Title
`
`Characteristics and chromatographic separation of astaxanthin
`and its esters from the microalga haematococcus pluvialis
`
`Advisor(s)
`
`Chen, SF
`
`Author(s)
`
`Yuan, Jianping.; 袁建平
`
`Citation
`
`Yuan, J. [袁建平]. (1999). Characteristics and chromatographic
`separation of astaxanthin and its esters from the microalga
`haematococcus pluvialis. (Thesis). University of Hong Kong,
`Pokfulam, Hong Kong SAR. Retrieved from
`http://dx.doi.org/10.5353/th_b3123971
`
`Issued Date
`
`1999
`
`URL
`
`http://hdl.handle.net/10722/35452
`
`Rights
`
`The author retains all proprietary rights, (such as patent rights)
`and the right to use in future works.
`
`RIMFROST EXHIBIT 1098 page 0001
`
`

`

`CHARACTERISTICS AND CHROMATOGRAPHIC
`
`SEPARATION OF ASTAXANTHIN AND ITS ESTERS
`
`FROM THE MICROALGA Haematococcus plu vialis
`
`by
`
`JIAN-FING YUAN
`
`B. Sc. (Dalian College of Light Industry, P. R. China)
`
`M. Sc. (South China University of Technology, P. R. China)
`
`A thesis submitted in partial fulfillment of the requirements
`
`for the degree of Doctor of Philosophy
`
`at The University of Hong Kong
`
`August 1999
`
`RIMFROST EXHIBIT 1098 page 0002
`
`

`

`DECLARATION
`
`I declare that this thesis represents my own work, except where due
`
`acknowledgment is made, and that it has not been previously included in a
`
`thesis, dissertation or report submitted to this University or to any other
`
`institution for a degree, diploma or other qualification.
`
`-s-: )
`
`L
`.7
`............................ :.
`
`Signed
`
`\C\
`
`Jian-Ping Yuan
`
`RIMFROST EXHIBIT 1098 page 0003
`
`

`

`ACKNOWLEDGMENTS
`
`I would like to express my deepest gratitude to my supervisor, Dr. Feng
`
`thoughtful advice and
`Chen for his moral encouragement, helpful guidance,
`I have benefited a lot during my study under his
`
`stimulation.
`
`supervision.
`
`I am also impressed by his efforts to supply me with necessary
`I would like to thank him for the
`
`instruments and conditions.
`
`numerous revisions of my manuscripts and for the substantial investment in time
`
`spent on my behalf.
`
`I wish to acknowledge that this research project was supported by the
`
`University of Hong Kong Committee on Research and Conference Grants and the
`
`Hong Kong Research Grants Council grants to Dr. Feng Chen, and that I was
`
`supported by a scholarship from the University of Hong Kong during my study.
`
`I am grateful to Professor Si-Yuan Guo of South China University of
`
`Technology for his great help.
`
`I also acknowledge Professor Sammy Boussiba of
`
`Ben-Gurion University of the Negev, Israel for the supply of canthaxanthin standard
`
`and dry biomass samples of the alga H. pluvialis.
`
`I wish to thank my colleagues in our laboratory, Miss Chui-Yee Yap, Prof.
`
`Lin Li, Prof. Jian-Jiang Zhong, Dr. Yi-Ming Zhang, Dr. Xian-Di Gong, Dr. Xian-
`
`Ming Shi, Dr. Xue-Wu Zhang, Dr. Xian-Zheng Li, Dr. Yue Jiang, Dr. Gui-Xing
`
`Ren, Dr. Rema Vazhappilly, Mr. Hui Chen, Miss Oi-Ha Lau, Miss Ping-Ping
`
`Lam, Miss Suk-Man Cheung, Mr. Zhi-You Wen, and Mr. Yin-Nm Ma for their
`
`helpful cooperation and discussion.
`
`Finally, I have to express my thanks to my parents, my wife and my son for
`
`their understanding and support.
`
`RIMFROST EXHIBIT 1098 page 0004
`
`Iv
`
`

