tetraploid species of the Achillea millefolium aggregate used as an aromatic and medicinal plant. Achillea collina is cultivated for its chamazulene-containing (30-67%) essential oil. Agroscope developed the homogenous and productive cultivar ‘Spak’ adapted to the cultivation in mountainous regions. In order to optimize the production of this species the harvesting stage for the best quality has to be refined. The kinetic of essential oil was studied in different countries to recommend the harvest period: in Germany, the optimal harvesting period is the stage ‘full-bloom’ [1], but in Czech Republic the optimal stage is ‘early-bloom’ [2]. The aim of the study was to define the optimal harvesting period for the cultivar ‘Spak’.

Seven harvests were observed between June 1st and August 19th by adapting the cutting height to this of green leaves, but not lower than 20 cm. Yields increased till the first part of ‘full-bloom’ (BBCH 65) with a peak at about 900 g/m2 but with low percentage of leaves (35%). Yields have subsequently decreased from second part of ‘full-bloom’ and at the end of blooming. This decrease was mainly due to the natural fall of basal leaves and consequently to a higher cutting height, above the ligneous stems. The highest amount of essential oil in the leaves was measured at the ‘early-bloom’ stage, which was with 0.22% the only one to be over the level that is prescribed by the Pharmacopoeia. The content in chamazulene was rather high (ca. 40%), which confirmed the high quality of the variety ‘Spak’, and it was not significantly influenced by the harvest stages.

In conclusion, it is recommended to harvest the upper 60 cm horizon of A. collina ‘Spak’ with inflorescences at ‘early-bloom’ or beginning ‘full-bloom’ stages (20-50% of blooming plants) without the basal and colourless leaves, and without woody stems or stems with over 4 mm in diameter. Under such conditions the leaf/stem ratio and essential oil content will be high. 1. Berghold H, Mandl M, Brantner AH, Wagner S, Prattes B, Pelzmann H, Boechzelt H. (2006) Atherischöl-

Gehalt von Schafgarbe (Achillea collina Becker) in Abhängigkeit vom Entwicklungsstadium. Arzn. Gew. Pfl. 1:59–63.

2. Karlova K, Petrikova K (2005) Variability of the content of active substances during Achillea collina Rchb. Alba ontogenesis. Hort. Sci., 32 (1):17–22.

57

Poster P 08

Isolation and purification of a minor constituent with apoptosis-inducing activity from soil cyanobacterium Nostoc sp.

José Cheel, Pavel Hrouzek, Kateřina Voráčová, Petra Kučerová, Aleksandra Kapuscik, Jiří Kopecký

Department of Autotrophic Microorganisms, Institute of Microbiology, Academy of Sciences of the Czech Republic, Opatovický mlýn, CZ-379 81 Třebon, Czech Republic

Aim: As a part of ongoing research to find natural compounds that can serve as potential anticancer drugs, an apoptosis-inducing compound was isolated from the filamentous cyanobacterium Nostoc sp. strain 183 by applying a method of sequential and selective fractionation monitored with UV and MS detection. Results: An extract of lyophilised cells of cultivated Nostoc sp. strain 183, previously screened for apoptosis-inducing activity towards HeLa cervical cancer cells and Hep2G human liver carcinoma cells, was fractionated by using HPLC coupled to UV and MS detectors. The most active HPLC fraction (IC50 = 0.92 µg/ml and 1.43 µg/ml in HeLa and Hep2G tests, respectively) exhibited a low yield in mass, and its MS analysis mainly revealed a minor compound with a molecular weight of 299.2329 Da (299.2335 calculated) and a molecular formula of C16H31N2O3

+, which has not been previously reported. For obtaining larger amounts of this compound (299C), 30 g of lyophilised biomass was extracted with 5% (v/v) acetic acid [1] using an ultrasound-assisted method. This acidic aqueous extract was neutralised with ammonia and then submitted to graded precipitation in ethanol to remove polysaccharides. The supernatant was purified by permeation on gel Sephadex LH-20 monitored by UV-MS detection and subsequently submitted to a column chromatography on Amberlite XAD-7 resin using a stepwise linear gradient from 50% to 100% methanol to afford a 299C-enriched fraction. The active compound was finally isolated by semi-preparative HPLC [2] (Figure 1), and its apoptosis-inducing activity, as assessed by the caspase 3/7 assay, was confirmed towards HeLa cell lines in a concentration range from 1 to 2 μg/ml (Figure 2). Conclusions: This study proposes a method of isolating a promising apoptogen from Nostoc sp. strain 183 and suggests 299C as a potential anticancer agent. Studies oriented at determining both its chemical structure and the mechanisms underlying its apoptosis-inducing activity are now in progress. Figure 1. Sequential purification of 299C from Nostoc sp. Strain 183

Figure 2. Apoptosis-inducing activity of 299C. Relative luminescence units (RLU).

1. Harada KI (2004) Chem. Pharm. Bull. 52: 889–899. 2. Cheel J, et al. (2005) J. Agric. Food Chem. 53: 8512–8518

58

Poster P 09

Estimation of Anti-inflammatory and Antinociceptive Activities of Verbascum pyramidatum Bieb.

Derya Civeleka, Ipek Suntarb Cigdem Kahramana, I.Irem Tatlic, Esra Kupeli Akkolb, Zeliha S. Akdemira

aDepartment of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Turkey;

bDepartment of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler 06330, Ankara, Turkey; cDepartment of Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Turkey.

Verbascum L. species have been used to treat respiratory problems, rheumatism,

hemorrhoids and several types of inflammatory conditions in traditional Turkish medicine [1]. In order to evaluate this traditional information, the anti-inflammatory and antinociceptive activities of the aerial parts and roots of V. pyramidatum Bieb. which is used for women’s health as haemostatic [2] in Turkish folk medicine were investigated.

In vivo inhibitory effect of extracts and fractions in the carrageenan-induced hind paw edema model in mice was studied for the assessment of anti-inflammatory activity [3] and p-benzoquinone-induced abdominal constriction test was performed on mice for the determination of antinociceptive activity [4]. The methanol, chloroform and the remaining water extracts prepared from the aerial parts (leaves and flowers, separately) and roots of Verbascum pyramidatum were investigated. The results showed that the aqueous extract of the flowers had the highest anti-inflammatory activity (25.3%, 90 min and 29.7%, 180 min) at 200 mg/kg as compared to indomethacin (29.0%, 90 min and 37.6%, 180 min) as well as antinociceptive activity (23.6%, 100 mg/kg and 30.5%, 200 mg/kg) as compared with acetyl salicylic acid (43.6%, 100 mg/kg and 49.1%, 200 mg/kg). For this reason the aqueous extract was fractionated on a polyamide column and the obtained fractions were evaluated the same activities. Fractions 3 and 7 were found to possess significant anti-inflammatory (27.9%, 180 min and 37.1%, 270 min; 27.4%, 180 min and 23.8%, 270 min, respectively) and antinociceptive (25.7%; 30.3%, respectively) potentials at 200 mg/kg, per os without inducing any apparent acute toxicity or gastric damage. Phytochemical studies on the active fractions 3 and 7 of the title plant are going on. 1. Ucar Turker and Gurel (2005) Pytotherapy Research 19: 733-739. 2. Alpınar K (1979) Bitki 6(3): 243-249. 3. Yesilada and Küpeli (2007) Journal of Ethnopharmacology 110: 504–515. 4. Okun et al. (1963) Journal of Pharmacology and Experimental Therapeutics 139: 107–109.

59

Poster P 10

Hydroxylated Xanthones from Garcinia succifolia KRUZ

Sutsawat Duangsrisaia, b, Madalena Pintoc, Anake Kijjoaa,d

aICBAS- Instituto de Ciêncas Biomédicas de Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; bFaculty of Science, Kasetsart University, Bangkok, Thailand;

cCEQUIMED-UP and Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto,; dCentro Interdisciplinar de Investigação Marinha e Ambiental

(CIIMAR), Rua dos Bragas, 289, 4050-123 Porto, Portugal

The genus Garcinia (family Clusiaceae), which includes more than 300 tropical species,

is widely known as rich source of xanthones. Simple as well as prenylated xanthones are characteristic constituents [1], besides triterpene derivatives which have been also reported from the representatives of this genus [2]. Since xanthone derivatives exhibit interesting biological activities, such as antioxidant, antifungal, anti-inflammatory and cytotoxic effects [3], Garcinia species are considered to be a valuable source for bioactive compounds.

In our on-going search for bioactive natural products for anticancer and antifungal activities from tropical plants, we have investigated secondary metabolites of many Garcinia species from Thailand. We now report on the isolation of 1,5-dihydroxyxanthone, 1,7-dihydroxyxanthone, 1,3,7-trihydroxyxanthone, 1,5,6-trihydroxyxanthone and 1,3,6,7-tretahydroxyxanthone from the wood of Garcinia succifolia, collected from Northern Thailand. The structures of these xanthones were established by 1D- and 2D-NMR spectral analysis.

1: R1 = R3 = R4 = H, R2 = OH 2: R1 = R2 = R3 = H, R4 = OH 3: R1 = R4 = OH, R2 = R3 = H 4: R1 = R3 = R4 = OH, R2 = H 5: R1 = R4 = H, R2 = R3 = OH

1. Pinto M, Castanheiro R (2009) Natural Products: Chemistry, Biochemistry and Pharmacology, Narosa Publishing House Pvt. Ltd., New Delhi, India.

2. Vieira L, Kijjoa A, Silva A (2004) Phytochemistry 65:393-398. 3. Pinto M, Sousa M, Nascimento M (2005) Curr Med Chem 12: 2517-2538.

60

Poster P 11

Cytotoxicity Assessment of Triterpene Saponins from Gypsophila trichotoma Wend. toward Human Leukemia Cells in Culture

Reneta Gevrenovaa, Maya M. Zaharievab, Laurence Voutquenne-Nazabadiokoc, Max Henryd, Spiro Konstantinove

aDepartment of Pharmacognosy, Faculty of Pharmacy, Medical University - Sofia, 2, Dunav street, 1000 Sofia, Bulgaria; b Institute of Microbiology, Bulgarian Academy of Sciences, 26, Georgi Bonchev street, Sofia 1113, Bulgaria; c Institut of Molecular Chimistry of Reims, UMR CNRS 7312, Bâtiment 18, BP 1039, 51687 Reims

cedex 2, France; d Groupe S.U.C.R.E.S., UMR 7565 CNRS, University of Lorraine, BP 239,54506 Nancy-Vandoeuvre, France; e Department of Pharmacology and Toxicology, Faculty of Pharmacy,

