Garlic Health ABOUT OUR NEW HPLC METHOD P

Garlic & Health ABOUT OUR NEW HPLC METHOD P 5 J. AUGER Ø We have identified a new compound in garlic HPLC profile, the S-allyl cystein (SAC) that has a potential therapeutic activity 0, 30 Alliin 0, 25 SAC GLUAl. CS Allicin 0, 20 GLUAl. CS Alliin 0, 40 GLUPe. CS 0, 30 0, 20 0, 15 0, 10 GLUPhe. Ala SAC 0, 10 0, 05 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) Ø We are now able to quantify 3 precursor compounds of Allicin H NH 2 N HOOC O S COOH γ-glutamyltranspeptidase H 2 O 2 H 2 N S Alliinase COOH O S-allyl-L-cysteine γ-glutamyl-S-allyl-L-cysteine (SAC) sulfoxide (alliin) (GLUAl. CS) Allicin biosynthesis from γ-glutamyl-S-allyl-L-cysteine Allicin ØWith this method we are now ready to compare the core collection

G&H April 2002, Leipzig Stimulating effect of i light on Alliin synthesis WP 3 / cultivation, P 4 0, 8 30 PRI Alliin (mmol/mg FW) 3, 0 * 60 2, 5 120 2, 0 1, 5 1, 0 0, 5 Mean bulb weight (g) 3, 5 0, 7 0, 6 0, 5 S 30 * S 60 S 120 0, 4 0, 3 0, 2 0, 1 0, 0 F F+i F F 27 * MES F+i F 27 * * Sucrose (in g/l) COOPD'OR R&D

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. Alliin concentration (nmole/mg) Bulbing conditions: Sucrose (S 30, S 60 and S 120). Cold induction period, 2 months, and F+i. Lichtwer HPLC method used ØThe sucrose 60 g/l seems to induce more Alliin contrary to the first results, and this HPLC method is more suitable for its quantification alone. Garlic & Health

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

G&H April 2002, Leipzig Sulphur is limiting for Alliin storage in garlic bulbs 0, 6 1, 0 Mean bulb weight (g) Alliin content (nmole/mg FW) WP 3 / cultivation, P 4 0, 8 0, 6 0, 4 0, 2 0, 5 0, 4 0, 3 0, 2 0, 1 0, 0 2 S_F+i 1 S_F+i 2 S_F 1 S_F L SO OR A E R AN OM O T M DR IN I R P S O ES AD M R O M COOPD'OR R&D 2 S_F+i 1 S_F+i 2 S_F 1 S_F

CONCLUSION OF IN VITRO EXPERIMENTS Among all the conditions tested, the better to induce more alliin in garlic bulbs are: • • Sucrose in the medium : Cold induction period : Environmental factor : Sulphur in the medium : 60 g/l 3 months F+i light 6 Meq SO 3 Garlic & Health P 5 J. AUGER

G&H April 2002, Leipzig S fertilization greatly influenced alliin content WP 3 / cultivation, P 4 Mean bulb weight (g) Alliin content (nmole/mg FW) COOPD'OR R&D

Garlic & Health PRINTANOR POWDERS 2001 P 5 J. AUGER Relation between alliin concentration (nmole/mg) and Sulphur fertilisation level (kg/ha) 18 360 16 320 14 280 12 10 240 8 200 0 100 200 400 France 4 Sulphur fertilisation levels 0 100 200 400 2000 Spain 5 Sulphur fertilisation levels Ø 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. Ø Alliin content increases in France with the S fertilisation (+25%) In Spain, the maximum was obtained even with the lowest S fertilisation (+8%).

G&H April 2002, Leipzig Soil quality may explain bulb quality WP 3 / cultivation, P 4 Treatment (S 04 unit) Sulphur analysis 0 Total Water soluble 100 Total Water soluble 200 Total Water soluble 400 Total Water soluble France 25/08/00 217 9 COOPD'OR R&D 21/02/01 149 7 14/05/01 189 5, 6 172 9, 7 168 12, 2 188 20, 8 Spain 20/06/01 156 8 187 11 127 16 114 22 14/08/01 208 10 186 9 213 15 185 20 14/05/01 234 9, 5 249 10, 1 238 8, 7 244 8, 4

G&H April 2002, Leipzig Powder production from Spain WP 3 / cultivation, P 4 %DM I II IV Mean 0 32, 94 35, 00 32, 39 33, 44 100 37, 21 33, 00 33, 74 33, 29 34, 31 200 34, 50 35, 78 31, 67 34, 50 34, 11 400 35, 05 32, 53 32, 83 35, 38 33, 95 Mean 34, 93 33, 77 33, 31 33, 89 33, 95 2000 37, 27 33, 60 37, 40 28, 58 34, 21 Powder (g) 0 100 I 548 698 II 207 815 III 590 1411 IV 578 793 Mean 481 929 Total (g) 1923 3717 Total powder available (g) 200 682 819 471 892 716 2864 400 656 603 955 912 782 3126 Mean 646 611 857 794 727 2000 405 330 370 738 461 1843 COOPD'OR R&D 13473

G&H April 2002, Leipzig Powder production from France WP 3 / cultivation, P 4 %DM I II IV Mean 0 42, 12* 40, 18 45, 00 37, 88 41, 30 100 42, 26 43, 50 42, 65 40, 29 42, 17 200 43, 55 42, 32 41, 33 40, 23 41, 86 400 40, 83 42, 84 40, 00 40, 51 41, 04 Mean 42, 19 42, 21 42, 24 39, 73 41, 59 400 2920 3270 3350 3210 3188 Mean 2930 3093 3373 3278 3168 * burned material with reduced alliin content Powder (g) 0 I 3050 II 3270 III 3950 IV 3200 Mean 3368 100 2830 3230 3440 3170 3168 Total (g) 7000 6670 Total powder available (g) 200 2920 2600 2750 3530 2950 5520 6130 25320

G&H April 2002, Leipzig WP 3 / cultivation, P 4 Deliverables P 4 ü DP 2 Chinese garlic powder to be analysed ü DP 6 Garlic powder issued from various S-fertilisation in field condition ü various Chemical distinction DP cultivated accessions between in garlic 14 when sulphur nutrition regimes and under distinct temperatures in vitro ü DP 15 Physiological (growth traits) and chemical distinction (CSO) between accessions when cultivated in various sulphur nutrition regimes over two years ü Chemical DP distinction various between 19 accessions (CSO) cultivated when in 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 various sulphur regimes (greenhouse) and mineral nutrition regimes (in vitro) COOPD'OR R&D