`

`Abstract of thesis entitled
`
`CHARACTERISTICS AND CHROMATOGRAPHIC
`SEPARATION OF ASTAXANTHIN AND ITS ESTERS
`FROM THE MICROALGA Huematococcus pluvialis
`
`submitted by
`Jian-Pmg Yuan
`
`for the degree ofDoctor of Philosophy
`at The University of Hong Kong
`August 1999
`
`Reversed-phase high performance liquid chromatographic methods were
`
`developed for the separation, identification, and determination of astaxanthin esters
`
`and the isomers of astaxanthin in the unsaponified and saponified pigment extracts
`
`from the microalga Haeinatococcus pluvialis. HPLC was conducted on a Waters
`
`liquid chromatograph equipped with two 510 pumps and a 996 photodiode array
`
`detector set at 250-700 nm.
`
`Beckman Ultrasphere C column and Waters
`
`Symmetry C18 column were used to separate carotenoids and chiorophylls in
`
`pigments extracts . The mobile phase consisted of methanol , dichioromethane,
`
`acetonitrile, and water.
`
`The hydrolysis of astaxanthin esters and the degradation of astaxanthin during
`
`saponification of the pigment extract were investigated. Different concentrations of
`
`NaOH in methanol were used for the saponification under nitrogen in darkness. The
`
`concentration of NaOH was important for promoting the hydrolysis of astaxanthin
`
`esters and minimizing the degradation of astaxanthin during saponification. A high
`
`temperature should be avoided to minimize the degradation of astaxanthin.
`
`The purification method including extraction, saponification, and separation
`was established for preparing purified ¿ans-astaxanthin from a high-yielding
`
`RIMFROST EXHIBIT 1098 page 0005
`
`I
`
`

`

`astaxanthin ester-producing strain of the microalga Haematococcus pluvialis which
`
`contained 3 67% frans-astaxanthins and i .3 5% cis-astaxanthins ofthe dry cells. 94.4%
`free trans-astaxanthin was obtained from trans-astaxanthin esters after 12 h of
`
`saponification at 5 °C. With this method, 32.2 mg of trans-astaxanthin was obtained
`
`from i g ofdry algal cells.
`
`The isomerization of trans-astaxanthin in organic solvents was investigated.
`
`trans-Astaxanthin was dissolved in dirnethylsulfoxide, methanol, acetonitrile, acetone,
`
`chloroform, dichioromethane, and dichloromethane : methanol (25 :75), respectively,
`
`and heated at 3 5 °C, followed by analyzing cts and bans-astaxanthins in the solutions.
`
`In different solvents, the isomerization rates of Pans-astaxanthin and the relative
`
`content of 9-cis and i 3 -cis-astaxanthins formed during isomerization were different.
`
`In all cases, 13-cis-isomer was the main c/s-isomer from frans-astaxanthin,
`
`trans-
`
`Astaxanthin dissolved in dichioromethane or chloroform was readily isomerized to cis-
`
`isomers, especially for dichioromethane. A higher temperature could promote the
`
`isomerization of ans-astaxanthin.
`
`The contents of carotenoids and chiorophylls in the high-yielding astaxanthin
`
`ester-producing strain of the microalga Haematococcus pluvialis during cultivation
`
`were determined. The accumulation of astaxanthin esters and the changes in contents
`
`of chlorophylls and other carotenoids in this alga during cultivation were investigated.
`
`At the beginning of growth, chiorophylls and primary carotenoids (lutein and J3-
`carotene) were the main pigments and only a very small amount of secondary
`carotenoids, including astaxanthin and canthaxanthin, were found. At the end of the
`
`cultivation, astaxanthin esters were the maj or components and only very small amounts
`
`of J3-carotene, free
`
`ans-astaxanthin, canthaxanthirì, lutein, and chiorophylls were
`
`present in the deep red algal cells.
`
`The
`
`carotenoid composition in
`
`a new ketocarotenoid-producing alga
`
`Chlorococcum isolated from flora on rocky wall was analyzed and compared with that
`
`of the alga Haematococcus pluvialis. Chiorococcum could produce a large amount of
`
`ketocarotenoid esters
`
`like Haematococcus pluvialis.
`
`But,
`
`in Chlorococcum,
`
`astaxanthin and the other ketocarotenoids were the major carotenoids, whereas only
`
`astaxanthin was the major carotenoid in Haematococcus pluvialis.
`
`II
`
`RIMFROST EXHIBIT 1098 page 0006
`
`

