1
Extension scientific progress report
Garlic & Health project
QLK1-CT-1999-00498
2004
Table of contentspage number
Section 1: Project identification4
Section 2: Project progress report6
Annex I: project progress summary6
Annex II: progress report8
1.Objectives and expected achievements10
2.Project workplan10
2.1Introduction10
2.2Project structure, planning and timetable13
2.2.1Progress during the first reporting period13
2.2.1.1Discussion – conclusion15
2.2.1.2Future action15
2.2.1.3Action requested from the Commission15
2.2.2Progress during the second reporting period15
2.2.2.1Discussion – conclusion18
2.2.2.2Future action19
2.2.2.3Action requested from the Commission20
2.2.3Progress during the third reporting period24
2.2.3.1Discussion – conclusion29
2.2.3.2Future action30
2.2.3.3Action requested from the Commission31
2.2.4Progress during the fourth reporting period33
2.2.4.1Discussion – conclusion38
2.2.4.2Future action39
2.2.4.3Action requested from the Commission39
2.2.5Progress during the extension reporting period39
2.2.5.1Discussion – conclusion41
2.2.5.2Future action41
2.2.5.3Action requested from the Commission41
2.2.6Overall list of milestones41
2.2.7Overall list of deliverables48
2.3 Description of the workpackages53
3.Role of participants68
4. Project management and coordination 99
- Exploitation and dissemination activities 101
- Ethical aspects and safety provisions 107
- Appendix: report on ISHS Allium symposium109
The G&H partners who participated in the International ISHS Symposium on Edible Alliaceae in April 2004
Project Progress Summary
Section 1: Project identification / NOT CONFIDENTIALTitle of the project
Garlic & Health: the development of high quality garlic and its influence on biomarkers of atherosclerosis and cancer in humans for disease prevention
Acronym of the project
G&H
Type of contract SC / Total project cost (in euro)
6.122.383€
Contract number / Duration (in months) / EU contribution (in euro)
QLK1-CT-1999-0498 / 51Months / 3.939.136€
Commencement date
February 1 2000 / Period covered by the progress report
February 1 2004 – April 30 2004
PROJECT COORDINATOR
Name
C. Kik / Title
Dr. / Address
Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands
Telephone
31 317 477001 / Telefax
31 317 418094 / E-mail address
Key words
garlic, health, nutrition, food, metabolism
World wide web address
List of participants
P1: C. Kik. Plant Research International, Wageningen University & Research Center, P.O. Box 16, 6700 AA Wageningen, The Netherlands. tel. 31317 477278/477001; fax:31317418094; e-mail:
Subcontractor 1: H. de Groot, BGS, Westelijke Randweg 1, 1721 CH Broek op Langedijk, the Netherlands. tel. 31 226 316391; fax: 31 226 316291; e-mail: .
P2: B. Thomas. HRI, Wellesbourne, Warwick CV35 9EF, United Kingdom. tel: 44 1789 470382; fax:441789470552; e-mail:
P3: H.A. Collin. The University of Liverpool, School of Biological Sciences, Donnan Laboratories, L69 7ZD Liverpool. United Kingdom. tel: 44 151794 3612, fax: 44 151794 3655; e-mail:
P4: O. Huchette, COOPD’OR , B.V. 1540, 21034 Dijon, France. tel: 33-380693127; fax: 33-380693263; e-mail:
Subcontractor 2: P. Besset, GIE UNISEM DROM, Z.A. de Brunelle, 26400 Eurre, France. Tel. 33 475250284; fax: 33 475768692
Subcontractor 3: C. Hug, S.T.L., route de Chevigny, 21130 Auxonne, France. Tel. 33 380774747; fax: 33 380774759; e-mail:
P5: J. Auger. Universite de F. Rabelais, IRBI, Faculte des Sciences et Techniques, Avenue Monge 37200 Tours, France. tel: 33 247366970; fax: 33 247366966; e-mail:
P6: J. Martin Sanchez. Dto. de Microbiologia, Universidad de Cordoba. San Alberto Magna S/n 14004 Cordoba, Spain. tel: 34 957 218603; fax: 34957218650; e-mail: and X. Barandiaran tel: 34 957 293333/206; e-mail: (current e-mail: )
P7: R. Kamenetsky. The Volcani Center, P.O. Box 6, Bet Dagan, 50250, Israel. tel: 972 3 9683511; fax:97239660589; e-mail: or
Subcontractor 4: O. Ashurmetov, Institute of Botany, Uzbek Academy of Sciences, Khojaev street 32, 7000143 Tashkent, Uzbekistan.