Medical University - Sofia, 2, Dunav street, 1000 Sofia, Bulgaria

Saponins are considered as the major bioactive components of the roots of Gypsophila species (Caryophyllaceae). Saponins with an aldehyde function at C-4 from Gypsophila oldhamiana Miq. exhibited cytotoxic activity against different human cancer cell lines [1]. In the present study, the cytotoxic activity of a crude plant extract of Gypsophila trichotoma was compared to that of a purified saponin mixture and new triterpenoid aminoacyl saponins isolated from the same extract. After a mild acid hydrolysis of the hydroalcoholic extract, two representative prosaponins of the Gypsophila species, gypsogenin 3-O-glucuronide and quillaic acid 3-O-glucuronide, were isolated by solid-phase extraction (SPE) and identified by LC-ESI/MS. The quantitative analysis of prosaponins by RP-HPLC was performed using linear gradient elution and UV detection at 210 and 254 nm. The gypsogenin 3-O-glucuronide was the dominant prosaponin, being present in amount up to 11.27 ± 0.96 mg/g dry weight whereas quillaic acid 3-O-glucuronide was detected in much lower quantity 0.56 ± 0.07 mg/g. The saponin mixture comprised bidesmosides of gypsogenin substituted at C-3 with trisaccharide and at C-28 with oligosaccharide substituted with aminoacyl or acetyl and (or) sulfate groups [1]. The cytotoxic activity was analyzed in the concentration range from 0.01 to 1000 μg/ml toward the chronic myelogenous leukemia (CML) cell line BV-173, the Hodgkin lymphoma cell line HD-MY-Z and the acute myelogenous leukemia (AML) cell line MV4-11 for 24 h of incubation. The results obtained show that at concentration of the crude extract > 10 µg/ml, cell viability was significantly reduced in the MTT test with IC50 38.08 μg/ml and 74.03 µg/ml for BV-173 and HD-MY-Z, respectively. The saponin mixture of bidesmosides had a lower cytotoxic activity on BV-173 with IC50 67.11 µg/ml as compared to that of the crude extract but showed the same effect on HD-MY-Z (IC50 = 60.66 μg/ml). Its activity on the AML cell line MV4-11 was similar to that of the other two cell lines (IC50 = 76.98 µg/ml). Less potent were the purified aminoacyl saponins (IC50 = 261.40 µg/ml for BV-173, 97.11 µg/ml for HD-MY-Z and 385.1 μg/ml for MV4-11). The combination between the aminoacyl saponins and the classical cytostatic etoposide was tested on the resistant HD-MY-Z cell line for an incubation period of 48 h. Results showed a significant additive effect by concentrations of the aminoacyl saponins higher than 100 µg/ml. Aminoacyl saponins were found to be less cytotoxic to assayed cell lines as compared to the crude plant extract and the saponin mixture of bidesmosides. Possibly the argenin substituent reduced their cytotoxicity. In G. trichotoma, gypsogenin 3-O-glucuronide derivatives could be responsible for the toxicity toward leukemia cell lines.

1. Bai H, Zhong Y, Xie YYet al. (2007) Chem Biodivers 4: 955–960. 2. Voutquenne-Nazabadioko L, Gevrenova R, Borie N et al. (2013) Phytochemistry 90: 114-127.

61

Poster P 12

Ecological cultures of medicinal and aromatic plants commercialized in food supplements

Elvira Gillea, Dana Bobitb, Georgiana Gavrila, Irina Bozc,d, Monica Hancianue

aNIRDBS / The „Stejarul” Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt, Romania; bS.C. Dacia Plant S.R.L., Harmanului fn, 507015-Bod, Brasov, Romania; cNIRDBS - Institute of Biological

Research, Lascar Catargi Street, no. 47, Iasi, Romania; dFaculty of Biology, “Alexandru Ioan Cuza” University, Carol I Bd., 20A, Iasi, Romania; eFaculty of Pharmacy, “Gr. T. Popa” University, Universitatii

16, 700115 Iasi, Romania

The aim: These studies aim in the ecological cultures set up by Dacia Plant (SME) to commercialize the raw material obtained under controlled conditions and to use them in different types of food supplements. Applied methods: By the phytochemical study we intended to determine the quality of the plant material obtained from our own cultivation. The obtained extracts were analyzed by means of spectrophotometry, TLC, HPLC and GC-MS to determine the amount of active principles such as total polyphenols, polyphenol carboxylic acids, flavonoids, antocyans, procyanidins, polysaccharides, iridoids, terpene compounds, and volatile oils/volatile oil fractions.

Results and conclusions: The project was developed from the necessity of our own strategy for medicinal plants species preservation. Since 2006, Dacia Plant company has established its own cultivation, and beginning with 2009 they have passed over to the completion of the experimental model by means of the concept of ecological culture. 4 ha were certified BIO in 2013 according to EU criteria [1,2,3]. In the present study we refer to the types of established cultures: interspersed cultures, associated cultures (Trigonellla foenum-graecum associated with Angelica archangelica or with Heracleum mantegazzianum). From the wild flora we took into culture species such as: Angelica archangelica (angelica), Chelidonium majus (greater celandine), Helleborus purpurascens (hellebore) or Eupatorium cannabinum, acclimatized allochthonous species as: Satureja montana (winter savory), Leuzea carthamoides (maral root) and Heracleum mantegazzianum (giant hogweed). At present, Dacia Plant has over 250 products in its portfolio in which there are about 200 aromatic and medicinal plants that are commercialized (80% are from Romania) in the form of: remedies, syrups, powders, teas, tablets, Tinctures. In the project to evaluate the effects of the adjuvant treatment with food supplements given to patients with lung affections between the following institutions are collaborating: NIRDBS/ “Stejarul” Biological Research Centre (the Qualitative and Quantitative Phytochemical Evaluation of Some Food Supplements); The Pneumophysiological Hospital, Bisericani, Neamt (evaluation of the patients due to a study regarding clinical investigations in the medicine field - approved of by the Ministry of Health, Nr. 75/2012); Dacia Plant SME Brasov (the food supplements producer). 1. Commission Regulation (CE) No. 834/2007. 2. Commission Regulation (EC) No 537/2009. 3. Commission Regulation (EU) No 1161/2011.

62

Poster P 13

Extraction processes to obtain some extracts enriched in hypericin from Hypericum perforatum species from natural populations

Ruxandra Cretua, Elena Ionescua, Gabriela Mitroia, Elena Iacoba, Carmen Tebrencua, Catrinel Giurescua

aComercial Society for Medicinal Plant Research and Processing PLANTAVOREL S.A., Cuza Voda 46, 610019

Piatra-Neamţ, Romania

St John’s Wort has a chemical composition well studied: flavonoids, naphthodianthrones (hypericin, pseudohypericin and isohypericin, protohypericin and protopseudohypericin), biflavonoids, phloroglucinol derivatives, and polyphenols [1]. Standardized extracts of H. perforatum are effective for the treatment of mild to moderate cases of depression [2], and also represent a natural source of antifungal and antibacterial agents [3].

Vegetal material was collected from the natural population from Brasov Depression, Barsa Country, conditioned in dry form according to Ph.Eur.6.0 and processed in order to perform extraction.

The paper presents various extraction processes of hypericin from Hypericum perforatum in order to obtain an extract with high content of hypericin. We used different methods and solvent for extraction: ethanol 50%, 60%, 70%, 80% v/v (1:10) at room temperature, for 10 days (T50%, T60%, T70%, T80%); ethanol 50%, 60%, 70%, 80% v/v (1:15), by reflux for 90 minutes (E50%, E60%, E70%, E80%); n-hexane and thereafter extraction of hypericin from leftover residue of the above extraction, with ethanol 70% v/v (1:15), by reflux for 90 minutes (EHX 70%) [2].

All extracts were qualitatively and quantitatively analyzed. The qualitative analysis consisted in spectroanalytical profile by HPTLC [1]. Detection of hypericin - equipment: CAMAG LINOMAT IV, CAMAG TLC 3 Scanner, WINCATS Planar Chromatography Manager. Chromatographic conditions: stationary phase - HPTLC plates G60F254 10 x 10 cm; wavelength - 366 nm after derivatization; mobile phase - ethyl acetate: formic acid: glacial acetic acid: water = 20:2,2:2,2:5,4 v/v; derivatization - 1% diphenylboryloxyethylamine (Natural Product, NP) in methanol, followed by 5% polyethylene glycol (PEG) in ethanol 96%; reference – hypericin. Total naphthodianthrones (as hypericin) were quantitatively evaluated. The absorbance of reaction mixture was measured at 590 nm with a CARY 50 UV/VIS spectrophotometer.

The results of the phytochemical analysis of hypericin in the extracts are presented in the following table:

Parameter Ethanol 50% Ethanol 60% Ethanol 70% Ethanol 80%

T50% E50% T60% E60% T70% E70% EHX70% T80% E80% Hypericin, %g/ml 0,0022 0,0033 0,0032 0,0032 0,0043 0,0031 0,0059 0,0033 0,0030

Hypericin, %g/g dry matter

0,086 0,176 0,116 0,167 0,224 0,162 0,335 0,139 0,174

According to our above-mentioned results, we can observe that the hydroalcoholic extract EHX70% has the highest content in naphthodianthrones expressed in hypericin, and may represent an important source of active principles with bioactive potential.

1. Nuevas-Paz L., Molina-Torres J., Prieto-González Sylvia (2005) Acta Farm. Bonaerense 24 (1): 89-90. 2. Koul V.K., Koul S. (2007) Natural Product Radiance, 6(4):293-296. 3. Arif T., Dabur M. (2011) Opportunity, Challenge and Scope of Natural Products in Medicial Chemistry, 283-311.

63

Poster P 14

Evaluation of Antioxidant and Antimicrobial Potential of Aerial Parts and Roots of Ferula caspica Bieb.

Çiğdem Kahramana, Didem Öztürkb, Melike Ekizoğlub, Zeliha Ş. Akdemira

aDepartment of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, 06100, Sıhhiye, Ankara, Turkey; bDepartment of Microbiology, Faculty of Pharmacy, Hacettepe University, 06100, Sıhhiye, Ankara, Turkey

Ferula species belong to the family Apiaceae and are used in traditional medicine. The genus Ferula L. is represented by 22 species, 12 of which are endemic in flora of Turkey [1-3]. These species, known as “çaşır”, have been used to treat hypercholesterolemia as well used as aphrodisiacs and immunostimulants in traditional Turkish medicine. The aerial parts of F. caspica Bieb. are also used for treatment of diabetes, stomach ache, and gynaecological diseases in Anatolia [4]. The aim of this study was to investigate antimicrobial and antioxidant properties 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of various extracts of F. caspica Bieb. While the aerial parts of F. caspica Bieb. were extracted with chloroform and then methanol, the roots of the title plant were extracted with petroleum ether, chloroform and methanol, respectively. Antimicrobial activity was determined by broth microdilution method against the bacteria Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus and the fungi Candida albicans, C. krusei, and C. parapsilosis [5,6]. Antioxidant properties were determined by DPPH radical scavenging activity assay [7,8]. The MeOH extract of aerial parts of F. caspica Bieb. showed a moderate antioxidant activity with 52.21 % inhibition of DPPH. The petroleum ether (64 μg/ml against E. faecalis, 128 μg/ml against S. aureus ) and the chloroform (64 μg/ml against E. faecalis, S. aureus) extracts of the roots and the chloroform (64 μg/ml against E. faecalis, 32 μg/ml against S. aureus) extract of the aerial parts of F. caspica Bieb. were found to be more effective against gram (+) bacteria than gram (-) bacteria. The chloroform extract of the plant showed antifungal activity against C. parapsilosis (64 μg/ml). These results may be used to explain the traditional usage of F. caspica Bieb., but further studies are required.