`

`PUBLICATIONS
`
`1. Yuan, J. P.; Gong, X. D.; Chen, F. Separation and identification of astaxanthin
`esters and chiorophylls in Haematococcus lacusfris by HPLC. Biotechnology
`
`Techniques, 1996, 10, 655-660.
`2. Yuan, J. P.; Gong, X. D.; Chen, F. Separation and analysis of carotenoids and
`
`chiorophylls
`
`in Haematococcus
`high-performance
`by
`liquid
`chromatography photodiode array detection. Journal ofAgricultural and Food
`
`lacustrEs
`
`Chemistry, 1997, 45, 1952-1956.
`3. Yuan, J. P.; Chen, F.
`lacustris by RPLC-photodiode array detection. Biotechnology Techniques, i 997,
`
`Identification of astaxanthin isomers in Haematococcus
`
`il, 455-459.
`4. Chen, H.; Yuan, J. P.; Chen, F.; Zhang, Y. L.; Song, J. Y.
`production in Ti-transformed Salvia miltiorrhiza cell suspension cultures. Journal
`
`Tanshinone
`
`ofBiotechnology, 1997, 58, 147-156.
`
`5. Shi, X. M., Chen, F.; Yuan, J. P.; Chen, H. Heterotrophic production of lutein
`
`by selected Chlorella strains. Journal ofAppliedPhycology, 1997, 9, 445-45 0.
`6. Yuan, J. P.; Yap, C. Y.; Chen, F.; Chen, H. Determination of anions in
`Haematococcus pluvialis
`photometric
`culture media
`using indirect
`ion
`Proceedings of Asia-Pac/ìc Biochemical Engineering
`Conference, 1997, 1164-1167.
`
`chromatography.
`
`7. Yuan, J. P.; Chen, F. Separation and identification offtiranic compounds in fruit
`juices and drinks by HPLC-PDA detection. Journal of Agricultural and Food
`
`Chemistry, 1998, 46, 1286-1291.
`
`8. Yuan, J. P.; Chen, H.; Chen, F. Simultaneous determination of rosmarinic acid,
`lithospermic acid B, and related phenolics in Salvia miltiorrhiza by HPLC.
`
`Journal ofAgricultural andFood Chemistry, 1998, 46, 265 1-2654.
`9. Yuan, J. P.; Chen, F. Chromatographic separation and purification of trans-
`Journal of
`astaxanthin from the extracts of Haematococcus pluvialis.
`Agricultural andFood Chemistry, 1998, 46, 3371-3375.
`
`RIMFROST EXHIBIT 1098 page 0007
`
`

`

`10. Yuan, J. P.; Chen, F. Degradation of ascorbic acid in aqueous solution. Journal
`
`qfAgricultural and Food Chemistry, i 998, 46, 5078-5082.
`i 1. Yuan, J. P.; Chen, F. Hydrolysis kinetics of astaxanthin esters and stability of
`Journal of
`astaxanthin of Haematococcus pluvialis during saponification.
`Agricultural andFood Chemistiy, 1999, 47, 3 1-35.
`12. Yuan, J. P.; Chen, F. Simultaneous separation and determination of sugars,
`ascorbic acid and fliranic compounds by HPLC-dual detection, Food Chemistry,
`
`1999, 64, 423-427.
`
`13. Yuan, J. P.; Chen, F. Isomerization of trans-astaxanthin to cis-isomer in organic
`
`solvents. Journal ofAgricultural andFood Chemistry (in press).
`
`14. Boussiba, S.; Bing, W., Yuan, J. P.; Zarka, A.; Chen, F. Changes in pigment
`profile in the green alga Haematococcus pluvialis exposed to environmental
`
`stresses. Biotechnology Letters (in press).
`
`RIMFROST EXHIBIT 1098 page 0008
`
`

