ANALELE ŞTIINŢIFICE ALE UNIVERSITĂȚII “ALEXANDRU IOAN CUZA” DIN IAŞI (SERIE NOUĂ) SECȚIUNEA II a. BIOLOGIE VEGETALĂ, 2016, TOMUL 62, Fascicula 1
Content of the printed abstracts book and the online edition is copyright of the MAPPPS 2016 and the papers accepted for publication will appear in: Scientific Annals of „Alexandru Ioan Cuza” University of Iaşi New Series, Section II a, Vegetal Biology and Annals of the „Alexandru Ioan Cuza” University Sect. II a, Genetics and Molecular Biology.
The authors are responsible for the scientific content of the papers.
This book of abstracts is edited by the NIRDBS/„Stejarul“ Biological Research Centre with the financial support of the Executive Agency for Higher Education, Research, Development and Innovation Funding (UEFISCDI) from Romania through the National Project code 16-190-401 (BIODIVERS).
Editing by Dr. Georgiana Luminita GAVRIL Revised by Dr. Irina IRIMIA – Production Editor of Analele Științifice ale Universității ”Alexandru Ioan Cuza” din Iași, Serie nouă, s. II a. Biologie vegetală
Copyright © NIRDBS/ „Stejarul“ Biological Research Centre Piatra Neamt, ROMANIA www.ccb-stejarul.ro
Print ISSN: 1223-6578 Online ISSN: 2247-2711 Analele Ştiinţifice ale Universităţii „Alexandru Ioan Cuza” din Iaşi (serie nouă) Secțiunea II a. Biologie vegetală, 2016, Tomul 62, Fascicula 1
www.incdsb.ro/p/CCB-Stejarul/MaPS2016/index.html
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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1 MAPPPS – 2016
COMMITTEES
Organizing Committee Dr. Elvira Gille – Chairman, NIRDBS/"Stejarul" Biological [email protected] Research Centre, Piatra Neamț, Romania Dr. Mihael Cristin Ichim - Co-Chairman, NIRDBS/"Stejarul" [email protected] Biological Research Centre, Piatra Neamț, Romania Dr. Anca Oancea, NIRDBS, Bucharest, Romania [email protected] Dr. Simona Carmen Litescu, NIRDBS, Bucharest, Romania [email protected] Prof. Dr. Anca Miron, "Gr. T. Popa" University of Medicine and [email protected] Pharmacy, Iași, Romania Assistant Prof. Dr. Oana Cioanca, "Gr. T. Popa" University of [email protected] Medicine and Pharmacy, Iași, Romania Dr. Adela Halmagy, Institute of Biological Research, Cluj Napoca, [email protected] Romania Dr. Valentin Grigoraș, NIRDBS/"Stejarul" Biological Research [email protected] Centre, Piatra Neamț, Romania Dr. Georgiana Luminita Gavril, NIRDBS/"Stejarul" Biological [email protected] Research Centre, Piatra Neamț, Romania Dr. Ruxandra Mihaela Crețu, NIRDBS/"Stejarul" Biological [email protected] Research Centre, Piatra Neamț, Romania Dr. Alexandru Amărioarei, NIRDBS, Bucharest, Romania [email protected]
Scientific Secretary PhD student Radu Necula, NIRDBS/"Stejarul" Biological Research [email protected] Centre, Piatra Neamț, Romania PhD student Paula Paraschiva Sosoi, NIRDBS/"Stejarul" Biological [email protected] Research Centre, Piatra Neamț, Romania Madalina Oana Popa, NIRDBS/"Stejarul" Biological Research [email protected] Centre, Piatra Neamț, Romania Larisa Elena Tomescu, NIRDBS/"Stejarul" Biological Research [email protected] Centre, Piatra Neamț, Romania Andreea Andrei, NIRDBS/"Stejarul" Biological Research Centre, [email protected] Piatra Neamț, Romania
Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1 MAPPPS – 2016
Scientific committee Prof. Dr. Robert Verpoorte, Leiden University, The Netherlands [email protected] Prof. Dr. Satyajit D. Sarker, Liverpool John Moores University, [email protected] United Kingdom Prof. Dr. Milen I. Georgiev, Institute of Microbiology, Bulgaria [email protected] Acad. Prof. Dr. Maria Duca, University of Academy of Sciences, [email protected] Moldova Dr. Manuela Elisabeta Sidoroff, National Institute of R&D for [email protected] Biological Sciences, Romania Dr. Hugo de Boer, University of Oslo, Norway [email protected] Prof. Dr. Ilkay Erdogan Orhan, Gazi University, Turkey [email protected] Prof. Dr. Monica Hancianu, "Gr. T. Popa" University of Medicine [email protected] and Pharmacy, Romania Prof. Dr. Zora Dajić Stevanovic, University of Belgrade, Serbia [email protected] Prof. Dr. Maria Magdalena Zamfirache, "Al. I. Cuza" University, [email protected] Iasi, Romania Acad. Cm. Dr. Gheorghe Coldea, Institute of Biological Research, [email protected] Cluj Napoca, Romania Prof. Dr. Adam Matkowski, Medical University of Wrocław, [email protected] Poland Prof. Dr. Wiesław Oleszek, Institute of Soil Science and Plant [email protected] Cultivation State Research Institute, Pulawy, Poland Prof. Dr. Gabriel Lucian Radu, National Institute of R&D for [email protected] Biological Sciences, Romania Prof. Dr. Chin-Kun Wang, Chung Shan Medical University, Taiwan [email protected] Prof. Dr. Ursula Stănescu, "Gr. T. Popa" University of Medicine [email protected] and Pharmacy, Iași, Romania
SECTIONS AND TOPICS
VALUABLE PHYTOCHEMICALS AS DRUGS AND NUTRACEUTICALS . Phytochemistry . Phytotherapy . Traditional medicine . Functional food and dietary supplements
CONSERVATION, BREEDING AND MOLECULAR FINGERPRINTING OF MEDICINAL PLANTS AND DERIVED PRODUCTS . Cultivation, breeding and biotechnology . Biodiversity, protection and conservation . Quality control and authentication . New technologies (e.g. -omics), advances and perspectives . Varia
Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
Dear Participants,
It is a great pleasure and an honour to welcome you to the 12th edition of the national symposium with international participation "MEDICINAL PLANTS – PRESENT AND PERSPECTIVES", which takes place September 06-09, 2016, Piatra Neamt, Romania. This edition of the symposium celebrates 60 years since the establishment of the "Stejarul" Biological Research Centre, branch of the National Institute of Research and Development for Biological Sciences Bucharest. We invite you to celebrate it together. In our picturesque town, surrounded by mountains, with a rich history and vibrant culture, you will have the possibility to exchange scientific knowledge and lay the foundation for further collaborations. Also, in addition to the scientific program, we propose a one day trip in the mountainous area of the Neamt County. We acknowledge the contribution of our colleagues from the Phytochemical Society of Europe (PSE) which kindly accepted to be part of our symposium. We are very grateful to all our sponsors for financial support and to all people involved in organisation of this symposium.
We sincerely hope that this symposium will meet all your expectations.
Welcome to Piatra Neamt and we wish you a pleasant stay!
On behalf of the Organizing committee,
Dr. Elvira GILLE, Chairman Head of the NIRDBS/"Stejarul" Biological Research Centre, Piatra Neamt, Romania
Dr. Mihael Cristin ICHIM, Co-chairman Scientific Researcher/NIRDBS/"Stejarul" Biological Research Centre, Piatra Neamt, Romania
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Dear Colleagues,
In the framework of the European regional development policies, the Europe-Asia research of the river basins, as well as that of the EU Strategy for the Danube Region that aims the sustainable development of the Danube-Danube Delta-Black Sea system, assuring the environment protection and an increase of the Danube Region welfare, Romania tries, by implementing far- reaching strategic projects, to actively contribute to the regional sustainability. The Romanian project of strategic importance, “The International Centre of Advanced Studies for the Râu-Mare Systems” DANUBIUS-RI, is the expression of this continuous approach. The project became, in 2013, the flagship project of the Danube European Strategy. In the same year, DANUBIUS-RI entered the common declaration of the Asia-Europe Meeting (ASEM) Water and River Basin Management in Can Tho City, Viet Nam. Yet the greatest achievement is that, since March, the 14th, 2016, the project has been included in the ESFRI (European Strategy Forum for Research Infrastructure Roadmap). The National Institute for Research and Development of Biological Sciences, Bucharest is one of the project coordinators being responsible for the Life Sciences domain. The ”DANUBIUS-RI” Centre also has a socio-economic component, so that the research results be taken over and put into practice. The investigation of the bio-economic plant potential is part of a large strategy at European level: in 2012, The European Commission made the call of the strategy „O Bio- economy for Europe”, namely the building of a bio-economy based on the sustainable use of the naturally regenerating resources, on competition, on socio-economic and environment problems, an economy to respond to some challenges such as food security, natural resource deficit, dependence on fossil resources and climatic changes. In Romania, The National Strategy for Research, Development and Innovation 2014–2020 settles the importance of the development of bio-economy as a national field of intelligent specializing, plant investigation in the Danube Basin. The investigation of the plant bio-economic potential in the Danube – Danube Delta – Black Sea area is a priority of the development strategy of the DANUBIUS-RI CENTRE. Taking into account the fact that bio-economy and modern bio- technologies are strongly linked, the innovation processes must be followed by economic growth. That is why DANUBIUS-RI proposes an interdisciplinary approach of the social, economic and scientific aspects to assure the integration of the synergic effects of the three areas and to make possible the optimum development of an innovating bio-economy on the Danube – Danube Delta – Black Sea sector. The cycle of Symposia “MEDICINAL PLANTS – PRESENT AND PERSPECTIVES”, organized by the Piatra Neamt Branch of our National Institute (INCDSB), is part of the strategies above mentioned. Combining all the favourable reasons such as, research (the investigation of the chemical and biological potentials of some vegetal species, taking into conventional and non-conventional (tissue cultures) cultures some autochthonous and allocthonous medicinal plant species, some standardizing procedures, both for the vegetal raw material and for the phytopreparations achieved from the extracts obtained to assure the quality and the chemical charge conformity, the safety and multiplication capacity of the biological action, as well as the aspects linked to demand, this cycle of symposia comes to meet the necessity of the society by the problems approached. The researches converge to a rational and sustainable development of the natural resources, a fact that leads to obtaining a good socio-economic status and a healthy environment. Even more,
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evaluating the commercial potential of the products obtained by processing vegetal resources, espacially the ones correlated to the challenges of the society: food security and safety, competition in agro-food and forestry industries, water supplies administartion, the pharmaceutical products (biomedicine, cosmetics), bio-fuels, the sustainability of the supply and demand processes and also the bio-remedial measures applied to the contaminated environment using plants (phytoremedial measures), all these frame an intelligent specialization strategy in the field of medicinal plants, strategy to which our contribution and that of the “Stejarul Biological Research Centre, Piatra Neamt is major.
I wish you all a very successful congress and a pleasant stay in Romania!
Dr. Manuela Elisabeta Sidoroff Director General National Institute of Research and Development for Biological Sciences, Bucuresti, ROMANIA
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Medicinal Plants - Present and Perspectives: behind the years
Prof. Dr. Ursula Helena Stănescu
Faculty of Pharmacy, University of Medicine and Pharmacy “Grigore T. Popa”, Iasi, Romania *Corresponding author, e-mail: [email protected]
The year of 2016 marks the 60th anniversary of the „Stejarul” Biological Research Centre (BRC) Piatra Neamt and also 30 years since the National Symposium Medicinal Plants - Present and Perspectives was first organized. In terms of scientific value we have no accounts from the first two editions, because at that time the summaries were not published. Starting with the IIIrd edition (1991) all plenary presentations were published as short papers. Also, the symposium was divided in two sections (Genetics and medicinal plant breeding and Phytochemistry and phytotheraphy); the two sections still exist, even if the topics covered were slightly changed in time. General reviews were presented by some personalities in biology (Prof. G. Gheorghita, Dr. M. Bodruc) and pharmacy (Prof. E. Grigorescu) The IVth edition was organized in 1993 when an abstracts volume was published - and the event was dedicated to the 70th anniversary of Prof. E. Grigorescu. The edition was attended by great personalities: Dr. E. Paun, Dr. Maria Gonceariuc, Prof. Dr. I. Ciulei, Prof. Dr. Viorica Istudor, Dr. G. Musteata. Also, some young researches that are attending this edition, will became in the next two decades important figures in Romanian research in the field of medicinal plant. Beginning with the Vth edition (1995), in the same time with plenary presentations the first posters are presented. The number of participants from abroad (especially from Rep. of Moldova) increases significatly and Piatra Neamt becames an important center for collaborations between researches on both sides of the Prut river. Now, Prof. S. Nicolae from BRC of Iasi, presents an overview on Medicinal plants from Danube Delta Biosphere, topic that remains in our biologists’ attention up to now. The VIth edition, organized in May 1997, was dedicated to the 40th anniversary of „Stejarul” BRC Piatra Neamt. In August 2000, the VIIth edition was held; the two sections (Genetics and Phytochemistry) were chaired by Acad. Prof. Dr. C. Toma and Prof. Dr. M. Tamas. As the two professors highlighted an important qualitative leap was made – English becoming the second official language of the symposium. The VIIIth edition, held in August 2003, was dedicated to the 80th anniversary of Prof. E. Grigorescu and included for the first time researchers from Serbia. The only edition of MaPPPS organized outside Piatra Neamt was held in 2006; this event was included in the AMAPSEEC Congress of Iasi 2006. In June 2011 the symposium returns to Piatra Neamt, where there are organized Xth and XIth edition as one single event. In 2014 of „Stejarul” BRC postponed the XIIth edition of MaPPPS, organizing instead the International Congress on Phytochemicals in Medicine and Pharmacognosy under the auspices of Phytochemical Society of Europe. Starting from the success of PSE Congress, today we are witnessing a new era of MaPPPS: a national symposium with international participation which adds important quality and visibility to the event.
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Distinguished Foreign And Romanian Participants To The XII Edition Of The National Symposium With International Participation ”MEDICINAL PLANTS – PRESENT AND PERSPECTIVES”
Dear Guests, Collaborators and Friends,
Today, we open, in this picturesque mountain town with a historical past rich in heroic deeds and a present of an authentic cultural and touristic state, the XIIth edition of the National Symposium with International Participation which will offer a moment of analysis of what we all achieved since the previous symposium; in this meeting we aim to know what the foreign and Romanian specialists materialized in such a beautiful and useful domain, to better understand our concerns, to know each other, to change ideas in an innovating spirit.
It is a feast of the spirit that honours not only the host, the organizers led by Dr. Elvira Gille, the manager of the “Stejarul” Biological Research Centre – Piatra Neamt, but also, as the rich agenda of the symposium proves, all those who, with competence and abnegation, produced works of a high scientific value and sometimes of a real socio-economical interest. The event we take part in, today, is coinciding with the 60th anniversary of the “Stejarul ”Biological, Geographical and Geological Research Station from Pângărati (Neamt County), founded by academician Petre Jitariu – “Al.I.Cuza” University, Iasi. At that moment, the “Stejarul“ Research Station was the fourth prestigious institution of the above mentioned University, along with the Natural History Museum (1834), The Botanical Gardens (1856), The Marine Research Station from Agigea (Constanta County) (1926).
The “Stejarul Biological Research Centre, Piatra Neamt has continued the activity of the Pangarati Research Station since 1983, being, in time, headed by well-known students and collaborators of mine: Ichim Ionită, Gogu Ghiorghită and Elvira Gille. This research centre was part of the Central Biology Institute from Iasi – The Biological Research Centre, Iasi. As the manager of this institution I have known well the activities of this team of specialists, their daily endeavour for the multiple point of view study of the medicinal plants, their capacity to approach experimental vegetal biology and genetics, the attempts to induce individual variability of some species, the actions of economically interesting plant melioration, and the complex study of the effects induced by chemical and physical mutagens, the selecting of forms and lines superior under the aspect of production characters, “in vitro” processing of medicinal plants to analyse the biosynthetic capacity of the tissues formed in artificial culture conditions, along with perfecting a valuable micro-propagation technology for the good biological material, all of these being only some of the research activities approached. In the last decade, the research teams from Piatra Neamt approached other directions of medicinal plant studying, such as: ▪ phytochemical aspects, ▪ ecology and molecular genetics. The achievements of these researches have been presented in the 12 national symposia “Medicinal Plants – Present and Perspectives” where I have been present together with my students and collaborators.
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Allow me, dear participants, to pay my respects to the collaborators of the “Stejarul” Biological Research Centre that prove, here at Piatra Neamt, a special care for their researches as here, 191 years back, was initiated the activity of the renowned pharmaceutic laboratory Vorel which is today the food supplement processing company “Plantavorel”, the main activity of which is medicinal plant processing. Hereby, I thank you for your invitation to take part in the prestigious international scientific reunion, and please allow me to bring – in the name of the Biology Faculty of the “Al. I. Cuza” University of Iasi and in that of the Biological Sciences Section of the Romanian Academy – greetings to all the participants to the Symposium, to congratulate the organizers led by the present director of the Centre, Dr. Elvira Gille, expressing my conviction and confidence that, in the following days, we shall have a fruitful exchange of ideas, we shall establish new links among those who perform variable research work, under different aspects, in the field of medicinal plants – our source of health.
I wish you the best of success in the works of the Symposium!
Piatra Neamt, September 6th, 2016
Academician Constantin TOMA Faculty of Biology,“Alexandru Ioan Cuza” University, Iasi, ROMANIA
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Academician Professor Doctor CONSTANTIN TOMA
DATE AND PLACE OF BIRTH - November 19th, 1935 , Gugesti village, Vaslui county,Romania; - Parents: Irimia and Virginia Toma (farmers and furriers).
WORKPLACE - ,,Alexandru Ioan Cuza” University, Faculty of Biology Carol I Bd., 11, RO-700506, Iaşi, Romania.
STUDIES - Primary school and gymnasium Gugesti village and Huşi (1943-1950); - ,,Cuza-Vodă” High School Huşi (1950-1953); - Faculty of Natural Sciences at ,,Alexandru Ioan Cuza” University of Iaşi (1953-1958).
SCIENTIFIC TITLE - PhD in Biology (1969), University of Bucharest, Romania.
FIELD OF ACTIVITY - Plant biology: floristics and chorology, plant anatomy and morphology, history of biology.
HONORS - “Profesor evidenţiat” Award of Romanian Ministry of Education (1982); - The Order "Merit for the Education” (2004); - Professor Emeritus (2005); - Doctor ,,Honoris Causa” at: ,,Vasile Alecsandri” University from Bacău (2005), Vasile Goldiş” University from Arad (2008), University of Oradea (2009), “Apollonia” University from Iaşi (2010).
AWARDS - Emanoil Teodorescu” Award of Romanian Academy (1978, 2000, 2001, 2016).
CAREER a) Scientific activity: Associate Instructor (1958 – 1959), Assistant (1959 – 1966), Assistant professor (1966-1972), Associate Professor (1972-1978), Professor (1978-2005), Consulting Professor (since 2005), since 1988 PhD supervisor for 35 PhD students, Organiser and President of the Biology Second National Congress (1992), Organiser and Leader of Photonic and Electronic Microscopy Laboratories (1958-2005), Founder and Leader of Plant Anatomy School (celebrated in 2008), Member of National Council for Academic Assessment and Accreditation (1994-2005), Member of Biology Committee of the National Council for Attesting Titles , Diplomas and Certificates (1995-2005), President of ,,Ştefan Lupaşcu” International Foundation for Sciences and Culture from Iaşi (since 1997).
b) Managerial activity Director of the Botanical Garden of Iasi (1970-1973), Scientific secretary of Academic Council (1973-1975), Vice-dean at Faculty of Biology-Geography (1975-1977), Head of Chair (1977-1986, 1990- 1996), Director at Biological Research Center of Iasi (1986-1990), Dean of the Faculty of Biology- Geography-Geology (1989-1990), Scientific secretary of „Alexandru Ioan Cuza” University Senate (1990-1992), Dean of the Faculty of Biology (1996-2001), Scientific secretary of Romanian Academy
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(Iasi branch) (2001-2011), Vice-president (1990-2007) and Honorary President (since 2007) of National Society of Biological Sciences, President of the Natural Monuments from Moldova Subcommission / Iasi Subsidiary of the Romanian Academy (since 1992).
ACADEMIC POSITIONS - Corresponding member of Romanian Academy (since 1991); - Member of Ecology Academy from Republic of Moldova (din 1999); - Member of Sciences Society from Republic of Moldova (din 2011); - Member of Romanian Academy (since 2012).
SOCIETIES AND ACADEMIC FOUNDATION Botanical Society of France, The Society of Physicians and Naturalists Iasi, National Society of Biological Sciences, Phytotherapy Society from Romania, National Society of Celular Biology, Association of Medicinal and Aromatic Plants of Southeast European Countries, Founder member of Romanian Scientists Association , Founder member of „Stefan Lupascu” International Foundation for Sciences and Culture, Founder member at Christian Pedagogues Association, Founder member of ,,August Treboniu Laurian”Association.
EDITORIAL ACTIVITY Director of COLUMNA Magazine (Romanian Committee for History and Philosophy of Science and Technology / Romanian Academy), editor-in-chief of: "Scientific Annals ,,Alexandru Ioan Cuza” University from Iaşi – section Plant biology" and ,,Romanian Journal of Biology. Plant Biology” (Romanian Academy), member of several editorial boards of journals in Romania (Bucureşti, Iaşi, Bacău, Craiova, Piteşti, Arad, Oradea, Huşi), member of Research Board of Advisors of the American Biogeographical Institute, Member in Botanical Magazine’s editorial collectiv (Chişinău).
SPECIALIZED TRAINING PROGRAMS - Hungary (1966), U.S.S.R (1968), Belgium (1971-1972).
DOCUMENTATION TRAININGS - France (1972), England (1995), Turkey (1996), Belgium (1996).
PARTICIPATION TO INTERNATIONAL SCIENTIFIC MEETINGS Republic of Moldova (1968, 1992, 1994, 1996, 2001, 2009, 2011, 2014, 2015), Czechoslovakia (1972), Germany (1973, 1975, 2008), Greece (1997, 2001, 2002), Italy (1997), Austria (2002), Slovakia (2004), Czech Republic (2008), Spain (2012), Turkey (2013), Portugal (2014).
PUBLICATION A. 29 courses, universitary manuales, atlases, treatises, monographies, as single author or in colaboration; B. 450 original articles (single author or in colaboration) published in national and international journals, 125 articles of popularization and history of biology , 35 reviews.
Acad. Constantin Toma
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ROBERT VERPOORTE
Natural Products Laboratory, Institute Biology Leiden, Leiden University, Leiden, The Netherlands
He holds a Pharmacists degree (1972) and a PhD (1976) from Leiden. He was lecturer at Leiden University 1976-1987, and since 1987 professor and head of the department of Pharmacognosy. He was guest professor in London (UK), Uppsala (Sweden), Amiens (France) and Reims (France). From 1992-1998 he was Vice-Chairman and Chairman of the committee of the Phytochemical Society of Eu¬rope (PSE). Since May 2011 he is Professor Emeritus at Leiden University.
He is author/co-author of 725+ scientific papers, 4 books and 6 patent applications and is Editor-in- chief of Journal of Ethnopharmacology (IF 3.055) and Phytochemistry Reviews (IF 2.686) and Executive Editor Biotechnology Letters (IF 1.639). He supervised 65 PhD-theses, and 150+ MSc theses.
He received an Honorary Doctorate University of Amiens, France (2004) and of the University of Uppsala, Sweden (2012). In 2007 he received the PSE Medal. He is a honorary professor at the Hong Kong Baptist University since 2015. In 2015 he was awarded the Gusi Peace Prize in Manila, The Philippines.
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Plenary Lecture – PL1
Natural products research: Quo Vadis?
R. Verpoorte
Natural Products Laboratory, Institute of Biology Leiden, Leiden University, PO Box 9505, 2300RA Leiden, The Netherlands, *Corresponding author, e-mail: [email protected]
Life Sciences are going through a rapid change. Since molecular biology started its advance some 30 years ago, it had a major landmark in obtaining the full sequence of the human genome, followed by that of various other organisms. We are now reaching the phase that the 1000 $ full sequencing of an organism becomes reality. It is almost cheaper to sequence again than to save the full sequence of an organism. At the same time it becomes clear that having a sequence does not help much to really understand a living organism. The high expectations for drug development, for example, have shown to be over optimistic, as so far no novel drugs have resulted from this knowledge. In fact a genome is like a blueprint, and a blueprint has only two dimensions, and not the four of life: 3 of space and 1 of time. Using these blueprints the research is now going to a more holistic approach: systems biology. That means in an integrated approach study organisms at all levels of phenotype, metabolome, proteome, transcriptome and genome. The importance of a systemic approach can be illustrated by the fact that plants can be considered to be super organisms in the sense that they are dependent on the collaboration of the plant with all kind of microorganisms, e.g. in the rhizosphere, but also endophytes in the plant itself. That means many new opportunities for natural products research. Plant interactions with their environment, health effects of our food, traditional medicine, biosynthesis, metabolic engineering are examples of areas where society expects us to translate basic research into novel products and concepts to the benefit of all of us. So we have many new opportunities but also many challenges. Close collaboration between all disciplines is a must in such systemic approaches
References: Y.H. Choi and R. Verpoorte. Metabolomics: What you see is what you extract. Phytochem. Anal. 25(2014)289-290. N. Dewi Yuliana, M. Jahangir, R. Verpoorte and, Y.H. Choi. Metabolomics for the rapid dereplication of bioactive compounds from natural sources. Phytochem. Rev. 12(2013)293–304. R. Verpoorte, From traditional use to clinical trials and meta-analyse.J. Ethnopharmacol. 164(2015)1.
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Dr. ADAM MATKOWSKI
Professor of Pharmaceutical Biology and Biotechnology, Department Head Dept. Pharmaceutical Biology, Botany and Biotechnology Medical University of Wroclaw, Poland
Education
MSc, in Molecular Biology from the Jagiellonian University in Cracow, Faculty of Biology, Poland.
PhD, in Plant Developmental Biology, Faculty of Natural Sciences, University of Wroclaw, Poland
DSc (habilitation), in Pharmaceutical Sciences, Pharmaceutical Biology and Biotechnology, Wroclaw Medical University
Full Professorship, Wroclaw Medical University, 2016
Research experience
1997, Research Fellow, Institute for Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany - genetic background of leaf and shoot morphogenesis in Antirrhinum majus;
2002, Postdoctoral Scientist at the University of California, Davis; Dept. Plant Sciences, Don J. Durzan Lab - Investigation of stress-induced nitric oxide bursts and taxanes production in cell cultures of Taxus brevifolia and somatic embryogenesis in the endangered recalcitrant conifer Araucaria angustifolia;
Visiting scientist at the Florida State University, Tallahassee, Dept. Biological Sciences. Visiting professor at the Ankara University, Faculty of Pharmacy, Turkey; Isfahan University, Dept. Biological Sciences, Iran.
Research interest Plant in vitro cultures and biotechnology, physiology of secondary metabolism and role of stress response in regulation of phenylpropanoid and terpenoid metabolites biosynthesis, phytochemistry of medicinal plants, pharmacological activity of plant natural products;
Memberships: Member of The Society for In Vitro Biology, Polish Pharmaceutical Society, Federation of Botanical Gardens and Arboreta in Poland, AMAPSEEC, Phytochemical Society of Europe.
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Plenary Lecture – PL2
How stressed plants can help stressed humans? On the role of oxidative stress response in biosynthesis of bioactive metabolites
Adam Matkowski1,*, Sylwia Zielińska1, Marta Libik-Konieczny2, Robert Konieczny3, Weronika Kozłowska1, Sylwester Ślusarczyk1,4
1 Dept. Pharmaceutical Biology and Botany, Medical University in Wroclaw, Borowska 211 Wroclaw, Poland 2Dept. Stress Biology, Institute of Plant Physiology, Polish Academy of Science, Niezapominajki 21; 3Dept. Plant Cytology and Embryology, Institute of Botany, Jagiellonian University, Gronostajowa 9, Cracow, Poland 4Dept. Biochemistry and Crop Quality, IUNG-Institute of Plant Cultivation and Soil Science, Pulawy, Poland.
An imbalance between the reactive oxygen species (ROS) production and antioxidant defense leads to oxidative stress in a plant organism. The paradox of aerobic life is that organisms cannot exist without oxygen though oxygen is dangerous to their existence. Oxygen serves as a terminal electron acceptor during cellular respiration, which provides a high yield of energy. In green plants, oxygen and oxidating molecules are also generated during photosynthesis. From the other hand oxygen can be considered toxic to life processes due to the products which are formed when oxygen is partially reduced. One of the most important evolutionary achievement of higher eukaryotic aerobic organisms is that they can cope with “double-edge sword” – the oxygen molecule. All aerobic organisms are equipped with a redox regulation system (antioxidant system) that can keep ROS level at the physiological concentration required for normal cellular functions. ROS have dual role that depend upon their concentration in the cell. Thus, the role of antioxidant systems is not to remove oxidants entirely, but of keeping them at an optimum level. ROS can serve as intra-, and intercellular messengers in physiological processes such as the activation of cell signaling cascades, gene expression or apoptosis. At low concentrations, ROS induce defense genes and adaptive responses. They are ideally suited to act as signaling molecules because of their small size and ability to diffuse over short distances. ROS participate in induction of plant cell death program which is an essential process in the plant’s life cycle and mechanism of defense against pathogens [1,2]. They also act as an intrinsic factor during signaling in plant growth and development processes [3,4]. ROS are byproducts of different metabolic pathways occurring in various cellular compartments of every living cell. Under physiological conditions mitochondria, chloroplasts peroxisomes and NADPH-oxidases (NOX), are potential powerful intracellular generators of ROS [5]. The generation of mitochondrial ROS is a consequence of oxidative phosphorylation. In chloroplasts, the primary sources of ROS production are the Mehler reaction and the antenna pigments [6]. Production of ROS by these sources is enhanced in plants by stressful conditions like for example limitation in CO2 fixation. In some plants, limiting CO2 conditions can also activate the photorespiratory pathway and as part of this pathway, H2O2 is generated in peroxisomes by the enzymatic activity of glycolate oxidase. Peroxisomes, are probably the major sites of intracellular H2O2 production. It is generated in the peroxisomal respiratory pathway by different flavin oxidases. ROS production in peroxisomes is attributed to the matrix-localized enzyme, xanthine oxidase which catalyzes the oxidation of xanthine or hypoxanthine to uric acid, releasing O2˙ˉ. The accumulation of ROS in response to biotic and abiotic stress can cause the metabolic changes and physiological damage of plant cell despite the activity of efficient antioxidant systems. Scavenging of ROS derived from molecular oxygen in plants is performed by avoidance processes, antioxidant enzymes, and non-enzymatic antioxidants activity. One of the most important regulatory role in plants tolerance to oxidative stress have components of ascorbate-glutathione pathway like ascorbate
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peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, ascorbate, glutathione, NADPH, hydrogen peroxide (H2O2). Environmental factors such as temperature stresses, osmotic stress, flooding, pollutants can affect components of ascorbate- glutathione pathway in crop plants, by the implementation of changes in size of the ascorbate and glutathione pools. Crosstalking of glutathione - mediated stress responses with other signaling pathways takes place via participating of glutathione in redox signaling and interaction of glutathione with hormonal systems. Plant organisms being devoid of motility and immune system have elaborated strategies resting on synthesis of variety of secondary metabolites playing important function in defense against oxidative stress. It gives them an advantage in comparison to animal kingdom. Plant-derived antioxidant compounds may be able to bolster significantly biological resistance against oxidants in animal cells. In the last decade secondary metabolites became a subject of increasing interest relevant to their significant practical implication for medicinal, nutritive and cosmetic purposes. The entire category of herbal adaptogens is considered as essential to improving the quality of life in the hectic modern societies. Plant antioxidants, such as flavonoids, carotenoids, tocopherols, betalains, resveratrol, rosmarinic acid, and other are involved in a set of reactions to protect cell functions. For instance, chelation of transition metals by flavonoids that interferes with the generation of reactive oxygen species (ROS), thus contributing to a powerful antioxidant/antiradical performance [7]. The presence of conjugated double bonds (delocalized π-electrons) in flavonoids, including anthocyanins, cinnamic and xanthophylls predispose them to photoprotective function. Stress-induced phenolic accumulation could be linked by transduction mechanisms that involves the proline redox cycle, the stimulated oxidative pentose phosphate pathway and successively the reduced growth of plant tissues. It follows that, in the response to stress conditions, the pool of phenolic compounds in plant tissues increase since the growth is inhibited more than photosynthesis [8]. Plant defensive mechanisms are costly for plants. Therefore, plants need to “decide” how to allocate the limited resources to different competing functions. The strategies of adaptation leading to plant metabolism modification to stress are based on the changes of cellular and molecular activities.
In our research program, we employ various experimental systems, reaching from conventional field cultivation, through hydroponics and in vitro cell, tissue and organ culture of medicinal plants. We use several non-model species of Asian origin and their European relatives, such as Salvia miltiorrhiza, S. przewalskii, S. glutinosa, Perovskia atriplicifolia, all of which are known as herbs rich in highly active tanshinones and polyphenolic acids. Other interesting species investigated within this program include Scutellaria baicalensis, Agastache rugosa, Belamcanda chinensis. The metabolic profiles of plants or in vitro cultures challenged by stress factors are monitored by targeted metabolomic approach using hyphenated chromatography-spectroscopy techniques as well as qNMR followed by multivariate statistics.
In conclusion, results of many advanced experiments on the oxidative stress responses in plants support the hypothesis that the trade-off between growth and defense in plant cells exist, and that it is mediated by the resources availability. In that way, plant important antioxidants production is orchestrated by the regulation mechanisms of plant response to environmental conditions. These mechanism can be exploited by humans in form of improving medicinal properties of herbs, especially those that are known as adaptogens.
Bibliography 1. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F. Reactive oxygen gene network of plants. Trends Plant Sci. 2004; 9(10):490-8.
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2. Overmyer K, Brosché M, Kangasjärvi J. Reactive oxygen species and hormonal control of cell death. Trends Plant Sci. 2003; 8(7):335-42 3. Konieczny R, Banaś AK, Surówka E, Michalec Ż, Miszalski Z, Libik-Konieczny M. Pattern of antioxidant enzyme activities and hydrogen peroxide content during developmental stages of rhizogenesis from hypocotyl explants of Mesembryanthemum crystallinum L. Plant Cell Rep. 2014; 33(1):165-77. 4. Libik-Konieczny M, Kozieradzka-Kiszkurno M, Desel C, Michalec-Warzecha Ż, Miszalski Z, Konieczny R. The localization of NADPH oxidase and reactive oxygen species in in vitro-cultured Mesembryanthemum crystallinum L. hypocotyls discloses their differing roles in rhizogenesis. Protoplasma. 2015; 252(2):477-87 5. Pastori GM, Del Rio LA. Natural senescence of pea leaves (an activated oxygen-mediated function for peroxisomes). Plant Physiol. 1997; 113(2):411-418. 6. Asada K. Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol. 2006; 141(2):391-6. 7. Leopoldini M, Russo N, Chiodo S, Toscano M. Iron chelation by the powerful antioxidant flavonoid quercetin. J Agric Food Chem. 2006; 54(17):6343-51. 8. Caretto S, Linsalata V, Colella G, Mita G, Lattanzio V. Carbon fluxes between primary metabolism and phenolic pathway in plant tissues under stress. Int J Mol Sci. 2015, 16(11):26378-94.
Acknowledgements S. Slusarczyk’s research is supported by the Polish National Center for Science (NCN) grant FUGA #DEC/2014/12/S/NZ9/00715. A. Matkowski and W. Kozłowska use funding from Wroclaw Medical University grant # ST-909.
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WIESŁAW OLESZEK
Institute of Soil Science and Plant Cultivation State Research Institute Ul. Czartoryskich 8 24-100 Pulawy Poland http://iung.pulawy.pl/staff/Oleszek/ e-mail: [email protected]
Education: MS in 1975, Maria Curie-Sklodowska University of Lublin; PhD in Agricultural Sciences with major in Biochemistry 1985, IUNG Pulawy; Habilitation in Biochemistry 1992, IUNG Pulawy; Full Professor 1998, President of Poland.
Work experience: In 1979-80 and 1987 (one year each) a Visiting fellow at Cornell University performing research on glycoalkaloids in tomato and its progenies received from the crosses with wild Solanum species related to tomato (Solanum penelli and Solanum lycopersicoides) and on phenolic compounds from apples and their function in enzymatic browning of fruits. In 1989 a visiting scientist in Food Research Institute, Norwich, UK working on membrane activity of alfalfa saponins and NMR techniques for their structure elucidation. In 1992 an INRA, Avignon, France, Postdoctoral Trainee working on pear phenolics. In 1992 I assumed position as a Head of Biochemistry and Crop Quality Department in the Institute of Soil Science and Plant Cultivation, Pulawy, Poland. In 2010 I was appointed Director of the Institute of Soil Science and Plant Cultivation, State Research Institute, Pulawy, Poland
Major research interests: Isolation, purification, structural and quantitative determination of plant glycosides (saponins, phenolics, glycoalkaloids, glucosinolates, resorcinols, cyanogenic glucosides), natural toxicants of plants - allelopathy, natural pesticides, post harvest quality of agricultural products, natural antioxidants and anticancerogens, nutraceuticals biosynthesis of secondary metabolites, food and feed stuff additives, functional food
Professional societies membership: Member of Polish Academy of Sciences; Phytochemical Society of Europe - Committee member (1994-1998), General Secretary 2002-2007, incoming V-ce Chairman 2008-2010, Chairman 2010-2012. ( http://www.phytochemicalsociety.org ) Polish Phytochemical Society - Secretary, 1995 Polish Biochemical Society – member Polish Botanical Society – member Allelopathy Journal - Editorial board member Phytochemistry Reviews - Editorial board member (http://www.kluweronline.com/issn/1568- 7767/current) Phytochemistry Letters - Editorial Board Member http://www.sciencedirect.com/science/journal/18743900 Journal of Food, Agriculture and Environment - Regional Editor (2010-2015) (http://www.isfae.org/scientificjournal.php)
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International Allelopathy Society - founding member (http://www-ias.uca.es/) (http://www.ashland.edu/~jweiden/ids350/newslett.2002.htm ) Open Journal of Biochemistry (http://www.rossscience.org/ojbioc/ ) Member of the Domain Committee “Food and Agriculture” COST, Brussels Participation in European projects: Fate and toxicity of allelochemicals (natural plant toxins) in relation to environment and consumer. Vth Framework Program of European Community www.fateallchem.dk Evaluating physiological and environmental consequences of using organic wastes after technological processing in diets for livestock and humans (SAFEWASTES), VIth Framework http://safewastes.bal-pm.com/ Ready-to-eat food for breakfast and sport with high content of nutraceutics preventing disease and promoting public health (NUTRA-SNACKS), VIth Framework Program http://www.mlib.cnr.it/nutra-snacks/ Healthy Feed for Safety – Dissemination of research results of EC funded research on feed quality (FEED-SEC), VIth Framework Program of European Community (http://www.feed-seg.net/) Strengthen IUNG’s proficiency on “Managing the Production of Food and feedstuff, their safety and quality under global Climatic Change, ProFiCienCy – VIIth Framework Program of European Community, coordinator http://proficiency-fp7.eu/ Edible, Medicinal and Aromatic Plants (EMAP) 7 FP EU; (2011-2014), Marie Curie Program Safe Food for Europe – Coordination of research activities and dissemination of research results of EC funded research on food safety (FOODSEG) 7 FP EU Program Optimising Subsidiary Crop Applications in Rotations (OSCAR) (2012-2015) 7 FP EU ProgramImpact of new technologies on the health benefits and safety of bioactive plant compounds, COST 926 action (number of European countries as participants), http://www.uochb.cas.cz/Zpravy/COST_926/ New Strategies on Bio-Economy in Poland (BioEcon), H2020, coordinator
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Plenary Lecture – PL3
Agriculture crops as the source of bioactive compounds
Wieslaw Oleszek
Institute of Soil Science and Plant Cultivation, State Research Institute, ul. Czartoryskich 8, 24-100 Pulawy, Poland *Corresponding author, e-mail: [email protected]
Living plants produce a great number of chemicals that are crucial for their function and development. Some of these chemicals are primary metabolites, which include proteins (aminoacids), carbohydrates, fats, nucleic acids etc. However, besides these primary chemicals, the plants also produce so called secondary metabolites, which are specific to some taxonomic groups (families, genera).
Their physiological function was questioned already in earlier work, but recent research has shown that they are important constituents of plants. They are formed under environmental pressure and play a crucial function in protecting plants against some environmental stresses.
This group includes classes of compounds such as phenolics, carotenoids, alkaloids, saponins, glucosinolates, cyanogenic glycosides, terpenes etc. Each of these groups contains compounds with different biological activities, which in traditional medicine and ethno-pharmacology were used for centuries to cure or to protect from diseases.
In Western Europe, the aging of population has been and currently is a driving force for the study of new phytochemical products, able to fight age-related conditions and prevent diseases such as high blood cholesterol level, high blood pressure, arthritis, obesity and cancer. In fact, only a few plants have been the subject of detailed investigations but more than 1000 species have been claimed to offer special benefits.
However, in many cases no certainty exists about the real effects of dietary phytochemicals and more epidemiological surveys and clinical studies should be performed. Moreover, in various cases contrary effects are reported.
Probably the main reason for this discrepancy is that many clinically tested “products” are, in fact, multicomponent mixtures since it is frequently very difficult to isolate only one component to study its biological effect.
Another important question in analysis of activity of secondary metabolites is recognition of their easy degradation by microorganisms or physical factors.
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In some cases the gradation products which are in many cases being neglected show much higher activity then the mother molecule. This will be presented in relation to cyanogenic glycosides as well as for benzoxazinones.
Present lecture focuses on a number of examples of secondary metabolite analysis and activity, their chemical complexity, and challenges of their separation and structure elucidation.
Literature 1. Pollier, J., Moses, T., Gonza´lez-Guzma, M., De Geyter, N., Lippens, S., Vanden Bossche, R., Marhavy, P., Kremer, A., Morreel, K.,. Gue´rin, C., Tava, A., Oleszek, W., Thevelein, J.M., Campos, N., Goormachtig, S.. Goossens, A. The protein quality control system manages plant defense compound synthesis. Nature, 504, 148-152, 2013. 2. Kowalska I., Cieśla Ł., Oniszczuk T., Waksmundzka-Hajnos M., Oleszek W., Stochmal A. Comparison of two TLC-DPPH-image processing procedures for studying free radical scavenging activity of compounds from selected varieties of Medicago sativa. Journal of Liquid Chromatography & Related Technologies, 36, 1–8, 2013. 3. Cieśla L., Kowalska I., Oleszek W., Stochmal A Free Radical Scavenging Activities of Polyphenolic Compounds Isolated from Medicago sativa and Medicago truncatula Assessed by Means of Thin-layer Chromatography DPPḢ Rapid Tests Phytochemical Analysis, 24 (1) , pp. 47-52, 2013. 4. Wallace R.J., Oleszek W., Franz C., Hahn I., Baser K.H., Mathe A., Teichmann K. Plant bioactives for poultry health and productivity. British Poultry Science 51 (4), pp. 461-487, 2010. 5. Oleszek W., Stochmal A., Janda B. Concentration of isoflavones and other phenolics in the aerial parts of Trifolium species. J. Agric. Food Chem. 2007, 55, 8095-8100.
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Professor Satyajit D Sarker BPharm (Hons) MPharm PhD FHEA
Official contact details: Director of School of Pharmacy and Biomolecular Sciences & Professor of Pharmacy
School of Pharmacy and Biomolecular Sciences Faculty of Science Liverpool John Moores University Room 905, James Parsons Building Byrom Street Liverpool L3 3AF, England, UK
Tel: 01512312096; Mobile: 07929999508 Fax: 01512312170 E-mail: [email protected]
Current role: As the Director of School of Pharmacy and Biomolecular Sciences at LJMU, I have been providing strategic, operational, financial, academic and research leadership to this multidisciplinary School.
In addition to chairing various committees within the School, I am actively involved in the activities of various committees at the Faculty and the University levels e.g., Faculty Management Team, Faculty Research, Knowledge Transfer and Enterprise Committee, Faculty Professors/Readers Applications Review Panel, Life Science Building Users’ Group, University Strategy Development Forum (SDF), University Education Committee, LJMU International Policy Committee, Equality and Diversity Steering Group, Professorial Pay Review Committee, STEM-funded Refurbishment Steering Group and South-Asian Recruitment Sub-Committee. I have also been managing the School budget, over 1500 students and around 130 members of academic and non-academic staff.
I am a member of the Pharmacy Schools Council (PhSC), Editor-in-Chief of Phytochemical Analysis, and the Honorary Vice-President of the Phytochemical Society of Europe.
Academic background: PhD in Pharmaceutical Sciences, University of Strathclyde, UK MPharm, 1st class and 1st in order of merit, University of Dhaka, Bangladesh BPharm (Hons), 1st class and 1st in order of merit, University of Dhaka, Bangladesh
Employment history: Director of School of Pharmacy and Biomolecular Science & Professor of Pharmacy (2013-present), Faculty of Science, Liverpool John Moores University Professor of Pharmacy, Deputy Head of Department of Pharmacy & Pharmacy Research Group Leader (2008-2013), Department of Pharmacy, University of Wolverhampton Reader in Pharmacy, Course Director and Chair of the MPharm Course Planning Team, Course Director of the BSc (Hons) Pharmacology and MSc in Pharmaceutical
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Sciences courses (2004-2008), School of Pharmacy and Pharmaceutical Sciences, University of Ulster Lecturer in Pharmaceutical Sciences, and Medicinal and Natural Products Chemistry Research Group Leader (2000-2004), School of Pharmacy, The Robert Gordon University Senior Research Scientist and Head of Spectroscopy Group (1998-2000), MolecularNature Ltd., Institute of Grassland and Environmental Research (IGER) BBRSC Post-doctoral Research Fellow (1995-1998), University of Exeter Lecturer in Pharmacy, Department of Pharmacy, University of Dhaka (1990-1991) Research Fellow in Pharmacy, Department of Pharmacy, University of Dhaka (1989- 1990)
Research interests: Drug Discovery, Design and Delivery: Pharmaceutical, Medicinal and Natural Products Chemistry Biosynthesis of pharmaceutically important plant secondary metabolites Application of hyphenated techniques in natural products research Metabolomics of medicinal plants Toxicological and pharmacological evaluation and quality control of herbal medicine Development of bioassays for screening plant extracts and isolated compounds Synthesis of pharmaceutically important compounds
Current research projects: Extraction, isolation, identification, structural modification (synthesis), bioactivity and mode of action of pharmaceutically important natural products, especially focussing on novel anticancer, antimicrobial, antimalarial, antioxidant and anti-inflammatory compounds Production of compound libraries of dereplicated natural products for high throughput Screening (HTS) Structure-Activity-Relationships (SAR) studies on bioactive compounds and their analogues Bioactivity and phytochemical studies on plants from the Bangladeshi, British, Cameroonian, Iranian, Iraqi, Kenyan, Libyan, Pakistani and Turkish flora
Membership of professional bodies: Fellow of the Higher Education Academy (FHEA) Member of: The Phytochemical Society of Europe (PSE) The American Society of Pharmacognosy (ASP), USA The Botanical Society of Scotland, UK The Pharmacy Graduates’ Association, Bangladesh The Bangladesh Pharmaceutical Society, Bangladesh
Honorary Treasurer of the Phytochemical Society of Europe (PSE) (2008-2013) Honorary Vice-President of the Phytochemical Society of Europe (Since 01 June 2016)
Membership of journal editorial board: Editor-in-Chief, Phytochemical Analysis (2010-present)
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Associate Editor, TANG Journal (May 2011-present) Guest Editor, special issue of Natural Product Communications in the honour of Professor Peter G Waterman (2008), and Special issue of Advances in Pharmacological Sciences on ‘Anti-inflammatory, antinociceptive and antipyretic activities of medicinal plants and their constituents’ (2012), and special issue of Medicines on Essential Oils: Chemistry and Bioactivity (2015-16) Member of the Editorial/Editorial Advisory Board: Current Medicinal Chemistry, Scientific Reports, and 26 other international journals
Publications:
Total number of publications: 406 Total citations: Well over 7710 (source: Google Scholar) h-index: 40 (source: Google Scholar) i10-index: 219 (source: Google Scholar)
Most popular books: 1. Sarker SD and Nahar L (2012) “Natural Products Isolation”, 3rd edition, Humana Press/Springer Verlag, USA (invitation from Professor J Walker, Series Editor), ISBN: 978- 1-617-79623-4. 2. Sarker SD and Nahar L (2007) “Chemistry for Pharmacy Students: General, Organic and Natural Product Chemistry”, John Wiley & Sons, London. ISBN 978-0-470-01780-7 and 978-0-470-01781-4, and its Greek, Japanese and Portuguese translations.
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Plenary Lecture – PL4
An overview on chemopreventive phytochemicals
Satyajit D. Sarker*, Georgiana Zavoianu, Fyaz M. D. Ismail, Kenneth J. Ritchie, Lutfun Nahar
Medicinal Chemistry and Natural Products Research Group, School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, James Parsons Building, Byrom Street, Liverpool L3 3AF, United Kingdom *Corresponding author, e-mail: [email protected]
It is a well-known fact that ‘prevention is better than cure’. This becomes even more relevant when it comes to prevention of various forms of cancer than any other diseases. According to the WHO, cancer is one of the leading causes of death worldwide, accounting for 8.2 million deaths in 2012, with lung (1.59 million deaths) and liver (745 000 deaths) cancers being the major ones. The number of cancer sufferers is set to increase considerably in the coming years because of changing environment and life-style resulting from increasing urbanisation and socio-economic changes. Cancer treatments, which are currently available, are costly, lengthy, have serious side-effects, and often have limited effectiveness. In the search for suitable cancer prevention measures, scientists have worked with dietary supplements, mainly plant-based, that have chemopreventive potential. Although attempts have been made to purify phytochemicals having chemopreventive potential, the main focus has always been on food plants; non-food plants have not been explored extensively in relation to chemoprevention. The search for new, cheaper, less toxic and more effective anticancer drugs is still one of the major areas of modern drug discovery operations; however, the importance of finding suitable cancer chemopreventive agents from plants is also well recognised, considering the huge spending in healthcare for the treatment of cancers. This talk aims to present an overview on available chemopreventive phytochemicals, their plausible mechanisms of actions, and the authors’ own research findings on chemopreventive agents from non-food plants, utilising the mechanistic approach of induction of Nrf2 activation as an indicator for cancer chemoprevention.
Further reading: Basar N, Nahar L, Oridupa OA, Ritchie KJ, Talukdar AD, Stafford A, Kushiev H, Kan A and Sarker SD (2016) Utilization of the ability to induce activation of the nuclear factor (erythroid-derived 2)- like factor 2 (Nrf2) to assess potential cancer chemopreventive activity of liquorice samples, Phytochemical Analysis (in press). Y-J (2003) Cancer chemoprevention with dietary phytochemicals, Nature Reviews - Cancer 3, 768- 780.
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Prof. Dr. ILKAY ERDOGAN ORHAN
Faculty of Pharmacy, Gazi University, Ankara, Turkey
Born in 1972 in Ankara (Turkey). Completed her B.Sc. (B. Pharm.) at Faculty of Pharmacy, Gazi University, Ankara, Turkey in 1993 and M.Sc. degree at Department of Pharmacognosy at the same faculty (1996) with young scientist scholarship provided by TUBITAK (Scientific and Technological Research Council of Turkey). Awarded her second M.Sc. degree in Marine Natural Product Chemistry (1998) at the University of the Ryukyus in Japan supported by Monbusho scholarship. Earned Ph.D. degree in Pharmacognosy at Faculty of Pharmacy, Gazi University in 2002 and visited Department of Chemistry at University of Winnipeg (Canada) in 2003 as NATO- TUBITAK fellow. Promoted to Assoc.Prof. position in 2004 and full professor in 2009. Received several awards such as o Young Scientist Award by Turkish Association of Pharmacists (2006) o Young Woman Scientist Award in Asia-Pacific region by OWSD & Elsevier (2010) o Science Award in Biology by COMSTECH (2010) o Young Woman Scientist Award by L’Oreal & Turkish Academy of Sciences (2011) o Honor Award by Gazi University (2011) o Innovation Award for Women by TUBITAK and Association of Woman & Democracy (2015) Served as “Dean” of Faculty of Pharmacy at Eastern Mediterranean University in the Northern Cyprus for the period of 2011-2014. Currently affiliated as full professor at Faculty of Pharmacy, Gazi University. Her research interests are enzyme inhibition, phytochemistry, natural products, and herbal cosmetics. Author of 184 papers published in reputed international journals, 41 papers in national journals and 13 book chapters. Dr. Orhan was also Editor of “Biotechnological Production of Plant Secondary Metabolites” published in 2012. Her h index is 25.
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Plenary Lecture – PL5
Our current verdicts into herbal bioactive molecules by means of molecular docking approaches
Ilkay Erdogan Orhan*
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey *Corresponding author, e-mail: [email protected]
Enzyme inhibition is one of the valid and popular experimental strategies in drug discovery and development process. Many enzyme inhibitors are successfully applied in clinic for the treatment of various diseases. Since plants as well as microorganisms, marine organisms, etc have been proven to be rich natural sources of biologically active compounds, an extensive research has been also going on these sources to discover new drug candidates. Many enzymes catalyze vital reactions in the body and play a critical role in pathology of several diseases. In our ongoing screening studies on medicinal plants and natural substances, we have screened many plant extracts and pure natural compounds for their possible enzyme inhibitory effects such as cholinesterase family, tyrosinase, elastase, collagenase, phosphodiesterase-1, carbonic anhydrase, thermolysin, etc. The experimental part has consisted of in vitro assays followed by in silico-based assays such as molecular docking (Figures 1 and 2). In this talk, our latest findings on active natural compounds obtained from our collaborative studies will be highlighted in details.
Figure 1. The 2D (left) and 3D (right) interactions of docked rutin against phosphodiesterase-1
Figure 2. The predicted docked poses of carbonic anyhydrase-II with some phenolic compounds that we tested and reference acetazolamide (stick red color).
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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
HUGO DE BOER PERSONALIA Born: May 24th, 1978, Citizenship: Swedish
EDUCATION 2006 – 2012 Uppsala University Uppsala, SE . PhD (Tekn. Dr.) in Systematic Botany 2009 – 2009 University of California, Berkeley Berkeley, USA . Postgraduate Research Fellow, Department of Integrative Biology 2003 – 2004 Uppsala University Uppsala, SE . 2nd Master of Science in Biology 1998 – 2003 Wageningen University Wageningen, NL BSc and MSc in Biology (Engineer), cum laude ACADEMIC MERITS POSITIONS . Leader of the Nordic Research School in Biosystematics – ForBio, Natural History Museum, University of Oslo (2015-06-01-ongoing) . Researcher, Natural History Museum, University of Oslo (2014-01-01- ongoing) . Associate Professor (Docent), Syst. Biology, Uppsala University (2014-10- 24 - ongoing) . Associated Researcher, Naturalis Biodiversity Center, the Netherlands (2015 - ongoing) . Associated Researcher, Natural History Museum, University of Oslo (2012- 2013) . Assistant Professor (Forskare), Systematic Biology, Uppsala University (2012-2014) . Researcher, Naturalis Biodiversity Center, Leiden, the Netherlands (2012- 2014) . Research Assistant, Dept of Systematic Botany, Uppsala University (2004- 2012) . Parental leave, 15 months (2007-2011) . Leave for commission of trust within Uppsala University, 6 months (2006- 2012) . Botanical expert for medicinal plants at the WHO Collaborating Centre for International Drug Monitoring, Uppsala (2003-2005)
BOARDS & . Scientific Committee for Food Security, Specialist Group in Exotic and CONSULTANCY Trade in CITES-listed Species, Ministry of Health and Care Services, Norway (2015-) . AllGenetics & Biology, SL: Expert consultant on DNA barcoding (2013-) . Faculty of Technical and Natural Sciences’ Scholarships Board (2009-2012) . Faculty Board of Technical and Natural Sciences, Uppsala University (2006- The symposium is dedicated to the 60-year celebration of the „Stejarul” Biological Research Centre – MAPPPS 2016, Piatra Neamt, ROMANIA – Page 28
Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
2008)
AWARDS & . Uppsala University’s Recruitment Group for Biology (2006-2009; 2010) PRIZES . Uppsala University’s Board for Stipends and Scholarships (2006-2012) . Center for Sustainable Development, UU-SLU, Board, locum (2011)
SELECTED . Konung Carl XVI Gustafs 50 årsfond för vetenskap, teknik, och miljö, prize PUBLICATIONS research on DNA barcoding of threatened species in trade. . NVGO van Os prize for research on herbal medicine. . Richard E. Schultes Award, Society for Economic Botany. . French-Swedish Prize for Young Researchers 2010: Biodiversity and Human Health. . Graduate Thesis Award 2004, Wageningen University Fund. . Osathanunkul, M., Suwannapoom, C., Osathanunkul, K., Madesis, P., de Boer, H.J., 2016. Evaluation of DNA barcoding coupled high resolution melting for discrimination of closely related species in phytopharmaceuticals. Phytomedicine 23 :156–165. IF = 3.1 . Osathanunkul, M., Suwannapoom, C., Ounjai, S., Rora, J.A., Madesis, P., De Boer, H.J. 2015. Refining DNA barcoding coupled high resolution melting for discrimination of 12 closely related Croton species. PLoS ONE 10(9): e0138888. doi:10.1371/journal.pone.0138888 . De Boer, H.J., Ichim, M.C., Newmaster, S. 2015. DNA Barcoding and Pharmacovigilance of Herbal Medicines. Drug Safety 38: 611-620. IF = 3.2 . Osathanunkul, M., Madesis, P., De Boer, H.J. 2015. Bar-HRM for authentication of plant-based medicines: evaluation of three medicinal products derived from Acanthaceae species. PLoS ONE 10.1371/journal.pone.0128476. IF = 4.1 . Kreziou, A., De Boer, H.J., Gravendeel, B. 2015. Harvesting of salep orchids in northwestern Greece continues to threaten natural populations. Oryx [online first] doi:10.1017/S0030605315000265 . Ghorbani, A., Gravendeel, B., Zarre, S., De Boer, H.J. 2014. Illegal wild collection and international trade of CITES-listed terrestrial orchid tubers in Iran. TRAFFIC Bulletin 26(2): 42-52 . Veldman, S., Otieno, J.N., van Andel, T., Gravendeel, B., De Boer, H.J. 2014. Flourishing trade in African orchids. TRAFFIC Bulletin 26(2): 54-58 . Ghorbani, A., Naghibi, F., Gravendeel, B., De Boer, H.J. 2014. Wild orchid tuber collection in Iran: A wake-up call for conservation. Biodiversity and Conservation 23: 2749-2760. IF = 2.3 . De Boer, H.J., Ouarghidi, A., Abbad, A., Martin, G.J., Kool, A. 2014. DNA Barcoding Reveals Limited Accuracy of Identifications Based on Folk Taxonomy. PLOS ONE 9 (1), e84291 IF = 4.1 . Ouarghidi, A., Powell, B., De Boer, H.J., Abbad, A., Martin, G. 2012. Species substitution in medicinal roots and possible implications for toxicity in Morocco. Economic Botany 66: 370-382. IF = 1.9 . Kool, A.*, De Boer, H.J.*, Rydberg, A., Kruger, A., Abbad, A., Björk, L., Martin, G. 2012. Molecular Identification of Commercialized Medicinal Plants in Southern Morocco. PLOS ONE 7 (6), e39459 *shared first authors. IF = 4.1 . Mati, E. De Boer, H.J., 2011. Trade and commercialization of herbal
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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
medicine in the Qaysari Market, Kurdish Autonomous Region, Iraq. Journal of Ethnopharmacology 133: 490-510. IF = 3.0
. De Boer, H.J., Hagemann, U., Ericsson, J., Meyboom, R., 2007. Allergic reactions to Pelargonium-derived medicines. Drug Safety 30: 677-680. IF = 3.7 . Farah, M., Olsson, S., Bate, J., Lindquist, M., Edwards, R., Simmonds, M.S.J., Leon, C., De Boer, H.J., Thulin, M. 2006. Botanical Nomenclature in Pharmacovigilance and a Recommendation for Standardisation. Drug Safety 29: 1023-1029. IF = 3.7
. Peer-reviews for 37 peer-reviewed journals. . Editorial boards: Phytokeys, Phytotaxa, J. Ethnobiology and Ethnomedicine
OTHER . English, Dutch, Swedish: Fluent . German: Intermediate LANGUAGES . French, Thai, Lao: Basic
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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
Plenary Lecture – PL6
Comparative authentication of Hypericum perforatum herbal products using amplicon metabarcoding
Ancuta Cristina Raclariu1,2, Ramona Paltinean3 , Laurian Vlase3, Aurélie Labarre1, Vincent Manzanilla1, Mihael Cristin Ichim2, Gianina Crisan3, Anne Krag Brysting4, Hugo de Boer1,*
1Natural History Museum, University of Oslo, P.O. Box 1172 Blindern, 0318 Oslo, Norway. 2NIRDBS/”Stejarul” Research Centre for Biological Sciences, Alexandru cel Bun St., 6, Piatra Neamt, Romania. 3Department of Pharmaceutical Botany, University of Medicine and Pharmacy “Iuliu Haţieganu”, Faculty of Pharmacy, V. Babeş Street, 8, Cluj-Napoca, 400012, Romania. 4Department of Biosciences, Centre for Ecological and Evolutionary Synthesis (CEES), University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway. *Corresponding author, e-mail: [email protected]
Abstract St. John’s wort (Hypericum perforatum L.) herbal products have limited oversight, frequent off-label use, and insufficient monitoring of adverse drug reactions. Many herbal products have a long history of use, but there are rising concerns over product efficacy, safety and quality in the wake of recent cases exposing discrepancies between labelling and constituents. In this study, we use amplicon metabarcoding (AMB) to authenticate 78 Hypericum perforatum herbal products sold in the European Economic Area (EEA) and evaluate its ability to detect substitution with European Pharmacopoeia standard methods using thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). AMB was carried out in triplicate using nrITS1 and nrITS2 on an Ion Torrent PGM sequencing platform. AMB, TLC and HPLC can all detect H. perforatum in herbal products. Furthermore, AMB is sensitive in detecting incongruence between constituent species and those listed on the label. TLC and HPLC are accurate methods for authenticating presence of the target species, but have limited efficiency in detecting infrageneric substitution and do not yield any information on the other plant ingredients in these products. Random post-marketing AMB of herbal products by regulatory agencies could raise awareness among consumers of substitution and would provide an incentive to manufacturers to increase quality control from raw ingredients to products.
Keywords: Drug safety; DNA metabarcoding; Herbal products; Herb-drug interaction; Hypericum perforatum (St. John’s wort); High-throughput sequencing; HPLC; TLC
Acknowledgements The research has received funding from the Romanian - EEA Research Programme operated by the MECS-ANCSI PO under the EEA Financial Mechanism 2009-2014 and Project Contract No 2SEE/2014.
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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
Dr. MILEN I. GEORGIEV
Milen Georgiev, PhD in biotechnology, is heading plant biotechnology, natural products chemistry and metabolomics work. He has 13+ years of experience in natural products and has published in excess of 90 papers (e.g. in Trends in Biotechnology, Biotechnology Advances, and Phytochemistry among others).
He has delivered invited lectures in 14 different countries. Dr. Georgiev holds several grants from the National Science Fund of Bulgaria and framework programs of EU (incl. H2020). He spent several years abroad as postdoctoral scientist in Germany (2005-2007) and in The Netherlands (2010- 2012), both supported by the Marie Curie program of EU. In 2011 and 2015 he was awarded by the Bulgarian Government with Pythagoras award for outstanding scientist, as at present the only scientist in Bulgaria to win twice.
Dr. Georgiev serves as an Associate Editor of Phytomedicine (Elsevier) and on the Editorial board of Biotechnology Letters (Springer), also prepared Guest editorials for Biotechnology Advances (2014), Phytochemistry Reviews (2014; 2016), Food Chemistry (2015), Molecules (2015) and Food and Chemical Toxicology (undergoing). He was a chairman of the International Conference on Natural Products Utilization: from Plants to Pharmacy Shelf (ICNPU-2013 with 230 attendees) and its second edition in Plovdiv, Bulgaria (ICNPU-2015 with 330 participants.
His current research focuses on the biosynthesis of fine molecules and the development of biotechnological tools for their sustainable mass production along the application of emerging modern platforms for comprehensive metabolite profiling (i.e. NMR-based metabolomics) and biochemometrics.