Table 1. Minimum Inhibitory Concentration (MIC) - ug/ml

Extracts/Fungi/bacteria C. albicans C. krusei C. parapsilosis E. coli E. faecalis S. aureus P. aeruginosa

Aerial Parts/CHCl3 Extract 256 256 128 512 64 32 512Aerial Parts/MeOH Extract 256 128 256 512 512 1024 512Root/ PE Extract 256 256 256 512 64 64 512Root/CHCl3 Extract 256 128 64 512 256 512 512Root/MeOH Extract 128 256 256 512 64 128 512Flukonazol 0.5 32 0.5 0.25 4 0.125 0.5

Table 2. DPPH radical scavenging activities of F. caspica extracts

200μg/ml 100μg/ml 50μg/ml 25μg/mlAerial Parts/CHCl3 Extract 28.16 17.35 14.27 11.62Aerial Parts/MeOH Extract 52.21 27.16 16.26 10.04Root/ PE Extract 13.26 8.02 6.62 3.53Root/CHCl3 Extract 31.19 19.14 12.33 6.19Root/MeOH Extract 28.04 14.93 9.90 4.70Ascorbic acid 94.10 94.22 94.17 94.17

1. Peşmen H (1972) Flora of Turkey and the East Aegean Islands, Vol 4, Edinburgh University Press, Edinburgh

2. Sagiroglu M, Duman H (2010) Ann. Bot. Fenn, 47: 293 – 300. 3. Duman H, Sagiroglu M (2005) Bot. J. Linn. Soc. 147: 357 – 361. 4. Altundag E, Ozturk M (2011) Procedia Soc. Behav. Sci. 19: 756 – 777. 5. Wayne P (2008) Reference method for broth dilution antifungal susceptibility testing of yeasts: approved

Standard, 3rd ed., M 27-A3 edn, Clinical and Laboratory Standards Institute. 6. Wayne P (2008) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically:

Approved Standard, 8th ed., M 07-A8 edn, Clinical and Laboratory Standards Institute. 7. Hatano T, Edamatsu R, Hiramatsu M, Mori A, Fujita Y, Yasuhara T, Yoshida T, Okuda T (1989) Chem.

Pharm. Bull. 37: 2016 – 2021. 8. Jensen SR, Gotfredsen CH, Harput US, Saracoglu I (2010) J. Nat. Prod. 73: 1593 – 1596.

64

Poster P 15

Use of LC-SPE-NMR and LC-MS to Characterise Caffeoylquinic Derivatives of Inositol from Dandelion Root

(Taraxacum officinale)

O. Kennya, b, T.J. Smytha, C.M. Hewageb, N.P. Bruntonc, P. McLoughlina

aDepartment of Food Biosciences, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland. bConway Institute of Biomolecular and Biomedical Research, UCD, Belfield, Dublin 4, Ireland.

cSchool of Agriculture & Food Science, UCD, Belfield, Dublin 4, Ireland.

Plant secondary metabolites are generally regarded as the biologically active components in many ancient and traditional medicinal plant remedies. For example, extracts and infusions of dandelion (Taraxacum officinale) root have been widely used in folk medicine for centuries. The present study reveals how in vitro antioxidant bioactivity guided fractionation of a dandelion root ethyl acetate extract has resulted in two fractions of interest (DF1 and DF2). LC-SPE-NMR and LC-MS were used to isolate and characterise a series of substituted inositol derivatives from each fraction. The 1H NMR spectra and MS data obtained for all peaks in DF1 revealed that these compounds were isomers of the chlorogenic acid derivative of inositol compound tetrahydroxy-5-[2-(4-hydroxyphenyl)acetyl]oxycyclohexyl-2-(4-hydroxyphenyl)acetate (C22H24O10, MW 448). Fraction DF2 consisted of tri-substituted inositol derivatives (C30H30O12, MW 582). For both fractions the compounds isolated are chlorogenic acid ester derivatives of inositols that vary in the number of chlorogenic groups present and the position and geometry of the inositol ring. DF1 is a group of di-substituted (Figure 1) and DF2 a group of tri-substituted inositols (Figure 2), which were determined both by comparison with previous inositol species isolated from the Taraxacum genus [1] and by comparison with the 1H NMR of other substituted inositols [2], with some of the compounds isolated in this case being previously unreported.

1. Gao, F., Wang, H., & Marby, T.J. (1990) Inositol derivatives and pseudoguaianolides from Hymenoxys

texana. Phytochemistry, 29 (7): 2273 – 76. 2. Zidorn, C., Ellmerer-Muller, E.P., & Stuppner, H. (1999) Eudesmanolides and inositol derivatives from

Taraxacum linearisquameum. Phytochemistry, 51 (8): 991 – 94.

65

Poster P 16

Chemical Composition and Antiglycoxidant Activities of French Organic Propolis extracts

Séverine Boisarda, Anne-Marie Le Raya, Marie-Christine Aumonda, Patricia Planchenaulta, Séverine Derbréa, Isabelle Péruchesb, Catherine Flurinb and Pascal Richommea

aEA 921 SONAS/SFR 4207 QUASAV, Université d’Angers,16 Bd Daviers ,49045 Angers Cedex 01, France;

bBallot-Flurin Apiculteurs, La miellerie, 65700 Lahitte-Toupière, France Propolis is a resinous natural substance collected by honeybees from buds and exudates of various trees and plants, mixed with bee wax and salivary enzymes. Bees generally use it as a sealer, to smooth out the internal walls of the hive as well as a protective barrier against intruders. Several pharmacological activities have been attributed to propolis extracts, mainly antibacterial, antiviral, antifungal as well as antioxidant properties [1]. A mixture of 24 batches of French propolis, supplied by “Ballot-Flurin Apiculteurs” (BFA), a company located in the South-West of France, was extracted with five different solvents: water, 70% ethanol, methanol, methylene chloride (DCM) and a mixture of solvents (DCM/methanol/water). Their chemical composition was determined by HPLC/DAD and HPLC/MS profiling followed, when necessary, by 1D and 2D NMR analysis (Fig. 1). Total polyphenol content and antioxidant activity were evaluated for these five extracts using respectively Folin-Ciocalteu, DPPH and ORAC assays. The antiglycation potential (anti-AGEs) of the same extracts was then evaluated using an automated test developed in our laboratory [2] and based on their ability to inhibit the formation of fluorescent Advanced Glycation End-products (AGEs).

Figure 1 : HPLC profiling (UV 254nm) of a DCM propolis extract All extracts from BFA organic propolis exhibited phenolic acids and esters as well as flavonoids as the predominant polyphenols, except for the aqueous one which predominantly contained phenolic acids. They all showed high antioxidant activities, about 2-5 times higher when compared to the rosemary extract which was recently approved as a food additive in Europe (E392) [3]. Except for the aqueous one they also exhibited a very good antiglycoxidant potential with anti-AGEs IC50 ≤ 0.06 mg/ml [reference : EtOH extract of Styphnolobium Japonicum (IC50 = 0.09 mg/ml)].

1. Castaldo S, Capasso F (2002) Fitoterapia 73: S1-S6. 2. Derbré S, Gatto J, Pelleray A, Coulon L, Séraphin D, Richomme (2010) Anal. Bioanal. Chem. 398: 1747-

1758. 3. Official Journal of European Union (Directive 2010/67/UE – L277/17).

66

Poster P 17

The Study of Volatile Oil in Chemovarieties of Origanum vulgare cultivated in Romania

Irina Mihalachea, Georgiana Gavrilb, Adrian Spaca, Catalina Drutuc, Radu Neculaa,b, Elvira Gilleb

aFaculty of Pharmacy, “Gr. T. Popa” University, Universitatii 16, 700115 Iasi, Romania;

bNIRDBS / The „Stejarul” Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt, Romania; cStation of Agricultural Research – Development Secuieni – Neamt, 617415, Romania

Aim: To identify some correlations between certain morphological characters and the composition of the volatile oil obtained from three chemovarieties of Origanum vulgare. The plants were cultivated at SCDA Secuieni, Neamt, in certified ecological cultures. The five year studies on the same cultures and the phytochemical investigations were made on samples harvested in the period of flowering. The selection criterion was the flower colour, the variants with pink, red and white flowers being selected. Applied methods: The separation of the volatile oil from the fresh vegetal material was made due to the European Pharmacopoeia (Ph. Eu.6, 2008,) by hydro distillation in Neo-Clevenger. The separation and identification of the volatile compounds was achieved by means of gas chromatography with mass spectrometry (GC-MS), Agilent Technologies type 6890N, Network GC System, MS Agilent Technologies type 5975 inert XL Mass Selective Detector. Results and conclusions: After the phytochemical analysis we noticed the presence of the following compounds: β-caryophyllene, germacrene D, β-sabinene, caryophyllene oxide in all three variants, camphene (6.20%) in in red flower Origanum vulgare and β-bisabolene (5.60%) in white flower O. vulgare, compounds with antioxidant action [1,2].

Tab.1. The main compounds identified in the volatile oils in chemovarities of Origanum vulgare

RT (min.) Compounds Samples Origanum vulgare – Area%

Pink flowers Red flowers White flowers 5.861 β-sabinene 11.71 5.29 10.37 6.087 β-myrcen 1.04 1.24 1.17 6.519 α-terpinene – 0.66 0.75 6.657 p-cymene 3.28 2.52 2.11 6.969 α-ocimene 12.67 – – 6.995 trans-β-ocimene – 11.14 11.50 7.160 γ-terpinene 2.98 3.27 4.18 7.748 β-linalool 1.28 2.48 9.211 camphene – 6.20 –

12.466 β-caryophyllene 20.24 13.78 14.85 11.963 β-bourbonene – 0.59 – 13.305 germacrene D 11.56 12.26 8.73 13.505 trans-α-farnesene 7.20 8.03 – 13.548 β-bisabolene – – 5.60 13.755 δ-cadinene 1.35 1.70 2.16 14.517 spathulenol 2.36 2.17 3.40 14.603 caryophyllene oxide 8.65 6.07 5.65

The results (tab.1) show the existence of a qualitative and quantitative variability, the pink plants have over 10% volatile fractions which give the oregano aroma, used in cosmetics and aroma therapy. The researches proved the correlation between the morpho-criteria and the volatile oil composition.

1. Mockutë D., Bernotienë G., Judpentienë A. (2004) Chemical composition of essential oils of Origanum vulgare L. growing in Lithuania Biologija 4: 44–49.

2. Lukas B., Schmiderer C., Novak J. (2013) Phytochemical diversity of Origanum vulgare L. subsp. Vulgare (Lamiaceae) from Austria Biochemical Systematics and Ecology 50:106–113.