`

`CONTENTS
`
`ABSTRACT.............................................................................................................. I
`
`DECLARATION ................................................................................................... ifi
`
`ACKNOWLEDGMENTS ..................................................................................... IV
`
`PUBLICATIONS ..................................................................................................... V
`
`CONTENTS .......................................................................................................... VII
`
`CHAPTER 1
`
`Literature Review and Introduction ....................................................................... 1
`
`i . i
`
`Importance of astaxanthin ................................................................................... i
`
`i . i . i Potential applications in medicine ................................................................ i
`
`i . i .2 Pigment additives in aquaculture ................................................................. 4
`
`i . i .3 Biological function in microorganisms ......................................................... 7
`
`i . 2 S ources of astaxanthin ........................................................................................ 9
`
`1.2.1 The green niicroalgaHaematococcuspluvialis ............................................ 9
`
`i .2.2 The red yeast Phaffia rhodozyrna ............................................................... i i
`
`i.3 Properties and biosynthesis ofastaxanthin .......................................................... 13
`
`1 .3 . i Properties of astaxanthin ............................................................................ 13
`
`1.3.2 Biosynthesis ofastaxanthin ......................................................................... 16
`
`1 .4 Analysis of carotenoids and chiorophylls ............................................................ 20
`
`i .4. 1 Spectrophotometry and thin-layer chromatography .....................................20
`
`1 .4.2 High-performance liquid chromatography ...................................................24
`
`1.4.2.1 ApparatusofHPLC ...........................................................................25
`
`1,4.2.2 Pumps ................................................................................................25
`
`1.4.2.3 Sampleinjectors .................................................................................26
`
`1.4.2.4 Column and guard column ..................................................................26
`
`1.4.2.5 Photodiode-array detector ..................................................................26
`
`1 .4.2. 6 Selection of separation column ........................................................... 27
`
`VII
`
`RIMFROST EXHIBIT 1098 page 0009
`
`

`

`1.4.2.7 Mobile phase
`
`14.2.8 Quantitative analysis
`
`28
`
`28
`
`1.4.2.9 Semi-preparative separation ...............................................................29
`
`i .4.3 HPLC separation of astaxanthins and other pigments .................................. 29
`
`L4.4 HPLC separation ofisomers ofcarotenoids .............................................. 31
`
`1.5 Introduction to this thesis .................................................................................. 33
`
`CHAPTER 2
`
`Determination Method of Carotenoids and Chiorophylls by HFLC .................... 36
`
`2.1 Abstract ............................................................................................................. 36
`2.2 Introduction ....................................................................................................... 36
`
`2.3 Materials and methods .......................................................................................37
`
`2.3.1 Chemicals and reagents .............................................................................. 37
`
`2.3.2 Preparation of standard solution ofpigments .............................................. 37
`
`2.3.3 HPLC analytical method ............................................................................. 38
`
`2.4 Results and discussion ........................................................................................ 38
`
`2.4. 1 Chromatographic conditions ....................................................................... 38
`
`2.4.2 Absorption spectra of carotenoids and chiorophylls .................................... 40
`
`2.4.3 Standard calibration curves ........................................................................ 44
`
`2.4.4 Recovery and reproducibility ...................................................................... 48
`
`CIIAPTER 3
`
`Change in Contents of Carotenoids in the Alga Haematococcus pluvialis during
`
`Cultivation .............................................................................................................. 50
`
`3.1 Abstract ............................................................................................................. 50
`3.2 Introduction ....................................................................................................... 50
`
`3.3 Materials and methods ....................................................................................... 51
`
`3 .3 . i Alga strain ................................................................................................. 51
`
`3 . 3 . 2 Culture conditions ...................................................................................... 51
`
`3 .3 .3 Chemicals and reagents .............................................................................. 52
`
`3.3.4 Pigment extraction and sample preparation ................................................. 52
`
`3.3.5 HPLCmethod ............................................................................................53
`
`VIII
`
`RIMFROST EXHIBIT 1098 page 0010
`
`

`

`3.4 Results .
`
`3 .4. 1 Development ofHPLC separation method
`
`3.4.1.1 Separation column
`
`3.4.1.2 Mobilephase
`
`3 .4.2 Changes in carotenoids and chiorophylls during growth .
`
`3.5 Discussion
`
`CHAPTER 4
`
`53
`
`53
`
`53
`
`54
`
`57
`
`64
`
`Identification ofAstaxanthin Isomers in the Alga Haematococcuspluvialis ........ 66
`
`4.1 Abstract .............................................................................................................66
`
`4.2 Introduction ....................................................................................................... 66
`
`4.3 Materials and methods ....................................................................................... 67
`
`4.3.1 Alga strain and culture conditions ............................................................... 67
`
`4.3.2 Chemicals and reagents .............................................................................. 68
`4.3.3 Pigment extraction ..................................................................................... 68
`
`4.3.4 HPLCmethod ............................................................................................ 68
`
`4.3.5 Saponification ofastaxanthin esters ............................................................ 69
`
`4.4 Results and discussion ........................................................................................ 69
`
`4.4.1 Separations ofisomers and esters ofastaxanthin ......................................... 69
`
`4.4.2 Identification method ofastaxanthin isomers ..............................................71
`
`4.4.3 Identification of astaxanthin isomers in astaxanthin esters ........................... 73
`
`CHAPTER 5
`
`Development of a Gradient Reversed-Phase ITPLC Method for the Separation of
`the Pigment Extract from the Alga
`Carotenoids and Chiorophylls
`Haematococcuspluvialis ......................................................................................... 78
`
`in
`
`5.1 Abstract ............................................................................................................. 78
`
`5.2 Introduction .......................................................................................................78
`
`5.3 Materials and methods ....................................................................................... 79
`
`5.3.1 Alga strain and culture conditions ............................................................... 79
`
`5.3.2 Chemicals and reagents .............................................................................. 79
`
`5.3.3 Pigment extraction ..................................................................................... 80
`
`Ix
`
`RIMFROST EXHIBIT 1098 page 0011
`
`

