CYAN EXHIBIT 1002
`
`Order No. 181
`
`kDO0\lChU'|-l>UJI\)I4
`
`THESIS
`
`presented at the
`UNIVERSITY of LYON FACULTY OF SCIENCES
`to obtain
`A PHYSICAL SCIENCES DOCTORATE
`
`by
`Rene GRANGAUD
`
`Specialist Assistant Professor at the Algeria Faculty of Medicine and Pharmacy
`
`1“ THESIS — Research on Astaxanthin, a New Vitamin A Factor
`
`2nd THESIS — proposals given by the school
`
`Presented orally on October 23, 1950 before the Examination Jury
`
`Mr. CORDIER................President
`Mr. BERNARD..............Examiner
`Mr. MEUNIER...............Examiner
`
`EDITIONS DESOER
`
`21, RUE SAINTE-VERONIQUE
`LIEGE
`
`1951
`
`

`

`I4
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`
`TABLE OF CONTENTS
`
`Introduction: Importance of astaxanthin among natural carotenoids.
`
`Chapter I: Constitution and physical and chemical properties of astacine, astaxanthin and their derivatives.
`
`Chapter II: Astaxanthin in living creatures — Study of the Aristaeomorphafoliacea (Penaeidae).
`
`Chapter III: Biological Importance of Astaxanthin
`
`A. — Origin and destination of astaxanthin in living creatures.
`B. Astaxanthin, the new Group A Vitamin:
`I. — Vitamin A Activity in the oils of Aristaeomorphafoliacea.
`II. — Antixerophthalmic activity of the Aristaeomorphafoliacea carotenoid pigment
`1. Activity of the pigment extracted from the hepato-pancreas oil
`2. Activity of the pigment extracted from the hypoderm.
`III. — Location of astaxanthin in the treated rats.
`Observation in the retina.
`
`Chapter IV: Discussion of Results
`
`1. Astaxanthin’s chemical constitution and vitamin activity.
`2. Dissociation of astaxanthin’s antixerophthalmic activity and its effect on growth rate in the
`vitamin A deficient white rat.
`
`3. Astaxanthin and vision problems.
`
`General Conclusions
`
`

`

`kDO0\IChU'|-l>UJl\)l4
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`Chapter I
`
`THE CONSTITUTION, CHEMICAL AND PHYSICAL PROPERTIES OF ASTACINE, ASTAXANTHIN AND THEIR
`DERIVATIVES
`
`A. — ASTACINE AND ITS DERIVATIVES
`
`The carotenoid nature of astacine and the presence of oxygen in each of the [3-ionone cores have been
`recognized by Kuhn and Lederer (45).
`
`In 1934, P. Karrer and colleagues (38) established the general formula, C4oH48O4 and the constitution of the
`pigment which is that of a [3-carotene-tetrone. This constitution was deduced from the general formula and
`oxygen group properties. Astacine, in fact, provides a dioxin with 4 active hydrogen atoms, two of which
`belong to oxime radicals, while the other two are fixed to two enolized carbonyls. The carotenoid has,
`therefore, 4 ketonic groups which are not equivalent. Ortho-phenylenediamine reacts and creates bis-
`phenazine astacine which indicates that each [3-ionone core encloses two carbonyl groups. Oxidation with
`permanganate yields malonic dimethyl acid and oxidation of the diphenazine derivative leads to oL-oL-dimethy|-
`succinate which establishes that the carbonyl groups occupy the 3, 4, 3’, 4’positions. The formula for the
`constitution of astacine is, therefore, as follows:
`
`CH:
`
`CH:
`
`CH,
`
`>C\/\
`
`CH:
`CH.
`CH.
`I
`I
`I
`c.cH =cH.r.:=cH.cH=cH.c=cH.cH=cH.cH = C.CH=CH.CH =C.CH=c1-]_c
`
`_CHa
`
`CH, QH:
`
`\ /
`/'°\
`G1,
`
`or:
`
`c.cH,
`\C°l
`
`\cdl
`
`Astacine (Ketonic Form)
`
`19
`
`