67

Poster P 18

In vitro evaluation of antioxidant and antiproliferative effects of Pinus cembra L. extracts

Anca Mironb, Cristina Lungu Apetreia, Cosmin Teodor Mihaic, Pincu Rotinbergc, Adriana Trifanb, Ana Clara Aprotosoaieb

aDepartment of Plant and Animal Biology, bDepartment of Pharmacognosy, Faculty of Pharmacy, University of

Medicine and Pharmacy "Gr. T. Popa" Iasi, 700115, Iasi, Romania; c National Institute of Research and Development for Biological Sciences/Institute of Biological Research Iasi, 700107, Iasi, Romania

Pinus cembra L. (cembran pine, Swiss stone pine, Arolla pine) is naturally spread in

the Central European Alps and Carpathian Mountains. It grows at high altitudes being permanently exposed to stress factors (low temperatures, elevated ozone levels, irradiance). Although cembran pine has high resistance to biotic and abiotic stress factors, few studies have investigated its pharmacological potential. The aim of the present work was to examine the phenolic content, antioxidant and antiproliferative effects on HeLa cells of the hydromethanolic extracts from cembran pine bark, needles, cones and twigs. The phenolic content and profile were studied using spectrophotometry and HPLC-DAD-ESI-MS [1]. The antioxidant activity was investigated by several in vitro assays: reducing power assay, DPPH and ABTS radicals scavenging assays, ferrous ion chelating ability assay [1,2]. The antiproliferative activity on HeLa cells was initially screened by a protein synthesis inhibition assay. Cell proliferation, viability, apoptosis and cell cycle assays were also performed by flow cytometry [3]. Bark extract had the highest total phenolic, flavonoid and proanthocyanidin contents (299.3±1.4, 125.3±1.2 and 74.3±0.5 mg/g, respectively). Bark extract was found to possess the highest reducing capacity (EC50= 26.0±0.3 μg/mL), DPPH and ABTS radicals scavenging effects (EC50=71.1±0.5 and 6.3±0.2 μg/mL, respectively). In addition, bark extract significantly inhibited the protein synthesis in HeLa cells; at 200 μg/mL, the inhibition on protein synthesis reached 96.66±0.73% as determined by methylene blue assay. The inhibition on HeLa cells proliferation was further confirmed by CFSE flow cytometry. In cell cycle analysis with NIM-DAPI reagent, a 48 h incubation with bark extract (200 μg/mL) considerably increased the number of dead/apoptotic HeLa cells in comparison with the control (81.78±1.73% vs. 6.03±0.55%); the percentages of HeLa cells in G0/G1-, S- and G2/M-phases were lower than those in the control (12.66±1.20% vs. 63.93±0.94%, 3.98±0.37% vs. 17.53±0.78%, 1.23±0.38% vs. 11.67±0.35%). Among the other cembran pine extracts, needle extract showed promising antiproliferative effects. In the same experimental conditions, it inhibited the protein synthesis in HeLa cells by 97.45±0.50% and increased the number of dead/apoptotic cells to 63.52±1.14%. The results of this study suggest that Pinus cembra L. bark and needles contain phytochemicals of therapeutic interest with respect to antioxidant and antiproliferative effects and therefore further investigation should be done to identify the active constituents and their way of action.

1. Wangensteen H, Samuelsen AB, Malterud KE (2004) Food Chem 88: 293 – 297. 2. Re R, Pellegrini N, Proteggente A et al. (1999) Free Radic Biol Med 26: 1231 – 1237. 3. Li H, Wang LJ, Qiu GF et al. (2007) Food Chem Toxicol 45: 2040 – 2046.

68

Poster P 19

New flavonoides from Helleborus caucasicus A.BRAUN.

Tamar Muzashvilia,b, Milena Masulloc, Mariusz Kowalczykb, Ether Kemertelidzea, Wiesław Oleszekb, Sonia Piacentec, Anna Stochmalb

aIovel Kutateladze Institute of Pharmacochemistry, 36 P. Sarajishvili st., 0159, Tbilisi, Georgia; bDepartment of

Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation State Research Institute, ul. Czartoryskich 8, 24-100 Puławy, Poland; cDipartimento di Scienze Farmaceutiche e Biomediche, via Ponte Don

Melillo, 84084 Fisciano, Salerno, Italy; E-mail:[email protected] Helleborus species (Ranunculacae) are studied mostly for their content in steroidal

compounds. In recent years phenolic glycoside derivatives and quercetin glycosides have been reported from the aerial parts of some European plant spp. [1-3].

In present work we report on the investigation of Helleborus caucasicus A. BR., a widespread in Georgia Caucasian endemic plant, for its phenolic composition.

Leaves of H. caucasicus were defatted with CHCl3 in a Soxhlet apparatus. Defatted plant material was extracted with 80% MeOH two times at 45°C. After removing solvent, dry residue was suspended in water and passed through a short preparative column (6 cm × 10 cm, LiChroprep RP-18, 40-63 µm, Merck) preconditioned with water, followed by washing with water to remove sugars and 40% MeOH to eluted phenolics. Freeze-dried phenolic fraction after suspending in water was applied onto a 4 cm × 50 cm, 40-63 µm LiChroprep RP-18 column (Millipore Corp., Bedford, MA), first washed with distilled water and then with increasing concentration of aqueous methanol (2.5 % increments from 0 % to 100% MeOH). Fractions containing 1-3 major compounds were further purified on a 2 cm × 50 cm, 25-40 µm RP-18 glass column using an isocratic system (ACN-1% AcOH) optimized for each fraction based on the analytical separation. This yielded isolation of six new flavonoides: quercetin-3-O-β-D-xylopyranosyl-(1→2)-O-β-D-galactopyranosyl-7-O-β-D-glucopyranoside (1), kaempferol-3-O-[2-(E-caffeoyl)]-β-D-xylopyranosyl-(1→2)-β-D-galactopyranosyl-7-O-β-D-glucopyranoside (2); quercetin 3-O-[2-(E-caffeoyl)]-β-D-xylopyranosyl-(1→2)-β-D-galactopyranosyl-7-O-β-D-glucopyranoside (3); quercetin-3-O-[2-(coumaroyl)]-β-D-xylopyranosyl-(1→2)-β-D-galactopyranosyl-7-O-β-D-glucopyranoside (4); quercetin-3-O-[3-(E-caffeoyl)]-β-D-xylopyranosyl-(1→2)-β-D-galactopyranosyl-7-O-β-D-glucopyranoside (5) and quercetin-3-O-β-D-galactopyranosyl-(3-hydroxy-3-methylglutaroyl)-7-O-β-D-glucopyranoside (6), along with the known quercetin-3-O-β-D-xylopyranosyl-(1→2)-β-D-galactopyranoside (7), 3-hydroxyl-2-methyl-4-H-pyran-4-one-3-O-(6-O-caffeoyl)-β-D-glucopyranoside (8) and cholest-7-en-3-one (9).

The structures of single compounds were established by spectrometric (ESI-MS) and spectroscopic (UV, NMR) means.

O

HO

HOO

HOH2C

OHO

HOOH

O

OOH

O

OH

OHO

HOOH

HOH2C

O

OH

1

3

O

HO

HOO

HOH2C

OHO

HOO

O

OOH

O

OH

OHO

HOOH

HOH2C

O

HO

O

OH

4

O

HO

HOO

HOH2C

OHO

OOH

O

OOH

O

OH

OHO

HOOH

HOH2C

O

HO

O

OH

5

O

HO

HOOH

O

OOH

O

OH

OHO

HOOH

HOH2C

O

OH

HO

OOH

O

O

6HO

O

HO

HOO

HOH2C

OHO

HOO

O

OOH

O

OH

OHO

HOOH

HOH2C

O

HO

HO

O

OR1

R = H

R = OH2

1. Braca A, Prieto J.M, Tommasi N.D., Tome F, Morelli I (2004) Phytochemistry 65: 2921-2928 2. Prieto J.M,Siciliano T, Braca A (2006) Fitoterapia 77: 203-207 3. Vitalini S, Braca A, Fico G (2011) Fitoterapia 82: 152-154

Acknowledgement: The work was supported by the Seventh Framework Program of European Community, PROFICIENCY (Contract No. 245751). The authors declare no competing financial interest.

69

Poster P 20

Contribution to the Phytochemical Study of Vaccinium vitis-idaea L. Wild Populations from Eastern Romanian Carpathians

Doina Danilaa, Camelia P. Stefanachea,b, Radu Neculaa,c, Valentin Grigorasa, Anca Mironc

a National Institute of R&D for Biological Sciences/ “Stejarul” Biological Research Centre, Piatra Neamt, Alexandru cel Bun 6, 610004, Romania; bFaculty of Biology, “Al. I. Cuza” University, Carol I 20 A, 700505,

Iasi, Romania; cFaculty of Pharmacy, “Gr. T. Popa” University, Universitatii 16, 700115 Iasi, Romania

The diversity of the wild plant resources continues to be an essential component in the strategies that envisage human nutrition and health, locally and regionally. By optimizing the use of the genetic resources and of the biodiversity in natural habitats and in agrosystems, their conservation and capitalization is stimulated.

Vaccinium vitis-idaea is a shrub species of the Ericaceae Family, with high value both for nutritional and health purposes. The phytochemical compounds belonging to polyphenols (e.g. proanthocyanidins) and others (e.g. arbutin), confers a wide applicability in phytopharmacy due to hypoglycemic, antioxidant, antiinflammatory and antimicrobial activity of the plant material and extract [1, 2].

The aim of our studies was the development of preliminary estimations of the phytochemical diversity in the natural populations of V. vitis-idaea from Romanian Eastern Carpathians. These will be of great importance both for the capitalization from the natural populations (plant material with known bioproductive features) and for the selection of the seeding plant in the establishment of cultures in agrosystems.

The plant material was collected from the wild populations in the Bistrita Valley. The qualitative analysis of methanolic extract was performed by TLC and RP-HPLC-UV and the quantitative one was made by VIS spectrophotometry. The samples consisted in leafs, preserved fruits (dried and frozen) and frozen juice (obtained from fresh fruits). In the dried fruits of V. vitis-idaea, the content of phenolic acids was ranging from 554.35 - 569.87 mg/100 g d.w. (caffeic acid equiv.), while the phenolic content was 3.788 - 4.048 g/100 g d.w. (gallic acid equiv.).

Table 1. Spectrophotometric analysis of the methanolic extracts of V. vitis-idaea samples

No. crt. Sample mg/100g dried plant material

Hiperoside Apigenin-7-O-glucoside Quercetin Luteolin 1. Frozen juice 78.92 27.98 47.82 <LD* 2. Frozen fruits 69.28 33.90 44.07 <LD* 3. Dried leafs (A) 1366.27 29.84 46.98 8.59 4. Dried fruits (A) 84.94 33.90 49.20 3.00 5. Dried leafs (B) 1431.33 35.67 46.71 7.92 6. Dried fruits (B) 100.60 45.05 49.76 3.84

<LD* = value below the detection limit; A,B-wild subpopulations near Ortoaia spring (North-East Romania)

The RP-HPLC-UV analysis revealed hyperoside as the major phenolic compound, with minimum and maximum values (mg/100 g d.w.) of 1366.27 - 1431.33 in leafs and of 78.92 - 100.60 in fruits. This was followed by quercetin and apigenin-7-O-glucoside, in similar amounts both in leafs and fruits. The luteolin was found only in the dried samples, in higher amounts in fruits than in leafs.

These results are part of a preliminary study, which will be further developed in order to assess the cultivation possibilities and capitalization of the plant material (food development). 1. Törrönen R, Kolehmainen M, Sarkkinen E, Mykkänen H, Niskanen L. (2012) Am J Clin Nutr. 96(3):527-33. 2. Mane C, Loonis M, Juhel C, Dufour C, Malien-Aubert C. (2011) J. Agric. Food Chem. 59: 3330–9.

