Extraction and characterisation of water soluble carotenoids of gardenia jasminoides ellis

d·· MINISTRY OF EDUCAnON & TRAINING HOCHIMINH CITY NATIONAL UNIVERSITY UNIVERSITY OF TECHNOLOGY THESIS OF DOCTORATE EXTRACTION AND CHARACTERISATION OF WATERSOLUBLE CAROTENOIDS OF Gardeniajasminoides Ellis Supervisors: Dr F'RANC;OIS CORMIER ~ Dr MARIE ROSE Dr VANCALSTEREN EDWARD FARNWORTH Dr CHI Bi\O Do Dr VAN HANG TONG Student: J'HA-NH QUAN PRAM 700692 School-years 1997·1999 DEDICATION To my country Vietnam and to my family for always encouraging me and always believing in me Thanh Quan Pham 11 ACKNOWLEDGEMENTS This work was in the scope of the cooperation project between the Food Research and Development Centre of the Canadian Ministry of Agriculture and Agrofood and the University of Technology of the Ho Chi Minh City National University of the Vietnamese Ministry of Education and Training Most of the work was carried out in the laboratories of the Food Research and Development Centre -Agriculture and Agrofood Canada * I would like to express the special thanks from my heart to: Dr Franc;ois Cormier, Head of Bio-Ingredient Section, Food Research & Development Centre-Agriculture & Agrofood Canada, Dr Chi Bao Do, Food Research & Development Centre-Agriculture & Agrofood Canada, Dr Van Hang Tong, Ho Chi Minh City National University, Vietnam for all of the advices, helps and teaching me during the time I lived, worked and studied in Canada ** I wish to express my thanks more specially to: Dr Claude B Aube, Director of Food Research & Development Centre- & Agrofood Canada, Prof Dr Minh Ve Truong, Rector of University of Technology - Ho Chi Minh City National University, Vietnam, for the financial support and the permission that helped me the conditions of working and studying *** I also wish to express my thanks to: Dr Marie Rose Vancalsteren, Dr Edward Farnworth, Madame Christian Dufresne, Mr Christopher Barr, Mr Brian Steward, for the advices and the helps in this work iii tt Finally, I would like express my indefinite thanks to: Prof Dr Quoc Dung Lam, Vice Director of HoChiMinh City National University, Prof Dr Van Bon Pham, Vice Rector of HoChiMinh City University of Technology, Prof Dr Minh Tan Phan, Dean of Chemical Engineering Faculty of HoChiMinh City University of Technology, Prof Dr Van Thanh Tran, Head of Organic Chemical Department of Chemical Engineering Faculty of HoChiMinh City University of Technology, All of my teachers, my professors and my friends in Vietnam, My parents, my wife and all of my sisters and my brother, For all of the advices, helps and teaching me for the years I have lived, worked and studied in Vietnam iv ABSTRACT Carotenoids are abundant in many fruits and vegetables and they plays diverse roles in photobiology, photochesrnistry and medicine Crocin and crocetin glycosyl esters are water soluble Carotenoids which are found in nature in stigmas of saffron (Crocus sativus Linne) and in the fruits of gardenia (Gardenia jasminoides Ellis) The best condition for the extraction of gardenia fruits was conducted at 30°C for 48 h in the dark using 50% EtOH as a extraction solvent for preventing the pigments against the degradation of enzymatic reaction and oxidation The content of crocin in gardenia fruit from Vietnam was 0.120 ± 0.016 mglg and of geniposide was 5.76 ± 1.01 % (w/w) of dried fruit of gardenia In most of the extract from gardenia fruits, crocin occupied up to 68 % of the total yellow crocetin glycosyl esters For further purification of crocin the aqueous solution of 50% acetone is used as a extraction solvent , washing with ether, ionic exchanging, and separation on preparative HPLC The purified crocin with purity more 99.6 % has been using as the standard and the materials for oxidation monitored by NMR and LC-MS and for the antioxidant assays In both thermostability assays at 25°C and at 95°C, crocin was stable at pH 6.0 Colour losses of crocin over 10 days at 25°C were 39 % in pH 6.0 medium and almost 99 % in pH 3.0 solution In the thermostability trials at 95°C the colour residue of crocin after 60 was 96 % at pH 6.0 in comparison with the initial colour at time = , but it was 73 % at pH 3.0 Attention has to be paid when carrying out the oxidation or other reaction of crocin Crocin has an antioxidative activity at concentrations of 10-40 ppm The antioxidative property of crocin as evaluated by thiocyanate method was better than with the thiobarbituric acid (TBA) method When crocin reacted with oxygen using FeS04 the intermediates such as monohydroperoxides, polyhydroperoxides, polyhydroxy-polyhydroperoxides of crocin were formed and detected by MS or LC-MS The main monohydroperoxides were mainly 15-monohydroperoxide and 14-monohydroperoxide based on the fragments at the C13-C14 and CI5-CI5' double bonds obtained by MS The adduct between linoleic acid radical and crocin was detected by LC-MS The semiempirical PM3 geometry optimization calculation and the semiempirical ZINDO/S spectral calculations have been carried out for crocetin , crocetin glycosyl esters - the water soluble carotenoids and the hydroperoxides of crocetin The 12-mono hydroperoxide of crocin is the most v stable and the 14-monohydroperoxide of crocin is the most unstable The maximum wavelengths of the crocetin glycosyl esters calculated by the semiempirical ZINDO/S spectral calculations