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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
Plenary Lecture – PL7
Pharmaceutically relevant molecules from plant origin and their sustainable bioproduction
Milen I. Georgiev 1,2*
1Group of Plant Cell Biotechnology and Metabolomics, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 139 Ruski Blvd., 4000 Plovdiv, Bulgaria; 2 Center of Plant Systems Biology and Biotechnology, Plovdiv, Bulgaria *Corresponding author, e-mail: [email protected]
Iridoid and phenylethanoid glycosides are natural compounds with remarkable and diverse biological properties. In the recent years, interest has been growing in phenylethanoid and iridoid glycosides, because of the significantly increasing volume of literature about their evident role in prevention and treatment of various human disorders. Harpagoside is an iridoid glycoside that was first isolated from devil’s claw, a medicinal plant in which it is the major constituent of the iridoids pool. Both harpagoside and devil’s claw extracts have shown potent anti-rheumatic, anti-inflammatory and analgesic properties [1]. Verbascoside – phenylethanoid glycoside – is among the most widespread of the disaccharide caffeoyl esters, possessing pharmacologically beneficial activities for human health, including anti-inflammatory, antineoplastic and cytoprotective ones, besides abundant wound-healing and neuroprotective properties [2]. This lecture will provide an overview on chemical (inlc. NMR-based metabolomics) and pharmacological aspects of research on phenylethanoid and iridoid glycosides along with some successful examples of their sustainable bioproduction [3-5].
References [1] Georgiev MI, Ivanovska N, Alipieva K, Dimitrova P, Verpoorte R (2013) Harpagoside: from Kalahari Desert to pharmacy shelf. Phytochemistry 92:8-15.
[2] Alipieva K, Korkina L, Erdogan Orhan I, Georgiev MI (2014) Verbascoside – A review of its occurrence, (bio)synthesis and pharmacological significance. Biotechnology Advances 32:1065-1076. [3] Dimitrova P, Milanova V, Alipieva K, Georgiev MI (2015) Regulations of neutrophil functions by verbascoside and isoverbascoside. ICNPU Abstracts Book pp. 39. [4] Marcoccia D, Georgiev MI, Alipieva K, Lorenzetti S (2014) Inhibition of the DHT-induced PSA secretion by Verbascum xanthophoeniceum and Serenoa repens extracts in human LNCaP prostate epithelial cells. Journal of Ethnopharmacology 155:616-625. [5] Georgiev MI, Radziszewska A, Neumann M, Marchev A, Alipieva K, Ludwig-Müller J (2015) Metabolic alterations of Verbascum nigrum L. plants and SAArT transformed roots as revealed by NMR-based metabolomics. Plant Cell, Tissue and Organ Culture 123:349-356.
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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
Dr. CHIN-KUN WANG
Personal Profile Dr. Chin-Kun Wang is a distinguished professor in Chung Shan Medical University, President of International Society for Nutraceuticals and Functional Foods, fellow of International Academy of Food Science & Technology, honorary president of Nutrition Society of Taiwan, council member of FANS, director for International Life Science Institute, Taiwan.
He got his Ph.D. degree from National Taiwan University and worked at Chung Shan Medical University in 1993. In 1996, he promoted as a full professor, and then took the positions of the chair, dean, vice president and president in Chung Shan Medical University. His research work is focused on human clinical trials and human metabolism of medicine, nutritional supplement, nutraceuticals, herbs, and functional foods. He got the National Award of Biomedicine for his great contribution to the medical education in 2008. He was also honored as 2012-16 Who’s who in the world, Who’s who in Asia, and 2009-2010, 2011-12 Who’s who in Medicine and Healthcare.
Dr. Chin-Kun Wang was the former president of Nutrition Society of Taiwan (from 2009 to 2012). For food safety and nutrition, he promoted the legislation for school sanitary law and national nutrition law. During the food safety problem in Taiwan, he jointed as a director of ILSI Taiwan and ambassador of Global Harmonization Initiative to communicate with the media and press. He believes that scientific evidence is the best support for food safety and world nutrition problem. In the future, he tries his best to work together with the scientists around the world by network.
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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
Plenary Lecture – PL8
Effect of the mixture of Dong Quai, Tang Shan, Linchi and citronellol on the infection and ulcer of Helicobacter pylori
Hsin-Lun Huang1, Chin-Kun Wang1,
1School of Nutrition & Medicine,Chung Shan Medical University, Taichung, Taiwan
Abstract. The extract of the mixture of Dong Quai, Tang Shan, Linchi and citronellol significantly inhibit the infection and ulcer induced by H. pylori. This effect was not to destroy the bacteria, but greatly suppress the adhesion of H. pylori and reduce Cag A secretion and the series of inflammation. In addition, this mixture could effectively promote the healing of ulcers. Key words: Dong Quai, Tang shan, Lichi, citronellol, H.pylori. Introduction. Helicobacter pylori (H. pylori) is a human gastric pathogen, which induces chronic inflammation to lead gastritis, gastric ulcer and gastric carcinoma. Thus, to obstruct colonization of H. pylori or relieve inflammation may decrease the risk of gastric diseases during H. pylori infection. Mixture of Dong Quai, Tang Shan, Linchi and citronellol provides strong antioxidant activity and anti-inflammation but the it’s protection on stomach during H. pylori infection remains unknown. Material and methods. H. pylori was measured by C13-urea breath test (UBT). The H. pylori positive subjects (UBT >10‰, n=36) were recruited. Sample or placebo was taken for two months and two weeks of follow-up period after without administration. The UBT, blood sample, antioxidant capacity, total phenol, and inflammatory markers were analyzed at the initial, 4th, 8th and 10th weeks. In cell model, the AGS cell was treated with the complex extract and H. pylori to investigate minimum inhibition concentration (MIC), cell viability (MTT) and anti-adhesion. Results and discussion
Conclusions. Great potential was found from traditional used food or herb to new application. Evidence-based is required. Bibliography 1. Shu-Ru Zhuang, Su-Lin Chen, Jih-Hsin Tsai, Hong-Sen Lee, Min-Chang Huang, Guang-Tzuu Shane, Cheng-Hua Yang, Yeong-Yu Yan, You-Cheng Shen and Chin-Kun Wang (2012) Effect of the Chinese medical herbs complex on cellular immunity and adverse effect of breast cancer patients. The British Journal of Nutrition 107: 712-718 2. Hsin-Lun Huang, Chien-Hui Ko CH, Yeiong-Yu Yan, Chin-Kun Wang (2014) Antiadhesion and antiinflammation effects of noni (Morinda citrifolia) fruit extracts on AGS cells during Helicobacter pylori infection. Journal of Agriculture and Food Chemistry, 62, 2374-2383.
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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
Short Lecture – SL1
In vivo and in vitro evaluation of the anti-diabetic nutraceutical potentials of Capparis spinosa
Adriano Mollica1,*, Azzurra Stefanucci1, Giorgia Macedonio1, Marcello Locatelli1, Olakunle Onaolapo2, Adejoke Onaolapo3, Ettore Novellino4
1 a Dipartimento di Farmacia, Università “G. d’Annunzio” di Chieti-Pescara, via dei vestini 31, 66100, Chieti, Italy 2a Department of Pharmacology, Ladoke Akintola, University of Technology, Osogbo, Osun State, Nigeria 3 a Department of Anatomy, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria. 4 a Dipartimento di Farmacia, Università “Federico II” di Napoli, via D. Montesano, Napoli, Italy
Abstract. In this study, we investigated Capparis spinosa leaf and commercial salted buds. This was with the aim of evaluating its usefulness as a valuable nutraceutical. We conducted a series of in vivo and in vitro tests. The leaf, fresh buds and salty buds (desalted 24 h in water) were obtained and processed to dry powder. 40% MeOH/H2O extract was obtained for HPLC analysis, to determine quantitatively the polyphenols, flavonoids and the presence of other relevant compounds. The inhibition of enzymes related to different dismetabolic pathologies were also assessed. To evaluate the anti-diabetic potential in rodents, dry powder of Capparis Spinosa leaf and bud were administered orally to streptozocin-induced diabetic rats over a period of 21 days, during which body weight and blood glucose were monitored weekly. At the end of the experimental period, animals were sarificed and blood taken for assessment of lipid profile, and liver/kidney biochemistry. Key words: HPLC, polyphenols, flavonoids, Capparis spinosa, diabetes. Introduction. Capparis spinosa L. is one of the most popular, aromatic, edible plants used in the Mediterranean cuisine, it also important for the preparation of frozen food and as appetiser. In Italy, the main production sites of Capparis is in Sicily and Sardinia. The floral bud is the commonly used part, which is stored under salt or acetic solution, before commercial packaging and shipping. Previous studies reported the presence of bioactive products (e.g. flavonoids, lipids, alkaloids and glucosinolates) as flavour compounds, antioxidant and anti-inflammatory agents [1] in the fresh buds. In this study, we focused on the fresh leaf (as alternative to the buds), and the salty buds, which is the real commercial product present in the market.
Figure 1. Lipari island and capparis spinosa plant. Material and methods. Capparis spinosa L. plants were collected in July, from Lipari isle, in the Aeolian Archipelago (Italy), and provided to us by Capersud Manufacture. Extraction. The leaf, fresh buds and salty buds (24 h desalted) were freeze-dried by a VirTis lyophilizer. The natural product was then powdered, and 500 mg of each sample was extracted by three different techniques: microwave extraction, soxhlet extraction and decoction by 40% MeOH/H2O. The extracts were subjected to lyophilization. The dry extract obtained was analyzed by HPLC and tested for enzymatic assays. HPLC analysis.We analysed extracts of Capparis spinosa through the quantitative and qualitative determination of polyphenols and flavonoids, which were performed by means of a reverse phase HPLC-UV analysis carried out using an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA, USA) comprised of a double solvent delivery system, an on-line degasser, an autosampler, a column temperature controller and UV-photodiode array detector (DAD). In vivo antidiabetic study. 48 rats have been divided into eight groups of six rats each. The eight groups were treated with
The symposium is dedicated to the 60-year celebration of the „Stejarul” Biological Research Centre – MAPPPS 2016, Piatra Neamt, ROMANIA – Page 36
Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
streptozocin in order to create a diabetic rat model. All the rats were monitored by glycemic strips assays to determine if the glycemic level was raised from the basal value. Then Group A was left untreated and monitored for a period of 21 days. Group B was treated with 100mg/kg of Capparis spinosa leaf, group C with 200 mg/kg of Capparis spinosa leaf, group D with 400 mg/kg of Capparis spinosa leaf, group E with 100 mg/kg of Capparis spinosa salty buds, group F with 200 mg/kg of Capparis spinosa salty buds and group G with 400 mg/kg of Capparis spinosa salty buds every day. Group H was treated with metformin at 1.8 mg/kg. Results and discussion. We have demonstrated that already at the lower dose tested, both Capparis spinosa buds and leaf are capable to efficaciously lowering the glycemic level in the rats blood. The effect is visible at the first week and it is complete during the second week. The test was conducted until 4 weeks and there was no mortality neither toxic effect. The final glycemic values were collected and the results clearly show that all the treated rats have a lower level of glucose in the blood with respect to the starting point, with the value comparable and in same cases better than that obtained with the commercial drug metformin. The analysis of the extracts content have revealed a large amount of the flavonoid rutine and other important bioactive substances. The enzymatic analysis have also demonstrated a strong inhibition activity of the glucosidase enzyme, an enzyme involved in glucose metabolism. However the single activity of each compound present is not enough to explain the anti-diabetic effect observed After one month, the rats were sacrificed and tissues analyzed. Blood tests were carried out to evaluate if there are any toxic effect or alteration of the liver and other tissue. Also lipidemic panel was evaluated, bearing in mind that lipid metabolism is also affected in diabetes mellitus. Table 1. In vivo studies. Streptozocin untreated (control group); group treated with 100 mg/kg of capparis leaf, Group treated with 100 mg/kg capparis buds, and group treated with Metformine 1.8 mg/kg. STZ control Capparis leaf 100 Capparis buds 100 Metformin 1.8 mg/kg mg/kg mg/kg Animals Week I* Week IV* Week I* Week IV* Week I* Week IV* Week I* Week IV* 1 278 343 288 50 600 97 405 100 2 174 428 281 56 313 72 446 71 3 284 301 600 82 554 45 338 66 4 166 335 527 96 275 99 398 dead 5 425 377 305 60 277 72 392 56 6 267 375 332 55 415 72 315 112 * mg/dL
Conclusions. Analytical studies on C. spinosa L. highlighted the presence of the flavonoidic type of compounds: kaempferol and quercetin derivatives, easily digestible in humans, and with potent antioxidant actions. The present findings demonstrated that C. spinosa flowering buds and leaf possess anti-diabetic activity possibly correlated to the inhibition of glucosidase and amidase enzymes in liver. We have also demonstrated that at 100 mg/kg, the daily assumption of Capparis buds or leaf help to maintain the glucose in the physiological range, in diabetic rodent model. In this study, rats were normally fedeed and the dry powder was simply administered by direct oral delivery.Other experiments are actually in course to establish the minimum active dose. Acnowledgement. We thank the Capersud manufacturing, Lipari (Italy) for kindly providing the raw material and salted caparis buds.
Bibliography 1. Santini, A.; Novellino, E., Nutraceuticals: Beyond the Diet Before the Drugs, Current Bioactive Compounds, 2014, 10, 1-12. 2. Tlili, N.; Khaldi, A.; Triki, S.; Munné-Bosch, S.; Phenolic compounds and vitamin antioxidants of caper (Capparis spinosa). Plant. Foods Hum. Nutr. 2010, 65, 260-5. 3. Rezzan, A.; Ozan, E. E.; Huseyin, S.; Oktay Y.; Nimet, B. Phenolic components, antioxidant activity, and mineral analysis of Capparis spinosa L. African Journal of Biotechnology, 2013, 12, 6643-6649.
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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
Short Lecture – SL2
Medicinal Plants in Bulgaria – Current State and Perspectives
Elena Genova, Marina Stanilova, Boryanka Traykova
Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences Department of Plant and Fungal Diversity and Resources 23, Acad. G. Bonchev str., Sofia, Bulgaria
Abstract: This communication presents the achievements of the Bulgarian scientists on the study and protection of the medicinal plants. Some measures for rational use and conservation of the natural habitats of the medicinal plants have been marked. Key words: medicinal plants, study and protection Bulgaria is well known with varied relief and geology, the specific microclimate conditions and these factors determine a great biodiversity. The Bulgarian flora is very rich – 4102 species vascular plants and 842 species from them are used in the traditional and folk medicine. For several decades Bulgaria is one of the leaders in the export list in Europe – between 13 000 - 17 000 t herbs annually (Lange, 1998; 2006). About 25 % of this export is obtained from the cultivated plants: Mentha piperita L., Lavandula vera L., Salvia officinalis L., Althaea officinalis L., Chamomila recutita L., Rosa canina L., etc. The climatic changes, pollution of the air and water, overexploitation of the resources and intensive urbanization are powerful negative factors affecting the rich and original biological diversity. Conservation of the biological diversity is object of more than 100 normative acts, some of them at European level: Bern Convention (1996), The European ecological network NATURA 2000, other at national level: Biological Diversity Act (2002), Protected Territories Act (1998), Medicinal plants Act (2000), Forest Act (2011), etc. In Bulgaria the legislation concerning the protection and conservation of threatened medicinal and aromatic plants has been regulated mainly by the Medicinal plants Act (2000) and Biological Diversity Act (2002). About 70 species are protected by the Medicinal plants Act or have a restricted regime of collection. The first edition of the Red Data Book of Bulgaria was published in 1984-1985 comprising two volumes. The new edition of the Red Data Book of the Republic of Bulgaria in three volumes: Plants and fungi, Animals, and Natural habitats (2015), was realized by the financial support of the Bulgarian Academy of Sciences and Ministry of Environment and Waters. This is a serious scientific basis for making correct decisions according the IUCN conception for the categories. In vol. 1. Plants and Fungi (Peev et al.eds., 2015), 810 species: algae, vascular plants and fungi have been included. Special attention was given of the “critically endangered“ species of the medicinal plants as Hippophae rhamnoides L., Rheum rhaponticum L., Sideritis syriaca L., Alchemilla mollis (Buser) Rotm. Some rare medicinal plants as Ruta graveolens L., Gentiana lutea L., Alkanna tinctoria L., Menyanthes trifoliata L. are included in the category “endanger” species. These medicinal plants are objects of the scientists for study of biology, ecology, reproductive capacity and protection of their natural habitats. As a result of the field studies and analyses of the existing habitats according to the EUNIS classification 166 habitats of conservation importance have been identified in Bulgaria (Biserkov et al., eds., 2015). The shapefiles of GIS models of the distribution of the habitats have been elaborated within the project “Development of the network of protected areas NATURA 2000 in Bulgaria”. They have been used for the preparation of the maps. The habitats of Arctostaphylos uva-ursi, Castanea sativa, Abies alba, Quercus pubescens have been included in category “endangered”.Twenty years ago the programme for cultivation of rare and threatened medicinal plants has been started (Evstatieva, 2005). Sideritis scardica Griseb., Rhodiola rosea L., Gentiana lutea L., Rheum rhaponticum L. etc. have been introduced in the experimental field of the former Institute of Botany, Bulgarian Academy of Sciences. This ex situ collection has been enriched with perspective forms of Alchemila spp., Betonica officinalis L, Melissa officinalis L., Origanum vulgare ssp. hirtum (L.) Ietsw., Ruta graveolens L., Leucojum aestivum L., Salvia officinalis L., Acorus calamus L., Valeriana officinalis L., etc. In the last
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years plantations have been created from Sidertis scardica Griseb., Valeriana officinalis L., Tribulus terrestris L., Origanum vulgare ssp. hirtum (L.) Ietsw. in some regions with suitable ecological conditions. In vitro propagation of some medicinal plants with resource deficit and market demand has been applied as well. For this purpose, a special Biotechnological laboratory of medicinal plants was organized in the former Institute of Botany, now a days a part of the Institute of Biodiversity and Ecosystem Research, at the Bulgarian Academy of Sciences. High productive individuals of some endangered species were selected and in vitro multiplied. One of them was Leucojum aestivum L., used in the pharmaceutic industry for its valuable alkaloid galanthamine. The obtained plants are a suitable plant material for both: establishment of commercial plantations, and reinforcement of their populations of origin (Stanilova et al., 2010). Another medicinal plant forbidden for collection from the wild, Valeriana officinalis L., was successfully in vitro multiplied starting from a single selected individual (Kozhuharova, 2015). Plants have been used for establishment of a pilot commercial plantation in cooperation with Bioprograma EAD as a business partner. The application of the in vitro techniques is appropriate also for the rapid propagation of some endemic or critically endangered medicinal plant species, such as the Bulgarian endemics Alchemilla achtarowii Pawł., A. jumrukczalica Pawł and A. bundericensis Pawł. (Gorgorov et al., 2011), and the critically endangered in Bulgaria Alchemilla mollis (Stanilova, 2012). The high mountain Alchemilla plants have been ex vitro adapted and successfully acclimated at experimental filed plots in different altitudes: 1500 m as well as 550 m, where they have kept the normal biosynthesis of their bioactive substances.Every year Ministry of Environment and Waters organizes discussion with experts of Institutions and Sofia University for discussion of the quotas of the medicinal plants with restricted regime of collection. The annual Order is published in the State gazette of Bulgaria at the end of January each year. The experts from the foundation “Information and environmental protection” obtained a financial support from the “Bulgarian-Swiss cooperation programme” for the project ”Model for protection and sustainable use of the medicinal plants at municipal level with participation of the local communities and mass media”. Scientists from several Universities and from the Bulgarian Academy of Sciences were included in this project. Besides, many lectures, education materials and discussions have been carried out with firms and people gathering the herbs. In November 2015 a Fair of Medicinal plants was organized in Plovdiv, with participation of scientists, business partners, and representatives from the Ministry of Environment and Waters. Major problems were identified and new strategies were discussed, related to the gathering of medicinal plants from their natural populations, cultivation of rare and valuable medicinal species, and manufacture of different products such as teas, essential oils, and patent medicines. A proposal for a new edition of the methodology for determination of the resources of medicinal plants was made. The Bulgarian scientists have to combine their efforts to prepare a new long-term strategy for monitoring of the natural resources of medicinal plants. For the successful protection and sustainable use of the rare and threatened medicinal and aromatic plants both in situ and ex situ measures have to be applied.
References Biserkov, V. et al. (Eds.) 2015. Red Data Book of the Republic of Bulgaria .volume 3. Natural habitats BAS & MoEW, Sofia Evstatieva, L. 2005. A review of the cultivation of endangered medicinal plants in Bulgaria. God. Sofiiski Univ. ”Kliment Ochridski” Biol. Fak.2, Bot., 97: 45-52. Gorgorov R., Stanilova M., Vitkova A. 2011. In vitro cultivation of some endemic and rare Alchemilla species in Bulgaria. Roman. Biotech. Let., 16(6): 65-70. Kozhuharova A. 2015. In vitro micropropagation of Valeriana officinalis L. (Valerianaceae). MSc Thesis. Lange, D. 2006. International trade in medicinal and aromatic plants. Actors, volumes and commodities. In: R.J. Bogers, L.E. Craker and D. Lange (eds.). Medicinal and Aromatic Plants. Netherlands: Springer. Lange, D. 1998. Europe's medicinal and aromatic plants. Their use, trade and conservation. TRAFFIC International, Cambridge, 77 p. Peev, D. et al. (Eds.) 2015. Red Data Book of the Republic of Bulgaria. Volume 1. Plants and Fungi. BAS & MoEW, Sofia Stanilova M., Molle E., Yanev S. 2010. Galanthamine Production by Leucojum aestivum Cultures In Vitro. In: The Alkaloids: Chemistry and Biology (G. Cordell, ed.) vol. 68, Chapter 5, 167-270. Stanilova M., Gorgorov R., Trendafilova A., Nikolova M., Vitkova A. 2012. Influence of nutrient medium composition on in vitro growth, polyphenolic content and antioxidant activity of Alchemilla mollis. Nat. Prod. Commun., 7(6): 761-766.
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Short Lecture – SL3
New insights regarding the biologic potential of a standardized chamomile extract
Radu Ionita2, Lucian Hritcu2, Adriana Trifan1, Monica Hancianu1*, Oana Cioanca1
1Faculty of Pharmacy, University of Medicine and Pharmacy “Gr. T. Popa”, 16 University Str., Iasi 700117, Romania 2 Department of Biology, Alexandru Ioan Cuza University, Bd. Carol I, No. 11, Iasi 700506, Romania *Corresponding author, e-mail: [email protected]
Abstract. The present study evaluated the effectiveness of a hydro-alcoholic extract of Chamomillae flos in experimental anxiety and amnesia model induced by administration of scopolamine to Wistar rats. Our aim was to obtain, standardize and biologically evaluate a the extract from chamomile flowers of known origin. Key words: HPLC, polyphenols, memory, anxiety Introduction. Cognitive impairment indicates a condition in which there are greater cognitive deficits than normally expected at a certain age. Nevertheless, the mild deficits are not severe enough to be included in dementia. For such patients it is important to intervene with specific therapy to help preserve and protect the neurons, thus increasing memory capacity and concentration. Among the risk factors frequently connected to cognitive problems are chronic stress, behavioural inhibition, and depression. The most typicall symptoms of mild cognitive impairment are memory problems, errors of judgement, lack of concentration, spatial motor coordination. Matricaria recutita L. syn. Chamomilla recutita (L.) Rausch., Matricaria chamomilla L. (Asteraceae) is an old herbal medicine, widely used in medical practice. Due to their mild sedative properties the water extracts of matricaria flowers are used for restlessness and specifically for gastrointestinal disturbance with associated nervous irritability in children. Also, most of the sedative effects and pharmacological properties are attributed to apigenin that is quantitatively the most abundant flavonoid found in chamomile flowers. The rational phytotherapy trends impose strict control of the plant material used to treat ailments. Therefore, the source and the quality of the raw material is highly important for obtaining a herbal medicinal product with certain biologic activity. Material and methods. The dry plant product of known and controlled origin was pulverized and subjected to extraction. The extract (ethanol 50 %; 2.5 g/100 mL) was obtained by repeated reflux extraction on a thermostated water bath. The final volume was brought to a level in a volumetric flask. Then, for stability purposes the obtained extract was concentrated to dryness in the oven at 40oC. Thus, we obtained a glossy mass, stable, easily soluble in water and alcohol, also easier to handle in the analyzes. The quantitative and qualitative determination of polyphenols was performed by means of thin layer chromatography (TLC) and liquid chromatography techniques (UPLC). UPLC was performed with a Thermo UltiMate3000 gradient chromatograph equipped with a quaternary pump controlled by Chromeleon interface, an autosampler and multydiode array detector (DAD). Solvents were filtered using a Millipore system and analysis was performed on an Accucore XL C18 column (150x4,6x4). Standard curves for authentic samples of the polyphenols were obtained from purchased reagents (Sigma Chemical Co.) of analytical or high-performance liquid chromatography (HPLC) grade. Each solution was injected in triplicate and the calibration curves were constructed with the averages. For the screening of the biological potential we used several in vitro (Folin –Ciocalteu assays, scavenging capacity against DPPH and ABTS radical) and in vivo on white Wistar male rats (b.w 200±50g). Radial plus maze, forced swimming, and Y test were used to indicate the neuroprotective effects by observing the animal’s behavioral activities (statistically analyzed with two-way analysis of variance (ANOVA)). The animal model was induced by intracerebroventricular The symposium is dedicated to the 60-year celebration of the „Stejarul” Biological Research Centre – MAPPPS 2016, Piatra Neamt, ROMANIA – Page 40
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injection of scopolamine and all surgical procedures were conducted under aseptic conditions with sodium pentobarbital anesthesia, to minimize animal suffering and to reduce the number of animal used. All ethical aspects were taken into consideration according to the guidelines of animal bioethics and all procedures were in compliance with the European Council Directive of 24 November 1986 (86/609/EEC). Results and discussion. TLC and UPLC results confirmed the presence of luteolin and apigenin glycosides, as well as caffeic and chlorogenic acids. Apigenin-7-glucoside amounted up to 0.42%, higher than the European Pharmacopoeial limit (minimum 0.25%). This glycoside will most probably suffer hydrolization in the body fluids, producing its aglycon – apigenin. Total polyphenol content of the extract was 68.70 ± 2.55 mg GAE/g. The investigated extract had a good scavenging activity both against DPPH radical (IC50 = 47,8 ± 1,4 µg/mL) and ABTS cation (IC50 = 21,4 ± 0,2 µg/mL), comparable with the IC50 values of the chosen standard (caffeic acid). The chemical analysis and in vitro tests indicated that the obtained extract is rich in components with recognized sedative effects. The scopolamine model was used due to already known amnesic effects of this alkaloid. Thus, the treated rats exhibited disorientation, a decreased exploratory activity, a low percentage of the time spent in the open arm within elevated plus-maze test and an increased immobility time within forced swimming test, suggesting that the rats are anxious, depressive and lack the will to survive. The observation of the animals included in the groups with chamomile extract was that they behaved similar to the positive control lot (diazepam). This is in accordance with literature that suggest that apigenin binds to GABA-A receptors, thus acting in a similar manner to diazepam. The advantage comes from the lack of side effects of the natural compound as compared to the drug.Moreover, the administration of chamomile extract in doses of 25 mg/kg b.w. or 75 mg/kg b.w. significantly induced anxiolytic- and antidepressant-like effects. Also, short memory was improved considerably as compared to the positive control group. Conclusions. In summary, the present study indicated that doses of 25 mg to 75 mg of chamomile extracts rich in flavonoids (especially apigenin derivatives) could effectively restore memory impairment by administration of scopolamine and anxious behavior. Therefore, chamomile standardized extracts could be potential candidates for further preclinical and clinical studies aimed at long-time prevention and treatment of cognitive deficits in neurological disorders.
Bibliography 1. Hritcu L., Noumedem J.A., Cioanca O., Hancianu M., Postu P., Mihasan M. (2015), Anxiolytic and antidepressant profile of the methanolic extract of Piper nigrum fruits in beta-amyloid (1–42) rat model of Alzheimer’s disease. Behavioral and Brain Functions, 11(1):13. 2. Viola H, Wasowski C, Levi de Stein M, Wolfman C, Silveira R, Dajas F, et al.(1995), Apigenin, a component of Matricaria recutita flowers, is a central benzodiazepine receptors-ligand with anxiolytic effects. Planta Med, 61(3):213–216. 3. Zanoli P, Avallone R, Baraldi M. (2000), Behavioral characterisation of the flavonoids apigenin and chrysin. Fitoterapia, 71:117–23.
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Short Lecture – SL4
The antimicrobial activity of medicinal plants depending on storage conditions
Steliana RODINO1, Alina BUTU1, Marian BUTU1*
1National Institute of Research and Development for Biological Sciences, Bucharest, Romania, Splaiul Independentei 296, P.O. Box 17-16, 060031, Bucharest, Romania, Tel. / Fax. +4 021 220 0880 *Corresponding author, e-mail: [email protected]
Abstract. This study presents a comparison of the antimicrobial activity of Humulus lupulus (hops) ethanolic extract, depending on the type of packaging and under different storage conditions. The evaluation was performed by agar diffusion method, with the use of the following standard strains: Staphylococus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis. The results showed that the prolonged storage in sealed and unsealed package influenced antimicrobial activity of the plant extract. Key words: medicinal plants, storage conditions, antimicrobial activity, plant extract Introduction. Many strains of microorganisms acquired drug resistance due to improper use of dosage and prescriptions. This greater resistance of the microbial population makes the search for new agents with antimicrobial activity one of the most important problems of modern medicine. Currently, research is more and more directed to find new raw sources containing biologically active substances. In recent years, there has been an increased interest in the therapeutic potential of medicinal plants as antioxidants, antimicrobial [1] and allelopathic agents [2]. The aim of this study was to compare the antimicrobial activity of a plant extract depending on storage conditions of the primary packaging. Material and methods. The plant material was harvested in accordance with the recommendations of the Good agricultural practice of medicinal plants and national guidelines [3]. The extraction was performed in ethanol, by ultrasonication. The nature of antimicrobial action of the hops extracts, produced on standard bacterial strains (Staphylococus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633) was tested after storage under various conditions. Evaluation was performed by agar diffusion method in Mueller-Hinton medium [4]. The extract was stored in dark recipients, at refrigerator (4 °C unsealed/ sealed), at room temperature (25 °C ± 2°C unsealed/ sealed) and accelerated storage conditions (40 °C ± 2 °C sealed), for 7, 14, 30 days, and 2, 4, 6, 8, 12 months, respectively before the use for the bioassay. Zero time samples were used as controls. Results and discussion. Any drug must be developed to meet the requirements for efficacy, safety and quality. During the period of validity, the claimed medicinal product must meet all the requirements of the registration dossier, including in terms of efficiency. This study aimed to assess the impact of external factors, such as temperature, period of storage and type of packaging, on the stability of the antimicrobial activity of a plant extract (Table1).
Table 1. Effect of storage conditions on the antimicrobial activity of hops extract Storage conditions 12 0 7 days 14 days 30 days 2 months 4 months 6 months 8 months months Staphylococus aureus 4 °C unsealed +++ +++ +++ +++ +++ ++ ++ ++ + 4 °C sealed +++ +++ +++ +++ +++ +++ +++ +++ +++ 25 °C ± 2 °C +++ +++ +++ +++ ++ + + + - unsealed 25 °C ± 2 °C sealed +++ +++ +++ +++ +++ +++ +++ +++ +++
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Storage conditions 12 0 7 days 14 days 30 days 2 months 4 months 6 months 8 months months 40 °C ± 2 °C sealed +++ +++ +++ +++ ++ ++ + + - Pseudomonas aeruginosa 4 °C unsealed ++ ++ ++ ++ ++ ++ + + - 4 °C sealed ++ ++ ++ ++ ++ ++ ++ ++ ++ 25 °C ± 2 °C ++ ++ ++ ++ + + - - - unsealed 25 °C ± 2 °C sealed ++ ++ ++ ++ ++ ++ ++ ++ ++ 40 °C ± 2 °C sealed ++ ++ ++ ++ + +/- - - - Escherichia coli 4 °C unsealed +++ +++ +++ +++ +++ ++ ++ ++ + 4 °C sealed +++ +++ +++ +++ +++ +++ +++ +++ +++ 25 °C ± 2 °C +++ +++ +++ +++ ++ ++ + + - unsealed 25 °C ± 2 °C sealed +++ +++ +++ +++ +++ +++ +++ +++ +++ 40 °C ± 2 °C sealed +++ +++ +++ +++ ++ +/- - - - Bacillus subtilis 4 °C unsealed ++++ ++++ ++++ ++++ ++++ ++++ +++ +++ ++ 4 °C sealed ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ 25 °C ± 2 °C ++++ ++++ ++++ ++++ +++ ++ ++ ++ + unsealed 25 °C ± 2 °C sealed ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ 40 °C ± 2 °C sealed ++++ ++++ ++++ ++++ +++ +/- - - - *++++ 20-15 mm diameter of inhibition; +++ 14-10mm; ++ 9-5 mm; +4-1mm; - No inhibition Stability of a pharmaceutical product may be defined as the capability of a particular formulation in a specific container/closure system to remain within its physical, chemical, microbiological, toxicological, protective and informational specifications [5]. Stability studies usually include testing of those attributes of the drug substance that are susceptible to change during storage and are likely to influence quality, safety, and/or efficacy. Our results showed the prolonged storage in sealed recipients, did not negatively influenced the antibacterial activity of the plant extract, for the tested range of time (12 months). The plant extract antimicrobial activity against the selected bacterial strains remained stable for at least two months (at room temperature) to four months (at refrigerator) in unsealed recipients. Conclusions. The results showed that the antibacterial activity of the hops extract remained stable for at least 12 month, when stored in sealed recipients. Future testing should cover, as appropriate, the physical, chemical, and biological attributes. Acknowledgements. This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation - UEFISCDI, research contract PN-II PT-PCCA 106/2012.
Bibliography 1. F. Karahan, C. Avsar, I. Ilker Ozyigit, I. Berber, Antimicrobial and antioxidant activities of medicinal plant Glycyrrhiza glabra var. glandulifera from different habitats, Biotechnology & Biotechnological Equipment, 30 (4), 2016 2. G. Brahmachari, Natural products in drug discovery: impacts and opportunities – an assessment, Bioact. Nat. Prod. (2011), 1 - 199 3. Good practice guide for cultivation and harvesting of medicinal and aromatic plants, approved by Order nr. 170/2011 4. S. Rodino, A. Butu, M. Butu, P. C. Cornea, Comparative studies on antibacterial activity of licorice, elderberry and dandelion, Digest J of Nanomaterials and Biostructures, 10 (3), 2015, 947 - 955 5. Bajaj S, Singla D, Sakhuja N, Stability Testing of Pharmaceutical Products, J Appl Pharm Sci. 2012; 2:129 – 38.
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Short Lecture –SL5
Comparison of total phenolic content and antioxidant capacity of seeds vs. sprouts of representative species from Republic of Moldova
Grigoriev Valeria1, Chiru Tatiana1
1State University of Medicine and Pharmacy „Nicolae Testemitanu” of the Republic of Moldova
Abstract. Antioxidant activity and total phenol contents of ethanol extracts of Zea mays, Triticum aestivum, Lens culinaris and Helianthus annuus for their seeds and sprouts were investigated. The results showed higher antioxidant abilities correlated with total phenols in the sprouts than their seeds. Sprouts described above could be used in our diet as a beneficial foods with antioxidant capacity. Key words: seeds, sprouts, antioxidant, phenols.
Introduction. Phenolic compounds are a group of above 8,000 phytochemicals that received considerable attention for being potentially protective factors against degenerative diseases, mostly because of their potent antioxidative properties and their omnipresence in consumed foods of plant origin. According to information in the Medline database, the past ten years have seen a 340% increase in manuscripts mentioning “antioxidants”[1]. Many of the biological functions, such as anticarcinogenicity,antiaging, and antimutacigenity, originate from antioxidant capacity [2,3] . Agriculture represents 16,2 % of Republic of Moldova GDP, 86,3 % of arable surface is cultivated with cereals (especially wheat and corn) and sunflower. Sprouting grains leads to increased activities of hydrolytic enzymes, improvements in the contents of total proteins, fat, certain essential amino acids, total sugars, B-group vitamins, and a decrease in dry matter, starch and anti-nutrients, on the face of it they have attracted much interest in recent years [4,5]. So, the purpose of this research paper work is to determine and analyze the content of polyphenols compounds and their antioxidant activity in seeds and sprouts harvested in Republic of Moldova and to find out the perspectives of their therapeutical use.
Material and methods. The extracts, obtained by ethanol extraction technique, were further analyzed to determine their total phenolic (Folin-Ciocalteau assay), and antioxidant (DPPH scavenging, using Ascorbic and Gallic acid as standards) capacity.
Results and discussion. From the results it was revealed that sprouts exhibited greater total phenolics that seed extracts in terms of gallic acid equivalent (Table 1). In all assays, the ethanolic extracts of all species of sprouts showed the highest values of antioxidant activity (Table 2). It was noticed that the highest antioxidant activity was obtained for sunflower sprouts. The evident correlation between the two experiments is the major increase of total phenolic content and antioxidant capacity in sprouts in relation to seeds.
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Table 1. Total phenol content in seeds and spouts1,2 Total phenol as gallic acid equivalent (mg GAE/g dried weight) Seeds Sprouts Wheat 17.35 ± 0.47a 22.05 ± 1.49a Lentil 2.05 ± 0.59b 3.65 ± 1.32b Sunflower 52.96 ± 3.16c 86.14 ± 0.47c Corn 20.87 ± 0.31a 48.44 ± 0.82d
1The values are mean ± standard deviation 2Values in the same column not sharing the same superscript were significantly different from each other (p < 0.05).
Table 2. Free radical scavenging ability of the seeds and sprouts by the DPPH method1,2,3
DPPH IC50 (μg/ml) Seeds Sprouts Wheat 766.26 ± 2.32a 653.21 ± 1.15a Lentil - 614.97 ± 5.13a Sunflower 428.21 ±2.54b 128.81 ± 1.68b Corn 347 ± 2.03c 169 ± 0.63b Ascorbic acid 0.60 ± 0.01 Gallic acid 1.50 ± 0.02 1The values are mean ± standard deviation 2Values in the same column not sharing the same superscript were significantly different from each other (p < 0.05) 3- No antioxidant activity.
Conclusions On the basis of the results of total phenolics, the ethanolic extracts of seeds and sprouts analyzed was placed in the following order: sprouted Helianthus annuus > seed Helianthus annuus > sprouted Zea mays > sprouted Triticum aestivum > seed Zea mays > seed Triticum aestivum > sprouted Lens culinaris > seed Lens culinaris. From the results it was revealed that sprouts exhibited higher total antioxidant capacity compared to seed extracts. The results showed that sprouts are a promising source of phenolic compounds with antioxidant potential.
Bibliography
1. David Swenson Oufnac (1999), Determination of antioxidant capacity in corn germ, wheat germ and wheat bran using solvent and microwave-assisted solvent extraction, B.S. Culinary Arts, Nicholls State University. 2. M. Marton1, Zs. Mandoki (2010), The role of sprouts in human nutrition. A review, Acta Univ. Sapientiae, Alimentaria. 3. Xu-Dan Guo , Yu-Jie Ma , John Parry , Jin-Ming Gao , Liang-Li Yu and Min Wang (2011), Phenolics Content and Antioxidant Activity of Tartary Buckwheat from Different Locations, Molecules. 4. Sutharut, J. (2012), Total anthocyanin content and antioxidant activity of germinated colored rice, International Food Research Journal. 5. Do Tan Khang, Tran Nhan Dung, Abdelnaser Abdelghany Elzaawely and Tran Dang Xuan (2016) ,Phenolic Profiles and Antioxidant Activity of Germinated Legumes, Foods MDPI.
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Short Lecture – SL6
Biological and chemical study of Lavandula sp. varieties cultivated in Dobrogea Plateau
Daniela Lupu1*, Radu Necula2,3, Georgiana Gavril2, Ruxandra Cretu2, Valentin Grigoras2, Elvira Gille2
1Lupu Vasile Agro Ii, 827170, Nufaru, jud.Tulcea, Romania 2NIRDBS/'Stejarul' Biological Research Centre, 610004, Piatra Neamt, Romania 3Faculty of Chemistry, "Alexandru Ioan Cuza" University, 700506, Iasi, Romania *Corresponding author, e-mail: [email protected]
Abstract. In the present study we analyzed the lavender essential oil from two cultivars of Lavadula sp. cultivated in Dobrogea Plateau — which is situated between the lower Danube River and the Black Sea, and south of the Danube Delta. The phytochemical analysis showed a small quantitative variation and no significant qualitative differences between the two varieties of lavender. The lavender oil product is of good quality for aromatherapy according to the European Pharmacopoeia standard. Key words: lavender, essential oils, gas chromatography, mass spectrometry.
Introduction. The private cultivator chose the lavender culture because it is a species widely used in cosmetics and food industry and due to the climate and soil conditions suitable for the culture. In the Dobrogea Plateau the climate is temperate continental with sub-Mediterranean influences with high thermal amplitudes, low rainfall, and high atmospheric humidity. The soil is mostly argillaceous. Therefore there are not many cultures that fit this type of soil, namely the more common ones are grape-vine and the fruit trees that give the best results for this type of soil. The Lavandula genus is part of the Lamiaceae family comprising 47 species of annual plants, herbaceous plants, and shrubs. Some of the pharmacological activities of Lavandula extracts have been documented, such as the hypnotic effect useful for anxiety-related restlessness and disturbed sleep [1,2].
Material and method. In our study the culture of lavender covers 2 hectares, near the village Victoria, Nufăru commune (Tulcea County, Romania). The private cultivator planted about 20,000 cuttings / ha in early November of 2014 using the planting scheme of 1.2 m distance between rows and 0.4 m distance between plants in a row. The cultivar Lavandula × intermedia Emeric ex Loisel. var. ‘Grosso’ was chosen since it is very well suited for the cultivation area. This cultivar is hybrid between the species Lavandula angustifolia Mill. (common lavender) – more resistant to frost, and Lavandula latifolia Medik. (spike lavender) – better tolerant of hot and dry summers. This lavender culture has been in full conversion to ecological agriculture since 2015. The lavender essential oil has been obtained by steam distillation by means of a professional semi-industrial equipment manufactured in France. On the area cultivated were obtained 3 tons of fresh floriferous spikes, which have been subject to steam distillation yielding the final 35 l of lavender essential oil. The oil was bottled in 1 L sealed and darkened bottles. The phytochemical analysis was performed at “Stejarul” BRC laboratory facility for the volatile oil extracted from fresh and dried plants cultivated in 2015 and 2016 by means of gas chromatography coupled with mass spectrometry and volatile fractions evaluation was made in accordance with European Pharmacopoeia 6.0 (2008).
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We have also performed analysis of the lavender essential oil obtained from a second cultivar Lavandula angustifolia Mill. var. 'Vera' for comparison purposes.
Results and discussion. GC-MS analysis revealed the presence of numerous volatile compounds in essential oil of Lavandula sp. varietes, the results obtained are shown in table 1.
Table 1. Main compounds of Lavandula sp.by GC/MS Area % Eur Pharma RT (min) Compound LAV-1 LAV-2 LAV-3 vol. 6.0 'Grosso' 'Grosso' "Vera" 01/2008: 1338 dried 2015 fresh 2016 6.49 3-Octanone 0.50 0.97 0.61 0.1-2.5% 7.83 Limonene 0.66 1.23 1.79 <1.0% 7.90 Eucalyptol 2.26 0.96 1.35 <2.5% 8.15 trans-β-Ocimene 1.63 4.78 5.02 8.48 cis-β-Ocimene 0.55 2.95 2.86 9.28 cis-Linalool oxide 1.32 0.20 0.18 9.85 trans-Linalool oxide 1.03 0.24 0.16 10.56 Linalool 25.10 22.58 20.18 20-45% 10.84 Octen-1-ol acetate 1.33 1.45 1.66 11.99 Camphor 0.73 0.24 0.43 <1.2% 12.85 Borneol 2.39 1.16 1.86 12.95 Lavandulol 1.05 0.65 0.65 >0.1% 13.36 Terpinen-4-ol 2.22 4.09 2.66 0.1-6.0% 13.84 α-Terpineol 0.27 0.32 0.39 <2.0% 13.94 Hexyl butyrate 0.37 0.37 0.32 16.93 Linalyl acetate 35.07 34.80 34.86 25-46% 17.84 Isobornyl acetate 0.38 0.17 0.40 18.15 Lavandulol acetate 5.51 4.90 3.92 >0.2% 21.09 Nerol acetate 0.12 0.22 0.19 23.33 β-Caryophyllene 4.17 6.50 7.54 29.61 Caryophyllene oxide 2.32 0.44 0.59
Conclusions. The essential oils obtained by steam distillation are characterized by a content of linalool, linalyl acetate, lavandulyl acetate, and lavandulol falling within the limits recommended by the European Pharmacopoeia 6.0 for characteristic components of the product which can be used in aromatherapy.
Bibliography 1. Hajhashemi, V., Safaei, A. (2015) Hypnotic effect of Coriandrum sativum, Ziziphus jujuba, Lavandula angustifolia and Melissa officinalis extracts in mice. Research in Pharmaceutical Sciences 10, 477–484. 2. Kasper, S., Anghelescu, I., Dienel, A. (2015) European Neuropsychopharmacology 25, 1960–1967. 3. European Pharmacopoeia 6.0: Directorate for the Quality of Medicines & Healthcare, Council of Europe, 2008. 4. Robert P. Adams (1989) Identification of Essential Oils by Ion Trap Mass Spectroscopy. Academic Press Inc., 1989.
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Short Lecture – SL7
Comparative study of essential oils of Heracleum species
Dana Bobit1*, Calin Garlea1, Andrei Paduraru2, Madalina Popa3, Radu Necula3,4, Oana Cioanca2
1SC Dacia Plant SRL, Hărmanului FN, Bod - Brașov, Romania 2Faculty of Pharmacy, “Gr. T. Popa” University of Medicine and Pharmacy, 16 Universitatii, 700115, Iasi, Romania 3 NIRDBS/"Stejarul" Biological Research Centre, 610004, Piatra Neamt, Romania 4Faculty of Chemistry, "Alexandru Ioan Cuza" University, 700506, Iasi, Romania *Corresponding author, e-mail: [email protected]
Abstract. This paper presents comparative studies performed on species of Heracleum sosnowskyi Manden from ecological culture (SC Dacia Plant, Romania) and Heracleum sphondylium subsp. sibiricum (L.) Simonk. from China. The investigations were made by gas-chromatography coupled with mass spectrometry (GC-MS), in order to determine the main volatile fractions in these species.
Key words: Heracleum sosnowskyi, Heracleum sphondylium, essential oils, gas chromatography, mass spectrometry.
Introduction. The species is widespread in the wild flora of Europe, being classified in the flowering plant phylum Magnoliophyta, Dycotiledonate class, Apiales order, Apiaceae family. Since ancient times it was known for action to protect the genitalia, as aphrodisiac, digestive, expectorant, and sedative [1-3]; it was studied by the team of „Dacia Plant“ Company from the beginning being present in the product portfolio even in the early years (2001) as “Hogweed”. It is native to the Transylvanian Plateau but the wild populations are spread quite unevenly and sparse, especially along roads and on the edge of clearings. Since this species is utilized in food supplements with therapeutical value, ecological cultures were initiated starting with a population from wild flora of Bod, Harman and of surroundings areas (Brasov County, Romania). Thus, the growth of this species was ensured under controlled conditions using an accessible cultivation technology, in order to improve the content in active principles and to increase the bioproductivity.
Material and method. The essential oils extracted from roots of Heracleum sosnowskyi and Heracleum sphondylium subsp. sibiricum were analysed by gas-chromatography coupled with mass spectrometry. GC–MS was performed with an Agilent 6890N gas chromatography instrument coupled to an Agilent 5975 mass spectrometer and an Agilent ChemStation software (Agilent Technologies, Palo Alto, CA). A capillary column (30 m×0.25 mm i.d.) coated with 0.25 µm film 5% phenyl methyl siloxane (HP-5 MS) was used for separation. High purity helium was used as carrier gas with flow-rate at 1.0 ml/min. The other GC conditions such as inlet mode, injection temperature and separation temperature program were optimized as follows: – inlet mode: split (100:1 split ratio); –injection temperature: 250°C; –separation temperature program: from 40°C (at 6°C/min) to 280°C (for 5 min) – total run time: 45 min. The spectrometer was operated in electron-impact (EI) mode, the scan range was 15–400 amu; the quadrupole and ionization source temperature were 200 and 250°C, respectively.
Results and discussion. The results of the GC-MS analysis of essential oils extracted from roots of H. sosnowskyi and H. sphondylium are presented in Tables 1 and 2, Figures 1 and 2. The essential oil obtained by steam distillation from root of H. sosnowskyi contains numerous volatile fractions, the main represented by p-cymene (40.73%), anethole (23.64%), p-anisaldehyde (3.99%) and α-
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terpinolen (3.63%). Trans-anethole is the major volatile fraction (86.69%) in the essential oil from root of H. sphondylium obtained by the same method, which limits its use in therapeutical purposes.
Tabel 1. Main compounds of H. sosnowskyi essential oil by Tabel 2. Main compounds of H. sphondylium essential GC-MS oil by GC-MS RT (min) Compounds Areas % 5.83 Heptanal 0.42 6.40 α-Thujene 0.33 6.55 α-Pinene 0.85 RT (min) Compounds Areas % 7.48 Sabinene 0.30 6.55 α-Pinene 1.63 7.55 β-Pinene 0.55 7.56 β-Pinene 0.18 7.88 β-Myrcene 1.00 7.89 β-Myrcene 0.08 8.16 Octanal 0.77 8.21 α-Phellandrene 0.26 8.71 p-cymene 40.73 8.70 p-Cymene 0.95 8.80 D-Limonene 1.81 8.80 D-limonene 2.10 9.00 trans-β-Ocimen 2.20 9.53 γ-Terpinene 0.12 9.26 cis-β-Ocimen 0.32 12.43 4-Terpineol 0.08 9.53 γ-Terpinen 8.50 12.67 Cryptone 0.06 10.26 α-Terpinolen 3.63 12.92 Estragole 0.62 12.42 4-Terpineol 0.50 13.65 Isothymol methyl 0.18 12.66 Cryptone 0.46 ether 12.74 α-Terpineol 0.28 13.76 Thymol methyl ether 0.35 12.92 Estragole 0.28 14.23 p-anisaldehyde 3.71 13.64 Isothymol methyl ether 0.64 14.79 4-Hydroxy-2- 0.68 13.76 Thymol methyl ether 1.39 methylacetophenone 14.22 p-anisaldehyde 3.99 15.06 trans-anethole 86.69 other compounds 2.31 14.78 4-Hydroxy-2- methylacetophenone 3.39 14.97 Trans-anethole 23.64 other compounds 4.02
Abundance Abundance
TIC: 2016_MAR_07.D\data.ms 8.711 TIC: 2016_MAR_06.D\data.ms 2.3e+07 15.061 4e+07 2.2e+07 3.8e+07 2.1e+07 3.6e+07 2e+07 3.4e+07 1.9e+07 1.8e+07 3.2e+07 1.7e+07 3e+07 1.6e+07 2.8e+07 1.5e+07 2.6e+07 1.4e+07 2.4e+07 1.3e+07 14.964 2.2e+07 1.2e+07 2e+07 1.1e+07 1.8e+07 1e+07 9000000 1.6e+07 8000000 1.4e+07 7000000 1.2e+07 9.532 6000000 1e+07 5000000 8000000 4000000 6000000 3000000 10.256 14.22314.781 14.234 9.007 4000000 2000000 8.799 6.557 8.805 13.756 35.010 8.697 6.555 7.552 7.8808.16210.45613.646 34.608 2000000 12.917 25.881 1000000 5.828 6.400 7.477 8.504 9.26412.42412.65412.74112.91616.18917.13019.200 7.559 8.209 9.53610.46013.65013.76014.797 19.181 0 0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Time--> Time--> Fig. 1. GC-MS chromatogram of H. H. sosnowskyi Fig. 2. GC-MS chromatogram of H. sphondylium essential oil essential oil
Conclusions. The comparative study of the essential oils from the two species, revealed that Heracleum sosnowsky, due to its reduced content in trans-anethol, can be used in phytotherapy. Also, this species can be cultivated in ecological conditions in the field of SC Dacia Plant, Romania.
Bibliography 1. Jakubska-Busse A, Śliwiński M, Kobyłka M (2013) Identification of bioactive components of essential oils in Heracleum sosnowskyi and Heracleum mantegazzianum (Apiaceae). Archives of Biological Sciences 65: 877–883. 2. Sefidkon F, Dabiri M, Mohammad N (2002) Analysis of the Oil of Heracleum persicum L. (Leaves and Flowers). J. Essent. Oil Res. 14: 295-297. 3. Maggi F, Quassinti L, Bramucci M et al. (2014) Composition and biological activities of hogweed [Heracleum sphondylium L. subsp. ternatum (Velen.) Brummitt] essential oil and its main components octyl acetate and octyl butyrate. Natural Product Research 28: 1354–1363.
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Short Lecture – SL8
Screening of microorganisms for the recovery of critical metals
Marian BUTU1, Steliana RODINO1*, Alina BUTU1
1National Institute of Research and Development for Biological Sciences, Bucharest, Romania, Splaiul Independentei 296, P.O. Box 17-16, 060031, Bucharest, Romania, Tel. / Fax. +4 021 220 0880 *Corresponding author, e-mail: [email protected]
Abstract. Given the general requirements for sustainable economic development, the increased urbanization and the rise of the demand for high technology applications, an important concern for most of the industrialized countries is securing the constant supply with mineral raw materials, strongly dependent on raw material imports. Therefore, sourcing secondary solutions for critical materials is a must. In this context, this work presents preliminary studies on the isolation of microorganisms existing in mining residues collected from Romania. Key words: critical metals, mine tailings, microorgamisms
Introduction. In order to apply the principles of sustainable development of economy and industry at national and European level is required the reconsidering of industry supply with raw materials. The security of supply with mineral materials represent a current issue of high priority, both within the EU countries and worldwide. Beginning with 2011, a list of Critical Raw Materials (CRMs) was drawn by the European Commission, with the intention to release an update every three years. CRMs combine a high economic importance to the EU with a high risk associated with their supply [1]. Critical metals are defined as those metals essential for economic development but which are associated with scarce availability, supply risk and implicitly a high price volatility [2]. The emergency is represented by finding secondary sources of critical materials, used especially in green technologies, biomedical and defense applications and high tech industry. One such source can be the mine tailings, sources once considered as low quality due to the technology constraints of the respective moment. This work presents preliminary studies performed for the screening and isolation of microorganisms existing in mining residues collected from Romania.
Material and methods. Horikoshi media, soil extract media, potato dextrose agar and potato dextrose broth were used for isolation and further maintenance of the fungal strains. The soil extract media contains 500 mL / L soil extract and 15 g / L agar. The soil extract was prepared by mixing 1000 g of soil with 2 L of double distilled water and incubating overnight at room temperature. The mixture was filtrated and centrifuged at 15000 rpm. The supernatant was sterilized three times by autoclaving and then used for the preparation of the soil extract media [3]. The Horikoshi medium was prepared as follows: 10 g glucose, 5 g yeast extract, 5 g polypeptone, 1 g K2HPO4, 0.2 g MgSO4 ·7 H2O, 15 g agar and 900 mL distilled water. It was autoclave sterilized at 121 °C for 15 min. Before the use it was aseptically added 100 mL of 10 % Na2CO3. Final pH was adjusted to10. For the microorganisms isolation and screening, an aliquot of mine waste sample was diluted in sterilized distilled water, and afterwards it was directly plated on soil extract media and Horikoshi media using the method of serial dilution.
Results and discussion. Up to this moment, bioreduction and bioaccumulation process of metals were highly studied in view of their potential to be used for bioremediation in the treatment of polluted soils and waters and only recent attention has turned to the biorecovery of precious, critical or strategic metals. The symposium is dedicated to the 60-year celebration of the „Stejarul” Biological Research Centre – MAPPPS 2016, Piatra Neamt, ROMANIA – Page 50
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After repeated transfer and purification of the initial cultures (more than 10 fungal strains), 4 fungal strains were isolated as pure cultures and then transferred to liquid media with 2% (w/v) mine tailing enriched with critical metals. The changes in the pH values of the media were monitored, and it was observed that a fungal strain reduced the pH value of the medium from 10 to 4, in 14 days. This strain (named BCrF-1) will be further used in the future experiments. Preliminary data obtained and the scientific literature existing at this moment, indicate that a large number of high volume waste rocks can contain sufficient critical metals (such as PGMs and REEs) to warrant their recovery from both an economic and an environmentally beneficial point of view [4].
Conclusions. Waste rock and tailings are usually considered wastes and deposited in the mining area, with negative impact on the environment, having the potential for acid rock drainage. These wastes can turn into valuable secondary sources of raw material of critical importance for the economic development. The biotechnologies using microorganisms to recover critical metals, stand up as a promising alternative for the future. Nevertheless, this microbe metal interaction, relying on substrates to thrive the microbial metabolism is often crucially influencing the economic feasibility of the process.
Acknowledgements. This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CCCDI – UEFISCDI, project number 18/2016, within PNCDI III.
Bibliography 1. European Commission. Critical raw materials for the EU – report of the Ad-hoc Working Group on defining critical raw materials. 2014 Available online at: http://eur-lex.europa.eu/legal- content/EN/TXT/?uri=CELEX:52014DC0297; 2. Glöser S., Tercero Espinoza L., Gandenberger C., Faulstich M., Raw material criticality in the context of classical risk assessment, Resources Policy, 44, 2015, 35–46; 3. Karelová E, Harichová J, Stojnev T, Pangallo D, Ferianc P, The isolation of heavy-metal resistant culturable bacteria and resistance determinants from a heavy-metal-contaminated site, Section Cellular and Molecular Biology, Biologia, 2011, 66 (1) 18-26; 4. De Corte S, Hennebel T, De Gusseme B, Verstraete W, Boon N. Bio-palladium: from metal recovery to catalytic applications. Microbial Biotechnol. 2012, 5, 5–17.
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Short Lecture – SL9
Possible action mechanisms involved in expression of the in vitro cytostatic and cytotoxic impact of some fractionated proanthocyanidin products obtained from grape seeds
Cosmin-Teodor MIHAI1,2, Gabriela Vochita2, Daniela Gherghel2, Pincu Rotinberg2
1Interdisciplinary Research Department – Field Science, "Al. I. Cuza" University of Iasi, Bd. Carol I, no. 20A, Iasi, Romania; 2NIRDBS/Biological Research Institute, Str. Lascar Catargi, nr. 47, Iasi, Romania
Abstract. In this paper are presented some preliminary results focused on the possible action mechanisms of some new proanthcyanidin products, extracted from grape seeds. Apoptosis triggering is the main interaction of these cytostatic and cytotoxic active agents, it being more intense expressed in the neoplastic cells than in normal ones. The progression of the cell cycle wasn’t importantly affected by the tested compounds. Key words proanthocyanidins, normal and neoplastic cells, apoptosis and cell cycle Introduction. Wastes from wine industry represent a large and accessible source of recovery of some bioactive products, useful in the organic agriculture, in animal farming or even in human therapy. Development of a highthroughput technology able to release, identify and concentrate the biological active compounds from wine waste could respond, among other things, at two important problems: identification and validation of new antineoplastic compounds and waste depletion. The polyphenolic compunds are secondary metabolites abundant in different vegetables, fruits, cereals, including and dimers, oligomers and polymers of catechins and epicatechis (monomers of flavan-3-ols, the most chemically complex subclass of flavonoids) (Fantini et al., 2015). The broad pharmacological spectrum and medicinal properties of the polyphenols from grape seed include benefits against the cardiovascular dysfunctions, acute and chronic stress, gastrointestinal distress, neurological disorders, pancreatitis, various stages of neoplastic processes (carcinogenesis). A significant cytotoxicity towards human breast, lung and gastric adenocarcinoma cells in parallel with the improvement of the growth and viability of normal cells was noticed (Bagchi, Bagchi & Stohs, 2002; Bagchi, Swaroop, Preuss, & Bagchi, 2014). Also, these chemical compounds are agents useful in the detoxification of carcinogenic metabolites, in the scavenging of free radicals, released as a result of oxidative stress. The antioxidant ability is significantly better than of vitamins C, E and beta- carotene oxidative stress scavangers. Therefore, Vitis vinifera grapes represent a generous and available source for obtaining new actively biopreparations with agricultural, biomedical and ecological capitalization. In this paper are presented our results focused on the identification of the possible action mechanisms of the proanthcyanidin bioproducts (PproF-f.l., and PProF-f.m.) fractionated from grape seed primary extracts and proved cytostatic and cytotoxic active. Thus, the in vitro experimental approach was oriented to investigation of the interaction of the fractionated proanthcyanidin bioproducts with the cell apoptosis cell cycle progression in neoplastic and normal cells in order to identify and understand their mechanism of action. Material and methods. The proanthocyanidin biopreparations were obtained from Vitis vinifera seeds by fractioning the crude extracts obtained from grape marc after the oil removal by cold pressing. Neoplastic HeLa and normal Vero cells were seeded in DMEM medium supplemented with fetal bovine serum (FBS) in 24 well plates at a density of 50.000 cells / well. After 24 hours from cell cultures initiation, the growth medium was replaced with fresh complete medium containing PProF- f.l. (denoting the final laboratory form) and PProF-f.m. (denoting the final micropilot form)
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proanthcyanidin bioproducts, in a dose of 1500 µg/mL. After 48 hours from adding the compounds, the apoptosis process (by Annexin V-FITC assay) and cell cycle (PI-RNAse assay) were investigated. The registration of ROS and apoptosis levels was performed with a Beckman Coulter Cell Lab QuantaSC flow cytometer, equipped with a 488 nm laser and with specific excitation and collection filters suitable for the selected fluorochromes. The collected data were exported as LMD files and analyzed with Flowing Software (developed by Perttu Terho, Turku University, Finland). Results and discussion. The intensity of the apoptotis process in tumoral HeLa and normal Vero cells was assayed after 48 hours of incubation with the PProF-f.l. and PproF-f.m. fractionated proanthocyanidin bioproducts. Table 1. Distribution of % live, % dead, % preapoptotic and % apoptotic cells into neoplastic and normal cell cultures treated with the PProF-f.l. and PProF-f.m. agents, in a cytostatic/cytotoxic dose of 1500 µg/mL HeLa cells Vero cells Control PProF-f.l. PProF-f.m. Control PProF-f.l. PProF-f.m. %Live cells 77.57±0.58 14.12±2.51 18.51±6.89 95.84±0.57 48.74±3.72 55.82±2.47 %Dead cells 16.57±0.65 20.19±2.35 11.04±1.99 1.75±0.50 27.10±2.45 14.43±2.07 %Preapoptotic cells 2.31±0.01 9.27±1.02 5.57±0.57 1.05±0.10 2.41±0.29 3.60±0.55 %Apoptotic cells 3.76±0.01 56.39±4.16 64.84±9.14 1.36±0.32 21.73±2.72 26.14±1.62 In HeLa cells, the frequency of preapoptotic and apoptotic cells was significantly increased, above the specific frequency of the control group. In the case of normal Vero cells, the intensity of the apoptotic process was intensified, but in a proportion much lower than in the case of neoplastic HeLa cells.
Table 2. Percentual repartition of neoplastic and normal cells after the treatment with PProF-f.l. and PProF- f.m. compounds, in a dose of 1500 µg/mL, into stages of cell cycle as determined by flow cytometry % HeLa cells % Vero cells Control PProF-f.l. PProF-f.m. Control PProF-f.l. PProF-f.m. G0/G1 46.51 43.66 42.78 67.19 54.83 49.55 S 35.87 32.73 36.24 9.01 13.55 19.94 G2/M 17.25 22.87 20.13 23.63 30.68 29.54 The progression of cell cycle in HeLa cells wasn’t major affected by submission of the cells to the treatment with both compounds. In the case of Vero cells, a consistent proportion of cells were in S phase, suggesting an active process of division. The study of cellular apoptosis process in the same cell cultures has shown that neoplastic cells were more sensitive to the tested compounds as compared with the normal cells. Our investigations, presented in this paper, have revealed that the impact of the proanthocyanidin compounds,fractionated from Vitis vinifera grape seed extracts, on the normal and cancerous cells is selective, the proapoptotic and cytotoxic effects being higher in neoplastic cells than in normal ones. Conclusions. Apoptosis triggering is the main effect of the proanthocyanidin extracts, being more intensively expressed in the neoplastic cells than in normal ones. The progression of the cell cycle wasn’t major affected by the treatment with the tested compounds.
Bibliography 1. Bagchi, D., Bagchi, M., & Stohs, S. J. (2002). Annals of the New York Academy of Sciences, 270, 260–270. 2. Bagchi, D., Swaroop, A., Preuss, H. G, Bagchi, M. (2014). Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 768, 69–73. 3. Fantini, M., Benvenuto, M., Masuelli, L., Frajese, G., Tresoldi, I., Modesti, A., & Bei, R. (2015). Internațional Journal of Molecular Sciences, 16(5), 9236–9282.
