Third year annual scientific report
Garlic & Health project
QLK1-CT-1999-00498
2002- 2003
Table of contents
Section 1: Project identification2
Section 2: Project progress report4
Annex I: project progress summary4
Annex II: progress report9
1.Objectives and expected achievements11
2.Project workplan11
2.1Introduction11
2.2Project structure, planning and timetable14
2.2.1Progress during the first reporting period14
2.2.1.1Discussion – conclusion16
2.2.1.2Future action16
2.2.1.3Action requested from the Commission17
2.2.2Progress during the second reporting period17
2.2.2.1Discussion – conclusion20
2.2.2.2Future action20
2.2.2.3Action requested from the Commission21
2.2.3Progress during the third reporting period26
2.2.3.1Discussion – conclusion30
2.2.3.2Future action31
2.2.3.3Action requested from the Commission31
2.2.4Overall list of milestones32
2.2.5 Overall list of deliverables39
2.3 Description of the workpackages44
3.Role of participants113
4. Project management and coordination 296
- Exploitation and dissemination activities 298
- Ethical aspects and safety provisions 302
Project Progress Summary
Section 1: Project identification/ NOT CONFIDENTIAL
Title 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 / 48Months / 3.939.136€
Commencement date
February 1 2000 / Period covered by the progress report
1 February 2002 – 31 January 2003
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: 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:
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:
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: or
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:
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.
Third year results and milestones:
Garlic:
WP 1 (Genetic resources)
P1, P7, and P10 - Collection and evaluation of garlic accessions and close relatives
Flowering evaluation of collected plant material
Phenological observations have been performed in Israel on ca. 300 landraces of garlic from collections or collected in the first and second years of the project. Garlic accessions were separated into two major sub-populations : semi-bolters (producing blind scapes) and bolters (producing flower scape), which differ in leaf number, leaf length and bulbing ability under short-day conditions. The bolting accessions were subdivided into two subgroups according to flower/topset ratio. Most of flower-producing accessions had fertile pollen and receptive stigmas. In Israel, flower development was recorded in ca. 30 accessions from Central Asia.The plants used to assess their seed-set capacity were selected on basis of their AFLP fingerprint and previously obtained info on flowering. In the Netherlands, the same accessions were transferred to greenhouse. In contrast to the last years, a large number of seeds were produced (around 1700) and 7 accessions produced most seeds. A provisional core collection of 53 accessions was then established.
Seed development and distribution of garlic clones for further use in the project
Following anthesis, pollination, and fertilization, true garlic seeds were obtained in 2001 in Israel from 5 accessions previously collected in Central Asia between 1996 and 1999.Germination rate reached ca 90%, and the emerged seedlings developed normally, mature bulbs consisted of a single clove and varied in color and size. They also differ in bulbing ability and ripening date. We were surprised by the extreme variation in phenotypic responses of garlic accessions from a single geographical region, especially with regard to flowering traits and seed production.
P5 - Screening for CSO in the existing and the new collected garlic collections
Typical chromatograms of garlic bulbs were obtained by HPLC-UVunder identical conditions. Concentrations of S-compounds in fresh bulbs from the Israeli and the Dutch collections were compared, some accession numbers clearly differentiated from the others. PCA analysis showed that the accessions of sativum group are separated from the accessions of thelongicuspisgroup. Cv. Morado is located closer to the sativumgroup, as compared to cvs. Morasol and Thermidrôme. Growth conditions affected S-compound contents of garlic clones. Cultivation under Israeli conditions increased GLUAlCS, IsoGLUALCS and allicin contents, while alliin and isoalliin contents were not affected.
WP 2 (Breeding)
P7and P10 - Sexual hybridisation
The effect of environmental conditions on sequential morphological processes during the vegetative and reproductive development of garlic have been studied. Under all growth conditions, the apical meristem underwent floral transition, and floral differentiation occurred in all plants. Further development was temperature- and day-length dependent. In the inflorescence, differentiation of newly formed flowers and topsets proceeded concomitantly with scape elongation. The development of topsets was dominated by photoperiod.
P9 - Sanitary aspects
Virus-free plants (ELISA controlled) have been produced in field under insect-proof tunnel for the other partners (300 bulbs/variety). Virus elimination by thermotherapy and meristem-tip culture was performed on 4 clones selected and supplied by the partners of WP1.