agreed with those obtained by spectroscopy in Calculations have also been performed for several torsion angles of the monohydroperoxides of crocetin All of the monohyd~operoxides have the minimum binding energy when the torsion angle is around 160-180° Calculations have also performed for all-trans and several cis crocetin The B-cis crocetin is the most stable cis-form with an enthalpy of cis-trans transformation from all-trans to B-cis crocetin of 0.15 kcal/mol The enthalpy of transformation from the singlet state to triplet state of crocetin is 11.59 kcal/mol corresponding to the energy of radiation with the wavelength of 2470 nm The process of extraction and production of yellow pigment from gardenia fruits was investigated Iridoids were eliminated from the extract of gardenia fruits Key words: Carotenoides, Gardenia, crocetin derivatives, antioxidative activity, molecular modelling, HPLC, NMR, MS vi TABLE OF CONTENTS Title Dedication ii Acknowledgements Ill Abstract v Table of Contents vii General Introduction Chapter I: Extraction, Purification and Identification of the main compounds from Gardenia jasminoides Ellis 1.1 Introduction Carotenoids Crocetin derivatives and iridoids from Gardenia jasminoides Ellis Objective and Direction of Study 11 1.2 Materials and Methods 12 I Materials 12 Extraction 12 Isolation and purification 15 HPLC methods 17 Determination of iridoid content 18 UVNisible spectroscopy 18 NMR analysis 19 MS analysis 20 1.3 Results and Discussions 21 I Extraction 21 Analysis 26 Purification and Identification 33 1.4 Conclusion 39 Chapter II: Stability of crocin from Gardeniajasminoides Ellis 41 II.l Introduction 42 Stability of carotenoids vii 42 Objective and Direction of Study 44 II.2 Materials and Methods 44 II.3 Results and Discussions 45 II.4 Conclusion 48 Chapter III: Antioxidative activity of crocin from Gardeniajasminoides Ellis 49 m.l Introduction 50 I Lipid oxidation and antioxidant action 50 Method for evaluation of antioxidant action 53 Objective and Direction of Study 57 m.2 Materials and Methods 58 I Materials 58 Assay of of superoxide dimutase-like activity Thiobarbituric assay ; , 59 60 Thiocyanate assay 62 m.3 Results and Discussions 64 m.4 Conclusion 74 Chapter IV: Determination of mechanism of antioxidative activity of crocin 75 N.I Introduction 76 I Mechanism of antioxidative activity of carotenoids 76 Molecular modeling 80 Objective and Direction of Study 83 N.2 Materials and Methods 84 Computational method 84 Crocin oxidation assay 87 N.3 Results and Discussions N.4 Conclusion 88 : 117 Chapter V: Elimination iridoids from the extract of gardenia fruits and Development of extraction process of colorants from Gardeniajasminoides 119 V.I Introduction 119 I Elimination iridoids 120 viii Objective and Direction of Study 123 V.2 Materials and Methods 124 V.3 Results and Discussions 125 V.4 Conclusion 132 General Conclusion 135 References 137 Annexes 144 Annex 144 Annex 153 Annex 155 Annex 157 Annex 158 Annex 160 Annex 168 Annex 171 Annex 174 Annex 10 176 Annex 11 177 Annex 12 178 Annex 13 179 Annex 14 188 Annex 15 193 List ofTables 197 List of Figures 198 List ofSymbols 20 I ix GENERAL INTRODUCTION There is considerable interest worldwide in the development of food colorants from natural sources A variety of fruits, vegetables and flowers have been studied as potential sources of food colorants The technological success of the pigments in anatto (Bixa orellana L.) and saffron (Crocus salivus Linne) combined with their relatively high price led to search for other plant sources with the same pigments The same pigments could be obtained in the greater quantities and at a lower price from the fruit of Gardenia lasminoides Ellis [1] The fruits of Gardenia jasminoides Ellis contain three major groups of pigments: water soluble carotenoids (crocetin derivatives), iridoids, and flavonoids [2-5] The water solubility of carotenoid crocin and their derivatives leads to more application in foods and other products The production of food colorants from gardenia fruits is apparently being investigated at the present time [66] The colorants from gardenia appear to have good potential because of their wide range of available colors and their apparent excellent stability [6] Being a carotenoid, crocetin derivatives have been shown to possess antioxidant properties [2,3] The use of natural antioxidants has received special attention because of the worldwide trend to avoid or reduce the use of synthetic food additives The application of natural antioxidants in foods and biological systems has received considerable interest because of their presumed safety and potentially nutritional and therapeutic effects [7] Another aspect of their application of the water-soluble carotenoid crocin and crocetin deri vati ves is that they can be used as an anticancer agent against different kinds of tumors and cancers [8] Among the most significant activities of crocin and crocetin derivatives are their free radical scavenger properties [9-12] The free radicals are known to be very harmful to cellar components as a precursor of more reactive species, contributing to the various diseases such as inflammation, multagenesis, carcinogenesis, tissue injury by circulatory disturbance and aging [13,14] Gardeniajasminoides Ellis plants which are called "danh danh" or "chi IU" or "son chi