`

`1
`2
`
`However, catalytic microhydrogenation reveals that there are not 11 but 13 double bonds. The extra two
`double bonds establish the possibility of the enolic form represented by the following diagram:
`
`CH:
`
`cn.
`
`CH
`c/
`1
`’
`/ \
`cu
`c.cH = an C-
`H°c\
`C CH.
`C0
`
`°“-
`
`.
`
`Astacine (enolic form)
`
`In reality, free astacine is only very slightly enolized as demonstrated by the slow etherification speed with
`diazo-methane and the determination of mobile hydrogens (Zerewitinoff). Astacine‘s acidic nature disappears
`after hydrogenation.
`
`Astacine crystallizes from a mix of pyridine and water into purple needles with metallic reflection which melt
`at 228°C (Kuhn, Stene and Sorensen (48) (1).
`
`Astacine is insoluble in water, is very difficult to dissolve in ether, petroleum ether or methane, is difficult to
`dissolve in benzene, ethyl acetate and acetic acid, is relatively easy to dissolve in carbon sulfur and is very easy
`to dissolve chloroform, pyridine (blood-red concentrated solution, diluted orangey solution) and dioxane.
`
`When the solution is agitated in petroleum ether with 90% methanol, astacine transforms into the hydro-
`alcoholic phase but after dilution with water, the pigment is easily dissolved in petroleum ether. In contrast, it
`remains in the aqueous phase if the latter is diluted with sodium or potassium after which it manifests its
`acidic nature. After cooling, the pigment separates from the alkaline solution in the form of red flakes which
`gather at the interphase. Acidification by acetic acid transforms the amorphous salt into shiny purple crystals
`of free astacine.
`
`Astacine dissolved in a mix of benzene and ligroin is absorbed rapidly in calcium carbonate. It is also absorbed
`in aluminum oxide from a benzene-ligroin or petroleum ether solution: it forms a purple area on the upper
`part of the column. The eluents are benzene and pyridine diluted with methanol as well as petroleum ether
`diluted with 5% potassium.
`
`(1) Other possible temperatures are 240 -243° C (Kuhn and Lederer [45]) and 241°C (Karrer and Benz [36]).
`
`20
`
`45 6
`
`7 8
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`9
`10
`11
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`31
`32
`
`cu
`‘
`CH.
`C.CH=vCH.Cl-I-C cH=cH C
`Hail.C\
`
`CH CH
`ix ‘
`
`H
`/LE0}:
`
`CO
`
`

`

`The absorption spectrum of astacine is characteristic and present in a large band with a single peak (500 nm in
`pyridine and 510 nm in carbon-sulfur). The absorption spectrum and the pigment’s behavior vis-a-vis sodium
`are the two main characteristics which identify it. Other, less specific, characteristics: in concentrated sulfuric
`acid, astacine dissolves into a deep blue color. The chloroform solution turns a blue-green color when diluted
`with antimony trichloride (R. de Carr and Price).
`
`Astacine is very resistant to oxidation in air. Kuhn, Lederer and Deutsch (46) have obtained astacine diacetate.
`Dipalmitate (astacene) has also been produced (Kuhn and colleagues [48]).
`
`B. —Astaxanthin and its Derivatives
`
`Kuhn and Sorensen (47) established that astaxanthin has four more hydrogen atoms than does astacine and
`has a general formula of C40H52O4. In an alkaline solution in the presence of air, they have demonstrated that
`astaxanthin transforms into astacine by means of an enediol auto-oxidation in which the enediol absorbs two
`oxygen molecules and forms two molecules of oxygenated water. Deriving the structure of astaxanthin from
`that of astacine, they assigned it the following formula which is that of 3,3'-dihydroxy-B-carotene-4,4‘-dione:
`
`CH‘
`
`cn
`CH
`CH‘
`{x '
`V’
`«
`my
`=.s~-
`‘i"'
`‘W’
`A
`«in. C.I:H=cH.c -cH.cH=::H.c-.cH.cu=4;H,.;;H,c cH_c,_,.c"_‘l_‘cH=cH Ct‘ \H
`.
`"ll
`j '
`HCIHC
`fc cu.
`WEI
`Hm
`W
`
`co
`
`Astaxanthin (Ketonic Form)
`
`on.
`c1-1.
`\/
`m
`E“
`G»
`/’\
`«.=-
`_
`_
`.
`l
`;
`cu.
`-:.cn=cn.-.
`[1
`per! cu-cl-I c cu cl-I_cI-1 c-4-: EH-cl-l.El-I-u0.‘..l:H=H'.‘H.!.‘.'
`I
`~==\,=-c~=
`...c.-
`
`*'-'-"H;-‘H:
`>
`
`H,
`4....
`
`A.
`
`Tw
`
`Astaxanthin (Enolic Form)
`
`The structural diagram for 3,3‘-dihydroxy-B-carotene-4,4‘-dione can also be created. Based on energy order
`criteria, however, Kuhn
`
`21
`
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`
`IJIJIJIJIJIJIJIJIJ
`
`19
`20
`21
`22
`
`23
`24
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`28
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`30
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`