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Short Lecture – SL10
The Evaluation of Some Natural Populations of Prunus spinosa L. and Lycium barbarum L. from Dobrogea, Romania
Elvira Gille1,*, Ruxandra Cretu1, Valentin Grigoras1, Radu Necula1,2, Manuela Elisabeta Sidoroff3, Georgiana Gavril1
1NIRDBS/„Stejarul” Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt, Romania 2Faculty of Chemistry, "Alexandru Ioan Cuza" University, 700506, Iasi, Romania 3National Institute R&D for Biological Sciences, 296, Splaiul Independentei, 060031, Bucharest, Romania *Corresponding author, e-mail: [email protected]
Abstract. This study evaluated some valuable populations of Prunus spinosa and Lycium barbarum from Dobrogea sites (Romania) in terms of bioproductivity (biomass and bioactive compounds), in order to be exploited by local populations. The main classes of active principles investigated are represented by polyphenols, flavonoids, vitamin C, anthocyanins and carotenoids. Key words: blackthorn, goji, polyphenols, flavonoids, carotenoids.
Introduction. Prunus spinosa L. (blackthorn), family Rosaceae, is a spontaneous species, used in phytotherapy as diuretic, laxative, in various forms of cough, as spasmolytic, anti-inflammatory antioxidant and antibacterial agent. It contains flavonoids, phenolic acids, coumarin derivatives, anthocyanins and type A proanthocyanidins, tannins, carotenoids, vitamins (C, E), organic acids [1-3]. Lycium barbarum L. (goji), family Solanaceae, is used as a traditional herbal medicine and functional food in Asian countries. It has a wide range of biological activities (anti-aging, neuroprotection, anti-fatigue, increased metabolism, glucose control, anti-glaucoma, antioxidant, immunomodulation, anti-tumor, radioprotective, and cytoprotection). Fruits contain various chemical compounds: flavonoids, carotenoids, β-sitosterol, p-coumaric acid, vitamins (A, B, C), minerals, aminoacids, glutathione, polysaccharides [4-6]. The aim of this study was to identify some valuable populations of P. spinosa from wild flora of Dobrogea (Romania) and of L. barbarum also from wild flora and ecological culture (the same area), in terms of bioproductivity (biomass and bioactive compounds), in order to be exploited by local populations.
Material and methods. Vegetal materials consisted in fruits and twigs from blackthorn, and fruits and leaves from goji respectively; they were collected during maturation and ripening of fruits (October 2015), and naturally dried. Thus: six populations of P. spinosa from wild flora of Tulcea county [Mircea Voda - Ps(MV); Slava Cercheza - Ps(SC); Slava Rusa - Ps(SR)/1, Ps(SR)/2; Nicolae Balcescu - Ps(NB); Somova - Ps(Sm)]; L. barbarum Tortoman (Constanta county) populations – 4 from wild flora [Lb(s)/1, Lb(s)/2, Lb(s)/3, Lb(s)/4], and 2 from ecological culture [Lb(c)/1 and Lb(c)/2]. The following parameters were evaluated: morphological (fresh and dried weight of fruits); biochemical by specific methods - TLC for flavonoids and polyphenolcarboxylic acids, HPLC for carotenoids, UV-VIS spectrophotometry for flavonoids, total polyphenols and polyphenolcarboxylic acids, anthocyanins and iodine titrimetry for vitamin C.
Results and discussion. A medium variability of biomass was revealed in goji berries, Lb(c)/2 and Lb(s)/3 populations presented the optimum values (41.61 g f.w. and 8.83 g d.w./100 fruits; 42.62 g f.w. and 10.16 g d.w./100 fruits, respectively). P. spinosa population from Mircea Voda [Ps(MV)] has a high value of fresh (157.36 g/100 fruits) and dry weight (77.28 g/100 fruits). Polyphenols (expressed as gallic acid), polyphenolcarboxylic acids (expressed as caffeic acid) and flavonoids (expressed as rutin and luteolin) recorded values with medium to very large variability, for both blackthorn (fruits and twigs) and goji (fruits and leaves). Goji samples from wild flora are richest, thus fruits have a high content in polyphenols -Lb(s)/3, while leaves are rich in polyphenolcarboxylic acids and flavonoids-Lb(s)/1. The level of polyphenols and polyphenolcarboxylic acids are higher in twigs of blackthorn- Ps(NB), while
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flavonoids are predominant in fruits – Ps(SR)/1. Ps(NB) sample is also rich in anthocyanidins (expressed as cyanidin-3-glucoside chloride) - 120.60 mg/100 g d.w.).
Fig. 1. TLC chromatogram for flavonoids and polyphenolcarboxilic acids in P. spinosa fruits
Samples: Ps(MV), Ps(SC), Ps(NB), Ps(Sm).
The TLC analysis of methanolic extracts from fruits and twigs of P. spinosa indicated that polyphenolcarboxylic acids are predominant. Fig. 2. TLC chromatogram for polyphenolcarboxilic acids in L. barbarum fruits
Samples: Lb(c)/1, Lb(c)/2, Lb(s)/1, Lb(s)/2, Lb(s)/3, Lb(s)/4.
The TLC analysis of methanolic extracts from goji revealed the predominance of polyphenolcarboxylic acids in fruits, and the
presence flavonoid derivatives in leaves. References: flavonoids – rutoside (R), quercetin (Cv), luteolin (L), luteolin-7-O-glucoside (L7g), apigenin (A), apigenin-7-O-glucoside (A7g), kaempferol (K); polyphenolcarboxylic acids - caffeic acid (Caf.); chlorogenic acid (Cl.), ferulic acid (Fer.), o-coumaric acid (oCum.), p-coumaric acid (pCum.), rosmarinic acid (Roz).
Fig. 3. Variation of the content in total polyphenols, Fig. 4. Variation of the content in total polyphenols, polyphenolcarboxylic acids and flavonoids in L. barbarum polyphenolcarboxylic acids and flavonoids in P. spinosa
Goji fruits from wild flora – Lb(s)/3 – are rich in vitamin C (111.300 mg/100 g d.w). For blackthorn populations, vitamin C recorded a great variability (32.93-116.51 mg/100 g d.w.): Ps(SC) - the highest content; Ps(Sm) - the lowest value. Fruits of L. barbarum collected from wild flora – Lb(s)/3- have higher values of β-carotene (1590,84 μg/100 g d.w.). HPLC analysis also revealed the presence of carotenoids in P. spinosa fruits, between 18.52 μg/100 g d.w. [Ps(SR)/1] and 82.19 μg/100 g d.w. [Ps(SC)].
Conclusions. The phytochemical analysis of Prunus spinosa and Lycium barbarum highlighted some differences between populations, plants from wild flora and culture, and vegetal organs (fruits and leaves, fruits and twigs respectively); also, an medium to very large intrapopulational variability was observed, due to the pedoclimatic conditions. Both species species collected from Dobrogea areas (Romania) are characterized by an important content in bioactive principles with "scavenger” properties, which will lead to further research from exploitation point of view: functional foods and nutraceuticals that can be attributed as "local brands" used by local people.
Bibliography 1. Veličković Jamina M, Kostić Danijela A, Stojanović Gordana S, Mitić Snežana S, Mitić Milan N, Ranđelović Saša S, Đorđević Aleksandra S (2014), Phenolic composition, antioxidant and microbial activity of the extracts from Prunus spinosa L. fruit., Hem. Ind. 68(3):297-303. 2. Sikora Elżbieta, Bieniek Małgorzata I, Borczak Barbara (2013), Composition and antioxidant properties of fresh and frozen stored blackthorn fruits (Prunus spinosa L.), Acta Sci. Pol., Technol. Aliment. 12(4):365-372. 3. Pinacho Raquel, Cavero Rita Y, Astiasarán Icíar, Ansorena Diana, Calvo María I (2015), Phenolic compounds of blackthorn (Prunus spinosa L.) and influence of in vitro digestion on their antioxidant capacity, Journal of Functional Foods 19:49-62. 4. Agamase H, Farnsworth NR (2011), A review of botanical characteristics, phytochemistry, clinical relevance in efficacy and safety of Lycium barbarum fruit (goji), Food Research International 44:1702-1717. 5. Agamase H, Sun B, Borek Carmia (2009), Lycium barbarum (goji) juice improves in vivo antioxidant biomarkers in serum of healthy adults, Nutrition Research 29:19-25. 6. Wang CC, Chang SC, Inbaraj SB, Chen BH (2010), Isolation of carotenoids, flavonoids and polysaccharides from Lycium barbarum L. and Evaluation of antioxidant activity, Food Chemistry 120:184-192.
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Short Lecture – SL11
Proanthocyanidins: new insight into their chemistry and biology
Anca Miron, Adriana Trifan, Vlad Simon Luca, Ana Clara Aprotosoaie
Department of Pharmacognosy, Faculty of Pharmacy, Grigore T. Popa University of Medicine and Pharmacy, Universitatii 16, 700115, Iasi, Romania
Abstract. Important advances in proanthocyanidin chemistry and biology have been made in recent years. Hyphenated techniques proved to be an important tool in chemical characterization of proanthocyanidins. Identification of bioactive microbial metabolites contributed to a partial elucidation of the molecular mechanisms involved in their bioactivity. Key words: RP-LC, NP-LC, HILIC, mass spectrometry, microbial metabolites. Proanthocyanidins, also known as condensed tannins, are widely spread in many plant species and have a huge chemical complexity and diversity derived from the hydroxylation pattern of their constitutive units, location and stereochemistry of interflavonoid bonds, esterification or glycosylation and different degrees of polymerization. Plants contain very complex mixtures of proanthocyanidin oligomers to polymers which are difficult to separate and analyze [1]. Hyphenated techniques coupling chromatographic and electroseparation methods with spectroscopic detection proved to be an important step forward in the analysis of proanthocyanidin complex mixtures. Reversed-phase liquid chromatography (RP-LC) hyphenated with tandem mass spectrometry (electrospray ionization in the negative ion mode) has been extensively used in proanthocyanidin analysis. The technique affords a good separation of monomers to trimers. The separation of proanthocyanidins with a degree of polymerization equal or higher than 4 is usually not possible; they co-elute in an unresolved peak. Using this technique, catechin and epicatechin ([M-H]- at m/z 289.0, [2M-H]- at m/z 579.4, fragment ion at m/z 245.1), three dimers ([M-H]- at m/z 577.3, [2M-H]- at m/z 1155.7, fragment ions at m/z 289.0, 286.9, 451.1, 425.3, 407.4) and three trimers ([M- H]- at m/z 865.3, fragment ions at m/z 288.6, 575.5, 287.2, 577.4, 739.2, 713.9) were tentatively identified on the basis of their MS fragmentation pattern in an ethyl acetate extractive isolated from Pinus sylvestris bark (Fig. 1A) [2-4]. In normal phase-liquid chromatography (NP-LC), proanthocyanidins are separated according to their degree of polymerization. The resolution decreases as the degree of polymerization increases, higher polymers coeluting as a single peak. Using NP-LC- MS coupling, proacyanidins up to heptamers were detected in the ethyl acetate extractive from Pinus sylvestris bark (Fig. 1B). Oligomers higher than tetramers ([M-H]- at m/z 1153.7) were detected by extracting their multiply charged ions from the total ion chromatogram (pentamers: [M-2H]2- at m/z 720.7, hexamers: [M-2H]2- at m/z 864.5, heptamers: [M-2H]2- at m/z 1009.0) [3, 4]. In order to improve the characterization of proanthocyanidins in complex mixtures, different techniques have been developed. Hydrophilic interaction chromatography (HILIC) affords the separation of proanthocyanidins according to their degree of polymerization with a better performance in comparison with other chromatography methods. In Hippophaë rhamnoides berries, proanthocyanidins occur as very complex mixtures. HILIC enabled the detection of more than 60 proanthocyanidins (dimers and trimers of procyanidins and prodelphinidins) [5]. Obviously, a good resolution of complex proanthocyanidin mixtures can not be achieved by conventional one-dimensional liquid chromatography methods. Comprehensive two-dimensional liquid chromatography, involving two separation methods (HILIC and RP-LC) coupled to photodiode array and tandem mass spectrometry detection, made possible the direct analysis of very complex proanthocyanidin mixtures. For instance, the technique enabled the tentative identification of 43
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proanthocyanidins with different degrees of polymerization and galloylation in a grape seed extract [6]. In recent years, there has been conclusive evidence from human studies that proanthocyanidin- rich extracts have health benefits in cardiovascular diseases, urinary tract infections, inflammatory diseases and cancer. For instance, oral administration of pine bark extract (65-75% procyanidins) have been reported to reduce osteoarthritis clinical symptoms, improve breathing ability in asthma, reduce edema and ulcerations in chronic venous insufficiency, reduce systolic blood pressure in mild hypertension, microangiopathy and retinopathy symptoms in type II diabetes mellitus [7].
25
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0 0 10 20 30 40 0 0 10 20 30 40 50 60 Retention time (min.) Retention time (min.) A B Fig. 1. RP (A)- and NP (B)-LC-UV trace (280 nm) of ethyl acetate extractive from Pinus sylvestris bark (1-monomers, 2-dimers, 3-trimers, 4-tetramers, 5-pentamers, 6-hexamers, 7-heptamers, tax-hex-taxifolin-O-hexoside)
Surprisingly, after ingestion, the majority of proanthocyanidins are poorly absorbed in the small intestine. In consequence, they reach the colon and undergo microbial degradation into phenylvalerolactones and phenolic acids. These metabolites are responsible for some of the health benefits of proanthocyanidins (atheroprotective, anti-inflammatory and chemopreventive effects). In addition, the ability of proanthocyanidins to affect the composition of human gut microflora seems to contribute, at least in part, to their health benefits [8]. Conclusions. The chemistry and biology of proanthocyanidins have not yet been fully elucidated. Further progress in chemical characterization of plant proanthocyanidin mixtures, identification of new proanthocyanidin microbial metabolites and their targets in the human body are prerequisites for a more appropriate therapeutic valorization.
Bibliography 1. Daneel Ferreira, Jannie P. J. Marais, Christina M. Coleman and Desmond Slade (2010), Proanthocyanidins: Chemistry and Biology in Comprehensive Natural Products II (Mander L., Liu H., eds.). 2. Roxana Laura Mihailescu Amalinei, Adriana Trifan, Oana Cioanca, Sorin Dan Miron, Cosmin Teodor Mihai, Pincu Rotinberg and Anca Miron (2014), Polyphenol-rich extract from Pinus sylvestris L. bark – chemical and antitumor studies, The Medical-Surgical Journal - The Society of Physicians and Naturalists of Iasi-Romania, 118(2):551-557. 3. Anca Miron, unpublished data. 4. Maarit Karonen, Jyrki Loponen, Vladimir Ossipov and Kalevi Pihlaja (2004), Analysis of procyanidins in pine bark with reversed- phase and normal-phase high-performance liquid chromatography-electrospray ionization mass spectrometry, Analytica Chimica Acta, 522:105-112. 5. Heikki Kallio, Wei Yang, Pengzhan Liu and Baouru Yang (2014), Proanthocyanidins in wild sea buckthorn (Hippophaë rhamnoides) berries analyzed by reversed-phase, normal-phase, and hydrophilic interaction liquid chromatography with UV and MS detection, Journal of Agriculture and Food Chemistry, 62(31):7721-7729. 6. Lidia Montero, Miguel Herrero, Marin Prodanov, Elena Ibáñez, Alejandro Cifuentes (2013), Characterization of grape seed procyanidins by comprehensive two-dimensional hydrophilic interaction × reversed phase liquid chromatography coupled to diode array detection and tandem mass spectrometry, Analytical and Bioanalytical Chemistry, 405(13):4627-4638. 7. Gabriele D'Andrea (2010), Pycnogenol: A blend of procyanidins with multifaceted therapeutic applications?, Fitoterapia, 81:724- 736. 8. Keqin Ou and Liwei Gu (2014), Absorption and metabolism of proanthocyanidins, Journal of Functional Foods, 7:43-53.
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Short Lecture – SL12
Polyphenolic compounds production in shoot cultures of two Romanian Hypericum species
Ana Coste1*, Elvira Gille2, Valentin Grigoraş2, Radu Necula2, Adela Halmagyi1, Gheorghe Coldea1, Constantin Deliu1
1NIRDBS/Institute of Biological Research, 48 Republicii St, 400015, Cluj-Napoca, Romania 2NIRDBS/„Stejarul” Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt, Romania *Corresponding author, e-mail: [email protected]
Abstract. The present paper is aimed at the quantification of polyphenolic compounds in H. richeri ssp. transsilvanicum Čelak and H. umbellatum A. Kern shoot cultures under the influence of two cytokinins, 6-benzyladenine (BA) and thidiazuron (TDZ), each tested in three different concentrations (0.10, 0.25 and 0.50 mg l-1). Key words: shoot cultures, cytokinins, polyphenols, spectrophotometry Introduction. Hypericum genus has been reported to contain many high value bioactive constituents: naphthodianthrones (hypericin and pseudohypericin), phloroglucinols (hyperforin and adhyperforin), flavonoids (hyperoside, rutin and quercitrin), benzophenones/xanthones (garcinol and gambogic acid) and essential oils, that are associated with several bioactivities including neuroprotection [3] and antitumor properties [4]. However, only one representative of the genus, Hypericum perforatum (St. John's wort), has been intensely exploited for the extraction of valuable secondary metabolites especially through intensive collection from the wild and through field cultivation. Since, conventional methods are time consuming and yield of field-grown plants is affected by genetic, physiological and environmental factors, in vitro culture methods have been attempted for the improvement of secondary metabolite production and the obtainment of high-quality standardized extracts under controlled conditions. Optimizing secondary metabolites production by plant growth regulator supplementation is an alternative approach. Several reports using different plant growth regulators, suggest that specific concentrations of different cytokinins, may improve or alter the production of secondary metabolites in shoot cultures of different Hypericum species [1]. So far, from the huge Hypericum genus only few representatives were introduced into in vitro culture system, most papers being focused on H. perforatum. The published research on the chemical composition and biological activity, showed that some Hypericum species are more valuable than H. perforatum. Therefore, phytochemical screening and introduction of unexplored Hypericum species into in vitro culture, optimisation of elicitation measures, and intensive biomass production are essential for further progress in this area. H. richeri ssp. transsilvanicum Čelak, an endemic species in Romania and H. umbellatum A. Kern, a rare and endangered Daco-Balkan species, are yet unexplored for secondary metabolites content neither in vivo nor under in vitro conditions. Material and methods. Establishment of shoot cultures - H. richeri ssp. transsilvanicum and H. umbellatum shoot cultures were initiated from aseptic seedlings as previously described by Coste et al. [2]. Nodal explants of each species were cultured in Erlenmeyer flasks (100 ml) on MS [7] basal medium supplemented separately with different concentrations of BA and TDZ (0.10; 0.25 and 0.50 mg l-1). Medium without PGRs was used as control. The media were fortified with 3% (W/V) sucrose, 0.75 % agar (W/V), and pH was adjusted to 5.6. Subcultures were performed every 45 days. Plantlets were cultured under light and dark conditions (16/8 h) at 25 ± 2oC in 36 μmol s-1 m-2 light intensity in a growth room at ambient humidity conditions. Quantification of polyphenols - Fresh shoot samples of H. richeri ssp. transsilvanicum and H. umbellatum were collected after 45 days of in vitro culture, dried at 60oC, weighed and then subjected to extraction of secondary metabolites. For culture media
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variants supplemented with TDZ, the basal parts of the shoots exhibited serious signs of hypertrophy, forming a callus-like massive conglomerates which were analysed separately from the upper side of the stems. The alcoholic extracts were analysed and we determined by spectrophotometry the total polyphenols (mg GAE g-1 DW), phenolic acids (mg CAE g-1 DW) and flavonoids (mg rutoside g-1) content. Results and discussion. The HPLC analysis displayed different chemical profiles and significant content variation among the investigated species and culture variants. Among the two tested cytokinins, TDZ induced a significant increase of total phenolics and phenolic acids content especially in the lower hypertrofied parts of the stems in both species compared to the control. However, H. richeri ssp. transsilvanicum shoots (both stems and hypertrofied basal parts) exhibited under TDZ the highest increment percentages for all investigated compounds compared to the control and to H. umbellatum. TDZ treatment induced an inhibitory effect in H. umbellatum shoots (upper side of the stems) for all tested compounds. Differential effects of TDZ on production of phenolic compounds were also reported by Khan et al. [5] in callus cultures of Fagonia indica. Regarding the effect of BA on secondary metabolites production in the two tested species, we’ve found that moderate and higher concentrations (0.25-0.50 mg l-1) stimulated the synthesis of all investigated compounds, including flavonoids in H. richeri ssp. transsilvanicum, while for H. umbellatum only total phenolics and phenolic acids increased exclusively at the maximum concentration (0.50 mg l-1). In H. umbellatum, BA at all concentrations inhibited flavonoids production compared to the control, even though these amounts were higher than levels detected for H. richeri ssp. transsilvanicum. Differences in total phenolics and flavonoids content under BA influence were reported for in vitro cultured Hypericum rumeliacum, where the exclusion of BA from the medium resulted in an increase of their total content [6]. Conclusions. The analysis of H. richeri ssp. transsilvanicum and H. umbellatum shoot cultures highlighted the presence of different types of polyphenolic compounds. Among tested cytokinins, TDZ elicited secondary metabolites production especially in the callus-like structures from the basal parts of the stems. H. richeri ssp. transsilvanicum shoot cultures exhibited the best response for enhancement of polyphenolic compounds. Acknowledgements. We acknowledge the support of a Core PN16-19-401 BIODIVERS project from the Ministry of National Education and Scientific Research and the National Authority for Scientific Research and Innovation.
Bibliography 1. Ana Coste, Laurian Vlase, Adela Halmagyi, Constantin Deliu and Gheorghe Coldea (2011), Effects of plant growth regulators and elicitors on production of secondary metabolites in shoot cultures of Hypericum hirsutum and Hypericum maculatum, Plant Cell, Tissue and Organ Culture, 106(2):279-288. 2. Ana Coste, Adela Halmagyi, Anca Livia Butiuc-Keul, Constantin Deliu, Gheorghe Coldea and Bogdan Hurdu (2012), In vitro propagation and cryopreservation of Romanian endemic and rare Hypericum species, Plant Cell, Tissue and Organ Culture, 110(2):213-226. 3. Ana I. Oliveira, Cláudia Pinho, Bruno Sarmento and Alberto C. P. Dias (2016), Neuroprotective activity of Hypericum perforatum and its major components, Frontiers in Plant Science, http://dx.doi.org/10.3389/fpls.2016.01004. 4. Wasundara Fernando and H. P. Vasantha Rupasinghe (2013). Anticancer Properties of Phytochemicals Present in Medicinal Plants of North America, Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century, Dr. Marianna Kulka (Ed.), InTech, DOI: 10.5772/55859. 5. Tariq Khan, Bilal Haider Abbasi, Mubarak Ali Khan and Zabta Khan Shinwari (2016), Differential effects of thidiazuron on production of anticancer phenolic compounds in callus cultures of Fagonia indica, Applied Biochemistry and Biotechnology, 179(1):46-58. 6. Kalina Danova, Eva Čellárová, Anna Macková, Zuzana Daxnerová and Veneta Kapchina-Toteva (2010), In vitro culture of Hypericum rumeliacum Boiss. and production of phenolics and flavonoids, In Vitro Cellular & Developmental Biology-Plant, 46(5):422-429. 7. Toshio Murashige and Folke K. Skoog (1962), A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol Plantarum, 15:473-497.
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Short Lecture – SL13
In vitro evaluation of the cytotoxic impact of some proanthocyanidin fractions extracted from grape seeds
Cosmin-Teodor Mihai1,2, Gabriela Vochita2, Daniela Gherghel2, Rodica Pașa3, Ancuța Nechita3, Pincu Rotinberg2
1Interdisciplinary Research Department – Field Science, "Al. I. Cuza" University of Iasi, Bd. Carol I, no. 20A, Iasi, Romania; 2NIRDBS/Institute of Biological Research Iasi, Str. Lascar Catargi, no. 47, Iasi, Romania; 3Research and Development Station for Viticulture and Vinification Iasi, Aleea Mihail Sadoveanu, no. 48, Iasi, Romania
Abstract. In this paper, are included the results obtained in a preliminary screening of the antitumoral potential of some proanthocyanidin bioproducts fractioned from different total polyphenolic extracts separated from Vitis vinifera grape seeds. In vitro evaluation of cell viability by MTT assay has proven that the proanthocyanidin preparations have diminished the cell viability of some types of cell cultures (HeLa neoplastic cells and Vero normal cells) with a more significant cytotoxic impact on the cancerous cells. The most remarkable impact on the cell viability was registered in the case of PProF-f.l. and PProF-f.m. phytoproducts. Keywords: proanthocyanidins, normal and neoplastic cells, cell viability, MTT. Introduction. In the last decades, the cancerous disease has become one of the most frequent cause of the human mortality. Despite the fact that the understanding of the mechanisms underlying the initiation, progression and spreading of the cancer in the animal organisms has registered important progressions, its treatment with nowadays methods is still ineffective in many cases (Miller et al., 2013). So, the actual chemotherapeutic research is oriented to the identification of new sources for antineoplastic compounds, with a more pronounced selectivity upon the cancerous cells, paying a special attention to the natural resources. Addressing to improvement of the antitumoral chemotherapy selectivity, the polyphenols are very promising in prevention of the DNA damages and to impairment of the steady state of the malignant transformed cells. Vitis vinifera seeds are very rich in bioactive compounds, especially polyphenols, they representing a generous and available source for obtaining new actively biopreparations with agricultural, biomedical and ecological capitalization (Akaberi and Hosseinzadeh, 2016). In our paper, are included the results obtained in the conditions of preliminary screening for establishment of antitumoral potential of different fractioned proanthocyanidin phytoproducts. Material and methods. The fractioned proanthocyanidin biopreparations were obtained, either in laboratory conditions (PproF-f.l.) or in micropilot conditions (PProF-f.m.), from Vitis vinifera seeds by fractioning the crude polyphenolic extracts separated from grape marc after the oil removal by cold pressing. Neoplastic HeLa and normal Vero cells were seeded in DMEM medium supplemented with fetal bovine serum in 96 well plates at a density of 10.000 cells / well. After 24 hours from cell cultures initiation, the growth medium was replaced with fresh complete medium containing different doses (presented in figures) of tested proanthocyanidin fractions. After 48 hours from treatment, MTT assay was performed for cell viability evaluation. Results and discussions. In vitro evaluation of cell viability by MTT assay has proven that proanthocyanidin phytoproducts have a cytotoxic effect upon both types of cell cultures (neoplastic and normal) with a more significant cytotoxic impact on neoplastic cells. Also, a dose-effect relationship was registered, cytotoxic impact being strongly amplified by the increase of the treatment dose. Also, the most remarkable impact on the cells viability was registered in the case of PProF-f.l. and PProF-f.m., those being characterized also by a lower cytotoxicity on normal cells (fig. 1). Conclusions. The tested proanthocyinidin bioproducts shown an important cytotoxic effect on neoplastic cells.The highest cytotoxicity on HeLa cells, correlated with a reduced negative impact on
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normal cells, was registered in the case of fractioned proanthcyanidin phytoproducts denoted PProF- f.l. and PProF-f.m.
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Bibliography 1. Miller MJ, Foy KC, Kaumaya PT (2013) Cancer Immunotherapy: Present Status, Future Perspective, and a New Paradigm of Peptide Immunotherapeutics. Discov. Med. 15:166–176. 2. Akaberi, M. and Hosseinzadeh, H (2016) Grapes (Vitis vinifera) as a Potential Candidate for the Therapy of the Metabolic Syndrome. Phyther. Res. 30:540–556.
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Poster Presentation – PP01
Trends in romanian medicinal plants scientific research
Alexandru Amărioarei, Raul Jibotean, Iris Tușa, Corina Ițcuș, Marian Buțu
1Bioinformatics Department, National Institute of Research and Development for Biological Sciences, Bucharest *Corresponding author, e-mail: [email protected]
Abstract. This paper presents results concerning the trends in romanian medicinal plants scientific research by extracting relevant information from research publications. Using text, data mining and web scrapping techniques we have obtained the distribution of the number of papers per year and the number of citations as a measure of scientific impact. Keywords: medicinal plants, scopus, citations, scientific impact Introduction. In this paper we present some results regarding the romanian medicinal plants scientific research potential. In order to identify and evaluate the trends in the huge amount of scientific data related to medicinal plants we have limited our search to scientific publications from Scopus database. Material and methods. To extract the relevant information from Scopus database we have used text and data mining techniques. The scientific publications list was generated using a set of 335 keywords which were obtained by web scrapping methods from two Wikipedia pages. In this study we have limited the search period over the last 16 years and over several relevant domains: Agricultural and Biological Sciences (AGRI), Biochemistry, Genetics and Molecular Biology (BIOC), Chemical Engineering (CENG), Engineering (ENGI), Environmental Science (ENVI), Immunology and Microbiology (IMMU), Materials Science (MATE), Multidisciplinary (MULT) and Pharmacology, Toxicology and Pharmaceutics (PHAR). To achieve the goal of this research1 we have used R language, a free enviroment for statistical computing and graphics. Results and discussion. In the present study, a total of 4517 scientific publications were obtained. The following map illustrates the number of publications in each county of Romania. We observe that the first three counties are Bucuresti, Cluj, Iasi which they cover 2759 papers representing 63% of total publications taken into consideration.
Figure 1: The distribution of publications per county
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Figure 2 presents the number of publications per year and domain (left) and the number of publications over the last 5 years per institution type (right). From the left figure, it can be observed that from 2008 the number of publications icreased abruptly for three out of nine domains (AGRI, BIO and ENGI). As expected, Universities and Research Institutes presents the largest contribution of research papers over the last 5 years representing a total of 92%, as one can see from the figure on the right.
Figure 2: The number of publications per year and domain (left) and the number of publications for the last five years per institution type (right) The use of citation counts is a well-established measure of scientific impact (e.g. [1] and [2]). In Figure 3 (left) one can observe that from the year 2008 the number of citations rise rapidly showing an increasing interest in medicinal plants scientific research. The distribution of the number of citations is illustrated in Figure 3 (right). As one can see, the data is clearly skewed to the right, the mean and the median being 7.3 and 3 respectively.
Figure 3: The cummulative number of citations per year and domain (left) and the distribution of the number of citations for the studied period (right)
Conclusions. The analysis highlighted an increasing trend in the scientific publications that have keywords related to medicinal plants over the last 16 years. An abrupt increment of the number of citations and scientific papers was observed starting with the year 2008 thus showing an elevated interest in the romanian medicinal plants scientific research.
Bibliography [1] Ball, P. (2007). Achievement index climbs the ranks. Nature, 448, 737. [2] Bornmann, L., Mutz, R., & Daniel, H-D. (2008). Are There Better Indices for Evaluation Purposes than the h Index? A Comparison of Nine Different Variants of the h Index Using Data from Biomedicine. Journal of the American Society for Information Science and Technology, 59, 830-837.
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Poster Presentation – PP02
Immunomodulatory potential of Inula helenium L.
Alice Grigore1, Georgeta Neagu1, Nicoleta Dobre1, Carmen Ionita2, Lucian Ionita2, Dana Bobit3
1National Institute of Chemical-Pharmaceutical Research and Development, ICCF Bucharest 2Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest 3 SC Dacia Plant SRL, Hărmanului FN, Bod - Brașov, Romania
Abstract. Our investigations refer to the immunomodulatory potential of Inula helenium root extract. HPLC analysis confirmed the presence of phenolic compounds and also of alantolactone as the main sesquiterpene lactone in the hydroalcoholic extract studied. The in vitro results highlighted the immunostimulatory as well as scavenger potential of the extract. Key words: HPLC, polyphenols, flavonoids, alantolactone, immunostimulation Introduction. Inula helenium L. (Asteraceae) and its active components have been widely used as anti-inflammatory, anti-microbial, and anti-cancer agents. Currently, it is possible to claim for the root and the rhizome of Inula helenium (as infusion, powder, wine, syrup) the following therapeutic indications: (i) antiseptic in exanthema, bacterial and fungal dermatitis; (ii) antipruritic in dry patches; (iii) to facilitate urinary and digestive functions; (iv) to treat symptomatic cough and bronchitis; (v) to treat failure by the dyspeptic hepatobiliary or biliary dyskinesia; (vi) as an adjunct in the fight against hyperglycemia and obesity (Ghedira et al., 2011). The aim of this study is to highlight the immunomodulatory potential of Inula helenium root extract. Material and methods. 100 g of dried and milled Inulae radix in 1000 mL of 50% ethyl alcohol were soaked for 10 days at room temperature in a dark place and then filtrated. The crude extract was concentrated under reduced pressure (72-74 mmHg), dissolved in 20% propylenglycol and used for further investigations. Chromatographic separation was performed on a HPLC ELITE – LaChrom system, with DAD detector and a Inertsil ODS 3 column (250 x 4.6 mm, 5μm) at 25ºC. Separation of polyphenols was performed using a mobile phase consisting of an A solution (water acidified with phosphoric acid, pH = 2.5) and a B solution (methanol) at an initial flow rate of 1 mL/min; with an injection of 20 μL. Separation of alantolactone was performed using a mobile phase consisting of water:methanol solution (40/60 v/v) at an initial flow rate of 1 mL/min; with an injection of 20 μL. Free radical (DPPH) scavenger activity was assayed according to Oms-Oliu et al., 2009). The viability of NR8383 alveolar macrophages incubated with different concentrations of extracts in the presence or absence of stimuli that trigger immune response (LPS) was carried out by a colorimetric method using kit CellTiter 96® Aqueous Non-Radioactive Cell proliferation Assay (Promega, USA). Lypopolysaccharide (LPS) was used due to its ability to induce a rapid dose- dependent response in the host - secretion of cytokines (TNF, IL-1, IL-6) mediated by TLR-4. This activity is similar to Gram-negative bacterial endotoxin. Alveolar macrophages NR8383 were cultivated in 96 well-plates 10 4 cells/ well în DMEM F12 medium (Sigma, Germania), supplemented with 15% fetal bovine serum (Gibco), 1% antibiotic, kept at 37°C with 5%CO2. The extract was dissolved în culture media without serum în concentrations of 25-150μg/ mL and incubated with cells for 21 hours with 10 μg / mL LPS (Sigma, Germany). All samples were tested în duplicate and levamisole 100μg/ mL was used as reference substance (Romvac, Romania). Results and discussion. Chemical composition of Inula extract (I5) as resulted by HPLC is presented in table 1 and reflect the existence of both phenolic compounds and sesquiterpene lactones.
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Table 1 – Chemical composition of the Inula helenium extract Compound mg% by HPLC Compound mg% by HPLC Alantolactone 2.765 Quercetin 0.674 Chlorogenic acid 27.78 Kaempferol 0.557 Caffeic acid 3.177 Gallic acid 9.327 Rutin 1.595 3,4-dihydrobenzoic acid (protocatechuic 0.768 acid) Rosmarinic acid 1.05 2,5-dihydrobenzoic acid (gentisic acid) 97.081 The extract exhibits a scavenger effect on DPPH radical of over 80% at 50-100μg/ mL.
As is it presented în figure 1, smaller doses of extract (25-50μg/ mL) enhance the cells proliferation rate.
Figure 1 – Viability of rat alveolar macrophages exposed to Inula extract
Simultaneous administration of Inula extract and LPS and exposure of rat alveolar macrophages for 21 hours to this combination maintain cellular viability to over 50% (figure 2).
Figure 2 – Viability of rat alveolar macrophages exposed to both Inula extract and LPS for 21 hours
Conclusions The analysis of Inula helenium hydroalcoholic extract highlighted the presence of phenolic compounds such as polyphenolcarboxylic acids (caffeic, rosmarinic, chlorogenic), flavonoids (rutin, quercetin, kaempferol) and also of a sesquiterpene lactone – alantolactone. In vitro assays carried out on macrophages a one of the most important cell type involved în the immune response confirmed the immunostimulatory potential of the extract. The studies should be continued in order to investigate the compound(s) resposible for this action and to elucidate the mechanism of action. Acknowledgements: The work was supported by a grant of UEFISCDI, Romania, PN-II-PT-PCCA-2 no. 134/ 2012.
Bibliography 1. K. Ghedira, P. Goetz, R. Jeune (2011), Inula helenium L. (Asteraceae) aunée, Phytothérapie, 9 176–179 2. Oms-Oliu G, Odriozola-Serrano I, Soliva-Fortuny R, Martín-Belloso O (2009). Effects of high-intensity pulsed electric field processing conditions on lycopene, vitamin C and antioxidant capacity of watermelon juice. Food Chemistry, 115:1312–1319.
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Poster Presentation – PP03
Cytotoxic potential of Helleborus purpurascens L.
Alice Grigore1, Georgeta Neagu1, Nicoleta Dobre1, Adrian Albulescu1, Carmen Ionita2, Lucian Ionita2, Dana Bobit3
1National Institute of Chemical-Pharmaceutical Research and Development, ICCF Bucharest 2Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest 3SC Dacia Plant SRL, Hărmanului FN, Bod - Brașov, Romania
Abstract. Our investigations refer to the cytotoxic potential of Helleborus purpurascens L.. The in vitro assays conducted on two cancer cell lines (Jurkat and BT-20) exposed to alcoholic extract for 24 and 48 hours, respectively, confirmed that this plant is a potential antitumoral agent. Key words: HPLC, MTS, viability, Jurkat, BT-20 Introduction. Many species of Helleborus are seen today as potential sources for anticancer drugs. Studies involving extracts or chemical compounds gave optimistic results related to cancer inhibition and cytotoxicity (1). Lindholm et al. (2) tested, through a large-scale screening protocol, 100 fractionated plant extracts, seven of them showing interesting cytotoxic properties. The aim of this study is to investigate the cytotoxic potential of Helleborus purpurascens alcoholic extract on two cancer cell lines. Material and methods. For this study, a Helleborus extract was used according to patent application A/00285/2016. The cytotoxic effect on Jurkat T cells (a leukaemic T-cell line) and BT-20 (breast cancer cell line) was analyzed by MTS assay, reflecting cell viability, as described in the manufacturer kit (Promega, USA). For the MTS assay, Jurkat T cells (2.5 × 103 per well) and BT-20 cells (7.5 × 103 per well) were incubated with extract of several dilutions and 5-fluorouracil (5-FU, 50μg/ mL) in 96- well plates (all samples were tested in duplicate). After incubation for 24 h and 48h, 50 μl of the MTS solution was added. The optical density (OD) values of the solutions were measured at 492 nm using a plate reader. All data are expressed as the mean + SD. Statistical analysis was done using student’s t-test. Results and discussion. Exposure of Jurkat cells to H. purpurascens alcoholic extract for 24 and 48 hours conducted to a strong decrease of cell viability for all concentrations tested (figure 1a and b).
Figure 1 – Cytotoxic effect of different concentrations of H. purpurascens alcoholic extract on Jurkat cells
a)24 h exposure;
b)48 h exposure
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Exposure of BT-20 cells to H. purpurascens alcoholic extract for 24 and 48 hours conducted to a decrease of cell viability only for higher concentration of the extract (60-250μg/ mL) (figure 2a and b).
Figure 2 – Cytotoxic effect of different concentrations of H. purpurascens alcoholic extract on BT-20 cells
a)24 h exposure;
b)48 h exposure
Conclusions In vitro assays carried out on two cancer cell lines confirmed the cytotoxic potential of the H. purpurascens alcoholic extract. The effect is dose-dependent and it is obvious that Jurkat cells are more sensible than BT-20 to exposure.The studies should be continued in order to investigate the compound(s) resposible for this action.
Acknowledgements:The work was supported by a grant of UEFISCDI, Romania, PN-II-PT-PCCA-2 no. 134/ 2012 and Nucleu no. 16-27 04 01/ 2016.
Bibliography 1. Maior M., Dobrota C. (2013). Natural compounds with important medical potential found in Helleborus sp. Cent. Eur. J. Biol. 8(3), 272-285. 2. Lindholm P., Gullbo J., Claeson P., Göransson U., Johansson S., Backlund A., (2002). Selective cytotoxicity evaluation in anticancer drug screening of fractionated plant extracts, J.Biomol. Screen., 7, 333-340.
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Poster Presentation – PP04
Crocus sativus studies on morphostructural analysis
Adina Segneanu1,2, Daniel Damian2, Ioan Grozescu2
1Scient –Research Center for Instrumental Analysis, Cromatec Plus, Tancabesti, Romania 2 University Politehnica Timisoara
Abstract. The paper aims to investigate an analytic study on Crocus sativus using advanced spectroscopy techniques: mass-spectroscopy, FT-IR spectrometer with Spotlight 400 microscopy, electronic microscopy SEM-EDAX. The research has been conducted mainly on morphology, elemental and chemical composition of dry sample of Crocus sativus. Key words: elemental composition, flavonoids, carotenoids, amino acids, spectroscopy Introduction.This exotic spice is a native plant used mostly in cuisine, but important health benefits recommend them successfully as herb with high therapeutic properties. Beyond the exorbitant price of saffron its health benefits are known from many centuries. Modern researche on this plant shown that through their complex chemical composition may underlie of new biomedical applications for the treatment of serious diseases: cancer, neurologic diseases, metabolic disorders, etc [1-3]. Material and methods. The morfo-structural characterization of solid samples (stigmas) of Crocus sativus was investigated by scanning electron microscopy (SEM) using Inspect S PANalytical model coupled with the energy dispersive X-ray analysis detector (EDX). Frontier MIR/NIR FT-IR spectrometer with Spotlight 400 microscope, Micro-Raman inVia BASIS, Fully automated chip-nano- ESI performed in a NanoMate 400 robot incorporating ESI chip technology (Advion BioSciences, Ithaca, USA) couplet on a High Capacity Ion Trap Ultra mass spectrometer (HCT Ultra, PTM discovery) (Bruker Daltonics, Germany). Results and discussion. The results obtained shown an complex chemical composition highlighting the presence of carotenoids, antocians, flavonoids and amino acids. The topografy and elemental composition was emphasized through spectroscopic methods used. The results gained in this study corroborates with the existing data from the literature [2-4]. Conclusions. This study was designed for investigating the chemical composition and morphostructural analysis of a valuable spice and also a medicinal plant with known therapeutic effects, saffran. The analysis results indicates the presence of a complex mixture of highly active compounds such as: antocians, flavoinoids, carotenoids and amino acids with multiples biomedical applications.
Bibliography 1. Anastasia Kyriakoudi, Stella A Ordoudi, Marta Roldán-Medina, Maria Z Tsimidou (2015), Saffron, A Functional Spice, Austin J Nutri Food Sci – Vol. 3 (1):1-5 2. Vijaya Bhargava K, (2011), Medicinal uses and Pharmacological Properties of Crocus sativus Linn (Saffron), International Journal of Pharmacy and Pharmaceutical Sciences, Vol 3, (3): 21-26. 3. S.R. Hassan-Beygy, D. Ghanbarian, M.H. Kianmehr, M. Farahmand (2010), Some Physical Properties Of Saffron Crocus Corm, Cercetări Agronomice în Moldova Vol. XLIII , No. 1 (141): 17-29. 4. Bilal Ahmad Wani, Amina Khan Rouf Hamza, F.A. Mohiddin, (2011), Saffron: A repository of medicinal properties, Journal of Medicinal Plants Research Vol. 5(11):2131-2135.
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Poster Presentation – PP05
Curcuma longa – An analytical study
Adina Segneanu1,2, Daniel Damian2, Ioan Grozescu2
1Scient –Research Center for Instrumental Analysis, Cromatec Plus, Tancabesti, Romania 2 University Politehnica Timisoara
Abstract. The current paper proposes the determination of curcumin and morpho-structural characterisation of powder of Curcuma longa using chromatography (HPLC, GC), MS spectroscopy and electronic microscopy methods. Key words: HPLC, electronic microscopy, curcumin,topografy. Introduction. Turmeric is an Indian spice used mostly in cuisine, but with important health benefits recommend as herb with high therapeutic properties.Turmeric as well its use in Ayurvedic practices. Modern research demonstrate that curcumine have important pharmacological applications such as: antimicrobian, anti-oxidant, antiinflamator [1-3]. Material and methods. The morfo-structural characterization of solid sample (powder extracts) of Curcuma longa and Curcuma longa was investigated by electronic microscopy using Inspect S PANalytical model coupled with the energy dispersive X-ray analysis detector (EDX). Frontier MIR/NIR FT-IR spectrometer with Spotlight 400 microscope, Micro-Raman inVia BASIS, FT_IR spectroscopy. The curcumin extracts were investigated using chromatographic techniques. The quantitative and qualitative determination of curcumin was performed by means of a uHPLC analysis carried out using an FX15 Liquid chromatography system (UHPLC) with DAD and RI detectors and post-column derivatization unit. Results and discussion. The results obtained releaved the fact that curcumin quantity depend on extraction parameters (solvent, time) The topografy and elemental composition was emphasized through spectroscopic methods used. The results gained in this study are according to data from the literature [2-4]. Conclusions. This research aimed to investigate the concentration of curcumin and the topography of turmeric sample. Together with curcumin were highlighted volatile oils, carotenoids, components with role in combating oxidative stress.
Bibliography 1. Hamid Nasri, Najmeh Sahinfard, Mortaza Rafieian, Samira Rafieian, Maryam Shirzad, Mahmoud Rafieian-kopaei (2014), Turmeric: A spice with multifunctional medicinal properties, Journal of HerbMed Pharmacology, 3(1): 5-8. 2. Julie S. Jurenka, (2009), Anti-inflammatory Properties of Curcumin, a Major Constituent of Curcuma longa: A Review of Preclinical and Clinical Research, Alternative Medicine Review Vol. 14 (2):141-153. 3. Duggi Shrishail, Handral Harish K, Handral Ravichandra, G.Tulsianand, S.D. Shruthi, (2013), Turmeric: Nature’s Precious Medicine, Asian J Pharm Clin Res, Vol 6 (3):10-16. 4. Jennifer R. Alambra, Rod Russel R. Alenton, Pia Clarisse R. Gulpeo, Christine L. Mecenas, Abigail P. Miranda, Rey C. Thomas, Maden Krista S. Velando, Lawrence D. Vitug, Mary Beth B. Maningas (2012), Immunomodulatory effects of turmeric, Curcuma longa (Magnoliophyta, Zingiberaceae) on Macrobrachium rosenbergii (Crustacea, Palaemonidae) against Vibrio alginolyticus (Proteobacteria, Vibrionaceae), Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society Vol.5(1): 13-17.
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Poster Presentation – PP06
The neuroprotective effects of Anemarrhena asphodeloides rhizome extract in the PC12 cell model
Nina Rembiałkowska1,2, Anna Maria Patrzałek2, Magda Lewińska2, Arnold Garbiec3, Zofia Marchewka2, Agnieszka Piwowar2, Anna Długosz2, Adam Matkowski4
1Department of Medical Biochemistry, Wroclaw Medical University, Chalubinskiego 10, 50-368, Wroclaw, Poland 2Department of Toxicology, Wroclaw Medical University, Borowska 211, 50-556 Wroclaw, Poland 3Department of General Zoology, Zoological Institute, University of Wroclaw, Sienkiewicza 21, 50-335 Wroclaw Poland 4Department of Pharmaceutical Biology and Botany, Wroclaw Medical University, Borowska 211, 50-556 Wroclaw, Poland
Abstract. The burden of neurodegenerative diseases is growing as the population ages. Alzheimer’s is an example of the incurable and eventually fatal condition. In a symptomatic therapy, plant natural products and complex extracts obtained from medicnal plants appear to be a promising class of therapeutics for the treatment of this type of diseases. Delivery of Anemarrhena asphodeloides can improve the condition of neuronal cells. Here, we report the protective effect of extract from Anemarrhenae asphodeloides rhizoma on PC12 neuronal cell line in model system of 3-NP induced toxicity. Key words: Anemarrhena asphodeloides, neuroprotection, 3-nitropropionic acid, PC12, xanthone glycosides.
Introduction. Nowadays, neurodegenerative diseases are among the most serious pathological conditions of modern societies. The most common disorders in this group are Alzheimer’s and Parkinson’s diseases. In a symptomatic therapy, plant natural products and complex extracts obtained from medicnal plants appear to be a promising class of therapeutics for the treatment of this type of diseases. [1]. The rhizomes of Anemarrhena asphodeloides (Chinese name zhi-mu), known for their stimulating effect on cognitive functions have been used in the Trraditional Chinese Medicine in treatment of neurological disorders. Recently, its monograph herb has been included in European Pharmacopoeia and requires standardization for xanthone glucoside (mangiferin) content. In this study, we show that delivery of Anemarrhena asphodeloides rhizome extract can improve viability of neuronal cells in vitro [2].
Material and methods. Rat pheochromocytoma cell line (PC12) were used. PC12 cells were cultured in RPMI 1640 with 10% FBS, 1 % penicillin-streptomycin. The effect of Anemarrhena asphodeloides rhizome extract in concentrations from 0.5 to 10 μg/mL was investigated. In order to evaluate the neuroprotective properties of Anemarrhena asphodeloides rhizome extract we have provoked the cytotoxicity by 3-nitropropionic acid (3-NP) in concentrations from 2.5 to 15 mM. The control group the PC12 cells was with/without extract of Anemarrhena asphodeloides, with/without 3-NP and without any additional compounds. After 24 hours the viability assay was applied. Cells were incubated for 45 min with Mitotracker® Deep Red for detecting the mitochondria and DAPI for detecting the nucleus. Cells were determined by confocal microscopy. Anemarrhena asphodeloides rhizomes were harvested from the plants cultivated in the Botanical Garden of Medicinal Plants of the Wroclaw Medical University in October 2015. The rhizomes were dried under ambient temperature and extracted using ultrasound-assisted extraction with 80%ethanol.
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The extract was dried under nitrogen flow and used for the experiment as well as characterized using TLC and HPLC.
Results and discussion. 3-NP in combination with Anemarrhena asphodeloides root extract affected positively the viability of PC12 cells. Extract found to be more effective after using lower concentration. 3-NP in combination with Anemarrhena asphodeloides root extract, caused increased viability when compared to the application of 3-NP alone. After 24 h incubation, the extract solution with a concentration of 15.0 μg/mL or 2.5 mM solution of 3-NP cells were observed with different morphology. After 24 h incubation with 10.0 mM 3-NP cells characterized by smaller nucleus, but after incubation with 15.0 mM of 3-NP have degenerate nucleus. It was observed that the cells were preincubated with the extract at low concentrations and incubated with 15.0 mM with 3-NP are not so much degenerate and are present cells with normal nucleus morphology. Thye main compounds in the ethanolic extract were the highly hydroxylated xanthone glycosides: mangiferin (2-C-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone), neomangiferin, and isomangiferin. A minor content of several saponins, typical for this plant, was also present.
Conclusions. Due to the use of low concentration of extract and relatively low toxicity, a potential of Anemarrhenae asphodeloides rhizoma extract can be further explored as an alternative source of therapeutic substance against neurodegenerative diseases [1]. The extract treatment following the irritating of the cells with 3-NP, caused an increase in cell survival, suggesting a regenerative action. Moreover, a positive effect was observed with respect to the morphology of cultured cells. Eventually, the zhi mu products can be used for alleviating or preventing of the pathological injuries associated with the development of the disease [2].
Bibliography 1. B. Mandavilli, I. Boldogh, B. Van Houten (2005), 3-Nitropropionic acid-induced hydrogen peroxide, mitochondrial DNA damage, and cell death are attenuated by Bcl-2 overexpression in PC12 cells. Mol. Brain Res., 133:215-223. 2. G. Kulasekaran, S. Ganapasam (2015), Neuroprotective efficacy of naringin 3-nitropropionic acid-induces mitochondrial dysfunction through the modulation of Nrf2 signaling pathway in PC12 cells. Mol. Cell Biochem., 409:199-211.
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Poster Presentation – PP07
LC-DAD-ESI-MSn profile of phenolic compounds in flowering stems of Sideritis raeseri from Republic of Macedonia, Albania and Greece
Bujar Qazimi1, Jasmina Petreska Stanoeva2, Gjoshe Stefkov1, Marina Stefova2, Svetlana Kulevanova1
1Institute of Pharmacognosy, Faculty of Pharmacy, University SS Сyril and Methodius, Skopje, R. Macedonia 2Institute of Chemistry, Faculty of Natural Sciences and Mathematics, University SS Сyril and Methodius, Skopje, R. Macedonia
Abstract. Phenolic compounds were determinated in methanolic extracts of Sideritis raeseri . With LC-DAD-ESI-MSn were identified 28 compounds with a total content of 62.70-131.11 mg/g DW, and were classified into four groups: hydroxycinnamic acids derivatives, phenylethanoid glycosides, flavonoid 7-O-diglycosides and flavonoid acetylglycosides aglycones. Key words: Sideritis raeseri, LC-DAD-ESI-MSn, phenolic compounds. Introduction. Mountain tea (Sideritis raeseri Boiss. et Heldr.) is endemic to the Balkan Peninsula and is reported to grow in Greece, R. Macedonia and Albania [1]. Flowering parts of this plant are widely utilized in Mediterranean folk medicine in the form of a decoction or infusion, to treat the common cold, to alleviate sinus congestion, pains and virus infections, including infuenza [2]. Phenolic compounds have several roles in the plants physiological processes and have demonstrated significant health beneficial effects [3]. The aim of this work was the determination of the phenolic compounds in the methanolic extracts of flowering stems of Sideritis raeseri using LC-DAD-ESI-MSn. Material and methods. Plant material: The flowering stems of S. raeseri were collected in different localities in R. Macedonia (Baba, Kazani and Korito in National Park Galichica), Albania (Gramoz and Tepelena) and Greece ( Lefkada and Parga) during the summer of 2012. The plant material was air dried, packed in paper bags and kept in a dark and cold place until analysis. Extraction of phenolic compounds: 0.2 g of powder plant material (homogenized samples from flower, leaf and stem) was extracted with 25 ml of 70% methanol, 30 min using US bath. The supernatant was filtered through 0.45 μm pore-size polyethersulfone filter before analysis.LC/DAD/ESI-MSn analysis: Chromatographic separations were carried out on 250 mm x 4.6 mm, 5 µm C18 Luna column (Phenomenix). The mobile phase consisted of two solvents: water – formic acid (1 % v/v) (A) and methanol (B). A linear gradient starting with 25% B was installed to reach 30% B at 7 min, 45% B at 30 min, 50% B at 50 min and 100 % B at from 55 to 60 min. The flow rate was 0.5 ml min-1 to 50 min and 0.8 ml min-1 from 50 min to 65 min, the injection volume 10 µL. The HPLC system was equipped with an Agilent 1100 series diode array and mass detector in series (Agilent Technologies, Waldbronn, Germany). Spectral data from all peaks were accumulated in range 190-600 nm and chromatograms were recorded at 290 and 300 nm from glycosides and acylated derivatives and at 330 nm for phenylethanoid glycosides and hydroxycinnamic acid. The mass detector was a G2449A Ion- Trap Mass Spectrometar equipped with an electrospray ionisation (ESI) system and controlled by LCMSD software (Agilent, v.6.1.). Nitrogen was used as nebulising gas at pressure of 65 psi and the flow was adjusted to 12 L min-1. The heated capillary and the voltage were maintained at 325 C and 4 kV, respectively. MS data were acquired in the negative ionization mode. The full scan covered the mass range at m/z 100-1200. Collision–induced fragmentation experiments were performed in the ion trap using helium as collision gas, with voltage ramping cycle from 0.3 up to 2 V. Maximum accumulation time of ion trap and the number of MS repetitions to obtain the MS average spectra were set at 300 ms and 5, respectively. The symposium is dedicated to the 60-year celebration of the „Stejarul” Biological Research Centre – MAPPPS 2016, Piatra Neamt, ROMANIA – Page 72
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The identification and peak assigmentation of all phenolic compounds was based on comparison of their retention times and mass spectral data with those of standards and published dates. Hydroxycinnamic acids were quantified using 5-caffeoylquinic acid external standard at 330 nm, phenylethanoid glycosides were quantified and expressed as verbascoside equivalent at 330 nm, hypolaetin glucosides were quantified with 4'-O-methylhypolaetin 7-O-[6′′′-O-acetyl]- allosyl(1→2)glucoside at 290 nm, whereas isoscutellarein glucosides were quantified and expressed as isoscutellarein 7-O-[6′′′-O-acetyl]-allosyl(1→2)glucoside equivalent at 300 nm. The stock solutions of phenolic standards were made up in 70 % methanol to a concentration of 1000 µg/mL. The corresponding calibration curves were constructed with five dilutions of the stock solutions. Results and discussion. Phenolic compounds in the methanolic extracts were identified by their UV spectra, their deprotonated molecular ions and their corresponding ion fragments, by using LC/DAD/ESI-MSn. Total of 28 individual components were identified in the methanolic extracts of flowering stems of S. raeseri, representing 62.70-131.11 mg/g DW of the total content. Phenolic components were classified into four groups: hydroxycinnamic acids derivatives (3), phenylethanoid glycosides (9), flavonoid 7-O-diglucosides (5) and flavonoid acetylglucosides aglycones (11). The total amount of hydroxycinnamic acid derivatives in all extracts ranged from 1.72-6.04 mg/g DW. 5- caffeoylquinic acid was found in all samples and it was dominant hydroxycinnamic acid (1.72-4.60 mg/g). Phenylethanoid glycosides (PHEG) were the abundant group of polyphenols in the studied samples with the content ranging from 36.10-80.46 mg/g. Verbascoside (15.86-32.70 mg/g), lavandulfolioside (12.59-22.63 mg/g), allysonoside (1,43-9.99 mg/g), leucoseptoside A (1.89-4.95 mg/g) and echinacoside (0.61-4.51 mg/g) were the most abundant compounds and represent around 90% of total phenylethanoid content. Total content of flavonoid glycosides (non acetylated and acetylated) ranged from 22.87-48.18 mg/g. The prevailing components were isoscutellarein 7-O-[6′′′- O-acetyl]-allosyl(1→2)glucoside (3.25-14.51 mg/g), 3'-O-methylhypolaetin 7-O-[6′′′-O-acetyl]- allosyl(1→2)glucoside (4.43-10.72 mg/g), hypolaetin 7-O-[6′′′-O-acetyl]-allosyl(1→2)glucoside (1.43-3.42 mg/g), 3'-O-methylisoscutellarein 7-O-[6′′′-O-acetyl]-allosyl(1→2)glucoside (0.70-14.42 mg/g) and 32 4'-O-methylhypolaetin 7-O-[6′′′-O-acetyl]-allosyl-(1→2)-[6′′-O-acetyl]-glucoside (0.48- 8.34 mg/g). Conclusions. Methanolic extracts of S. raeseri showed that extracts were rich in bound forms of phenolics such as hydroxycinnamic acids, phenylethanoid glycosides and flavonoid glycosides. In studied samples from R. Macedonia (Galichica), the content of total phenolics is variable depending on the place of collection (67.20-131.11 mg/g DW), while in samples from Greece prevalence ranged from 85.06-109.46 mg /g DW. The lower representation is detected in samples from southern Albania (74.77-95.90 mg /g DW).
Bibliography 1. Obon de Castro C, Rivera–Nunez D (1994), A taxonomic revision of the section Sideritis (genus Sideritis) (Labiatae), Cramer JED, Berlin-Stuttgart. 2. Marija Karapandzova, Bujar Qazimi, Gjoshe Stefkov, Katerina Baceva, Trajce Stafilov, Tatjana Kadifkova Panovska, Svetlana Kulevanova (2013), Сhemical characterization, mineral content and radical scavenging activity of Sideritis scardica and S. raeseri from R. Macedonia and R. Albania, Natural Product Сommunication, 8 (5): 639-644. 3. Jasmina Petreska, Gjoshe Stefkov, Svetlana Kulevanova, Kalina Alipieva, Vasya Bankova, Marina Stefova (2011), Phenolic compounds of Mountain tea from Balkans: LС/DAD/ESI/MSn profile and contents, Natural Product Сommunication, 6, 1305-1314.
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Poster Presentation – PP08
Biochemical and HPTLC Fingerprinting Identification of the Hypericum perforatum L.- Finished Products
Ţebrencu Carmen E.1,3*, Iacob Elena1, Ciupercă Oana T.1, Creţu Ruxandra M.2, Chiriac Maria1, Ionescu Elena1,3
1Medicinal Plants Research and Processing “PLANTAVOREL” S.A., Cuza Voda Street, no. 46, 610019, Piatra Neamt, Romania; 2NIRDBS / “Stejarul” Research Centre for Biological Sciences, Alexandru cel Bun St.,6, Piatra Neamt, Romania; 3Academy of Romanian Scientists, Splaiul Independentei , no.54, 050094 Bucharest, Romania *Corresponding author, e-mail: [email protected]
Abstract. Hypericum perforatum L., as single ingredient or in finished products with other medicinal plant species have been selected and tests correlated with biochemical and HPTLC fingerprinting of the plant species Hypericum perforatum L. have been performed. Based on the obtained data, the study is takes contributions along with other modern methods to the correct identification of this species and finished products identity. Key word: Hypericum perforatum L. identification, herbal supplements, biochemical fingerprinting, HPTLC fingerprinting Introduction. Hypericum perforatum L. is the official source of Hyperici herba as accepted by both the Romanian Pharmacopoeia and the European Pharmacopoeia. Hypericum perforatum contains at least ten classes of biologically active compounds, of which two of the more important bioactive compounds, hypericin and hyperforin. [1;2;3] The objective of this study is to identify the Hypericum perforatum L. in finished products ( food supplements) based on this species by specific biomarkers obtained with biochemical and HPTLC fingerprinting. Material and methods. St. John’s wort, aerial part (Hypericum perforatum L.) is used as raw powder, dry extract or mixed with othet plant species in finished products ( Hp -St. John's wort, powder; HEi -St. John's wort dry extract; HE-St John's wort obtained by atomization at Plantavorel; H-HEPATOBIL V, tablets; VR-VITA ROZ, tablets; G- GASTROVIT, tablets; F -Tonic herbs FEMINA, hydroalcoholic solution) produced by Medicinal Plants Research and Processing “PLANTAVOREL” SA Piatra Neamt , Romania . All the chemicals and reagents were of analytical grade or pure. Biochemical and chromatographic fingerprinting were performed through phytochemical methods according to the European Pharmacopoeia, Romanian Pharmacopoeia, own validated methods and instrumental methods ( UV-VIS spectrometry and thin layer chromatography HPTLC). Results and discussion. The biochemical and chromatographic analysis of the results was done by comparative evaluation and correlation of the content through the marker compounds in raw materials, intermediate and final product whose composition is found. Chromatographic polyphenols, flavons and naphtodianthrones fingerprint : for St. John's wort powder, dry extract (raw materials in the composition of the finished products) and finished products conditioned as tablets (PVR, PG) were clearly identified flavonoid and polyphenol compounds - rutin, hyperoside, chlorogenic acid and naphtodianthrones - hypericin. The phytochemical screening confirms the assignment of reference substances, with a good overlapping of spectra standards over spectra attributed to standards in samples for rutin and hyperoside; for hypericin, although there was a very good overlap of spectra, densitometric determination of standard samples confirms the assignment of standard in samples. As a result of tests performed on products PH (composition based on five plant species powders) and PF hydroalcoholic solution no attributable standards of interest couldn’t be done because of complexity of the phytochemical profile and the interactions with extraction solvents and development. It is obvious the correlation between identification of the markers and type of vegetal raw material (powder or processed plant material) and their concentration in vegetable complex from the finished products.( fig 1; fig 2). Quantitative analysis of selected products: values obtained from phytochemical compounds of interest dosage correspond exactly to
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qualitative assessment conducted by chromatographic fingerprinting for flavones, polyphenol compounds and naphtodianthrones. The highest concentration values of phytochemical compounds are found in St. John's wort dry extract (compared to St. John's wort powder as raw material), which also corresponds to higher values in finished products based on extracts (PVR, PG). If for solid forms of conditioning (tablets) qualitative and quantitative determinations have been conducted, in fluid form, where phytochemical profile is already selected by extraction solvent, identification of compounds could not be achieved by instrumental methods.( tab.1).
Tab. 1 Results of the qualitative analysis of selected samples
Phytochemical parameters Selected samples/(% g/g p.v.) Hp HEi H VR G F Total polyphenolcarboxylic acids (chlorogenic acid) 2.7832 5.7092 0.1164 5.1254 1.0248 0.2851 Total flavones (rutin) 1.6355 7.6253 0.1443 3.6023 1.0636 0.1589 Total flavones (hyperoside ) 1,6122 1.7287 0.1422 1.9454 0.1922 0.3420 Total naphtodianthrones ( hypericine) 0.0489 0.2535 0.0077 0.1233 0.0303 0.0214
Values obtained from phytochemical compounds of interest dosing corresponds exactly to qualitative assessment carried out by chromatographic fingerprinting for flavones, polyphenols compounds and naphtodianthrones.