Iu " have been cultivated widely in Vietnam for many years Far from being time the extract from the gardenia fruit has been applied traditionally in daily food products such as stick rice, cake, food colors and in traditional medicals Gardenia trees grow freely and are planted in many regions of the north of Vietnam (Ha Bac, Quang Ninh) and south of Vietnam (Ninh Thuan, Long An) Researches on fruit extract of Gardenia lasminoides Ellis need to be carried out to explore and 445 447.5 450 452.5 455 456 457 458 459 460 461 462 463 464 465 466 467.5 470 472.5 475 480 485 490 495 500 505 510 515 520 525 530 540 2.1274 2.0375 1.9671 1.9371 1.9520 1.96n 1.9888 2.0127 2.0374 2.0620 2.0850 2.1020 2.1134 2.1182 2.1135 2.0982 2.0555 1.9312 1.7524 1.5428 1.1015 0.7320 0.4331 0.2681 0.1674 0.1054 0.0704 0.0572 0.0397 0.0315 0.0252 0.0155 365 370 375 380 385 390 395 400 405 410 412.5 415 417.5 420 422.5 425 427.5 430 432 434 435 436 437 438 439 440 441 442 444 445 447.5 450 452 453 454 455 457.5 459 460 461 462 463 464 465 466 467.5 470 472.5 475 480 485 490 492.5 495 497.5 500 502.5 505 507.5 510 515 520 525 530 535 540 0.3544 0.4111 0.4821 0.5807 0.6907 0.8075 0.9300 1.0785 1.2604 1.4382 1.5067 1.5558 1.5938 1.6315 1.6837 1.7600 1.8631 1.9787 2.0661 2.1334 2.1581 2.1757 2.1870 2.1907 2.1867 2.1761 2.1572 2.1338 2.0725 2.0381 1.9518 1.8845 1.8567 1.8545 1.8594 1.8697 1.9158 1.9512 1.9735 1.9940 385 390 395 400 405 410 412.5 415 417.5 420 422.5 425 427.5 430 432 434 436 437 438 439 440 442 444 446 448 450 451 452 453 454 455 456 460 461 462 463 463.5 464 465 466 467 468 470 2.0100 2.0195 2.0267 2.0145 472.5 1.9541 477.5 1.9980 1.8287 1.6540 1.4512 1.0292 0.6808 0.3994 0.3130 0.2448 0.1932 0.1508 0.1184 0.0932 0.0747 0.0607 0.0428 0.0321 0.0245 0.Q185 475 480 482.5 485 490 492.5 495 497.5 500 502.5 505 510 515 520 525 530 535 540 0.6517 0.7558 0.8642 0.99n 1.1598 1.3155 1.3755 1.4181 1,4502 1.4837 1.5312 1.6000 1.6937 1.7970 1.8754 1.9345 1.9717 1.9801 1.9820 1.9754 1.9630 1.9212 1.8631 1.7967 1.7350 1.6892 1.6747 1.6665 1.6651 1.6700 1.6804 1.6947 1.7744 1.7927 1.8064 1.8134 1.8147 1.8137 1.8058 1.7887 1.7627 1.7271 1.6278 1.4660 1.2821 1.0930 0.9074 312 314 315 316 317 318 319 320 321 322 323 325 326 328 330 332.5 335 337.5 340 342.5 345 347.5 350 352.5 355 360 365 370 375 380 385 390 395 400 402.5 405 410 412.5 415 417.5 420 422.5 425 430 432.5 433 434 435 436 0.7366 0.5978 0.3518 436.5 0.2744 437 0.2155 437.5 0.1701 0.1327 0.1034 0.0820 0.0535 0.0375 0.0281 0.0212 0.0160 0.0115 0.0080 439 440 442.5 445 447.5 450 451 452 452.5 454 455 457.5 460 461 462 0.0138 0.0098 462.5 463 463.5 464 465 467.5 470 475 480 485 487.5 490 492.5 495 497.5 500 502.5 505 507.5 510 512.5 515 520 525 530 535 540 189 0.4672 0.4678 0.4681 0.4707 0.4722 0.4734 0,4738 0,4748 0.4757 0,4751 0,4727 0,4705 0.4684 0,4615 0.4530 0,4401 0.4268 0.4141 0.3991 0.3862 0.3757 0.3677 0.3637 0.3631 0.3650 0.3724 0.3854 0.4057 0.4351 0.4777 0.5280 0.5838 0.6451 0.7195 0.7635 0.8081 0.8902 0.9201 0.9404 320 321 322 322.5 323 324 325 326 328 330 332 335 337 340 345 350 352 355 357 360 362 364 366 368 370 372 374 376 378 380 382 384 386 390 395 400 405 407.5 410 0.9552 412.5 0.9715 415 0.9971 417.5 1.0361 1.1491 1.2015 1.2105 1.2248 1.2355 1.2427 1.2447 1.2452 1.2448 1.2375 1.2275 1.1880 1.1361 1.0850 1.0467 1.0375 1.0327 1.0321 1.0350 1.0411 1.0678 1.0975 1.1067 1.1138 Ln~~ 1.1147 1.1131 1.1058 1.0635 0.9867 0.7712 0.5430 0.3585 0.2700 0.2128 0.1662 420 422.5 425 427.5 430 432 434 435 436 436.5 437 437.5 438 439 440 442 444 446 448 450 452 452.5 453 454 456 458 460 462 462.5 463 464 465 466 468 470 472 475 480 485 0.1311 0.1041 487.5 0.0422 497.5 0.0350 0.0294 0.0252 0.0195 500 502.5 505 0.0825 0.0647 0.0518 0.0154 0.012' 0.0097 0.0074 490 492 495 507.5 510 515 520 525 510 0.5488 0.5491 0.5495 0.5490 0.5488 0.5474 0.5457 0.5412 0.5345 0.5251 0.5144 0.4974 0.4868 0.4681 0.4387 0,4210 0.4178 0,4155 0.4148 0.4144 0.4140 0.4145 0,4157 0.4175 0.4207 0.4251 0.4318 0.4411 0.4498 0.4590 0.4701 0.4834 0.4971 0.5267 0.5687 0.6194 0.6788 0.7071 0.7315 0.7498 0.7612 0.7688 0.n87 0.7955 0.8225 0.8604 0.9021 0.9320 0.9534 0.9595 0.9632 0.9638 0.9637 0.9625 0.9605 0.9547 0.9470 0.9217 0.8895 0.8571 0.8264 0.8050 0.7955 0.7952 0.7954 0.7982 0.8092 0.8260 0.8424 0.8520 0.8525 0.8527 0.8492 0.8422 0.8308 0.7958 0.7448 0.6822 0.5788 0.4060 0.2672 0.2021 0.1587 0.1307 0.0980 0.0784 0.0618 0.0505 0.0388 0.0314 0.0258 0.0188 0.0142 0.0111 00 linoleic acid at 37'C and pH 6.0 (40 mM Fig IV 3: Changes in absorbency during the oxidation of phosphate buffer) without FeSO, Absorbance (T= h) Wavelength (nm) Absorbance (T=48 h) Wavelength (nm) Absorbance (T=24 h) Wavelength (nm) (nm) Absorbance (T=72 h) 200 202 204 206 208 209 210 211 212 213 214 215 216 217 217.5 218.5 219 219.5 220 220.5 221 222 223 224 225 226 227 228 229 230 231 232 233 234 236 238 239 240 242 244 246 248 250 252 254 256 258 260 262 264 266 268 270 272 280 285 290 300 310 330 340 0.0161 0.0375 0.0691 0.1055 0.1557 0.1705 0.1904 0.1814 0.1631 0.1518 0.1292 0.1017 0.0811 0.0678 0.1018 0.1311 0.0182 0.0007 0.0091 0.0444 0.0464 0.0421 0.0421 0.0407 0.0395 0.0397 0.0402 0.0380 0.0382 0.0381 0.0380 0.0375 0.0382 0.0370 0.0360 0.0348 0.0344 0.0330 0.0314 0.0298 0.0274 0.0254 0.0232 0.0217 0.0207 0.0197 0.0195 0.0191 0.0184 0.0181 0.0185 0.0175 0.0170 0.0165 0.0140 0.0117 0.0101 0.0098 0.0110 0.0104 0.0097 