P8: R. Kahane. CIRAD-FLHOR, Ta50/PS4, Boulevard de la Lironde, 34398 Montpellier Cedex 5, France. tel: 33 4 676 15562, fax: 33 4 67615688; e-mail:
(P9): V. Chovelon, INRA, Pathologie Vegetale, BP 94, 84143 Montfavet, France. tel: 33 4 32 72 28 87/40; fax: 33 4 32 72 28 42; e-mail:
P10:H.D. Rabinowitch, The Hebrew University, Faculty of Agriculture, Food & Environmental Quality Sciences, Department of Field Crops, Vegetables & Genetics. P.O. Box 12, Rehovot 76100, Israel. tel: 972 8 9489292/306; fax: 972 8 9462384; e-mail:
P11: R. Gebhardt. University of Leipzig, Dept. of Biochemistry. Liebigstrasse 16, 04103 Leipzig. Germany. tel: 49 341 9722100; fax: 49 341 9722109; e-mail:
Subcontractor 5: K. Burggraaf, Centre for Human Drug Research, Zernikedreef 10, 2333 CL Leiden, the Netherlands; tel: 31 71 5246404; fax: 31 71 5246499; e-mail:
P12: M.H. Siess. UMR de Toxicologie Alimentaire INRA-ENSBANA, 17 rue Sully, BP 86510, 21065 Dijon Cedex, France. tel. 33 3 80693221; fax: 33 3 80693225; e-mail: (current e-mail: )
P13: A.M. Vollmar. University of Munchen, Lehrstuhl fur Pharmazeutische Biologie, Zentrum fur Pharmaforschung, Butenandstrasse 5, 81375 Munchen, Germany. tel: 49 89 2180 7172/7168; fax: 49 89 2180 7173; e-mail:
P14:J.M.G. Princen. TNO- Prevention and Health, Gaubius Laboratory, Zernikedreef 9, 2333 CK Leiden, The Netherlands; tel: 31 71 5181471 (5181478); fax: 31 71 5181904; e-mail:
P15:Th. Haffner, Lichtwer Pharma AG, Wallenroder Strasse 8-10, D-13435 Berlin, Germany; tel. 49-30 40370 502; fax 49 30 40370 581; e-mail: (current e-mail: )
All partners are contractors. P9 and P12 are linked.
Annex I: Project Progress Summary
Section 2: Project Progress Report NOT CONFIDENTIALObjectives:
Garlic: The general objective of the plant part of the G&H project is to generate knowledge which will enable the production of high quality European garlic material with a high concentration of health beneficial components. This will be achieved by: a. the development of a highly variable core collection for S-alk(en)yl cysteine sulphoxides (ACSOs), b. the development of new breeding, mass propagation and cultivation methods and c. the elucidation of the sulphur pathway of garlic. The specific objectives of the first year in plant part were characterised by the development of protocols (plant, cell, tissue cultures; molecular, biochemical and physiological analyses) and the build-up of material (garlic accessions or powders) with distinct properties.
Health: Cardiovascular diseases and cancer are by far the leading causes of death and morbidity in the EU. Garlic has been used for a long time as food with many therapeutic effects. However, detailed studies on the mechanisms underlying the interference of garlic with cardiovascular diseases and cancer are lacking. Moreover, only few is known about the compounds of garlic responsible for these effects. Consequently, experimental and clinical studies using defined garlic preparations and compounds are poorly found. Therefore, the main objective of the health part of the project is the analysis of the role of garlic for the prevention of chronic diseases like artherosclerosis and cancer through cellular, molecular and clinical studies in cells, animals and humans. In particular, molecular criteria will be worked out for quality testing of garlic preparations and biomarkers will be identified as indicators for the modulation of theses diseases in humans.
Extension period results and milestones:
Garlic:
WP1 Genetic resources
The level of organo S-compounds in thirty-eight garlic accessions grown in the Netherlands were analysed. Chromatographic separations were performed, using an UV detector. Principal Component Analysis was carried out on concentrations of alliin, isoalliin, GLUAlCS and isoGLUAlCS. It was shwon that some accessions had a very deviant composition in organo-S compounds. Furthermore a paper and a poster on the ISHS Allium symposium was prersented.
