ABOUT OUR NEW HPLC METHOD

1. GLUAlCS 0,30 Alliin GLUAlCS GLUPeCS 0,40 Alliin SAC Allicin 0,25 0,30 0,20 GLUPheAla 0,15 SAC 0,20 0,10 0,10 0,05 0,00 0,00 4,00 8,00 12,00 16,00 20,00 24,00 28,00 0,00 4,00 8,00 12,00 16,00 20,00 24,00 28,00 Chromatogram of 4 standards (left) or of a garlic bulb in inhibited alliinase conditions (right) H N H 2 N H2O2 Alliinase H N 2 γ-glutamyl-transpeptidase S H O O C S C O O H O C O O H O S-allyl-L-cysteine (SAC) S-allyl-L-cysteine sulfoxide (alliin) Allicin Garlic & Health P5 J. AUGER ABOUT OUR NEW HPLC METHOD • We have identified a new compound in garlic HPLC profile, the S-allyl cystein (SAC) that has a potential therapeutic activity • We are now able to quantify 3 precursor compounds of Allicin γ-glutamyl-S-allyl-L-cysteine (GLUAlCS) Allicin biosynthesis from γ-glutamyl-S-allyl-L-cysteine • With this method we are now ready to compare the core collection

2. 3,5 0,8 S30 30 0,7 3,0 S60 0,6 60 2,5 S120 0,5 120 2,0 0,4 Mean bulb weight (g) Alliin (mmol/mg FW) 1,5 0,3 1,0 0,2 0,1 0,5 0,0 0,0 F F+i F27 F F+i F27 G&H April 2002, LeipzigStimulating effect of i light on Alliin synthesisWP3 / cultivation, P4 * * PRI * * * Sucrose (in g/l) MES COOPD'OR R&D

3. Garlic & Health P5 J. AUGER IN VITRO EXPERIMENTS : REP. OF SERIE I THE AIM OF THIS EXPERIMENT : to verify the results of the first experiment with an other hybrid, Morasol. Bulbing conditions: Sucrose (S30, S60 and S120). Cold induction period, 2 months, and F+i. Lichtwer HPLC method used Alliin concentration (nmole/mg) • The sucrose 60g/l seems to induce more Alliin contrary to the first results, and this HPLC method is more suitable for its quantification alone.

4. MES MOR G&H April 2002, LeipzigIn the absence of i light, erratic response to SWP3 / cultivation, P4 Alliin content (nmole/mg FW) 1,0 0,065 0,060 0,8 0,055 0,050 0,6 0,045 Bulb fresh weight (g) 0,040 0,4 0,035 0,030 0,2 0,025 0,0 0,020 0 S 3S 6 S 12 S 0 S 3S 6S 12S COOPD'OR R&D

5. G&H April 2002, LeipzigSulphur is limiting for Alliin storage in garlic bulbsWP3 / cultivation, P4 0,6 1,0 0,5 0,8 0,4 0,6 Alliin content (nmole/mg FW) Mean bulb weight (g) 0,3 0,4 0,2 0,2 0,1 0,0 0,0 2S_F+i 1S_F+i 2S_F 1S_F 2S_F+i 1S_F+i 2S_F 1S_F MORASOL PRINTANOR MESSIDROME MORADO COOPD'OR R&D

6. Garlic & Health P5 J. AUGER CONCLUSION OF IN VITRO EXPERIMENTS • Among all the conditions tested, the better to induce more alliin in garlic bulbs are: • Sucrose in the medium : 60g/l • Cold induction period : 3 months • Environmental factor : F+i light • Sulphur in the medium : 6 Meq SO3

7. G&H April 2002, LeipzigS fertilization greatly influenced alliin contentWP3 / cultivation, P4 Mean bulb weight (g) Alliin content (nmole/mg FW) COOPD'OR R&D

8. 18 360 16 320 14 280 12 240 10 200 8 0 100 200 400 0 100 200 400 2000 Garlic & Health P5 J. AUGER PRINTANOR POWDERS 2001 Relation between alliin concentration (nmole/mg) and Sulphur fertilisation level (kg/ha) France 4 Sulphur fertilisation levels Spain 5 Sulphur fertilisation levels • Alliin content in Spanish PRI is 3-6 times that in French PRI It seems that the treatment 2000 had no effect on Alliin content. • Alliin content increases in France with the S fertilisation (+25%) In Spain,the maximum was obtained even with the lowest S fertilisation (+8%).

9. G&H April 2002, LeipzigSoil quality may explain bulb qualityWP3 / cultivation, P4 Treatment (S04 unit) Sulphur analysis 25/08/00 21/02/01 14/05/01 20/06/01 14/08/01 0 Total 217 149 189 156 208 Water soluble 9 7 5,6 8 10 100 Total 172 187 186 Water soluble 9,7 11 9 200 Total 168 127 213 Water soluble 12,2 16 15 400 Total 188 114 185 Water soluble 20,8 22 20 France Spain 14/05/01 234 9,5 249 10,1 238 8,7 244 8,4 COOPD'OR R&D

10. G&H April 2002, LeipzigPowder production from SpainWP3 / cultivation, P4 %DM 0 100 200 400 Mean 2000 I 32,94 37,21 34,50 35,05 34,93 37,27 II - 33,00 35,78 32,53 33,77 33,60 III 35,00 33,74 31,67 32,83 33,31 37,40 IV 32,39 33,29 34,50 35,38 33,89 28,58 Mean 33,44 34,31 34,11 33,95 33,95 34,21 Powder (g) 0 100 200 400 Mean 2000 I 548 698 682 656 646 405 II 207 815 819 603 611 330 III 590 1411 471 955 857 370 IV 578 793 892 912 794 738 Mean 481 929 716 782 727 461 Total (g) 1923 3717 2864 3126 1843 Total powder available (g)13473 COOPD'OR R&D

11. G&H April 2002, LeipzigPowder production from FranceWP3 / cultivation, P4 %DM 0 100 200 400 Mean I 42,12* 42,26 43,5540,83 42,19 II 40,18 43,5042,32 42,84 42,21 III 45,0042,65 41,33 40,00 42,24 IV 37,88 40,29 40,23 40,51 39,73 Mean 41,30 42,17 41,86 41,04 41,59 * burned material with reduced alliin content Powder (g) 0 100 200 400 Mean I 3050 2830 29202920 2930 II 3270 32302600 3270 3093 III 39503440 2750 3350 3373 IV 3200 3170 3530 3210 3278 Mean 3368 3168 2950 3188 3168 Total (g) 7000 6670 5520 6130 Total powder available (g)25320

12. G&H April 2002, LeipzigWP3 / cultivation, P4Deliverables P4 • DP 2 Chinese garlic powder to be analysed • DP 6 Garlic powder issued from various S-fertilisation in field condition • DP 14 Chemical distinction between garlic accessions when cultivated in various sulphur nutrition regimes and under distinct temperatures in vitro • DP 15 Physiological (growth traits) and chemical distinction (CSO) between accessions when cultivated in various sulphur nutrition regimes over two years • DP 19 Chemical distinction (CSO) between accessions when cultivated in various sulphur (greenhouse) and mineral nutrition regimes (in vitro) • DP 22 Paper on the interaction genotype x environment on the sulphur content of garlic plants grown in vitro , in greenhouse and in field conditions • DP 30 Paper on the effect of combined sulphur, nitrogen and selenium nutrition on garlic growth, flavour precursor content and on biological potential of garlic for disease prevention • DP 34 Chemical distinction (CSO) between garlic accessions when cultivated in various sulphur regimes (greenhouse) and mineral nutrition regimes (in vitro) COOPD'OR R&D