`

`5.3.4 Saponification ofastaxanthin esters
`
`5.3.5 Analytical HPLC method
`
`5.3.6 Semipreparative IJIPLC method
`
`80
`
`80
`
`81
`
`5.4 Results and discussion ........................................................................................ 82
`
`5.4.1 Extraction and saponification ..................................................................... 82
`
`5.4.2 Analytical HPLC ........................................................................................ 83
`
`5 .4. 3 Purification of b'ans-astaxanthin from the alga H. pluvicilis ......................... 91
`
`CHAPTER 6
`
`Hydrolysis of Astaxanthin Esters in the Pigment Extract and Degradation of
`Astaxanthin during Saponification ........................................................................ 93
`
`6.1 Abstract ............................................................................................................. 93
`
`6.2 Introduction ....................................................................................................... 93
`
`6.3 Materials and methods ....................................................................................... 94
`
`6.3 . i Alga culture and pigment extraction ........................................................... 94
`
`6.3 .2 Chemicals and reagents .............................................................................. 95
`
`6.3.3 High-performance liquid chromatography ................................................... 95
`
`6 . 3 .4 S aponification of pigment extracts and stability of carotenoids ....................96
`
`6.3.5 Kinetic study ofthe hydrolysis ofastaxanthin esters .................................... 97
`
`6.4 Results and discussion ........................................................................................99
`
`6.4.1 Optimization ofsodium hydroxide concentration ........................................99
`
`6.4.2 Hydrolysis kinetics ofastaxanthin esters ................................................... 103
`
`6.4.3 Degradation reaction ofastaxanthin .......................................................... 105
`
`6.4.4 Effect oftemperature on astaxanthin productivity ..................................... 108
`
`6.4.5 Stability ofcarotenoids to NaOH .............................................................. 109
`
`6.4.6 Degradation ofchlorophylls ..................................................................... 109
`
`CHAPTER 7
`
`Isomerization of trans-Astaxanthin to cis-Isomer in Organic Solvents ............... 111
`
`7.1 Abstract ........................................................................................................... ill
`
`7.2 Introduction ..................................................................................................... 112
`
`7.3 Materials and methods ..................................................................................... i 14
`
`RIMFROST EXHIBIT 1098 page 0012
`
`