WP2 Breeding systems
The main activities that took place in the extension period were the coordination and writing of the scientific reports for the plant side of the G&H project. The other activities included the writing of research papers and the participation in the Allium symposium in Beijing in April 2004. On this symposium a paper, a key note and a posters were presented.
WP3 Cultivation
During the extension peiod a paper was presented and a poster co-presented on the ISHS Allium symposium in Beijing. Furthermore the writing of scientific papers took place.
WP4 Sulphur biochemistry
The extension was used to confirm the characterization of the cysteine synthase enzyme in garlic by repeating the analysis of the transformed Arabidopsis and making use of the E.coli in vitro system. Arabidopsis plants which had been transformed with the putative gene for cysteine synthase in garlic were examined for expression of this gene by a bioassay for cysteine synthase involving HPLC analysis. The repeat experiments confirmed those run earlier that although the plants were transformed successfully the gene was silenced. The problem of gene silencing in transformed plants is quite common. No expression of cysteine synthase could be detected. When the ability of the gene to form cysteine synthase was examined in vitro using E.coli lysate, the gene isolated from garlic was in fact able to synthesise cysteine synthase thus confirming the earlier analyses.
Furthermore considerable time was spent on the writing of papers and the participation in the ISHS Allium symposium in Beijing. At this symposium a paper was presented and a poster co-presented.
WP8 Human intervention study
The human intervention study was initiated according to schedule, but was delayed because of unforseen reasons could be finished only during the extension period. It was carried out as a double-dummy placebo-controlled trial in parallel design with 3 groups treated with a garlic medication versus control and a statin as positive control. With this design, careful analytisis of the garlic medication and a total of 90 subjects of either sex, this study is the most advanced, thoroughly performed and reliable clinical trial ever realized on a worldwide scale. The study convincingly shows that use of garlic in a dose of 2.1 mg daily for 12 weeks is not associated with a beneficial effect on a variety of markers characteristic for atherosclerosis in a population with a couple of risk factors for cardiovascular disease. Furthermore, garlic administration for 90 days neither protected against lymphocyte DNA alteration nor reduced urine mutagenicity in smoker subjects suggesting that garlic consumption would also not protect against the first stages of carcinogenesis. In conclusion, the disease preventive potential of garlic in humans appears much less than believed before.
Benefits and Beneficiaries:
Garlic
The international scientific community benefitted for the first time from the G&H results as they were presented at the Symposium on Edible Alliaceae in Beijing China. As a feedback effect, the publicity for the project was immediate and it became clear for the scientific community, mainly plant scientists, that the EU project on garlic was the largest one in the world, and that the main references, experience and expertise are at present in Europe. This should give an advantage to European research laboratories and teams when competing for grants and funding. This should enhance the interaction between research and private initiatives, as mostly SME are involved in garlic industry, with poor access to research results and innovative information. The large impact of the Plant part outputs has been acknowledged, opening new opportunities for looking at plant diversity, for breeding with conventional or unconventional methods, including quality traits based on flavour, N- or S-fertilization, or stress resistance, and should be converted into new projects and applied collaborations in the next months
Health:
The carefully performed human intervention study has helped considerably in a realistic assessment of garlic’s health care potential. Even if the outcome suggests that the disease preventive potential of garlic may be lower than previously thought, such a solid discrimination of facts and fancy is of inestimable value for the consumer and even more for the patient in Europe as well as in the whole world.
Future Actions (if applicable):
Annex II: Progress report
Progress Report
Title of the projectGarlic & Health: the development of high quality garlic and its influence on biomarkers of atherosclerosis and cancer in humans for disease prevention
Acronym of the project
G&H
Type of contract
SC / Total project cost
6.122.383 €
Contract number / Duration (in months) / EU contribution
QLK1-CT-1999-00498 / 51Months / 3.939.136€
Commencement date
1 February 2000 / Period covered by the progress report
February 1 2004 – April 30 2004
PROJECT COORDINATOR
Name
C. Kik / Title
Dr. / Address
Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands
Telephone
31 317 477278 / Telefax
31 317 418094 / E-mail address
Key words
garlic, health, nutrition, food, metabolism
World wide web address
List of participants
P1: C. Kik. Plant Research International, Wageningen University & Research Center, P.O. Box 16, 6700 AA Wageningen, The Netherlands. tel. 31317 477278/477001; fax:31317418094; e-mail:
Subcontractor 1: H. de Groot, BGS, Westelijke Randweg 1, 1721 CH Broek op Langedijk, the Netherlands. tel. 31 226 316391; fax: 31 226 316291; e-mail: .