P1 and P6 - Genetic transformation
An efficient callus induction and plant regeneration system have been developed from segments taken along the entire length of the roots. This system was used for garlic transformation. A reliable transformation system has been established using different sources of explant and different constructs. A number of transgenic garlic plants have survived the hardening in greenhouse. Additional transgenic plants are growing in the chamber room. Regeneration experiments were carried out to study the effect of light and flurprimidol, or the efficiency of basal plates as explants. Garlic cell suspensions provided by P8, have expressed excellent material for the biolistic device. High levels of GUS expression have been obtained and selection protocols were more efficient with this type of explant.
P8 - Embryogenesis
Callus production was optimal on explants derived from root sections, but callus from young leaves expressed higher embryogenic potential. Up to 90% of such embryogenic callus differentiated globular somatic embryos after 2 months. Embryogenic calluses were used to establish cell suspension cultures. Friable calluses were induced from compact ones, and could
give rise the production of cell suspension cultures composed of small aggregates of embryogenic cells. This type of explant was shared with partners for genetic transformation.
P1, P5, P8 and P9 - Acclimatisation and genetic characterisation
A large number of somatic embryos (potentially 8.109 to 1011 per year) could be produced for each tested variety. The conversion rate into plantlet was ca. 50%. Plants were successfully hardened in greenhouse. The final products of somatic embryogenesis derived from callus or from cell suspension have been tested for each variety. Four tetraploid plantlets from 100 tested were detected by flow cytometry. Such a surprising result has to be repeated and the stability of this material to be checked.
WP 3 (Cultivation)
The experimentation in vitro and in greenhouse was carried out according to the programme and the respective Alliin analyses were performed on the bulbs produced. Furthermore, a methodological issue could be published. A synthesis of the data from field experiments could be done from additional analysis on the powders produced in 2000 and 2001, analysis performed following the latest protocol. Confirmation of the effect of light spectrum quality on Alliin synthesis in garlic bulbs in vitro gives arguments for an environmental effect in field condition. Greenhouse experiments on these factors will be launched for 2003.
P4 and P5 – In vitro experiments
On the opposite to last year results, an interaction has been revealed between sucrose feeding and Alliin production in Morasol bulbs produced in vitro : 6% sucrose produced larger bulbs, which were higher concentrated in Alliin. This may be due to the variety, or to the new method for Alliin analysis. However, a similar strong effect of the far red light has been reported throughout the years and the varieties. Bulbing experiments on plant material issued from somatic embryogenesis were performed according to the previous experiments. This milestone belongs to WP2, but the results will contribute to WP3 deliverables.
P4 and P5 - Greenhouse experiments
The scheduled experiment was carried out, and the next one is launched from October 2002. Similarly to the preliminary experiment, and to the field trials, no morphological or yield difference could be observed between S-treated plants, although S-fertilization treatments differed strongly. The Alliin concentrations were also not much different between treatments, leading to two hypotheses : i) S was not available by the plants, ii) S was incorporated into the plants but not as ACSOs. The data from total S of the plants are expected.The virus contaminated material (OYDV or LYSV) expressed higher Alliin concentration than virus-free bulbs, but was much lower yielding. Healthy plants had a better synthesis in Alliin, and we proved the hypothesis of higher Alliin production due to stressing conditions is wrong. This will be published as an additional deliverable (milestone for year 4).
P4 and P5 - Field experiments
No experiment took place in the field this year. The data collected from 2000 and 2001 are still to be synthesized and published. The results from the experiments in vitro and in greenhouse are needed to comfort the environmental hypothesis of light factor for Alliin differences between locations and genotypes.
WP 4 (Sulphur biochemistry)
P3 - Analysis of pattern of labeling in later stage intermediates
A series of further experiments were undertaken to build on the results obtained. In earlier experiments we faced variation in callus growth, which has been now reduced by selecting one rapidly growing callus as a source for all subsequent subcultures. The aim was to repeat the precursor feeding with garlic callus so as to establish whether the serine pathway was functional in garlic. A range of potential precursors was incorporated in callus medium prior to incubation. The production of any alk(en)yl cysteine conjugate and/or alk(en)yl cysteine sulphoxide was measured by HPLC after extraction of the callus. There was an increase in alliin, propiin, allyl cysteine and propyl cysteine when the relevant intermediates were used which indicated the network nature of the metabolic pathways. There were no obvious differences between the sampling times. It appeared that thiols provided an intermediate for the synthesis of alliin or propiin confirming the possibility of the existence of this pathway.