`

`and Sorensen admit that it's the carbonyl group that is conjugated with the polyenic chain.
`
`In the absence of oxygen, astaxanthin with potassium yields a dark blue salt to which Kuhn and Sorensen, by
`analogy with the potassium derivative of stilbenediol and the methyl diesters of dihydrocrocetin and
`dihydrobixin, give the following formula:
`
`1
`
`‘\f"
`/X
`IOU.
`|.(”OC”.
`Ix
`(.1211.
`i
`5
`
`*7”
`/\
`7''
`ll‘
`7"‘
`I§‘.Q‘OCH.C:m.CNlCK.CHOC.(“'a..CHV(-(H-Qflfi
`‘fir.’
`u.¢,¢;‘l/(pg
`i
`Q
`
`Khun and Sorensen admit that lobster egg ovoverdin can also be considered to be a derivative of the enolic
`form and can be represented by the following diagram:
`
`an /00:
`:14. cm
`>t
`05:
`iii
`‘III!
`Clio
`€51.
`‘l.(l-I'tI-II:-(H.041-OIC‘lC‘IT.Of=I.l¢.¢H-{,.<l-Inc-I,q_|q-1!a|;g' gf \'°~
`I
`I
`C
`€.C‘H
`:1‘.
`1:
`IR!
`1/;
`‘°
`‘E
`5 9-
`-". _.p'
`"Q :'-
`
`This representation, which admits an ionic bond between the protein and the carotenoid explains the change
`in color which is produced when the prosthetic group is detached from the chromo-protein. This method of
`bonding does not, however, explain the auto-oxidation resistance of the protein complex. Kuhn and Sorensen
`believe that other, unknown, bonds between the protein and the carotenoid must be present to protect the
`latter from oxidation: the prosthetic group might be embedded in the protein (1).
`
`In reality, the color which develops from this potassium action on astaxanthin in the absence of oxygen must
`actually be interpreted as the bathochromic effect of a halochromic reaction (73) (maximum absorption
`around 700 nm as in
`
`(1) Kuhn and Sorensen use the expressions "anchored” and "embedded”.
`
`CDU'|-DUJIUIJ
`
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`the Carr and Price reaction, cf. Fig III). This reaction corresponds to the following structure which implies an
`oscillation of six electronic pairs [P. Meunier (1)]:
`
`
`
`With heteroprotein edifices such as ovoverdin, it may be that the union between astaxanthin and protein is
`
`also of the same type, the end of a polypeptic chain
`
`- CO — CH- NH3+
`
`occupying the place of a K ion.
`
`The molecular weight of ovoverdin has been determined by various researchers, Wyckoff (112) assigned a
`value of approximately 300,000, Kuhn and Sorensen have described a method for preparing ovoverdin. The
`pigment is precipitated from its aqueous solution by ammonium sulfate at saturation. It can be purified by
`dissolving it in water and absorbing it with aluminum hydroxide then eluting it with ammonium sulfate at 40%
`saturation. The concentration of ammonium sulfate only needs to be increased to re-precipitate
`chromoprotein. By repeating these operations several times a very pure product is obtained in which the
`carotenoid and the protein are united in the ratio of 1 to 242 which yields a molecular weight of 144,000. Stern
`and Salomon (93) have stated that the ovoverdin protein has the characteristics of albumin. Under these
`conditions, two protein molecules might be united with one astaxanthin molecule.
`
`(1) P. Meunier, personal communication.
`
`-hUJl\)l4
`
`LDO0\lChU'|
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`10
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`The color of the chromoprotein varies depending on the species: blackish-blue (Astacus gammarus shell),
`green (Astacus gammarus eggs and ovaries), blue (Heterocope saliens), brownish-green (Gamarus pulex),
`reddish-purple (Dendrodoa grossularia) (40), bluish purple (Aristaeomorphafoliacea), reddish purple (Aristeus
`antennatus).
`
`Stern and Salomon (92) studied "ovoverdin” in eggs and ovaries of Homarus americanus: the chromoprotein
`has an approximate pHi of 7 and its color is stable when the pH is between 4 and 8. It is reversibly dissociable
`into astaxanthin and protein if mildly heated for short intervals in the absence of air.
`
`Ball (1) has also isolated eggs of other species of crustaceans (Lepasfascicularis and L. anatifera) a blue
`chromoprotein which represents the same thermally-reversible dissociation phenomenon which can also be
`obtained by acidification performed in a cooled aqueous solution ofthe chromoprotein in the presence of
`ammonium sulfate.
`
`Recently, Wald and colleagues (107) succeeded in extracting the lobster she||’s blackish-blue chromoprotein by
`treating it with a solution of citric acid. They named it "crustacyanin”. Crustacyanin in solution in a pH 7
`veronal buffer heated to 60°C also undergoes a reversible dissociation and the color changes from blue to
`purple.
`
`Heat, strong acids, alkalis, acetone, alcohol, by denaturing the protein, facilitate easy extraction of astaxanthin
`from these various chromoproteins.
`
`It is easily dissolved in pyridine and, after the addition of water, it crystallizes into shiny tablets [F = 216°C
`(40)].
`
`ABSORPTION SPECTRUM
`
`The first spectroscopic data is that of Kuhn and Sorensen (47) who described an astaxanthin spectrum with
`three maxima (476, 493 and 513 nm in pyridine). According to Wald (105), these measurements are certainly
`erroneous: Tischer (97), by spectrophotometry, established that the spectrum of free and esterified
`astaxanthin has a large, single band. it is comparable to that of astacine but, compared to the latter, it is
`shifted towards the shorter wavelengths of 4 to 7 nm depending on the type of solvent used; for free
`astaxanthin, the maximum is located at 470 nm in hexane and approximately 492 nm in pyridine.
`
`-§UJl\)I4OLD00\lCDU'|-bUJl\)l4OLD00\lCDU'|-bUJl\Jl4OLD00\lCDU'|-PUJIUIJ
`
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`
`24
`
`