P2: B. Thomas. HRI, Wellesbourne, Warwick CV35 9EF, United Kingdom. tel: 44 1789 470382; fax:441789470552; e-mail:
P3: H.A. Collin. The University of Liverpool, School of Biological Sciences, Donnan Laboratories, L69 7ZD Liverpool. United Kingdom. tel: 44 151794 3612, fax: 44 151794 3655; e-mail:
P4: O. Huchette, COOPD’OR , B.V. 1540, 21034 Dijon, France. tel: 33-380693127; fax: 33-380693263; e-mail:
Subcontractor 2: P. Besset, GIE UNISEM DROM, Z.A. de Brunelle, 26400 Eurre, France. Tel. 33 475250284; fax: 33 475768692
Subcontractor 3: J.P. Vauchon, S.T.L., route de Chevigny, 21130 Auxonne, France. Tel. 33 380774747; fax: 33 380774759; e-mail:
P5: J. Auger. Universite de F. Rabelais, IRBI, Faculte des Sciences et Techniques, Avenue Monge 37200 Tours, France. tel: 33 247366970; fax: 33 247366966; e-mail:
P6: J. Martin Sanchez. Dto. de Microbiologia, Universidad de Cordoba. San Alberto Magna S/n 14004 Cordoba, Spain. tel: 34 957 218603; fax: 34957218650; e-mail: and X. Barandiaran tel: 34 957 293333/206; e-mail: (current e-mail: )
P7: R. Kamenetsky. The Volcani Center, P.O. Box 6, Bet Dagan, 50250, Israel. tel: 972 3 9683511; fax:97239660589; e-mail: or
Subcontractor 4: O. Ashurmetov, Institute of Botany, Uzbek Academy of Sciences, Khojaev street 32, 7000143 Tashkent, Uzbekistan.
P8: R. Kahane. CIRAD-FLHOR, Ta50/PS4, Boulevard de la Lironde, 34398 Montpellier Cedex 5, France. tel: 33 4 676 15562, fax: 33 4 67615688; e-mail:
(P9): V. Chovelon, INRA, Pathologie Vegetale, BP 94, 84143 Montfavet, France. tel: 33 4 32 72 28 87/40; fax: 33 4 32 72 28 42; e-mail:
P10:H.D. Rabinowitch, The Hebrew University, Faculty of Agriculture, Food & Environmental Quality Sciences, Department of Field Crops, Vegetables & Genetics. P.O. Box 12, Rehovot 76100, Israel. tel: 972 8 9489292/306; fax: 972 8 9462384; e-mail:
P11: R. Gebhardt. University of Leipzig, Dept. of Biochemistry. Liebigstrasse 16, 04103 Leipzig. Germany. tel: 49 341 9722100; fax: 49 341 9722109; e-mail:
Subcontractor 5: K. Burggraaf, Centre for Human Drug Research, Zernikedreef 10, 2333 CL Leiden, the Netherlands; tel: 31 71 5246404; fax: 31 71 5246499; e-mail:
P12: M.H. Siess. UMR de Toxicologie Alimentaire INRA-ENSBANA, 17 rue Sully, BP 86510, 21065 Dijon Cedex, France. tel. 33 3 80693221; fax: 33 3 80693225; e-mail: (current e-mail: )
P13: A.M. Vollmar. University of Munchen, Lehrstuhl fur Pharmazeutische Biologie, Zentrum fur Pharmaforschung, Butenandstrasse 5, 81375 Munchen, Germany. tel: 49 89 2180 7172/7168; fax: 49 89 2180 7173; e-mail:
P14:J.M.G. Princen. TNO- Prevention and Health, Gaubius Laboratory, Zernikedreef 9, 2333 CK Leiden, The Netherlands; tel: 31 71 5181471 (5181478); fax: 31 71 5181904; e-mail:
P15:Th. Haffner, Lichtwer Pharma AG, Wallenroder Strasse 8-10, D-13435 Berlin, Germany; tel. 49-30 40370 502; fax 49 30 40370 581; e-mail: (current e-mail:
All partners are contractors. P9 and P12 are linked.