`

`7.3.1 Chemicals and reagents
`
`7.3 2 Isomerization of trans-astaxanthiri in organic solvents
`
`7.3.3 High-performance liquid chromatography
`
`i 14
`
`i 14
`
`114
`
`7.4 Results and discussion ...................................................................................... 115
`
`7.4. 1 Isomerization oftrans-astaxanthin ............................................................ i 15
`
`7.4.2 Effect of solvents on the isomerization offrans-astaxanthin ...................... i 17
`
`7.4.3 Effect oftemperature on the isomerization ............................................... 122
`
`7.4.4 First order kinetics with a reverse reaction ................................................ 123
`
`7.4.5 Effect ofinorganic solute on the isomerization ......................................... 127
`
`CHAPTER 8
`
`Purification of trans-Astaxanthin from a High-Yielding Astaxanthin Ester-
`Producing Strain of the Alga Haematococcuspluvialis ....................................... 130
`
`8.1 Abstract ........................................................................................................... 130
`
`8.2 Introduction ..................................................................................................... 130
`
`8.3 Materials and methods ..................................................................................... 132
`8 .3 . i Microalga Haematococcuspluvialis ......................................................... I 32
`
`8.3 .2 Chemicals and reagents ............................................................................ 132
`
`8.3.3 Analysis ofastaxanthin esters ................................................................... 132
`
`8.3.4 Extraction ofpigments ............................................................................. 133
`
`8.3.5 Hydrolysis ofastaxanthin esters ................................................................ 133
`
`8 . 3 . 6 Purification of trans-astaxanthin ............................................................... 134
`
`8.4 Results and discussion ...................................................................................... 134
`
`8.4.1 Analysis ofpigments ................................................................................ 134
`
`8.4.2 Extraction ofpigments ............................................................................. 136
`
`8.4.3 Hydrolysis ofastaxanthin esters ................................................................ 137
`
`8.4.3.1 Effectoftemperature ....................................................................... 137
`
`8.4.3.2 Effect ofNaOH concentration .......................................................... 138
`
`8.4.3.3 Hydrolysis process ........................................................................... 139
`
`8 .4.4 Purification of trans-astaxanthin ............................................................... i41
`
`RIMFROST EXHIBIT 1098 page 0013
`
`

`

`CHAPTER 9
`
`Accumulation of Astaxanthin Esters in a High-Yielding Astaxanthin Ester-
`Producing Strain of the Alga H. pluvialis during Cultivation ............................. 144
`
`9.1 Abstract ........................................................................................................... 144
`
`9.2 Introduction ..................................................................................................... 144
`
`9.3 Materials and methods ..................................................................................... 145
`
`9 .3 . I Dry biomass of the alga Haematococcus pluvialis .................................... i 45
`
`9.3.2 Chemicalsandreagents ............................................................................ 145
`
`9.3 .3 Pigment extraction and sample preparation ............................................... 146
`
`9.3.4 HPLC analytical method ........................................................................... 146
`
`9.4 Results and discussion ...................................................................................... 147
`
`9.4. 1 HPLC analysis of carotenoids and chiorophylls ......................................... 147
`
`9.4.2 Accumulation ofastaxanthin esters ........................................................... 150
`
`9.4.3 Change offree trans-astaxanthin .............................................................. 152
`
`9.4.4 Change ofcanthaxantliin .......................................................................... 152
`
`9.4.5 Change of3-carotene ............................................................................... 153
`
`9.4.6 Change oflutein ....................................................................................... 153
`
`9.4.7 Change ofchlorophylls ............................................................................. 153
`
`CHAPTER 10
`
`Summaryand Recommendation for Further Work ............................................ 157
`
`10.1 HPLC analysis ofpigments ............................................................................. 157
`
`10.1.1 TheHPLC ............................................................................................. 157
`
`10.1.2 The isocratic reversed-phase HPLC method ........................................... 157
`
`10.1 .3 The gradient reversed-phase HPLC method ............................................ I 58
`
`1 0.2 Pigment compositions in H pluvialis .............................................................. i 59
`
`i O . 3 Accumulation of astaxanthins in H. pluvialis .................................................. i 59
`
`10.4 Hydrolysis ofastaxanthin esters ...................................................................... 160
`
`10.5 Isomerization oftrans-astaxanthin .................................................................. 160
`
`1 0. 6 Purification of trans-astaxanthin ..................................................................... i 61
`
`10.7 Recommendation for further work .................................................................. 161
`
`XII
`
`RIMFROST EXHIBIT 1098 page 0014
`
`

`

`APPENDIX
`
`Biosynthetic Pathway of Astaxanthin in the Alga Haematococcus pluvialis and a
`
`New Ketocarotenoici-Producing Alga Chiorococcum .......................................... i63
`A.1 Abstract .......................................................................................................... 163
`
`A.2 Introduction .................................................................................................... 163
`
`A.3 Materials and methods ..................................................................................... 165
`
`A.3 . i Algal species and culture ......................................................................... i 65
`
`A.3 .2 Chemicals and reagents ........................................................................... 165
`
`A.3 .3 Pigment extraction .................................................................................. 166
`
`A.3.4 Saponification ......................................................................................... 166
`
`A.3 . 5 F!IPLC analysis ......................................................................................... i 67
`
`A.4 Results and discussion ..................................................................................... 167
`
`A.4. i Isolation and culture ofthe ketocarotenoid-producing alga ...................... 167
`
`A.4.2 Separation ofpigments in the alga Chiorococcum .................................... 168
`
`A.4.3 Effect ofglucose on production ofpigments ............................................ 174
`
`A.4.4 Biosynthetic pathways of astaxanthin ...................................................... 177
`
`LITERATURE CITED ......................................................................................... 182
`
`XIII
`
`RIMFROST EXHIBIT 1098 page 0015
`
`