70

Poster P 21

Ergostane Derivatives from Termitomyces microcarpus (Lyophylaceae)

Alice W. Njueab, Peter K. Cheplogoia, Josiah O. Omoloa, Moses K. Langatb, Dulcie A. Mulhollandb

aDepartment of Chemistry, Egerton University, 536-20115, Egerton, Kenya; bDepartment of Chemistry, Faculty

of Engineering and Physical Sciences, University of Surrey, GU2 7XH, United Kingdom

Termitomyces is a genus of basidiomycete fungi and there are about 30 species in the genus. Termitomyces microcarpus is one of the species of this fungus. It is an edible species, found in Africa and Asia, where it grows in groups or clusters in deciduous forest near the roots of tree stumps associated with termite nests [1]. They are a food source for a sub family of termites, the macrotermites, who enjoy an obligate symbiosis and are also a popular wild food for people. A highly degraded sterol dimethylincisterol (1) [2] was isolated as a colourless oil, 5α,8α–epidioxyergosta-6,9(11),22-trien-3β-ol (2) [3] and 5α,8α–epidioxyergosta-6,22-dien-3β-ol (3) [4] were also isolated from the methanol extract. The structures were determined using 1D and 2D NMR spectroscopic studies and the spectroscopic data showed agreement with those reported in the literature. These cytotoxic sterols have been isolated previously from a marine sponge, Homaxinella sp. and herbaceous plants, Helianthus tuberosus but not from this family. The compounds will be submitted to National Cancer Institute (NCI) for anticancer screening against NCI 59 human tumour cell line panel. 1. Zhishu B, Zheng G, Taihui L (1993) The Macrofungus Flora of China’s Guangdong Province. Columbia

University Press, New York. 2. Mansoor TA, Hong J, Lee CO, Bae SJ, Kwang SI, Jung JH (2005) J. Nat. Prod. 68: 331-336. 3. Liu XH, Tang XZ, Miao PF, Ji NY (2011) Nat. Prod. Commun. 6: 1243-124 4. Li XD, Miao PF, Ji NY (2011) Molecules 16: 8646-8653.

O

H

OHO

2

O

O

H1

HO

O

H

OHO

H

3

71

Poster P 22

Synthesis of 2,3-disubstituted quinazolin-4-one by condensation of a nucleophile with 2-methyl-6,7 disubstituted-1, 3- benzo-oxazine-

4-one

P.O. Osarumwensea,b, M.O. Edemab, O, Usifohc

a,b*Faculty of Physical Sciences, University of Benin, cFaculty of Pharmaceutical Chemistry, University of Benin

The condensation of 2-amino-methyl-4, 5-dimethoxybenzoate with acetic anhydride yielded the cyclic compound 2-methyl-4, 5-disubstituted-1, 3-benzo-oxazine-4-one which further produce a novel 2,3-disubstituted quinazolin-4 ones via the reaction with hydrazine hydrate. The compounds synthesized were unequivocally confirmed by means of Infrared, Nuclear Magnetic Resonance (1H and 13C), Gas Chromatography Mass Spectrophotometer and Elemental analysis.

Keywords: Quinazoline-4-one, 2-methyl-6, 7-disubtituted 1, 3-benzo-oxazine-4-one, Nucleophile.

Scheme 2:

Where: R1=OCH3, R2=OCH3 and R3=H

72

Poster P 23

New Triterpenoid Glycosides from the Aerial Parts of Alsike Clover (Trifolium hybridum L.)

Andy J. Péreza, Mariusz Kowalczyka, Ana M. Simonetb, Francisco A. Maciasb, Wiesław Oleszeka, Anna Stochmala

aDepartment of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, ul. Czartoryskich 8, 24-100, Puławy, Poland; bGrupo de Alelopatía, Departamento de Química Orgánica, Facultad

de Ciencias, Universidad de Cádiz, C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain.

Five azukisapogenol glycosides (1-5) have been isolated from the aerial parts of Alsike Clover (Trifolium hybridum L.) and their structures were elucidated by combined spectroscopic (1D and 2D NMR), spectrometric (HRESIMS, ESI-MS/MS) and chemical methods. Three of them are new compounds and were identified as 3-O-[-α-L-arabinopyranosyl(1→2)]-β-D-glucuronopyranosyl azukisapogenol (1), 3-O-[-β-D-glucuronopyranosyl(1→2)-β-D-glucuronopyranosyl]-29-O-β-D-glucopyranosyl azukisapogenol (2) and 3-O-[-α-L-arabinopyranosyl(1→2)-β-D-glucuronopyranosyl]-29-O-β-D-glucopyranosyl azukisapogenol (3) [1]. The remaining two (4, 5) are known compounds but have not been previously described as saponin constituents of the genus Trifolium.

An interesting feature of these compounds is the identification of azukisapogenol as a new aglycone for saponins of the Trifolium species. Soyasapogenols were not found in the aerial parts of this plant, while as previously reported they were constituents of saponins occurring in the seeds [2]. Thus, this difference in content of specific triterpenoid saponins in different parts of the plant may suggest a specially differentiated biosynthetic pathway, perhaps as a result of different oxidation steps of β-amyrin and/or different biological functions.

This work has recently been published (Isolation and structural determination of triterpenoid glycosides from the aerial parts of alsike clover (Trifolium hybridum L.) by Pérez, Andy J.; Kowalczyk, Mariusz; Simonet, Ana M.; Macías, Francisco A.; Oleszek, Wiesław; Stochmal, Anna. Journal of Agricultural and Food Chemistry (2013), 61(11), 2631-2637).

Compound R1 R2

1 α-L-Arap H

2 β-D-GlcAp β-D-Glcp

3 α-L-Arap β-D-Glcp

4 H H

5 H β-D-Glcp

Structures of saponins 1-5 isolated from T. hybridum aerial parts.

1. Pérez A.J, Kowalczyk M, Simonet A.M, Macías F.A, Oleszek W, Stochmal A (2013) J. Agric. Food Chem.

61: 2631 – 2637. 2. Oleszek W, Stochmal A (2002) Phytochemistry 61: 165 - 170.

73

Poster P 24

Bromoindoles from Iotrochota baculifera

Chadaporn Prompanyaa, b, Sindhchai Keokitichaib, Madalena Pintoc, Anake Kijjoaa

aICBAS- Instituto de Ciêncas Biomédicas de Abel Salazar and CIIMAR, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; bFaculty of Pharmaceutical Sciences, Burapha University, Bangsaen, Chonburi, Thailand; cCEQUIMED-UP and Laboratório de Química Orgânica e Farmacêutica,

Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal

The oceans encompass about 71% of the surface of our planet, and they represent the greatest extremes of temperatures, light, and pressure encountered by life. The immense biodiversity of the marine biosphere has proved to be an extraordinary source of new metabolites isolated from numerous organisms, including algae, sponges, molluscs, tunicates and phytoplanktons [1].

Marine sponges have been recognized as a rich source of structurally novel compounds and a large number of sponge-derived bioactive metabolites have been isolated and identified so far, some of which have pharmaceutical potential as anticancer, antiviral and anti-inflammatory drugs or as biomedical tools [2]. Consequently, our group has investigated secondary metabolites from the marine sponges, collected from the Gulf of Thailand. Examination of the ethyl acetate extract of the marine sponge Iotrochota baculifera, collected from the Gulf of Thailand, Chonburi Province, led to isolation of methyl (2E)-3-(6-bromo-1H-indol-3-yl)-prop-2-enoate (1), 6-bromo-1H-indole-3-carbaldehyde (2) and sterols so far. The structures of 1 and 2 were established by 1D and 2D NMR as well as HRMS spectral analysis.

1. Napolitano J, Daranas A, Norte M (2009) Anti-cancer Agents in Medicinal Chemistry 9, 122-137. 2. Blunt J, Copp B, Hu W (2009) Nat Prod Rep 26: 170-244.

1

5'

6'

7'

1

2

3

2

3'

5'

6'

7'

4'

3'

2'

4'

2'1'1'

74

Poster P 25

Major compounds and antimicrobial activity of Cynara scolymus and Rosmarinus officinalis against Bacillus subtilis ATCC 6633

strain

Steliana Rodinoa,b, Alina Butub, Marian Butuc, Petruta Calina Corneaa aDoctoral School – Engineering and Resources Management, University of Agronomic Sciences and Veterinary

Medicine, Marasti 59, Bucharest, Romania, bBiotechnology Department, National Institute of Research and Development for Biological Sciences, Splaiul Independentei 296, Bucharest, Romania, cBioinformatics

Department, National Institute of Research and Development for Biological Sciences, Splaiul Independentei 296, Bucharest, Romania, [email protected]

The main target of this presentation is to report on the preliminary study of the in vitro

antibacterial activity of the extracts of Cynara scolymus (artichoke) and Rosmarinus officinalis (rosemary) against Bacillus subtilis. The artichoke and rosemary from the spontaneous flora have been used in traditional medicine in Romania since ancient times. The crop establishment of Cynara scolymus and Rosmarinus officinalis took place due to higher demand in national and international markets and in the same time, due to favourable pedoclimatic conditions. Cynara scolymus has a rich composition in polyphenols (chlorogenic acid, cynarin, gallic acid, etc.) [1, 2], inulin [3], vitamin C [4] and does not accumulate toxic compounds [5]. Rosmarinus officinalis contains phenolic acids, monoterpenes, diterpenes, triterpenes, sesquiterpenes, triterpenoid saponins (betulinic acid).

The extracts were obtained from whole aerial part of plants by cold maceration in 50% ethanol. The vegetal materials were collected from Southern Romania. In order to assess the antibacterial activity of the studied plant extracts it was used the Kirby-Bauer diffusion technique, based on the properties of the solutions to diffuse in the culture medium at different distances from the point of application. The Muller Hinton culture medium was inoculated with Bacillus subtilis ATCC 6633 strain, from a 20 hours old culture. On the culture medium were added 6 mm sterilised filter paper discs (Whatman no 1), impregnated with the selected plant extracts. The disc impregnated with solvent was considered control.

The radius of the inhibition area for the tested vegetal materials was 7 and respectively 9 mm (figure: 10 - Cynara scolymus extract, 11 - Rosmarinus officinalis extract, M - control), which proves the antibacterial activity of the extracts used in the experiments on Bacillus subtilis ATCC 6633 strain.

The results obtained confirm the use of these plants in traditional medicine as antibacterial treatment and encourages us to continue studies in this direction.

1. Lattanzio V, Kroon P A, Linsalata V, Cardinali A (2009) J. Funct. Foods 1: 131-144. 2. Mutalib A, Nasser A G (2012) Pharm Sci, 21: 6-13. 3. Pandino G, Lombardo S, Mauromicale G, Williamson G (2011) J. Food Compos. Anal. 24: 148-153. 4. Orlovskaya T V, Luneva I L, Chelombit¢ko V A (2007) Chemistry of Natural Compounds, 43: 239-240. 5. Ceccarelli N, Curadi M, Picciarelli P, Martelloni L, Sbrana C, Giovannetti M (2010) Mediterr. J. Nutr.

Metab. 3: 197-201.

75

Poster P 26

Determination of tRNA-bound cytokinins in microalgae and cyanobacteria using UHPLC-MS/MS

Jan Šimuraa,b, Ondřej Nováka, Ladislav Nedbalc, Miroslav Strnada,b

aLaboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR,

Šlechtitelů 11, 783 71 Olomouc, Czech Republic; bCentre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 21, CZ-783 71 Olomouc, Czech

Republic; cGlobal Change Research Centre AS CR, Drasov 470, 664 24 Drasov, Czech Republic

For cytokinin biosynthesis two different pathways have been described: 1) de novo biosynthesis of free cytokinins and 2) the liberation of cytokinins from tRNA. Endogenous levels of free cytokinins and tRNA-bound cytokinins were quantified in different representatives of microalgae and cyanobacteria over phylogenetic tree.