P2 and P3 - Analysis of second-year field experiment and whole plant labelling studiesAn experiment was carried out to examine the patterns of sulphur uptake and partitioning as a repeat of a similar experiment carried out in 2001. In addition, labelled sulphur was fed to developing garlic plants to attempt to determine the patterns of partitioning of organic sulphur during garlic growth and clove formation. From the data so far available, the pattern of sulphur accumulation was similar to that in the previous years, although some detailed aspects of the timing of changes in development were observed, mainly reflecting variability in the environmental conditions experienced year on year.
P2 - Purification of PCR amplified cDNA and sequencing
This milestone concentrated on genes for Cysteine synthase (Csase) and serine acetyl transferases (SATases). Screening for a novel CSase that exhibits S-allyl-CSase activity led to the isolation of four clones encoding cysteine synthase. Functional studies on these genes were attempted using an inducible transgenic expression system based on ethanol induction. For technical reasons, this strategy was unsuccessful and alternative inducible systems are being investigated. A plasmid cDNA library was constructed from combined total RNA extracted from garlic clove, leaf and root organs. An aliquot of the cDNA library was enriched with a PCR product derived from a cDNA fragment with the aim to detect at least one SATase isoform. Among the 1.6 x 104 colonies screened one full-length SATase cDNA was identified.
P2 - Expression studies on alliinase clones
Alliinase expression was measured using a RT-PCR approach, using the 18S rRNA as an internal standard. Alliinase expression in both clove and leaf tissue did not vary significantly with stage of development. These experiments were initiated in 2002 and are still in progress.
Health:
WP5 (Cardio-vascular diseases)
P13 – The interaction of garlic with mediators of inflammation
Neither garlic powders nor garlic constituents (including new metabolites such as AMSO2) showed a significant effect on the transcriptional activity of transcription factor NF-B determined by reporter gene assays. These results correspond to the data obtained for the adhesion molecules ICAM-1, VCAM-1 and E-selectin during the second reporting period. It can be concluded that NF-B is not the main target of garlic at least in endothelial cells, in vitro. Using whole human blood samples, garlic extracts and compounds lead to the release of several pro- and antiinflammatory cytokines from blood cells. The extent and kind of cytokine, however, varied with extracts from different years and locations. This does not seem to result from microbial contamination (i.e. LPS), but may reflect the different composition of these extracts revealed by HPLC analysis performed by P5 and P15. Apparently Spanish garlic had a slightly better impact on cytokine liberation.
P11 – interactions of garlic with signal transduction and cholesterol metabolism
The inhibitory mode of action of garlic compounds (DADS) on hepatocellular cholesterol biosynthesis could successfully be attributed in this and the last year to an activation of AMP-dependent kinase. This result propted additional studies on the interference of garlic compounds on other intracellular signaling events in muscle and liver cells from diabetic rats. It could be shown that AMPK-mediated neoformation of mitochondria and insulin sensitivity are two other events that may be enhanced by garlic compounds such as DADS at reasonably low concentrations. Apparently, garlic may provide a health benefit under a various conditions of stress. In addition, endothelial cells responded to DADS by selectively reducing the secretion of matrix metalloproteinases and TIMP-1 although at somewhat higher concentrations. These results indicate that also endothelial cells may be affected by garlic compounds but via other mechanisms than NFkB. In a third complex it could be shown that garlic powders from the second harvest again showed a transition from stimulating to inhibiting effects on cholesterol metabolism depending on the degree of fertilization.
P14 – first and second intervention study performed with ApoE3 Leiden transgenic mice
The first study was completed with the following conclusions: Garlic powder and garlic isolated compounds do not have an effect on plasma levels of cholesterol and triglycerides in line with recent human intervention studies. Neither cholesterol synthesis nor platelet activation even under atherogenic conditions was observed. The garlic preparations, however, may have liver protective effects, and one powder and allicin appear to have a beneficial effect on the inflammation marker serum amyloid A. The second atherosclerosis study in transgenic ApoE3-Leiden mice that considered the experiences of the first study and involved milder hyperlipidemic conditions is not yet completed. The main focus there remained on the influence of garlic powders on atherosclerosis but antiinflammatory and hepatoprotective effects were also considered. The results of these in vitro and in vivo studies will have impact on the design of the Human intervention study.