`

`Among the carotenoids whose constitution is established in a certain manner, astaxanthin and astacine are the
`only ones which have an absorption spectrum with a single maximum (1).
`
`Agitation of the astaxanthin solution in petroleum ether with 85% alcohol drives the entire pigment into the
`alcoholic phase: astaxanthin is completely hypophasic.
`
`The blue potassium salt obtained by the action of the potassium in the absence of air is characteristic of
`astaxanthin. In the presence of oxygen, the color instantly turns to red by transformation into astacine. This
`property facilitates the identification of astaxanthin.
`
`In a 1:4 benzene and petroleum ether solution, astaxanthin is absorbed on cane sugar (Kuhn and Sorensen)
`and can be eluated with benzene. The aluminum oxide can also be used as an absorbent if the solvent is
`
`petroleum ether and the eluent is petroleum ether mixed with 1% methanol.
`
`Many astaxanthin esters have been obtained by partial synthesis: Kuhn and Sorensen prepared diacetate,
`dicaprylate and dipalmitate, the latter having been isolated from the lobster hypoderm by Karrer, Loewe and
`Hubner (41) and, at first, considered to be an astacine dipalmitate.
`
`Of these derivatives, acetate alone is hypophasic while all the others are epiphasic (40).
`
`(1) Glycymerin, isolated by Fabre and Lederer (17) from Pectunculus glycymeris has an absorption spectrum
`that is very close to that of astacine but shifted with respect to it by about ten nm towards the shorter
`wavelengths. Glycymerin, which is distinct from astacine by the absence of any acidic properties, is of yet
`unknown constitution.
`
`Euler and Helstrom (16) isolated Asteria rubens, a carotenoid with acidic properties, which they called asterinic
`acid. The latter has an absorption spectrum with a single maximum (A = 480 nm in pyridine) but Rubel (84)
`showed that it is probably astacine.
`
`Mytiloxanthin, located by Scheer (85) in the muscle of Mytilus californianus alongside zeaxanthin, only seems
`to be distinguishable from astacine by its lower melting point.
`
`Ix)I4OLD00\lChU'|-bUJl\)l4OLD00\lChU'|-b(.Ul\)l4OLD00\lCDU'|-bUJl\)l4
`
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`
`25
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`kDO0\lChU'|-l>UJl\)l4
`
`Chapter II
`
`Astaxanthin in Living Organisms
`
`First considered to be a carotenoid restricted to fauna, astaxanthin has now been detected in flora as well. In
`organisms, astaxanthin is found in its free form, esterified or bonded to a protein. In a certain number of cases,
`astacine, not astaxanthin, has been characterized and, as a result, it is not possible to say whether the latter
`represents the natural pigment or its oxidation product.
`
`On the other hand, chromoproteins of the type found in lobster egg ovoverdin have not been found in plants
`[McKinney] (59)].
`
`The following table provides a list of animal and plant species in which astaxanthin (or astacine) has been
`found.
`
`Tablel (1)
`
`Distribution of astaxanthin (or astacine) in living beings.
`
`I. — GREEN ALGAE
`
`Haematococcus p|uvia|is* [Kuhn, Stene and Sorensen (48), Tischer (98)].
`
`ll. — PROTOZOA
`
`Euglena he|iorubescens* [Tischer (97)].
`
`Ill. — SPONGES
`
`Axinella crista-galli [Karrer and Solmssen (44)].
`
`(1) Most of the data in this table were taken from Kuhn, Stene and Sorensen (48). The organisms in which
`astaxanthin has been identified, without any ambiguity, are marked with an asterisk.
`
`27
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`