A new method for extraction and purification of tRNA-bound cytokinins was developed based on phenol/m-cresol treatment followed by alkaline hydrolysis and enzymatic dephosphorylation of nucleotides.[1] Samples were then purified using MCX solid phase extraction column[2] and analyzed on UHPLC-MS/MS.[3] Samples for free cytokinins analysis were prepared using two ion-exchange chromatography (IEC) steps (SCX, DEAE-Sephadex combined with SPE C18-cartridges) and immunoaffinity chromatography (IAC).[4,5]

The predominant free cytokinins present in the dry samples were isopentenyle-type (iP) in Cyanobacteria and cZ-type (cis-zeatin) in Chlorophyta. Four cytokinins [cis-zeatin riboside (cZR), N6-(2-isopentenyl) adenosine (iPR), trans-zeatin riboside (tZR), dihydrozeatin riboside (DHZR)] were detected in the tRNA extracts and these generally occurred in higher concentrations compared to the free cytokinin forms. In Chlorophyta, the cZ-type cytokinins were the prevalent form in tRNA-bound cytokinins, whereas iP-type cytokinins were the prevalent cytokinins in Cyanobacteria. Cytokinin homeostasis is regulated by biosynthetic rates, interconversion between conjugate forms and degradation is controled by CKX (cytokinin oxidase/dehydrogenase). These results show that there are the differences in cytokinin metabolism between cyanobacteria and chlorophyta. The finding that cZ-type and iP-type cytokinins are dominant is fully compatible with the assumption that tRNA-mediated cytokinin biosynthesis can be an important source of cytokinins.

1. Maass, H.; Klaembt, D. (1981) Planta 151: 353-358 2. Dobrev,P; Kamínek, M. (2002) Journal of Chromatography A 950: 21–29 3. Svačinová, J. (2012) Plant Methods 8:17 4. Novák, O. (2003) Analytica Chimica Acta 480: 207–218 5. Novák, O. (2008) Phytochemistry 69: 2214–2224

76

Poster P 27

Comparative Phytochemical Analysis for Agastache rugosa Kuntze Experimental Variants in Conventional Cultures

Camelia Stefanachea,b, Doina Danilaa, Radu Neculaa,c, Valentin Grigorasa, Elvira Gillea

aNational Institute of R&D for Biological Sciences Bucharest, “Stejarul” Biological Research Centre Piatra Neamt, Alexandru cel Bun 6, 610004, Piatra Neamt, Romania; bFaculty of Biology, “Alexandru Ioan Cuza”

University of Iasi, Carol I 20 A, 700505, Iasi, Romania; cFacultaty of Pharmacy, „Grigore T. Popa” University of Medicine and Pharmacy, 600388, Iasi, Romania

Agastache rugosa Kuntze, Lamiaceae family, is a medicinal, aromatic and melliferous plant with traditional use in China to treat fever, stomach ailments, and angina aches. More recent studies highlighted the antitumour, antifungal, antiviral and cytotoxic activities of the extract [1]. It has a wide distribution in Eastern Asia, in Romania is found only in cultures, especially for decorative purposes.

The aim this study is the evaluation of the content of polyphenolcarboxylic acids of A. rugosa experimental variants in conventional cultures established in the experimental field of “Stejarul” Biological Research Centre, Piatra Neamt in order to determine the optimum cultivation characteristics for the production of a plant material rich in bio-active compounds.

The plant material (herb and flowers), harvested in August, September and October, consisted of 2 plants regenerated in vitro, 2nd year of vegetation (1A, 1S; 2A, 2S) [2] and their offspring generatively generated (3S, 3O; 4S, 4O) – with blue flowers, and plants obtained from generatively propagated plants, with white (which further generated plants with white flowers – 5S, and blue flowers – 6S, 6O) and blue flowers (7O). The variants 3-7 consisted in plants in the first year of vegetation.

For the phytochemical analysis of the polyphenols, the methanolic extracts of the plants material were subjected to TLC, RP-HPLC-UV and spectrophotometry analysis.

The spectrophotometric methods where used in order to determine the total content of phenols, expressed as rosmarinic acid equiv. (mg/100 g d.w.) – with values between 1317 for variant 4O and 3513 for the variant no. 7O, or as caffeic acid equiv. (mg/100 g d.w.) 1045 for variant 4O and 2597 for variant 7O. For the total polyphenolic content (expressed in gallic ac. equiv. mg/100 g d.w.) the values varied between 1688 in variant 4O and 3665 in variant 7O.

The RP-HPLC-UV analysis highlighted the rosmarinic acid as the major phenolic compound, the minimum and maximum values with values (mg/100 g d.w.) varying from 38.85 for variant 4O and 2095.87 for variant 7O. This was followed by chlorogenic acid (mg/100 g d.w.), with values of 50.19 for variant 5S and 344.05 for variant 7O, p-coumaric acid with values of 20.81 for variant 5S and 24.35 for variant, caffeic acid with values of 15.02 for variant 4O and 26.37 for variant 1S. The flavonoid apigenin and its glycosylated derivatives were found in amounts varying from 909.69 in variant 1A and 3275.13 for variant 3O acid (mg/100 g d.w.).

For the plants generatively derived from the plant with white flowers, the ones with white flowers had a higher amount in total phenols compared with the descendants with blue flowers, regardless of the harvesting time. The generative descendants of the in vitro regenerated plants had a higher content in total polyphenols, compared with their mother plants.

A high diversity in the content of polyphenols was observed both between the plants regenerated in vitro – 2nd of vegetation and the plants obtained generatively – 1st year of vegetation. The experiment will be continued in order to identify, characterize, and isolate the chemotypes within these experimental variants.

1. Lee C, Kim H, Kho Y (2002) J. Nat. Prod., 65: 414-416. 2. Stefanache C, Danila D, Gille E, Necula R, Falticeanu M (2012), Proc. of 7th CMAPSEEC, 74 – 80.

77

Poster P 28

Molecular mechanisms of brassionosteroid action and receptor interactions in human carcinoma cells

Jana Steigerováa,b, Lucie Rárovác, Kateřina Křížováb, Jana Oklešťkovád, Michaela Šváchováe, Zdeněk Kolářa,b and Miroslav Strnadc,d

aLaboratory of Molecular Pathology, Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15 Olomouc, Czech Republic;bInstitute of Molecular and

Translation Medicine, Faculty of Medicine and Dentistry, Palacký University and Faculty Hospital in Olomouc, Puškinova 6, 77 20 Olomouc, Czech Republic; cCentre of the Region Haná for Biotechnological and Agricultural Research, Department of Growth Regulators, Faculty of Science, Palacký University,

Šlechtitelů 11, 783 71 Olomouc, Czech Republic; dLaboratory of Growth Regulators, Palacký University & Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic; eDepartment of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacký University and University

Hospital Olomouc, I. P. Pavlova 6, 775 20 Olomouc, Czech Republic The study of plant-derived compounds with effect at the molecular level has become

an important approach in the selection of new agents with antitumour activity in humans. Brassinosteroids (BRs) are plant growth regulators representing a group of newly-discovered agents with relatively wide-ranging effects in plants. Like steroid hormones in animals, structurally BRs consist of a cholesterol skeleton with various hydroxyl substitutions and functional groups required for biological activity. To date, over 70 BRs have been identified in 50 plant species and currently 42 brassinosteroid metabolites and their conjugates are known. Natural BRs and their synthetic derivatives caused growth inhibition, cell cycle arrest and initiation of apoptosis in different human cancer cell lines. Based on the structural motifs of these agents, a possible explanation for their cytotoxic effects is that they may bind to steroid receptors. In hormone-sensitive cancer cells, BR treatment resulted in alterations of localization and expression of the steroid hormone receptors (ER-α, ER-β, AR). The comparison of the effects of natural BRs and their analogues on steroid receptors, and detailed characterization of their influence on hormone-sensitive and hormone-insensitive human carcinomas, could both provide extend fundamental knowledge of hormone-receptor interactions and have valuable medical applications.

This work was supported by grant from the Ministry of Health of the Czech Republic (NT11060), the Biomedicine for Regional Development and Human Resources Project (BIOMEDREG) C.1.05/2.1.00/01.0030, grant ED0007/01/01 of Centre of the Region Haná for Biotechnological and Agricultural Research, grants No. IAA400550801 and 1M06030.

78

Poster P 29

Phytochemical screening of Urtica dioica extracts: preliminary study for their utilization as antimicrobial and antifungal agents

Carmen Tebrencu a Ruxandra Cretua, Gabriela Mitroia, Elena Iacoba, Maria Chiriaca Valentin Grigorasb

aComercial Society for Medicinal Plant Research and Processing PLANTAVOREL S.A., Cuza Voda 46, 610019

Piatra-Neamţ, Romania; bThe National Institute of R&D for Biological Science / The „Stejarul” Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt

Urtica dioica (stinging nettle) is traditionally used as an herbal medicine approved by

German Commission E for internal and external use. Aerial part contains flavonoids, acids (caffeic, chlorogenic, silicic), minerals, coumarin, triterpenes, tannin, vitamins, and aminoacids [1]. Recent studies have demonstrated the antimicrobial activity of some Urtica dioica extracts on bacteria strains (Streptococcus sp., Bacillus cereus, B. subtilis, and Staphylococcus aureus) [2].

The paper presents different processes of extraction for the main constituents in nettle (Urtica dioica) in order to obtain an extract rich in aminoacids, flavonoids and polyphenols, with potential utilization in crops phytopathology. Selective extraction solvents for the above-mentioned active principles were used: distilled water, ethanol 30%, 50%, 70%, 95% v/v, and methanol, in different temperature conditions: at room temperature (EA1, EE31, EE51, EE71), and by reflux (characteristic for each extraction solvent) (EA1, EE31, EE51, EE71).

All extracts were qualitatively and quantitatively analyzed, by different chemical and instrumental methods. The qualitative analysis consisted in spectroanalytical profile by HPTLC, equipment CAMAG LINOMAT IV, TLC 3 Scanner, WINCATS Planar Chromatography Manager, and stationary phase HPTLC plates G60F254. Quantitative analyses consisted in determination of: total aminoacid content (as glutamic acid) by ninhydrin reaction, at 570 nm; flavonoid content (as rutin) by following colorimetric aluminum chloride method, at 430 nm; and polyphenol content (as caffeic acid) by Arnow method, at 500 nm. For each determination, the absorbance of the reaction mixture was measured with a CARY 50 UV/VIS spectrophotometer.

The most relevant results are presented in the following table: Parameter

%g/ml Distilled water Ethanol 30% Ethanol 50% Ethanol 70%

EA1 EA2 EE31 EE32 EE51 EE52 EE71 EE72 Aminoacids (as glutamic acid) 0,0957 0,0487 0,0416 0,0447 0,0530 0,0846 0,0683 0,0795

Flavonoids (as rutin) 0,0100 0,0158 0,0101 0,0353 0,0165 0,0377 0,0269 0,0380 Polyphenolcarboxylic acids

(as caffeic acid) 0,0396 0,0264 0,0211 0,0489 0,0232 0,0481 0,0280 0,0382

The extraction by reflux ensured the obtaining of important amounts of the analyzed active principles, compared to those in extraction condition at room temperature. Also, we can observe that ethanol 50% v/v assured the obtaining of an nettle extract, rich in aminoacids and polyphenol substances (polyphenolcarboxylic acids and flavonoid compounds), mentioned in literature for their antimicrobial and antifungal activities [3].