200 202 204 206 208 209 210 211 212 213 214 215 216 217 217.5 218 219.5 220 220.5 222 223 224 225 226 227 228 230 230.5 231 232 233 234 235 236 238 240 242 244 246 248 250 252 254 256 258 260 265 270 280 290 300 320 340 0.0370 0.0635 0.0952 0.1310 0.1621 0.1661 0.1761 0.1705 0.1698 0.1810 0.1865 0.1881 0.1924 0.2038 0.2461 0.2991 0.1702 0.0518 0.0915 0.2502 0.2595 0.2681 0.2750 0.2821 0.2860 0.2907 0.2950 0.2967 0.2941 0.2925 0.2901 0.2850 0.2780 0.2695 0.2487 0.2251 0.2000 0.1707 0.1381 0.1050 0.0772 0.0564 0.0421 0.0328 0.0262 0.0217 0.0170 0.0147 0.0132 0.0112 0.0100 0.0050 0.0055 200 202 204 206 208 210 212 214 216 218 220 222 224 226 228 230 232 234 236 237 238 240 242 244 245 246 247 248 249 250 251 252 253 254 255 256 258 260 262 264 266 268 270 272 274 275.5 276 278 280 282 284 286 288 290 292 294 296 298 300 302 304 306 308 310 315 320 330 0.1312 0.1005 0.1607 0.2044 0.2780 0.4131 0.5442 0.7687 0.9858 1.2877 1.5707 1.7195 1.9352 2.1105 2.2514 2.4072 2.5322 2.6071 2.6431 2.6432 2.6268 2.5354 2.3557 2.0557 1.8620 1.5665 1.3655 1.2521 1.0674 0.9015 0.7588 0.6377 0.5387 0.4581 0.3934 0.3420 0.2672 0.2212 0.1934 0.1791 0.1722 0.1691 0.1685 0.1691 0.1697 0.1700 0.1694 0.1691 0.1661 0.1621 0.1577 0.1501 0.1411 0.1301 0.1185 0.1068 0.0957 0.0848 0.0755 0.0654 0.0565 0.0488 0.0427 0.0374 0.0284 0.0232 0.0190 200 202 204 206 208 210 212 214 216 218 220 222 224 226 227 228 229 230 231 232 233 234 235 236 237 238 240 242 244 246 248 250 252 254 256 258 260 262 264 266 268 270 272 274 276 278 280 282 284 286 288 290 292 294 296 298 300 302 304 306 308 310 312 315 320 330 340 350 0.1595 0.1762 0.2024 0.2638 0.3030 0.3392 0.4581 0.6461 0.8355 1.1377 1.2350 1.3624 1.4525 1.5110 1.5262 1.5322 1.5217 1.5104 1.4860 1.4530 1.3772 1.1941 1.2188 1.2597 1.1990 1.1374 1.0120 0.8834 0.7494 0.6170 0.4970 0.3982 0.3218 0.2687 0.2328 0.2085 0.1911 0.1807 0.1741 0.1698 0.1675 0.1654 0.1631 0.1608 0.1575 0.1532 0.1482 0.1425 0.1342 0.1261 0.1161 0.1070 0.0971 0.0885 0.0804 0.0732 0.0661 0.0590 0.0534 0.0485 0.0444 0.0411 0.0385 0.0358 0.0281 0.0238 0.0207 0.0172 Wavelength 190 Fig.lV 4: Changes in absorbency during the cooxidation of crocin with linoleic acid (crocin/linoleic acid mole ratio - 1: 2) at 37°C and pH 6.0 (40 mM phosphate buffer) containing FeSO• Wavelength Absorbance (T=Oh) Wavelength (om) 200 2025 207'5 210 215 217 218 219 220 222.5 225 226.5 228 229 230 231 232 233 235 237.5 240 242.5 245 247.5 250 252.5 255 256 257 258 259 259.5 260 261 262 263 264 265 266 268 270 272.5 275 2n.5 280 281 282 282.5 283 284 285 287.5 290 295 300 305 310 315 317 320 322.5 325 327.5 330 335 337.5 340 342.5 345 347.5 350 352.5 355 357.5 360 362.5 365 370 375 S421 0:6622 0.8088 09054 1:1712 1.2760 1.2894 1.1675 1.1517 1.1037 1.0150 0.8004 0.8782 0.8804 0.8580 0.8358 0.8157 0.7964 0.7672 0.7371 0.7117 0.6972 0.6930 0.6995 0.7120 0.7331 0.7651 0.7785 0.7915 0.8014 0.8057 0.8061 0.8048 0.7955 0.7797 0.7587 0.7340 0.7102 0.6871 0.6492 0.6241 0.6060 0.5999 0.5988 0.6032 0.6045 0.6054 0.6057 0.6052 0.6040 0.6024 0.5958 0.5844 0.5531 0.5125 0.4658 0.430' 0.4090 0.3931 0.3840 0.3754 0.3655 0.3518 0.3357 0.3040 0.2940 02875 0.2832 0.2847 0.2900 0.3002 0.3142 0.3325 0.3555 0.3777 0.4164 0.4531 0.5437 0.6582 200 205 208 210 212 214 215 216 216.5 217 217.5 218 218.5 219 220 222 224 225 226 228 230 232 234 232.5 235 236 238 240 242 244 245 248 250 252 253 254 254.5 255 256 257 258 259 260 261 262 263 264 265 266 268 270 272 273 274 275 276 277 277.5 278 279 280 281 282 283 284 285 286 288 290 292.5 295 297.5 300 302.5 305 307.5 310 312.5 314 (om) Absorbance T=3h Wavelength 0.5365 0.6471 0.7604 0.7394 0.7504 200 202 205 208 210 212 214 216 217 217.5 218 219 220 222 224 226 227 228 229 230 231 231.5 233 234 235 236 237 237.5 238 239 240 242 245 247 250 252 254 256 258 259 260 262 264 286 268 270 272 274 276 278 279 280 281 282 283 284 285 286 288 290 292 295 297 300 302 305 307 310 312 314 315.5 317 318 320 322 325 327 330 332 0.7594 0.7711 0.7712 0.8291 0.8864 0.9665 0.9235 0.8797 0.8262 0.8371 0.8671 0.8957 0.8664 0.7388 0.8717 0.9062 0.9131 0.9077 0.9135 0.9000 0.890' 0.8632 0.8337 0.7992 0.7584 0.7362 0.6742 0.6465 0.6230 0.6174 0.6148 0.6134 0.6134 0.6162 0.6211 0.6265 0.6322 0.6357 0.6367 0.6348 0.6281 0.6185 0.6068 0.5931 0.5635 0.5411 0.5264 0.5217 0.5181 0.5152 0.5127 0.5114 0.5118 0.5115 0.5112 0.5114 0.5115 0.5111 0.5110 0.5098 0.5082 0.5051 0.5005 0.4954 0.4880 0.4812 0.4735 0.4662 0.4592 0.4521 0.4458 0.442' 0.4410 0.4414 (om) Absorbance T-6 h 0.5550 0.5814 0.6504 0.8151 0.8152 0.8964 0.9910 1.0745 1.1968 1.2694 1.2605 1.1985 1.2361 1.3187 1.3925 1.4338 1.4512 1.4600 1.4721 1.4785 1.4865 1.4894 1.4467 1.2667 1.2905 1.3267 1.3574 1.3735 1.3574 1.3204 1.2807 1.1960 1.0400 0.9287 0.7890 0.7245 0.6842 0.6645 0.6590 0.6605 0.6625 0.6697 0.6762 0.6807 0.6858 0.6935 0.7040 0.7134 0.7222 0.7301 0.7327 0.7357 0.7384 0.7391 0.7402 0.739' 0.7378 0.7358 0.7288 0.7218 0.7122 0.6997 0.6881 0.6758 0.6672 0.6534 0.6454 0.6351 0.6305 0.6281 0.6254 0.6264 0.6264 0.6247 0.6212 0.6100 0.5977 0.5720 0.5531 191 Wavelenglh (nm) 200 205 210 215 216 217 217.5 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 242.5 245 250 252.5 255 255.5 257 260 262.5 265 267.5 270 272.5 275 276 277 278 279 280 281 281.5 282 283 285 287.5 290 295 297.5 300 302.5 305 307.5 310 312.5 315 317 318 319 320 321 322 324 326 327.5 330 332.5 335 337.5 340 Absorbance