`

`kDO0\lChU'|-l>UJl\)I4
`
`1. Crustaceans
`
`IV — ARTHROPODS
`
`a) Malacostraca
`1. Schizopoda Euphausia [Drummond and Walter (11)].
`2. Decapods:
`Asatacus gammarus* [Kuhn and Lederer (45), Kuhn and Sorensen (47)];
`Homarus americanus* [Stern and Salomon (92)];
`Maja squinado [Kuhn, Lederer and Deutsch (46)];
`Palinurus vulgaris [Fabre and Lederer (17)];
`Portunus puber [Fabre and Lederer (17)];
`Nephrops Species [Fabre and Lederer (17)];
`Leander serratus [Fabre and Lederer (17)];
`Cancer pagurur [Fabre and Lederer (17)];
`Nephrops norvegicus [Burkhardt, Heilbron, Jackson, Parry and Lovern (5)];
`Potamobius astacus [Wi||staedt (111)];
`Aristaeomorpha fo|iacea* [Grangaud, Chechan, Miss Massonet (25)];
`Eupagurus Prideauxii [Lederer (54)].
`
`b) Copepods:
`Calanus finmarchicus [Lederer (54)];
`Heterocope saliens [Sorensen (89)].
`
`c) Phyllopodsz
`Holopedium gibberum [Sorensen (89)].
`
`d) Arthrostracians:
`Gammarus pu|ex* [Sorensen (89)].
`
`e) Cirripedes:
`Lepas fascicu|aris* [Ball (1)];
`Lepas anatifera* [Ball (1)].
`
`2. Insects
`
`Locusta* [Goodwin and Srisukh (23)];
`Schistocerca* [Goodwin and Srisukh (23)];
`Leptinotarsa* [Manunta (62)].
`
`Lima excavata [Sorensen (89)];
`
`V. MOLLUSKS
`
`28
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`

`

`VI: - ECHINODERMS
`
`Ophidiaster ophidianus [Karrer and Benz (36)];
`Echinaster sepositus [Karrer and Solmssen (44)].
`
`Dendrodoa grossularia [Lederer (54)];
`Halocynthia papillosa [Lederer (54)].
`
`Vll. —TUNlCATE
`
`V|l|.—F|SHES
`
`Beryx decadactylus (skin) [Lederer (52)];
`Carassius auratus (skin) [Lederer (52)];
`Cyclopterus Iumpus (liver) [Sorensen (88)];
`Lophius piscatorius (liver) [Sorensen (87)];
`Regalecus ge|sne* (liver) [Sorensen (87), Kuhn, Stene and Sorensen (48)];
`Salmo salar (muscle) [Sorensen (88)];
`Sebastes marinus (skin) [Lederer (53)];
`Perca fluviatilis (fins) [Lederer (53)].
`
`IX. — REPTILES
`
`Clemmys insculpata (retina) [Wald and Zussman (109)].
`
`X. — BIRDS
`
`Phasianus co|chicus* [Brockmann and Volker (4), Kuhn, Stene and Sorensen (48)].
`
`XI. — MAMMALS
`
`Balaenoptera musculus (fat) [Schmidt-Nielsen, Sorensen and Trumpy (86), Burkhardt, Heilbron, Jackson, Parry
`and Lovern (5)].
`
`STUDY OF THE PIGMENT OF THE ARISTAEOMORPHA FOLIACEA
`
`The Aristaeomorpha foliacea, a decapod crustacean in the Penaeidae family, is a large dark-red shrimp found
`in the Mediterranean at depths of 2 to 300 meters and beyond. Sold in large quantities in the Algerian port
`markets from June to October, a period in which trawl-fishing is only authorized outside of territorial waters, it
`is much rarer in winter.
`
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`l\)I4OLD00\lChU'|-bUJl\Jl4OLD00\lChU'|-PUJIUIJ
`
`IUIUIUIJIJIJIJIJIJIJIJIJIJ
`
`23
`24
`
`In order to look for the pigment which, as far as we know, had not been identified previously, we were inspired
`by the techniques, developed for other crustaceans, by Kuhn and Lederer (41), Lederer (53), Kuhn and
`Sorensen (47), Kuhn, Stene and Sorensen (48)].
`
`We studied the pigment in the oil extracted from hepato-pancreas and that extracted from the red hypoderm
`which lines the shell interior and the stomach pocket.
`
`1. Preparation of the Hepato-Pancreas Oil
`
`Hepato-pancreases extracted from the cephalothoraxes of 1kg ofAristaeomorpha were crushed with
`anhydrous sodium sulfate then the mass was stripped with acetone (200, 100 and 100 mL). The acetone
`solution was placed in a decantation beaker was diluted with petroleum ether (50 mL) and water (75 mL).
`After agitation and settling, the petroleum ether, which had drawn out the entire pigment, was separated,
`washed with water and dried on anhydrous sodium sulfate. The solvent was then flushed by heating in a
`double-boiler (bain-marie) (without exceeding 40°C) and distillation in a reduced-pressure nitrogen
`atmosphere.
`
`Using this process, 5 to 8 grams of oil was obtained per kg of shrimp. The oil extracted between May and
`October was dark-red while that extracted between May and October was orangey with a much lower
`concentration of pigment. The absorption spectrum (Fig. 1) show these qualitative and quantitative
`differences between the summer and winter oils (24).
`
`Aristaeomorpha folicea Oil in
`3%
`
`Thickness: 1cm
`
`30
`
`