`

`Chapter 1. Literature review and introduction
`
`Chapter 1
`
`Literature Review and Introduction
`
`1.1 Importance of Astaxanthin
`
`1.1.1 Potential applications in medicine
`
`Carotenoids have important biological functions, including conversion to
`
`vitamin A, enhancement of the immune response, and protection against cancer by
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`scavenging of oxygen radicals (Johnson and An, 1991). The possible role of dietary
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`carotenoids in the prevention of cancer has been suggested in many epidemiological
`
`studies (Gradelet et al. 1997). Epidemiological investigations have shown that
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`cancer risk is inversely related to the consumption of green and yellow vegetables
`
`and fruits, in which j3-carotene is present in abundance and has been investigated
`
`extensively as a possible cancer-preventive agent (Nishino, 1998). Carotenoids
`anticarcinogenic properties and can act on the different phases of
`
`have
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`carcinogenesis, for example, initiation, promotion, and progression (Astorg et al.,
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`1997). Dietary lutein may increase tumor latency, suppress mammary tumor
`
`growth and enhance lymphocyte proliferation (Chew et al. , 1996).
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`Extended
`
`studies on the cancer-preventive activities of natural carotenoids indicate that a-
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`carotene,
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`lutein, zeaxanthin,
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`lycopene, phytoene,
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`fucoxanthin, peridinin,
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`and
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`astaxanthin seem to be promising and some of them show a higher potency than 3-
`
`carotene to suppress carcinogenesis (Nishino, 1998).
`
`It has been noted that nutritional
`
`factors can modulate photochemical
`
`damages, in particular the common carotenoids present in food, which can be
`
`considered as potential prophylactic agents against carcinogenesis (Savoure et al.,
`
`i
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`RIMFROST EXHIBIT 1098 page 0016
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`

`

`Chapter 1 Literature review and introduction
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`i 995) . Savoure et al . ( i 995) investigated the effect of solar radiations (UV A and
`
`B), which could cause epidermis photoaging and skin cancers, on the skin of the
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`SKFT i hairless mouse fed a diet either lacking in vitamin A or supplemented with
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`retinol , n-carotene, or astaxanthin. The results show that astaxanthin has a stronger
`
`inhibitory effect on putrescine accumulation than retinol and decreases spermidine
`
`and spermine concentrations, suggesting a specific action on transg!utaminases.
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`Tanaka et al. (1994 and 1995) investigated the chemopreventive effects of
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`astaxanthin
`
`on
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`urinary
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`bladder
`
`carcinogenesis
`
`induced
`
`by N-butyl--N(4-
`
`hydroxybutyl) nitrosamine (OH-BBN) in male ICR mice and oral carcinogenesis
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`induced by 4-nitroquinoline I-oxide (4-NQO) in male F344 rats,
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`found that
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`astaxanthin was a possible chemopreventive agent for bladder carcinogenesis and
`
`oral carcinogenesis and suggested that such an effect of astaxanthin might be partly
`
`due to suppression of cell proliferation.
`
`Gradelet et al.
`
`(1997) tested the effects of several carotenoids on the
`
`initiation of hepatocarcinogenesis by aflatoxin Bi. The results clearly demonstrate
`
`the preventive effects of J3-carotene, canthaxanthin, and astaxanthin against aflatoxin
`
`B i carcinogenicity.
`
`f3-Carotene, canthaxanthin, and astaxanthin are also very
`
`efficient in reducing the number and the size of liver preneoplastic foci (Gradelet et
`
`al., 1998). Okai and Higashi-Okai (1996) analyzed the effects of 3-carotene,
`
`canthaxanthin, and astaxanthin on the proliferation and functions of murine
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`immunocompetent cells by in vitro cell culture experiments and suggested that
`
`carotenoids such as
`
`3-carotene, canthaxanthin, and astaxanthin had possible
`
`immunomodulating activities to enhance the proliferation and o