1.OBJECTIVES AND EXPECTED ACHIEVEMENTS
The production of garlic (Alliumsativum L.) in Europe is concentrated in Mediterranean countries, Spain, France and Italy. The price of European garlic is high, especially in comparison with Chinese garlic. This price difference could severely threaten the European garlic growers if there was a free market in Europe for garlic. However, to protect the European garlic growers the European Commission issued in 1993 a regulation that only 12000 tons of Chinese garlic can be imported annually. The main objective of the plant part of the project is to develop methods, through improved seed technology and genetics, which will enable the European garlic growers to meet the internal demand for high quality products and to compete on the international level.
Cardiovascular diseases and cancer are by far the leading causes of death and morbidity in the EU. The health cost spent on the treatment of these diseases reaches many billion ECUs. Garlic has been used for a long time as food with many therapeutic effects. Its beneficial effects are directly linked to its sulphur metabolism. Detailed studies on the mechanisms underlying cancer and cardio-vascular diseases are lacking, especially studies with defined garlic preparations and compounds are poorly found. Therefore the main objective of the health part of the project is the analysis of the role of garlic for the prevention of chronic diseases like atherosclerosis and cancer through cellular, molecular and clinical studies in cells, animals and humans. Specific biomarkers will be identified as indicators for the modulation of these diseases on the cellular and tissue level and in humans as a basis for new intervention studies.
Two technological objectives are pursued for industrial potential applications: garlic mass propagation of elite clones, to enable cropping and processing of high quality products, and the development of new garlic pharmaceutical formulations with improved properties and quality for the therapy and prevention of chronic diseases.
2.PROJECT WORKPLAN
2.1Introduction
The main targets of this proposal are to improve the quality of garlic and derived products and to understand sulphur metabolism in the plant as well as in the human body, in order to strengthen the European garlic-based food industry and public health. To reach these targets, the work of the proposed project will focus on:
-establishing a highly variable core collection of garlic for CSOs (S-alk(en)yl cysteine sulphoxides) and fertility.
-developing new breeding, mass propagation and cultivation methods for garlic.
-analysing the biochemistry of the garlic sulphur pathways.
-the molecular mechanisms of garlic bio-active sulphur compounds on cancer and cardio vascular diseases as atherosclerosis.
-garlic properties in humans relating to cancer and atherosclerosis.
-establishing pharmaceutical requirements for new garlic formulations.
The project outline is presented in the following figure:
TARGET: Understanding the mechanism of the sulphur metabolism in garlic and in human health for the development of high quality garlic and the improvement of human health.
The programme is organised into nine interrelated workpackages (wp):
-WP one concerns the evaluation of the gene pool for organo-sulphur traits and fertility and the construction of a core collection; P1, P5, P6, P7 and P10 are involved.
-in WP two a reliable genetic transformation system and a mass propagation method will be developed and the sexual hybridisation system studied; P1, P6, P7, P8, P9 and P10 are involved.
-in WP three the interaction between genotype and environment will be evaluated; P4, P5, P6, P8 and P9 are involved.
-WP four involves the analyses of the biochemistry of the sulphur pathway in garlic; P2 and P3 are involved.
-WP five will examine the influence of garlic compounds on the patho-mechanisms of cardio-vascular diseases like atherosclerosis, especially the immunologically mediated inflammatory process of atherosclerosis with identification of inflammatory bio-markers, further the enzymes involved in signal transduction regulating cholesterol metabolism and the action and efficacy of garlic preparations in transgenic mice as models for human-like hyper-lipidemia and atherosclerosis with experiments in cell cultures, in animals, and finally in humans; P11, P13, P14 are involved.
-WP six deals with the influence of garlic on the development of cancer, especially the activation and detoxication of carcinogens and prevention of carcinogen geno-toxicity as well as inhibition of the initiation of hepato-carcinogenesis, and the metabolism of garlic compounds in tissues, animals, further the signalling pathway of apoptosis induction is investigated; P5, P12 and P13 are involved.