`

`2. Preparation of the Hypoderm Extract (1) and the Peristomal Connective Tissue.
`
`The hypoderm lining the internal face of shells, and the connective tissue lining the stomachal pocket, can
`easily be detached by curettage. Acetone-stripping yields a red liqueur which is then processed by water and
`
`petroleum ether. The latter is separated, washed, dried and evaporated as indicated above, The resulting
`extract is much less abundant than that obtained from the hepato-pancreas but is considerably richer in
`pigment.
`
`Pigment Identification:
`
`2 grams of hepato-pancreas oil (July extract) were dissolved in 200 mL of petroleum ether.
`
`The solution was slowly poured and then ingested into an aluminum oxide column of 20 cm in height and 2 cm
`in diameter. The pigment gathered in the upper part of the column. After development by petroleum ether
`
`(200 mL), the following chromatogram was obtained (2) (3):
`
`a) pink zone
`b) red zone
`c) pink zone
`d) purplish-red zone
`e) orangey-yellow zone
`
`(2 cm thick)
`(0.2 cm thick)
`(1 cm thick)
`(0.5 cm thick)
`(0.15 cm thick)
`
`The elution was performed by agitation with colored aluminum oxide with petroleum ether diluted with 1%
`methanol, then the solvent was flushed by distillation in a reduced-pressure, inert atmosphere. The residue
`was then saponified with 15% potassium hydroxide in alcohol for four hours at 20°C. The pigment was then
`separated by the addition of water and petroleum ether: after settling in a refrigerator, it gathered at the
`interphase by dissolution in hot alcohol. It was then cooled and filtered and diluted with a 10% sodium
`carbonate solution then agitated with petroleum ether. The pigment gathered again at the contact surface of
`the two liquids; it was then separated and purified by placing into an alcohol solution followed by salting out
`using sodium carbonate. This sequence of operations was repeated
`
`(1) The term "hypoderm” seems to be dedicated by use (40), (48). In reality, the pigment is located in the
`chromatophores located in the dermus.
`(2) The winter oils provide a very similar chromatogram; only the relative proportions of the various
`constituents seem to vary.
`(3) Aluminum oxide for chromatography according to Brockmann.
`
`ChU'|-bUJl\)
`
`10
`11
`
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`31
`32
`33
`
`31
`
`