T=10h Wavelenglh 0.4180 0.5317 0.7348 0.9678 1.0257 200 205 210 215 220 225 226 227 228 229 230 230.5 231 231.5 232 233 233.5 234 234.5 235 235.5 236 236.5 237 237.5 238 239 240 241 242 243 244 245 247.5 250 252 254 255 256 257 258 259 260 262 264 266 268 270 272 274 275 27E 277 278 279 279.5 280 281 282 283 284 285 286 288 290 292 294 296 300 305 310 312 314 315 316 317 318 319 320 1.1785 1.2825 1.2445 1.1874 1.2301 1.2822 1.3267 1.3725 1.4081 1.4408 1.4630 1.4758 1.4960 1.5127 1.5244 1.5381 1.5418 1.5390 1.4921 1.3115 1.3324 1.3644 1.3867 1.3724 1.3341 1.2185 1.0734 0.7941 0.7107 0.6665 0.6621 0.6538 0.6562 0.6715 0.6894 0.7084 0.7282 0.7475 0.7620 0.7648 0.7690 0.7731 0.7750 0.7757 0.7758 0.7764 0.7760 0.7748 0.7687 0.7575 0.7424 0.7095 0.6938 0.6788 0.6648 0.6488 0.6337 0.6194 0.6097 0.6051 0.6038 0.6032 0.6032 0.6027 0.6020 0.6002 0.5952 0.5860 0.5757 0.5578 0.5382 0.5188 0.4997 0.4780 (om) Absorbance T=23h 0.2804 0.3864 0.6070 0.9281 1.2688 1.4695 1.4878 1.5021 1.5074 1.5175 1.5218 1.5244 1.5272 1.5248 1.5201 1.5102 1.4660 1.3051 1.2955 1.2991 1.3181 1.3360 1.3518 1.3650 1.3781 1.3584 1.3175 1.2760 1.2340 1.1864 1.1352 1.0804 1.0247 0.8884 0.TT70 0.7132 0.6770 0.6641 0.6565 0.6525 0.6510 0.6525 0.6557 0.6697 0.6875 0.7075 0.7300 0.7502 0.7688 0.7841 0.7885 0.7942 0.7985 0.8008 0.8020 0.8024 0.8021 0.8011 0.7974 0.7945 0.7892 0.7900 0.7760 0.7595 0.7402 0.7195 0.6980 0.6790 0.6421 0.5972 0.5544 0.5427 0.5342 0.5318 0.5302 0.5287 0.5270 0.5250 0.5230 380 385 390 395 400 405 410 412.5 415 417.5 420 422.5 425 427.5 430 432 434 436 437 438 439 439.5 440 441 442 443 445 446 448 449 450 451 452 454 455 456 457 457.5 458 459 460 461 462 464 466 468 470 472.5 475 476 480 482.5 485 490 492.5 495 500 502.5 505 0.8097 0.9692 1.1258 1.3090 1.5467 1.8097 1.9891 2.0444 2.0810 2.1224 2.1897 2.2921 2.4180 2.5204 2.5330 2.5377 2.5450 2.5450 2.5464 2.5480 2.5508 2.5524 2.5520 2.5487 2.5261 2.4855 2.4107 2.3795 2.3408 2.3361 2.3408 2.3578 2.3818 315 316 318 320 321 322 323 324 326 328 330 332.5 335 337.5 340 342.5 345 347.5 350 350.5 352 355 357.5 360 365 370 375 380 385 390 395 400 405 2.4437 407.5 2.4742 410 412.5 415 416 2.5057 2.5208 2.5235 2.5220 2.5165 2.5107 2.5037 2.4960 2.4732 2.3878 2.2167 2.0010 1.7015 1.4087 1.2992 0.9087 0.7132 0.5651 0.3225 0.2520 0.1995 0.1285 0.1044 ,10 0.0852 0.0612 515 520 525 530 540 550 0.0388 0.0318 0.0258 0.0475 0.0167 0.0115 417.5 420 422.5 425 427.5 430 432 434 435 436 437 438 439 440 442 444 446 448 450 452 453 454 455 456 458 460 461 462 463 464 465 466 468 470 472 475 477.5 480 485 490 495 500 505 510 515 520 0.4420 0.4440 0.4465 0.4471 0.4458 0.4455 0.4445 0.4432 0.4387 0.4311 0.4210 0.4065 0.3912 0.3770 0.3608 0.3478 0.3379 0.3314 0.3290 0.3287 0.3297 0.3351 0.3431 0.3538 0.3830 0.4241 0.4785 0.5548 0.6435 0.7395 0.8408 0.9640 1,1164 1.1932 1.2651 1.3225 1.3650 1.3780 1.3952 1.4252 1.4670 1.5298 1.6160 1.7157 1.7920 1.8540 1.8764 1.8941 1.9045 1.9078 1.9052 1.8957 1.8607 1.8048 1.7415 1.6781 1.6301 1.6041 1.6012 1.6032 1.6111 1.6240 1.6607 1.7031 1.7211 1.7314 1.7457 1.7498 1.7448 1.7318 1.6771 1.5890 1.4711 1.2660 0.4348 0.3992 335 340 345 350 352 355 357 360 362 365 367 370 372.5 375 378 380 385 390 395 400 405 410 412 415 417 420 422 424 426 428 430 432 434 435 436 437 0.5242 0.4767 0.4295 0.3948 0.3854 0.3758 0.3707 0.3645 0.3605 0.3552 0.3524 0.3498 0.3495 0.3510 0.3560 0.3608 345 350 355 360 365 370 372.5 375 380 385 390 395 400 405 0.3791 412.5 0.4042 0.4338 415 0.2792 0.2798 0.2782 417.5 0.2747 420 422.5 425 0.2711 437.5 0.7520 438 439 440 442 444 446 448 450 452 0.7515 0.7492 0.7432 0.4711 0.5172 0.5605 0.5737 0.5862 0.5911 0.5980 0.6055 0.6181 0.6364 0.6605 0.6890 0.7142 0.7350 0.7432 0.7484 0.7514 0.7262 0.7027 0.6764 0.6507 0.6301 0.6180 0.6152 453.5 0.6178 455 457 459 460 461 462 463 463.5 464 465 466 468 470 472 475 477 480 482 485 487 490 492 495 500 510 520 0.6274 0.6408 0.6485 0.6551 0.6602 0.6635 0.6641 0.6640 192 0.2742 0.2771 0.2720 0.2738 0.2802 0.2888 0.2958 0.3010 0.3025 0.3034 0.3035 0.3034 0.3025 0.3004 0.2978 0.2957 0.2924 0.2868 0.2888 0.2698 0.2570 0.2484 0.2430 0.2415 0.2421 0.2434 0.2477 0.2525 0.2548 0.2565 0.2575 0.2572 0.2562 0.2540 0.2464 0.2330 0.2107 0.1854 0.1590 0.1321 0.1081 482.5 0.4778 0.4227 0.3405 490 0.0882 0.0638 0.0500 492.5 0.0387 495 500 50S 510 0.0181 0.0110 0.0067 0.1105 0.0537 0.0382 430 432 434 435 436 436.5 437 438 439 440 44' 442 443 444 446 448 450 452 454 455 456 458 460 461 462 463 464 465 466 468 470 472.5 475 0.2635 0.2604 0.2631 0.2675 477.5 0.0827 0.0511 0.0212 0.0117 oO?Rs ·~27.5 0.2977 0.2885 0.2722 0.6362 0.6022 0.5562 0.1797 0.1341 0.2150 0.1330 0.0824 410 0.3555 0.3317 0.3081 0.6620 0.6574 0.2888 0.2250 1.0834 0.9014 0.5994 0.3501 407.5 0.3757 480 485 487.5 0.0305 321 322 323 324 326 328 330 332.5 335 337.5 340 345 350 355 360 365 370 375 380 385 390 395 400 405 410 415 420 425 430 435 440 445 450 455 460 0.5198 0.5170 0.5131 0.5072 0.4958 0.4815 0.4057 0.4445 0.4237 0.4034 0.3795 0.3337 0.2934 0.2625 0.2331 0.2017 0.1690 0.1411 0.1132 0.0937 0.0787 0.0678 0.0568 0.0482 0.0410 0.0345 0.0288 0.0237 0,0191 0.0158 0.0141 0.0121 0.0095 0.0084 0.0075 Annex 15 Croci"n concentration in Fig.V.I-3 was calculated as follows: A 440 Crocin concentration Kdil 10-3 [mg/I] = -=:::.-. 