Conclusions. They identified the common “biomarkers” for identifying the species Hypericum perforatum L. in raw powder, dry extract or mixed with othet plant species in finished products, respectively rutin, hyperoside and hypericin through biochemical and HPTLC fingerprinting. In the case of finished products as plant complex compositions (with many plant species / in fluid extract) is required completing investigations with other modern analytical methods for the safety of authentication plant species.
Acknowledgements:The research leading to these results has received funding from the Romanian - EEA Research Programme operated by the MECS-ANCSI PO under the EEA Financial Mechanism 2009-2014 and Project Contract No 2SEE/2014.
Bibliography : 1. Avato, P., 2005. A survey of the Hypericum genus: secondary metabolites and bioactivity. Studies in Natural Product Chemistry, 30:603–634 2. EMEA, 2012,“ Risk profile-Hypericum perforatum, extract and oil, European Agency for the Evaluation of Medicinal Products (EMEA), London 3. Nicoletti Marcello, 2010. HPTLC fingerprint: a modern approach for the analytical determination of Botanicals, Revista Brasileira de Farmacognosia Brasilian Journal of Pharmacognosy, 21(5): 818-823.
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Poster Presentation – PP09
Seed germination of Geum urbanum depending on storage conditions (low temperatures)
Catană Rodica1, Florescu Larisa2
1Plant and Animal Cytobiology Department, Institute of Biology Bucharest, Romanian Academy, 296 Splaiul Independenței St., 060031 Bucharest, P.O. Box 56-53 2Ecology, Taxonomy and Nature Conservation Department, Institute of Biology Bucharest, Romanian Academy
Abstract. G. urbanum is a perennial herb used in traditional medicine. This species is characterized by a low physiological dormancy and a low seedling survival rate. Here we investigate how the germination of G. urbanum is affected by the year of seed collection and storage at low temperatures in the aim of conservation. Key words: germination, medicinal plant, storage, low temperatures, Geum. Introduction. Geum (Rosaceae family) is a genus of about 50 species of perennial herbaceous plants. Geum urbanum L. occurs naturally in shaded habitats in Europe. This species has a low outcrossing rate (5 - 20%) (Ruhsam et al., 2010). While, G. urbanum has a high germination rate (Taylor, 1977), a low survival rate at the seedling stage was reported (Endels et al., 2004). In Romania, the Geum species is represented by 6 taxa, G. reptans and G. aleppicum being sporadic spreading. G. urbanum is a widespread plant species and may be used as indicator species for nitrogen soil supply and as medicinal plant (Ciocârlan, 2009). This species is mentionated as endemic for Europe in the European Red List of Medicinal Plants (Allen et al., 2014). The main biologically active compounds (tannins and phenolic acids) are found in herb and underground parts (Kuczerenko et al., 2011). Around of 69% of eugenol (main compound of the essential oil) is found in the roots (Owczarek et al., 2013). Especially the roots are anti-inflammatory, antiseptic, aromatic, astringent, being used in traditional medicine (Vogl et al., 2013). Interest for the conservation of medicinal plants is increasing all over the world, due to a growing recognition of their role. Storage of the seeds is a technique accessible to a large part of higher plants, serving as a safe and relatively inexpensive method of conservation. The maintaince of the seeds quality until it is used is one of the seed storage goals. Due to the lack of knowledge of medicinal plant reproduction biology and seed behavior (Liza et al., 2010), these species are poorly represented in seedbanks (Heywood 2000). Material and methods. The experiments were conducted to study the effects of the maintenance at low temperatures on the germination of seeds from a medicinal plant G. urbanum. The mature seeds (collected in 2012 and 2014) were obtained from Botanical Garden, Bucharest in 2014. After collecting, seeds were maintained at room temperature in paper bags. The experiments were carried out in the laboratory conditions in 2014. No seed sterilization protocol was applied. Seeds were washed 2 hours in tap water and placed on double layered filter paper moistened with distilled water in Petri dishes. 4 replicetes of 50 seeds per each treatment were used. The dishes were placed in a chamber room Weiss Gallekamp Fititron, with a photoperiod of 16/8 hrs (two fluorescent lamp of 36W with maximum intensity of ~ 90µmol m-2 s-1) for 4 weeks. After germination, the seedlings were transferred in a ground-perlite mix. In the first step we checked the seed germination of two lots of seeds, collected in 2012 (two years of room temperature storage) and 2014 (freshly collected). For seed storage at low temperatures (4º; -20º; -75ºC) experiment we used seeds collected in 2014. In the aim of dehydration of the seeds before storage and to promote synchronous germination seeds after storage, treatments with polyethylene glycol (PEG) solutions were used. Two concentrations of PEG 6000 solutions (T1 - 4%; T2 - 10%) were applied for 20 minutes at room temperature before seed storage. After treatments seeds were placed in Petri dishes covered with aluminium foil, and maintained at 4º, -20º and -75ºC for 3 weeks. The control was represented by seeds untreated with PEG 6000 and stored at room temperature (~25 ºC).The thowing was realised at room temperature for 10 minutes. The estimated parameter was the final germination percentage (%) expressed like total number of germinated seeds/total number of seeds X 100. Results and discussion. Seeds collected in 2014 start to germinate from the 44th days, while the seeds collected in 2012 germinated after 50 days. The germination percentage of the seeds collected in 2012 and 2014 are significantly different (p<0.05), those collected in 2014 germinating more than 4 times than those maintained for two years at room temperature. Our data are confirming those of McDonald (2005), which emphasized that Geum seeds are short storage life category (less than 1 year). There are some studies concerning the decrease of
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seed germination with age: Inula helenium (Bilińska and Buchwald, 2015), Agastache foeniculum (Matei et al., 2011). Baskin and Baskin (1998) showed that G. urbanum seeds are characterized by a low physiological dormancy, which may be broken by cold stratification. For this reason we stored seeds at different low temperatures. To promote the synchronous seed germination and to protect them of ice forming, seeds were pretreated with PEG solutions. Germination of the seeds stored at different low temperatures (4º, -20º and -75ºC) starts after 7 days, comparing with control temperature, when seeds start to germinate after 44 days. These results are confirming the ideea that the dormancy may be broken through cold stratification. The germination percentage varied significantly from 8 to 100%, under different storage temperatures. The seeds kept at 4º and - 75ºC presented an increase of the germination (at 68%, respectivly at 100%) comparing with room temperature condition (46%). The lowest percentage of germination was noticed at -20ºC. An explication may be that in the refrigerators, the humidity fluctuates too much and may affect the life of seeds. It is known that storege of seeds at room temperature affects them causing low germination, deterioration and loss of vigor and viability (Müller et al., 2011). These effects are natural phenomena during storage (Schmidt, 2002). In our case, storage of untreated seeds at room temperature assure a 46% of germinated seeds, while treatments with PEG and stored at 4º and -75ºC assure more than 60%. There are many studies concerning germination of medicinal plant seeds after storage at low temperatures. In 2012, Kholina and Voronkova showed that seeds of wild medicinal legume species germinated better after storage at low temperatures, surviving after cryostorage with results better than control. The same effect was recorded in the case of Nardostachys jatamansi, a flowering plant from Himalaya, were seeds stored at 0 to -5ºC in refrigerator presented a better germination percentage than those at room temperature (10 - 35ºC) (Chauhan and Nautiyal, 2007). Many techniques have been used for improving seed germination at low temperatures, including PEG pretreatments. Being a non-toxic and inert chemical which does not damage the seed metabolism, PEG solutions are commonly used to control water potential in the study of seed germination (Emmerich and Handegree 1990). In our case, significant diferences (R= 0.208, p= 0.0001) were observed between germination of the seeds treated with 4% and 10% PEG solutions. Geum seeds stored at - 75°C and pretreated with PEG 4% (T1 treatment) showed the highest seed germination percentage (100%) than those treated with PEG 10% (66%). At 4°C and -20°C, T2 treatment (78% and respectively 42%) was more eficient than T1 treatment (68% and respectivly 8%). Developing vigorous seedlings and enhancing seed germination are crucial phenomena for ex situ conservation and for cultivation. Seedlings obtained from Geum seeds treated with PEG solutions and stored at -75ºC were better developted and more vigurouses than them stored at 4°C. The explication may consist in the effect of PEG on the tannins. PEG interact with tanins forming PEG-tannin complexes which inactivates them. It is well known that the action mechanism of tannins is complex, acting like germination inhibitor (Varga and Koves, 1959) and interfering with radicle elongation, root activity, hormonal action, membrane permeability, mineral uptake, photosynthesis (Muthukumar et al., 1985). Conclusion. Our results show that freshly mature seeds germinated better than the two years older and maintained at room temperature. Seeds pretreated with PEG 4% and stored at -75ºC had the best seed germination percentage and seedlings vigurosity.
Selective references 1. Allan D., Bilz M., Leaman DJ., Miller RM., Timoshyna A., Window J. (2014), European red List of Medicinal plants. Luxembourg: Publications Office of the European Union. 2. Baskin C.C. & J.M. Baskin (1998), Seeds. Ecology, Biogeography, and Evolution of Dormancy and Germination. Academic Press, London. 3. Ciocarlan V. (2009), Flora ilustrata a Romaniei Pteridophyts et Spermatophyta, Edit. CERES, pp. 320-321. 4. Matei Cristina Firuţa, Duda Marcel M., Olar Marius V., Ardelean Anca Eva, Mădaş Mariana Niculina (2011), Results Regarding Seed Germination of Agastache Foeniculum (Pursh) Kuntze, Bulletin UASVM Agriculture, 68(1): 207-211. 5. Bilińska Elżbieta, Buchwald Waldemar (2015), Biology of germination of medicinal plant seeds. Part XIXb. Diaspores of Inula helenium L. from Asteraceae family, Herba Polonica, 61(3):7-12. 6. Emmerich W. E. and S. P. Hardegree (1990), Polyethylene glycol solution contact effect on seed germination. Agronomy Journal, 82: 1103-1107. 7. Endels P., Adriaens D., Verheyen K., Hermy M. (2004), Population structure and adult plant performance of forest herbs in three contrasting habitats, Ecography, 27:225–241. 8. Heywood V. (2000). Management and sustainability of the resource base for medicinal plants. In: Honnef S. and Melisch R. (eds) Medicinal Utilization of Wild Species: Challenge for Man and Nature in the New Millennium. WWF Germany/TRAFFIC Europe-Germany, EXPO 2000, Hannover, Germany.
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Poster Presentation – PP10
Phytochemical Screening and Chromatographic Fingerprint Studies on Ethanolic Extracts of Arnica Montana L.
Ciupercă Oana T.1,Ţebrencu Carmen E.1,3*, Iacob Elena1, Creţu Ruxandra M.2, Chiriac Maria1, Ionescu Elena1,3
1Medicinal Plants Research and Processing “PLANTAVOREL” S.A., Cuza Voda Street, no. 46, 610019, Piatra Neamt, Romania; 2NIRDBS/“Stejarul” Research Centre for Biological Sciences, Alexandru cel Bun St.,6, Piatra Neamt, Romania; 3Academy of Romanian Scientists, Splaiul Independentei , no.54, 050094 Bucharest, Romania *Corresponding author, e-mail: [email protected]
Abstract. This study was aimed to develop the fingerprint profile of ethanolic extracts of average samples from the 3 wild population of Arnica montana L. using high performance thin layer chromatography (HPTLC). The phytochemical evaluation and HPTLC fingerprint analysis on flowers, leaves, roots and rhizomes showed the presence of important chemical constituents like flavonoids, polyphenols, sterols etc. Key word: Arnica montana L., phytochemical screening, HPTLC fingerprint, polyphenols. Introduction. Arnica montana L (Asteraceae) is a rare plant under strict protection in several European countries. Due to its intensive collection from nature, it is a vulnerable species in Romania [1,2]. It is attractive for its therapeutic properties: antiseptic, antifungal, antimicrobial, antibiotic, anti-inflammatory, antioxidant and cytotoxic . Chemists give special attention due to its complex chemical composition: volatile oils, terpenoids, sesquiterpene lactones, flavonoids, polyphenols, bitter principals, inulin, polysaccharides, carotenoids and tannins[3]. It is used in phytotherapy in external applications. Phytochemical screening and HPTLC fingerprint analysis on flowers, leaves and roots of A. montana showed the presence of this bioactive compounds. Material and methods. Average samples from the 3 wild population of Arnica montana L. (flowers, leaves, roots and rhizomes) harvested from Northern area of the Romanian Eastern Carpathians were conditioned in dry form according to Ph. Eur. 6.0. The reference substances were purchased from Sigma- Aldrich, Roth. All other reagents were of analytical grade or pure. The samples were prepared by extraction with methanol and etanol 30,50,70,80,95%(v/v)-vegetal material/solvent rate-1/10 m/v for 2 hour under reflux (C) and 7 days at room temperature (R). Each extract was filtered through a textile filter, and used as a stock solution for further analyses. The samples were qualitative and quantitative analyzed, by different chemical and instrumental investigations. Biochemical and chromatographic fingerprinting were performed through phytochemical methods according to the European Pharmacopoeia, Romanian Pharmacopoeia, own validated methods and instrumental methods ( UV-VIS spectrometry and thin layer chromatography HPTLC). Results and discussion. The ethanolic extracts were qualitatively and quantitatively analyzed in order to highlight the main constituents (flavones, polyphenols, sterols). Results are presented in figures 1, 2. In HPTLC chromatograms for flavonoids and polyphenols in ethanolic extracts of Arnica montana – flowers, cynarin, chlorogenic acid, caffeic acid, luteolin-7-glucoside, apigenin-7-glucoside and izoquercitrin were clearly identified in all samples, after derivatization and examination at 366 nm; the spots are more intense in samples E50Ar, E70Ar, E80Ar (R) and E30Ac, E50Ac, E70Ac , E80Ac (C); rutin was not identified in this samples. In the ethanolic extracts of Arnica montana – leaves, cynarin is identified in all samples with greater intensity in E50hAr, E70hAr, E30hAc, E50Ahc, chlorogenic acid in MAhr, E95Ahr, E80Ahc, E95Ahc samples, and hyperoside in E70Ahr, E80Ahr, E80Ahc samples; luteolin and apigenin-7-glucoside were not identified. In ethanolic extracts of Arnica montana- roots and rhizomes, chlorogenic acid is distinctly identified in E30Arc, E50Arc, E50Arc, E80Arc samples, at Rf=0,50 (fluorescent-blue spots); cynarin at Rf=0,89 (fluorescent- blue spots) in all samples with greater intensity in E30Ahc, E50Ahc; gallic acid and ferulic acid were not identified. In HPTLC chromatograms
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for sterols in ethanolic extracts of Arnica montana – flowers were identified specific coloured spots in samples MAr, E70Ar, E80Ar, E95Ar, MAc, E70Ac, E80Ac, E95Ac.
a) b) c)
Fig.1 HPTLC cromatograms for flavonoids and polyphenols in ethanolic extracts of Arnica montana flowers (a), leaves (b), roots and rhizomes (c) a) b) c)
Fig. 2. HPTLC cromatograms for phytosterols in ethanolic extracts of Arnica montana flowers (a), leaves (b), roots and rhizomes (c); examination at 366 nm
In ethanolic extracts of Arnica montana – leaves were identified spots also representing sterols in E70Ahr, E80Ahr, E95Ahr, MAhc, E70Ahc, E80Ahc, E95Ahc; the ethanolic extracts of roots and rhizomes of Arnica montana also present spots according to sterols for the samples MArr, E80ARr, E95ARr, E80ARc, E95Arc; screening at 540 nm indicates the presence of stigmasterol in E80ARr, E95ARr, E80ARc, E95Arc samples. The quantitative analysis shows a high level of hyperoside in the flowers of A. montana (2.0102- 2.5372% w/w d.m.) and polyphenols expressed as caffeic acid (6.0042 to 8.3402% w / w d.m.) in extracts obtained with 30%, 50%, 80% v/v ethanol solvent (C) and 50% and 70% v/v ethanol (R); the leaves contain polyphenols expressed as caffeic acid (from 11.2110 to 15.9098% w /w d.m.) in the 30% and 50% v/v ethanolic extracts, while roots and rhizomes have a high content of polyphenolic compounds (9.757% w/w d.m caffeic acid and 14.3002% g/g d.m. chlorogenic acid) in E70Arc and E80Arc extracts. Flavonoid compounds were absent. Conclusions. In all extracts obtained from A. montana L. (flowers, leaves, roots and rhizomes) were identified polyphenolic compounds, flavones and phytosterols with some exceptions: in roots - flavones were not identified. The richest extracts in polyphenolic compounds (as caffeic acid) are those obtained from the roots and rhizomes of A. montana (7.7839- 15.9098% w/w d.m.) with ethanol 70% and 80% v/v under reflux extraction (C). The highest content in flavones (as hyperoside) was obtained for extracts of A. Montana flowers and leaves (2.578-2.7518% w/w d.m.) when the solvent was ethanol 50% and 70%v/v under reflux extraction (C). Acknowledgements. The work was sustained from the Project ARMOREC/74-2014-PT/PCCA-2013 financed by the Executive Agency for Higher Education, Research, Development and Innovation subordinated to the Ministry of Education and Science, Romania. Bibliografie [1] Craciunescu O, Constantin D, Gaspar A, Toma L, Utoiu E, Moldovan L (2012) Evaluation of antioxidant and cytoprotective activities of Arnica montana L. and Artemisia absinthium L.ethanolic extracts. Chemistry Central Journal 6 (97): 1-11. [2] Gowda Jyothi S, Veerabhadrappa Somashekaraiah B (2013) Study of in vitro antioxidant activity and HPTLC fingerprint of quercitin in Cassia auriculata L., Asian Journal of plant Sciences and Research, 3(4): 162-169. [3] Stefanache C, Danila D, Necula R, Gille E (2010) Studies regarding in vitro regeneration of Arnica montana L. from natural population – Bistrita Valley (Eastern Carpathians), Annals of “Al. I. Cuza” University, Iasi, Tomul LVI, fasc. 1, s. II a. Vegetal Biology: 33-34.
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Poster Presentation – PP11
HPTLC identification of bioactive compounds from Ganoderma lucidum and Flammulina velutipes hydroalcoholic extracts
Corina Bubueanu1*, Popa Gabriela2, Alice Grigore1, Colceru – Mihul Svetlana1, Petruta Calina Cornea2
1National Institute for Chemical-Pharmaceutical R&D (ICCF-Bucharest), Vitan Road 112 Sector 3, Bucharest, ROMANIA, 2University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59 Mărăşti Blvd, District 1, 011464, Bucharest, Romania, Phone: +4021.318.25.64, Fax: + 4021.318.25.67, *Corresponding author, e-mail: [email protected]
Abstract. Mushrooms are considerate functional foods, due to their chemical composition in principal and secondary metabolites. In this paper are analyzed the composition in polyphenols and triterpenes in Ganoderma lucidum (reishi) and Flammulina velutipes, mushrooms species collected from Romania. Keywords: HPTLC, phenolic compounds, mushrooms, functional food. Introduction. Mushrooms have become attractive as functional foods and as a source of bioactive compounds with beneficial to the human health. Ganoderma lucidum is a medicinal mushroom which has many biologically active compounds like triterpenes, polysaccharides, ganodermic acids and also antimicrobial, antioxidant, antiviral and anticancer properties. Flammulina velutipes is an edible mushroom with a mild delicious flavour. This mushroom has been shown to have anti-tumor and anti- lymphoma activity, as well as immuno-boosting, cholesterol-lowering and antihypertensive potentials. The present study was carried out to identify the phytochemicals (polyphenols and triterpenes) and evaluate antioxidant activity of the extracts. Identification of the polyphenolic and triterpenes compounds was carried out by HPTLC (High-Performance Thin Layer Chromatography) techinque. HPTLC is a simple and accurate method that can provide important information regarding the chemical composition and the chromatographic fingerprints are unique to each species. Materials and Methods. Raw material – Ganoderma lucidum and Flammulina velutipes (fruiting body - wild) samples were obtained from University of Agronomic Sciences and Veterinary Medicine of Bucharest, Faculty of Biotechnologies - mushroom collection. A voucher specimen is deposited in INCDCF-ICCF Plant Material Storing Room. Sample preparation: Ganoderma lucidum and Flammulina velutipes samples were prepared by extraction with 50% (v/v) ethanol, 1/20 raw material/solvent ratio, at boiling temperature, for 2 hours. The solutions were filtered and kept frozen until analysis. HPTLC Analysis: The densitometric analysis (HPTLC) was made according to TLC Atlas - Plant Drug Analyses (1) and the characteristic fingerprint profile for chemical compounds was determined. 3-3.5µl of the samples and 1-3µl of references substances (10-3M ferulic and caffeic acid-Sigma- Aldrich) were loaded as 10mm band length in the 20 x 10 Silica gel 60F254 TLC plate using Hamilton- Bonaduz, Schweiz syringe and CAMAG LINOMAT 5 instrument. Polyphenolic compounds: the mobile phases (A) consisted in 100:11:11:27 (v/v/v/v) ethyl acetate-acetic acid- formic acid-water and (B) consisted in 7:6:1(v/v/v) toluene-acetone-formic acid . The TLC twin chamber was pre-saturated with mobile phase for 30 min at ~20°C. The plate was developed in the mobile phase up to 90mm. After development, plates were dried and derivatized in Natural Product followed by PEG4000 reagent. The fingerprints were evaluated at UV with a WinCats and VideoScan software. Triterpenes according to (2): 3-12µl of the samples were loaded as 10 mm on band length in the 20 x 10 Silica gel 60F254 TLC. The mobile phase (C) consisted in dichloromethane:methanol
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(9:1). The plate was developed in the mobile phase up to 70mm. The plate was dried and derivatized in vanillin–sulphuric acid reagent. The fingerprint was evaluated in visible light, comparative with literature (2). Total phenol content- Total phenol content was determined according to Folin – Ciocalteu method (3). Briefly, 1ml of the extract was transferred to a 25ml volumetric flask, 10ml of water and 1ml of Folin Ciocalteu reagent was added. The volume was made to 25ml with 5% sodium carbonate (w/v). The blend was left at room temperature for 30 minutes. Then the absorbance of the samples was read at 760nm with a UV/VIS spectrophotometer (Helios λ, Thermo Electron Corporation). Distilled water was used as blank. Total phenol content was determined from the extrapolation of the calibration curve (y=0.0525x-0.020, R2 = 0.992), which was obtained for gallic acid (Sigma Chemical Co., St. Louis, USA) The results were expressed as milligrammes of gallic acid equivalents (GAE) per gramme of dried material.
Results and Disscutions
System: B A C HPTLC chromatograms of Ganoderma lucidum and Flammulina velutipes
Track 1 - caffeic acid, Track 2 – ferulic acid, Track 3 – Ganoderma lucidum extract, Track 4 – Flammulina velutipes extract, Track 1a – Ganoderma lucidum fruiting body (culture). Track 2a - Ganoderma lucidum fruiting body (culture), Track 3a Ganoderma lucidum fruiting body (wild) (2,4) In the extracts, ferulic acid (system B - Rf=0.78, system A – Rf= 0.97) was the main polyphenolic compound. Giving the fact that both extracts contain a wide range of triterpenes, is complicated to choose one as a marker compound. The chromatograms of reishi mushroom differ among the various samples. In the fruiting body sample (track 3), prominent red bands are seen at Rf = 0.23 and Rf= 0.4. Flammulina velutipes chromatogram show a prominent red band at Rf=0.5. According to American Herbal Pharmacopoeia, “the chromatograms of reishi mushroom differ among the various samples". Our results are showing for the first time, the polyphenols and triterpenes chromatographic fingerprints, for the two mushroom species, collected from Romania. The total phenolic content (TPC) was 0.455mg/ml GAE for Fammulina velutipes extract and 0.472mg/ml GAE for Ganoderma lucidum extract. Mushrooms are used both for nutritional and therapeutic properties all over the world. Knowing the chemical composition of our natural resources can be a first step in superior valorification of this wild species.
Bibliography 1. Wagner H., Bladt S., (1996) - Plant Drug Analysis, Second Edition, Springer. 2. American Herbal Pharmacopoeia® • Reishi Mushroom • 2006; 3. European Pharmacopoeia 6,0 4. Hildebert Wagner et al., 2011.HPTLC Identification of Reishi Mushrooms (Ganoderma lucidum) – CAMAG.
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Poster Presentation – PP12
Antioxidant activity of extracts of Armillaria mellea
Corina Bubueanu1*, Alice Grigore1, Ecaterina Serban1, Colceru – Mihul Svetlana1
1National Institute for Chemical-Pharmaceutical R&D (ICCF-Bucharest), Vitan Road 112 Sector 3, Bucharest, ROMANIA, *Corresponding author, e-mail: [email protected]
Abstract. All over the world edible mushrooms are considered a delicacy, being a good substitute for meet, because of the protein content. Mushrooms have, also, an important composition in chemical compounds with biologic activity, such as polysaccharides, polyphenols, triterpenes, microelements, vitamins, etc. In this paper are analyzed the polyphenolic composition and antioxidant activities of extracts obtained from Armillaria mellea mushroom. Key words: phenolic compounds, antioxidant, Armillaria mellea Introduction. Armillaria mellea - honey fungus - is a mushroom that belongs to the Armillaria genus (Basidiomycetes). Armillaria mellea is a parasitic fungus that live on trees and woody shrubs. It has been used for food and traditionally medicine. In different studies, the following compounds were identified: carbohydrates, peptides, sphingolipids, sterols, sesquiterpenoids, phenolics. This mushroom has been shown to have immunomodulatory, anti-inflammatory and antioxidant properties (1,2,3). The aim of this study is to evaluate the total polyphenolic composition (Folin-Ciocalteu method) and the antioxidant activity (Dpph and total antioxidant activity assays) of some extracts obtained from the Armillaria mellea mushroom. Material and methods. Raw material – Armillaria mellea (fruiting body - wild) sample was harvested from Dambovita region, Romania. The identification was done by the botanist’s team of National Institute of Chemical-Pharmaceutical R&D (ICCF), Bucharest, Romania. A voucher specimen is deposited in INCDCF-ICCF Plant Material Storing Room. Sample preparation: Armillaria mellea extracts were prepared by extraction with 50% (v/v) ethanol, acetone and ethyl acetate, 1/20 raw material/solvent ratio, at boiling temperature, for 1 hours. The solutions were filtered and kept frozen until analysis. Total phenol content- Total phenol content was determined according to Folin – Ciocalteu method (4). Briefly, 1ml of the extract was transferred to a 25ml volumetric flask, 10ml of water and 1ml of Folin Ciocalteu reagent was added. The volume was made to 25ml with 5% sodium carbonate (w/v). The blend was left at room temperature for 30 minutes. Then the absorbance of the samples was read at 760nm with a UV/VIS spectrophotometer (Helios λ, Thermo Electron Corporation). Distilled water was used as blank. Total phenol content was determined from the extrapolation of the calibration curve (y=0.01322x+0.0272, R2=0.995), which was obtained for gallic acid (Sigma Chemical Co., St. Louis, USA) The results were expressed as milligrammes of gallic acid equivalents (GAE) per 100 gramme of dried material. Free radical scavenging assay- was evaluated using the Sanchez-Moreno et al. (1998) assay (5). The extracts concentration were 1%, 0.1% in methanol. 50μl aliquots of the extract were mixed with 2950μl of the DPPH methanolic solution (0.0025g/l). The radical scavenging activity of the extracts against 2,2-diphenyl-1-picryl hydrazyl radical (Sigma-Aldrich) was determined by measuring UV absorbance at 517nm. A blank solution was prepared containing the same amount of methanol and DPPH, and measured after standing at room temperature 30 minutes. The radical scavenging activity (RSA) was calculated using the following formula: % inhibition = {(AB – AA)/AB} x 100. Where AB is the absorption of blank sample and AA is the absorption of tested extract solution.
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Total antioxidant capacity assay. Was assessed by phosphomolybdenum method, according to Prieto et al. (6). To 0.3 ml ethanolic solution of the sample (1%, 0.1% ) was added 2.7 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium molybdate, and 4 mM ammonium phosphate). The mixtures were incubated at 950C for 90 minutes. After cooling the samples to room temperature, their extinction was measured at 695 nm at UV-VIS spectrophotometer. Ethanol was used as negative control. The antioxidant capacity was expressed as ascorbic acid equivalent to 1 mg of active substance. The calibration curve is linear for ascorbic acid in the range of 0.001 to 1 mg / ml, n = 6, r2 = 0.999. Results and discussion. Table 1 shows the total phenol content of the extracts expressed as gallic (GAE) acid equivalents per 100g of raw material.
Table 1. Total phenol content of mushroom extracts No Extract mg (GAE)/100g 1 Ethanolic 50% 0.640 2 Acetone 0.305 3 Ethyl acetate 0.031
The mushroom extracts show antioxidant activity in a dose-dependent manner in both assays (table 2)
Table 2. Antioxidant activity of mushroom extracts AA (Dpph) mg (AAE)/g No Extract 0.1%(conc 0.1%(conc 1% (conc) 1% (conc) ) ) 1 Ethanolic 50% 76.32 27.52 0.25 0.79 2 Acetone 18.9 - 0.28 0.70 3 Ethyl acetate 1.53 - 0.42 0.90
The obtained results show that all extracts proved to have antioxidant properties, namely radical scavenging activity and total antioxidant activity. The solvent used influences directly the content and bioactivity of the extracts. Conclusions. Because mushrooms are considered functional food, having both nutritional and therapeutic properties, is important to take into consideration the wild species. Antioxidants are natural substances that may prevent or delay some types of cell damage.
Bibliography 1. Daniela Elena Zavastin, Cornelia Mircea, Ana Clara Aprotosoaie, Simona Gherman, Monica Hancianu, Anca Miron ARMILLARIA MELLEA: PHENOLIC CONTENT, IN VITRO ANTIOXIDANT AND ANTIHYPERGLYCEMIC EFFECTS Rev. Med. Chir. Soc. Med. Nat., Iaşi –2015–vol. 119, no. 1 2. Wu SJ, Tsai J -Y, Lai M-N, Ng L-T. Armillaria mellea Shows Anti-inflammatory Activity by Inhibit-ing the Expression of NO, iNOS, COX-2 and cytokines in TNP-1 Cells. Am J Chin Med 2007; 35 (3): 507-516 3. LW Gao, WY Li, YL Zhao, JW Wang, The cultivation, bioactive components and pharmacological effects of Armillaria mellea African Journal of Biotechnology VOl.8 (25), pp 7383-7390, 2009 4. European Pharmacopoeia 6,0 5. Sanchez-Moreno C., Larrauri J.A., Saura-Calixto, F., 1998 A procedure to measure the antiradical efficiency of polyphenols. Journal of Agricultural and Food Chemistry 76, 270-276. 6. PRIETO P. , PINEDA M. , AQUILAR M. 25. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. //Analytical. phosphomolybdenum complex: specific application to the determination of vitamin E. //Analytical Biochemistry, 1999, p. 337-341
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Poster Presentation – PP13
Rose-scented Geraniums cultivated in Romania – essential oil profile
Cristina Elena Iancu1, Oana Cioanca1, Cornelia Mircea1*, Adrian Spac1, Silvia Robu2, Monica Hancianu1
1Faculty of Pharmacy, University of Medicine and Pharmacy “Gr. T. Popa”, 16 University Str., Iasi 700117, Romania 2Faculty of Pharmacy, Dunarea de Jos” University, Galati, Romania *Corresponding author, e-mail: [email protected]
Abstract. This study is part of a broader research that will conclude with a PhD thesis and investigates for the first time the Pelargonium species cultivated in Romania. The current paper presents the some morphologic and chemical characteristics that are connected to the essential oil produced by these species. Key words: essential oil, glandular trichomes, geranium, Pelargonium. Introduction. High-value perennial aromatic shrubs, Pelargonium species are known today as rose- scented geraniums originating from South Africa, Egypt and Morocco. The species cultivated in this areas are used to obtain the geranium oil commercially used for perfumes and soaps. The chemical composition of geranium oil is very complex and varies due to intrinsic and extrinsic factors, especially the origin. Today, the most used commercial geranium oils include Bourbon, Chinese, Algerian, Egyptian, and Moroccan types. On the other hand, Pelargonium species are highly adaptive and can be cultivated in varied climates. Therefore they are easily spread in tropical, subtropical, temperate, and Mediterranean areas. The literature contains different data regarding the chemical profile of geranium oil, but there is no reference to the cultivars grown in Romania. Material and methods. Pelargonium hispidum, P. grandiflorum and P. radens specimens were obtained from the Botanical Garden "Anastasie Fatu" from Iasi. Initially the macroscopic and microscopic features of the leaves were observed. For macroscopy, fully grown, healthy leaves were selected from each specimen and then they were analyzed with the naked eye and with the magnifying glass under natural light. The hystoanatomy of the leaf parts were observed under a photonic microscop (NOVEX, Holland) after sections were made through leaf blade surface and cross sections of leaf stalk and blade. The essential oils were obtained by steam-distillation of fresh aerial parts of the investigated Pelargonium species. The chemical characterization of essential oils was performed using capillary gas chromatography coupled with mass spectrometers (GC/MS) and flame ionization detector (GC/FID). The GC-MS analysis of the oil was carried out on an Agilent type 7890A gas chromatograph,equipped with an Agilent 5975C mass spectrometer selective detector with electron impact ionization The gas chromatography with flame ionization detector (GC-FID) analysis was performed using an Agilent 6890 gas chromatograph equipped with a flame ionization detector. HP- 5MS capillary column (30 m x 0.25 mm internal diameter, 0.25 µm film thickness) was used. The volume of 0.2 µL of rssential oil was injected in the split mode (split ratio 1:50). Helium was used as carrier gas at a flow rate of 1 mL/min. The analysis was performed using the following temperature program: 4°C/min from 60°C to 250°C, 10°C/min from 250°C to 300°C; the final temperature was held for 7.5 min. The identification of the volatile compounds imply a correlation between their retention times (RT), mass spectra and Kovats indices with those obtained from authentic samples and/or NIST/NBS, Wiley libraries and literature.
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Results and discussion. The macroscopic features showed notable interspecific variations, maintained for the organoleprtic characteristics (different color, concistency, flavor and smell) of the leaves. The hysto-anatomical analysis gave important information about the structure of the leaf for each investigated species. P. hispidum leaf surface presented many surface and glandular trichomes. The secretory hairs had either short or medium lenght pedicle under a very large, spherical gland. In contrast, P. grandiflorum leaf had multiple long, unicellular surface hairs and short (3-4 cells pedicle) glandular thrichomes with a pyriform gland. P. radens had leaves covered with short, long and thick surface hairs, along with multiple short secretory hairs with a spherical gland, similar to a disk from the top view. In Romanian scientific literature data referring to Pelargonium species grown in this area are dated before 1980’s. Therefore our results have a great taxonomic value, although the notable differences between the samples were mainly quantitative. Thsese aspects are an indicator to the presence of essential oil for the investigated samples. The GC chromatograms (figure 1) showed the presence of some marker constituents such as isomenthone, menthone, cis-rose oxide, α-pinene, myrcene, and β-phellandrene.
Fig.1. Chromatograms for geranium oils: P. hispidum, P. grandiflorum, P. radens (from left to right)
Specifically, linalool, cis-rose oxide and geranyl derivatives were found in P. grandiflorum oil, whereas P. hispidum aetheroleum contained pinene, thymol and eucalyptol in small amounts, and menthone in higher quantity (15.70%). Both essential oils have a greater number of compounds (over 150) than the identified components found in P. radens sample (17). Nevertheless, the major component of P. radens oil (about 85 %) is a monoterpene: isomenthone. P. radens oil sample id represented by small amounts of pinene, terpinene, limonene, myrcene, cymene and piperitone. Our results are partially similar to other researchers data, but we also noted much higher wuantities of menthone/isomenthone in our samples. As compared to the commercially used geranium oils, only the essential oil extracted from Pelargonium grandiflorum comes close to the requirements. The other two are extremely different. Conclusions. The analysis of three Pelargonium essential oils obtained from specimens grown/cultivated in Romania revealed important differences between the number and the type of volatile compounds. Moreover, such variations were to be expected since the flavor and smell of the leaves of these species is different. The low extraction yield of the essential oil for these species (0.1- 0.22 %) did not allow further chemical or biological studies, but in the near future we intend to extend our research much further.
Bibliography 1. Lancu CE, Cioanca O, Mircea C, Hăncianu M. (2013), Contributions regarding the leaf histo-anatomy of some Pelargonium species. Rev Med Chir Soc Med Nat Iasi, 117(3):812-8. 2. Lis-Balchin, M., (2002), Essential oils from different Pelargonium species and cultivars: their chemical composition (using GC, GC/MS) and appearance of trichomes (under EM). In: Lis-Balchin, M. (Ed.), Geranium and Pelargonium. Taylor and Francis, London. 3. Singh P., Srivastava B., Kumar A., Kumar R., Dubey N.K., Gupta R. (2008), Assessment of Pelargonium graveolens oil as plant- based antimicrobial and aflatoxin suppressor in food preservation. J. Sci. Food Agric, 88:2421–2425. 4. Slima A.B., Ali M.B., Barkallah M., Traore A.I., Boudawara T., Allouche N., Gdoura R. (2013), Antioxidant properties of Pelargonium graveolens L’Her essential oil on the reproductive damage induced by deltamethrin in mice as compared to alpha- tocopherol. Lipids Health Dis, 12:30.
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Poster Presentation – PP14
Assessment of toxicity and inflammatory activities of mud extracts
Elena Codrici1, Cristiana Tanase1, Ionela Daniela Popescu1, Simona Mihai1, Ana-Maria Enciu1/2, Nicu Stoica3, Radu Albulescu1/4
1 Victor Babes National Institute of Pathology, Biochemistry-Proteomics Department, No. 99-101 Splaiul Independentei, 050096 Sector 5, Bucharest, Romania 2 Carol Davila University of Medicine and Pharmacy, Cellular and Molecular Medicine Department, No. 8 Bd Eroilor Sanitari, 050474 Sector 5, Bucharest, Romania 3SC Pellamar Cosmetics SRL, Str. Dr. Stefan Ionescu Calinesti, Nr. 14, Balta Alba, Buzau, Romania 4National Institute for Chemical Pharmaceutical R&D, 112 Calea Vitan, 031299 Sector 3, Bucharest, Romania
Abstract. Mud extracts represent valuable therapeutic adjuvants and therapeutic alternatives to synthetic drugs. Cytotoxicity tests on 13 mud extracts indicated the relatively low-cytotoxic effects of mud extract. Furthermore, we demonstrated that these extracts modulate cytokine release, generating profiles that are characteristic to anti-inflammatory effects/activity. Key words: mud extracts, cytotoxicity tests, cytokines, inflammation. Introduction. Mud extracts represent valuable therapeutic adjuvants and therapeutic alternatives to synthetic drugs; especially in chronic diseases, such as arthritis and knee osteoarthritis (1). The use of mud extract contributes to a long term stability of therapeutic effects, thus avoiding common inconveniences of conventional drugs, like installation of therapeutic resistance and adverse effects (2). Active fractions obtained from mud were investigated using in vitro methods regarding cytotoxicity and therapeutic efficacy. The real effects of mud bath applications on the inflammatory processes are still not clarified. The purpose of the investigations is to analyze the use of such extract in more addressed applications, like injection, besides the classical use of mud and its extract in topical applications. Material and methods. Cytotoxicity testing was performed in vitro using ATCC-CRL-9855 cell cultures, in standard conditions, at different cells concentrations (5000 and 10000 cells), at different times of exposure (48h/72h) and at concentrations of 75 mM, 15 mM, 6 mM and 3 mM using the MTS (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega). The 0.15 M mud solutions were filtrated using sterile syringe filter with pore size 0.2 µm, 25 mm diameter (GE Healthcare, Whatman). Anti-inflammatory effects: based on our preliminary data, the anti-inflammatory action was present in the mud fractions. Cytokine measurements were performed using Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel kit (IL-10, IL-1beta, IL-6, IL-8, RNTS, TNF-alpha, G- CSF, GM-CSF, IL-12P70, IL-1a, IL-4, MIP-1a) and the 96-well plate was analyzed using Luminex 200 system (Luminex Corp., TX, USA). Results and discussion. Cytotoxicity tests: In the first part of our study, we focused to establish if these 13 mud extracts have cytotoxic effects (MTS assay) and to what extent. The extracts were provided by Pell-Amar Cosmetics as spray-dried powders. For this purpose, we used different concentrations - ranging 3 to 75 mM, considering an “average” MW of 90 for extracts, at different cell densities (5000/10000 cells/well) and incubation times (48/72h). Preliminary results showed - for 10000 cells incubated for 72 hours – IC50 were 247 mM for sample 1, 386 mM for sample 3, 410 mM for sample 5 and 373 mM for sample 7. For 5000 cells at 72 hours – IC50 were 440 mM for both samples 3 and 5. IC50 could not be calculated for 48 hrs exposure, although a dose-effect relation could be observed.
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Our results indicated the relatively low-cytotoxic effects of the mud extract analyzed.
1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 Optical density Optical (mean)density 0,1 0 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 CTRL
Figure 1. MTS assay; exposure 48 hrs; Figure 2. MTS assay; exposure 48 hrs; (5000 cell/well) (10000 cell/well).
0,6
0,5
0,4
0,3
0,2
0,1 Optical density Optical density (mean) 0 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 CTRL Figure 3. MTS assay; exposure 72 hrs; Figure 4. MTS assay; exposure 72 hrs; (5000 cell/well). (10000 cell/well).
Anti-inflammatory effects: The mud extracts were demonstrated to modulate cytokine release, generating profiles that are characteristic to anti-inflammatory activities (incresed level of IL-10) and decrease of pro-inflammatory cytokines release (IL-1beta, IL-4, IL-6 and IL-8) with statistical significance.
Conclusions. Using a combination of in vitro assays, mud extracts could be classified and ranked for their cytotoxiciy and specific activity, providing an effective screening system for the discovery of potential therapeutic compounds.
Acknowledgment: Grants PNII 265/2014 and PN 16.22.04.01/2016.
Bibliography: 1. Ceccarelli F, Perricone C, Alessandri C, Modesti M, Iagnocco A, Croia C, et al. Exploratory data analysis on the effects of non pharmacological treatment for knee osteoarthritis. Clinical and experimental rheumatology. 2010;28(2):250-3. 2. Sarsan A, Akkaya N, Ozgen M, Yildiz N, Atalay NS, Ardic F. Comparing the efficacy of mature mud pack and hot pack treatments for knee osteoarthritis. Journal of back and musculoskeletal rehabilitation. 2012;25(3):193-9.
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Poster Presentation – PP15
Generating Diversity in Natural Product Scaffolds. Efficient C-17 Alkylations of ent-Kaur- 16-enic Derivatives
Elena Pruteanu1,2, Vladilena Gîrbu1,2, Nicon Ungur1,2, Veaceslav Kulciţki1,2, Philippe Renaud3
1Institute of Chemistry of the Academy of Sciences of Moldova, Chişinău, Republic of Moldova 2University of the Academy of Sciences of Moldova, Chişinău, Republic of Moldova 3Department of Chemistry and Biochemistry, University of Bern, Switzerland
Abstract. The current work presents the first results on the application of the radical addition methodology for the simultaneous attachment of a C-2 synthon and a functional group to the ent- kaurenoic acid methyl ester and its C-15 hydroxylated derivative at the C-17 carbon atom. Key words: Carboazidation, radical chemistry, diterpene, kaurane. Introduction. Many representatives of tetracyclic ent-kauranic diterpenoids, which occur broadly in the plant kingdom, display a diverse biological activity. Investigations of active principles of medicinal plants, especially those used in non-traditional Chinese medicine, have shown that a large spectrum of biological activities, including anti-microbial, anti-inflammatory, the cardio-vascular, diuretic, cytotoxic and ant-AIDS are conditioned by the presence in these plants of ent-kauranic diterpenoids [1]. The diversity of this family of compounds is impressive and it stems on a whole plethora of functional groups attached to the ent-kauranic backbone. Surprisingly, very recent studies have revealed a group of totally unprecedented hybrids of ent-kauranic skeleton with highly oxygenated sesquiterpenes [2]. This example has prompted us to initiate an investigation towards the synthesis of ent-kauranic alkylated conjugates through a linkage, involving the C-17 carbon atom. Material and methods. Ent-Kaur-16-en-19-oic 1 and 15α-angeloyl-ent-kaur-16-en-19-oic 2 acids were isolated from the wastes of sunflower (Helianthus Annuus) as described previously [3, 4]. Methyl-ent-kaurenoate 3 and 15α-angeloyl-methyl-ent-kaur-16-en-19-oate 4 were obtained on methylation of 1 and 2 with an etherial solution of diazomethane. Synthesis of 15α-hydroxi-methyl- ent-kaur-16-en-19-oate 5 was performed on refluxing 4 with a ethanolic solution of sodium hydroxide. Radical carboiodination or carboazidation of 3 and 5 was performed according to the described procedures [5, 6]. Shortly, the substrate was treated under reflux with ethyl iodoacetate (excess) in the presence of a radical initiator (Bu6Sn2 or dilauroyl peroxid, DLP) and an azide source (phenylsulfonylazide). Catalytic amounts of a second initiator (di-tert-butyl hyponitrite, DTBHN) was added periodically to the reaction mixture in the case of carboazidation. The reaction course was monitored by TLC. Usual aqueous workup and flash chromatography provided pure reaction products. Their structural characterization was performed based on spectral data. Results and discussion. Formation of new C-C bonds remains one of the major challenges in organic synthesis. The apparently simple synthetic problem has a separate significance in the natural product chemistry. Usually, compounds isolated from natural sources represent relevant structural complexity and sometimes, high chemical reactivity. In consequence, elaboration of selective methods for new C- C bond formation requires considerable efforts involving long sequence of transformations and protective group manipulations. Therefore, elaboration of new mild and selective alkylation methods represents a permanent scientific priority. Among the vast arsenal for selective alkylations, methods based on free radical chemistry are emerging now as powerful tools for mild and selective functional group transformations. Visible light - catalyzed red-ox processes and remote functionalizations of inactivated C-H bonds are among the „hottest” area of research in this context. But in the same time, free radical processes can be also efficiently used for generation of new C-C bonds, introducing new functionalities in the molecules of
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interest under mild reaction conditions that do not affect other functional groups. Such methods are very relevant tools for natural product modifications within diverse SAR studies. Ent-Kaurenoic acid 1 can be readily isolated from the wastes of sunflower (Heliathus Annuus L.), along with other functionalized derivatives, like C-15 hydroxylated compound 2. Following chemical modifications of such ent-kauranes can bring about new compounds with unknown bioactivities. Using radical chemistry processes for such purposes represents an approach which is underexplored for this class of diterpenoids. Therefore, we embarked on a research project aimed to the modification of readily available ent-kauranes with free radical processes. We report here our preliminary results on carboiodination and carboazidation of ent-kaurenic esters 3 and 5 (scheme below). N3 H 11 17 1 b 9 a 15 O (86%) 3 (50 %) O O 1 R=H O CO2Me 8 CO2R 3 R=Me H CO2Me 6 11 17 1 a 9 15 (57%) 3 O OR2 OH O CO2R1 O CO2Me 7 2 R1=H; R2=Ang Ang: 4 R1=Me; R2=Ang 5 R1=Me; R2=H Reagents and conditions: (a) ICH2CO2Et, DLP, Ph-H, 24 hrs. reflux; (b) ICH2CO2Et, Bu6Sn2, PhSO2N3, DTBHN, Ph-H, 2 hrs. reflux. Carboiodination of both substrates 3 and 5 was relatively sluggish and expected tertiary iodides were not isolated. The basic alkylation products from both substrates were compounds 6 and 7, which represent products of dehydroiodination of the initially formed iodides. On the contrary, carboazidation of 3 proceeded with an excellent yield over a much shorter period of time. The obtained products 6 - 8 will be used for following structural modifications. Conclusions. The present work demonstrates utility of the free radical transformations for efficient structural modification of ent-kauranic derivatives. Very convenient alkylation processes based on radical carboiodination and carboazidation can be used for spanning the structural diversity of this class of compounds. This functionalization method allows a one step, high yielding generation of a new C-C bond with simultaneous introduction of an additional functional group. Both can be used for following transformations within diverse SAR investigations.
Acknowledgements The presented work was performed within the project “Radical mediated modifications of natural products” supported financially by the Swiss National Science Foundation (SCOPES program, project No. IZ73Z0_152346/1).
Bibliography 1. Ghisalberti, E. L. Fitoterapia 1997, 63, 303. 2. Torres, A.; Molinillo, J.M.G.; Varela, R.M; Casas, L.; Mantell, C.; Martínez de la Ossa, E.J.; Macías, F.A. Org. Lett., 2015, 17 (19), 4730–4733. 3. Ungur, N.; Grinco, M.; Kulciţki, V.; Barba, A.; Bîzîcci, T.; Vlad, P.F. Chem. J. Mold. 2008, 3(2), 105-108. 4. Grinco, M.; Chetraru, O.; Kulciţki, V.; Barba, A.; Boico, A.; Vlad, P.F.; Ungur, N. Chem. J. Mold. 2010, 5(1), 106- 108. 5. Ollivier, C.; Bark, T.; Renaud, P. Synthesis 2000, 11, 1598–1602. 6. Panchaud, P.; Ollivier, C.; Renaud, P.; Zigmantas, S. J. Org. Chem. 2004, 69, 2755-2759.
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Poster Presentation – PP16
Consumer survey on medicinal tea consumption habits, practices and attitudes in Eastern Romania
Elisabeta Oprea1, Oana Cioanca1*, Cristina-Corina Bentea2, Ana Clara Aprotosoaie1, Ursula Stanescu1, Monica Hancianu 1
1Faculty of Pharmacy, University of Medicine and Pharmacy “Grigore T. Popa”, Iasi, Romania 2“Dunarea de Jos” University, Galati, Romania *Corresponding author, e-mail: [email protected]
Abstract. Our investigations refer to tea consumption habits, practices and attitudes for medicinal plants commonly used in the domestic environment in Romania. Therefore, we conducted a survey within pharmaceutical network on a representative sample of patients. Tilia cordata, Matricaria chamomilla, Achilea milefollium, Hypericum perforatum and Calendula officinalis are amongst the preferred species for daily consumption. Key words: consumption practices, chamomile, marigold, lime flowers, St. John’s wort Introduction. Medicinal and aromatic plants are used worldwide both for curative and preventive purposes. Nevertheless, in the last two decades there is a tendency to expand these boundaries towards socializing - meeting for a cup of tea to interact with others. This is a healthier and a “bio” choice that is inspired by the higher development and education of the present society. Thus, the demand for quality medicinal and aromatic plants has increased significantly. Along the way, the guidelines in regards to these plants/products were modified accordingly by inclusion of requirements for proper botanical identification, microbial contamination and even standardization (where applicable). On the other hand, quality is defined as the status of the plant product identification, purity or manufacturing processes. Safety of herbal preparations consumption depends mainly on the quality of raw plant material, which in turn is determined by multiple internal and external factors. Therefore it is absolutely necessary to ensure and maintain the quality of the plant material that is either frequently used as medicinal tea or it is included in food preparations. Material and methods. We used a survey with 12 questions in order to investigate the food consumption practices of medicinal teas in Romania. Initially, we pre-tested all the questions by interviewing a sample of 20 people. All the interviews took place in a confined space that allowed the privacy in discussion with each participant. After validation, the questionnaires were spread in 10 pharmacies from Bacau, Vaslui and Galati and the results were collected over two month’s period. Moreover, on the same participants (400 respondents from the mentioned counties) we assessed the impact of medicinal tea use to maintain health. Each respondent has agreed to anonymous data processing. The distribution of the participants in the survey was analyzed in terms of respondents' gender, age, and residence, instructional and educational level. Noteworthy is that to avoid confusion the questions included the vernacular name of the medicinal plants. The parameter used to analyze internal consistency was Cronbach Alpha index, whose value (0.703) was in the optimal range (0.7- 0.9), thus highlighting the true extent and the validation of the questionnaire. The hypothesis verification was investigated with Chi-Square, Crosstabs and Pearson Chi-Square coefficient. Statistics were calculated with SPSS-version 16.0i. Results and discussion. The results of the survey indicated that our respondents were mainly women (63.20%) as compared to man (36.80%). The largest group age (63.2%) comprised people between 25 and 60 years old, followed by youngsters below 25 (28.5%) and elderly over 60 (8.3%). The residence of our respondents was both situated in rural (32.2%) and urban areas (67.8%), whereas in terms of educational level we tried to select a balanced distribution of the sample (secondary school - 29.2%,
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high school – 36.2%, university – 34.6%). In regards to the intake, higher frequency of over 50% was noticed for lime tea (Tilia cordata), marigold (Calendula officinalis), chamomile (Matricaria chamomilla), yarrow (Achilea milefollium) and St. John’s wort (Hypericum perforatum). Besides these, basil (Ocimum basilicum) is also preferred especially as a flavoring agent for food, but also as a tea. The survey revealed that, in terms of reasons for herbal tea intake, the number of respondents is similar: therapy (53.2%) and enjoyment (the taste and flavor likeness) – 42%. Medicinal teas are used both for therapeutic purposes, but also for enjoyment, and to a lesser extent in need of socialization (4.8%). Moreover, the favorite source to purchase medicinal teas is represented by specialist shops and pharmacies (43.2%), followed by harvesting from the wild flora by the consumer (23.5%), grocery stores, markets and cultivating its own. The quantity and frequency of tea intake is shown in figure 1. From a quantitative perspective, both respondents who drink tea every day, and those who drink tea occasionally, mainly drink a cup of tea.
Fig.1.Herbal tea intake: quantity and frequency
In terms of frequency of medicinal tea use, the obtained results have not highlighted major differences between the three parameters selected (daily, 2-3 times a week, and occasionally). Also, the statistic results for the frequency of consumption of herbal teas by age (χ2 (4) =10.35, p<0.05) and according to health (χ2 (2) =25.179, p<0.001) indicated that there are differences in the age groups. Thus, the consumption habits and practices for respondents under 25 years include occasional consumption, while those over 60 years prefer a daily consumption of herbal tea. There are also statistically significant differences between the health status and the frequency of tea intake. The data obtained showed the preponderance of daily consumption for those with health problems, and occasional consumption for those with a general state of good health. The statistic correlation (χ2 (1) =18.647, p<0.001) showed that tea intake for therapeutic purpose is adopted mainly by respondents with health problems, but also by the healthy respondents. There are no similar surveys conducted in Romania, therefore all the results represent an original study that reveals the habits, practices and attitudes in regards to medicinal and aromatic plants intake in the form of tea. Conclusions. The sociological study revealed statistically significant relationships between tea consumption and the state of health of the respondents, highlighting the daily consumption of a cup of tea from species such as Tilia cordata, Matricaria chamomilla, Achilea milefollium, Hypericum perforatum and Calendula officinalis both for therapeutic purposes and pleasure. Moreover, consumer educational level and motivation of medicinal tea intake can be correlated, all categories of respondents preferring herbal remedies.
Bibliography 1. Oprea E (2015) Contributions to the quality assessment of pharmaceutical interest plant products. PhD thesis. Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy “Grigore T. Popa”, Iasi, Romania. 2. Oprea E, Tuchiluș C, Aprotosoaie AC, Cioancă O, Trifan A, Grădinariu V, Miron A, Hăncianu M. (2015) Assessment of the microbial load of some medicinal plants commonly used in Romania, Rev Med Chir Soc Med Nat Iasi, 119(1):267-272. 3. Cioancă O., Mircea C, Poiată A, Stănescu U, Hăncianu M. (2010), Microbial contamination of commercial samples of Matricariae flos from Romania, Planta Med., 76(12):1245.
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Poster Presentation – PP17
Total polyphenols, flavonoids and antioxidant activity of Romanian propolis samples
Florentina Gatea1, Eugenia Dumitra Teodor1, Gabriel Lucian Radu2, Elvira Gille3
1NIRDBS, Centre of Bioanalysis, 296 Spl. Independentei, 060031, Bucharest, Romania; 2University”Politehnica”, Faculty of Applied Chemistry and Materials Science, 1-7 Polizu Str., 011061, Bucharest, Romania; 3NIRDBS/„Stejarul” Biological Research Centre, Alexandrucel Bun 6, 610004, Piatra Neamt, Roamnia
Abstract. Forty-four propolis samples collected from entire Romaniawere assessed for total polyphenols and flavonoids content and the results were correlated with their free radical scavenger activities. Key words: Propolis, polyphenols, flavonoids, antioxidant activities
Introduction. The propolis is one of hive products with a complex structure used in complementary medicine because of its remarkable properties: anti-oxidant, anti-inflammatory, immunomodulatory, antimicrobial, antiviral etc. [1]. The great variability of propolis is given by the diversity of plant sources visited by bees when they harvested lipophilic material from the leaves and leaf buds. In this study, 44 samples of propolis collected from different regions of the country were compared in terms of total content of polyphenols, flavonoids and antioxidant activity.
Material and methods. Ethanolic extracts (EEPs) of propoliswere obtained by extraction of crude materials with 70 % ethanol (1:10 w/v), for 10 days, at 25oC, in dark. The total phenolic contents of EEPs were determined using the Folin-Ciocalteau reagent solution [2]. The flavonoid contents were determined by the method described by Woisky&Salatino [3]. The antioxidant capacities of propolis extracts were tested by DPPH, ABTS and FRAP methods [4, 5, 6]. All the methods were adapted as micromethods. For spectrometric assessmentsit was used a Biochrom Anthos Zenyth 340 Microplate reader (England).
Results and discussion. The amounts of total phenolics in the Romanian EEPs ranged between 179.97 ± 4.09 and 342.88 ± 15.08 mg GAE g-1 of EEP. The concentrations are higher, but are similar with those reported for Greece and Cyprus propolis, which are between 80.2 and 338.5 mg GAE g-1of EEP [7].The results are also comparable with those reported by Kumazawaet all for ethanolic extracts of propolis from different countries including those from the temperate zone, which ranging from 174 ± 6.4 to 299 ± 0.5 mg GAE g-1[2]. Similar results were reported for the propolis extracts belongs to different regions of China [8]. Total flavonoids found in the EEPs varied from 23.83±0.4to 143.58±0.60 mg EQ g-1 of EEP. Compared to others studies, our results regarding the total flavonoid contents are higher, but in accordance with those reported for Iranian propolis (12.2±0.33 mg EQ g-1 to 77.9±0.39 mg EQ g-1), or for Algerian propolis( 10±1 mg EQ g-1to 69±1 mg EQ g-1)[9,10]. In poplar propolis are reported 344 chemical compounds, therefore we can say that scavenger capacity of propolis is the summed action of many individual compounds [11]. All the samples investigated by DPPH method exhibited scavenger properties. There were obtained very good correlations between polyphenols concentrations and scavenger activity against DPPH radicals (r = 0.939). Similar high correlation was reported for Greece and Cyprus propolis [7]. The antioxidants may be defined as substances which when are present at low concentrations compared to those of the oxidizable substrate, significantly delays or inhibits oxidation of that substrate [12]. The low values of IC50 obtain for EEPs, ranged between 12.44±0.6 µg mL-1and 29.14±0.3 µg mL-1 with an average of 16.35±3.48 µg mL-1, which confirms
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their radical scavenger ability. These results are similar with those reported for propolis from Algarve (Portugal) andmuch lower thanthose from Croatia (from 29.0 ±2.2 to 36.5±3.8 µg mL-1) [13, 14]. A positive and good r coefficient was obtained for the correlations between the values of TEAC (Trolox Equivalent Antioxidant Activity) obtained for EEP samples and polyphenols concentrations (r=0.830). The TEAC values of EEPs varied between 2.711±0.008 and 4.696±0.029 mMTrolox g- 1EEP. Close values of TEAC were reported for propolis from Basque Country (Northeastern Spain) [15]. FRAP assay was used to measure the antioxidant capacity from a wide range of biological samples. In this assay procedure, comparing with DPPH and ABTS scavenger methods, no free radicals or oxidants are used. It seems that the antioxidant capacity of an antioxidant against a free radical does not necessarily match with its ability to reduce Fe3+to Fe2+ [16]. FRAP values for EEPs -1 -1 varied from 3.56±0.04 mM FeSO4g EEP to 12.74±0.09 mM FeSO4g EEP. A good correlation was obtained between polyphenols concentration of samples and FRAP activities results (r=0.724). We could noticed also a good correlation between the results obtained by DPPH, ABTS and FRAP, respectively 0.794 FRAP versus DPPH, 0.670 for FRAP and ABTS, and 0.768 for DPPH and ABTS.
Conclusions. The propolis samples analysed showed high antioxidant capacity correlated with high polyphenolics and flavonoids content. These results show that Romanian propolis is high quality and is suitable for its use in complementary medicine and the pharmaceutical industry.
Bibliography 1. Lotfy, M. (2006). Biological Activity of Bee Propolis in Health and Disease. Asian Pacific Journal of Cancer Prevention, 7, 22-31. 2. Kumazawa, S., Hamasaka, T., & Nakayama, T. (2004). Antioxidant activity of propolis of various geographic origins.Food Chemistry, 84, 329–339. 3. Woisky, R. G., &Salatino, A. (1998). Analysis of propolis: some parameters and procedures for chemical quality control. Journal of Apicultural Research, 37, 99–105. 4. Litescu, S. C., Oprea, E., Diaconu, M., &Radu, G. L. (2011). A rapid determination of radical scavenger properties of plant extracts using electrochemical approach. Revue Roumaine de Chimie, 56, 25-32. 5.Erel, O. (2004).A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation.Clinical Biochemistry, 37, 277-285. 6. Benzie, I. F. F., & Strain, J. J. (1999). Ferric reducing/antioxidant power assay: Direct measure of Total Antioxidant Activity of Biological Fluids and Modified Version for Simultaneous Measurements of Total Antioxidant Power and Ascorbic acid concentration. Methods in Enzymology, 299, 15-27. 7. Kalogeropoulos, N., Konteles, S. J., Troullidou, E., IoannisMourtzinos, I., &Karathanos, V. T. (2009). Chemical composition, antioxidant activity and antimicrobial properties of propolis extracts from Greece and Cyprus. Food Chemistry, 116, 452–461. 8. Ahn, M.-R., Kunimasa, K., Kumazawa, S., Nakayama, T., Kaji, K., Uto, Y., Hori, H., Nagasawa, H., &Ohta, T. (2009). Correlation between antiangiogenic activity and antioxidant activity of various components from propolis.Molecular Nutrition Food Research. 53, 643–651. 9. Mohammadzadeh, S., Sharriatpanahi, M., Hamedi, M., Amanzadeh, Y., Ebrahimi, S. E. S., &Ostad, S. N. (2007). Antioxidant power of Iranian propolis extract. Journal of Agricultural Food Chemistry, 103, 729-733. 10. Boufadi, Y.,M., Soubhye, J., Riazi, A., Rousseau, A., Vanhaeverbeek, M., Nève, J., Karim ZouaouiBoudjeltia, K. Z., & Van Antwerpen, P. (2014). Characterization and Antioxidant Properties of Six Algerian Propolis Extracts: Ethyl Acetate Extracts Inhibit Myeloperoxidase Activity. International Journal of Molecular Sciences, 15, 2327-2345. 11. De Groot, A. C., Popova, M. P., &Bankova, V. S. (2004).An update on the constituents of poplar-type propolis.Wapserveen, The Netherlands: acdegroot publishing, 11 pages. ISBN/EAN: 978-90-813233-0-7 12. Halliwell, B. (1997). Antioxidants: the basics – what they are and how to evaluate them. Advances in Pharmacology, 38, 3–20. 13. Miguel M. G., Nunes S., Anahi Dandlen S. S., Cavaco A. M., &Antunes M. D. (2011). Antioxidant Activity of Propolis from Algarve.Advances in Environmental Biology, 5, 345-350. 14. Jug, M., Koncic, M. Z., &Kosalec, I. (2014). Modulation of antioxidant, chelating and antimicrobial activity of poplar chemo-type propolis by extraction procures. LWT - Food Science and Technology, 57, 530-537. 15. Bonvehı, J. S., & Gutierrez, A. L. (2011). Antioxidant Activity and Total Phenolics of Propolisfrom the Basque Country (Northeastern Spain). Journal of American Oil Chemistry Society, 88, 1387–1395. 16. Cao, G., & Prior,R. L. (1998). Comparison of different analytical methods for assessing total antioxidant capacity of human serum.Clinical Chemistry, 44, 1309-1315.