`

`three times then the flakes were placed back into suspension in 50% alcohol. After acidification with acetic
`acid, the red precipitate turned blackish-purple. It was then re-processed with petroleum ether and
`chromatographed on aluminum oxide. The pigment rigorously remained in the upper part of the column,
`forming a purple zone with a thickness of only 1.5 to 2 mm. It was developed with petroleum ether then
`eluated with methanol containing 5% potassium. After the addition of water and acetic acid, the pigment was
`extracted using chloroform; evaporation of the solvent under reduced pressure left a deposit of blackish-
`purple crystals inside a red amorphous mass. The enormous mass of lipids, which accompany the pigment in
`hepato-pancreas oil, preventing us from performing any further purification.
`
`Using the extract taken from the hypoderm and connective tissues of stomachal pockets, it was possible to
`obtain this same pigment in a well-crystallized form: after extraction and saponification, the potassium salt
`was washed with some 50% alcohol then petroleum ether, re-stored to suspension in aqueous alcohol and
`acidified with acetic acid: the red flakes transformed into a blackish-purple mass. The pigment was then
`separated and re-crystallized using aqueous pyridine.
`
`The acidic nature of the pigment, its behavior vis-a-vis potassium and petroleum ether, the nature of the
`crystals, their solubility in pyridine (blood-red solution), in concentrated sulfuric acid (dark-blue color), in
`chloroform (orangey-red solution turning to greenish-blue after reacting with antimony trichloride) can be
`used to identify the isolated carotenoid as astacine. This fact was confirmed by the melting point (228°C), the
`wide-band absorption spectrum and the single maximum (A = 500nm in pyridine).
`
`It was likely that the natural pigment was not astacine itself but, as in lobster, astaxanthin in the form of
`esters. In effect, the extract from the hypoderm of shells placed in a pyridine solution provide an absorption
`spectrum that is characteristic of astaxanthin esters with a maximum at 490 nm (25) (Fig ll) (1).
`
`(1) An attempt to isolate the pigment from oil by condensation with the Girard and Sandulesco "T” reagent
`was not successful. This negative result was confirmed at the Lyon Faculty of Sciences Biological Chemistry Lab
`by Mr. Mallein whom we would like to thank.
`The inability of the pigment to condense with the "T” reagent seems to indicate the absence of free ketonic
`functions and the most likely hypothesis that the astaxanthin esters in the Aristaeomorpha oil are esters in the
`enolic form and not esters in the ketonic form.
`
`NJIJOLD00\lChU'|-bu.)l\J|4OLD00\lCDU'|-bUJl\)l4OLD00\lChU'|-PUJIUIJ
`
`UJ(JJ(JJl\)l\Jl\)l\Jl\Jl\Jl\)l\Jl\Jl\Jl4l4l4l4l4l4l4l4l4l4
`
`32
`
`

`

`\lChU'|-bUJl\)l4
`
`10
`11
`12
`
`13
`
`14
`15
`
`With hepato-pancreas oil, the difference in the chromatograms before and after saponification also shows that
`its natural pigment was not astacine but transformed into the latter during the isolation operations.
`Nevertheless, the maximum of the hepato-pancreas oil spectrum is not located at 490 nm as in the case of the
`hypoderm pigment but, rather, at 484 nm. In addition, the peak, which is flatter, is practically flat between 480
`and 490nm indicating the existence of several constituents. The chromatogram features, formed with five
`individual zones, provides a first confirmation of this hypothesis.
`
`
`
`Each of the chromatogram zones was separated and the pigment was eluated by colored aluminum agitation
`with petroleum ether diluted with 1% methanol. After evaporation of the solvent in an inert atmosphere and
`placed into a pyridine solution, the spectrums of each fraction was recorded (7).
`
`(1) This spectrum and that presented in Figure III were recorded at the Lyon Faculty of Sciences Biological
`Chemistry Lab thanks to Mr. Jouanneteau and Mr. Zwingelstein whom I would like to thank.
`
`33
`
`

`

`OO\lCDU'|-bUJl\)|4
`
`10
`11
`12
`13
`14
`15
`16
`
`The spectrophotometric curves are all bell-shaped. Those corresponding to zones a and b have a maximum
`spread between 465 and 468 nm. In zone c, the more pronounced peak is located at 482 nm and, for fraction
`d, its spectrum has a flat peak from 480 to 490 nm which suggests its heterogeneity; it is likely that it was
`formed by the mix of the predominant constituents in fraction c (}\max = 482 nm) and the constituent
`characterized in shell hypoderms (}\max = 490nm). In zone :2, its minimal thickness (0.15 cm), has not facilitated
`an isolation without dragging some zone d along with it; its constitution appears to be intermediate between
`that of c and d.
`
`Hypoderm pigment.
`Carr and Price Reaction
`
`Chromatography, therefore, can be used to dissociate the pigment from the hepato-pancreas oil (1) in at least
`three different constituents
`
`(1) Chromatography on aluminum oxide of the hypoderm extract in petroleum ether solution produces a
`chromatogram where the identified zones are the same as for the hepato-pancreas oil but in which zone d
`dominates.
`
`34
`
`