This is a preliminary study to identify the active principles with anti-pathogenic activity. Next step is to demonstrate this activity by biological screening.

1. Barnes Joanne, Anderson Linda A., Phillipson J.D. (2007) Herbal Medicines, Pharmaceutical Press, 452. 2. Modarresi-Chahardehi A., Ibrahim D., Fariza-Sulaiman S., Mousavi L. (2012) Rev.Biol.Trop. 60 (4):1567-76. 3.Abad MJ., Ansuategui M., Bermejo P., (2007), Active antifungal substances from natural sources”, Department ARKIVOC , 116-145

79

Poster P 30

NF-κB inhibitors from Eurycoma longifolia

Tran Thi Van Anha, c, Stefan Schwaigera, Clemens Malainerb, Atanas Georgiev Atanasovb, Elke H. Heissb, Verena M. Dirschb, Hermann Stuppnera

a Institute of Pharmacy / Pharmacognosy, Center for Molecular Biosciences Innsbruck, University Innsbruck, Innrain 80/82, A-6020, Austria; bDepartment of Pharmacognosy, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria; cDepartment of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy

of HoChiMinh city, 41 DinhTienHoang street, HoChiMinh city 70000, Vietnam

Aim of the study: Eurycoma longifolia Jack. is used in traditional medicine of Vietnam and Southeast Asian countries for alleviation of various illnesses (e.g. indigestion, diarrhoea, malaria, dysentery, arthritis, and sexual insufficiency) [1]. In a screening for NF-κB inhibitors from Vietnamese medicinal plants, the methanol extract of E. longifolia showed 66.9% ± 3.2 inhibition at a concentration of 10 µg/mL. Therefore, a bio-guided isolation was conducted to identify the compound(s) responsible for the observed activity.

Materials and methods: Anti-inflammatory activity was assessed by the ability to inhibit the activity of the NF-κB pathway in TNF-α-stimulated HEK-293 cells stably transfected with a NF-κB-driven luciferase reporter gene. The methanolic root extract of E. longifolia was separated by liquid-liquid extraction with water and solvents of decreasing polarity (n-hexane, diethyl ether, ethyl acetate, and n-butanol). Bioactive compounds were purified by chromatographic methods (silica gel, sephadex column chromatography) and their structures were elucidated using spectroscopic methods (ESI-MS and 1D/2D-1H/13C-NMR).

Results: The diethyl ether and ethyl acetate extract fractions showed the most promising inhibitory effects against NF-κB at a concentration of 10 µg/mL. With the aid of different chromatographic techniques, 12 quassinoids, 6 alkaloids, 2 coumarins, 2 squalenes, and 6 phenolic compounds were isolated from these extracts. Evaluation of the pharmacological activity revealed that C-19 type, C-20 type quassinoids and canthin-6-one alkaloids are potent NF-κB-inhibitors with IC50s in the low micromolar range, while C-18 type quassinoids, phenolic compounds and squalenes turned out to be inactive when tested at a concentration of 30 µM.

Conclusion: The observed bioactivity of the isolated compounds rationalizes the traditional use of the root of Eurycoma longifolia against inflammation.

Acknowledgements: The authors want to thank the Austrian Federal Ministry for Science and Research for financing the scholarship in frame of the ASEA-Uninet granted by the OeAD and the Austria Science Foundation (FWF; DNTI S 10703 and S 10704). 1. National Institute of Medicinal Materials (2004) Medicinal animals and plants, Science and Technology, HaNoi, 116-118.

80

Poster P 31

Polyacetylenes from Daucus carota - Biosynthesis and Function

Marion Wiggermann, Ute Wittstock

Institute of Pharmaceutical Biology, Technical University, Mendelssohnstraße 1, 38106 Braunschweig,

Germany Typically, polyacetylenes are aliphatic, unbranched C11-C17 hydrocarbons with several C-C-triple bonds that usually also possess C-C-double bonds. More than 1400 polyacetylenes have been identified in higher plants. They are widely distributed especially in the families Apiaceae, Araliaceae and Asteraceae and are known for their antifungal, cytotoxic and antibacterial effects. Feeding experiments with labelled precursors have shown that polyacetylenes are derived from fatty acids [1]. Very little is known about the enzymes involved. The aim of our project is to get a better insight into polyacetylene biosynthesis using Daucus carota (Apiaceae) as a model species. Falcarinol and falcarindiol (Fig.1) are the two most important polyacetylenes from D. carota. They are formed from linoleic acid with crepenynic acid and dehydrocrepenynic acid as the supposed key intermediates. In different plants and fungi, desaturases termed acetylenases have been shown to introduce triple bonds by dehydrogenation of existing double bonds [2].

As a first step, we are in the process of establishing an experimental system that will allow us to perform our studies with biosynthetically active tissue. We have therefore analyzed the falcarinol and falcarindiol-content in the root of D. carota (cultivated and wild form) grown in soil. In total the cultivated carrot contains mainly falcarinol, whereas falcarindiol predominates in the wild form. While the wild carrot has been analyzed as a whole so far, we divided the root of the cultivated carrot in different parts. Here we detected the highest concentration of polyacetylenes in the cortical parenchyma with falcarinol as the major compound. Falcarindiol had its highest concentration in the top part of the root. This is in agreement with previous findings that the polyacetylenes of the carrots are localized as an extracellular oil in the periderm or the pericycle [3]. As falcarinol and falcarindiol have also been reported as phytoalexins, we conduct elicitation experiments with fungal pathogens (Mycocentrospora acerina) and chemical elicitors (e.g. methyl jasmonate, arachidonic acid) using tissue cultures (normal root cultures, hairy root cultures, callus suspension cultures) and soil grown plants. Once elicitation has been achieved, we are going to use the system to detect enzyme activities in cell-free extracts.

Fig.1 1. Bohlmann, F. (1971) Botan. J. Linn. Soc. 64 Suppl.1: 279-292. 2. Minto, R.E., Blacklock, B.J. (2008) Prog. Lipid. Res. 47: 233-306. 3. Garrod, B., Lewis, B.G. (1979) Trans. Br. Mycol. Soc. 72: 515-517.

OH

falcarinol

OH

falcarindiol

OH

81

Poster P 32

Cytotoxic Effect of Arnebia purpurea Root Extract and Its Naphthoquinone Content Against L20B Cell Line

Merve Yuzbasioglua, Yasin Genca, U. Sebnem Harputa, Zuhal Guvenalpb, Iclal Saracoğlua, L. Omur Demirezera, Ayse Kuruuzum-Uza

aDepartment of Pharmacognosy, Faculty of Pharmacy, University of Hacettepe, 06100 Ankara-Turkey;

bDepartment of Pharmacognosy, Faculty of Pharmacy, University of Ataturk, 25240 Erzurum-Turkey

The genus Arnebia (Boraginaceae) is represented by 5 species in the Flora of Turkey [1]. Arnebia species are used as dyestuff and for healing of eczema, callus and burnt in traditional medicine [2]. Earlier investigations on different Arnebia species showed that they have important cytotoxic and antitumoral activity [3]. Napthoquinones are main components of these species. Their cytotoxic activity has showed against different cell lines [4]. In our previous researches, major napthoquinone derivatives: acetylalkannin and isovalerylalkannin isolated from the roots of Arnebia purpurea which is an endemic species growing in Turkey had found strong cytotoxic activity against L929 fibrosarcoma cell line [5]. In this study, the cytotoxic activity of the n-hexane extract of Arnebia purpurea root and its major napthoquinone derivatives: acetylalkannin and isovalerylalkannin against L20-B cell line using MTT assay have been studied for the first time. This cell line was genetically engineered mouse cell line expressing the human poliovirus receptor, derived from a human rhabdomyosarcoma. Our results showed that the extract and its major compounds have very strong cytotoxic activity in 10-50 µg/ml concentration range. These results indicated that the extract and the compounds may be important in cancer theraphy especially for the sarcomas. 1. Edmondson JR, Davis PH (1988) Arnebia Forssk. in Flora of Turkey and the East Aegean Island Supll. I,

Edinburgh University Press, Edinburgh. 2. Baytop T (1999) Theraphy with Medicinal Plants in Turkey, Nobel Tıp Kitapevleri, İstanbul. 3. Yuzbasioglu M, Kuruuzum-Uz A (2012) Hacettepe Univ J Fac Pharm 33: 59-74. 4. Cui XR, Tsukada M, Suzuki N, Shimamura T, Gao L, Koyanagi J (2008) Eur J Med Chem 43: 1206-1215. 5. Yuzbasioglu M (2010) Pharmacognostical Studies on Arnebia purpurea Master Thesis, Hacettepe University

Health Sciences Institute, Ankara.

82

Poster P 33

Flavonoid Profiles and Antioxidant Activity of Bupleurum Species

Reneta Gevrenova, Nikolay Denkov, Dimitrina Zheleva-Dimitrova

Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Sofia, Dunav str. 2, 1000, Bulgaria

Bupleuri radix [roots of Bupleurum L. spp. (Apiaceae)] is one of the most frequently used herbs in Chinese herbal medicine [1]. Flavonoids are a major class of secondary metabolites present in all species of the genus Bupleurum. Most flavonoids in the genus are derivatives of the flavonol aglycones kaempferol, isorhamnetin or quercetin; the diglycoside rutin is the most common [2]. Recently, few quantitative HPLC-UV and UPLC-PDA methods have been established for simultaneous determination of flavonoids in the aerial parts of several Bupleurum species for quality assessment of the raw materials of traditional Chinese medicines [3, 4]. However, no quantitative data are available on the flavonoid content of European Bupleurum species.

The aim of this study was to investigate HPLC flavonoid profiles of aerial parts of Bupleurum flavum, B. affine and B. baldense, growing in Bulgaria. Ultrasound assisted extraction of aerial parts with 80 % methanol aqueous solution at room temperature allowed good extraction of all compounds of interest. Subsequent HPLC separation was performed on a Luna C18 column by linear gradient elution with mobile phase comprised acetonitrile and water (0.1% phosphoric acid), and with UV detection at 360 nm. In addition, the radical scavenging activities of methanolic extracts of aerial parts of Bupleurum species was evaluated in vitro using the DPPH and ABTS methods.

The developed analyses had good linearity (R2>0.999). The LOD and LOQ of rutin were 1.10 and 3.51 µg/ml, respectively. The RSD of the repeatability was estimated to be ≤3.2%. The accuracy values varied between – 16.2 and 1.5% (rutin). The mean recoveries ranged from 92.6% (isoquercitrin) to 99.67% (rutin) (RSD<5.9%).

Five flavonoid glycosides – rutin, quercetin-3-glucoside (isoquercitrin), isorhamnetin-3-rutinoside (narcissin), kaempferol-3-glucoside (astragalin), isorhamnetin-3-glucoside as well as five flavonol aglycones – luteolin, kaempferol, quercetin, isorhamnetin and kaempferide were determined in assayed samples. Flavonoids showed differential quantitative distributions in Bupleurum species. The highest content of rutin was found in B. baldense (28.63 ± 1.57 mg/g dry weight), followed by B. flavum (21.94 ± 0.82 mg/g), whilst B. affine had the lowest amount (1.30 ± 0.04). B. flavum was found to posses the highest DPPH and ABTS activity with IC50 of 22.12 µg/ml and 118.15µg/ml, respectively.