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Poster Presentation – PP18
Evaluation of accelerated solvent extraction (ASE) for obtaining chlorogenic acid from Cynara scolymus L. (artichoke) leaves
Ibrahim Ahmed Saleh1, Mohamed-Elamir Fathy Hegazy1, Tarik Abdelhalim Mohamed1, Khaled Ahmed Shams1, Elsayed Aboutabl2, Nahla Sayed Abdel-Azim1 and Faiza Mohamed Hammouda1
1Phytochemistry Department, National Research Centre (NRC), 33 El Bohouth St. (former El Tahrir St.), 12622 Dokki, Giza, Egypt 2 Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr-el-Aini Street, 11562 Cairo, Egypt
Abstract. The objective of this study was to evaluate extractability of chlorogenic acid form Cynara scolymus L. leaves using Accelerated Solvent Extraction (ASE) under different extraction parameters. The highest chlorogenic acid yields (67.4 and 66.8 mg/5g DM) were obtained after extraction for 10 min at 120 °C using one and three static cycles of extraction, respectively. Keywords: Cynara scolymus, HPLC, chlorogenic acid, accelerated solvent extraction
Introduction. Finding new techniques to enhance the extraction efficiency of bioactive compounds from medicinal and aromatic plants (MAPs) is important due to their potential health and economic benefits. Recent studies on the development of MAPs extraction process have mainly focused to minimize solvent and time of extraction and to maximize the obtained yield of bioactive compounds. The use of innovative extraction techniques, ultrasonic-assisted extraction, microwave-assisted extraction and accelerated solvent extraction (ASE) is considered as a green extraction approach to have the lowest possible impact on the environment (lower energy and solvent consumption, etc.). These methods are widely replacing the traditional ones (soxhlet, maceration, heat reflux and hydro- distillation). The use of green methods for MAPs extraction on both small and large scale of production with lesser impact on the environment has been of great interest among many researchers to meet the challenges of the twenty-first century [1]. (ASE) is an extraction technique which combines both elevated pressure and temperature in order to increase the efficiency of the extraction process. Increased temperature accelerates the extraction kinetics and elevated pressure keeps the solvent in the liquid state, thus enabling safe and rapid extractions [2]. The objective of this study was to evaluate extractability of chlorogenic acid form Cynara scolymus L. leaves using solvents under high pressure and temperature.
Material and methods. Chlorogenic acid standard (purity 95.0%, CAYMAN Chemical) was purchased from VWR International (East Grinstead, West Susses, UK). Cynara scolymus L. leaves (Romanian strain) were collected from the farm of the National Research Centre–Giza–Egypt. Collected leaves were dried at 40 °C for 48 h in a tray dryer with air circulation; dried leaves were ground into fine powder and graded material with a particle size distribution 500–90 μm was used for extraction. The moisture content of this material was found to be 7.5% [3]. Extraction method: Extraction of the plant material was performed using a Dionex ASE 350 extraction system with dionium components smartrum and solvent saver system and applying one and three static cycles of extraction at 40, 80, and 120 °C. Static period of extraction was carried out for 5, 10 and 15 min using 80% methanol/water (v/v) as the extraction solvent. Analytical procedure: Chlorogenic acid quantification was carried out using High performance liquid chromatography (Shimadzu Prominence series). HPLC device comprised of a DGU-20A5 degasser, LC-20AD pump, SIL-20A injector, CTO-20AC oven, SPD-M20A detector, HiChrome C18
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250x4mm 5 µm column and a CBM-20Alite controller. The data was analysed using Shimadzu LCsolution version 1.23 software. Chromatograms were recorded at 330 nm. The analytical method was adapted from British Pharmacopeia [4]. Gradient elution was performed using (solvent A) 0.5% phosphoric acid in water and (solvent B) 0.5% phosphoric acid in acetonitrile at a flow rate of 1.2 mL/min. Chlorogenic acid quantification was carried out using standard calibration curve of chlorogenic acid as an external standard with the above mentioned HPLC method. All data presented herein are average values ± standard deviation of three independent experiments and expressed as mg/5g dried leaves of Cynara scolymus L. leaves.
Results and discussion. Applying one and three static cycles of extraction, increasing the extraction temperature form 40 °C to 120 °C significantly increased the extraction efficiency of chlorogenic acid from Cynara scolymus L. leaves. Moreover, increasing extraction time from 5 to 15 min at 40 °C, applying one and three static cycles, increased significantly the chlorogenic acid yields obtained. On the other hand, extraction at 80 and 120 °C using one and three static cycles of extraction, increasing the extraction time over 10 min did not affect the extraction of chlorogenic acid; however, there was insignificant decrease in the yields of chlorogenic acid obtained. The highest chlorogenic acid yields (67.4 and 66.8 mg/5g DM) were obtained after extraction for 10 min at 120 °C applying one and three static cycles of extraction, respectively, and (56.5 and 64.2 mg/5g DM) for 10 min at 80 °C applying one and three static cycles of extraction, respectively (Fig 1).
Fig 1: Effect of accelerated solvent extraction on chlorogenic acid yields from Cynara scolymus leaves
Conclusions ASE has proven to be an effective extraction technique. Using ASE the best results for the extraction of chlorogenic acid from Cynara scolymus L. leaves (artichoke) were obtained after 10 min of ASE extraction applying one and three static cycles of extraction at 120 °C. However its recommended not to exceed the extraction time over 10 min at elevated temperatures, due to the possible degradation of chlorogenic acid for prolonged extraction at extreme conditions.
Bibliography 1. Farid Chemat, Natacha Rombaut, Anne-Sylvie Fabiano-Tixier, Jean Pierson and Antoine Bily (2015), Green Extraction: From Concepts to Research, Education, and Economical Opportunities. In Green Extraction of Natural Products Theory and Practice. Farid Chemat and Jochen Strube (Eds.), First Edition, Wiley-VCH Verlag GmbH & Co. KGaA. Weinheim, Germany, 1-36 2. Arwa Mustafa and Charlotta Turner (2011), Pressurized liquid extraction as a green approach in food and herbal plants extraction: A review, Analytica Chimica Acta, 703: 8–18 3. Ibrahim Ahmed Saleh, Mircea Vinatoru, Timothy Mason, Nahla Abdel-Azim, Elsayed Aboutabl and Faiza Hammouda (2016), A possible general mechanism for ultrasound-assisted extraction (UAE) suggested from the results of UAE of chlorogenic acid from Cynara scolymus L. (artichoke) leaves, Ultrasonic Sonochemistry, 31: 330–336 4. British Pharmacopoeia (2009), Herbal drugs and herbal drug Preparations, The Stationery Office, London.
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Poster Presentation – PP19
The action of two kinds of lingonberry (Vaccinium vitis-idaea L.) extracts on liver function in Paracetamol - induced toxicosis
Ioana Roman1, Vlad Toma1,2,3, Ana Coste1, Adela Halmagyi1, Anca Farcaş1,2,3
1 NIRBDS/Institute of Biological Research, 48 Republicii Str, 400115, Cluj-Napoca, Romania 2Faculty of Biology and Geology, Babes - Bolyai University, 400028, Cluj-Napoca, Romania 3National Institute for Research and Development of Isotopic and Molecular Technologies, 400293, Cluj-Napoca, Romania *Corresponding author, e-mail: [email protected]
Abstract. In the present study we intend to highlight the lingonberry protective effects depending on the extract preparing mode (hydro or dry) on liver function by analysis of biochemical metabolic and stress parameters as well as histo-enzymological study of rat liver. Key words: lingonberry extracts, paractemol intoxication, liver, blood parameters. Introduction. Acetaminophen (paracetamol) is a widely used analgesic, antipyretic medication and is safe at therapeutic dose but accidentally or intentionally overdose determine kidney and liver malfunction [1]. It is activated and converted by the cytochrome P450 enzyme to the NAPQI metabolite which causes oxidative stress and glutathione (GSH) depletion. Vaccinium vitis-idaea fruits are rich in antioxidant activity. Suggested benefits of these fruits include the maintenance of vascular and eyesight health, preventing or reducing the severity of cardiovascular diseases, diabetes and cancer and antimicrobial action. Restoration of the liver affected by paracetamol intoxication, following the administration of hepatoprotective products such as vegetal extracts, in our case the Vaccinium vitis idaea L extracts do not benefit of many data. Material and method. Experiments were performed on white female Wistar rats*, weighing 150 ± 20 g, divided into 6 groups of 6 animals each: control group (C); Paracetamol intoxicated group, 75 mg/100 g bw (P); lingonberry dry extract treated group (Lp) (100 mg extract/100 g bw); lingonberry hidroalcoholic extract treated group (LEx) (200 mg extract/100 g bw); P + Lp treated group (PLp) and P + LEx treated group (PLEx). Plant extracts were administered by gavage, á jeun, for a period of 15 days. The lingonberry fruit extract was obtained from fresh fruit harvested at Mount Băişorii (Cluj county) at the end of August 2014 at the UMF ”Iuliu Hațieganu” Cluj-Napoca.. It was obtained 500 ml of a hydroalcoholic extract of 45° respectively an extract of 1: 1, which means that 1 ml of extract corresponds to 1 g of fresh fruit (content of benzoic acid was 40mg / 100g) and the dried extract was obtained from the fluid 1: 1 extract in an installation of fluid bed into the Aeromatic-Strea-1 (Gea- Switzerland) apparatus, by the adsorption of the fluid extract on a mixture of microcrystalline cellulose-lacose (2:1), so that 1 g of the dry extract corresponds to 3 g of 1: 1 fluid extract (or 1 g of fresh fruits), that we finally obtained a dry extract 3: 1 (content of benzoic acid eas 112mg / 100g). In the 16th day, animals were killed by decapitation after an anesthesia with ketamin-xylazine cocktail (60:7,5 mg). Blood was collected in order to obtain blood serum. For histological analyses, the kidneys were fixed in neutral formalin solution (10%) for 24 h. *Animals were obtained from the biobasis of „Iuliu Hatieganu” MPU, Cluj-Napoca and kept under standardized zoohigienical conditions according to the Law no. 43/2014 on the protection of animals used for scientific purposes and to the 2010/63/UE Directive with the approval of the Ethics Committee from the Institute of Biological Research, Cluj-Napoca, Romania Results and discussion. The results obtained are in table 1. In P group significantly increase serum cholesterol, alkaline phosphatase (ALP) and decreases in other groups except LEx group. Serum transaminases (GOT/GPT) increase in paracetamol intoxication. Oral administration of the both lingonberry extract seems to protect the possible hepatic tissue damage caused by paracetamol as it
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reduces the serum levels of GOT, GPT. An overdose of paracetamol causes depletion of glutathione and excessive metabolites reacts with the liver macromolecules and cause hepatic cell death leading to an elevated level of hepatic cellular enzyme ALP in serum [2]. In addition, in P group total cholesterol increases in serum, extracts administered under paracetamol intoxication normalizes its level and extracts administered simply lower cholesterol significantly (p<0.1). Elevated levels of total cholesterol may be due to cholesterolemia, a condition commonly occurs in hepatocellular diseases [3]. Tabel 1. Level of serum cholesterol, alkaline phosphatase (ALP) and transaminases (TGO/TGP) C P Lp LEx PLp PLEx Cholest (mg/dL) x±ES 90,86±4,02 112,86±3,67 72,33±3,69 79,17±3,08 95,5±5,89 94,17±14,43 D% - 24,21% -20,39% -12,86% 5,10% 3,64% ALP (U/L) x±ES 258,67±12,27 295,0±32,57 183,83±16,96 296,25±17,81 212,6±37,57 226,0±16,85 D% - 14,04 -28,93 14,52 -17,81 -12,62 GOT (U/L) x±ES 728,8±40,96 854,0±50,29 220,67±13,32 588,5±81,35 368,5±66,2 494,4±57,89 D% - 17,17 -69,72 -19,25 -49,43 -32,10 GPT (U/L) x±ES 97,50±4,29 124,67±5,9 47,50±3,43 96,75±8,11 72,5±13,09 107,0±9,56 D% - 27,86 -51,28 -0,76 -25,64 9,74 Note: mean ± S.E. (standard error), Student “t” test, significant differences from p<0.05) – marked values
Hepatoprotective effect of the polyherbal extract was further confirmed by the histopathological study of the liver sections, which supported the results obtained from the serum biochemical assays. Thus, in group P it can remark specific necroptotic hotspots associated with vacuolar degeneration. These morphopathological issues are uncommon in PLp and PLEx groups, suggesting the differential hepatoprotective effect of these extracts amid paracetamol intoxication. Histoenzymologic, administration of both lingonberry extracts (Lp and LEx) induces a slight increase in liver SDH activity compared to group C. Liver SDH activity sharply decrease in group P being enhanced by extracts administration. Conclusions. Both biochemical and the histo-enzymological results shows both the structure and the liver function damages by the Paracetamol sub-chronic treatment and their recovery following the administration of the two types of lingnberry extract, more intense to dried lingonberry extract.
Acknowledgements We acknowledge the support of a Core PN 16-19 BIODIVERS project from the Ministry of National Education and Scientific Research and the National Authority for Scientific Research and Innovation.
Bibliography 1. Anne M. Larson, Julie Polson, Robert J. Fontana, Timothy J. Davern, Ezmina Lalani, Linda S. Hynan, Joan S. Reisch, Frank V. Schiødt, George Ostapowicz, A. Obaid Shakil and William M. Lee (2005), Acetaminophen- induced acute liver failure: results of a United States multicenter, prospective study, Hepatology, 42: 1364-1372. 2. Dewasya Pratap Singh, Harshika Awasthi, Suaib Luqman, Saudan Singh and Dayanandan Mani (2015), Hepatoprotective effect of a polyherbal extract containing Andrographis paniculata, Tinospora cordifolia and Solanum nigrum against Paracetamol induced hepatotoxicity, Pharmacogn Mag., 11(Suppl 3): S375–S379. 3. Neil McIntyre (1978), Plasma lipids and lipoproteins in liver disease, Gut, 19:526-530.
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Poster Presentation – PP20
The effect of harvesting time on essential oils composition of Thymus pannonicus L.
Irina Boz1, Ioan Burzo2, Corneliu Tanase3
1Department of Experimental and Applied Biology, Institute of Biological Research, Iasi, Romania, 2Department of Horticulture, University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania, 3University of Medicine and Pharmacy of Tirgu Mureș, Faculty of Pharmacy, Tirgu Mures, Romania
Abstract. In this paper the authors investigating the possible effect of harvesting time on essential oils composition at Thymus pannonicus L., a species that grows wild in the Romanian flora. In this sense the material was collected in 2 different phenophases (vegetative and anthesis), during 2 consecutive years. The main chemical components of essential oils are Germacrene D, nerolidol, Farnesol and α-Terpinyl acetate. Key words: thyme, essential oils, GC-MS, phenophases Introduction. Thymus pannonicus is a perennial herbaceous plant, distributed in central and eastern Europe. It grows over open dry meadows, grasslands and rocks. In Romania these plant is spread all over the country, including two subspecies pannonicus and auctus (Oprea, 2005). The plant present robust stems, ascending or decumbent on basis, very branched, covered all around with hair. The leaves are elliptic or lanceolate, covered with hairs on both epidermises, with prominent ribs (Guşuleac, 1961). Thymus pannonicus L. is commonly used as herbal tea, flavoring agent and medicinal plant due to his biological active substances (such as thymol, carvacrol, geraniol, linalool and other compounds from the essential oil) (Stahl-Biskup and Saez, 2002). The harvesting date, time of day and weather conditions is very important for the quality and quantity of essential oils. The main objective of this paper is to highlight the possible effect of harvesting time on essential oils composition of Thymus pannonicus. For this purpose individuals of this species were collected in vegetative and full flowering phases. Material and methods. The vegetal material was represented by Thymus pannonicus, a species that grows wild in the Romanian flora. The species was collected in 2 different phenophases (vegetative and anthesis), during 2013 and 2014, from Fălticeni, Suceava County, Romania. The identification of taxa was made by Dr. Ioan Sârbu from the Botanical Garden “Anastasie Fătu”, Iasi. The collected material was registered and stored in „Alexandru Ioan Cuza” Universityʼs Herbarium from Iaşi. The chemical composition of the essential oil was established by GC-MS analysis with the help of a gas-chromatograph Agilent Technologies tip 6890N coupled to a mass detector (MSD) of the 5975 inert XL Mass Selective Detector type. The conditions for chromatography were: column HP 5MS, mobile phase Helium – discharge: 1 mL/min, injector temperature: 250°C, detector temperature: 250°C, temperature regime from initial 40°C (10 degrees/min.) to 280 degrees, injected volume: 0.1-0.3 µl, splitting ratio-1:100. The DB5 chromatographic column has a length of 30 m an interior diameter of 0.25 mm and a film diameter of 0.25 μm. The separated compounds were identified by means of the Nist spectrum database, and the peak position was confirmed by the Kovats retention index. Results and discussion. Following our analysis of essential oils, a total of 58 compounds were identified, representing between 92.26% and 97.58% of the total number of identified compounds. The highest number of chemicals (46 compounds) was identified in the volatile oil derived from individuals collected in 2013, in the vegetative stage. The lowest number of compounds (31) was identified in the volatile oil derived from plants collected in 2013 in the anthesis stage. The main chemical components are Germacrene D (between 8.05% and 17.31%), nerolidol (between 7.74% and 18.49%), Farnesol (between 12.95% and 14.77%) and α-Terpinyl acetate (between 6.56% and 9.58%). Major differences were registered at the taxa collected in 2013 in anthesis stage, where the main components were carvacrol (42.32%) and thymol (13.98%). In generally, a high chemical variability and diversity is observed in the essential oils of Thymus species: at least 20 different chemotypes in the genus have been established until now (Tepe et al., 2005). According to Karuza-Stojaković et al., the principal constituents of Thymus pannonicus essential oil from southern parts of Vojvodina province were terpinyl acetate, terpinen-4-ol, thymol, carvacrol and geranyl acetate (listed in order of descending quantity). Recent studies on chemical variability of essential oils of Thymus pannonicus have shown that the main constituents are thymol and p-cymene (Pluhár, 2007).
Table 1. Chemical composition of the essential oil of Thymus pannonicus, collected in various phenophases in two consecutive years (2013-2014), from Fălticeni, Suceava County, Romania
Vegetative stage Anthesis stage Vegetative stage Anthesis stage Compound Year Year Compound Year Year
2013 2014 2013 2014 2013 2014 2013 2014 α-Pinene 0.21 0.23 0.16 0.91 α- Cariophyllene - 1.50 1.14 2.10 The symposium is dedicated to the 60-year celebration of the „Stejarul” Biological Research Centre – MAPPPS 2016, Piatra Neamt, ROMANIA – Page 98
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Camphene 0.21 0.25 0.22 - Alloaromadendren 0.44 - - - Octen-3-ol 0.18 0.26 0.30 0.28 β- Cariophyllene 1.30 - - - Myrcene 3.29 3.15 5.20 2.53 Farnesene 0.18 - - - o-Cymene 0.37 0.33 0.25 0.62 τ-Murolen 0.30 0.51 - 0.74 Limonene 0.93 0.50 0.09 0.58 Germacrene D 17.08 17.31 8.05 14.51 Eucalyptol 0.90 0.98 0.27 - β-Elemene 0.39 - - - cis-β-ocimene 2.31 1.26 - 2.15 γ-Elemene 2.10 1.03 - 1.32 γ-Terpinene 0.33 - 0.68 - β-Bisabolene 2.56 1.53 0.12 2.72 cis- Sabinene hydrate 0.78 0.85 0.39 0.58 γ-Cadinol 0.89 - - 2.32 Linalool 3.05 - 0.31 2.55 τ-Cadinol 0.45 - - - Octen-3-ol- acetate 0.175 0.31 - 0.41 τ-Cadinene 0.36 - 0.35 - Camphor 0.42 1.09 - 0.54 γ-Cadinene 1.27 - 0.61 0.54 Borneol - 0.41 0.45 0.33 Elemol 4.30 2.59 - 1.59 Terpinen-4-ol 0.45 0.317 0.13 - Nerolidol 7.74 18.49 - 12.51 α-Terpineol 0.69 0.261 - 0.62 Spathulenol 7.70 1.18 0.31 - Caryophyllene Nerol 0.46 0.47 0.51 0.26 oxide - 2.36 0.73 2.79 Linalyl acetate - - - 3.73 Leden 1.36 - 0.13 0.35 Neral 0.46 0.51 0.32 Cubenole 0.47 - - - Methyl thymol - - 2.50 - γ-Eudesmol 0.85 - - - Geraniol 2.21 0.74 13.45 0.48 Spatulenol - - 0.14 1.48 Geranial 0.67 - 0.15 - τ-Muurulol 3.68 - 0.15 - Aromadendrene Thymol - - 13.98 - epoxyde - 1.31 0.14 1.13 Carvacrol - - 42.32 - Eudesmol - 0.55 - α-Terpinyl acetate 8.52 6.56 - 9.58 τ-Murolol - 1.47 - 1.27 Cis-Trans- Neril acetate 0.32 0.65 - 0.33 Farnesol - 2.03 - 1.06 Linalil acetate 0.45 - - - Farnesol 14.18 14.77 - 12.95 Geranyl acetate - 1.37 4.21 -- Farnesal 0.49 0.68 - 1.23 β-Burbonene 0.79 4.11 0.14 5.11 Farnesil acetate - 0.35 - 0.14
TOTAL % 96.26 92.26 97.58 92.66
Conclusions Our studies have shown that in the case of Thymus pannonicus the main chemical components of essential oils are Germacrene D, nerolidol Farnesol and α-Terpinyl acetate. The changes due to the harvesting period are found only in percentage variations of the compounds, except the plants collected in anthesis in 2014. Acknowledgement This work was supported by a grant of the Romanian Ministry of Education, CNCS– UEFISCDI, project number PN- II-RU-PD-2012-3-0307.
References 1. Mihail Guşuleac (1961), Thymus, In Flora Republicii Populare Române, VIII, Ed. Acad. RPR, Bucureşti, pp. 301-334 2. Adrian Oprea (2005), Lista critică a plantelor vasculare din România. Ed. Univ. Al. Cuza, Iaşi, pp. 306-311. 3. Elisabeth Stahl-Biskup and Francisco Saez (2002), Thyme. Taylor and Francis. London. p. 293 4. Bektas Tepe, Munevver Sokmen, H. Askin Akpulat, Dimitra Daferera, Moschos Polissiou, Atalay Sokmen (2005), Antioxidative activity of the essential oils of Thymus sipyleus subsp. sipyleus var. sipyleus and Thymus sipyleus subsp. sipyleus var. rosulans, J. Food Eng., 66:447-454. 5. Lj. Karuza-Stojaković, S. Pavlović, P. Živanović, B. Todorović (1989), Količina i sastav etarskih ulja različitih vrsta roda Thymus L., Arh. farm., 39:105- 111 (in Serbian). 6. Zsuzsanna Pluhár, Éva Héthelyi, Gabriella Kutta, Livia Kamondy (2007), Evaluation of environmental factors influencing essential oil quality of Thymus pannonicus All. and Thymus praecox Opiz, J. Herbs Spices Med. Plants, 3:23-43.
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Poster Presentation – PP21
Contributions to the knowledge regarding the structure of vegetative organs of Thymus dacicus Borb.
Irina Boz1,3, Constantin Crăciun2, Andrei Lobiuc3,4
1Department of Experimental and Applied Biology, Institute of Biological Research, Iasi, Romania, 2Electron Microscopy Centre, Babeş-Bolyai University, Cluj-Napoca, Romania, 3Faculty of Biology, “Alexandru Ioan Cuza” University, Iasi, Romania; 4Stefan cel Mare University, Faculty of Food Engineering, Suceava, Romania.
Abstract. Thymus L. is one of the most important genera of the Lamiaceae family regarding the species and varieties. Among these species, Thymus dacicus Borb. is less studied from structurally and biochemically point of view. In this paper the authors investigate the structure o vegetative organs of Thymus dacicus in order to complete the existing gaps from the scientific literature. Key words: thyme, anatomy, electron microscopy Introduction. The genus Thymus L. contains over 300 species of plants, mainly distributed in the Mediterranean region (Sunar et al., 2009). Thymus is represented in Romania flora by 17 species, one being cultivated (Thymus vulgaris) and 16 being species that grow spontaneously (Ciocârlan, 2009). The species of this genus are commonly used as spices, herbal tea, insecticide and flavoring plants (Özgüven and Tansi, 1998). Also, Thymus it is used in traditional herbal medicine due to its antiseptic, carminative, expectorant, antispasmodic, antiinflamatory properties (Sunar et al., 2009). Thymus dacicus Borb. is a perennial plant with initial vigorous recumbent stems, then ascendents, very branched. The leaves are elliptic or prolonged, green in color, both faces are covered with hairs, nervures little proeminent. The inflorescence is capitate. The calyx is 3-4 mm long and the corolla is lilac-red, 6-7 mm long (Guşuleac, 1961). Thymus dacicus is a species less studied from structurally and biochemically point of view. In this regard, the authors investigate the histo-anatomy of these species, in order to complete the existing gaps from the scientific literature. Material and methods. The vegetal material is represented by Thymus dacicus, a species that grows wild in the Romanian flora. The species was collected in 3 different phenophases (vegetative, anthesis and fruiting), from Novaci, Gorj County, Romania, during 2014. The identification of taxa was made by Dr. Ioan Sârbu from Botanic Garden “Anastasie Fătu”. The collected material was registered and stored in „Alexandru Ioan Cuza” Universityʼs Herbarium from Iaşi. For histo-anatomical research they were used traditional and modern methods. The vegetal material was firstly fixed and preserved in ethylic alcohol 70%. They were made cross sections at the aerial vegetative organs, sections lately colored by iod-green and carmine-red. Transmission electron investigation was conducted on fresh leaves and stems. The material was prefixed in 2.7% glutaraldehyde, dehydrated in successive, increasing in concentration, acetone solutions. The samples were embedded in Epon 812 epoxy resin and polymerized at 60° C. The blocks were sectioned with a Leica Ultramicrotome, to obtain semithin and ultrathin sections, for analyses under the optical microscope and electron microscope respectively. Results and discussion. The stem (Fig. 1). The contour of cross-section through the stem is quadratic with prominent ribs to the top of the stem. The epidermis is form by isodiametric cells, sometimes elongated tangentially, covered by a thin cuticle, with light streaks. The cuticle is thicker in the fruiting phase. The cortex is relatively thin (depends on the stage of plant development), consists of collenchyma cells in the ribs and parenchyma cells in the rest. The central cylinder is quite thick and is form by a ring of secondary phloem (form by sieve tubes, annex cells and phloem parenchyma cells) and a thick ring of secondary xylem (form by vessel elements irregularly
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distributed and libriform). Close to marrow are vessels of primary xylem, separated by cells of xylem parenchyma. The pith is relatively thick and includes at the exterior an area with parenchyma cells with cellulose walls, meatic type, and an aeriferous central cavity Figure 1. Cross section through the stem of Thymus dacicus (anthesis stage): a. overall picture; b. detail
Figure 2. Cross section through the leaf of Thymus dacicus (anthesis stage): a. foliar blade; b. stomata; c. vascular bundle; d. secretory hair
The leaf (Fig. 2). The epidermal cells of the leaf, like those of the stem, are compactly arranged and covered with a cuticle that reduces water loss. The stomata are present on both side of the leaf, protruding slightly, so the limb is amphistomatic. From place to place secretory and tectory hairs are presents. The secretory hairs can be divided into three categories: 1. Hairs with unicellular gland (present in all aerial organs). 2. Hairs with bicellular gland (observed more rarely, in particular at the species that are in the vegetative stage). 3. Hairs with pluricellular gland (present in all aerial organs, in all stages of vegetation). The mesophyll, the ground tissue of the leaf, is differentiated into palisade tissue and spongy tissue, so the limb presents a bifacial heterofacial structure.
Conclusions. By analyzing the species taking in work it was highlighted a structural uniformity with the others species of this genus. In generally, transition to the secondary structure of the stems occurs early, especially due to the activity of cambium. The cortex is relatively thin (depends on the stage of plant development), consists of collenchyma cells in the ribs and parenchyma cell in the rest. The mesophyll, the ground tissue of the leaf, is differentiated into palisade tissue and spongy tissue.
Acknowledgement. This work was supported by a grant of the Romanian Ministry of Education, CNCS– UEFISCDI, project number PN-II-RU-PD-2012-3-0307. We are also gratefully to project CERNESIM – POS CCE-O 2.2.1, SMIS-CSNR 13984-901, No. 257/28.09.2010-for the infrastructure used to complete this work.
References 1. Vasile Ciocârlan (2009), Flora ilustrată a României. Pteridophyta et Spermatophyta. Ed. Ceres, Bucureşti, pp. 662. 2. Mihail Guşuleac (1961), Thymus, In Flora Republicii Populare Române, VIII, Ed. Acad. RPR, Bucureşti, pp. 301-334 3. Serap Sunar, Ozkan Aksakal, Nalan Yildirim, Guleray Agar, Medine Gulluce and Fikrettin Sahin (2009), Genetic diversity and relationships detected by FAME and RAPD analysis among Thymus species growing in eastern Anatolia region of Turkey, Romanian Biotechnological Letters, 14(2):43134318. 4. Menşure Özgüven and Sezen Tansi (1998). Drug yield and essential oil of Thymus vulgaris L. as in influenced by ecological and ontogenetical variation, Turkish Journal of Agriculture & Forestry, 22:537-542.
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Poster Presentation – PP22
Antioxidant properties of extracts from Crataegus pentagyna assesing by flow cytometry for potential applications in blood stotrage
Iris Tusa1,2, Andreea Toader1, Valentin Grigoraş1, Elvira Gille1, Daniela Bratosin1,2
1National Institute for Biological Science Research and Development (INCDSB), Romania 2”V. Goldis” Western University of Arad, Faculty of Medicine, Pharmacy and Dental Medicine, Arad, Romania
Abstract. Reactive oxygen species (ROS) are the cause of numerous pathologies of plants and represent an important source of antioxidants. The aim of this study was to investigate, by flow cytometry on human erythrocytes, the antioxidant properties of extracts from Crataegus pentagyna, from fruits and sprig (branchlets). Our results indicate that Crataegus pentagyna is a promising source of natural antioxidants. Flow cytometric analysis of ROS generated by H2O2 in human erythrocyte is a available and sensitive method to indirectly determine the antioxidant properties of plant extracts. Keywords: Crataegus pentagyna, ROS, antioxidant activity, flow cytometry, human erythrocyte. Introduction. Reactive oxygen species (ROS), unstable reactive molecular species possessing an unpaired electron are produced continuously in cells as product of metabolism and they have focused attention on physiological and non-physiological mechanism for their generation. (De Zwart et al.,1999). Inside a cell, their high levels can oxidise various molecules leading to cell death and tissue damage that can be the cause of numerous pathologies (Hershko et al., 1998). The oxidative stress generation is characterized by: i) depletion of intracellular antioxidants (largely GSH) and free-radical scavengers (vitamins E and C), ii) inhibition of the activity of various enzymes that contribute to metabolism and detoxification of reactive oxygen species (ROS), such as glutathione peroxidase (GPx), GSH-reductase, GSH-transferase, catalase (CAT) and superoxide dismutase (SOD), and iii) increased production of ROS (superoxide anion radical, hydrogen peroxide, peroxyl radical, hydroxyl radical, nitric oxide, peroxynitrite radical, etc.). Measurement of reactive oxigen species (ROS) is extremely difficult, due to the short lifetime of these species (De Zwart et al., 1999) and methods such as electron spin resonance and spin trapping are complicated and provide average values that can skew results when heterogeneous populations are being studied. Flow cytometry has been used to measure oxidative stress in various cell types, including human normal and thalassaemic erythrocytes. (Bass et al., 1983; Amer et al., 2003). Plants are a promising source of natural antioxidants used as ingredients in dietary supplements for cardiovascular disease and other pathological conditions associated with oxidative stress (Giurescu Bedreag et al., 2014). In the present study, we investigated the ROS generation. Materials and methods. Extracts obtained from Crataegus pentagyna, fruits and sprig (branchletses) were harvested in autumn (15 October) from different points, namely: Ciucurova, Ciucurova –point Oala, located 1km between them, Tulcea and Cuza Voda, Constanta county. The method of extraction used was methanolic method: 2.5 g material was mixed with 100 ml of methanol. 20 ml from each extract was taken to dryness and taken up in 5 ml saline (SF) and filtered through syringe filter "Millex- GP-Syringe Filter Unit 0,22 mm". The antioxidant activity of different extracts obtained from Crataegus pentagyna was performed by measuring of ROS generated in red blood cells after stimulation with 2 mM H2O2 in the presence of 5-fold dilutions from each extract compared to the amount of ROS generated in erythrocytes subject only to oxidative stress (positive control). Crataegus pentagyna extracts have proved a very high content in flavonoids and polyphenol acids, according to Table 1. The five serial dilutions for each sample were: dilution 1: 25 mg/ml, dilution 2: 2.5 mg/ml, dilution 3: 0.25 mg/ml, dilution 4: 0.025 mg/ml, dilution 5: 0.0025 mg/ml. The level of the intracellular ROS was measured by flow cytometry using oxidation of sensitive fluorescent product, 2´,7´-dichlorofluorescein- diacetat (DCFH-DA), according the method described by Bass et al., 1983 and Amer et al, 2003. In the
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presence of various intracellular reactive oxygen species, 2', 7'-dichlorofluorescein-diacetate (DCFH- DA) is oxidized to a highly fluorescent compound, 2´,7´-dichlorofluorescein (DCF). Red blood cells (5x105/ml PBS) were incubated for 1h at 37°C with 5 mM DCFH-DA dissolve in DMSO. ROS species occurrence was measured in FL1, compared with a positive control sample.
Table 1. Total phenolic and flavonoids contents in Crataegus pentagyna extract. Sample Extracts s.u. polyphenolcarboxylic acids Content of flavones expressed in ph. caffeic acid equivalent of luteolin P1 fruits 453.3 mg/100g 58.97 mg/100g P2 fruits 453.3 mg/100 40.58 mg/100g P3 fruits 347.0 mg/100g 84.78 mg/100g P4 sprig (branchlets) 495.7 mg/100g 39.84 mg/100g P5 sprig (branchlets) 384.2 mg/100g 26.47 mg/100g P6 sprig (branchlets) 416.9 mg/100g 44.72 mg/100g Results and discussions. Figure 1 shows inhibition % for the six samples of Crataegus pentagyna (P1-P6), reported to RBCs sample that has not been subjected to oxidative stress by H2O2 and in which were dosed natural level of ROS, and to RBCs sample subjected to oxidative stress in absence of plants extracts. P1 P2 P3 P4 P5 P6
100 100 93 95 95 98 90 96 95 91 90 80 85 82 76 75 76 70 73 75 70 64 60 66 60 58 60 58 50 58 50 45 48 40 38 35 30 28 24 20
10
0 RBC dilu dilu dilu dilu dilu tia 1 tia 2 tia 3 tia 4 tia 5 Fig. 1. Antioxidant activity of the extracts obtained from of Crataegus pentagyna (P1-P6) determined by flow cytometry on human red blood cells subjected to oxidative stress induced by H2O2 in presence of 5-fold serial dilutions for each sample. A: MFI: mean of fluorescence intensity in FL1 of DCF fluorescence resulting from cleavage of the DCFH-DA non-fluorescent substrate by cellular esterases and subsequently oxidized by reactive oxygen species (ROS); B: inhibition % for the 6 Crataegus pentagyna extracts at different dilutions reported to the sample of RBCs with natural level of ROS.
Percentage (%) of inhibition ranges from 98% for P6 and 93% for P5 to the primary dilution, reaching to 66% for P5 and only 24% for P1, which represent a very high percent inhibition of the oxidative stress. It also notes that the P5 sample maintains the most inhibitory capacity to the dilution 5 comparatively with dilution 1, between 93% and 66% respectively. Conclusions. The results reported in the present study indicate that flow cytometric analysis of ROS generated by H2O2 in human erythrocyte is a very rapid and sensitive method to determine the antioxidant properties of plant extracts and the few last dilutions maintain this activity. It is very evident that Crataegus pentagyna is a promising source of natural antioxidants with wide range of applications.
References 1. Amer J, Goldfarb A, Fibach E, Flow cytometric measurement of reactive oxygen species production by normal and thalassaemic red blood cells, Eur. J. Haematol., 70, 84-90, 2003 2. Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M, Flow cytometric studies of oxidative product formation by neutrophils: A grades response to membrane stimulation, J. Immunol. 130, 1910-1917, 1983 3. De Zwart LL, Meerman JH, Commandeur JN, et al., 1999, Biomarkers of free radical damage applications in experimental animals and in humans, Free Radic. Biol. Med., 26, 202-226. 4. Giurescu CF, Bedreag I, Trifan A, Bucur LA, Arcus M, Tebrencu C, Miron A, Costache I, Chemical and antioxidant studies on Crataegus pentagyna leaves and flowers, Romanian Biotechnological Letters, 19, 6, 9859-9767, 2014 5. Hershko C, Link G, Cabantchik I, Pathophysiology of iron overload, Ann NY Acad Sci, 850, 191-201, 1998.
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Poster Presentation – PP23
Collagen membranes enriched with plant extracts used as scaffold in wound healing
Elena Iulia Oprita, Rodica Tatia, Larisa Calu, Agnes Toma, Lucia Moldovan
NIRDBS, 296, Splaiul Independentei, 060031, Bucharest
Abstract. The present work refers to the development of scaffolds based on type I collagen (COL), conditioned as membranes, enriched with bioactive compounds from Arnica montana L. and Urtica dioica L. extracts in two ratios, 5:1 and 10:1. Biological properties of the scaffolds were studied. Key words: collagen, A. montana, U. dioica, membrane, biocompatibility, wound healing.
Introduction. Collagen is the protein that gives tensile strength to the skin and plays a critical role in each phase of wound healing. It attracts cells, such as fibroblasts and keratinocytes to the wound, which encourage debridement, angiogenesis and reepithelization. In addition, collagen provides a natural scaffold or substrate for new tissue growth. In recent years, the development of wound dressings has changed from positive to active types after improving them with different herbal plant extracts.
Material and methods. Preparation of COL-plant extract membranes. Type I COL was isolated from bovine tendon by pepsin treatment using diluted acetic acid, as previously described (Oprita et al., 2006). Hydroalcoholic extracts of A. montana L. (AR) and U. dioica L. (UR) were obtained by maceration of plants in 70% alcohol, at room temperature, for 72 h. Mixtures of COL-plant extract were obtained in two ratios of 5:1 and 10:1 and were conditioned as membranes by drying in oven at 30 0C. In vitro enzyme degradation was achieved by incubation of the COL-plant extract membranes with collagenase type IA (Clostridium hystoliticum) and analysis of aminoacids content by ninhidrin method (Atala and Lanza, 2002). The results were calculated as percentage from the completely degraded membrane (100%) and were reported as mean of three independent experiments ± SD. A COL membrane processed in the same conditions served as control. A Cell Counting Kit 8 (CCK-8) assay (Sigma – Aldrich, St. Louis, MO, USA) was used to measure the cytotoxicity of COL-plant extract membranes in human HaCaT keratinocytes cell culture, after 24 and 48h, according to the manufacturer’s instructions. The untreated cell culture and the cells cultivated with a COL membrane in the same conditions served as controls. Cell morphology was observed by light microscopy (Haematoxylin – Eosin staining) after 48h of incubation. Cell apoptosis was observed by fluorescence microscopy by labelling cells with Hoechst 33258 (Molecular Probes, Invitrogen). Cell adhesion was analyzed by Scanning Electron Microscopy (SEM) after 24h of cell incubation with membranes. Fluorophore labelling of actin filaments (focal adhesions) was highlighted using Phalloidin Alexa Fluor 488 (Invitrogen) according to manufacturer's instructions.
Results and discussion. Both variants of COL–plant extract membranes, COL-AR and COL-UR showed high resistance to enzymatic degradation (65-90%) compared with COL membrane (100%). Composites rich in plant compounds (5:1 variant) had a lower degree of biodegradation. By comparison, COL-AR membranes were more resistant to enzymatic degradation (65-80%) than COL- UR membranes (72-90%).
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Cell viability and apoptosis analysis (human HaCaT keratinocytes) show that both COL-plant extract membranes (COL-AR and COL-UR) were non-cytototoxic. It was observed the high cell viability (more than 90%) with a very low number of apoptotic cells (different stages of apoptosis), especially in the case of COL-UR membranes compared with COL-AR membranes. Human HaCaT keratinocytes morphology was not affected after 48 h of cultivation with analyzed COL - plant extract membranes (5:1 and 10:1), compared to control culture. There was observed a high cell density, especially in the case of COL-UR membranes. Cells showed a normal morphology (polygonal), with circular nucleus and more nucleoli, a granular cytoplasm, similar to that of untreated cells (negative control). After 48 h of cultivation, cell cultures were in different stages of sub- confluence (70-90%) (5:1) or confluence (10:1), compared to controls that were at sub-confluence (80-90%). Adhesion tests carried out by SEM and focal contacts analysis showed that all analyzed COL-plant extracts membranes were good scaffolds for cell attachment and spreading. Composite membranes presenting a higher adhesion were the COL-UR membranes. Number of focal contacts is relevant for cell adhesion to the surface of various scaffolds. Labelling of actin filaments showed a higher cellular adhesion to the surface of COL-plant extract membranes (5:1 and 10:1) than that of cells adhered to the culture plate and to the COL membranes, used as control.
Conclusions. Both COL – plant extract membranes, COL-AR and COL-UR and both variants 5:1 and 10:1 had non-cytotoxic effect and provided good scaffolds for cell attachment. Taking into account all the results, we conclude that COL-UR membranes (5:1 and 10:1) were more biocompatible than COL-AR membranes. They have potential for future application in wound care treatment.
References 1. EI Oprita, L Moldovan, O Craciunescu, W Buzgariu, C Tardei, O Zarnescu (2006), A bioactive collagen- tricalcium phosphate scaffold for tissue engineering, Central European Journal of Biology, 1(1):61-72. 2. Atala A. and Lanza RP, Eds., Methods of Tissue Engineering, Academic Press, 1st edition, 2002.
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Poster Presentation – PP24
Antioxidant properties of the Sanguisorbae herba and Sanguisorbae radix extracts.
Izabela Nawrot-Hadzik1, Marta Szandruk2,3, Anna Kazana2 ,Sylwester Ślusarczyk1,4, Adam Matkowski1
1Dept. Pharmaceutical Biology, 2Student Scientific Group No. 84 at the Dept. Pharmaceutical Biology, 3Dept. Pharmacology, Wroclaw Medical University, Poland; Dept. Crop Biochemistry, IUNG-Institute of Plant Cultivation and Soil Science, Puławy, Poland.
Abstract. The solvent extracts of Sanguisorba officinalis L. (great burnet) aerial parts and roots were studied for content polyphenol content and antioxidant properties. High in vitro activity was noticed in deoxyribose, nitric oxide scavenging. DPPH, phosphomolybdenum and linoleic acid peroxidation assays. The methanol and acetone extracts and ethyl acetate fraction contained high amounts of total polyphenols, tannins and other phenolics. Keywords: Sanguisorba officinalis, great burnet, free radical, proanthocyanidin.
Introduction. In traditional medicine, aerial parts and roots are mainly used as astringent, haemostatic and anti-inflammatory drug. Some reports suggest also its anti-allergic, stomachic and antimicrobial activities [1, 2]. Traditionally it has been applied topically to burns and wounds. Previously isolated compounds include Tannins (oligomeric proathocyanidins and gallotannins): sanguiines H-3 through H-11, phenolic acids, flavonols as well as several triterpenes and triterpene saponins [2]. Plant material. Sanguisorba officinalis L. (Rosaceae) (Great Burnet), herb (aerial parts) supplied by Herbapol (Poland) as Sanguisorbae herba, was purchased in a local pharmacy store. Roots were harvested from plants cultivated in the Botanical Garden of Medicinal Plants, Wroclaw Medical University. MeOH extract with 80% aqueous methanol obtained by reflux extraction. The extract was vacuum dried and dissolved in 10% MeOH for further study. Total phenolics and tannins were determined by the Folin-Ciocalteu method with or without hide powder [3], total proanthocyanidins by acid butanol method of Porter et al. [4]. Chlorogenic acid and flavonol glycosides were determined by spectrophotometry and HPLC [5]. All measurements were expressed as reference compound equivalents per g of extract or dried herb. Studied activity. Hydroxyl radical scavenging was studied with deoxyribose degradation assay [6], nitric oxide scavenging by the method of Sreejayan and Rao [7] and phosphomolybdenum, lipid peroxidation and DPPH assays as described in the previous paper [8]. The concentrations tested were within the range of 1-250 μg/ml. The results were expressed as the maximum inhibition percentage, the EC50 and the reaction rate constant with OH• radical was calculated. Results. In all assays the free radical scavenging and antioxidant activity was high but different depending on the applied method. The extracts from S. officinalis aerial parts and roots are effective reactive oxygen species scavengers and antioxidants. The antioxidant activity is dose dependent. In the deoxyribose degradation assay, the minor prooxidant activity is possible above the concentration of 50µg/ml due to the slightly inverted U-shape of the dose response curve. The ability of nitric oxide scavenging is an important feature of the potential anti-inflammatory drug.
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The bioavailability studies are needed to confirm the in vivo therapeutic potential in this respect. The total polyphenol content was higher in the roots than in the aerial parts, but the differences in the antioxidant activity were moderate. Tannin content in the aerial parts was average when compared to the published results on related Rosaceae [5, 9] but at the same time, there is a high amount of proanthocyanidins which are likely to contribute to the most of the observed antioxidant capacity. In the roots, the tannin content was higher and liquid-liquid extraction allowed to obtain a concentrated fraction using ethyl acetate. The large difference between the active compound amount in extract and in the crude drug suggests solvent extraction as a preferred mode of antioxidant recovery from S. officinalis. Conclusion. The results of the present study confirm the strong antioxidant potential of Sanguisorbae herba, comparable to the more frequently reported Sanguisorbae radix.
References 1. Duke JA. Handbook of phytochemical constituents of GRAS herbs and other economic plants. Boca Raton, FL: CRC Press, 1992. 2. Xia HM. Sun LL.; Sun JY.; Zhong Y. Progress on chemical ingredient and pharmacological activity of Sanguisorba officinalis L. Food Drug 2009, 11, 67–69Liu X, Cui Y, Yu Q, Yu B. Phytochemistry 2005;66:1671. 3. Singleton VL, Orthofer R, Lamuela-Raventos RM. Methods in Enzymology 1999;299:152. 4. Porter LJ, Hrstich LN, Chan BG. Phytochemistry 1986;25:223. 5. Swiader K, Kowalczyk A, Matkowski A, Lamer-Zarawska E. Herba Pol 2003;49:157. 6. Halliwell B, Gutteridge J, Aruoma OI. Anal Biochem 1987;165:215. 7. Sreejayan N, Rao MN. J Pharm Pharmacol 1997;49:105. 8. Matkowski A, Piotrowska M. Fitoterapia 2006;77:346. 9. Kiselova Y, Ivanova D, Chervenkov T, Gerova D, Galunska B, Yankova T. Phytother Res 2006;20:961.
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Poster Presentation – PP25
Contributions to the knowledge of vegetative organs structure of Iris halophila Pall.
Lacramioara Ivanescu1, Marius Nicusor Grigore1, Constantin Toma1
1„Alexandru Ioan Cuza” University Faculty of Biology, Carol I Bd., 20A, zip code: 700506, Iasi, Romania
Abstract. This paper presents several aspects regarding the anatomy of the vegetative organs of Iris halophila Pall., a salt-tolerant plant, more or less known as a medicinal plant. Key words: Iris halophila, anatomy, salt-tolerant plant
Introduction. Iris halophila Pall. (IUCN category: rare) is grown on wet grasslands or meadows, on hillsides, beside rivers and on wet salty soils or salt marshes. The seeds are used in Chinese Herbal medicines to treat Hematochezia and various other problems [2, 3].
Material and methods. Our analysis refers to vegetal material from Carniceni (Iasi county), being collected in anthesis stage, fixed and preserved in ethanol (70°), sectioned in the microtome, stained with iodine green and Ruthenium red and photographed using a NOVEX (Holland) photonic microscope with a Canon digital photo camera.
Results and discussion. Rhizome. The epidermis presents isodiametric cells with an external wall slightly more thickened than the others. The cortex is very thick, differentiated into two sub-areas: - one external (five to seven layers), with cells rich in starch granules, having moderately thick walls; - one more thicker internal area with cells rich in tannin; here and there, this thick layer of tannin cells appears with deeply embedded projections into the fundamental parenchyma. The central cylinder is very thick, with a fundamental cellulosic parenchyma, of meatic type, in wich are dispersed numerous vascular bundles. Some are of collaterally closed type outwards, others of leptocentric concentric types inwards, all with xylem vessels with thick and intensely lignified walls. In the thickness of the fundamental parenchyma, we have noticed numerous cells with simple crystals of calcium oxalate; the component cells are rich in starch granules, being of very different sizes and shapes. In some sections, in the thickness of the parenchymatic-cellulosic cortex, we have seen many vascular bundles and simple crystals of calcium oxalate, some intensely radially elongated. Aerial stem. The epidermis (upper level) presents isodiametrics and isomorphic cells with internal and external walls that are thicker than the others; the external wall is covered by relatively thick cuticle; here and there, on can see stomata with a relatively deep stomatal antechamber. The cuticle is thicker and stomata no longer have a stomatal antechamber (lower level). The fundamental parenchyma of the central cylinder is mostly lignified, the compounding cells having thin or moderately thickened walls (upper level), is visibly sclerified and lignified (middle level) or more intensely lignified (lower level). The pericycle ring is multilayered and sclerenchymatous, with elements having intensely thickened and intensely lignified walls (upper and middle levels) and is thicker, with fewer bundles embedded in it at lower level. Leaf. In some places, the cells of the central colorless parenchyma are undergoing an ongoing disintegration, thus appearing air-storing lacunae of an irregular outline (at upper level with monofacial structure); the central colorless and aqueous parenchyma is thicker, with larger cells, often The symposium is dedicated to the 60-year celebration of the „Stejarul” Biological Research Centre – MAPPPS 2016, Piatra Neamt, ROMANIA – Page 108
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in ongoing disintegration (at middle and lower levels with bifacial structure), some of the composing cells presenting simple crystals of calcium oxalate (lower level) [1].
Conclusions Iris halophila is a species collected from a salty field predisposed to clogging and flooding conditions; however, at that time the land was dry. The plant has thick rhizomes that are resistant to asphyxia. It could not be excluded that the tannin-filled cells from the internal cortex confer rotting resistance to this plant’s rhizome during waterlogging or choked periods when it is also exposed to asphyxia by lack or oxygen deficiency. Therefore, it seems that this species has a wide ecological spectrum; it resists both during periods of high soil moisture, such as in spring, as well as in dry periods in summer. In this respect, the xeromorphic adaptations (stomata with suprastomatic cavity, water- storing formations in the foliar lamina) can be observed.
Bibliography 1. Grigore M.N., Lacramioara Ivanescu, C. Toma (2014), Halophytes: An Integrative Anatomical Study, Springer, ISBN 978-3-319-05728-6. 2. Kaššák P. (2012), Secondary metabolites of the choosen genus Iris species, Acta univ. agric. et silvic. Mendel. Brun., LX, 8: 269-280. 3. Wang Yong-Qiang, Tan Jun-Jie, Tan Chang-Heng, Jiang Shan-Hao, Zhu Da-Yuan (2003), Halophilols A and B, two new stilbenes from Iris halophila, Planta Medica, 69 (8): 779–781. doi:10.1055/s-2003-42792.
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Poster Presentation – PP26
Effect of culture medium supplementation with phytochemicals on human gingival fibroblast cells proliferation
Ana-Maria Seciu1,2, Lucia Moldovan1, Coroiu Viorica1, Oana Craciunescu1*, Otilia Zarnescu2
1National Institute R&D for Biological Sciences, 296, Splaiul Independentei, 060031, Bucharest, Romania, 2University of Bucharest, Faculty of Biology, 91-95, Splaiul Independentei, Bucharest, Romania *Corresponding author, e-mail: [email protected]
Abstract. Our investigation refers to human gingival fibroblast cells proliferation in a culture medium supplemented with phytochemicals instead of fetal bovine serum. We have established a primary culture of human gingival fibroblasts and characterized their phenotype by flow cytometry and immunofluorescence. We have analyzed the influence of the new culture medium on cell proliferation using MTT assay and light microscopy. Key words: quercetin, culture medium, fibroblast cells, cell proliferation. Introduction. Human gingival fibroblasts could be used for in vitro experimental models or in cell therapies. In both cases there is the need of cultivation in animal protein-free medium. Several reagents of plant origin were lately developed to replace standard culture medium supplemented with bovine fetal serum, such as VegetaCell or Prolifix [1, 2]. Besides ethics, the medium containing phytochemicals could offer several advantages for cell culturing, like cellular death decrease and usage in cell therapies. This study aims to investigate the effect of a new culture medium supplemented with quercetin or rutin on cell proliferation of human gingival fibroblasts, used in many labs for periodontitis drug testing. Material and methods. Cell culture of human gingival fibroblasts was obtained by explant technique from gingival tissue, surgically removed during orthodontic treatment of healthy patients, with their consent and according to bioethics regulations. The obtained culture was characterized by cell morphology, immunophenotyping and immunofluorescence of specific markers. For experiments, the cells were seeded in culture plates and cultivated in DMEM culture medium, medium supplemented with 10% bovine fetal serum or with different concentrations of quercetin/rutin ranging between 0-50 μM. The plates were incubated in standard conditions, at 37 °C, in 5% CO2 atmosphere. Cell proliferation and the doubling time were evaluated for 1-6 days of cultivation by MTT assay [1, 3]. Cell morphology was observed and the presence of apoptotic cells was evaluated by light microscopy. Results and discussion. The cell culture obtained by explant technique from gingival tissue fragments was characterized by daily tracking of cell migration and proliferation. Inverse phase contrast microscopy observations showed that the cells migrated from the tissue, at 24 h of cultivation of tissue fragments, and proliferated after 72 h of cultivation. The first passage was performed after 7 days of cultivation and the cell morphology was fusiform, fibroblast-like. Immunophenotyping of human gingival fibroblasts by flow cytometry allowed quantification of surface markers expression. The data indicated positive expression of CD29, CD44, CD73, CD90 and CD105 (Table 1). Cells were negative for CD34 hematopoetic cell marker and STRO-1 stromal cell marker. These results demonstrated a typical profile of fibroblast cells.
Table 1. Evaluation of surface markers expression in human gingival fibroblast cells by flow cytometry Marker CD 29 CD 44 CD 73 CD 90 CD 105 CD 34 STRO-1 % 98.98 % 98.2 % 99.9 % 80.6 % 74.9 % 3.8 % 1.4 %
Since some of the surface markers (e.g., CD 90) expressed in fibroblast cells are also present in mesenchymal stem cells, it were necessary other studies to confirm their fibroblastic phenotype.
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Synthesis of fibroblast specific markers, fibronectin and vimentin, was highlighted using immunofluorescence technique. The images showed that the obtained culture was positive for both markers. All these results demonstrated that it was obtained a viable cell culture of human gingival fibroblasts presenting a characteristic morphology and expressing specific markers. The study has also investigated the cell proliferation rate in three different conditions of cultivation: standard, without fetal serum and with quercetin/rutin supplementation, using MTT assay. The results showed that cells cultivated in standard conditions grew better than in medium without serum (Figure 1). The later presented 36% decrease of the optical density in the 6th day of cultivation.
0.3 M+SF 0.25 M-SF 0.2 0.5 µM Q 2.5 µM Q 0.15 5 µM Q 0.1 10 µM Q
OD at 570 nm0.05 25 µM Q 0 50 µM Q 24 48 72 144 Time of cultivation (h)
Figure 1. Cell proliferation in different culture systems: standard, without serum and medium supplemented with different concentrations of quercetin
In medium supplemented with 0.5 μM quercetin or rutin, cells presented a metabolic activity similar to standard conditions after 72 h of cultivation. After 6 days of cultivation, the cell number decreased, but it was significantly higher (p<0.05) than cells cultivated in medium without serum. The doubling time was comparable at 72 h and 144 h of cell cultivation in standard and quercetin/rutin supplemented culture medium. Cell morphology confirmed the data obtained by MTT assay. Presence of apoptotic cells was not observed in medium without serum and 50 μM quercetin/rutin supplemented medium. The lower proliferation rate was not correlated to apoptosis level. Using bovine fetal serum is controversial due to composition differences between batches, high demand and ethical aspects [4]. Its replacement with plant-derived compounds could expand the use of in vitro cell cultures in tissue engineering and regenerative medicine, allowing autologous cell expansion and their reimplantation. Conclusions. Viable cultures of human gingival fibroblast cells were obtained in optimal lab conditions. Complex tests resulted in confirmation of characteristic fibroblast morphology and expression of specific markers at cell surface and extracellular matrix. A new protocol of cell cultivation and proliferation of human gingival fibroblasts was established using culture medium supplemented with 0.5 μM quercetin or rutin. Acknowledgment. This work was supported by ANCSI, National Programme Nucleu, Project BIODIVERS 16-190/2016.
Bibliography 1. M. Kunova, K. Matulka, L. Eiselleova, P. Trckova, A. Hampl and P. Dvorak (2010), Development of humanized culture medium with plant-derived serum replacement for human pluripotent stem cells, Reproductive BioMedicine, 21:676-686. 2. P. Pazos, M. Boveri, A. Gennari, J. Casado, F. Fernandez and P. Prieto (2004), Culturing cells without serum: lessons learnt using molecules of plant origin, Altex, 21:67-72. 3. O. Craciunescu, A. Gaspar, M. Trif, M. Moisei, A. Oancea, L. Moldovan and O. Zarnescu (2014), Preparation and characterization of a collagen-liposome-chondroitin sulfate matrix with potential application for inflammatory disorders treatment, Journal of Nanomaterials, 2014:903691. 4. D. Brunner, J. Frank, H. Appl, H. Schoffl, W. Pfaller, G. Gstraunthaler (2010), Serum-free cell culture: the serum- free media interactive online database, Altex, 27:53-62.
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Poster Presentation – PP27
Biopolymer hydrogel coupled with indigenous herbal extract potentially therapeutics
Tcacenco Luminita1, Iordachel Catalin1, Berteanu Elena1*, Paraschiv Maria1, Dinu Ecaterina Liliana1, Enache Mihaela-Ionica1, Zuav Adina-Lidia1, Tusa Iris Maria1, Geanta Mariana1
1National Institute of Research and Development for Biological Sciences, Bucharest, Department Biomaterials Bioproducts, *Corresponding author, e-mail: [email protected]
Abstract. It was achieved a biocompatible, hygroscopic, emollient product, form of hydrogel, based on chitosan, a biopolymer naturally coupled with plant extracts, known for their anti-inflammatory effect: melilot (Melilotus officinalis), birch (Betula verrucosa), pansy (Viola tricolor), lemon balm (Mellissa officinalis), lady's mantle (Alchemilla vulgaris) and the method of obtaining it. At the stage of obtaining aqueous plant extracts were determined extraction parameters and some properties thereof: organoleptic properties (according to Romanian Pharmacopoeia, Edition IX, Chapter 2) and content in amino acids, identified by TLC method. It was also determined stretch and plasticity capacity of hydrogel by extensiometrica Ojeda-Arbussa method, both before and after incorporation of active substances from concentrated plant extracts. Evaluation of results show a good ability to stretch of hydrogels in the presence of incorporated plant extracts, which they recommended as bioproducts used for skin diseases. Key words: hydrogel, chitosan, plant extracts, potentially therapeutics. Introduction. Over the past decade, it has been tremendous progress in developing advanced biomaterials for tissue repair and regeneration. Chitosan is a natural environmentally friendly material found in various hybrid compounds. It is a biocompatible and biodegradable biomaterial used to obtain biomaterials. In this work, we developed a new method for obtaining a hydrogel based on chitosan, a natural biopolymer and plant extracts with anti-inflammatory effect. Materials and methods. Materials: Chitosan (CHI) obtained from crab shells, with molecular weight M=150,000 and degree of deacetylation DD = 84.5% (FLUKA BioCHEMIKA), viscosity high, <12% loss on drying (SIGMA ALDRICH). Sodium acetat, acetic acid, were all of reagent grade. Preparation of chitosan gel: 1% chitosan gel was prepared using 1 g of chitosan diluted in acetic solution (acetic acid 2M and sodium acetat 1M) and stirred with the magnetic agitator at 50ºC till a homogenous gel (pH=5.4) was obtained. Obtaining aqueous extracts: They were obtained by soaking dried plants in bidistilled water, extraction duration was 10 days with periodic agitation at ambient temperature. Extraction ratio was 1:13.2 g/v. The aqueous extracts obtained were analyzed in terms of content in biologically active substance. The extracts were kept at room temperature in a dark place and without using any preservatives [1, 2, 3]. The content of amino acids present in the obtained extracts was determined by the TLC (thin layer chromatography), according to Romanian Pharmacopoeia [4]. As materials were used silica gel plates type ALUGRAM SIL (Macherey-NAGEL), G / UV 254, 20x20cm. Mobile phase was: butanol: acetic acid: water (4: 1: 1); revelation was performed using 0.3% ninhydrin solution dissolved in acetone. Formulation gels in ointment: the gels based on chitosan and aqueous plant extracts (with anti- inflammatory effect and cellular regeneration) were formulated as ointments in three types, by changing the amount of carbopol [5,6]. For these samples were determined, besides the organoleptic properties,
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some physicochemical properties, important for this type of biomaterials, namely: pH by potentiometric method and plasticity control by Ojeda-Arbussa extensiometric method. Results and discussion. Regarding amino acids identified by TLC method, results that aqueous extracts of the studied plants, contain a number of important amino acids ranges from 11 to 5 amino acids and essential amino acids between 6 - 3. The aqueous extract from Viola tricolor contains the largest number of amino acids, and the lowest, Betula verrucosa. Due to the high amino acid content of the studied plants extracts, according to the data from traditional medicine, regenerative effect of damaged tissues is improved. Organoleptic characteristics of ointments. Table 1. Organoleptic characteristics of ointments. No. type Appearance Consistency Smell Color pH 1. translucent viscous characteristic Yellow - light brown 5.5-5.7 2. translucent viscous characteristic Slightly white - brownish 5.5 3. translucent glassy viscous characteristic Yellow - light brown 5.7-6
The control of stretching capacity was performed before and after incorporation of active substances in concentrated plant extracts. The results shown in Fig.1:
45 35 35
40 30 30 35 25 25 30
25 20 20
20 15 15
15 10 10 ) s m c ( y t i c a p a c g n i h c t e r t s ) s m c ( y t i c a p a c g n i h c t e r t s ) s m c ( y t i c a p a c g n i h c t e r t S 10
5 5 5
0 0 0 1 2 3 4 1 2 3 4 1 2 3 4 Weight (g) Weight (g) weight (g)
Fig. 1.a,b,c. Stretching capacity (Type 1,2,3) Evaluation results demonstrate a good ability to stretch hydrogels, which is influenced by the presence of plant extracts. It has been found that the organoleptic properties and the plasticity is not changed in the range of 30-60 days. Conclusions. Known as anti-inflammatory plants, their extracts with high content of amino acids, they also have a regenerative effect of damaged tissues. Evaluation of results show a good ability to stretch of hydrogels in the presence of incorporated plant extracts, which they recommended as bioproducts used for skin diseases.
Bibliography: 1. Temelie M., Enciclopedia plantelor medicinale cultivate in Romania, Editura Rovimed Publishers, 2008, ISSN: 973-7719-80-5. 2. Farmacopeea Romana, Editia a IX-a. Editura Medicala, Bucuresti, capitolul 2, 1993. 3. Vicas L. Evaluarea calitativa a unor hidrogeluri cu extracte vegetale, Practica Farmaceutica, Facultatea de Medicina si Farmacie, Universitatea din Oradea, vol.5, nr. 1-2, 2012. 4. Farmacopeea Romana, Editia a X-a. Editura Medicala, Bucuresti, pag. 996, 1044, 1993. 5. Jones A., Vaughan D., Hydrogel dressings in the management of a variety of wound types: A review, Journal of Orthopaedic Nursing, 9, S1-S11, 2005. 6. Ungureanu C., Ioniță D., Berteanu E., Tcacenco L., Zuav A-L., Demetrescu I.,“Improving Natural Biopolymeric Membranes Based on Chitosan and Collagen for Biomedical Applications Introducing Silver”, J. Braz. Chem. Soc. 2015.
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Poster Presentation – PP28
The influence of different extraction solvents on the yield and concentration of anthocyanins and polyphenols from bilberry (Vaccinium myrtillus L.)