0.0022 Kdil = 0.1+1.9 = 20 (koefficient of dilution for HPLC) 0.1 Geniposide concentration in Fig V.I-3 was calculated as follows: A Geniposide concentration 240 Kdil.lO- [gil] = 0.0039 Kdil = 0.1+1.9 0.1 =20 (koefficient of dilution for HPLC) Fig.V 2: Effect of concentration of acetone on the separation of iridoids by acetone solution using Amberlite XAD-16 absorbent (20g), gardenia extracV absorbent ratio = 1:2 (v/w) % Acetone geniposide content in geniposide content in geniposide content in Yield of elimination of the beginning extract geniposide crocin fraction iridoids fraction (v/v) (gil) (gil) (gil) (%) 9.18 ± 0.61 10 15 20 5.98 ± 0.08 3.11 ± 0.00 1.47 ± 0.00 0.28 +0.02 2.90 6.34 7.78 8.45 34.9 66.2 84.0 96.9 ±O.OO ±O.OO ±.O.OO + 0.00 Fig.V.3: Effect of concentration of acetone on the elution of iridoids by ethanolic solution using Amberlite XAD-16 absorbent (20g), gardenia extracV absorbent ratio = 1:2 (v/w) % EtOH (v/v) geniposide content in crocin fraction (gill geniposide content in geniposide content in Yield of elimination of iridoids fraction the beginning extract geniposide (g/I) (gil) (%) 8.20±0.14 2.5 5.0 10.0 15.0 8.13±0.08 7.51 ± 0.02 0.00 0.00 0.008 ± 0.000 0.031 ± 0.000 8.19 ±0.03 8.17 ± 0.04 193 0.87 ± 1.02 8.34 ± 0.20 100 100 Fig.V.4: Effect of concentration of ethanol on the elution of crocin by ethanolic solution using Amberlit£ XAD-16 absorbent (20g), gardema extracV absorbent ratio = 1:2 (v/w) Crocin concentration A440 of crocin 0/0 EtOH (vlv) (ro!!!!) II 0.01708 0.10033 0.15655 0.15312 0.16991 I 0.01378 0.10768 0.16677 0.15681 60 Sample BAH 0.17032 ([Omll 30 40 50 III v 0.09181 0.15699 0.15497 0.16362 Average 0.01543 0.09994 0.16010 0.15497 0.16795 E1TOr 0.00233 0.00794 0.00578 0.00185 0.00376 Average 0.140 0.909 1.455 1.409 1.527 Error 0.021 0.072 0.053 0.017 0.034 Yield of recovery of crocin ('!o) Average Error 9.17 1.38 59.53 4.72 95.28 3.47 92.27 1.11 The saturated amount of crocin and geniposide of the crude extract as adsorbed on adsorben Amberlite XAD-16 using 50% EtOH for eluting were calculated as follows: Crocin amount A X 440 = x.v.Kdil m 10 = ' [mg/g of absorbent] [mgll] 0.0022 Kdil v = 200 (koefficient of dilution for HPLC) = 0.2 L (final volume of eluant) m = 20.0 g (weight of adsorbent Amberlite XAD-16) x.v.Kdil Geniposide amount = - - - - - m A 240 x= -0.0039 [mg/g of absorbent] [mg/I] Kdil = 200 (koefficient of dilution for HPLC) v =0.2 L (final volume of eluant) m = 20.0 g (weight of adsorbent Amberlite XAD-16) 194 ) Death volume of the column (10 mm i.d x 250 mm length) of Amberlite XAD-16 absorbent ( 20 g) Replicate Death volume 10.60 11.80 11.30 10.30 10.10 10.82 0.71 (ml) Average Error Table V.2: The amount of crocin of the crude ext;act (90 ml) as adsorbed on adsorb.ent Amberlite XAD-16 using the procedure Replicate A440 of crocin 0.03817 0.03682 0.03642 0.03714 Average 0.00092 Error K dilute = 500 weight of absorbent: m= 20 g v = 0.2 I ( volume of the eluant) Crocin amount (mg/g of absorbent) 0.0868 0.0837 0.0828 0.0844 0.0021 Table V.3: The amount of crocin of the crude extract (30 ml x 3) as adsorbed on adsorbent Amberlite XAD-16 using the procedure Replicate A440 of crocin 0.06010 0.06726 0.06520 Average 0.06419 Error 0.00369 K dilute - 500 weight of absorbent: m= 20 g v = 0.2 I ( volume of the eluant) 195 Crocin amount (mg/g of adsorbent) 0.1366 0.1529 0.1481 0.1459 0.0084 Development of procedure: Parameters ofadsorption column in experiments: Weight of Amberlite XAD-16 ads rbent: m = 20 g Pore volume of adsorbents: p = 1.82 mUg = 1.82 cm'/g = 1.82 lIkg Volume of adsorbent: v d = p.m = 1.82 x 20 = 36.4 em' Column lengthli.d ratio: 11 i.d = 220: 15 = 14.67 : = 15: I Average rate of circulation = average rate of elution: v =4 mJ/min =4 cm'/min 1=220 mm Column across area: S = 7r 47r d = 41.5 = 1.766 em' Specific average rate of circulation and elution: v = v Is = 4/1.766 = 2.265 cm'/minlcm' -+ ;ri.d =15 mm Parameters ofadsorption in scale-up using procedure 2: Weight of Amberlite XAD-16 adsorbent: m = 40 kg Volume of adsorbent: V '" = p.m = 1.82 x 40 = 72.8 liters = 72.8 dm ' = Column length/i.d ratio = Ud = 15:1 = L = IS d ~d! L (0) (00) Inner diameter of column ( from (0) and (00)): d= 72.8xIO' x4 3.14xl5 =18.3cm=183mm=Chosed=200mm Length of column: L = IS d = IS x 200 = 3000 mm = m Column across area: S = 7r -d = 7r -20 = 314 em' Average rate of circulation and elution: V = v • S = 2.265 x 314 = 71lcm'/min = 0.71 literl 196 LIST OF TABLES Table Name of Table Page 1.1 The composition yellow pigments of gardenia fruit 1.2 Result of analyzing geniposide from gardenia fruit 22 1.3 Crocin content analyzed by HPLC 28 m.1 Measurement of degree of oxidation in accelerated tests 44 IV.I Optimized geometry and atomic charge for crocetin as predicted by the PM3 method 85 IV.2 Binding energy, heat of formation, and enthalpy of rrans-cis transformation as predicted by PM3 method 87 IV.3 Binding energy, heat of formation, maximum wavelength of crocetin derivatives as predicted by PM3 method 90 IVA Binding energy, heat of formation, maximum wavelength of crocetin and hydroperoxides of crocetin as predicted by PM3 method 93 IV.5 Optimized geometry and atomic charge for 15-S-hydroperoxides crocetin as predicted by the PM3 method 96 IV.6 Optimized geometry and atomic charge for 14-hydroperoxides of crocetin as predicted by the PM3 method 97 IV.7 Optimized geometry and atomic charge for 13-S-hydroperoxide crocetin as predicted by the PM3 method 98 IV.8 Optimized