`

`characterized by distinct absorption maxima located around 470, 482 and 490 nm. Nevertheless, the similar
`shape of the various curves and the fact that all of these constituents transform into astacine through auto-
`oxidation, show that it can only be very similar substances. It should be asked whether the absorption of the
`various lipid substances at the various levels in the aluminum oxide column is the cause for this apparent
`heterogeneity in the chromatogram and whether these lipid substances, eluted at the same time as the
`pigment of each of the zones, are responsible for a spectrum shift. It does not, however, seem necessary to
`accept this interpretation; in fact, the addition of 10% castor oil to each of the eluates only produces a general
`reduction in the optical density; it does not produce any appreciable shift in the maxima.
`
`The presence of a mix of several esters of the same pigment cannot explain the large shifts between the peaks
`of the different curves, either.
`
`The possibility ofthe existence of several pigments, chemically different but with a constitution that is similar
`enough to explain their common oxidation into astacine must also be examined. This oxidability no doubt
`implies the presence of the
`_CH_co_
`groups in the molecule, which might indicate the presence
`IOH
`
`of position isomers alongside the astaxanthin, the composite having, among other things, the structure of 3,3‘-
`dihydroxy-B-carotene-4,4‘-dione.
`
`Although it is not yet possible to definitively discard this interpretation, recent progress on the structure of
`carotenoids suggests a more appealing hypothesis that several astaxanthin stereoisomers or esters are present
`in the hepato-pancreas oil. The possibility of a single pigment in several stereoisomer forms (cis-trans isomers)
`is an indisputable fact established for several carotenoids and it is known, today, that, in the provitamin A
`series, the steric configuration is not without quantitative influence on the biological activity (113), (114).
`
`The following table provides a list of the carotenoids for which the existence of several stereoisomers have
`been recognized and studied:
`
`\lChU'|-bUJl\)l4(DLD00\lChU'|-hUJl\)|4OLD00\lChU'|-PUJIUIJ
`
`l\)l\)l\Jl\Jl\)l\Jl\Jl\Jl4l4l4l4l4l4l4l4l4l4
`
`28
`ix) LD
`
`35
`
`

`

`Table II
`
`kDO0\lChU'|-l>UJl\)l4
`
`Bixin [Herzig and Faltis (33), Karrer and colleagues (37)].
`Crocetin [Kuhn and Winterstein (50)].
`[3-carotene [Gillam and El Ridi (19), (20), Zechmeister and colleagues (115)].
`oL-carotene [Gillam and El Ridi (21), Zechmeister and colleagues (115), Zscheile and colleagues (115)].
`Capsanthin [Zechmeister and colleagues (115)].
`Lycopene [Zechmeister and colleagues (115)].
`Cryptoxanthin [Zechmeister and colleagues (115)].
`Xanthophyll [Strain (95), Zechmeister and colleagues (115)].
`Zeaxanthin [Zechmeister and colleagues (115)].
`Physalien [Zechmeister and colleagues (115)].
`Taraxanthin [Zechmeister and Tuzson (115)].
`Capsorubin [Zechmeister and Cholnoky (115), Polgar and Zechmeister (78)].
`oL-carotene [Hunter and Scott (34), Zechmeister and colleagues (115)].
`Pro-y carotene [Zechmeister and colleagues (115)].
`Celaxanthin [Le Rosen and Zechmeister (55)].
`Fucoxanthin [Strain and Manning (96)].
`Prolycopene [Zechmeister and colleagues (115)].
`Gazaniaxanthin [Zechmeister and Schroeder (115)].
`Spirilloxanthin [Zechmeister and colleagues (115)].
`
`Until now, the existence of stereoisomer forms has not been identified in the case of astaxanthin. To be able
`to definitively confirm that Aristaeomorpha oil pigment contains a new type of stereoisomer in the carotenoid
`series, conversion from one form to another must be demonstrated. Nevertheless, the various methods used
`for other carotenoids (iodine, light, boiling and reflux of the pigment in an organic solvent solution) risks
`profound alterations in the astaxanthin due to its extreme fragility. It is not at all certain that, in our follow-up
`research, we will succeed in creating this conversion.
`
`In the absence of this decisive data, the existence of stereoisomer forms of astaxanthin in Aristaeomorpha
`foliacea remains hypothetical.
`
`36
`
`

`

`In summary, the essential carotenoid pigment of Aristaeomorphafoliacea is astaxanthin. We have identified it,
`in fact, in the hypoderm of shells and the peristomal connective tissues. It is also present in the hepato-
`pancreas oil accompanied with similar forms which are likely stereoisomers of the completely transformed
`configuration.
`
`It was interesting to find, in the Aristaeomorpha, alongside astaxanthin, the possible presence of vitamin A and
`carotenes. In crustaceans, in fact, that while vitamin A is not generally detectable, carotenes are, however,
`present but in trace amounts: Lederer (54) could not characterize vitamin A in Calanusfinmarchicus, small
`copepods in the North Sea, and only found 0.4 mg of carotenes in 500 grams of animal.
`
`With Chechan, Miss Massonet and Odier (26), Chechan and Miss Massonet (25), we have, in Aristaeomorpha,
`looked for these factors in the hepato-pa