Maria Chiriac1*, Elena Iacob1, Carmen Elena Tebrencu1,2, Elena Ionescu1,2, Oana Teodora Ciuperca1
1Medicinal Plants Research and Processing “PLANTAVOREL” S.A., Cuza Voda Street, no. 46, 610019, Piatra Neamt, Romania; 2Academy of Romanian Scientists, Splaiul Independentei , no.54, 050094 Bucharest, Romania *Corresponding author, e-mail: [email protected]
Abstract. The objective of the study is to determine the optimum extraction conditions of anthocyanins and polyphenols from bilberry (Vaccinium myrtillusL,) so as to obtain concentrated extracts, stable and safe for human consumption. Extraction used as solvent ethyl alcohol from cereals acidified with citric acid and hydrochloric acid and performed under identical conditions using the conventional method. Keywords: bilberry, antocyanins, poliphenols, extraction, citric acid. Introduction. Used as such or processed medicinal and aromatic plants are an important source of raw materials for food supplements and pharmaceuticals. Today bilberries are a rich source of anthocyanins and polyphenols, powerful antioxidant active ingredients that are used as an adjuvant in the treatment of metabolic disorders. Material and methods. In this study it was used as raw material bilberries (Vaccinium myrtillus L.) collected from natural population (Zalau, Romania), conditioned in dried form, and its has been applied the classical method of liquid-liquid extraction. To obtain the proposed results of extraction conditions (number of steps of extraction, temperature, time, plant: solvent ratio, established in the other experiments) was held constant, the variable being the solvent (type and concentration). The qualitative analysis consisted in fingerprint by HPTLC fingerprinting for antocyaninins, polyphenols and flavones compounds. Quantitative assessment was evaluated by UV/VIS spectrophotometric method. Reference substances was purchased from Sigma-Aldrich Co. All the other reagents were of analytical grade or pure. Results and discussion. Extraction – the extraction tests were conducted by the conventional method, by Soxhlet in two successive steps and then concentration of the extracts under the vacuum pump to the rotavapor, using dried bilberries. For extraction of anthocyanins and polyphenols its was used ethanol in different concentrations (EtOH 30% EtOH 50% EtOH 70%) acidified with hydrochloric acid solution 0.5% and citric acid solution 1% - 5%. The samples were kept in cold and dark conditions. Choosing the optimal solvent was made on the following criteria: extraction efficiency (high yield, high concentration of active ingredients, namely anthocyanins and polyphenols), extract stability and its safety for human consumption. Phytochemical analysis methods – all the extracts were qualitatively and quantitatively analysed. The qualitative analysis consisted in fingerprint by HPTLC using as anthocyanins standards glycosides of delphinidin, cyanidin, peonidin and polyphenols standards cafeic, chlorogenic and gallic acids, as sample bilberry extracts. Quantitative assessment was evaluated by UV VIS spectrophotmetry method using CINTRA 101 and CARRY spectrophotometers. Total anthoyanins (as cyanidine-3-o-glycoside chloride) was quantitatively evaluated using methanol acidified 1% HCl and reading the absorbance at 528 nm wavelength. For the calculation, it has been used the specific absorbance (718) of cyanidine-3-o-glucoside at 528 nm. Total polyphenols (as chlorogenic acid) was quantitatively evaluated using the Arnow reagent method, reading the absorbance at a wavelength of 540 nm. For calculation, it has been used the calibration curve of chlorogenic acid. The results are shown in the figures and the following table.
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Fig. 1. Chromatographic fingerprint of Fig. 2. Chromatographic fingerprint of flavones and polyphenols antocyanins Table. 1. Results of quantitative extraction Extraction Monitored parameters steps Dry pH Antocyanins, %, related to Poliphenols,%, related to matter, % solution dry matter solution dry matter Test 1- extraction with alcohol 70c SE1 7.62 4.16 0.347 4.55 0.23 2.99 SE2 1.93 4.4 0.096 4.98 0.06 2.93 SC 10.6 3.77 0.47 4.38 0.20 1.86 Test 2- extraction with alcohol 70c acidified with HCl 1% SE1 6.04 1.96 0.31 5.16 0.19 3.16 SE2 3.5 1.85 0.14 4.10 0.12 3.54 SC 9.75 1.51 0.50 5.15 0.31 3.22 Test 3- extraction with alcohol 70c acidified with citric acid 1% , SE1 8.1 3.18 0.34 4.14 0.33 4.10 SE2 3.04 3.03 0.13 4.15 0.14 4.64 SC 10.32 2.71 0.48 4.64 0.28 2.55 Test 4- extraction with alcohol 30c acidified with citric acid 1% , SE1 8.96 3.78 0.39 4.37 0.27 2.96 SE2 3.04 3.62 0.1 3.24 0.1 3.37 SC 12.15 3.37 0.55 4.53 0.21 1.65 Test 5- extraction with alcohol 30c acidified with citric acid 3% SE1 10.69 2.82 0.394 3.72 0.24 2.26 SE2 4.66 2.5 0.085 1.83 0.19 3.94 SC 16.55 2.27 0.513 3.10 0.23 1.85
Conclusions. The results suggest as optimal solvent extraction for bilberries 30c acidified alcohol solution with citric acid 1% (test 4). By choosing citric acid as acidifying its can obtain an extract rich in active principles (double standardized in anthocyanins- cyanidin-3-glycoside and polyphenols- chlorogenic acid), stable, and safe for human consumption. Using a diluted alcohol solution (30c) it was obtain a significant decrease of cost price. Acknowledgments. This work was sustained from the project HIVEGRES/Ctr. 145/2013 (PN II-PT- /PCCA-2013) financed by Executive Agency for Higher Education, Research, Development and Innovation subordinated to the Ministry of Education and Science, Romania.
References: 1Georgiana Cretu, Gertrud Morlok, Gheorghe Nechifor, Development of a quantitative high performance thin layer cromatographic method for analysis of delphinidin 3-glucoside in berry extracts, 2013, U.P.B.Sci. Bull, Series B, Vol 75, Iss.4, 2013 2Fera Amelia, Galih Nur Afnani, Arini Musfiroh, Alia Nur Fikriyani, Sisca Ucche and Mimiek Murrukmihadi, 2013, Extraction and Stability Test of Anthocyanin from Buni Fruits (Antidesma Bunius L) as an Alternative Natural and Safe Food Colorants, J.Food Pharm.Sci. 1, 49-53 3 Azza A. Abou-Arab, Ferial M. Abu-Salem and Esmat A. Abou-Arab, Physico- chemical properties of natural pigments (anthocyanin) extracted from Roselle calyces (Hibiscus subdariffa), 2011, Journal of American Science; 7(7):445-456], ISSN: 1545- 1003.
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Poster Presentation – PP29
Preliminary phytochemical investigations of two new Romanian Ocimum basilicum L. cultivars
Marian BURDUCEA1, Andrei LOBIUC1,2*,Vasilica ONOFREI3, Zenovia OLTEANU1, Mirela ARDELEAN4,Marin ZAGNAT5, Maria-Magdalena ZAMFIRACHE1
1„Alexandu Ioan Cuza” University of Iasi, Carol I Bd., 11, 700506, Iaşi, Romania; 2„Stefan cel Mare” University of Suceava, Universitatii Street, 13, Suceava, Romania; 3„Ion Ionescu de la Brad” University of Agricultural Sciences and Veterinary Medicine of Iaşi, Mihail Sadoveanu Str. 3, Iaşi, 700490, Iaşi, Romania; 4The Institute of Life Sciences: Plant Biotechnology Dep. “Vasile Goldis” Western University of Arad No.86, Liviu Rebreanu Street, Arad 310414, Romania; 5University of Medicine and Pharmacy “Grigore T. Popa”, Iasi, Romania, Kogalniceanu Str., 9-13, 700454 *Corresponding author, e-mail: [email protected]
Abstract. Ocimum basilicum L. is a largely used plant in alimentation, cosmetics and medicine due to its aromatic and therapeutic properties. The present paper analyses the total phenolic content, flavonoids content and antioxidant activity using the DPPH method for two new Romanian basil cultivars, the green leaved “Aromat de Buzau” and the purple leaved “Violet de Buzau”. Key words: basil, total polyphenolics, total flavonoids, antioxidant activity Introduction. Ocimum basilicum L. is a well known specie due to the volatile oils and phenolic compounds it synthesizes. It is widely used in therapeutic products formulations and in cosmetics for its antioxidant, antimicrobial, antiinflammatory etc. activities, as well as in food preparation, where it is considered to bring health benefits. More than 150 cultivars have been developed over the years and there is still interest in creating new, higher quality cultivars [1]. The present paper analyses the total phenolic content, flavonoids content and antioxidant activity in the DPPH method for two new Romanian basil cultivars, “Aromat de Buzau” with green leaves and “Violet de Buzau” with purple leaves. The analyses were carried in order to preliminary evaluate the bioactive potential of the newly developed cultivars. Material and methods. Plants of Aromat de Buzau and Violet de Buzau cultivars were obtained from seeds provided by the Station for Vegetables Research and Development, Buzau, Romania. Plants were grown in laboratory conditions, in soil in 4 L pots, under 16: 8 hours photoperiod, using 4200 K fluorescent lamps, at 25±2 °C, 55% relative humidity for 2 months. Extracts were prepared by macerating 5 g of ground fresh plant in 95 ml of distilled water or 30% w/v ethanol for 24 hours. Total phenolic content was assessed using the Folin Ciocalteu reagent method, by spectrophotometric readings at 760 nm of the colour of incubated extracts [2]. Total flavonoid content was determined according to the method described in [2], by evaluating the absorbance at 510 nm of extracts reacted with 5% NaNO2 and 10% AlCl3. Free radical scavenging activity was determined in the DPPH method [3], measuring the decolouration of DPPH solution reacted with extracts at 515 nm for 3 hours. Statistical analyses performed were ANOVA and Tukey tests. Results and discussion. Total phenolic content (as mg/g galic acid equivalents fresh weight) was 1.99±0.15 and 2.56±0.65 in the green cultivar extracts and 2.44±0.039 and 3.72±0.18 in the purple cultivar for the two types of extract, respectively (Fig. 1). Flavonoid content reached 9.70±0.27 mg/g quercetin in the green cultivar and 12.58±0.69 mg/g quercetin in the purple cultivar on fresh weight basis. Free radical scavenging activity was 29% for the aqueous extract and 48% for the ethanolic extract from the green cultivar and 38% and 87% from the purple cultivar. The values of the phenolic content are close or higher compared to those obtained in other studies: Sweet Dani cultivar had 3.47
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mg galic acid equivalents (GAE)/g dry weight (d.w.), Spice cultivar 17.58 mg GAE/g d.w. [4] and Cinnamon cultivar 35,6 mg GAE/ g d.w. [5]. Flavonoid content was close to values of other studies, such [6] where 9.91 mg quercetin equivalents (QE)/g d.w. in basil leaves were obtained or [7] 7 mg QE/g d.w. in Grant Vert cultivar. Regarding the antioxidant activity we obtained similar values to other studies: 57% inhibition in control plants and 63% in compost mixture treatment for Grant Vert cultivar [7] and 69% inhibition for basil from market in Turkey [8].
Figure 1. Total polyphenolic and flavonoid contents of basil cultivars
Conclusions. These results, especially considering the fresh weight basis, suggest that the two investigated basil cultivars “Aromat de Buzau” and “Violet de Buzau” have valuable characteristics and should be further analyzed from a chemical composition point of view.
Acknowledgements. This work was conducted using infrastructure provided by the CERNESIM Project (SMIS/CNMR Grant Nr. 13984/901).
Bibliography 1. Makri O, Kintzios S (2008), Ocimum sp. (Basil): Botany, cultivation, pharmaceutical properties and biotechnology, Journal of Herbs, Spices & Medicinal Plants, 13(3): 123-150. 2. Herald TJ, Gadgil P, Tilley M (2012), High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour. Journal of the Science of Food and Agriculture, 92(11), 2326- 2331. 3. Molyneux P (2004), The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity, Songklanakarin Journal of Science and Technology, 26(2) : 211-219. 4. Juliani HR and Simon JE (2002), Antioxidant activity of basil. p. 575–579. In: J. Janick and A. Whipkey (eds.), Trends in new crops and new uses. ASHS Press, Alexandria, VA. 5. Kwee EM, Niemeyer ED (2011), Variations in phenolic composition and antioxidant properties among 15 basil (Ocimum basilicum L.) cultivars, Food Chemistry, 128(4): 1044–1050. 6. Chandra S, Khan S, Avula B, Lata H, Yang MH, ElSohly MA , Khan IA (2014), Assessment of total phenolic and flavonoid content, antioxidant properties, and yield of aeroponically and conventionally grown leafy vegetables and fruit crops: a comparative study, Evidence-Based Complementary and Alternative Medicine, Article ID 253875, 9 pg. 7. Taie HAA, Salama ZA R, Radwan S (2010), Potential activity of basil plants as a source of antioxidants and anticancer agents as affected by organic and bio-organic fertilization, Notulae Botanicae Horti Agrobotanici Cluj- Napoca, V. 38(1):119-127. 8. Gülçin I, Elmastat M, Aboul-Enein HY (2007), Determination of antioxidant and radical scavenging activity of basil (Ocimum basilicum L. Family Lamiaceae) assayed by different methodologies, Phytotherapy Research, 21: 354–361.
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Poster Presentation – PP30
Histo-anatomical characterization of vegetative organs from two species of Inula from Romanian flora
Marinela Afemei1, Irina Boz2, Constantin Toma2
1 Sanitary Postgraduate School, Piatra Neamț, 2 Faculty of Biology, ”Alexandru Ioan Cuza” University of Iași
Abstract. In this paper are presented the histo-anatomical aspects of vegetative organs from two species of Inula L. from Romanian flora - Inula bifrons (Gou.)L. and Inula conyza DC. From histo-anatomical point of view the species taken in study are characterized by: numerous tectory hairs and rare secretory hairs on the level of stem and foliar blade epidermises, numerous vascular bundles of collateral open type on the level of central cylinder of aerial stem, rare stomata on the upper epidermis and numerous on the lower epidermis. Key words: Inula, helenin, tectory hairs, secretory hairs, vegetative organs Introduction. In our country vegetates nine species belonging to the genus Inula L (Nyárády, 1964; Ciocârlan, 2000; Oprea, 2005). From the species of this genus, Inula helenium L. called popular high grass is considered an medicinal plant appreciated for his active principles contained. (Perrot and Paris, 1971; Takhtajan, 2009). However, recent research of foreign specialists shows that also other species of Inula L. contain active principles with pharmaceutical value: Inula conyza DC. (Perrot and Paris, 1971), Inula viscosa (Nikolakaki and Christodoulakis, 2004). On the other hand, the anatomical structure of the Inula L. species has been little studied, Metcalfe and Chalk (1972), Napp-Zinn (1973, 1974) refer only to the genus Inula L., and in some atlases microscopy of medicinal plants (Toma and Rugină, 1998) it is taken into consideration only Inula helenium L. The two species of Inula conyza DC. and Inula bifrons (Gou.) L. are herbaceous plants, perennials, with the following morphological characteristics: fusiform roots, stem robust, hairy and up to 100 cm high, leaves subsessile and short attenuated at Inula conyza DC. and lanceolate leaves, sessile, decurente, hairy on the lower face, on the ribs and on the edges at Inula bifrons (Gou.) L., flowers grouped in anthodia and fruits short hairy called achenes (Nyarady, 1964). Material and methods. The vegetal material taken in study belong to two species of Inula L.: Inula bifrons (Gou.) L. and Inula conyza DC. from Romania flora and originates from Faculty of Biology Herbarium, ”Alexandru Ioan Cuza” University of Iași. Fragments of plant material were preserved in alcohol 70%, then were sectioned using botanical razor and hand microtome. Sections results were discolored with sodium hypochlorite for 30 minutes, and then stained with ruthenium red and green iodine. Results and discussion. The root -in cross section through the root of Inula conyza DC. is observed that the cortex is relatively thin, form by 6-7 layers, the internal layer having cells rich in helenin. The helenin is a mixture of lactones sesquiterpenes used in pulmonary diseases, and external use against ulcers and arthritis (Grigorescu et al., 2001). The central cylinder is thick, form by xylem tissue with numerous vessels of different size irregularly dispersed and phloem tissue placed as a discontinuous ring, of a different thickness. The stem - on both species takin in study, the aerial stem presents the epidermis form by cells with external and internal walls thicker than the side, the extenal wall being covered by a thin cuticle. From place to place they are visible tectory hairs with different length, pluricellular, uniseriats. Much rarer are the secretory hairs, with pluricellular gland form by cells disposed in 2-3 level. At Inula bifrons (Gou.) L., the tectory hairs are numerous on the unit area particularly at the level of wings form by the decurent limb. The cortex is relatively thin, plurilayers with the cells of hipodermic layer tangentially collenchimated. The central cylinder it is very thick with numerous vascular bundles of different size, collateral open type, separated by parenchymal lignified medullary rays. All the vascular bundles from both species present on the phloem periphery a very thick belt of sclerenchimatic fibers with strongly
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thickened walls and intense lignified. The pith is thick form by cellulosic parenchyma cells at Inula bifrons (Gou.) L. and mostly lignified parenchyma cells at Inula conyza DC. The leaf -at Inula conyza DC., the leaves shows short petiole. On the petiole level, the epidermis is formed by isodiametrical cells, with external and internal walls visibly thickened than the side. The tectory hairs are rare on adaxial face and numerous on the unit area on the abaxial face and on the two wings. On abaxial face, a part of cells from fundamental parenchyma are disorganized resulting 2-3 large air cavities. The conductive tissue form five vascular bundles arranged on a spring, all with a cord of sclerenchimatic fibers on the exterior part of phloem and on the internal part of xylem. The foliar blade - on both Inula conyza DC. and Inula bifrons (Gou.) L., the upper epidermis shows cells of irregular outline, with sinuous lateral walls, rare stomata and numerous tectory hairs especially at Inula bifrons (Gou.) L. The foliar blade present tectory and secretory hairs on the both faces, evident on Inula bifrons (Gou.) L., the both category being pluricellular formations, secretory hairs being more rare. The stomata are located in both epidermises, being very rare on the unit area in the upper epidermis. Along ribs are visible elongated epidermal cells with straight side walls and thick. In cross sections the foliar blade presents, on both investigated species, prominent median rib and lateral veins (first order) on inferior face and a little on superior face, especially median rib at Inula bifrons (Gou.) L. At Inula conyza DC., under the both epidermises is 1-2 layers of tangential collenchymas and fundamental parenchyma with a open collateral vascular bundles. At Inula conyza DC., under the both epidermises is 1-2 layers of tangential colenchyma and fundamental parenchyma with a fascicular bundles open collateral type, with primary structure, without sclerenchimatic fibers. At Inula bifrons (Gou.) L., under the both epidermises is of angular type collenchymas and the parenchyma shows vascular bundles, open collateral type, with primary structure, each provided with a cord of sclerenchimatic fibers on the phloem periphery. At Inula bifrons (Gou.) L., the mesophyll is poorly differentiated in unilayer palisade tissue and multilayers lacunos tissue, so the limb shows a bifacial-heterofacial structure. At Inula conyza DC., the mesophyll is thin and homogeneous lacunar type, only the cells under superior epidermis are arranged orderly, taking the appearance of a tissue in subpalisade. Conclusions. The species taking under study it shows a similar anatomical structure: tectory hairs and secretory hairs on the level of stem and foliar blade, vascular bundles of open collateral type surrounded by a thick cordon of sclerenchimatic fibers, amfistomatic limb with visible prominent stomata at the exterior of inferior epidermis. However, there are some differences: the presence of helenin in the root of Inula conyza DC., secretory hairs more evident at Inula bifrons (Gou.) L. or a greater number of vascular bundles on foliar blade at Inula bifrons (Gou.) L. Acknowledgement. We are also gratefully to project CERNESIM – POS CCE-O 2.2.1, SMIS-CSNR 13984-901, No. 257/28.09.2010 - for the infrastructure used to complete this work.
Bibliography 1. Adrian Oprea (2005), Lista critică a plantelor vasculare din România, Editura Univ. ,,Al.I. Cuza”, Iași: 361-362 2. A. Nikolakaki, Nicolaos Christodoulakis (2004), Leaf structure and cytochemical investigation of secretory tissues in Inula viscosa, Botanical Journal of the Linnean Society, 144: 437–448 3. A. Takhtajan (2009), Flowering Plants (second ed.), Springer Science, Business Media B.V. 4. Constantin Toma, Rodica Rugină (1998), Anatomia plantelor medicinale. Atlas., Editura Acad. Rom., București: 110-113 5. C.R Metcalfe, L. Chalk (1972), Anatomy of the Dicotyledons, Clarendon Press, Oxford, 2: 782-785 6. Emil Grigorescu, M.I. Lazăr , Ursula Stănescu, Ioan Ciulei (2001), Index fitoterapeutic, Editura Cantes, Iași: 275- 277 7. E.I. Nyárády (1964), Fam Compositae. In: Flora R. P. Române, Editura Academiei Române, București: 9: 264-291 8. E. Perrot, R. Paris (1971), Les plantes medicinales, Presses Universitaires de France, Paris: 24 9. K. L. Napp-Zinn (1973, 1974), Anatomie des Blattes. II. Angiospermen, In Handbuch der Pflanzenanatomie, VIII, 2 A1-2, Gebrüder Borntraeger, Berlin, Stuttgart 10. Vasile Ciocârlan (2000), Flora ilustrată a României (Pteridophyta et Spermatophyta), Editura Ceres, București, (Ediția a II-a revizuită și adăugită): 782-784
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Poster Presentation – PP31
Differentiated seabuckthorn treatment influences the graft rejection in rabbits
Mihaela Niculae, Carmen Dana Şandru, Gheorghe Florinel Brudaşcă, Pall Emoke, Spînu Marina
University of Agricultural Sciences and Veterinary Medicine, Str. Manaştur 3-5, 400372, Cluj-Napoca, Romania
Abstract. The effect of three different oral administration protocols of a H. rhamnoides whole fruit preparation on xenograft rejection in vaccinated rabbits (n=21) was investigated. Although statistically non significant (p<0.05, d=9.8±3.27mm), the increase in the group treated from day 0 to day 14, indicated the potential of both the extract and this protocol in augmenting the adaptive response in this species. Key words seabuckthorn, rabbits, vaccination, graft rejection.
Introduction. „Polivotarom”, a powdered product produced by PLANTAROM MICROPROD SRL, for human and veterinary use was investigated for its potential to stimulate adaptive immunity in rabbits. It was obtained from the fruit of Hippophae rhamnoides, a deciduous shrub in the family Elaeagnaceae. Its indications, due to the high content vitamins, carotenoids, flavonoids, oil, carbohydrates, organic acids, amino acids and minerals, are multiple, including anti-stress, immunomodulatory, hepatoprotective, radioprotective, anti-atherogenic, anti-tumor, anti-microbial activities (4). The aim of this research was to evaluate the specific immunological activity of a whole Hippophae rhamoides fruit extract in the graft rejection in rabbits.
Material and methods. The experiments were carried out on three equal groups (n= 28) of Supercunirom rabbit broiler males, aged two years. All animals were accomodated in individual cages, fed with the same diet supplemented with 2 ml/animal of a 30% suspension in tap water of the Polivitarom powder for the entire duration of the experiment (30 days)(group I, protocol I), from day 0 to day 14, including the period between the two vaccinations (group II, protocol II) and once, on the day following the vaccination (group III, protocol III). The antigenic stimulation was carried out twice, 7 days apart with a subcutaneous injection of 1.5 ml/animal of a 5% sheep red blood cell suspension in saline with addition of Freunds complete adjuvant. The graft rejection test was performed by injecting in the abdominal region, intradermally, 0.1 ml of a chicken lymphocytes suspension (8 x 106 cells/ml in RPMI culture medium) and measuring with callipers the skin thickness in mm before, 24, 48 and 72 h after the injection. The results were subject to statistical analysis by Excel program. The significance of the differences was calculated depending on reading intervals.
Results and discussion. Some researches cited that seabuckthorn seed oil has significant anti- atherogenic and cardioprotective activity in rabbits (1) and also that it protected 15 days old chickens against the immunosuppressant action of T-2 toxin (3). Other authors observed the significant anti- inflammatory activity and the potential for the treatment of arthritis in a rat model (2). There is no information on the adjuvant activity of the whole fruit extract in rabbits. The results of this experiment indicated the most pronounced activity at the first reading for the powdered seabuckthorn fruit extract administered around the two vaccination moments (days 3 and 10 of the experiment). Nevertheless, in this group the decrease in response was the fastest, a statisticall significant (p<0.01) decline being present (Table 1).
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There were statistically significant differences between the administration protocols I and II (p<0.05), the graft rejection being supported the most by the continuous administration of Polivitarom, which caused a very low decrease percentage from the first to the 72h reading. An intermediate decrease in the graft rejection response was observed for the single administration of the extract, in spite of the hypothesized weakest effect.
Table 1. Dynamics of the skin thickness in the graft rejection test in Polivitarom treated rabbits Group 24 h 48 h 72 h Decrease (%) I 8.75±3.09 6.91±4.02 5.50±3.53 37.14 II 9.80±3.27 6.40±3.64 2.20±1.33 77.55 III 8.41±4.92 6.11±4.02 4.13±2.07 50.83
Conclusions. These results suggested that the powdered seabuckthorn fruit extract not only could augment the adaptive immune response, but its activity could be an immune modulating one based on the administration protocol.
Bibliography 1. Basu M, Prasad R, Jayamurthy P, Pal K, Arumughan C, Sawhney RC. (2007) Anti-atherogenic effects of seabuckthorn (Hippophaea rhamnoides) seed oil. Phytomedicine. ; 14(11):770-7. 2. Ganju L, Padwad Y, Singh R, Karan D, Chanda S, Chopra MK, Bhatnagar P, Kashyap R, Sawhney RC. (2005) Anti-inflammatory activity of Seabuckthorn (Hippophae rhamnoides) leaves. Int Immunopharmacol. 5(12):1675-84. 3. Ramasamy T., Varshneya C., Katoch V.C. (2010) Immunoprotective Effect of Seabuckthorn (Hippophae rhamnoides) and Glucomannan on T-2 Toxin-Induced Immunodepression in Poultry. Vet Med Int. 2010 1; 2010:149373. 4. Suryakumar, Geetha; Gupta, Asheesh (2011). Medicinal and therapeutic potential of Sea buckthorn (Hippophae rhamnoides L.. Journal of Ethnopharmacology, 138 (2): 268–78.
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Poster Presentation – PP32
Anthocyanin contents in Sedum telephium ssp. maximum L. callus under growth regulators supplementation
Mirela Ardelean1*, Aurel Ardelean1, Mihaela Duțu1, Călin Lădașiu 1, Andrei Lobiuc2,3, Elida Rosenhech2, Burducea Marian2
1„Vasile Goldiş” Western University from Arad, Institute of Life Science, Romania; 2,, Alexandru Ioan Cuza" University of Iasi, Romania, Plant Biology Department, Carol I Bd., Romania; 3„Stefan cel Mare” University, Universitatii Str. 13, Suceava, Romania *Corresponding author, e-mail: [email protected]
Abstract. Environmental factors may influence organogenesis and metabolism of phytoinoculi. Anthocyanin production was assessed in Sedum callus cultivated under fluorescent tubes white light on Murashige and Skoog basal medium supplemented with 2,4-dichlorphenoxyacetic acid (2,4-D) and benzilaminopurine (BAP). Benziladenine added in 2.5 mg/l concentration in the medium induced higher synthesis of cyanidin-3-glucoside and petunidin-3-galactoside. Keywords: Sedum callus, vitrocultures, fluorescent tubes, anthocyanin pigments, BAP Introduction. The range of plant species used in phytotherapy was amplified every day and their valorification through vitro cultures techniques are in constant expansion. In this context, our attention has been directed towards the investigation possibilities of obtaining callus culture from a medicinal species which is still in a small scale recovered in terms of therapeutic, namely the plant Sedum telephium ssp. maximum L., that nowadays , awakens growing interest in herbal therapy. Our research revealed that callus cells contain anthocyanins Sedum whose concentrations differ in the nature of growth regulators present in the culture medium and their concentration. The process for the synthesis of anthocyanins was more intense in samples inoculated and grown on 1.5 mg / l 2,4- dichlorphenoxyacetic acid (2,4-D) plus 2.5 mg / l benzylaminopurine (BAP). In phytotherapy anthocyanins are regarded as antioxidants use as phytopharmaceutical products. Material and methods. Sedum telephium ssp. maximum L. was generated in primary culture from ,,in vitro” shoot apices inoculated on Murashige-Skoog (1962) basal medium supplemented with BAP and 2,4-D (V0- 2.5 mg/l 2,4 D; V1-1.5 mg/l 2,4 D; V2- 1.5 mg/l 2,4 D+2.5 mg/l BAP; V3- 1.5 mg/l 2,4D; V4- 1.5 mg/l 2,4D +1.5mg/l BAP), cultivated for 60 days under white light (16h : 8h photoperiod) supplied by white fluorescent tubes. Anthocyanins extraction was performed from 500 mg plant material in 9 ml acidified methanol (2% hydrochloric acid in methanol and 10% phosphoric acid) for 10 minutes under room temperature (Socaciu, 2007). A 2 ml extract aliquot was filtered through 0.45 µm filter (Chromafil Macherey-Nagel) and analysed through HPLC-DAD methods. Identification and quantification of anthocyanins was performed using Dionex Ultimate 3000 HPLC system with a DAD Ultimate 3000 attached, in a Thermo Scientific Acclaim 120 C18, 5 µm (4,6 x 250 mm) column coupled with a Acclaim C18 guard column, at 520 nm. Cyanidin-3-glucoside was used as an external standard for quantification of pigments (Azevedo și colab., 2010). Results and discussion. The nature and concentration of growth regulators may influence the amount of anthocyanin pigments synthesized by vitrocultivated cells, especially those in callus cultures (Fig. 1). According to our results, benziladenine (BAP), a synthetic cytoquinine, when added in 2.5 mg/l concentrations to the culture medium induced and sustained bud generation, stimulating meanwhile the synthesis of cyanidin-3-glucoside and petunidin-3-galactoside.
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Figure 1. Anthocyanin contents in 60 days old Sedum telephium ssp. maximum L. cultures illuminated with white fluorescent light
When added separately, 2,4-D and BAP stimulated cyanidin-3-arabinoside synthesis. At equal concentrations of 2,4-D and BAP, a higher callus growth rate was recorded, while the total anthocyanin pigments content was reduced compared to BAP 2.5 mg/l variant. These results are similar to those already described in the literature (Abeda și colab., 2014). The best results were obtained on MS basal medium supplemented with 1.5 mg/ml 2,4-D and 2.5 mg/l BAP, when the concentration of anthocyanin pigments was 7.9 mg/l, compared to control variants, where anthocyanin pigments concentration was 4.2 mg/l.
Conclusions. Increased contents of total anthocyanin pigments was recorded when benziladenine was added in 2.5 mg/l concentration to Sedum telephium ssp. maximum culture medium. Higher concentrations of petunidin-3-galactoside and cyanidin-3-glucoside were produced in cultures supplemented with 1.5 mg/l 2,4-dichlorophenoxyacetic acid and 2.5 mg/l benziladeninepurine.
References 1.Abeda, H. Z., Kouassi, M. K., Yapo, K. D., Koffi, E., Sie, R. S., Kone, M., Kouakou H. T. 2014. Production and Enhancement of Anthocyanin in Callus line of Roselle (Hibiscus sabdariffa L.) Int. J. Rec. Biotech. 2 (1): 45-56. 2.Azevedo, J., Fernandes, I., Faria. A., Oliveira, J., Fernandes, A., Freitas, V., Mateus, N. 2010. Antioxidant properties of anthocyanidins, anthocyanidin-3-glucosides and respective portisins. Food Chem., 119:518-523. ***, 2011, Thermoscientific Application Note 281. Rapid and Sensitive Determination of Anthocyanins in Bilberries Using UHPLC.
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Poster Presentation – PP33
Data and knowledge on the importance of Dracocephalum moldavica L. species (dragon’s head) to introduce and develop the cultivation technology
Naie Margareta, Trotus Elena, Lupu Cornelia, Popa Diana
Agricultural Development Research Resort Secuieni
Abstract. The dragon’s head is an annual plant cultivated for its aerial parts (Dracocephali herba) which are normally distilled as fresh weight obtaining 0.08-0.09% esteric oil. At present, it is used in food aromaization (canned fish, gems, candies, syrups), perfumery, alcoholic drinks industry, soaps and detergents, park decorations. Also used in medicine entering the composition of teas used for the digestive tract affections and for the nervous system. The volatile oil is antiseptic and carminative. The main properties of the dragon’s head herba are, astringent, antidiarrhoeic, inhibitor of the development of intestinsl microbial patogene flora and of the intestinal fermentation. It is a good meliferoue plant producing an average of 300-400 kg/ha honey. In fresh weight, it contains volatile oil between 0.078-0.8%,and in dry weight, between 0.25-2.8%. The volatile oil is rich in citral (40-50%) which produces vitamine A, alcohols (12-14% geraniol and les nerol and limonen), acids (caffeic, succinic), triterpens,one flavonoid (moldavoside), a bitter principle, minerals and other substances. The plant also contains 20% of sictive fatty oil. Key words: Dracocephalum moldavica, important species, cultivation technology. Introduction. The studies of the Dracocephalum moldavica species, to obtain and introduce cultivation technology are part of the ADER 2.4.1. project (2015-2018) with the title „Maintaining the medicinal and aromatic plant biodiversity by preserving and enriching the genetic resources and producing seeds from superiour biological categories for the representative species in the hill and mountain areas”, the plant being known for its medicinal and industrial properties. Materials and method. The experiment was organized at the Agricultural Research and Development Station – Secuieni, Neamt County, after the method of randomized blocks, in four repetions. The soil type was black earth (SRTS, 2012) with a slightly acid pH (6.29), the humus content was 2.55-3.10%, with an average of N and rich in P2O5 şi K2O. It was sown in early spring at a distance of 50 between the rows and at a depth of 2 cm. The seed norm was of 6 kg/ha, depending on the seed quality indices. The biological material used to set up the experimental field was the local cultivar (population) „De Militari” and originates from the Agricultural National Research and Development Institute, Fundulea. To elaborate the cultivation technology of the Dracocephalum moldavica species for Central Moldavia we shall monitor the technological links as the sowing period and the establishing of the optimum nutrition space (the distance between plants in a row and the distance between the rows), important steps to obtain a new technology to maximize the herba yield. Results. As a result of the observations and determinations, we noticedthat the plant has an erect stem, with a sweet lemmon like fragrance, about 60-80 cm high, with a rich basal branching.In the soil, it forms a fascuiculated root which reaches the depth of 35-40 cm. The leaves are opposed, spare shaped, and long of 1.5-7 cm, 0.7-2 cm wide, glabrous or very dispersedly hairy, glandulous spotted on the inferior part, with a short serrated petiole, crenelated on the brims. The flowers are 6-10 grouped in verticils, placed at the inferior part of the leaves from the superior part of the stem and the branching. The flower corolla has two lobs, of a blue violet color, very rarely white. It is with flower
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in June-August. At S.C.D.A. Secuieni, Pînzaru G. had made researches regarding the improving of the technological links of this species. Thus, there were experimented nine variants treated with simple and combined herbicides, which, applied in optimum doses had to assure weed control in the dragon’s head culture. There dominated the monocotyledonous Echinochloua sp. and Setaria species, and from the dicotyledonous species there were the Amaranthus retroflexus, Chenopodium album and Brassica sp. We monitored the phytotoxicity marking the species due the scale established by EWRS (European Weed Research Society). The marks regarding the weed control degree, this being done three times in the vegetation period. From the obtained data analysis, one can assert that the smallest mark (1.67) was given to the variant treated with Eradicane 5 l/ha. The number of weeds on the square meter was between 40 (Mt. II – un-hoed) and 8 (Dual EC 960 – 2l/ha). As to the herba, one noticed that all the tested herbicides were very phytotoxic for the cultivated plants, resulting in very low yields in case of herbicide treated variants. Thus, the yield varied from 260 kg/ha herba at the variants treated with Eradicane 5 l/ha and 2813 kg/ha at the Control II variant – un-hoed. The highest production of herba was obtained by the Control I variant – three times hoed, of 14756 kg/ha.
Conclusions 1. As a result of our determinations, we noticed that plant has a high stem of de 60-80 cm, and the fasciculate root reaches a depth of 35-40 cm. 2. The highest herba yield was obtained by the Control I variant – 3 times hoed, of 14756 kg/ha. 3. To extend the Dracocephalum moldavica species in agriculture, its cultivation asks for the elaboration and introduction of specific cultivatio technologies in Central Moldavia.
Bibliography 1. CRACIUN F. et al, 1994, Ghidul plantelor medicinal uzuale, Edit. Bucurestii-Noi, Bucuresti. 2. MUNTEAN L.S. et al, 2003, Fitotehnie, Edit. Ion Ionescu de la Brad, Iasi. 3. MILICA C. et al, 2012, Flora medicinal a Romaniei, Edit. Doxologia, Iasi. 4. MUNTEAN L.S. et al, 2007, Tratat de plante medicinal cultivate şi spontane, Edit. Risoprint, Cluj. 5. ŞTEFANACHE et al, 2011, “Tehnici de conservare utilizate la speciile Nepeta cataria L. si Dracocephalum moldavica L.”. Vol. - In situ and ex situ Plant Diversity Conservation, Edit. Univ. ” Alexandru Ioan Cuza” Iasi, pg. 30.
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Poster Presentation – PP34
Towards Mimics of Forskolin. Efficient Free Radical Alkylations of Manoyloxides
Olga Morarescu1,2, Vladilena Gîrbu1,2, Elena Pruteanu1,2, Nicon Ungur1,2, Veaceslav Kulciţki1,2, Philippe Renaud3
1Institute of Chemistry of the Academy of Sciences of Moldova, Chişinău, Republic of Moldova 2University of the Academy of Sciences of Moldova, Chişinău, Republic of Moldova 3Department of Chemistry and Biochemistry, University of Bern, Switzerland
Abstract. The current work presents the first results on the application of the radical addition methodology for the simultaneous attachment of a C-2 synthon and a functional group to the manoyloxide framework, wich resembles the well known bioactive natural product forskolin. Key words: Carboiodination, carboazidation, radical chemistry, diterpene, labdane. Introduction. Labdanic diterpenoids represent an important group of natural products with relevant biological activities. One of the most known compound of this series is forskolin 1 – a secondary metabolite isolated from Coleus forskohlii plant and showing a mirad of therapeutical activities [1]. Its main mechanism of action relates on the ability to penetrate the cell membranes and stimulate the enzyme adenylate cyclase. A lot of work has been done on the chemical synthesis of 1 and diverse strategies have been demonstrated for its total synthesis [2]. All the described procedures involve multiple step synthetic transformations and are not so atractive for preparative purposes. That’s why elaboration of simplier analogs of 1 on the basis of readily available manoyloxides can be an alternative avenue towards compounds with similar activities. Material and methods. Manoyloxide 2 and epi-manoyloxide 3 have been obtained by synthesis [3] from sclareol 4 – a diterpenoid readily available from the wastes of Salvia sclarea essential oil production. Separation of 2 from 3 was acheived by flash chromatography on silica gel impregnated with silver nitrate. Radical carboiodination or carboazidation of 2 and 3 was performed according to the described procedures [4, 5]. Shortly, the substrates have been treated under reflux with ethyliodoacetate in the presence of a radical initiator (Bu6Sn2 or dilaroylperoxid, DLP). An azide source (phenylsulfonylazide) and catalytic ammounts of a second initiator (di-tert-butyl hyponitrite, DTBHN) were also added to the reaction mixture in the case of carboazidation. The reaction course was monitored by TLC. Usual aqueous workup and flash chromatography provided pure reaction products. Their structural characterization was performed on the basis of spectral data. Results and discussion. Manoyloxide 2 represents the carbon skeleton of forskolin 1 but lacks the rich „decoration” with oxygenated functional groups. Their selective direct introduction by organic synthesis methods still represents a unrealistic task. Current remote functionalization procedures can be hardly implemented on the relatively fragile 2, due to the labile allylic ether functionality. We decided to apply a free radical methodology for the chemical modification of 2 and its epimer 3. Treatment of 2 with ethyliodoacetate led to the quick consumption of the starting material in the presence of dilaroylperoxide (DLP) as radical initiator in refluxing benzene. The main reaction product was isolated and identified on the basis of spectral data. It represets the iodinated alkylation product 5, which is expected in accordance with the suggested free radical alkylation mechanism (scheme below). Surprisingly, we were able to isolate a minor alkylated compound which lacks the iodine in its structure. A careful examination of NMR data led to the elucidation of its structure 6, which represents a product of 1,5-radical migration, followed by elimination of hydrogen iodide.
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The epi-manoyloxide 3 reacted under similar conditions to provide a mixture of epimeric iodides 7 as basic products.
Reagents and conditions: (a) ICH2CO2Et (or ICH2CO2Me for 7), DLP, Ph-H, 24 hrs. reflux; (b) ICH2CO2Et, Bu6Sn2, PhSO2N3, DTBHN, Ph-H, 8 hrs. reflux.
Submittion of 3 to radical carboazidation conditions led to the synthesis of the correspoding mixture of azides 8. No elimination products have been detected in this case. The obtained products 5 - 8 will be used for following structural modifications and SAR studies. Conclusions. The present work demonstrates application of the free radical transformations for efficient structural modification of manoyloxide 2 and its epimer 3 –important diterpenic compounds which posess the identical carbon skeleton of forskolin 1. Advanced functionalizations of 2 and 3 can lead to biological activities that mimic the relevant profile of 1 – a known plant secondary metabolite with broad therapeutical applications. Acknowledgements. The presented work was performed within the project “Radical mediated modifications of natural products” supported financially by the Swiss National Science Foundation (SCOPES program, project No. IZ73Z0_152346/1).
Bibliography 1. Dewick, P. M. Medicinal Natural Products (3rd ed.) 2009, Wiley. p. 232. 2. Bhat, S. V. Forskolin and congeners. In: Herz W, Kirby GW, Moore RE, Steglich W, Tamm CH, eds. Progress in the Chemistry of Organic Natural Products. Springer-Verlag, New York. 1993, 1-74. 3. Alvarez-Manzaneda, E.J.; Chaboun, R.; Alvarez, E.; Cabrera, E.; Alvarez-Manzaneda, R.; Haidour, A.; Ramos, J.M. Sylett.; 2006, 12, 1829–1834. 4. Ollivier, C.; Bark, T.; Renaud, P. Synthesis 2000, 11, 1598–1602. 5. Panchaud, P.; Ollivier, C.; Renaud, P.; Zigmantas, S. J. Org. Chem. 2004, 69, 2755-2759.
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Poster Presentation – PP35
Research on the knowledge of the harmful entomofauna for the milk thistle culture ((Silybum marianum L)
Elena Trotuş, Paula-Lucelia Ursache, Margareta Naie
Agricultural Development Research Resort Secuieni
Abstract. Silybum marianum L. is grown for its fruit (Fructus cardui material) containing a specific compound, insoluble in water, with hepatoprotective properties (2.5). The average yield of seeds (fruits) is between 6-12 q/ha, if the harvest is done when 80% of the inflorescences are dry, delaying the harvest involves loss of seeds, which spread easily due to the presence of papus. Loss of seeds are produced frequently and by a large range of specific and polyphagous pests (3,4,6,7,8,9). After conducting the observations and determinations was found that the dangerous entomofauna for the Silybum marianum L crops was composed of 28 species of insects that totaled averaged over the entire period between sowing and harvesting (April-July) a total of 702 specimens/sqm. The average density of collected species ranged from 3 specimens/sqm which as totaled at Anomalous solid, Decticus verucivorus, Tettigonia viridissima species and up to 377 specimens/leaf at Tetranychus urticae. Analyzing the species collected and determined regarding the systematic inclusion in orders was found that 11 species belong to the Coleoptera order, six species to Lepidoptera order, four species to Orthoptera order, three species to Heteroptera and Homoptera and 1 species to Acari order. Key words: Silybum marianum, insect, pests, species Introduction. Silybum marianum L is grown for its fruit (Fructus cardui material) containing a specific compound, insoluble in water, with hepatoprotective properties (2,5). The average yields seed (fruits) are between 6-12 q/ha, if the harvest is done when 80% of the inflorescences are dry, delaying the harvest involves loss of seeds, which spread easily due to the presence of papus. Loss of seeds are produced frequently and by a large range of specific and polyphagous pests (3,4,6,7,8,9). Materials and method. The researches were conducted at Secuieni- Neamt unit, located in the Southeast of Neamț County, between the geographical coordinates 26°5' east longitude, 46°5' latitude and at an altitude of 205,7 m above sea level. The biological material gathering was conducted with the help of Barber traps, ground surveys using metric frame 25/25 cm and mowing using entomological net. The colections and determinations were conducted at every ten day, starting from the plant rising phenophase until the harvest phenophase. For each species were calculated the average density/sqm for the entire plant vegetation period (April-August) and the ecological parameters representative like: abundance (A), constance (C), dominance (D) and ecological significance index (W). Results. After conducting the observations and determinations was found that the dangerous entomofauna for the Silybum marianum L crops was composed of 28 species of insects that totaled averaged over the entire period between sowing and harvesting (April-July) a total of 702 specimens/sqm. The average density of collected species ranged from 3 specimens/sqm which totaled at Anomalous solid, Decticus verucivorus, Tettigonia viridissima species and up to 377 specimens/leaf to Tetranychus urticae. Analyzing the species collected and determined regarding the systematic inclusion in orders was found that 11 species belong to Coleoptera order, six species to Lepidoptera
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order, four species to Orthoptera order, three species to Heteroptera and Homoptera order and 1 species to Acari order.
By calculating the parameters for the species collected was found that: - Abundance (A) ranged between 3 specimens/sqm (Anomalous solid, Decticus verrucivorus, Tettigonia viridissima) to 377 specimens/leaf (Tetranychus urticae): - Dominance (D) of the collected species ranged from 0,28% (Aphis fabae, Macrosiphoniella sanborn) to 53,13% (Tetranychus urticae). - The constancy (C) index that expresses the continuity of the species occurrence in the analyzed habitat ranged from 16,6% at Polyphylla fulla, Loxostege stiticalis, Aphis fabae, Macrosiphoniella sanborn, Decticus verucuorus, Tettigonia viridissima species to 66,6% at Agriotes ustulatus, Macrosteles sexnatatus species; - Ecological significance index (W) which represent the relationship between structural and productive indicator it ranged between 0,05% at Macrosiphoniella sanborn, Aphis fabae species and 30,9% at Tetranychus urticae species.
Conclusions. 1.The harmful entomofauna from Silybum marianum L. crops is consisted of 28 species that totalized 702 specimens/ sqm for the entire vegetation period. 2.The highest density of insects, 293 specimens/sqm was recorded in the stem elongation – blossom phenophases. 3.The insects collected were placed in the systematic orders: Coleoptera, Lepidoptera, Orthoptera, Heteroptera, Homoptera and Acari.
Bibliography 1. PerjuT. si colaboratorii, 1988 - "Entomofagii şi utilizarea lor în protecţia integrataă a ecosistemelor agricole", Ed. Ceres, Bucureşti 2. Trotuş Elena, Druţu A.C., Găucă C., Popa D. L., Lupu C.,Pochişcanu S., Naie Margareta, Leonte A. , 2015 - "Tehnologii de cultivare a unor plante de camp pentru zona centrală a Moldovei", Ed. Ion Ionescu de la Brad, Iaşi, pag. 198 3. Trotuş Elena, Doina Dănilă, 2007 – "Date privind entomofauna specifică pajistilor din Lunca Siretului – SCDA Secuieni- Neamţ 1962-2007", 45 de ani de activitate ştiinţifică, Volum omagial, Ed. Ion Ionescu de la Brad, Iaşi, pg. 175.
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Poster Presentation – PP36
Bioinformatic approaches regarding the Salvia sclarea investigation
Rodica Martea1*, Ana Mutu1, Maria Duca1
1University of the Academy of Sciences of Moldova, University Center of Functional Genetics, Chisinau, Republic of Moldova *Corresponding author, e-mail: [email protected]
Abstract. The investigation of biological databases has revealed a high volume of data on medicinal and aromatic plants (MAPs), which has to be systematically evaluated, analyzed and compared. With UDaCoT – UnASM Data Collecting Tool it was investigated the information from databases of the three major portals in the field: NCBI, EMBL-EBI, ExPASy. Key words: databases, EMBL-EBI, ExPASy, NCBI, Salvia sclarea L. Introduction. The association of the genetic variability with valuable traits ensured by use of the bioinformatics tools allow a comprehensive analysis of the genetic-molecular and physiological processes and determines the rapid evolution in the field of plant biology. At present, the emphasis is on the creation and development of databases, statistical techniques for solving formal and practical problems generated by operating and analyzing biological data [1] and on information systems destined for the process of collecting, storing and analyzing biological information [6]. Bioinformatic approaches facilitate the management of data in the aspect of the biological material, being the proper tools in breeding programs and obtaining perspective varieties and hybrids [2, 3, 5]. Nowadays, the traditional methods of describing the Salvia sclarea has been supplemented by new researches, which are based on the implementation of the modern methods of molecular biology and the high-performance technologies [7]. All these elements generate a large volume of information that needs analyzed in the research process Material and methods. The UDaCoT tool developed within the University of the Academy of Sciences of Moldova [8], is destined for the extraction and analysis of data from different scientific resources, that are important for a wide range of areas of interest. The exploratory analysis of the Salvia sclarea was focused on extracting information based on keywords. The search by keywords was simultaneously realized in all databases of the NCBI, EMBL-EBI, and ExPASy portals) using the UDaCoT tool [4]. Results and discussion. The bioinformatics study shows that the ExPASy portal contains 1827 registrations referring to Salvia sclarea, and the EMBL – 30812. Most information (48116365 registrations) are revealed in the NCBI portal, which includes 39 databases. Most notifications were revealed in SNP, PubChem Compound, GEO Profiles, GSS, Gene, dbVar, BioSystems, NLM Catalog, Probe, dbGa databases. The results showed a considerable fluctuation in the NCBI categories. Most of registrations were revealed, by HealthDb (60,6%), followed by LiteratureDb with 30,4%, GenomesDb – 8,9%, GenesDb – 0,1%, ChemicalsDb – 0,007%, ProteinsDb – 0,005%. The bioinformatics analysis has allowed us to highlight publications regarding the systematic and ecological description at morphological and phenotypical level (37%), also biochemical researches (44%) on the use of biologically active compounds as well as genetic-molecular information (19%) referring to the genetic structure of Salvia sclarea.
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Conclusions. The bioinformatics analysis has revealed that most information on the description at morphological and phenotypical level, as well as biochemical results on the use of the biologically active compounds in medicine and traditional medicine. The number of publications and genetic- molecular data referring to the genetic structure is rather limited.
Bibliography 1. Barnes M.R., Gray I.C., eds., (2003), Bioinformatics for Geneticists, first edition. Wiley, 408 p. ISBN 0-470- 84394-2. 2. Dekkers J.C.M., Hospital F. (2002), The use of molecular genetics in the improvement of agricultural populations. In: Nature Review Genetics, nr. 3, p. 22-32. 3. Lamkey K.R., Lee M. (2006), Plant Breeding The Arnel R. Hallauer International Symposium. Wiley- Blackwell. 379 p. ISBN 978-0-8138-2824-4. 4. Levițchi A. (2012), (UnASM Data Collecting Tool): Principii de căutare şi utilizare a informaţiilor din bazele de date bioinformatice. UnAŞM, CBM, Lab. de Bioinformatică; Chişinău: S.n. T-PAR SRL, 148 p. ISBN 978- 9975-4280-0. 5. Martea R. (2013), Management of information for medicinal and aromatic plant. Abstract book of the Vth Symposium of Ethnopharmacology, Ethnopharmacology, in support of the human health and the environment, Braşov, România, p. 29. ISSN 1844-6604. 6. Hogeweg P.S., David B. ed. (2011), The roots of bioinformatics in theoretical biology, PLoS Computational Biology, vol. 7, nr. 3, p.1-5. 7. Sharma V., Sarkar I.N. (2012), Bioinformatics opportunities for identification and study of medicinal plants. In: Briefings in bioinformatics, vol. 14, nr. 2, p. 238-250. 8. udacot.unasm.asm.md.
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Poster Presentation – PP37
Phenolic contents and antioxidant activity in Viola odorata L., V. tricolor L. and V. arvensis (L.) Murray
Rosenhech Elida1, Lobiuc Andrei1,2*, Zamfirache Maria-Magdalena1
1”Alexandru Ioan Cuza” University of Iasi, Faculty of Biology, Romania 2”Stefan cel Mare” University, Faculty of Food Engineering, Suceava, Romania *Corresponding author, e-mail: [email protected]
Abstract. The genus Viola comprises approximately 30 species in Romania, of which the most well known for their therapeutic properties are V. tricolor and V. odorata. The current paper aims to evaluate some phytochemical parameters in V. tricolor, V. odorata and V. arvensis collected from wild populations from the eastern part of the country to assess their therapeutic potential. Key words: pansy, polyphenols, free radical scavenging Introduction. Approximately 500 species are included in the genus Viola (Violaceae), while in Romania there are about 30 species present. The chemical composition of these species includes mainly saponins, flavonoids, mucilages, salicylic derivatives, carotenoids, and coumarins, and the plants are used in traditional medicine for their anti-inflammatory, expectorant, diuretic properties, to treat bronchitis, cystitis as well as various skin conditions (Tamas, 1999; Toiu et al., 2009). Studies in Romania on the Viola genus were mainly concerned with populations from the western part of the country (Toiu et al., 2007a; Toiu et al., 2007b). In the current paper, the total phenolic contents, flavonoid and anthocyanin pigments contents and antioxidant activity were evaluated in V. tricolor, V. odorata and V. arvensis, collected from spontaneous populations from eastern part of Romania. Materials and methods. The plant mateial was collected from spontaneous populations from Breazu forest, Iasi county (V. odorata) and from Cornu Luncii, Suceava county (V. tricolor and V. arvensis). Total polyphenolic contents were assessed in aqueous and 50% ethanolic leaves extracts prepared in a 5 g/95 ml solvent concentration. The spectrophotometric method described in Herald et al. (2012) was used, evaluating color development of extract-Folin reagent mixture at 760 nm. Total flavonoid contents were determined using the AlCl3 method while antioxidant activity was evaluated through the DPPH free radical method (Herald et al., 2012) in the same extracts as described previously. Anthocyanin pigments contents was evaluated in acidified ethanolic flowers extracts (Fuleki and Francis, 1968). Dry matter content and ash content were evaluated gravimetrically in pansy leaves and flowers. Results and discussions. The content of polyphenolic compounds, as determined spectrophotometrically, varied among the investigated species, with the highest values in V. tricolor ethanolic extracts, followed by V. odorata and V. arvensis (Fig. 1). The flavonoid contents were, similarly, highest in V. tricolor ethanolic extracts (Fig. 2). Antioxidant activity, however, was highest in V. odorata extracts (Fig. 3). Anthocyanin pigments content was highest in V. odorata flowers, while similar values were found in V. arvensis and V. tricolor (Table 1). Considering the accumulation of organic and mineral substances in plants, the highest amounts were found in V. arvensis and V. tricolor, while lower quantities were recorded in V. odorata (Table 1). The recorded values for polyphenolic contents are similar to those described in the literature for V. tricolor, however in methanolic extracts (Chandra et al., 2015). The higher phenolic contents in V. tricolor compared to V. arvensis was also reported for other Viola populations (Toiu et al., 2008). However, comparing the results with those previously presented, the investigated material presented higher amounts of flavonoids in prepared extracts. The elevated values of free radical scavenging activity for V. odorata suggest that other compounds present in this specie besides polyphenolic substances may
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impart such activity. V. odorata is a specie known to synthesize volatile compounds (Akhbari et al., 2012), which may be responsible for the values obtained.
Conclusions. The analyzed plant material recorded similar or higher values for the content of total polyphenolic and flavonoids contents compared to other available studies. The highest amounts of these bioactive substances was found in V. tricolor, followed by V. odorata and V. arvensis. The analyzed plant populations represent a potential source of material for extraction of substances of interest from Viola species.
Bibliography Akhbaria M., Batoolib H., Kashi F.J. (2012) Composition of essential oil and biological activity of extracts of Viola odorata L. from central Iran, Natural Product Research, 26(9): 802-809 Deepak C., Gunjan K., Kundan P., Bisht G., Deep P.V., Khetwal K.S., Kumar D.J., Pandey H.K. (2015) Phytochemical and Ethnomedicinal Uses of Family Violaceae, Current Research in Chemistry, 7(2). p.44 Fuleki T., Francis F. J. (1968), Quantitative methods for anthocyanins, Journal of food science, 33: 72–77 Hatieganu”, Cluj-Napoca, 1999, p. 137–138. Herald T.J., Gadgil P., Tilley M. (2012) High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour, Journal of the science of food and agriculture, 92(11):2326-31 M. Tamas, Botanica farmaceutica, Vol. III. Sistematica-Cormobionta, Ed. Medicala Universitara “Iuliu Tamas M. (1999) Botanica farmaceutica, Vol. III. Sistematica-Cormobionta, Ed. Medicala Universitara “Iuliu Hatieganu”, Cluj-Napoca, p. 137–138. Toiu A., Muntean E., Oniga I., Tămaş M. (2009) Pharmacognostic research on Viola declinata Waldst. et Kit. (Violaceae), Farmacia, 57(2): 218-222 Toiu A., Pârvu A.E., Oniga I., Tămaş M. (2007a) Evaluation of anti-inflammatory activity of alcoholic extract from Viola tricolor, Revista medico-chirurgicală a Societăţ̜ii de Medici ş i Naturaliş ti din Iaş i; 3(2): 525-529 Toiu A., Vlase L., Oniga I., Tămaş M. (2007b) HPLC-MS study of flavonoids from Viola arvensis and V. declinata (Violaceae). Rev. Med. Chir. Soc. Med. Nat., Iaşi; 3(2, suppl. 2): 103-107 Toiu A., Vlase L., Oniga I., Tămaş M. (2008) Quantitative analysis of some phenolic compounds from Viola species tinctures, Farmacia, 56(4): 440-445.
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Poster Presentation – PP38
Lavandula hybrida: assessment of the essential oil properties
Silvia Robu1, Monica Hancianu2*, Dana Tutunaru1, Adrian Spac2, Cristina Tuchilus2, Adriana Trifan2, Ursula Stanescu2, Elvira Gille3, Oana Cioanca2
1“Dunarea de Jos” University, Galati, Romania 2Faculty of Pharmacy, University of Medicine and Pharmacy “Grigore T. Popa”, Iasi, Romania 3 NIRDBS/„Stejarul” Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt, Romania *Corresponding author, e-mail: [email protected]
Abstract. Lavandin (L. hybrida) is part of Lamiaceae family, but for which little data exists to confirm its use in therapy. The purpose of the study was to reveal the biological potential of volatile fractions isolated from lavender, especially on standard microorganisms. Key words: GC, essential oil, antimicrobial. Introduction. Lavender has been used in cosmetic and therapeutic purposes since ancient times. The Romans added the branches of lavender to bath water, the plant's name deriving from the Latin word lavare, which means to wash. In modern times, oil and lavender inflorescences have a wide range of uses: therapeutic aromatherapy, perfumery, cosmetics, pharmaceuticals, soap, detergent and even food industry. The importance of Lavandula sp. is shown by the fact that 2008 has been declared the International Year of lavender. The most known representatives of Lavandula Genus are: Lavandula angustifolia (L. officinalis), L. latifolia, L. stoechas, L. burnatii, L. dentata, L. canariensies, L. abrotanoides, L. lanata, L. multifida, L. pinnata, L. viridis, L. x intermedia, L. luisierii. L. hybida (sin. L. x intermedia) is common in Romania and has a highly productivity in regards to essential oil (60-150 kg essential oil/ha). The aim of our study was to evaluate the chemical composition and the antibacterial properties of Romanian origin lavandin essential oil. Material and methods. Dried plant product originating from the Biological Research Centre in Piatra Neamt, was crushed and extracted with water vapors. The essential oil was obtained hydro distillation for 3 hours in a Clevenger-type apparatus. The essential oil was dried on anhydrous sodium sulfate and stored at 4 ° C until analysis. Initially, the qualitative characteristics of the essential oil were established by TLC as compared to Lavandula officinalis aetheroleum, linalool and linalyl acetate as standards. The semi quantitative analysis employed GC-MS-FID techniques (Agilent Technologies 6890N/5975) with the following parameters: injection volume 1 µL (Column: HP, 5MS bonded phase 5% phenylmethylsiloxane; 0.25 mm i.d.; 30 m length; 0.25 µm film thickness); ratio 1:100, carrier gas Helium, temperatures: injector 250◦C, detector 280◦C, column 50◦C, 2 min; 10 ◦C/min to 250 ◦C for 10 min. The identification of the volatile compounds was based on comparison of their retention indices (RI), and mass spectra with those obtained from authentic samples and/or NIST/NBS, Wiley libraries and literature. The antimicrobial activity of the essential oil (10 µL) was measured by the disc-diffusion in Mueller - Hinton agar, on the following strains: Staphylococcus aureus (ATCC 25923), Streptococcus pyogenes (ATCC 19615), Pseudomonas aeruginosa (ATCC 27853), Eschcherichia coli (ATCC 25922) and Candida albicans (ATCC 10231). The analysis was in accordance to (National Committe for Clinical Laboratory Standards), NCCLS, 2009. Results and discussion. The TLC results showed that lavandin essential oil has a similar spectra to lavender essential oil, meanwhile the GC analysis indicated the presence of the compounds included in table 1 and. Most of the identified compounds were in accompliance with European Pharmacopeia
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(Ph.Eur.) requirements for Lavandula aetheroleum. Moreover, such coponents (4-terpinen-ol, α- terpineol) may suggest the existence of antibacterial properties.
Table 1. Selective compounds identified in Lavandula hybrida essential oil Requirement Compound Area % Ph.Eur. limonene less than 1.0 % 0.8 cineol less than 2.5 % tr* camphor less than 1.2 % tr linalool 20.0 – 45.0 % 21.5 linalyl acetate 25.0 – 46.0 % 22.5 4-terpinen-ol 0.1 – 6.0 % 16.7 lavandulyl acetate more than 0.2 % 8.4 lavandulol more than 0.1 % tr α-terpineol less than 2.0 % 7.5 * tr - traces
In regards to the antimicrobial properties the investigated essential oil showed no activity against Gram-negative strains. Moreover, our results showed that the anti staphylococcal activity is reduced, while there is a moderate antifungal activity. The calculated values against C. albicans strain were situated slightly below the measured values for nystatin used as standard. Since the investigated sample did not show a marked antibacterial action, although it contains components such potential (especially α-terpineol), we might presume that the ratio between each individual substance is of great importance in expressing the biological potential.
Conclusions. All in all, our research revealed that a lavandin essential oil of the type we investigated is not recommended as an antimicrobial in serious infections. Considering all aspects regarding possible antidepressant and anxiolityc effects, one might sustain that L. hybrida essential oil of Romanin origin can be used in rooms with ventilation systems or air conditioning to reduce stress condition and to give comfort, possibly reducing bacterial contamination in the spreading area.
Bibliography 1. Robu S, Aprotosoaie AC, Miron A, Cioancă O, Stănescu U, Hăncianu M. (2012), In vitro antioxidant activity of ethanolic extracts from some Lavandula species cultivated in Romania. Farmacia, 60(3):307-314. 2. Aprotosoaie AC, Hăncianu M, Costache I-I, Miron A. (2014), Linalool: a review on a key odorant molecule with valuable biological properties. Flavour and Fragrance Journal, 29(4):193-219. 3. Hritcu L., Cioanca O., Hancianu M. (2012), Effects of lavender oil inhalation on improving scopolamine-induced spatial memory impairment in laboratory rats. Phytomedicine, 19:529-534.
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Poster Presentation – PP39
The chemical composition of some grain sorghum (Sorghum bicolor L. Moench.) varieties experimented in A.R.D.S. Secuieni – Neamt pedoclimatic conditions
Simona – Florina Pochişcanu1*, Alexandra – Andreea Buburuz1, Lorena - Diana Popa1
1Agricultural Research and Development Station Secuieni – Neamt *Corresponding author, e-mail: [email protected]
Abstract. In this paper we present the scientific results obtained from A.R.D.S. Secuieni at grain sorghum, experimented for its increased resistance to drought and to determine the grain chemical composition at the most representative varieties that are on the market in Romania. The obtained results showed a high content in protein of the species, smaller than wheat but higher compared to the rest of the cereals. Key words: sorghum, protein, sugar, starch Introduction. With improvements, sorghum may become the "future global cereal" (2) being at this moment one of the most important cereal in the world (5). It is considered necessary for the survival of mankind due to different usage areas and due to high adaptability in all areas of the world (6). Sorghum is the main bread cereal in Africa, South Europe, Central America and South Asia (1), and in Asia is very appreciated and used in cooking. It is appreciated by nutritionists because it provides a number almost double of proteins compared to rice, and in Occident is highly sought by people intolerant to gluten (4). The spectacular benefits which sorghum has it, has convinced a number of businessmen, to allocate funds for conducting scientific researches on it. An example would be the founder of Microsoft (Bill Gates) who decided to dedicate a project that aimed to develop new plant species which contain everything necessary to the human body, from vitamins to minerals. These funds are earmarked for genetic researches on sorghum, a cereal that he believes to be the mankind superfood and future food (4). In the research conducted in Romania highlights that the grains of sorghum have a chemical composition similar to the maize grain. As a result of the performed determinations it was showed that sorghum has in its content a high percentage of protein, reaching 12.7%, cellulose is found in 1.5% and 1.6% ashes (3). Materials and method. The researches were conducted at Secuieni- Neamt unit, located in the Southeast of Neamț County, between the geographical coordinates 26°5' east longitude, 46°5' latitude and at an altitude of 205,7 m above sea level. The biological material used in the experiment was represented by three hybrids representative for our country, a Romanian hybrid created at I.N.C.D.A. Fundulea (Fundulea 32) and two hybrids created in France by the Euralis Semences S.A.S. company, one of them occupying the largest area of land cultivated in Romania (Alize) and another representing the company's latest creation at the level of 2014 (Albanus). The biochemical analyzes performed on the sorghum grains were made in the laboratory of Plant Breeding belonging to the Agricultural Research - Development Station Turda. For each individual compounds were made three determinations and the final results were the average of these determinations. In the laboratory was followed the percentage content of grain sorghum in: ADF, NDF, fiber, ash, saturated and unsaturated fats, protein, starch and sugar. These determinations were made using the NIR analyzer type, John Dickey brand, Installable 600. The obtained results were statistically calculated and interpreted according to the variance analysis (7.).