geometry and atomic charge for 12-S-hydroperoxide crocetin as predicted by the PM3 method 99 IV.9 Optimized geometry and atomic charge for II-S-hydroperoxide crocetin as predicted by the PM3 method 100 IV.1O Optimized geometry and atomic charge for lO-hydroperoxide crocetin as predicted by the PM3 method 101 V.I The saturated amount of crocin and geniposide of the crude extract as adsorbed on adsorbent Amberlite XAD-16 using 50% EtOH for eluting 129 V.2 The amount of crocin of the crude extract (90 mI) as absorbed on adsorbent Amberlite XAD-16 using the procedure I 130 V.3 The amount of crocin of the crude extract (30 mI x 3) as absorbed on adsorbent Amberlite XAD16 using the procedure 130 197 LIST OF flGURES Fig Name of Figure Page 1.1 Some carotenoid compounds 1.2 Structure of crocetin derivatives 1.3 Structure of iridoids of gardenia 10 1.4 Structure of flavanoid compounds of gardenia 10 1.5 Spectrum of crude extract of gardenia fruit after 16 hours of extraction at 25'C with 50%, gardenialEtOH50% ratio = 1:3 (w/v), k"","= 1000 22 1.6 Changes in absorbency at 440 nm during the extraction of gardenia fruits with water and ethanolic solutions at 25'C , gardenia/solvent ratio = 1.0 I 3.0 (w/v) 23 1.7 Crocin contents and % crocin in total yellow pigments of the extract of gardenia fruits at 25'C with different gardenia/solvent ratio ( analyzed by HPLC) 24 1.8 Changes in absorbency at 440 nm of the extract of gardenia fruit at different temperatures, gardenia/ 50%EtOH ratio = I : (w/v) 25 1.9 Standard curve of geniposide analyzed by HPLC Column 00S5:4.6 mm 1.0 x 250mm, photodiode array detector: 200nm-500nm, flow rate: Imllmin, solvents: A: water, B: MeOH, 0-10 min: % B to 50%B,10-40 min: 50%B to loo%B, 40-50min: loo%B, 50-65 min: O%A 27 1.10 The profile of analytical HPLC of the crude extract of gardenia in the region UV 28 1.11 The profile of analytical HPLC of the standard geniposide 29 1.12 The profile of co-chromatography of the crude extract of gardenia and standard geniposide in the region UV 30 1.13 Standard curve of absorbance at 440 nm vs content of crocin in 0.1 M citrate phosphate buffer (pH 7.0) 31 1.14 Standard curve of crocin analyzed by HPLC (method SCAN) 31 1.15 The profile of preparative HPLC for the crude extract of gardenia fruits 34 1.16 The profile of analytical HPLC of the crude extract of gardenia fruits 35 1.17 Profile of analytical HPLC of purified crocin from gardenia fruits 36 1.18 NMR spectrum of purified crocin from gardenia fruits 37 1.19 MS spectrum of purified crocin from gardenia fruit 38 II I Isomerisation of p-p-carotene under the actions of heat, light or radiation 42 198 II.2 % Colour remain of crocin in thermodegradation at 25°C in different pH medium of 0.1 M citrate _phosphate buffer (analyzed by HPLC) 45 II.3 % Colour remain of crocin in thermodegradation at 95"C in different pH medium of 0.1 M citrate-phosphate buffer, crocin concentration of 30 ppm (w/v) 47 III I General mechanism of lipid oxidation 50 II1.2 Lipid oxidation scheme 51 III.3 Structure of antioxidants used in the assays 58 III.4 Changes in absorbency at 500 nm vs time in oxidation of linoleic acid with crocin at different concentration using thiocyanate assay 65 III.5 Antioxidant activity of crocin at different concentration after days of oxidation using thiocyanate assay 66 III.6 Changes in absorbency at 500 nm vs time of oxidation linoleic acid with different antioxidants at the same concentration of 20 ppm (w/v) 67 III.7 Antioxidative activity of crocin, crude extract from gardenia fruit and different antioxidants at the same concentration of 20 ppm after days of oxidation using thiocyanate method 68 II1.8 Effect of crocin on the production of malonaldehyde from linoleic acid oxidized by FeSO, for 16 h at 37°C in 0.25 mM Tris-HCI buffer (pH 7.4 , containing 0.2% SOS and 0.75 mM KCI) 70 IIJ.9 Effect of crocin and other antioxidants on the production of malonaldehyde from linoleic acid oxidized by FeSO, for 16 h at 37°C in 0.25mM Tris-HCI buffer (pH 7.4, containing 0.2 % SOS and 0.75mM KCI ) Concentration of each antioxidant = 10 ppm 71 III.IO Superoxide Oismutase-like activity of crocin 73 IV.I Proposed mechanism of inhibition by carotenoid of peroxyl of radical-mediated lipid peroxidation 79 IV.2 Changes in UV/visible spectrum during the oxidation of crocin at 37°C and pH 6.0 (40 mM phosphate buffer) containing FeSO, 89 IV.3 Changes in UV/visible spectrum during the oxidation of linoleic acid at 37°C at pH = 6.0 (40 mM phosphate buffer) without FeSO, 90 IV.4 Changes in UV/visible spectrum during the co-oxidation of crocin with linoleic acid (crocin/linoleic acid mole ratio = I : 2) at 37'C at pH 6.0 (40 mM phosphate buffer) containing FeSO, 91 IV.s Numbering systems and optimized geometries of crocetin and 15-S-monohydroperoxide of crocetin 93 IV.6 Isomerisation mechanism of crocetin 97 IV.7 Binding energy of monohydroperoxide of crocetin with different bond torsion angles predicted by PM3 method: a IO-hydroperoxide, b II-hydroperoxide c 12-hydroperoxide, d 13hydroperoxide, e.14-hydroperoxide, and r.15-hydroperoxide 101 IV.8 Numbering systems and optimized geometries of monohydroperoxides of crocetin 103 199 IV.9 Numbering systems and optimized geometries of monohydroperoxides crocetin.