In conclusion, a HPLC method was developed for quantitative estimation of ten flavonoids in Bupleurum species for the first time. As regards the antioxidant activity, B. flavum represents a significant potential in safeguarding against various induced oxidative stress. 1. Bauer R, Franz G (2010) Planta Med 76: 2004-2011. 2. Ashour M, Wink M (2011) J Pharm Pharmacol 63: 305-321. 3. Mei Z, Yang J, Fan H-J, Yang H-J, Lin F, Wang Q (2011) Chin J New Drugs 20: 932-935. 4. Lin H, Wang S, Wang Y, Sun C (2012) Zhongguo Shiyan Fangjixue Zazhi 18: 76-79.

Authors

Akdemir Z.S. ............. 58, 63 Akkol E.K. .......................58 Aldhaher A. .....................50 Alqahtani A. ....................51 Antognoni F. ...................12 Apetrei C.L. .....................67 Appendino G. ................... 9 Aprotosoaie A.C. .............67 Atanasov A.G. ........... 41, 79 Ateba S. .........................52 Aumond M-C. ..................65 Awang K. ........................36 Backlund A. ..................... 8 Balabanova V. .................53 Bankova V. .....................14 Baroffio C.A. ...................56 Basar N. ................... 11, 23 Bauer J. ..........................41 Bauer R. .......................... 2 Billah Md.M. ....................11 Birlirakis N. .....................36 Bisio A. ...........................19 Bley T. ..................... 13, 15 Bobit D. ..........................61 Bodur M. ........................29 Boisard S. .......................65 Bonneau N. .....................54 Botha C. .........................34 Boz I. ....................... 55, 61 Braun H. .........................38 Brunelle A. ......................54 Brunton N.P. ............. 17, 64 Bucar F. ................... 25, 42 Butu A. ...........................74 Butu M. ..........................74 Cala A. ............................ 7 Carlen C. .................... 3, 56 Carron C-A. .....................56 Champy P. ......................54 Chan-Bacab M.J. .............46 Chantrapromma K. ..........28 Cheel Horna J. ................57 Cheplogoi P.K. ................70 Chinchilla N. ..................... 7 Chiriac M. .......................78 Cho S-H. ................... 16, 40 Civelek E. .......................58 Cornea P.C. ....................74 Costa J. ..........................39 Cretu R. .................... 62, 78 Csábi J. ..........................45 Csupor D. .......................21 Cvak L. ...........................26 Dai Y. .............................16

Danila D. .................. 69, 76 Demirezer L.O. ......... 29, 81 Denkov N. ...................... 82 Derbré S. ....................... 65 Didarul Islam K.Md. ......... 11 Dirsch V.M. .......... 32, 41, 79 Driscoll D.J. .................... 50 Drutu C. ......................... 66 Duangsrisai S.................. 59 Dumontet V. ............. 36, 39 Ecker G. ........................... 6 Edtbauer M..................... 31 Efrose R. ........................ 55 Einsenreich W. ................ 46 Ekizoğlu M. ..................... 63 Eldridge G. ..................... 10 Eloff J. ........................... 24 Emdadul Islam Md. ......... 11 Engström M. ................... 22 Ersöz T. ......................... 18 Escalante-Erosa F. ........... 46 Fernandez X. .................. 20 Fitzgerald R.J. ................. 17 Flurin C. ......................... 65 Galindo J.L.G. ................... 7 García-Sosa K. ................ 46 Gavril G. ................... 61, 66 Genc Y. .................... 27, 81 Georgiev V. .............. 14, 79 Gerstmeier J. .................. 41 Gevrenova R. ....... 53, 60, 82 Gille E. .......... 55, 61, 66, 76 Giurescu C. ..................... 62 Gornik A. ........................ 42 Grafenstein von S. .... 37, 38 Grienke U. ................ 37, 38 Grigoras V. .......... 69, 76, 78 Gröblacher B. ................. 25 Grúz J. ........................... 26 Guéritte F. ................ 36, 39 Guerrero-Vásquez G. ......... 7 Gurbuz P. ....................... 29 Guvenalp Z. .............. 29, 81 Haas C. .................... 13, 15 Hae Choi Y. .................... 16 Hagerman A.E. ............... 22 Hancianu M. ................... 61 Harput U.S. .............. 27, 81 Heiss E.H. ................ 41, 79 Henry M. ........................ 60 Hering S. ........................ 31 Hewage C.M. .................. 64 Hidalgo Bucheli W. .......... 35 Hohmann J. .................... 21

Hrouzek P. ..................... 57 Hunyadi A. ..................... 45 Hwang B.Y. .................... 40 Iacob E. .................... 62, 78 Iannello C. ..................... 12 Igartuburu J.M. ................ 7 Ionescu E....................... 62 Ivanov I. ........................ 14 Jamil S. ..................... 11, 23 Jegorov A. ..................... 26 José L. G. ................ 3, 7, 56 Kaehlig H. ...................... 52 Kahraman C. ............. 58, 63 Kapuscik A. .................... 57 Kaserer T. ...................... 33 Kaya D. ......................... 18 Kemertelidze E. .............. 68 Kenny O. ....................... 64 Keokitichai S. ................. 73 Kijjoa A. .................... 59, 73 Kim E.S. ......................... 40 Kim S. ........................... 40 Kirchmair J. ............... 37, 38 Kiss R. ........................... 43 Klančnik A. ..................... 42 Kolář Z........................... 77 Konstantinov S. .............. 60 Kopecký J. ..................... 57 Kostova N. ..................... 14 Kowalczyk M. ............ 68, 72 Kratz J.M. ...................... 31 Krenn L. ......................... 52 Křížová K. ...................... 77 Kučerová P. .................... 57 Kuruuzum-Uz A. ............. 81 Kutluay V. ...................... 27 Langat L.C. .................... 49 Langat M.K. . 28, 30, 49, 50,

51, 70 Lazic M. ......................... 33 Le Ray A-M .................... 65 Lee Ch. .......................... 40 Lee M. ........................... 40 Lefranc F. ...................... 43 Leyssen P. ..................... 39 Liedl K.R. .................. 37, 38 Litaudon M. ............... 36, 39 Lorenzi B. ...................... 12 Lovász N. ....................... 21 Ludwig B........................ 13 Maas W. ........................ 10 Macias F.A. .................... 72 Macias M.,........................ 7 Mair Ch.E. ................. 31, 37

Malainer C. ............... 41, 79 Mandrone M. ..................12 Marchev A. .....................14 Martins A. .......................45 Masullo M. ......................68 Mathieu V. ......................43 Mcloughlin P. ..................64 Mele G. ..........................19 Merck F. .........................20 Mihai C.T. .......................67 Mihalache I. .............. 55, 66 Miles Ch. ........................34 Miron A. ................... 67, 69 Mitroi G. ................... 62, 78 Molinillo J.M.G. ................. 7 Muffler K. .......................13 Muhammad N.A. .............23 Mulholland D.A. .. 28, 30, 49,

50, 51, 70 Muzashvili T. ...................68 Nahar L. .........................11 Necula R. ............ 66, 69, 76 Nedbal L. ........................75 Nesawar Miah Md. ...........11 Njamen D. ......................52 Njue A. ...........................70 Nothias L. .......................39 Novák O. ........................75 Oklešťková J. ..................77 Oleszek W. ............... 68, 72 Omolo J.O. .....................70 Omur Demirezer L. .... 29, 81 O'Neil-Johnson M. ...........10 Osarumwense O. .............71 Ozenver N. .....................29 Öztürk D. ........................63 Paolini J. .........................39 Pauw L. ..........................24 Pavlov A. ........................14 Peña-Rodríguez L. ...........46 Péruches I. .....................65 Piacente S. .....................68 Pinto M. .................... 59, 73 Piskernik S. .....................42

Planchenault P. ............... 65 Poli F. ............................ 12 Prasch S. ........................ 25 Prompanya C. ................. 73 Ramírez-Torres F.G. ........ 46 Rárová L. ................. 44, 77 Reichelt M. ..................... 35 Retailleau P. ............. 36, 39 Richomme P. .............. 4, 65 Richter M. ...................... 38 Rodino S. ....................... 74 Rolén E. ......................... 34 Rollinger J.M. 31, 32, 37, 38,

41 Romussi G. ..................... 19 Rotinberg P. ................... 67 Roza O. .......................... 21 Sabrin F. ........................ 11 Salminen J-P. .................. 22 Saracoğlu I. .................... 81 Sarker S. ........................ 11 Schabus K. ..................... 25 Schmidtke M. ............ 37, 38 Schmitz Afonso I. ............ 54 Schneider B. ............. 35, 47 Schulz S. .................. 13, 15 Schuster D. ......... 31, 32, 33 Schwaiger S. ............ 33, 79 Sebnem Harput U. .... 27, 81 Sezerman O.U. ............... 29 Shahar S. Binti ................ 23 Shilpi J.A. ....................... 11 Simon A. .............. 3, 45, 72 Simonet A.M. .................. 72 Smyth T.J. ........... 17, 48, 64 Soler-Villa A. ............. 17, 48 Spac A. .......................... 66 Spronsen Van J. .............. 16 Stefanache C.P. ........ 69, 76 Steingroewer J. ......... 13, 15 Stochmal A. .............. 68, 72 Strnad M. ...... 26, 44, 75, 77 Stuppner H. ... 32, 33, 41, 79 Suber M.P. ..................... 22

Suntar I. ........................ 58 Šváchová M. ................... 77 Tatli I.I. ......................... 58 Tatsis E.C....................... 47 Tebrencu C. .............. 62, 78 Temml V. .................. 32, 33 Tierney M, ..................... 48 Tommasi De N. .............. 19 Tóth G. .......................... 45 Touboul D. ..................... 54 Trifan A. ........................ 67 Uhlig S. .......................... 34 Ukowitz K. ..................... 52 Ulber R. ......................... 13 Usifoh O. ....................... 71 Uz A.K. .......................... 29 Varela R.M. ...................... 7 Verpoorte R. .................. 16 Voráčová K..................... 57 Vouillamoz J.F. ........... 3, 56 Voutquenne-Nazabadioko L.

................................. 60 Vrålstad T. ..................... 34 Waltenberger B. ............. 41 Waratchareeyakul W. ...... 28 Warskulat A. .................. 47 Wetschnig W. ............ 49, 51 Wiechmann K. ................ 41 Wiggermann M. .............. 80 Witkamp G-J. ................. 16 Wittstock U. ................... 80 Wolfender J. ..................... 5 Wu Z. ............................ 42 Yalçın F.N. ..................... 18 Yam-Puc A. .................... 46 Yuzbasioglu M. ............... 81 Zaharieva M.M. ............... 60 Zahler S. ........................ 44 Zamfirache M-M. ............ 55 Zehl M. .......................... 52 Zhang Q. ....................... 42 Zheleva-Dimitrova D. . 53, 82 Zupkó I. ......................... 21

Acknowledgements

The members of the Organizing Committee gratefully acknowledge the support of the symposium on "Trends in natural products research: a young scientists meeting of PSE and ÖPhG" by:

Supported by

University of Innsbruck, University Centre Obergurgl

ISBN-13-978-0-9565472-3-1

university centre obergurgl/tyrol, austria 21 – 25 july...

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