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Results. In recent years, concentrated feeds are tested in laboratories to assess soluble fiber content in neutral detergent (NDF) and soluble fiber content in acid detergent (ADF). The pooled fractions in NDF case are hemicellulose, cellulose and lignin, and in ADF cellulose and lignin. The obtained results showed that these two components differ from one hybrid to another, with values between 7.26% (Fundulea 32) and 8.76% (Albanus) for ADF and 2.69% (Fundulea 32) - 3.13% (Alize), in NDF case. The fiber content ranged from 2.02% (Albanus) and reached 2.27% (Fundulea 32), and the percentage of ash ranged from 1.38% (Alize) to 1.45% (Fundulea 32). The fats in feed are very important because they have the capacity to produce twice as much energy than carbohydrates or protein. In addition, fats make the feed to be more juicy. The obtained results showed that the grain of sorghum is more rich in saturated fats compared with the unsaturated ones. Thus, the percentage of saturated fats ranged from 4.61% (Albanus) and up to 5.04% (Alize) and the unsaturated were present in a proportion of 3.71% (Fundulea 32) and 3.85% (Alize). The experimented variants were characterized by a fairly high protein content, which ranged from 8.33% (Alize) and 9.36% (Albanus) and the percentage of starch was between 64.78% ( Albanus) and 66.72% (Fundulea 32). The sorghum experienced at A.R.D.S. Secuieni was characterized by a sugar content that ranged between 2.29% (Fundulea 32) and 2.54% (Albanus).
Conclusions 1. Sorghum is one of the most important cereal in the world and is a pity that it does’nt worthwhile its place that it deserves in our country as an alternative to the climatic conditions to aridity of the late period. 2. The chemical composition of sorghum seeds ranged from a hybrid to another, seeing a greater variation regarding the protein and starch content. The protein content ranged between 8.33% and 9.36% and the starch content was between 64.78% and 66.72%. 3. From the qualitative point of view, it has been noted that Albanus hybrid, which is characterized by a low fiber, starch and saturated fats content, but with higher values in the other five elements analyzed.
Bibliography 1. Cope T. A., 2000 – Postharvest Treatment of sorghum (Sorghum bicolor) in Botswana. Flora of West Tropical Africa, vol. 3, part 2. 2. Maunder B., 2006 – SORGHUM: The Global Grain of the Future", from National Sorghum Producers. 2006. What is Sorghum? 3. Mogârzan Aglaia, Morar G. şi Ștefan M. colab., 2004 – Phytotechny. Ed. Ion Ionescu de la Brad, Iaşi 4. Onciu Camelia, 2012 – Sorghum, rich in vitamins and minerals. Monitorul Expres – 21.08.201 5. Pochişcanu Simona – Florina, 2015 – Research regarding the aplication of some modern technological sequences at sorghum (Sorghum bicolor L.) in the pedoclimatic conditions of the Center of Moldova. Teză de doctorat. U.Ş.A.M.V. Iaşi 6. Uptomoor R., Wenzel W., Friedt W., Donaldson G., Ayisi K. and Ordon F., 2006 – Comparative analysis on the genetic relatedness of Sorghum bicolor accession from Southern Africa by RAPDs, AFLPs and SSRs. Journal Theoretical and Applied Genetics 106 (7) * ANOVA, 2013
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Poster Presentation – PP40
Phytochemical investigations, structural and ultrastructural aspects of the Passiflora caerulea L. plants cultivated in Romania
Simona Savin1, Agnes Toma1, Oana Craciunescu1, Anca Oancea1, Sorin Manoiu1, Tatiana Eugenia Şesan2,3, Anca Sarbu3, Daniela Smarandache3, Georgeta Negru4
1National Institute of Research-Development for Biological Sciences, 296, Splaiul Independentei, sector 6, 060031, Bucharest, Romania;; 2INCDCP (ICECHIM) Bucharest; 3University of Bucherest – Biology Faculty 4S.C. HOFIGAL Export Import S.A. România
Abstract. The present work describes the morpho-anatomical structure and ultrastructural aspects of Passiflora plants cultivated and acclimatized in Romania, associated with a phytochemical screening of some bioactive compounds. Key words : Passiflora, morpho-anatomy, TEM, phytochemical screening Introduction.The family Passifloraceae belongs to the order Malpighiales, class Magnoliopsida, and phylum Magnoliophyta [1], having approximately 630 different species. Many species of Passiflora are used for their medicinal properties (mild sedative and anxiolytic). In this study we have analyzed Passiflora cultivated in Romania in greenhouses, provided by S.C. Hofigal Export Import S.A.. The major phytoconstituents of the Passiflora plants are the alkaloids, phenolics, flavonoid, glycosides, cyanogenic compounds, passifloricins, polyketides and alpha-pyrones. We also studied some morphological and ultrastructural aspects of these plants. Material and methods. For morphological studies, fixed leaves and stem were dehydrated in ethanol and stained with Alum-Carmin and Green iodine [2]. For ultrastructural aspects, the fixed samples (glutaraldehyde 3%, paraformaldehyde 1,5% in phosphate buffer) were post-fixed with osmium tetroxide in phosphate buffer, dehydrated through ethanol and acetone series and embedded in epoxy resin. The specimen blocks were cut in ultrathin sections and analyzed with an electron microscope Philips EM208S (TEM). Phytochemical content: Passiflora plants were dried, grinded and extracted in ethanol / petroleum ether, in a ratio of 1,5:10 (w/v) and soak for 10 days. The extracts were qualitatively analyzed for different phytoconstituents, like tannins, polysaccharides, glycosides, triterpenoides, saponins and alkaloids. We also performed quantitative analysis (total polyphenolic content and flavonoids content) [3]. The antioxidant capacity of Passiflora extracts was evaluated by measuring the scavenging of the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the inhibition of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) cationic radical. [4] . Results and discussion. Morpho-anatomical aspescts of Passiflora plants: woody climbing plant, glabrous, with tendrils, growing to a heigth of 5-15 m. Leaves: sectate-palmate, palmate-lobed, generally 5-lobed, with entire margin. Petiole: 150-40 mm long, 2-4 until 6 petiol glands. Stipules: falciforme, semi-ovate, size: 10-20 mm x 5-10 mm. Bracts: 3 oval large bracts, green pale. Flowers: Blue and pink, time of flowering V-IX. Fruit: bright orange when ripes. The morphology of leaves (incuding petiole) and stem is presented in the figure 1. At ultrastructural level of Passiflora leves, we observed normal cells with cell wall, mitochondria, lysosomes, Golgi apparatus and chloroplasts that contain thylacoid system wich is suspended in stroma (Fig. 2). Concerning the phytochemical screening, we observed that the alcoholic extracts contained more bioactive compounds than petroleum ether extracts and saponins were detected only in ethanolic extracts.
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Fig. 1.: Morphology of leaves (incuding petiole) and stem. a) Cross-section through petiole; b) Cross section through the median foliar segment; c) Cross-section through the stem, highlighting epidermis, cortex and central cylinder elements.
Fig. 2. : Ultrastructural aspects of passiflora leaf Quantitative data showed that high amount of total polyphenols and flavonoids were found in ethanolic extracts of Passiflora leaves. Data obtained by the DPPH method shows that ethanol extract of Passiflora leaves had the largest content of free radical DPPH , with a concentration IC50 value of 54.01 µg/ml, similar to the standard BHT ( 57.16 µg/ml). Similar results were obtained for the antioxidant capacity studied through inhibition of the radical ABTS assay. Passiflora ethanol extract of leaves showed the highest antioxidant activity (79.77 ± 6.76 µmol TE / GDW). Conclusions. The morpho-anatomical and ultrastructural analysis allow a better understanding to the characteristics of the leaf and stem in Passiflora plants acclimatized in Romania. The highest concentration in polyphenols and flavonoids was recovered in the ethanoloic extracts of Passiflora. A correlation between the polyphenols/flavonoids content of Passiflora extracts and their antioxidant activity was observed. The highest values of antioxidant activity were calculated for the ethanolic plant extracts. Acknoledgement. This research was financially supported by the projects PN-II-PT-PCCA-2013-4- 0995-160/2014 (MAIA).
Bibliography 1. * * * Integrated Taxonomic Information System – IT IS – Report. http://www.itis.gov 2. O’Brien T. P & McCully M. E., 1981, The study of plant structure: principles and selected merthods, Melbourne, Termarcarphy Pty: 352 pp. 3. Oancea A., et al, 2013, Phytochemical screening of the bioactive compounds in the most widespread medicinal plants from Calarasi-Silistra cross-border area, Buletin of Transilvania University of Brasov, 6(55): 133-136. 4. Pourmorad F., et al (2006), Antioxidant activity, phenol and flavonoid contents of some selected Iranian medicinal plants, African Journal of Biotechnology, 5(11): 1142-1145.
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Poster Presentation – PP41
Centaurea cyanus L. extracts – source of antioxidants
Tatiana Chiru1, Anatolie Nistreanu1, Nicolae Ciobanu1, Ungureanu Ion2
1State University of Medicine and Pharmacy „Nicolae Testemitanu” of the Republic of Moldova 2Scientific Center for Cultivation of Medicinal Plants of SUMPh „Nicolae Testemitanu”
Abstract. We evaluated the in vitro antioxidant capacity of polyphenols extracts, employing DPPH, FRAP, ABTS methods and ferrozina test for iron chelating capacity. As part of this study, we have determined the total content of phenols, flavonoids, polysaccharides and the influence of different factors on extraction efficacy. Key words: Antioxidant activity,Centaurea cyanus L. Introduction. Literature data indicate polyphenols and polysaccharides as the main secondary metabolites of Centaurea cyanus L. [1,2,3,4]. As interesting compounds, various flavonoids [2], phenyl carboxylic acids [3] derivates were found. These substances are responsible for the majority of the oxygen capacity in most plant-delivered products [5]. Some works also mention polysaccharides as being responsible for pharmacological activities of Centaurea cyanus L. The polysaccharides fraction from Cyani flores developed anti-inflammatory effects in several models of inflammation and anti-complementary activity in rat serum in vitro [1]. However, up to date, no study has reported antioxidant activity of Centaurea cyanus L. polysaccharides. Material and methods. The extracts, obtained by different extraction techniques, were further analyzed to determine their total phenolic (Folin-Ciocalteau assay), flavonoids (with aluminum chloride), polysaccharides (phenol-sulfuric acid method) contents and antioxidant (DPPH scavenging, FRAP, ABTS assays; ferrozina test) activity. Results and discussion. In extracts the total phenolic content ranged from 156.63 to 388.81 mg/g dried weight, expressed as gallic acid equivalents. The content of flavonoids varied from 115.96 to 351.06 mg/g dried weight, expressed as rutin equivalents. The polysaccharides content was 2.66 % in Cyani herba and 1.59 % in Cyani flores extracts, expressed as glucose equivalents (Table 1). In all assays, the ethanolic extracts of Cyani herba showed the highest values of antioxidant activity (Table 2). A high correlation was found between the values for the total phenolic content and antioxidant activity. The results revealed that polysaccharides extract had a significantly (P < 0.05) higher Iron chelating activity (95.66 %) than phenolic extracts.
Table 1. Total polysaccharides and iron chelating activity of Centaurea cyanus L. extracts1,2,3 Total of polysaccharides, % Dried extract Iron chelating capacity, % expressed in glucose polysaccharides 2.66±0.02a 95.66±1.99a Cyani herba polyphenols N.T.- not tested 10.86±0.48b polysaccharides 1.59±0.18b 82.33±3.71c Cyani flores polyphenols N.T.- not tested 13.26±0.01d EDTA 95.37±3.37a The symposium is dedicated to the 60-year celebration of the „Stejarul” Biological Research Centre – MAPPPS 2016, Piatra Neamt, ROMANIA – Page 140
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1Mean of three replications ± standard error 2Means followed by the different small letters within a column denote significant differences (P<0.05) 3N.T.- not tested
Table 2. Antioxidant activity of the Cyani herba extracts1,2 DPPH, IC ABTS, µM TE/g FRAP, µM TE/g Cyani herba dried extract 50 µg/ml DW DW Methanol (after n-hexane 88.68± 0.63a 0.31±0.01a 26.15±5.31a and chloroform) Methanol 118.51±0.83b 0.51±0.01b 31.59±1.03b Ethanol 60% 71.88±0.51c 0.54±0.02b 52.43±0.06c 1Mean of three replications ± standard error 2Means followed by the different small letters within a column denote significant differences (P<0.05)
Conclusions. The present study was performed to determine the total phenolic, flavonoids, polysaccharides contents and antioxidant activities, together with their correlation values, for polyphenols and polysaccharides extracts of C. cyanus L. It was noticed that the highest concentration of polyphenols and flavonoids was obtained using 60% aqueous ethanol as a solvent. The highest concentration of polysaccharides was found in Cyani herba extract. In respect of all obtained results, it can be concluded that C. cyanus L. extracts could be regarded as a source of polyphenols and polysaccharides with powerful antioxidant activity.
Bibliography 1. Garbacki Nancy, Gloaguen V., Damas J. et al.(1999), Anti-inflammatory and immunological effects of Centaurea cyanus flower-heads, Journal of Ethnopharmacology, 68(1-3): 235-241. 2. Litvinenko V. I., Bubencikova V. N. (2007), Phytochemical study of Centaurea cyanus L., Chemistry of Natural Compounds, 24(6): 672-674. 3. Muravieva D. A., Bubencikova V. N.(2007), Phenolcarboxylic acids of the flowers of Centaurea cyanus L., Chemistry of Natural Compounds, 22(1): 102. 4. Pîrvu L., Coprean D., Schiopu D. et al. (2012), Vegetal extracts with gastroprotective activity. Part I. Extracts obtained from Centaurea cyanus L. raw material, Romanian Biotechnological Letters, 17 (2): 7169–7176. 5. Atoui A.K., Mansouri A. et al.(2005), Tea and herbal infusions: their antioxidant activity and phenolic profile, Food Chemistry, 89: 27-36.
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Poster Presentation – PP42
Effects of foliar ecological fertilization on inflorescence yield and chlorophyll parameters of Calendula officinalis L.
Vasilica Onofrei1*, Bernd Honermeier2, Andrei Lobiuc3,4, Marian Burducea3, Gabriel-Ciprian Teliban1, Carmenica Doina Jităreanu1, Remus Ciprian Cotunoaea3, Teodor Robu1
1"Ion Ionescu de la Brad" University of Agricultural Sciences and Veterinary Medicine of Iaşi, Mihail Sadoveanu Str. 3, Iaşi, 700490, Iaşi, Romania; 2Institute of Agronomy & Plant Breeding I, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, Schubertstr. 81, D-35392 Gießen; 3„Alexandru Ioan Cuza” University of Iasi, Carol I Bd., 11, 700506, Iaşi, Romania; 4”Stefan cel Mare" University of Suceava, Universitatii Street, 13, Suceava, Romania *Corresponding author, e-mail: [email protected]
Abstract. The present paper aims to evaluate the marigold inflorescence yield, chlorophyll content and chlorophyll fluorescence of Calendula officinalis L. under ecological fertilization with four different foliar fertilizers (Fylo®, Geolino Plants&Flowers®, Cropmax®, Fitokondi®). The flowers yield was increased in all fertilized plants, compared to unfertilized ones, but the content of chlorophyll pigment and chlorphyll fluorescence values were not significantly influenced. Key words: organic, marigold, yield dynamic, medicinal plants, chlorophyll Introduction. Cultivation of medicinal plants is wide spred today due to their outstanding properties owing to their biological active compounds [9,5] Calendula officinalis L. is an annual herb bellonging family Asteraceae, with yellow to organe flowers, native to Mediterranean region [3,6]. It is also known as pot marigold, a name historically associated with its use in soups and stews to combat illnesses [8]. Calendula officinalis L. is an important medicinal plant with antiphlogistic, choleretic, antibacterial, antimicrobial, antidermatitic, antimutagenic and anticancer effects, therapeutic properties determined by a diverse range of biologically active substances they contain (carotenoids, triterpenoids, flavonoids, glycosides, flavones, volatile oil, coumarins, minerals, mucilages, vitamin C, cholesterol esters, amino acids) [1,4]. As agriculture has a significant impact on the human health and the environment, recent years have seen growth in sustainable agricultural aproaches including products marketed as “organic” [2]. The EU recognizes the benefits offered by organic farming, with The Common Agricultural Policy (CAP) considering organic farming an important element for the development of the European agricultural systems. The objective of the current study was to evaluate the effects of selected ecological foliar fertilizers on the inflorescence yield and functioning of the photosynthetic apparatus of marigold. Material and methods. Seeds of Calendula officinalis L. cv. Orangefarbige were sown on 8th of May, 2015 in the research field of the U.A.S.V.M. Iaşi, Romania. The foliar fertilizers used are complex solutions, containing plant growth stimulators (auxins, cytokinins, gibberellins), organic acids, vitamins, plant enzymes, trace elements (Mg, Zn, Mn, Cu B, Ca, Mo, Co, Ni). The experimental variants were: control, Fylo® (0,25%), Geolino Plants & Flowers® (0.1%), Cropmax® (0.1%) and Fitokondi® (0.1%). Some of the characteristics of fertilizers are given in Table 1. Fertilization was applied twice (26.06 and 18.07.2015). Assimilatory pigments were measured with a portable CCM-200 Plus device, while chlorophyll fluorescence (Fv/Fm) was measured using a FMS2 portable fluorometer (Hansatech Ltd., UK). Statistical analyses performed: ANOVA and Tukey tests. Table 1. Physico-chemical parameters of the ecological foliar fertilizers Foliar fertilizer pH N% P% K% FYLO 4.37 32.33 1.28 1.04 GEOLINO 4.94 18.72 0.64 7.2 CROPMAX 4.5 0.2 0.4 0.02 FITOKONDI 4.5 0.02 0.01 0.26 Results and discussion. The highest flower yield was recorder for all treatments, at the end of July and at the end of August, with maximum values in Cropmax treatment (Fig. 1). The highest average yield was
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obtained in Fylo treatment (249.2 kg/ha) followed by Cropmax (227.1 kg/ha), Fitokondi (213.7 kg/ha) and Geolino (200.8 kg/ha) treatments, all fertilizers increasing yield compared to control plants (188.6 kg/ha). Our results are similar to other research, Rafie et al. 2013 [7], found that foliar application of Humiforte, 1.5 l/ha-1 caused an increase of flower dry weight with 36.92%.
Figure 1. Yield of marigold cultivated under foliar ecologic fertilization Considering that chlorophyll fluorescence may reflect different types of stress, no such effect were recorded on marigold plants under ecological foliar fertilization as Fv/Fm values are not significantly different between treatment (Table 2). Moreover, the photosynthetic apparatus was not influence considering that chlorophyll content values are also similar among treatments (Table 2). The most pronounced fertilization effect was recorded for the fertilizer with the highest nitrogen content, however two other fertilizers containing lower amounts of nitrogen led to yield increases in marigold. This suggests that not only macronutrient inputs may elevate yield, but also micronutrients and growth stimulators play an important part in plant fertilization. Table 2. Assimilatory pigments contents and chlorophyll fluorescence of marigold under ecologic foliar fertilization Treatment / Parameter Assimilatory pigments (CCI units) Chlorophyll fluorescence (Fv/Fm) CONTROL 20.41±1.17 0.89±0.02 FYLO 21.5±1.5 0.89±0.01 GEOLINO 20.4±0.87 0.91±0.01 CROPMAX 22.26±2.52 0.86±0.03 FITOKONDI 21.03±1.2 0.9±0 Conclusions Foliar fertilizers treatments influenced the culture of Calendula officinalis L. in the first year of cultivation. These partial results are the starting point for future analysis and experiments regarding quality and yield of marigold. Such foliar fertilizers can be recommended for ecological cultivation of marigold as a medicinal plant with important therapeutic properties. Bibliography 1. Britton G., Liaaen-Jensen S., Pfander H. (1995), Carotenoids, Basel, Birkhauser Verlag, Vol. 1A. 2. Butnariu M. and Coradini C.Z., (2012), Evaluation of Biologically Active Compounds from Calendula officinalis flowers using Spectrophotometry, Chemistry Central Journal, 6:35. 3. Crăciun, F., O. Bojor, M. Alexan (1997), Farmacia naturii vol I-II, Ed. Ceres Bucuresti. 4. Hawkins G., Burnett S.E., Stack L.B. (2012), Survey of Consumer Interest in Organic, Sustainable, and Local container-grown Plants in Maine. Horttechnology 22 (6):817-825. 5. Honermeier B., Ali S., Leschhorn B., Mahmood A., Ijaz M., Russo M., Hajiabad S.M., Ullah H., Zeller S. (2013), Cultivation of Medicinal and Spice Plants in Germany, International Journal of Agriculture&Biology, 15: 1379-1388. 6. Păun E., Mihalea A., Dumitrescu A., Verzea M., Coșocariu O. (1988),Tratat de plante medicinale și aromatice cultivate, vol. II, Editura Academiei, București. 7. Rafiee H., Mehrafarin A., Qaderi A., Kalate Jari S., Naghdi Badi H. (2013), Phytochemical, Agronomical and Morphological Responses of Pot Marigold (Calendula officinalis L.) to Foliar Application of Bio-stimulators (Bioactive Amino Acid Compounds), Journal of Medicinal Plants, 3(47): 48- 61. 8. Ramos A., Edreira A., Vizoso A., Betancourt J., López M., Décalo M. (1988), Genotoxicity of an extract of Calendula officinalis L., J. Ethnopharmacol., v. 61. 9. Robu T., Milică C. (2004). Plante medicinale autohtone, Editura Institutul European. Iași.
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Poster Presentation – PP43
Generating Diversity in Natural Product Scaffolds. Synthesis of ent-Kauranic Derivatives Functionalized with Triazole Fragments
Vladilena Gîrbu1,2, Marina Grinco1, Nicon Ungur1,2, Veaceslav Kulciţki1,2, Philippe Renaud3
1Institute of Chemistry of the Academy of Sciences of Moldova, Chişinău, Republic of Moldova 2University of the Academy of Sciences of Moldova, Chişinău, Republic of Moldova 3Department of Chemistry and Biochemistry, University of Bern, Switzerland
Abstract. The current work presents the first results on the application of the radical hydroazidation methodology for the functionalization of ent-kaurenic derivatives which represent an important family of natural diterpenoids with relevant biological activities. Following conversion of obtained azide to different triazoles containing hybrids was acheived via a click-chemistry procedure involving several alkynes. Key words: Hydroazidation, click reaction, radical chemistry, diterpenes. Introduction. Organic azides have been paid a relevant attention in chemistry, biology and medicine. Different transformations are employed in order to convert azides, including reduction to amines, aza- Wittig reaction and Staudinger ligation. But the most explored field connected to the azide functionality relates to the famous “click” reaction [1] – a cycloadition of an azide to alkynes leading to triazoles. This transformation is extreamily facile and represents an efficient tool for natural product modification, conjugation or covalent immobilization that is of relevant importance both for academia and pharmaceutical industry. In particular, natural products of terpenic structure have been in the focus of click chemistry procedures [2] due to the chemical stability and stereoelectronical properties of the triazole moieties, which are mimics of amide bonds. For this reason, 1,2,3-triazole ring in medicinal chemistry is now often considered as an active pharmacophore rather than a neutral linkage. On the other hand, diterpenoids of ent-kauranic structure occur broadly in plants and are known for their diverse biological activities. Unfortunately, one of the most abundant representatives available from the waste of sunflower (Helianthus Annuus L.) – ent-kaurenoic acid 1 is relatively poorly decorated with functional groups that limits its potential applications. Therefore, chemical functionalization of the ent-kauranic framework using incorporation of triazole fragment represents a potential way for broadning the activity spectrum of this family of diterpenoids. Material and methods. ent-Kaur-16-en-19-oic acid 1 was isolated from the wastes of sunflower as described previously [3]. Methyl-ent-kaurenoate 2 was obtained on methylation of 1 with an etherial solution of diazomethane. Radical hydroazidation of 2 was performed according to the described procedure [4]. Shortly, hydroboration of 2 was performed with 3 equivalents of catecholborane (HBCat) in dichloromethane, using N,N-dimetilacetamide (DMA) as catalyst. Excess borane was neutralized with tert-butanol, and solvents have been removed in vacuo. The crude organoboron compound was treated in situ with 3 equiv. of 3-pyridylsulfonyl azide and 0.1 equiv. of radical initiator - di-tert-butylhyponitrite (DTBHN) in DMF. As a result, the ent-kaurenic azide 3 was obtained in a 67% yield. Click reaction of 3 was performed with a set of alkynes using CuI and DIPEA as catalysts in DCM as described elswere [5]. Results and discussion. Due to its abundancy from plant material, ent-kaurenoic acid derivatives have been broadly used for chemical modification and structure-activity relationship (SAR) studies [6]. In the most examples, the reactivity of the carboxilic group, as well as oxidation ability of the
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exomethylenic double bond were used in order to generate structural diversity. We have identified an additional opportunity for structural modification of the ent-kaurenic scaffold by incorporation of a new C-N bond at the C-17 exomethylenic group by a free radical azidation procedure. This kind of transformation is now considered a very promising late stage functionalization method in natural product chemistry and following conversion of the azide moiety to other nitrogen containing functionalities is challenging. In particullar, conversion of the azide to triazoles by cycloaddition to diverse alkynes can be a path for very flexible structural modification and generation of an infinite number of derivatives for SAR studies. Such a functionalization pattern, based on carbon chain elongation at C-17 position have been recently reported in ent-kauranes from sunflower [7] and our efforts come to broaden the mollecular diversity in this direction basing on radical chemistry procedures. We have performed attachment of the triazole fragment in a two-step sequence, including hydroboration-azidation of the starting ester 2, followed by click reaction with several alkynes (scheme below). In order to confirm the structure of the azide 3, we have performed an alternative synthesis, based on a hydroboration-oxidation of 2, followed by mesylation and nucleofilic displacement for an azide group. The spectral data of azide 3 obtaied by both paths were identical.
Reagents and conditions: (a) HBCat, DMA, DCM, reflux, 5h; (b) 3-PySO2N3, DTBHN, DMF, 80°C, 2h, 67%; (c) BH3Me2S, THF, 0°C, 2h; (d) NaOH 15%, H2O2, 12h, t.c., 92%; (e) MsCl, Et3N, DCM, 0°C, 2h, 91%; (f) NaN3, DMF, 80°C, 12h, 87%; (g) CuI, DIPEA, AcOH, DCM, alkynes 4-7, t.c., 3h.
Cycloaddition to alkynes 4-7 occured with good to excellent yields and the resulting triazoles 8-11 have been characterized by the full set of phisico-chemical data (1H- 13C NMR, IR, optical rodation and elemental analysis). Following studies on the activity profile of new ent-kauranoic triazoles are on the way. Conclusions. The present work demonstrates utility of the radical hydroazidation of terpenic olefins. This functionalization method allows a one step, high yielding introduction of the azide functional group in the molecule of the terpenic substrate. The following transformation of the azide functionality was demonstrated by an efficient “click” reaction, leading to a triazole-linked prenylated hybrid. Acknowledgements. The presented work was performed within the project “Radical mediated modifications of natural products” supported financially by the Swiss National Science Foundation (SCOPES program, project No. IZ73Z0_152346/1). Bibliography 1. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128. 2. Kacprzak, K.; Skiera, I.; Piasecka, M.; Paryzek, Z. Chem. Rev. 2016, 116 (10), 5689–5743. 3. Ungur, N.; Grinco, M.; Kulciţki, V.; Barba, A.; Bîzîcci, T.; Vlad, P.F. Chem. J. Mold. 2008, 3(2), 105-108. 4. Kapat, A.; Kӧning, A.; Montermini, F.; Renaud, P. J. Am. Chem. Soc. 2011, 133, 13890–1389. 5. Shao, C.; Wang, X.; Zhang, Q.; Luo, S.; Zhao, J.; Hu, Y. J. Org. Chem. 2011, 76, 6832–6836. 6. Morărescu, O. Chem. J. Mold. 2015, 10(1), 9-19. 7. Torres, A.; Molinillo, J.M.G.; Varela, R.M; Casas, L.; Mantell, C.; Martínez de la Ossa, E.J.; Macías, F.A. Org. Lett., 2015, 17 (19), 4730–4733.
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Poster Presentation – PP44
Comparison of essential oil compositions of fresh and dried plant of endemic Salvia cadmica Boiss. var. bozkiriensis Celep, Kahraman & Doğan, in Turkey
Süleyman Doğu1, Sadiye Ayşe Çelik2, Yavuz Bağcı3
1Dept of Biology, Faculty of Education Meram, Necmettin Erbakan Univ., TR 42090 Konya, Turkey 2 Department of Field Crops, Faculty of Agricultural, Selcuk University, Agriculture Faculty, 42049, Konya, Turkey 3Department of Biology, Faculty of Science and Art, Selcuk University, Konya, Turkey
Abstract. In this study, essential oil compositions of Salvia cadmica Boiss. var. bozkiriensis Celep, Kahraman & Doğan, (dried and fresh aerial parts) collecting from type locality was investigated. Essential oil was obtained by hydrodistillation for 3 h using Clevenger type apparatus and the compositions was determined in GC-MS. In this research, it was observed that the Essential Oil compositions varied with respect to be fresh or dry of the plant parts. Key words: Essential oil, endemic, fresh parts, dried parts, Salvia cadmica var. bozkiriensis Introduction. The genus Salvia, with about 700 species and represented in Turkish flora by 88 species and 45 endemics, is one of the most widespread members of the family Lamiaceae. An unusually large number of useful secondary metabolites belonging to various chemical groups, such as essential oils, terpenoid compounds, and phenolic derivatives, have been isolated from the genus, which features prominently in the pharmacopoeias of many countries throughout the world. Material and methods. Fresh aerial parts of Salvia cadmica var. bozkiriensis was collected during the flowering period from Bozkır Konya in 2011 and the aerial parts were dried in the shade at room temperature. Plant was identified by Dr. Bağcı, and a voucher specimen (Doğu 3421 & Bağcı) is kept at the herbarium of the Biology Department, University of Selçuk, Turkey. Aerial parts (dried and fresh branch, leaf and herb) of the Salvia cadmica Boiss. var. bozkiriensis were subjected to hydrodistillation for 3 h using Clevenger type apparatus to produce essential oil. The essential oils were stored at -200C until analyzed. Results and discussion. Results was indicated that there were significant (p<0.01) differences between the the aerial parts of dried and fresh Salvia cadmica var. bozkiriensis with respect to their essential oil compositions. The oil yields of the the plants was determined to be in amount trace. The LSD test results revealed that the highest EO content was 2-nonanone (29.59 %), followed by 6- methyl-3,5-heptadien-2-one (12.64 %), 2-nonanol (7.90), β-ocimene (7.81 %) and delta-decalactone (4.92 %) in fresh aerial part of the plant. essential oil composition may vary considerably between aromatic plant species and varieties, and within the same variety from different geographic areas [1] Conclusions. Some components were observed in the fresh aerial parts, while it was not found in dried parts of the plant (for example; lindoxide, 2-oxapropanoic acid, α-terpineol, α-terpinene, carvone, geranyl butyrate, valealdehyde and safranol).
Bibliography 1. J. A. Zygadlo and H. R. Julianı (2003). In: Majunder DK, Govil, JN, Singh VC (eds.) Phytochemistry and Pharmacology. 8, 273.
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Poster Presentation – PP45
The Medical Plants of Karaman-Yeşildere Village and Its Surroundings
Turan Akdağ1, Süleyman Doğu2
1Dept of Animal and Plants Production, Seydişehir Vocational School, Necmettin Erbakan University, Konya, Turkey 2Department of Biology, Ahmet Keleşoğlu Faculty of Education, Necmettin Erbakan University, Konya, Turkey
Abstract. Present study was carried out between 2014-2015 years at area of Karaman Yeşildere residents and surrounding villages in order to determine the plants which used for medicinal purposes. A total of 21 taxa belonging to 8 families have been identified in the end of the research. According to the survey, Lamiaceae family is the most taxa family for used treatment. Key words: Medical plants,Yeşildere, Turkey Introduction. Today, the majority of the world population still uses raw material medicine plant for treatment. Especially in developing countries, a large part of the population try to solve the health problems from traditional medicinal plants, firstly. Approximately 80% of the world's population constituted from developing countries, so more than half of the total world population use plants for therapeutic purposes. Material and methods. The material of the study were collected between April and September of the 2014-2015 years who gathered at Yeşildere village (Karaman) and around the village. Examples of plants used as treatment against diseases. Local names given by villagers, parts of plants used and usage patterns have been identified with face to face interviews. Plant specimens dried in the herbarium and identified from the book of the "Flora of Turkey and the East Eagen Islands" [1]. Results and discussion. The villagers used the plants for treatment diseases such as; gastrointestinal diseases, cough corrector, opening of the abscess, painkiller, skin diseases, urine enhancer, tonic, antipyretic, depression, sinusitis, human diarrhea, wound mouth, hemorrhoid, heatstroke and rheumatic diseases. The medicinal use of two Salvia species which have not been previously identified as therapeutic use has been demonstrated for the first time in the study. Conclusions. A total of 21 taxa belonging to 8 families have been identified in the research. As a result, Lamiaceae family is the most taxa family for used treatment.
Bibliography 1. Davis PH (1965-1985) Flora of Turkey and the East Aegean Islands. Vol. 1-9. Edinburgh: Edinburgh University Press.
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Poster Presentation – PP46
Antioxydant activity and preventive possibility of Algerian medicinal plant Matricaria pubescens on hepatic toxicity
1Boubellouta Houria, 2Khelifi Touhami Fatima, 3Kermandji Mohamed Azed, 4Abidli Nacira, 5Bellatrache Cherifa
1Department of Biology. University of Bejaia. Bejaia. Algeria; 2Department of Animal Biology. University of Constantine 1. Constantine. Algeria; 3Department of Veterinary Medicine. University of Constantine 1. Constantine. Algeria; 4ENS Kouba. USTHB. Algiers. Algeria; 5Department of Clinical Biochemistry. University of Constantine 1. Constantine. Algeria
Abstract. The present survey is centered on protective effect of Matricaria pubescens against liver injury induced by carbon tetrachloride in rats. In our study, we indicated that these plants treatment have a potent protective effect as revealed by remarkable decrease in MDA contenent, additionally, methanolic extract could ameliorate acute liver damage to a high degree, as demonstrated by reduction of serum AlT levels. Introduction. The liver is a target organ for the many chemical products. CCL4 is a classically known compound that causes hepatotoxicity by an acute exposer (Recknagel, 1967 ). Matricaria pubescens plant of family compositea used in Algeria for the treatment of hepatic diseases. The main objective of the present work was to evaluate the hepatoprotective and antioxidant activity of Matricaria pubescens against liver injury induced by carbon tetrachloride in rats. Materials and methods. Extraction of methanolic extract: The aerial parts (soft twigs and leaves) of Matricaria pubescens is air-dried and extracted with methanol. Determination of free radical scavenging activity: The free radical scavenging activity of the extract was determined by the method described by (Burits and Bucar, 2000). Determination of total phenolic content and flavonoids: Total soluble phenolics in the methanol extract of Matricaria pubescens were determined with Prussian bleau according to the method of Price and Butler (1977), with slight modifications of (Graham 1992), using gallic acid as a standard compound. The flavonoids content in the methanol extract of Matricaria pubescens was determined by (Boharun et al., 1996) method using quercetin and rutin as a reference compound. Pharmacological essays (Study carry out on rats): Table 1: Distribution of animals and administration of the tested substances
Receive Control 1 3 ml/ kg of olive oil by a single intraperitoneal injection Control 2 3 ml/kg of CCL4 by a single intraperitoneal injection Group 3 Matricaria pubescens alone (800 mg/kg) Matricaria pubescens (800 mg/kg) + 3 ml/kg of CCL4 by a single Group 4 intraperitoneal injection after 4 weeks of treatment. All of the animals were sacrificed 24 h after administration of CCl4, and blood was collected, serum separated and stored at −20 ◦C. Hepatotoxicity assessment: The hepatic enzyme ALT, were used as the markers for early acute hepatic damage. The serum activities of ALT was determined by using Auto analyzer (Architedt c system). Estimation of hepatic thiobarbituric acid reactive substances (TBARS): The hepatic TBARS level, an index of malonyldialdehyde (MDA) production was determined by the method of Ohkawa et al. (1979). Results and discussion. The scavenging effect of methanol extract of Matricaria pubescens and standards with the DPPH radical is in the following order: rutin (0.361 mg/ml)> quercetin (0.34 mg/ml)> Matricaria pubescens (0.162 mg/ml) > gallic acid (0.058 mg/ml). The experimental data of these species reveal that this extract have the effect of scavenging free radical.
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In the Matricaria pubescens (1g), 53.27 mg gallic acid equivalent of phenols respectively was detected. Phenolic compounds are known as powerful chain breaking antioxidants. Phenols are very important plant constituents because of their scavenging ability due to their hydroxyl groups.
The total flavonoid contents present in Matricaria pubescens was shown in the Matricaria pubescens (1g), 20.00 mg quercetin and 43.44 mg rutin equivalent of flavonoids / weight dry plant respectively was detected. The activities of serum ALT in the CCl4 group were much higher than those in the control group. However, pre-treatment with Matricaria pubescens significantly prevented the elevation of serum ALT activities induced by CCl4 treatment. So the pre-treatment with Matricaria pubescens at a dose of 800 mg/kg prevented the elevation of ALT levels.
As shown in Fig. the concentration of MDA, an end product of lipid peroxidation, in the rats treated with CCl4 was increased 2.7-fold when compared with the vehicle control rats. Consistent with the serum ALT activitie, pre-treatment with Matricaria pubescens for 4 weeks to the rats resulted in a significant decrease in the concentration of hepatic MDA when compared with the CCl4 group.
Conclusion. Our investigation provided convincing data that Matricaria pubescens have an impressive hepatoprotective effects on acute liver injuries induced by CCL4. The mechanisms underlying hepatoprotection of the methanolic extract of Matricaria pubescens may be related to both its radical scavenging properties and indicate effects as a regulator of antioxidative systems, which might be considered to be therapeutic in clinical situations.
References: Bouharun, T., Gressier, B., Trotin, F., Brunet, C., Dine, T., Vasseur, J., Gazin, J.C., Pinkas, M., Luyckx, M. and Gasin, M. (1996). Oxygen species scavenging activity of phenolic extracts from Hawthorn fresh plant organs and pharmaceutical preparation. Arezneim-Forsh/ Drug Res. Burists, M., Bucar, F. (2000). Antioxidant activity of Nigella sativa essential oil. Phytother Res. 14: 323-328. Okhawa, H., Ohishi, N. and Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Add. Biochem. 95: 351-358. Pereira. R. P., Fachinetto.R ., Prestes.A.P., Puntel.R.L., Silva.G.N.S., Heinzmann.B.M., Boschetti.T.K., Athayde.M.L., Bu¨rger.M.E., Morel.A.F., Vera Maria Morsch.V.M and Rocha.J.B.T. (2008).Antioxidant Effects of Different Extracts from Melissa officinalis, Matricaria recutita and Cymbopogon citrates. Springer Science+Business Media, LLC. Price, M.L. and Butler, L.G. (1977). Rapid visual estimation and spectrophotometric determination of the tannin content of sorghum grain. J Agric Food Chem. 25: 1269-1273. Prosovskii M. A and Oleshko.G.I. (1986). Phenolic compounds of Matricaria discoidea. Plenum Publishing Corporation pp. 671. Recknagel, R.O. (1967). Carbon tetrachloride hepatotoxicity. Pharmacol. Rev. 19, 145-/208.
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Poster Presentation – PP47
Phenolic contents and antioxidant activities in Phaseolus coccineus L. flowers
Gabriel Teliban1, Marian Burducea2, Andrei Lobiuc2,3*, Elida Rosenhech2, Vasile Stoleru1, Vasilica Onofrei1, Maria-Magdalena Zamfirache2, Neculai Munteanu1
1„Ion Ionescu de la Brad” University of Agricultural Sciences and Veterinary Medicine of Iaşi, Mihail Sadoveanu Str. 3, Iaşi, 700490, Iaşi, Romania; 2„Alexandu Ioan Cuza” University of Iasi, Carol I Bd., 11, 700506, Iaşi, Romania; 3„Stefan cel Mare” University of Suceava, Universitatii Street, 13, Suceava, Romania *Corresponding author, e-mail: [email protected]
Abstract. Phaseolus coccineus is an important crop due to its high amount of protein and it is largely cultivated worldwide. Little is known about the contents of health related compounds in runner bean flowers, although they are edible, but rarely used. The present paper aims to assess the amounts of total polyphenolics, flavonoids and anthocyanins as well as antioxidant activity in several runner bean cultivars flowers. Key words: runner bean, total polyphenolics, total flavonoids, free radical scavenging, anthocyanins Introduction. Legumes are a rich source of proteins and contribute to soil conditioning [1], with Phaseolus coccineus L. (runner bean) offering high beans and pods yields. Although not the main plant parts consumed, flowers are edible, in some cuisines representing a delicacy [2]. In food preparation, flowers are used for improving aesthetics, odor and taste of foods. Aside from these properties, flowers in food are regarded as supplements in treating disorders related to the respiratory, gastric, skin etc. systems, due to the bioactive substances contained in flowers such as vitamins, minerals, flavonoids, anthocyanins, carotenoids etc. Examples of such species used in foodstuffs are chrysanthemum, dianthus, marigold, rose and tulips [3]. For Phaseolus coccineus, few reports indicate the use of flowers in foods, although several cultivars with different colours of flowers exist [4], including red and purple varieties, which indicate the presence of phenolic compounds. Therefore, the present study investigated the phenolic contents and antioxidant activity of different Phaseolus coccineus cultivars in order to justify the possible gastronomical usage of flowers of this specie. Material and methods. Flowers of Phaseolus coccineus L. were obtained from the University of Agricultural Sciences and Veterinary Medicine, Iaşi. Plants belonged to 4 cultivars raised for pods (“Lady Di”, “Desiree”, “Polestar”, “Tenderstar”) and 4 local populations raised for beans (P1, P2, P3 and P4). Total phenolic and flavonoid contents and antioxidant activity were determined in 5 g fresh petals/95 ml extracts macerated in 70% v/v ethanol for 1 hour. Total phenolic content was assessed using the Folin Ciocalteu reagent method, while total flavonoid content was determined using the AlCl3 method, according to [5]. Free radical scavenging activity was determined by the DPPH method [6]. Anthocyanin content was determined as described in [7], in 5 g of fresh petals in 95 ml of ethyl alcohol:distilled water:hydrochloric acid mixture (75:24:1) extracts. Results were expressed as means ± standard errors. Results and discussion. Anthocyanin contents varied among cultivars and populations (Table 1) with highest values in red flowered cultivars, intermediate values in red and white flowered cultivars, while no anthocyanins were found in the white flowered cultivars petals. White flowered cultivars had the highest flavonoid contents, up to two times higher than other cultivars (Table 1). Total polyphenolic content was highest in populations P2 and P3 of the cultivar raised for beans (Table 2). Generally, higher contents were registered in red and red and white flowered cultivars, suggesting the contribution of anthocyanins to the elevated values. Antioxidant capacity followed, generally, the results of the total polyphenolic content, with higher values in red and red and white flowered plants, especially in P2 and P3 populations of the cultivar raised for beans (Table 2). The total phenolic content and flavonoid content of analysed runner bean material are comparable with those of other edible flowers with high medicinal and nutritional value such as those of tagetes, marigold, bougainvillea [8]. Antioxidant activity registered high values, up to 51
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%, similar to other flowers with high antioxidant capacity such as multiflower rose [9]. The presence of phenolic compounds, such as anthocyanins and flavonoids are associated with important health benefits such as antioxidant, antimicrobial, antiinflammatory, antihyperglycaemic, antigenotoxic or astringent activities [10]. Table 1. Anthocyanin and flavonoid contents in Phaseolus coccineus flowers extracts Anthocyanin content mg cyanidin- Flavonoid content mg Phaseolus coccineus material Flower color 3-glucoside/g fresh weight quercetine/g fresh weight “Lady Di” cultivar Red 1.36±0.04 0.68±0.07 “Polestar” cultivar Red 1.21±0.04 0.5±0.23 “Tenderstar” cultivar Red and white 0.73±0.02 0.34±0.07 “Desiree” cultivar White 0±0 1.25±0.16 Population P1 Red 1.34±0.04 0.39±0.12 Population P2 Red 1.23±0.08 0.2±0.05 Population P3 Red and white 0.43±0.01 0.43±0.12 Population P4 White 0±0 1.09±0.23
Table 2. Total polyphenolic content and antioxidant capacity of Phaseolus coccineus flowers extracts Total polyphenolic content mg DPPH antioxidant Phaseolus coccineus material Flower color galic acid/g fresh weight capacity % “Lady Di” cultivar Red 3.56±0.01 38.46±2.37 “Polestar” cultivar Red 3.48±0.11 43.86±0.91 “Tenderstar” cultivar Red and white 3.98±0.12 47.03±0.88 “Desiree” cultivar White 2.38±0.08 19.1±1.11 Population P1 Red 3.31±0.07 41.8±0.87 Population P2 Red 4.05±0.13 47.49±1.46 Population P3 Red and white 5.18±0.54 51.12±1.79 Population P4 White 2.15±0.05 20.35±1.41
Conclusions. The results recommend the use of flowers of runner bean cultivated for beans and pods in food preparations, having valuable characteristics and should be further analyzed from a chemical composition point of view.
Acknowledgements. This paper was published under the frame of European Social Fund, Human Resources Development Operational Programme 2007-2013, project no. POSDRU/159/1.5/S/132765. Some of the infrastructure used was provided by CERNESIM Project (SMIS/CNMR Grant Nr. 13984/901).
Bibliography 1. Linnemann A.R., Dijkstra D.F. (2002), Toward Sustainable Production of Protein-Rich Foods: Appraisal of Eight Crops for Western Europe. PART I. Analysis of the Primary Links of the Production Chain, Critical Reviews In Food Science And Nutrition Vol. 42 , Iss. 4, 377-401. 2. Facciola S. (1990), Cornucopia - A Source Book of Edible Plants. Kampong Publications, 470 3. Mlcek J., Rop O. (2011), Fresh edible flowers of ornamental plants – A new source of nutraceutical foods, Trends in Food Science & Technology, 22(10):561-569. 4. Sicard, D., Nanni, L., Porfiri, O., Bulfon, D., Papa R. (2005), Genetic diversity of Phaseolus vulgaris L. and P. coccineus L. landraces in central Italy. Plant Breeding, 124: 464–472 5. Herald T.J., Gadgil P., Tilley M. (2012), High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour. Journal of the Science of Food and Agriculture, 92(11):2326-2331. 6. Molyneux P. (2004), The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity, Songklanakarin Journal of Science and Technology, 26(2):211-219. 7. Lee J., Durst R.W., Wrolstad R.E. (2005), Determination of Total Monomeric Anthocyanin Pigment Content of Fruit Juices, Beverages, Natural Colorants, and Wines by the pH Differential Method: Collaborative Study, Journal of AOAC International, 88(5):1269-1278. 8. Petrova I., Petkova N., Ivanov I. (2016) Five Edible Flowers – Valuable Source of Antioxidants in Human Nutrition, International Journal of Pharmacognosy and Phytochemical Research, 8(4):604-610. 9. Kwak C.S., Choi H.I., Yang J. (2016), Antioxidant activity of Rosa multiflora Thunb. flower extract and suppressive activity on proinflammatory mediator production in lipopolysaccharide- stimulated RAW 264.7 macrophages, Functional Foods in Health and Disease, 6(5):265-278. 10. Kaisoon O., Siriamornpun S., Weerapreeyakul N., Meeso N. (2011), Phenolic compounds and antioxidant activities of edible flowers from Thailand, Journal of Functional Foods, 3(2):88-99.
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Poster Presentation – PP48
Preliminary phytochemical study of Prunus spinosa L. buds harvested from natural populations of Dobrogea area
Georgiana Gavril1*, Valentin Grigoras1, Ruxandra Cretu1, Radu Necula1, Elvira Gille1, Ursula Stanescu2 *corresponding author: [email protected]
1NIRDBS/„Stejarul” Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt 2Faculty of Pharmacy, University of Medicine and Pharmacy “Grigore T. Popa”, Iasi, Romania
Abstract. This paper refers to the analysis of phenolic fraction separated with methanol from buds of Prunus spinosa L. species. A preliminary qualitative and quantitative chemical analysis of components such as flavonoids and polyphenolic acids was performed, using thin-layer chromatography and spectrophotometry methods. Key words: Prunus spinosa L., buds, flavonoids, phenolic acids.
Introduction. In a project that performs researches on flora of the Danube Delta areas, we have chosen for this study the Prunus spinosa L. species, wide-spread and commonly used in traditional medicine from the area. According to current knowledge, various organs (fruits, flowers, bark and roots) are used in phytotherapy for treatment of respiratory diseases, as antispasmodic, diuretic, laxative and anti-inflammatory [1]. The phytochemical researcheas carried out till now refer to the chemical composition, especially for fresh fruits, which contain polyphenols, vitamin C, anthocyanins and beta-carotene [2], while fresh flowers are rich in flavonoids, the major compounds identified being kaempferol, quercetin, kaempferol-3-O-arabinofuranoside [1], with a cyanogenic glycosides. Due to the content in polyphenols, the fruits and fresh flowers develop antioxidant activity [3]. Recent research has demonstrated the antimicrobial action of the extracts obtained from blackthorn fruit [4]. According to these, we tried to perform a qualitative and semi-quantitative chemical analysis of some bioactive components from various organs of Prunus spinosa (fruits, twigs and fresh foliar buds). In this paper, we present the results for extracts obtained from foliar buds. Material and method. The plant material, consisting of tips of twigs with foliar buds, harvested on 30/03/2016 from three natural populations (two of them located in Slava Cercheza, and the third from Slava Rusa/Tulcea), was processed as broaiat immediately after harvesting, followed by freezing (maximum 2 hours after collection). After refreezing, the plant material was extracted with 70% methanol (three successive extractions, final volume 100 mL) at boiling. After extraction, the hydro- methanol solutions were analyzed by TLC [5], then the flavonoids and phenolic derivatives were determinated by spectrophotemetrically method [6], using rutin, luteolin, caffeic and gallic acids as standards.
Results and discussion. TLC study revealed for the three methanol extracts of Prunus spinosa foliar buds, collected from Slava Cercheza (populations 1 and 2) and Slava Rusa (population 3), the presence of large number of spots. Luteolin-7-O-glycoside is present in Slava Cercheza samples (populations 1 and 2), while rutin and luteolin are highlighted in all three extracts (Figure 1). Regarding existing phenolic acids in the three extracts, we can appreciate that there are differences, at least quantitatively, between the identified compounds in samples from Slava Cercheza, comparative with the one from Slava Rusa.
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Figure 1. TLC chromatogram for flavonoids in in methanolic extracts from foliar buds of Prunus spinosa: 1,2=Slava Cercheza populations, 3= Slava Rusa population
Standards: Cv.=quercetin, R.=rutin, L.=luteolin, L7gl.=luteolin-7-O- glucoside, A.=apigenin, A7gl.= apigenin-7-O-glucoside
We noticed the presence of an intense spot of chlorogenic acid in Slava Rusa sample, while the for other two samples, this component appears in a smaller amount. In addition to the identified components, one can see the presence of four to five other spots belonging to some flavonoid derivatives, but also of some phenolic acids whose structure could not be specified because of the lack of standards. There are differences between the two populations from Slava Cercheza, but also between these and the one from Slava Rusa in terms of TLC chromatogram.
Table 1. Spectrophotometric determination of some polyphenols in methanol extracts of freezed foliar buds of blackthorn mg galic acid / mg caffeic acid / mg luteolin/ mg rutin/ No. Sample 100 g fpm 100 g fpm 100 g fpm 100 g fpm 1 Slava Cercheza 213.8 159.4 127.8 111.6 2 Slava Cercheza 399.8 375.0 236.8 201.9 3 Slava Rusa 342.9 316.3 201.9 179.2 * fpp = fresh plant material As shown in the table, the chromatographic differences observed for the two populations of blackthorn from Slava Cercheza confirmed the population 1 has a lower content in phenolic derivatives, compared with population 2. The sample of population 3 from Slava Rusa is between the two samples from Slava Cercheza.
Conclusions. The study highlighted that for natural populations located very small distance from one another (Slava Cercheza 1 located approximately 1000 m of Slava Cercheza 2), chemical composition may vary, especially quantitatively, while populations located at bigger distances, may present also quantitative and qualitative differences.
Bibliography 1. Velickovic J., Kostic D., Stojanovic G., Mitic S., Mitic M., Randjelovic S., Djordjevic A. (2014) Phenolic composition, antioxidant and antimicrobial activity of the extracts from Prunus spinosa L. fruit. Hemijskaindustrija 68, 297–303. 2. Jablońska-Ryś E., Zalewska-Korona M., Kalbarczyk J. (2009) Antioxidant capacity, ascorbic acid and phenolics content in wild edible fruits. J. Fruit Ornam. Plant Res17, 115–120. 3. Sikora Elżbieta, Bieniek Małgorzata I., Borczak Barbara (2013) Composition and antioxidant properties of fresh and frozen stored blackthorn fruits (Prunus spinosa L.). Acta Sci. Pol., Technol. Aliment. 12(4): 365-372. 4. Gegiu G., Branza A.-D., Bucur L., Grigorian M., Tache T. (2015) Contributions to the antimicrobial and antifungal study of the aqueous extract of Prunus spinosa L., Farmacia 63(2):275-279. 5. Wagner H., Bladt S., Plant Drug Analysis: A Thin Layer Chromatography Atlas, ed.a II-a, 1996, Springer Verlag Berlin, Heidelberg, NY. 6. Ghita G., Cercetări privind variabilitatea metabolomului, determinat taxonomic, la plante medicinale şi aromatice, Teza de doctorat, Universitatea Al. I. Cuza Iasi, 2012.
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Poster Presentation – PP49
Some effects induced by the treatment with a hydroalcoholic extract of Crataegus monogyna Jacq. in Triticum aestivum L.
Ruxandra Cretu1*, Iuliana Csilla Bara2, Georgiana Gavril1, Gogu Ghiorghita3, Gabriela Vochita4
1*NIRDBS/„Stejarul” Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt, Romania; 2Faculty of Biology, „Alexandru Ioan Cuza”University of Iasi, Carol I Bd., 20A, 700505, Iasi, Romania 3Academy of Romanian Scientists, Splaiul Independentei 54, 050094 Bucharest, Romania 4NIRDBS/Institute of Biological Research Iasi, Lascar Catargi Street, 47, Iasi, Romania *Corresponding author, e-mail: [email protected]
Abstract. A hydroalcoholic extract from hawthorn (Crataegi folium cum flos) was tested on Triticum aestivum L. (Dropia cultivar) caryopses, in a laboratory experiment. Some physiological (caryopses germination; growth, biomass, the biosynthetic capacity of plantlets) and cytogenetic parameters (mitotic index, mitotic phases and chromosomal aberration frequency) were investigated. Key words: hawthorn extract, biometric parameters, mitotic index, chromosomal aberrations
Introduction. Medicinal plants provide a vast number of pharmacologically active compounds that can act against many diseases being an important niche for discovering of new alternative therapies more effective and safer [1,2]. Therefore, it is extremely important to perform cytotoxicity assays for identifying the mutagenic potential of different vegetal compounds used in human therapy [3]. Crataegus monogyna Jacq. (hawthorn), family Rosaceae, is used in tradition medicine, especially in cardiovascular disorders (heart failure, arrhythmia, hypertension). Hawthorn leaves, flowers and berries possess a wide range of biological activities (free radical scavenging, anti-lipoperoxidation and anti- inflammatory) due to various constituents such as flavonoids, proanthocyanidins, phenolic carboxylic acids, vitamin C, saponins, tannins, cardiotonic amines, triterpene acids etc. [4]. The study aimed the action of a hydroalcoholic extract of hawthorn (leaves and flowers) on early ontogenetic stages of wheat plantlets. It was performed in order to highlight the physiological and cytogenetic effects induced by the extract, on the assumption that some plants along with bioactive compounds could also contain potentially toxic, carcinogenic and teratogenic substances.
Materials and methods. Xenobiotic agent: a hydroalcoholic extract of C. monogyna (CMEx) obtained from dried material (leaveas and flowers), under reflux conditions with ethanol 70% v/v (1:10), characterized by the presence of tannins, flavonoids, polyphenols, flavonoid glycosides and anthocyanosides [5]. The extract was applied in three concentrations: 1.5% (CMEx1), 1% (CMEx2) and 5% v/v (CMEx3). Distilled water was used as control (C). Biological material: caryopses of Triticum aestivum L. (Dropia cultivar). Treatment: caryopses were treated with CMEx test concentrations, for 12 hours, washed with distilled water and placed on an inert material, in hydroponic system and maintained at room temperature (23±10C) and natural light, with a photoperiod of 12 hours, for 10 days. After 10 days of ontogenesis, the following physiological parameters were evaluated: germination percent; length, fresh and dry biomass of plantlets (roots and shoots separately), and phytochemical profile of wheat shoots. For cytogenetic experiment the newly emerged roots (10-15 mm) were used and microscopic slides (five/variant) were obtained by squash method. The main cytogenetic parameters assesed were: mitotic index, frequency of mitotic division phases, type and frequency of cells with aberrations.
Results and discusion. The control treatment produced a germination rate of 96%. According to this, only CMEx2 induced a slight stimulation with 1% of germination capacity was obtained, while CMEx1 produced a significantly reduction with 8% (this could indicate a certain cytotoxic effect). All three concentrations of CMEx determined an elongation (with 11%-23%) of wheat root, compared to control. CMEx2 had a positive effect on shoot growth – stimulation with 4%, while the other two concentrations
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induced a minor reduction (<2%). Generally, the acumulation of fresh biomass in roots (CMEx1 and
CMEx2) and shoots (at all concentrations) increased after these treatments. Samples: T1-C; T5-CMEx1, T6-CMEx2, T7-CMEx3 References: rutin, chlorogenic acid, caffeic acid (fig.1); E1 (D-rafinose, D-fructose), E2 (sucrose, glucose) (fig.2); E1 (tirosine, alanine, L- serine), E2 (leucine, treonine), E3 (asparagine, valine, triptophan) (fig.3).
The qualitative analysis by HPTLC of wheat shoots revealed the presence of: flavonoids and polyphenolcarboxylic acids; carbohydrates; Fig. 1. HPTLC Fig. 2. HPTLC Fig. 3. HPTLC chromatogram for chromatogram for chromatogram for aminoacids. The extract did not modified the flavonoids and polyphenols carbohydrates aminoacids biosynthetic capacity.
The hawthorn extract has induced an intensification of mitotic activity in all treated variants, the maximum mitogenic effect being revealed in CMEx2 sample (18.95%, compared to 10.93% for control), this increase was made, especially by the accumulation of prophases that proves the low possibility of CMEx extracts to interact with DNA when it presents a high degree of spiralization and condensation. Even in low percentage, identification of micronuclei, between 0.13% (CMEx3) to 0.46% (CMEx2), proves both clastogenic effect and alteration of mitotic apparatus expressed as aneugenic action of the extract (Photo 1). The ana-telophase aberrations frequency, varying from 3.23% CMEx2 to 4.48% CMEx3, is not significantly higher than control (3.62%) but even lower in CMEx2 variant.
Photo 1. Interphase with one Photo 2. Ana-telophase with Photo 3. Lagging Photo 4. Multipolar and micronucleus (CMEx2) single bridge (C) chromosome (CMEx1) multiple bridges (CMEx3) As shown in photos, the main types of A-T aberrations were: simple and multiple bridges (higher in CMEx3), fragments (CMEx3), lagging chromosomes (CMEx1 and CMEx2), complex aberrations (especially in CMEx1). The amount of bridges and complex aberrations was reduced under the treatment with hawthorn extract; we can talk about the beneficial effect of this extract on the repair of some damages occurred in DNA macromolecule.
Conclusions. There is no clear correlation between extract concentration and evaluated physiological effects. Thus, CMEx2 variant determined the increase of root and shoot length (amplification with 23% and 4%, respectively). This phenomenon was associated with intensification of mitotic activity, the highest MI being registered to the same variant. Generally, the applied treatments affected the accumulation of dry biomass in roots and shoots, this data could not be correlated with plantlets growth.The extract did not induced negative effects on phytochemical composition of wheat shoots, at least in terms of flavonoids, polyphenolcarboxylic acids, carbohydrates and amino acids. The frequency of cells with aberrations is not significantly higher than control, but there are a wider range of chromosomal alterations in treated samples that indicate the clastogenic and/or aneugenic effect of the extract.
Bibliography 1. Zulkipli IN, David SR, Rajabalaya R, Idris A (2015) Medicinal Plants: A Potential Source of Compounds for Targeting Cell Division. Drug Target Insights. 9: 9–19. 2. Kumar VL, Singhal A (2009) Germinating seeds of the mung bean, Vigna radiata (Fabaceae), as a model for the preliminary evaluation of cytotoxic effects of drugs. Biocell, 33(1):19-24. 3. Schlegelmilch R (1994) Toxicity of Crataegus (Hawthorn) Extract (WS 1442). International Journal of Toxicology, 13(2):103-111. 4. Dahmer S, Scott Emilie (2010) Health Effects of Hawthorn. Am.Fam.Physician, 81(4):465-469. 5. Cretu R, Mihailescu R, Mitroi G, Iacob E, Verdes R, Tebrencu C (2011) Phytochemical investigation of Crataegi folium cum flos (hawthorn leaves and flowers) and Hyperici herba (St John's wort aerial parts) hydroalcoholic extracts. Scientific Annals of „Al. I. Cuza” University of Iasi (New Series), Section IIa. Genetics and Molecular Biology: XII (4):133-138.
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Poster Presentation – PP50
Satureja montana L.: Ecological Culture and Essential Oil Quality Assessment
Dana Bobit1, Calin Garlea1, Ruxandra Cretu2, Elena Larisa Tomescu2, Radu Necula2,3*
1SC Dacia Plant SRL, Hărmanului FN, Bod - Brașov, Romania 2NIRDBS/"Stejarul" Biological Research Centre, 610004, Piatra Neamt, Romania 3Faculty of Chemistry, "Alexandru Ioan Cuza" University, 700506, Iasi, Romania * Corresponding author, e-mail: [email protected]
Abstract. In this present study the essential oil of Satureja montana L. from ecological culture, in two years (2014, 2015), from SC Dacia Plant SRL (Brasov, Romania) was evaluated by gas- chromatography coupled with mass spectrometry (GC-MS), in order to determine the main volatile fractions. The phytochemical analysis highlighted a small quantitative variation between samples harvested in these two years.
Key words: winter savory, essential oils, gas chromatography, mass spectrometry.
Introduction. Satureja montana L. (winter savory), Lamiaceae, is traditionally used for various diseases and complications (gastrointestinal cramps, nausea, diarrhea, muscle pains, infections), as anti-spasmolytic, anti-diabetic, antitussive and expectorant. This species is characterized by a rich and diverse composition of secondary metabolites as well as diverse biological activities (antioxidative, antibacterial and antiviral). Literature data presents the chemical composition of the essential oil of winter savory, with the following main constituents, like γ-terpinene, p-cymene, thymol and carvacrol [1-3]. In this study it was analysed the essential oil obtained from Satureja montana species, resulting from ecological culture (2014, 2015) - SC Dacia Plant SRL Brasov, Romania.