(continued) 104 IV.1O MS analysis of the products of crocin oxidized at 37'C at pH 6.0 after 16 h with FeSO, 113 IV.II Proposed oxidation products of crocin when the C 13-CI4 double bond is cleaved 114 IV.12 Proposed oxidation products of crocin when the CIS-CIS' double bond is cleaved 114 IV 13 LC - MS analysis of the products of crocin oxidized at 37'C at pH 6.0 after 16 h with linoleic acid (crocin/linoleic acid mol ratio = I: 1) and FeSO, lIS IV.14 LC - MS analysis of the products of crocin oxidized at 37'C at pH 6.0 after days and FeSO, 116 V.I Chemical pathway for the formation of blue or red colour from geniposide 119 V.2 Effect of concentration of acetone on the separation of iridoids by acetone solution using Amberlite XAD-16 absorbent (20g), gardenia extract! absorbent ratio = 1:2 (v/w) 126 V.3 Effect of concentration of ethanol on the elution of iridoids by ethanolic solution using Amberlite XAD-16 absorbent (20g), gardenia extract! absorbent ratio = 1:2 (v/w) 127 VA Effect of concentration of ethanol on the elution of crocin by ethanolic solution using Amberlite XAD-16 absorbent (20g), gardenia extract! absorbent ratio = 1:2 (v/w) 128 V.S Flowchart of production of yellow pigment from gardenia fruits 133 200 LIST OF SYMBOLS AMI Austin Model I BHA Butylated hydroxy anisol BHT Butylated hydroxy toluene Car Carotenoid CroOOH Hydroperoxide of crocetin CNDO Complete Neglect of Differential Overlap INDO Intermediate Neglect of Differential Overlap LH Linoleic acid or unsaturated fatty acid LOOH Hydroperoxide of unsaturated fatty acid I.P Induction period Dilution koefficient MA Malonaldehyde MIND03 Modified Intermediate Neglect of Differential Overlap, version MNDO Modified Neglect of Differential Overlap Mw Molecular weight ODS CH, octadecylsilanyl group bonded to silica ( -0- Si-(CH,ll1 CH, l CH, PM3 TBA 2-Thiobarbituric acid TBARS TBA reactive substance Trolox Water soluble alpha-tocopherol SDS Sodium dodecyl sulfate 201 Agroalimentaire Canada Agri-food Canada Direction generale de la recherche Centre de recherche et de developpement sur les aliments Research Branch Food Research and Development Centre 3600, boulevard Casavant Ouest 3600 boulevard Casavant Ouest Saint-Hyacinthe (Ou~c) J2S 8E3 T~I.: (514) 773-1105 Fax: (514) 773-846' Saint-Hyacinthe (O~bec) J2S 8E3 Tel.: (514) 773-1105 Fax: (514) 773-8461 Voffe reference Your fife Notre reference Our file e-mail: connierf@em.aer.ca Le 26 octobre, 1998 OBJET: Evaluation de M Pbaro Tbanb Quan A qui de droit: 11 me fait plaisir de vous transmettre mon evaluation de M Pham Thanh Quan Cet eleve de l'Universite Polytechnique de HCM-Ville a sejourne dans mon laboratoire du 30 septembre 1996 au 28 septembre 1998 pour y effectuer un projet de recherche qui doit servir a I'obtention d'un doctorat dans son Universite M Pham a demontre beaucoup d'habiletes intellectuelles dans Ie deroulement de ses etudes 11 a puise dans la litterature I'information pertinente et a developpe et a valide plusieurs methodes de fayon autonome 11 a appris a maltriser des logiciels de modelisation moleculaire et a en interpreter les resultats grace aux lectures et aussi au soutien du Dr Marie-Rose Van Calsteren 11 a communique ses resultats ainsi que son interpretation des resultats au Dr Van Calsteren, au Dr Edward Farnworth ainsi qu'a moi Monsieur Pham communique par ecrit de fayon efficace tel que demontre par la redaction d' articles Sa thematique de recherche est tres originale Le but d'un doctorat est d'augmenter la connaissance des phenomenes pour essayer de les expliquer Ainsi, il ne s' est pas borne a effectuer des essais sur I'activite antioxidante de la crocine mais a aussi cherche a apporter une explication au phenomene par l'usage de methodes spectroscopiques (spectrophotometrie, RMN et spectrometrie de masse) et de la modelisation molecu1aire_ Soucieux que ses resultats trouvent une utilisation non seu1ement a long terme mais aussi a court terme, il a etabli les bases d'un procede de production de colorants issus du fruit de gardenia_ La de de ce procede reside dans l'elimination d'iridoides hepatotoxiques qui empechent l'utilisation d'extraits de gardenia en Amerique du Nord et en Europe Ainsi, il a trouve les conditions d'utilisation d'une resine absorbante qui elimine les iridoides de fayon efficace Une demonstration du procede a plus grande echelle n'a pu etre effectuee au CRDA pour des raisons de securite En effet, il existe au CRDA une limitation sur Ie volume de solvant qui peut y etre manipule J'estime neanmoins, qu'il a etabli les parametres critiques du procede, lesquels faciliteront la mise a I' echelle Canada Somme toute, je suis fier du travail de Quan que j 'estime etre digne d 'une these de doctorat Si vous avez des questions relativement a cette affaire, n'hesitez pas a me les demander ~/i~FraniYo{s Cormier, PhD Chercheur ... greater quantities and at a lower price from the fruit of Gardenia lasminoides Ellis [1] The fruits of Gardenia jasminoides Ellis contain three major groups of pigments: water soluble carotenoids (crocetin... and medicine Crocin and crocetin glycosyl esters are water soluble Carotenoids which are found in nature in stigmas of saffron (Crocus sativus Linne) and in the fruits of gardenia (Gardenia jasminoides. .. I: Extraction, Purification and Identification of the main compounds from Gardenia jasminoides Ellis 1.1 Introduction Carotenoids Crocetin derivatives and iridoids from Gardenia jasminoides Ellis