Material and method. The phytochemical analysis was performed at “Stejarul” BRC laboratory facility for the essential oil extracted from dried aerial parts from plants cultivated in 2014 and 2015, by means of gas chromatography coupled with mass spectrometry (GC-MS). This was performed with an Agilent 6890N gas chromatography instrument coupled to an Agilent 5975 mass spectrometer and an Agilent ChemStation software (Agilent Technologies, Palo Alto, CA). A capillary column (30 m×0.25 mm i.d.) coated with 0.25 µm film 5% phenyl methyl siloxane (HP-5 MS) was used for separation. High purity helium was used as carrier gas withflow-rate at 1.0 ml/min. The other GC conditions such asinlet mode, injection temperature and separation temperature program were optimized as follows: –inlet mode: split (100:1 split ratio); –injection temperature: 250°C; –separation temperature program: from 40°C (at 6°C/min) to 280°C (for 5 min) – total run time: 45 min. The spectrometer was operated in electron-impact (EI) mode, the scan range was 15–400 amu; the quadrupole and ionization source temperature were 200 and 250°C, respectively. Volatile fractions evaluation was made in accordance with the literature [3,4].
Results and discussion. The results of the GC-MS analysis of essential oil extracted from the aerial parts of S. montana are presented in Table 1 and Figure 1. It revealed the presence of numerous volatile fractions, which are listed in order of their retention time (min.). The main components were carvacrol (48.81%-2014; 49.20%-2015), γ-terpinene (22.60%-2014; 24.01%-2015), and p-cymene (12.98%-2014; 9.70%-2015), with slight differencies between plants harvested in 2014 and 2015. Carvacrol is responsible for some biological activities such as antimicrobial, antitumor,
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antimutagenic, antigenotoxic, analgesic, antispasmodic, anti-inflammatory etc.[5]. Also, we notice the low content of thymol (0.22 %), a positive result, because thymol in large quantities causes epileptic seizures.
Tabel 1. Main compounds of Satureja montana essential oil by GC-MS
Abundance
Area% TIC: 2015_NOV_04.D\data.ms RT 9.679 15.532
Compound Year Year 4.5e+07 (min) 2014 2015 4e+07 6.46 α-Thujene 1.26 1.01 8.809 3.5e+07 6.62 α-Pinene 1.41 1.84 3e+07 6.96 Camphene 0.14 0.13 2.5e+07 7.62 β-Pinene 0.60 1.04 2e+07
7.95 β-Myrcene 1.96 1.99 8.585 8.28 α-Phellandrene 0.36 0.36 1.5e+07 7.949 1e+07 8.59 α-Terpinene 3.82 3.82 6.4636.621 5000000 19.821 7.622 8.886 18.051 8.276 12.511 8.81 p-Cymene 12.98 9.70 6.964 8.41910.33610.59112.23512.92014.27415.13116.959 21.385 4.556 4.927 6.355 7.545 8.056 8.956 9.0829.2489.344 9.8319.88410.97811.15411.49211.59412.68712.75412.82513.08413.35214.05214.43014.78314.84614.98515.01915.21815.88615.93816.47417.25618.32718.45618.63618.76019.30419.37419.60719.94920.14320.48721.25823.00523.19625.96526.39027.01227.46927.66929.01029.55530.62230.696 0 8.88 Limonene 0.66 0.57 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Time--> 9.68 γ-Terpinene 22.60 24.01 Fig. 1. GC-MS chromatogram of Satureja montana essential oil - 2014
10.34 α-Terpinolen 0.14 0.20 Abundance TIC: 2016_APR_15.D\data.ms 9.582 15.389 10.59 Linalool 0.16 0.25 3.5e+07
12.23 Borneol 0.23 0.25 3e+07
12.51 4-Terpineol 0.75 0.96 2.5e+07
12.82 α-Terpineol 0.20 0.22 2e+07 8.716
15.02 Anethole 0.07 0.43 1.5e+07
1e+07 15.13 Thymol 0.22 0.22 8.511
7.882 15.53 Carvacrol 48.81 49.20 5000000 6.558 6.403 7.555 12.430 17.95919.734 8.210 8.807 4.883 5.165 6.903 7.481 7.992 8.353 8.880 9.275 9.7579.80410.26410.52210.70611.08011.51711.87912.15612.30712.61312.78212.92613.53913.97714.10414.28914.79714.94415.05315.14216.38216.87217.54518.24418.67119.20819.52519.60320.00020.05720.40420.79121.16521.293 27.378 18.05 β-Caryophyllene 0.66 0.85 0 6.00 8.00 10.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.00 Time--> 19.82 β-Bisabolen 0.69 1.04 Fig. 2. GC-MS chromatogram of Satureja montana essential oil - 2015
Conclusions. The essential oil obtained from Satureja montana L. resulting from ecological culture (years 2014 and 2015) by steam distillation is characterized by high quantities of carvacrol, γ- terpinene, p-cymene. We can notice a reduced variability of volatile compounds quantity in wintersavory obtained in the two years of cultivation. Also, it presents small quantities of thymol. Due to these results, this species can be recommended as nutraceutical and in food industry.
Bibliography 1. Hajdari A, Mustafa B, Kaçiku A, Mala X, Lukas B, Ibraliu A, Stefkov G, Novak J (2016) Chemical composition of the essential oil, total phenolics, total flavonoids and antioxidant activity of methanolic extracts of Satureja montana L. Records of Natural Products 10: 750–760. 2. Jafari F, Ghavidel F, Zarshenas MM (2016) A Critical Overview on the Pharmacological and Clinical Aspects of Popular Satureja Species. JAMS Journal of Acupuncture and Meridian Studies 9: 118–127. 3. European Pharmacopoeia 6.0: Directorate for the Quality of Medicines & Healthcare, Council of Europe, 2008. 4. Adams RP. (1989) Identification of Essential Oils by Ion Trap Mass Spectroscopy. Academic Press Inc., 1989. 5. Husnu Can Baser K (2008) Biological and Pharmacological Activities of Carvacrol and Carvacrol Bearing Essential Oils. Current Pharmaceutical Desing, 14:3106-3120.
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Poster Presentation – PP51
Progress towards efficient and cost effective molecular authentication of complex herbal food supplements through biochemical fingerprinting and DNA barcoding
Mihael C. Ichim1*, Andreea Andrei1, Madalina O. Popa1, Ancuța C. Raclariu1,4, Aliona Roșca1, Paula P. Sosoi1, Elena L. Tomescu1, Carmen Țebrencu2, Gianina Crișan3, Hugo J. de Boer4
1NIRDBS/'Stejarul' Biological Research Centre, 610004, Piatra Neamt, Romania 2Medicinal Plants Research and Processing “PLANTAVOREL” S.A., 610019, Piatra Neamt, Romania 3Department of Pharmaceutical Botany, Iuliu Hațieganu University of Medicine and Pharmacy, 400010, Cluj-Napoca, Romania 4Natural History Museum, University of Oslo, Oslo, Norway *Corresponding author, e-mail: [email protected]
Abstract. In the current paper we present the progress of our collaborative study on testing and developing suitable and cost-effective approaches for the molecular authentication of complex herbal food supplements and to directly address, investigate and evaluate the safety concerns posed to consumers by this large category of food supplements, i.e. plant dietary supplements and phytopharmaceutical products.
Key words: herbal food supplements, authentication, biochemical fingerprinting, DNA barcoding, Echinacea, Hypericum, Gentiana, Veronica, Dactylorhiza.
Introduction. The herbal food supplements are typically labeled as natural foods and a variety of claims are made regarding their possible health benefits for the consumers. These supplements can be bought all across Europe over the counter in pharmacies, supermarkets, specialist shops and via the internet. While some of these products have a long history of use in Europe, some concerns exist with regard to efficacy, safety and quality [1]. The EU legal system, including the European Medicines Agency (EMA), does not set out any kind of authorization procedure centralized at EU level for the use botanicals and derived preparations in food. This inter alia assigns primary legal responsibility for the safety of the commercialized products to business operators. The lack of standardized methods for quality assessment and the highly competitive market has increased the incentive for the use of substitutes and unlabelled fillers [5]. Adulteration is not necessarily intentional, and herbal products are also altered due to accidental adulteration and misidentification and confused nomenclature of ingredients of plant origin [1]. The main aim of the PhytoAuthent colaborative research project is to directly address, investigate and evaluate the safety concerns posed to consumers by a large category of food supplements, i.e. plant dietary supplements and phytopharmaceutical products [2]. Specifically, using and comparing traditional botanical identification, electronic microscopy (e.g. SEM), biochemical fingerprinting techniques (e.g. HPLC, HPTLC, TLC, LC-MS) with new DNA fingerprinting techniques such as DNA barcoding (e.g. high-throughput sequencing based amplicon metabarcoding) we intend to go beyond the current state-of-the-art when the authentication of the herbal products is done by high performance biochemical fingerprinting, and we will find and propose the most suitable and cost- effective approach for the molecular authentication of complex herbal food supplements.
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Material and method. Plant material belonging to the five plant cases taken into our study - Echinacea sp., Hypericum perforatum, Gentiana lutea, Veronica sp., and Dactylorhiza maculata - was collected from Romania and abroad for biochemical and genetic analyses, and as voucher specimens. More than 200 herbal food supplements were purchesed from Romania, Norway, Spain, Germany, UK, Czech Republic, Poland, Austria, France, Italy, Sweden, Netherlands, Slovakia, and Irland. These products present themselves under different forms: teas, capsules, tablets/pills, tinctures/drop dispensing bottles, and others (bombons, juice, sachets of powder). The number of ingredients ranges from one to even more them 10 different ingredients in a sigle herbal product. The products were purchsed from pharmacies, herbal shops/health shops, supermarkets, local food markets, and through e-commerce.
Results and discussion. The composition in caffeic and chlorogenic acids of aerial parts of relevant Veronica species (V. officinalis, V. teucrium and V. orchidea) harvested from the Romanian spontaneous flora. The quantitative content of the two phenolic acids was estimated by using a newly developed, short and rapid LC/MS method [4,5]. A genomic DNA extraction protocol from plant and dietary supplements was established and the testing of eight DNA gene regions as barcodes to identify plant species of interest namely: psbA-trnH, matK, rbcL, rpoC1, ycf5, trnL, ITS2 si ITS is curerently under way. The use of high-throughput sequencing based amplicon metabarcoding (AMB) is tested on a significant number of herbal food products.
Conclusions. The current authentication of the products is done by high performance biochemical fingerprinting allowing also the identification of new metabolic markers for the correct authentication of the herbal ingredients. Comparing these techniques with the high-throughput sequencing based barcoding we intend to go beyond the state-of–the-art and propose efficient and cost effective molecular authentication of complex herbal food supplements
Acknowledgements. The research leading to these results has received funding from the Romanian - EEA Research Programme operated by the MECS-ANCSI PO under the EEA Financial Mechanism 2009-2014 and Project Contract No 2SEE/2014.
Bibliography 1. De Boer, H.J., Ichim, M.C., Newmaster, S.G. (2015). DNA barcoding and pharmacovigilance of herbal medicines. Drug Saf., 38, 611–620. 2. De Boer, H.J., Raclariu, A.C., Ichim, M.C. (2015) Metabarcoding of european complex herbal food supplements. iBOL Barcode Bulletin, 6(2), 6-7. 3. Mocan, A., Vlase, L., Arsene, A.L., Vodnar, Bischin, C., Silaghi-Dumitrescu, R., Crisan G. (2015) HPLC/MS analysis of caffeic and chlorogenic acids from three Romanian Veronica species and their antioxidant and antimicrobial properties. Farmacia, 63(6), 890-896. 4. Mocan, A., Vodnar, D.C., Vlase, L., Crisan, O., Gheldiu, A.M., Crisan G. (2015) Phytochemical characterization of Veronica officinalis L., V. teucrium L. and V. orchidea Crantz from Romania and their antioxidant and antimicrobial properties. Int J Mol Sci, 16, 21109-21127. 5. Newmaster, S.G., Grguric, M., Shanmughanandhan, D., Ramalingam, S., and Ragupathy, S. (2013). DNA barcoding detects contamination and substitution in North American herbal products. BMC Med,. 11, 222.
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Poster Presentation – PP52
Evaluation of main volatile components from new formulae prepared by Anca pharmacy
Anca Boldea1*, Radu Necula2
1Anca Farm srl, 710028 Botosani, Romania 2NIRBDS/'Stejarul' Biological Research Centre, 610004 Piatra Neamt, Romania * Corresponding author, e-mail: [email protected]
Abstract. Based on standardized volatile oils three formulations were evaluated, formulations that can be used in different disorders. The oils were coded as BT01–Weight Loss Formula, BT02– Intensiv Hepatoprotectors Formula, BT03–Antiparasitic Formula. The formulations were exclusively prepared by Anca pharmacy and were analyzed in the laboratories of BRC "Stejarul" Piatra Neamt. Key words: essential oils, phytotherapy, new formulations, gas chromatography.
Introduction. BT01: Fats are an important component of food but also of a balanced diet, being the nutrients with the highest energy (9 kcal/kg). At the same time, an excessive consumption might lead to obesity – considered the most common metabolic and nutrition disorder in which the body fat accumulates in excess. This embedded oil contains citron oil which helps the immune system, regulates the metabolism and being a nervous and digestive tonic. Weight Loss Formula also helps all digestive and hepato-pancreatic functions, being a very good detoxifying and water regulating (by diuresis). BT02: At the moment there is a real industry in terms of detoxification by using juices, teas and food supplements. A complete and effective detoxification program should minimize the ingestion of toxins and also to accelerate the elimination of toxins. The most important stage is considered the first one, the pre-detox or preparing the human body which must not go suddenly from a lifestyle based on simple carbohydrates, sugars and animal protein to detoxification. This transition can be sustained by therapy using "Intensiv Hepatoprotectors Formula" capsules which contains essential oils, standardized in active principles such as rosemary, carrot or celery. These capsules stimulates metabolism, cleanse the liver by removing the excess of cholesterol and triglycerides, combat free radicals, reduce fluid retention etc. Intensiv Hepatoprotectors Formula also revitalize the cell function by improving tissue oxygenation and blood circulation and restores the bacterial flora of the intestine,combating the constipation. BT03. Mostly, these essential oils have a wide spectrum of antibacterial action: antiviral, anti-fungal, microbial, anti-viral and anti-parasitic. Also, the presence of chamomille and ajwain induce carminative properties (remove the gas accumulated in the intestines) and relieves pain sensation. Tea tree oil is a good disinfectant and is suitable against parasites such as amoebas and roundworms. Carrot oil reduces cholesterol, regenerates liver cells, being a very good hepatoprotective and detoxifying. Drug administration: BT01, BT02 si BT03 - according to the prospectus.
Material and method. Essential oils were analysed by gas-chromatography coupled with mass spectrometry. GC–MS was performed with an Agilent 6890N gas chromatography instrument coupled to an Agilent 5975 mass spectrometer and an Agilent ChemStation software (Agilent Technologies, Palo Alto, CA). A capillary column (30 m×0.25 mm i.d.) coated with 0.25 µm film 5% phenyl methyl siloxane (HP-5 MS) was used for separation. High purity helium was used as carrier gas with flow-rate at 1.0 ml/min. The other GC conditions such as inlet mode, injection temperature
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and separation temperature program were optimized as follows: – inlet mode: split (40: 1 split ratio); – injection temperature: 220 °C; – separation temperature program: from 60 °C (at 3 °C/min) to 246 °C (for 8 min) – total run time: 70 min. The spectrometer was operated in electron-impact (EI) mode, the scan range was 41–415 amu; the quadrupole and ionization source temperature were 200 and 250 °C, respectively.
Results and discussion. The chromatograms obtained by GC-MS analysis are shown in figure 1. Abundance Abundance Abundance
TIC: 2016_AUG_05.D\data.ms TIC: 2016_AUG_06.D\data.ms TIC: 2016_AUG_04.D\data.ms 4e+07 7.901 8.919 3.8e+07 7.782 5.205 4000000 3.8e+07 3.6e+07 3.6e+07 3.4e+07 7.734 3500000 3.4e+07 3.2e+07 A 3.2e+07 B 13.545 C 3e+07 3e+07 18.339 30.342 3000000 2.8e+07 2.8e+07 7.452 2.6e+07 2.6e+07 13.414 30.302 12.468 2.4e+07 2500000 2.4e+07 13.317 21.047 6.184 2.2e+07 2.2e+07 2e+07 2e+07 2000000 1.8e+07 1.8e+07 6.282 1.6e+07 10.37912.389 1.6e+07 1500000 1.4e+07 5.187 1.4e+07 35.482 6.180 7.8497.917 1.2e+07 25.547 1.2e+07 12.837 25.942 17.359 16.731 23.285 9.875 32.395 1e+07 16.606 1000000 1e+07 18.163 24.044 8.58713.931 5.541 8.802 6.629 8000000 26.726 8000000 9.118 17.211 25.983 17.780 6.273 23.225 27.682 16.542 6000000 5.010 10.245 6000000 7.043 500000 6.260 6.615 21.945 14.611 21.629 18.577 8.78413.25819.13019.92222.167 4000000 13.38515.824 32.971 4000000 21.671 17.01623.19625.268 55.910 7.674 26.25627.348 4.676 11.09312.777 11.088 23.059 24.60024.82126.841 5.544 18.424 24.83126.32027.39831.747 7.40710.25113.86014.073 26.419 11.766 17.879 24.01026.844 2000000 5.669 9.83113.58616.42120.62922.13722.71725.69628.82331.57831.77034.825 2000000 12.22214.542 24.58928.76329.646 36.164 5.004 8.081 9.06711.73911.94917.33221.86225.24628.60528.755 37.25438.864 5.818 6.713 8.44114.076 21.50123.93024.30125.267 4.916 6.7227.03011.03711.21714.37015.99817.14118.69718.88020.38821.449 29.61630.57231.23232.13933.75234.511 6.500 7.222 8.08610.47513.64415.82218.86019.31722.84823.58123.82625.41625.69526.47729.46434.769 0 0 0 5.00 10.0015.0020.0025.0030.0035.0040.0045.0050.0055.0060.0065.00 5.00 10.0015.0020.0025.0030.0035.0040.0045.0050.0055.0060.0065.00 5.00 10.0015.00 20.0025.00 30.0035.00 40.0045.00 50.0055.00 60.0065.00 Time--> Time--> Time-->
Fig.1 The GC-MS chromatograms for samples BT01 (A), BT02 (B), and BT03 (C).
The main compounds detected in sample BT01 (Weight Loss Formula AncaFarm) are: limonene - 30.76%, p-menthane-1,2,3-triol - 15.71%, trans-β-ionone - 8.30%, linalyl acetate - 5.44%, linalool - 3.82%, and 4-terpineol - 3.33%.
The main compounds detected in sample BT02 (Intensiv Hepatoprotectors Formula AncaFarm) are: limonene - 13.93%, menthol - 10.39%, carotol - 10.11%, menthone - 9.48%, 5,7,8-trime- dihydrocoumarin - 8.13%, α-pinene - 5.54%, 2-isopr-5-me-cyclohexanone - 4.18%, patchouli alcohol - 3.01%, sabinene - 2.93%, β-eudesmene - 2.76%, carvacrol - 1.96%, and β-pinene - 1.95%.
The main compounds detected in sample BT03 (Antiparasitic Formula AncaFarm) are: thymol - 10.24%, eugenol - 8.79%, carotol - 8.74%, γ-terpinene - 7.69%, 4-terpineol - 6.68%, isomenthol - 5.98%, p-cymene - 5.29%, linalool - 4.86%, trans-cinnamaldehyde - 4.27%, α-terpinene - 3.57%, isomenthone - 2.27%, nerol - 2.22%, geraniol - 2.14%, limonene - 1.7%, β-caryophyllene - 1.66%, sabinene - 1.37%, and eucalyptol - 1.22%.
Conclusions. Identification of the chromatographic peaks was carried out using the NIST database 2008 and the confirmation of the mass spectrum and retention time by Adams (1989). Formulations with standardized volatile oils are characterized by dominance of volatile fractions with significant pharmacological activity.
Bibliography 1. Adams, Robert P. (1989) Identification of Essential Oils by Ion Trap Mass Spectroscopy. Academic Press Inc., 1989. 2. European Pharmacopoeia 6.0: Directorate for the Quality of Medicines & Healthcare, Council of Europe, 2008. 3. Festy, D. (2008) Ma Bible des huiles essentielles. Leduc.s Editiones, Paris. 4. Franchomme, Pierre, L'Aromathérapie : Thérapeutique de pointe en médecine naturelle, éd. Amyris, 1999.
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In memoriam: Profesor Dr. Emanoil Grigorescu
On April 10, 2016, Professor Dr. Emanoil Grigorescu, fellow of the Romanian Academy of Medical Sciences, left us as quietly as he lived his entire life. Prominent figure of the Romanian pharmaceutical community, Professor Dr. Emanoil Grigorescu was born on May 19, 1924. He attended the Faculty of Pharmacy in Bucharest in the difficult years after 1945, and the Faculty of Chemistry; the first he graduated in 1948, and the second in 1947. He began his career as a pharmacist practitioner in 1949, being attracted by the multitude of ways to interact and communicate with patients offered by this profession, but also by the importance of rational thinking in developing magisterial formulations at a time when standard medications were less numerous (about 80), and people rarely visited a doctor but frequently a pharmacy. This "freedom" to use good judgment attracted him to pursue pharmacy as a career, convinced even at the end of his career that this was what he was ment to do, his chemistry training offering additional data of help in finding rational solutions for pharmacy-specific problem solving. In 1953 he joined the teaching staff of the "Carol Davila", Institute of Medicine and Pharmacy in Bucharest, first at the Department of Biochemistry, then at the Department of Pharmacognosy, where in recognition of the his standing and promise he was promoted to lecturer, also becoming Deputy Director at the National Institute for Drug Control and Pharmaceutical Research (ICSMCF), where he served between 1961-1963. In 1962, having already a solid experience in teaching and research, he was offered and accepted a position at the Institute of Medicine in Iasi, where he was to take the lead of the group of initiators (Prof. dr. C. Ichim, Prof. A. Pastia) of the new Pharmacy Department, with the first entrance exam held in September 1961. Moving together with his family to the capital town of Moldova, in pioneering circumstances, Professor Dr. Emanoil Grigorescu began organizing in 1962 not only the Department of Pharmacognosy, but the whole structure which in the coming years would become the nowadays the Iasi Faculty of Pharmacy. Promoted to Associate Professor in 1963, he then became (in 1967) the youngest Professor in Pharmacognosy in Romania and obtained his PhD in 1972. Starting with 1967 he was a PhD scientific supervisor and has contributed to forming an impressive number of Romanian and foreign scientists in the field of medicinal plant research and has an important contribution to the knowledge of both native and exotic species in Romania and abroad. Resigning his position as Deputy Director of ICSMCF in 1963, he worked in Iasi first as head of the Department, then as Dean of the Faculty of Pharmacy until 1975. Getting himself noticed for his dedication and modernity of his research, sound knowledge in obtaining plant-based medicines (contributed significantly to the development of a platform for plant extracts at the Antibiotice Iasi Pharmaceutical Company and held lectures and practical courses on developing phytodrugs at Plantavorel in Piatra Neamt), he was appointed in 1979 as United Nations expert in medicinal plants, participating in this capacity in UNIDO two missions in Burundi (1979) and Ruwanda (1984). During these missions, Professor Dr. Emanoil Grigorescu obtained from medicinal herbs harvested by local research teams some plant extracts which he then processed into medicines, according to recepies devised by him, these medicines being manufactured until the civil war in the region as the only affordable medicines for the majoritarily poor population. Continuing to take part in UNIDO activities, during 1980- 1985, in the pharmacognosy laboratory of the Iasi University of Medicine and Pharmacy he organized annual international summer courses in which students selected from African and Asian countries participated. They were pharmacists, doctors, biologists and chemists working in state laboratories for the production and analysis of medicinal plants in their countries of origin, attending a theoretical course and practical training in the field in Iasi.
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Since 1970, Professor Dr. Emanoil Grigorescu was involved as a director in over 22 projects/ research contracts with various institutions in the country: ICECHIM, ICCF; ICSMCF; ASM; BIOTEHNOS; ANTIBIOTICE IASI PHARMACEUTICAL COMPANY; BIOFARM; CHEMICAL & BIOCHEMICAL ENERGETICS INSTITUTE BUCHAREST, IASI AGRONOMIC INSTITUTE. Professor Dr. Emanoil Grigorescu’s rich inovative activity has materialized into 22 technological processes, 12 original drugs in microproduction, 7 original drugs in industrial production and 56 patents; for this last achievement, in 1991, he was awarded the title ELITE INVENTOR class IV ( for 30 patented inventions). As a dean, he was actively involved in obtaining funding for the contruction of the present building of the Faculty of Pharmacy in Iasi, inaugurated in 1974. As head of the Department of Pharmacognosy (for 32 years) he was the mastermind who set the foundations of a modern scientific approach for obtaining, analysis, control and conditioning of drugs of plant origin, who knew how to guide, but also to temper, when necessary, his "wizard disciples", who shared with great joy and generosity his professional knowledge, both with his disciples and students, making the days of all of us brighter with his optimism, good moods and humor. For both students and pharmacists, Professor Dr. Emanoil Grigorescu remains legendary for the wonderful courses on Pharmacognosy, Phytotherapy and History of Pharmacy. Blessed with the gift of oratory and ability to select and arrange words into sentences, he had an exceptional editorial activity, materialized in the publication of 288 papers (of which 74 indexed before 1992 in SCI Finder Scholar), 8 courses volumes and teaching materials and 11 books published by well- known publishing houses. The two volumes of the book entitled "Medicinal Plants, Phytochemistry and Phytotherapy" (authors I. Ciulei, Em. Grigorescu, U. Stanescu) were published by Ed. Medical Bucuresti in 1993, and has received the “Iuliu Hatieganu" Award of the Romanian Academy in 1993. Gifted painter during his little spare time, Professor Dr. Emanoil Grigorescu was a dreamer who was sure that everyone he knew was good and treated him/her as such; he was convinced that the only way to prove your mission in life is to leave a palpable trace, and if we look closely at many of those who were his students, we realize that Professor Dr. Emanoil Grigorescu made a mark on what they have become.
Prof. Emanoil Grigorescu was, for BRC Stejarul team, the chance of opening new directions in medicinal plant research, but also genetic and breeding – which were the subject of his studies until early 80’s. For all of us, as biologists, he was a patiently and persistently mentor on our way to discover the secrets of phytochemistry and pharmacognosy; those of us, who were lucky enough to know him and collaborate with him, consider Prof. Grigorescu not just our scientific mentor but also a good friend who passed away recently. He was part of our group and he was involved through his ideas and advices in our scientific activity until his last days.
For these reasons and many more, we all keep a vivid memory of Professor Dr. Emanoil Grigorescu.
The symposium is dedicated to the 60-year celebration of the „Stejarul” Biological Research Centre – MAPPPS 2016, Piatra Neamt, ROMANIA – Page 163
Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
Multumiri Primariei Piatra Neamt pentru Parteneriatul Public Privat
Primăria Municipiului Piatra Neamţ, ROMÂNIA (City Hall Neamt) www.primariapn.ro
The symposium is dedicated to the 60-year celebration of the „Stejarul” Biological Research Centre – MAPPPS 2016, Piatra Neamt, ROMANIA –
About Dacia Plant Dacia Plant is one of the top Romanian producers of natural supplements. The company was founded 15 years ago and lays its success on top of efficient natural supplements crafted after unique recipes. Dacia Plant’s natural supplements are well known on Romanian market for their 100% natural composition and for the original recipes that combine traditional phytotherapy and the latest studies in the field. The standing proof of success is represented by all the powerful brands that Romanian company is creating for over 15 years. The company’s vision is that the beneficent force of plants can help modern people to maintain health and energy. The lack of any preservatives and chemical compounds make the company’s natural products a premium choice for a healthy and long lifetime. The symbol of Dacia Plant, the Dacian Draco, rebirths continuously from nature and underlines the company mission: to bring nature and people together. Dacia Plant respects tradition and pays respect to traditional medicine, inspiring from popular wisdom and creating modern and scientifically tested products. The natural supplements contain an optimal quantity of nutritive substances, vitamins and minerals, necessary for the proper function of all human body systems.
Top 10 advantages of Dacia Plant products: • Designed, tested and produced in Romania; • 100% natural, with no preservatives, pigments or chemical sweeteners; • No adverse reactions; • The natural substances in the composition are scientifically studied and tested; • Help the organism to quickly balance itself; • Help the organism to quickly return to its proper functions; • Contain optimal amounts of active substances for an immediate and long lasting effect • Contain plants and standardized extracts collected and stored under maximum safety conditions; • Based on complex recipes created by experts in phytotherapy; • Brands dedicated to different categories of people and health conditions, for a correct and quick choice.
SC ANCA FARM SRL is a local chain of pharmacies operating since the summer of year 2000. This pharmacy offers for over 16 years expert advices to patients from city of Botosani, and more recently, to patients across the country through dedicated sales website opened in March 2016. The pharmacy was highlighted in the pharmaceutical market from Botosani with prompt service and quality at low prices. Also, following the recommendations of Ms. Dr. Pharm. Anca Boldea, the pharmacy is equipped with a laboratory for preparing pharmaceutical formulations. The pharmacy has a contract with the National Health Insurance House and HIHDPONSLA (Health Insurance House for Defense, Public Order, National Safety and Legal Authority) offering a wide range of products. To be a good pharmacist, Ms. Anca Boldea, throughout her whole career, she was motivated to maintain at the highest level the performance and the professional and personal behaviour through continuous updating her knowledge related to her professional activity. Mrs. Anca Boldea has defended her doctoral thesis in research, in the scientifica area of Phytotherapy, becoming Doctor in Pharmacy in 2010, through the work Study contributions native species of Galium and Ajuga, under the guidance of Mrs. Prof. Dr. Ursula Stanescu and Mrs. Dr. Elvira Gille, Head of NIRDSB / „Stejarul“ Biological Research Centre, Piatra Neamt. She continued many other specializations, masters and scientific works in order to bring new pharmaceutical insights to patients, because for her, the most important is the patient. For each patient is required to prepare an individualized treatment plan. This plan must be with maximum effectiveness, easier to monitor and economically for the pacient, aiming to achieve better results with simple means and minimal risks. Therapy will be complex and since always, the aim of ANCA FARM pharmacy was to treat the patient and not the disease itself. In the same time, Dr. Farm. Anca Boldea brought a fresh breeze in this area through aromatherapy. We completed our offer with over 300 essential oils formulated to be more performant through their simplicity, used in synergy to prevent or to treat (within various situations) things in a simpler way. In view of the results, there is no doubt that one will understand how, in nature, nothing is more powerful and effective than oil herbs, standardized active principles. They allow intervention if necessary, without resorting to drugs (whose very effective molecules can have side effects in the short or long terms). These essential oils can be used rectal, vaginal, internal, or even easier, directly on the skin (but always diluted with vegetable oils). In principle, pharmacotherapy can be used both in prophylactic and curative treatment, as well as for diagnostics. It is indicated prevention better than treatment, which is much more complex and difficult for a patient. Therefore, the slogan of ANCA FARM pharmacy supports this: "Health is expensive! Prevention is cheaper!". This is the pharmacist's role: to advise, to open the patient's mind about the infinite possibilities of formulated essential oils used since antiquity, found in the Bible (to bring comfort and healing). Within the ANCA FARM pharmacy, represented by Dr. Farm. Anca Boldea, we respect the fundamental principles of the pharmacist profession, through respect for life and human being, and in any cases outweigh the patient’s interests by respecting the their rights, by proving loyalty and solidarity towards each other, behaving with honor and professional honour.
5-star quality!
Carrying on the tradition of milk processing in Botosani, Five Continents Group has attracted in 2005 experienced specialists and invested in a new and modern milk processing plant. With the latest technology at that time, the factory was the first one in Romania that received „L1“ intra-Community trademark.
Our products are distributed in Romania and also exported to many countries, being known and appreciated by consumers. Therefore, we are proud of our best quality products and our traditionally Romanian cheese, made according to European requirements.
All our products are made from Romanian milk of the highest quality produced in Botosani county.
„We do it with love to ensure you what’s best“ it’s not just a slogan but it’s our daily concept.
[email protected] [email protected] Fax. 0231.544250 / Phone. 0231.544217
Charme Cosmetics SRL, Company's description,
Charme Cosmetics SRL was founded 14 years ago from its own funds and has as its main activity wholesale natural foods like teas, tinctures, tablets, syrups, cold-pressed oils, natural cosmetics. This assure a distribution by salespeople, employees of the company. In all these years of exploring the natural products market we noticed the degree of absorption and gradual increase in the number of consumers. People slowly but surely turning their attention to natural products, much healthier for our body being aware of the benefits of medicinal plants. The essence of everything we do is to guide people towards nature, because we are responsible for what containing our products, but also for the results they have. I became an ally of nature along the way, always contributing to building harmony between beauty and health. We focus on the real benefits of plants. Our features are: 1. Purity : use natural formulas, some of them with over 98 % natural ingredients 2. Quality: our products have high concentration of active ingredient and no synthetic fragrance , artificial colors, preservatives and petrochemical derivatives 3. Ethics: respect for both the environment and people , and do not realize tests on animals 4. Caring for Body and Soul: rely on moments of delight, comfort, and quiet evenings warmer, and bring more light to the face and soul of consumers. While every woman is unique, each appreciate when you're with her. Our products are for women who understand the importance of a balanced life and enjoy the moments that really make life special. Our products are aimed at women who appreciate the moments of serenity and happiness, and who truly know that health means beauty, and beauty is health Thus was born the desire to produce herbal cosmetics. We want to distinguish ourselves within those produced by larger quantity of active principle, no dyes, fragrances and parabens. Also we try to help human beings to relate more harmoniously with nature. These products have both Uses Beauty and treatment, helping skin to recover from cuts, bites, and cellulite, treat rheumatic pain and inflammatory muscle pain caused by colds, improve immunity level capillary treating acne and other skin conditions. For our products we chose herbs coming from unpolluted areas, so they en offers great quality and richness of their active ingredients. In areas close to the new farms are lavender and calendula, basic raw materials for herbal treatments. This field not only pollute the environment but not more than that creates a strong connection between man and nature. Also pursue the development of a modern laboratory for physicochemical and microbiological analysis and research in this area. With this laboratory will be ensured quality of products.
Charme Cosmetics SRL – Our Products
As regarding our products, we are in constant expansion, creating up to the present three ranges. Main, one of shampoos natural which focused on introducing formulas surfactant natural, gentle friendly to the scalp.
Active ingredients: propolis, calendula officinalis, symphytum officinalis Applications: antibacterial, antifungal, antiviral, strong healing effects, strong anti-inflammatory, strong epithelial regenerator. External applications: infectious eczema, hemorrhoids, unaesthetic scars, excoriations, minor burns (1st degree), superficial APICALEND lesions, anal clefts, cracked heels, insect stings, tegumentary Keeps skin young lesions.
Cosmetic: for irritant skin with inflammatory tendency, dry and rough skin, skin protection (wind, frost).
Active ingredients: thuya occidentalis extract Applications: skin tonic, antifungal, antiviral, antibacterial, balsamic, healing, antitumoral, stimulant of local immunity. Infections sensitive complexure and skin – cream is applied daily for 10 days, mornings and evenings, on the most exposed areas. Skin ulcerations: In a first stage use thuja alcoholic extract, 1-3 times a day, depending on the gravity, to clean the infected area by Thuya cream help of a cotton pad. After cicatrization, go to apply the cream on Passion for your skin the affected area, for a more complete and aesthetic healing. Anal and venereal vegetations – Anoint affected places with thuja cream, 2-3 times a day. The specialist may inject large verruca with thuja alcoholic extract, which will lead to them to disappear. Skin and tegument infectious eczema, fungus – Anoint the affected area twice a day with thuja cream.
Active ingredients: volatil oils: mint, thyme, juniper Actions: strong antirheumatic, anti-inflammatory, very good joint anti-inflammatory, antiallergic, antiseptic, bronchodilator, relaxing. External applications: rheumatic pains, joint stiffness, back pain caused by cold, post-traumatic pain, muscle pain, muscle pain after exercise, physical and nervous exhaustion.
FLEXIMAX Cosmetic: In very small quantities used to treat withered complexions. Used as body cream for people with skin sensitivity to cold, cyanotic skin (rapidly turning blue by exposal to the cold).
Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
MAPPPS – 2016
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PLENARY LECTURES
1. Natural products research: Quo Vadis? 13 R. Verpoorte 2. How stressed plants can help stressed humans? On the role of oxidative stress response in 15 biosynthesis of bioactive metabolites Adam Matkowski, Sylwia Zielińska, Marta Libik-Konieczny, Robert Konieczny, Weronika Kozłowska, Sylwester Ślusarczyk 3. Agriculture crops as the source of bioactive compounds 20 Wieslaw Oleszek 4. An overview on chemopreventive phytochemicals 25 Satyajit D. Sarker, Georgiana Zavoianu, Fyaz M. D. Ismail, Kenneth J. Ritchie, Lutfun Nahar 5. Our current verdicts into herbal bioactive molecules by means of molecular docking 27 approaches Ilkay Erdogan Orhan 6. Comparative authentication of Hypericum perforatum herbal products using amplicon 31 metabarcoding Ancuta Cristina Raclariu, Ramona Paltinean , Laurian Vlase, Aurélie Labarre, Vincent Manzanilla, Mihael Cristin Ichim, Gianina Crisan, Anne Krag Brysting, Hugo de Boer 7. Pharmaceutically relevant molecules from plant origin and their sustainable bioproduction 33 Milen I. Georgiev 8. Effect of the mixture of Dong Quai, Tang Shan, Linchi and citronellol on the infection and ulcer 35 of Helicobacter pylori Hsin-Lun Huang, Chin-Kun Wang
SHORT LECTURES
9. In vivo and in vitro evaluation of the anti-diabetic nutraceutical potentials of Capparis spinosa 36 Adriano Mollica, Azzurra Stefanucci, Giorgia Macedonio, Marcello Locatelli, Olakunle Onaolapo, Adejoke Onaolapo, Ettore Novellino 10. Medicinal Plants in Bulgaria – Current State and Perspectives 38 Elena Genova, Marina Stanilova, Boryanka Traykova 11. New insights regarding the biologic potential of a standardized chamomile extract 40 Radu Ionita, Lucian Hritcu, Adriana Trifan, Monica Hancianu, Oana Cioanca 12. The antimicrobial activity of medicinal plants depending on storage conditions 42 Steliana RODINO, Alina BUTU, Marian BUTU 13. Comparison of total phenolic content and antioxidant capacity of seeds vs. sprouts of 44 representative species from Republic of Moldova Grigoriev Valeria, Chiru Tatiana 14. Biological and chemical study of Lavandula sp. varieties cultivated in Dobrogea Plateau 46 Daniela Lupu, Radu Necula, Georgiana Gavril, Ruxandra Cretu, Valentin Grigoras, Elvira Gille 15. Comparative study of essential oils of Heracleum species 48 Dana Bobit, Calin Garlea, Andrei Paduraru, Madalina Popa, Radu Necula, Oana Cioanca 16. Screening of microorganisms for the recovery of critical metals 50 Marian BUTU, Steliana RODINO, Alina BUTU 17. Possible action mechanisms involved in expression of the in vitro cytostatic and cytotoxic 52 impact of some fractionated proanthocyanidin products obtained from grape seeds Cosmin-Teodor MIHAI, Gabriela Vochita, Daniela Gherghel, Pincu Rotinberg Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
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18. The Evaluation of Some Natural Populations of Prunus spinosa L. and Lycium barbarum L. 54 from Dobrogea, Romania Elvira Gille, Ruxandra Cretu, Valentin Grigoras, Radu Necula, Manuela Elisabeta Sidoroff, Georgiana Gavril 19. Proanthocyanidins: new insight into their chemistry and biology 56 Anca Miron, Adriana Trifan, Vlad Simon Luca, Ana Clara Aprotosoaie 20. Polyphenolic compounds production in shoot cultures of two Romanian Hypericum species 58 Ana Coste, Elvira Gille, Valentin Grigoraş, Radu Necula, Adela Halmagyi, Gheorghe Coldea, Constantin Deliu 21. In vitro evaluation of the cytotoxic impact of some proanthocyanidin fractions extracted from 60 grape seeds Cosmin-Teodor Mihai, Gabriela Vochita, Daniela Gherghel, Rodica Pașa, Ancuța Nechita, Pincu Rotinberg
POSTER PRESENTATIONS
22. Trends in Romanian medicinal plants scientific research 62 Alexandru Amărioarei, Raul Jibotean, Iris Tușa, Corina Ițcuș, Marian Buțu 23. Immunomodulatory potential of Inula helenium L. 64 Alice Grigore, Georgeta Neagu, Nicoleta Dobre, Carmen Ionita, Lucian Ionita, Dana Bobit 24. Cytotoxic potential of Helleborus purpurascens L. 66 Alice Grigore, Georgeta Neagu, Nicoleta Dobre, Adrian Albulescu, Carmen Ionita, Lucian Ionita, Dana Bobit 25. Crocus sativus studies on morphostructural analysis 68 Adina Segneanu, Daniel Damian, Ioan Grozescu 26. Curcuma longa – An analytical study 69 Adina Segneanu, Daniel Damian, Ioan Grozescu 27. The neuroprotective effects of Anemarrhena asphodeloides rhizome extract in the PC12 cell 70 model Nina Rembiałkowska, Anna Maria Patrzałek, Magda Lewińska, Arnold Garbiec, Zofia Marchewka, Agnieszka Piwowar, Anna Długosz, Adam Matkowski 28. LC-DAD-ESI-MSn profile of phenolic compounds in flowering stems of Sideritis raeseri from 72 Republic of Macedonia, Albania and Greece Bujar Qazimi, Jasmina Petreska Stanoeva, Gjoshe Stefkov, Marina Stefova, Svetlana Kulevanova 29. Biochemical and HPTLC Fingerprinting Identification of the Hypericum perforatum L. 74 Finished Products Ţebrencu Carmen E., Iacob Elena, Ciupercă Oana T., Creţu Ruxandra M., Chiriac Maria, Ionescu Elena 30. Seed germination of Geum urbanum depending on storage conditions (low temperatures) 76 Catană Rodica, Florescu Larisa 31. Phytochemical Screening and Chromatographic Fingerprint Studies on Ethanolic Extracts of 78 Arnica Montana L. Ciupercă Oana T., Ţebrencu Carmen E., Iacob Elena, Creţu Ruxandra M., Chiriac Maria, Ionescu Elena 32. HPTLC identification of bioactive compounds from Ganoderma lucidum and Flammulina 80 velutipes hydroalcoholic extracts Corina Bubueanu, Popa Gabriela, Alice Grigore, Colceru – Mihul Svetlana, Petruta Calina Cornea 33. Antioxidant activity of extracts of Armillaria mellea 82 Corina Bubueanu, Alice Grigore, Ecaterina Serban, Colceru – Mihul Svetlana Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
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34. Rose-scented Geraniums cultivated in Romania – essential oil profile 84 Cristina Elena Iancu, Oana Cioanca, Cornelia Mircea, Adrian Spac, Silvia Robu, Monica Hancianu 35. Assessment of toxicity and inflammatory activities of mud extracts 86 Elena Codrici, Cristiana Tanase, Ionela Daniela Popescu, Simona Mihai, Ana-Maria Enciu, Nicu Stoica, Radu Albulescu 36. Generating Diversity in Natural Product Scaffolds. Efficient C-17 Alkylations of ent-Kaur-16- 88 enic Derivatives Elena Pruteanu, Vladilena Gîrbu, Nicon Ungur, Veaceslav Kulciţki, Philippe Renaud 37. Consumer survey on medicinal tea consumption habits, practices and attitudes in Eastern 90 Romania Elisabeta Oprea, Oana Cioanca, Crsitina-Corina Bentea, Ana Clara Aprotosoaie, Ursula Stanescu, Monica Hancianu 38. Total polyphenols, flavonoids and antioxidant activity of Romanian propolis samples 92 Florentina Gatea, Eugenia Dumitra Teodor, Gabriel Lucian Radu, Elvira Gille 39. Evaluation of accelerated solvent extraction (ASE) for obtaining chlorogenic acid from Cynara 94 scolymus L. (artichoke) leaves Ibrahim Ahmed Saleh, Mohamed-Elamir Fathy Hegazy, Tarik Abdelhalim Mohamed, Khaled Ahmed Shams, Elsayed Aboutabl, Nahla Sayed Abdel-Azim, Faiza Mohamed Hammouda 40. The action of two kinds of lingonberry (Vaccinium vitis-idaea L.) extracts on liver function in 96 Paracetamol - induced toxicosis Ioana Roman, Vlad Toma, Ana Coste, Adela Halmagyi, Anca Farcaş 41. The effect of harvesting time on essential oils composition of Thymus pannonicus L. 98 Irina Boz, Ioan Burzo, Corneliu Tanase 42. Contributions to the knowledge regarding the structure of vegetative organs of Thymus dacicus 100 Borb. Irina Boz, Constantin Crăciun, Andrei Lobiuc 43. Antioxidant properties of extracts from Crataegus pentagyna assesing by flow cytometry for 102 potential applications in blood stotrage Iris Tusa, Andreea Toader, Valentin Grigoraş, Elvira Gille, Daniela Bratosin 44. Collagen membranes enriched with plant extracts used as scaffold in wound healing 104 Elena Iulia Oprita, Rodica Tatia, Larisa Calu, Agnes Toma, Lucia Moldovan 45. Antioxidant properties of the Sanguisorbae herba and Sanguisorbae radix extracts. 106 Izabela Nawrot-Hadzik, Marta Szandruk, Anna Kazana ,Sylwester Ślusarczyk, Adam Matkowski 46. Contributions to the knowledge of vegetative organs structure of Iris halophila Pall. 108 Lacramioara Ivanescu, Marius Nicusor Grigore, Constantin Toma 47. Effect of culture medium supplementation with phytochemicals on human gingival fibroblast 110 cells proliferation Ana-Maria Seciu, Lucia Moldovan, Coroiu Viorica, Oana Craciunescu, Otilia Zarnescu 48. Biopolymer hydrogel coupled with indigenous herbal extract potentially therapeutics 112 Tcacenco Luminita, Iordachel Catalin, Berteanu Elena, Paraschiv Maria, Dinu Ecaterina Liliana, Enache Mihaela-Ionica, Zuav Adina-Lidia, Tusa Iris Maria, Geanta Mariana 49. The influence of different extraction solvents on the yield and concentration of anthocyanins 114 and polyphenols from bilberry (Vaccinium myrtillus L.) Maria Chiriac, Elena Iacob, Carmen Elena Tebrencu, Elena Ionescu, Oana Teodora Ciuperca 50. Preliminary phytochemical investigations of two new romanian Ocimum basilicum L. cultivars 116 Marian Burducea, Andrei Lobiuc,Vasilica Onofrei, Zenovia Olteanu, Mirela Ardelean, Marin Zagnat, Maria-Magdalena Zamfirache Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
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51. Histo-anatomical characterization of vegetative organs from two species of Inula from 118 Romanian flora Marinela Afemei, Irina Boz, Constantin Toma 52. Differentiated seabuckthorn treatment influences the graft rejection in rabbits 120 Mihaela Niculae, Carmen Dana Şandru, Gheorghe Florinel Brudaşcă, Pall Emoke, Spînu Marina 53. Anthocyanin contents in Sedum telephium ssp. maximum L. callus under growth regulators 122 supplementation Mirela Ardelean, Aurel Ardelean, Mihaela Duțu, Călin Lădașiu, Andrei Lobiuc, Elida Rosenhech, Burducea Marian 54. Data and knowledge on the importance of Dracocephalum moldavica L. species (dragon’s 124 head) to introduce and develop the cultivation technology Naie Margareta, Trotus Elena, Lupu Cornelia, Popa Diana 55. Towards Mimics of Forskolin. Efficient Free Radical Alkylations of Manoyloxides 126 Olga Morarescu, Vladilena Gîrbu, Elena Pruteanu, Nicon Ungur, Veaceslav Kulciţki, Philippe Renaud 56. Research on the hnowledge of the harmful entomofauna for the milk thistle culture (Silybum 128 marianum L) Elena Trotuş, Paula-Lucelia Ursache, Margareta Naie 57. Bioinformatic approaches regarding the Salvia sclarea investigation 130 Rodica Martea, Ana Mutu, Maria Duca 58. Phenolic contents and antioxidant activity in Viola odorata L., V. tricolor L. and V. arvensis 132 (L.) Murray Rosenhech Elida, Lobiuc Andrei, Zamfirache Maria-Magdalena 59. Lavandula hybrida: assessment of the essential oil properties 134 Silvia Robu, Monica Hancianu, Dana Tutunaru, Adrian Spac, Cristina Tuchilus, Adriana Trifan, Ursula Stanescu, Elvira Gille, Oana Cioanca 60. The chemical composition of some grain sorghum (Sorghum bicolor L. Moench.) varieties 136 experimented in A.R.D.S. Secuieni – Neamt pedoclimatic conditions Simona – Florina Pochişcanu, Alexandra – Andreea Buburuz, Lorena - Diana Popa 61. Phytochemical investigations, structural and ultrastructural aspects of the Passiflora caerulea 138 L. plants cultivated in Romania Simona Savin, Agnes Toma, Oana Craciunescu, Anca Oancea, Sorin Manoiu, Tatiana Eugenia Şesan, Anca Sarbu, Daniela Smarandache, Georgeta Negru 62. Centaurea cyanus L. extracts – source of antioxidants 140 Tatiana Chiru, Anatolie Nistreanu, Nicolae Ciobanu, Ungureanu Ion 63. Effects of foliar ecological fertilization on inflorescence yield and chlorophyll parameters of 142 Calendula officinalis L. Vasilica Onofrei, Bernd Honermeier, Andrei Lobiuc, Marian Burducea, Gabriel-Ciprian Teliban, Carmenica Doina Jităreanu, Remus Ciprian Cotunoaea, Teodor Robu 64. Generating Diversity in Natural Product Scaffolds. Synthesis of ent-Kauranic Derivatives 144 Functionalized with Triazole Fragments Vladilena Gîrbu, Marina Grinco, Nicon Ungur, Veaceslav Kulciţki, Philippe Renaud 65. Comparison of essential oil compositions of fresh and dried plant of endemic Salvia cadmica 146 Boiss. var. bozkiriensis Celep, Kahraman & Doğan, in Turkey Süleyman Doğu, Sadiye Ayşe Çelik, Yavuz Bağcı 66. The Medical Plants of Karaman-Yeşildere Village and Its Surroundings 147 Turan Akdağ, Süleyman Doğu 67. Antioxydant activity and preventive possibility of Algerian medicinal plant Matricaria pubenscens on hepatic toxicity 148 Boubellouta Houria, Khelifi Touhami Fatima, Kermandji Mohamed Azed, Abidli Nacira, Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
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Bellatrache Cherifa 68. Phenolic contents and antioxidant activities in Phaseolus coccineus L. flowers 150 Gabriel Teliban, Marian Burducea, Andrei Lobiuc, Elida Rosenhech, Vasile Stoleru, Vasilica Onofrei, Maria-Magdalena Zamfirache, Neculai Munteanu 69. Preliminary phytochemical study of Prunus spinosa L. buds harvested from natural populations 152 of Dobrogea area Georgiana Gavril, Valentin Grigoras, Ruxandra Cretu, Radu Necula, Elvira Gille, Ursula Stanescu 70. Some effects induced by the treatment with a hydroalcoholic extract of Crataegus monogyna 154 Jacq. in Triticum aestivum L. Ruxandra Cretu, Iuliana Csilla Bara, Georgiana Gavril, Gogu Ghiorghita, Gabriela Vochita 71. Satureja montana L.: Ecological Culture and Essential Oil Quality Assessment 156 Dana Bobit, Calin Garlea, Ruxandra Cretu, Elena Larisa Tomescu, Radu Necula 72. Progress towards efficient and cost effective molecular authentication of complex herbal food 158 supplements through biochemical fingerprinting and DNA barcoding Mihael C. Ichim, Andreea Andrei, Madalina O. Popa, Ancuța C. Raclariu,Aliona Roșca, Paula P. Sosoi, Elena L. Tomescu, Carmen Țebrencu, Gianina Crișan, Hugo J. de Boer 73. Evaluation of some volatile oil components from formulations of Anca pharmacy 160 Anca Boldea, Radu Necula
Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
MAPPPS – 2016
Index of authors
A Corina Bubueanu 80, 82 Abidli Nacira 148 Corina Itcus 62 Adam Matkowski 15, 70, 106 Cornelia Lupu 124 Adejoke Onaolapo 36 Cornelia Mircea 84 Adela Halmagyi 58, 96 Corneliu Tanase 98 Adina Segneanu 68, 69 Cosmin-Teodor Mihai 52, 60 Adina-Lidia Zuav 112 Cristiana Tanase 86 Adrian Albulescu 66 Cristina Elena Iancu 84 Adrian Spac 84, 134 Cristina Tuchilus 134 Adriana Trifan 40, 56, 134 Cristina-Corina Bentea 90 Adriano Mollica 36 D Agnes Toma 104, 138 Dana Bobit 48, 64, 66, 156 Agnieszka Piwowar 70 Dana Tutunaru 134 Alexandra – Andreea Buburuz 136 Daniel Damian 68, 69 Alexandru Amărioarei 62 Daniela Bratosin 102 Alice Grigore 64, 66, 80, 82 Daniela Gherghel 52, 60 Alina Butu 42, 50 Daniela Lupu 46 Aliona Rosca 158 Daniela Smarandache 138 Ana Clara Aprotosoaie 56, 90 E Ana Coste 58, 96 Ecaterina Liliana Dinu 112 Ana Mutu 130 Ecaterina Serban 82 Ana-Maria Enciu 86 Elena Berteanu 112 Ana-Maria Seciu 110 Elena Codrici 86 Anatolie Nistreanu 140 Elena Genova 38 Anca Boldea 160 Elena Iacob 74, 78, 114 Anca Farcas 96 Elena Ionescu 74, 78, 114 Anca Miron 56 Elena Iulia Oprita 104 Anca Oancea 138 Elena Larisa Tomescu 156, 158 Anca Sarbu 138 Elena Pruteanu 88, 126 Ancuta Cristina Raclariu 31, 158 Elena Trotus 124, 128 Ancuta Nechita 60 Elida Rosenhech 122, 132, 150 Andreea Andrei 158 Elisabeta Oprea 90 Andreea Toader 102 Elsayed Aboutabl 94 Andrei Lobiuc 100, 116, 122, 132, 142, 150 Elvira Gille 46,54,58,92,102, 134, 152 Andrei Paduraru 48 Ettore Novellino 36 Anna Długosz 70 Eugenia Dumitra Teodor 92 Anna Kazana 106 F Anna Maria Patrzałek 70 Faiza Mohamed Hammouda 94 Anne Krag Brysting 31 Florentina Gatea 92 Arnold Garbiec 70 Fyaz M. D. Ismail 25 Aurel Ardelean 122 G Aurélie Labarre 31 Gabriel Lucian Radu 92 Azzurra Stefanucci 36 Gabriel Teliban 142, 150 B Gabriela Popa 80 Bellatrache Cherifa 148 Gabriela Vochita 52, 60, 154 Bernd Honermeier 142 Georgeta Neagu 64, 66 Boryanka Traykova 38 Georgeta Negru 138 Boubellouta Houria 148 Georgiana Gavril 46, 54, 152, 154 Bujar Qazimi 72 Georgiana Zavoianu 25 C Gheorghe Coldea 58 Calin Garlea 48, 156 Gheorghe Florinel Brudască 120 Călin Lădasiu 122 Gianina Crisan 31, 158 Catalin Iordachel 112 Giorgia Macedonio 36 Carmen Dana Sandru 120 Gjoshe Stefkov 72 Carmen Elena Tebrencu 74, 78, 114, 158 Gogu Ghiorghita 154 Carmen Ionita 64, 66 H Carmenica Doina Jităreanu 142 Hsin-Lun Huang 35 Chin-Kun Wang 35 Hugo J. de Boer 31, 158 Constantin Craciun 100 I
Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
MAPPPS – 2016
Index of authors
Constantin Deliu 58 Ibrahim Ahmed Saleh 94 Constantin Toma 108, 118 Ilkay Erdogan Orhan 27 Ioan Burzo 98 Nicoleta Dobre 64, 66 Ioan Grozescu 68,69 Nicon Ungur 88, 126, 144 Ioana Roman 96 Nicu Stoica 86 Ion Ungureanu 140 Nina Rembiałkowska 70 Ionela Daniela Popescu 86 O Irina Boz 98, 100, 118 Oana Cioanca 40, 48, 84, 90, 134 Iris Tusa 62, 102, 112 Oana Craciunescu 110, 138 Iuliana Csilla Bara 154 Oana Teodora Ciuperca 74, 78, 114 Izabela Nawrot-Hadzik 106 Olakunle Onaolapo 36 J Olga Morarescu 126 Jasmina Petreska Stanoeva 72 Otilia Zarnescu 110 K P Kenneth J. Ritchie 25 Pall Emoke 120 Kermandji Mohamed Azed 148 Paula P. Sosoi 158 Khaled Ahmed Shams 94 Paula-Lucelia Ursache 128 Khelifi Touhami Fatima 148 Petruta Calina Cornea 80 L Philippe Renaud 88, 126, 144 Lacramioara Ivanescu 108 Pincu Rotinberg 52, 60 Larisa Calu 104 R Larisa Florescu 76 Radu Albulescu 86 Laurian Vlase 31 Radu Ionita 40 Lorena - Diana Popa 124, 136 Radu Necula 46, 48, 54, 58, 152, 156, 160 Lucia Moldovan 104, 110 Ramona Paltinean 31 Lucian Hritcu 40 Raul Jibotean 62 Lucian Ionita 64, 66 Remus Ciprian Cotunoaea 142 Luminita Tcacenco 112 Robert Konieczny 15 Lutfun Nahar 25 Robert Verpoorte 13 M Rodica Catana 76 Madalina Popa 48, 158 Rodica Martea 130 Magda Lewińska 70 Rodica Pasa 60 Manuela Elisabeta Sidoroff 54 Rodica Tatia 104 Marcello Locatelli 36 Ruxandra Cretu 46, 54, 74, 78, 152, 154, 156 Margareta Naie 124, 128 S Maria Chiriac 74, 78, 114 Sadiye Ayse Çelik 146 Maria Duca 130 Satyajit D. Sarker 25 Maria Paraschiv 112 Silvia Robu 84, 134 Maria-Magdalena Zamfirache 116, 132, 150 Simona – Florina Pochiscanu 136 Marian Burducea 116, 122, 142, 150 Simona Mihai 86 Marian Butu 42, 50, 62 Simona Savin 138 Mariana Geanta 112 Sorin Manoiu 138 Marin Zagnat 116 Steliana Rodino 42, 50 Marina Grinco 144 Süleyman Doğu 146, 147 Marina Spînu 120 Svetlana Colceru – Mihul 80,82 Marina Stanilova 38 Svetlana Kulevanova 72 Marina Stefova 72 Sylwester Ślusarczyk 15, 106 Marinela Afemei 118 Sylwia Zielińska 15 Marius Nicusor Grigore 108 T Marta Libik-Konieczny 15 Tarik Abdelhalim Mohamed 94 Marta Szandruk 106 Tatiana Chiru 44, 140 Mihael Cristin Ichim 31, 158 Tatiana Eugenia Sesan 138 Mihaela Dutu 122 Teodor Robu 142 Mihaela Niculae 120 Turan Akdağ 147 Mihaela-Ionica Enache 112 U Milen I. Georgiev 33 Ursula Stanescu 90, 134, 152 Mirela Ardelean 116, 122 V Mohamed-Elamir Fathy Hegazy 94 Valentin Grigoras 46, 54, 58, 102, 152 Monica Hancianu 40, 84, 90, 134 Valeria Grigoriev 44
Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
MAPPPS – 2016
Index of authors
N Vasile Stoleru 150 Nahla Sayed Abdel-Azim 94 Vasilica Onofrei 116, 142, 150 Neculai Munteanu 150 Veaceslav Kulcitki 88, 126, 144 Nicolae Ciobanu 140 Vincent Manzanilla 31 Viorica Coroiu 110 Vlad Simon Luca 56 Vlad Toma 96 Vladilena Gîrbu 88, 126, 144 W Weronika Kozłowska 15 Wieslaw Oleszek 20 Y Yavuz Bağcı 146 Z Zenovia Olteanu 116 Zofia Marchewka 70
Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
MAPPPS – 2016
No. Name E-mail address 1. 2. Adam Matkowski [email protected] 3. Adela Halmagyi [email protected] 4. Adina Segneanu [email protected] 5. Adrian Spac [email protected] 6. Adriana Trifan [email protected] 7. Adriano Mollica [email protected] 8. Alexandru Amărioarei [email protected] 9. Alice Grigore [email protected] 10. Alina Butu [email protected] 11. Aliona Roșca [email protected] 12. Ana Clara Aprotosoaie [email protected] 13. Ana Coste [email protected] 14. Anca Farcas [email protected] 15. Anca Miron [email protected] 16. Anca Oancea [email protected] 17. Ancuta Cristina Raclariu [email protected] 18. Andreea Andrei [email protected] 19. Andrei Paduraru [email protected] 20. Bujar Qazimi [email protected] 21. Calin Garlea [email protected] 22. Carmen Ţebrencu [email protected] 23. Catană Rodica [email protected] 24. Chin-Kun Wang [email protected] 25. Constantin Deliu [email protected] 26. Corina Bubueanu [email protected] 27. Cornelia Mircea [email protected] 28. Corneliu Tanase [email protected] 29. Coroiu Viorica [email protected] 30. Cosmin-Teodor Mihai [email protected] 31. Cristiana Tanase [email protected] 32. Cristina Elena Iancu [email protected] 33. Cristina Tuchilus [email protected] 34. Dana Bobit [email protected] 35. Daniel Damian [email protected] 36. Daniela Bratosin [email protected] 37. Daniela Gherghel [email protected] 38. Daniela Lupu [email protected] 39. Elena Berteanu [email protected] 40. Elena Codrici [email protected] 41. Elena Genova [email protected] 42. Elena Ionescu [email protected] 43. Elena Iulia Oprita [email protected] 44. Elena Larisa Tomescu [email protected] Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
MAPPPS – 2016
45. Elena Pruteanu [email protected] 46. Elida Rosenhech [email protected] 47. Elvira Gille [email protected] 48. Enache Mihaela-Ionica, [email protected] 49. Gabriel Lucian Radu [email protected] 50. Gabriel Teliban [email protected] 51. Gabriela Vochita [email protected] 52. Georgiana Gavril [email protected] 53. Gianina Crisan [email protected] 54. Gianina Crișan [email protected] 55. Hugo de Boer [email protected] 56. Ibrahim Ahmed Saleh [email protected] 57. Ilkay Erdogan Orhan [email protected] 58. Ioan Burzo [email protected] 59. Ioan Grozescu [email protected] 60. Ioana Roman [email protected] 61. Iordachel Catalin [email protected] 62. Irina Boz [email protected] 63. Iris Tusa [email protected] 64. Iuliana Csilla Bara [email protected] 65. Khelifi Touhami Fatima [email protected] 66. Lacramioara Ivanescu [email protected] 67. Larisa Calu [email protected] 68. Laurian Vlase [email protected] 69. Lobiuc Andrei [email protected] 70. Lucia Moldovan [email protected] 71. Madalina Popa [email protected] 72. Manuela Elisabeta Sidoroff [email protected] 73. Maria Chiriac [email protected] 74. Maria Duca [email protected] 75. Maria-Magdalena Zamfirache [email protected] 76. Marian Burducea [email protected] 77. Marian Butu [email protected] 78. Mariana Geanta [email protected] 79. Marina Spînu [email protected] 80. Marinela Afemei [email protected] 81. Mihael Cristin Ichim [email protected] 82. Milen I. Georgiev [email protected] 83. Mirela Ardelean [email protected] 84. Monica Hancianu [email protected] 85. Naie Margareta [email protected] 86. Oana Cioanca [email protected] 87. Oana Ciupercă [email protected] 88. Oana Craciunescu [email protected] 89. Olga Morarescu [email protected] Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 1
MAPPPS – 2016
90. Paraschiv Maria [email protected] 91. Paula P. Sosoi [email protected] 92. Paula-Lucelia Ursache [email protected] 93. Pincu Rotinberg [email protected] 94. Popa Diana [email protected] 95. Radu Necula [email protected] 96. Robert Verpoorte [email protected] 97. Rodica Martea [email protected] 98. Ruxandra Cretu [email protected] 99. Satyajit d Sarker [email protected] 100. Simona – Florina Pochişcanu [email protected] 101. Steliana Rodino [email protected] 102. Süleyman Doğu [email protected] 103. Tatiana Chiru [email protected] 104. Tcacenco Luminita [email protected] 105. Teodor Robu [email protected] 106. Toma Vlad [email protected] 107. Trotus Elena [email protected] 108. Turan Akdağ [email protected] 109. Ursula Stanescu [email protected] 110. Valentin Grigoras [email protected] 111. Valeria Grigoriev [email protected] 112. Vasilica Onofrei [email protected] 113. Vladilena Gîrbu [email protected] 114. Wiesław Oleszek [email protected] 115. Zenovia Olteanu [email protected]
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