CYAN EXHIBIT 1016
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`TITLE: Antixerophthalmic Activity of the Carotenoid Pigment of the Aristeomorpha foliacea
`(Penzeidze)
`Authors: GRANGAUD, Rene.; MASSONET, Renee.
`Journal: Comptes Rendus de l'Academie des Sciences, March 27, 1950 Vol. 230 pp. 1319-1321
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`CHEMICAL BIOLOGY — Antixerophthalmic Activity of the Carotenoid Pigment of the
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`Aristeomorpha foliacea (Penaeidae). Note by Mr. Rene Grangaud and Ms. Renee Massonet,
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`presented by Mr. Maurice Javillier.
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`Astaxanthin, present in the oil of Aristeomorpha foliacea, behaves like vitamin A whose
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`antixerophthalmic activity is much more marked than the action on growth.
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`We have shown (1) that the antixerophthalmic activity of oil from the shrimp, Aristeomorphafoliacea,
`is much more acute than the action on the deficient white rat’s growth can predict. The dissociation
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`between these two characteristics, whose coexistence is considered to be specific to vitamin A
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`activity, led us to admit that “It is, therefore, likely that, in this oil, there is a constituent, other than
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`the carotenes and vitamin A, which play a role in the antixerophthalmic activity.”(2). The study of
`summer and winter oils also show that the first, deep red, are active, the second, light red with more
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`yellow, were practically devoid of activity (3). These results suggest that the antixerophthalmic
`principle could be identified in the red carotenoid pigment of the summer oil. We isolated this
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`pigment in the astacine state (B-carotene-3,3’,4,4’-tetrone)(4). Nevertheless, the differences between
`the chromatograms obtained before and after saponification and the fact that the addition of potassium
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`to the oil solution in ethanol caused the appearance of a violet tint which turned to orange, showed
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`that the pre-existing pigment is probably not astacine but its precursor, astaxanthin (3,3'-dihydroxy-IS-
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`carotene-4,4'-dione)(5) which exists in many crustaceans in the form of esters or in association with
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`proteins(6).
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`In a first test, the oil was saponified by 15% alcoholic potassium for four hours at 20°C. With the
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`addition of water and petrol ether and storage in the refrigerator, the major portion of pigment
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`gathered at the surface of separation of the two liquids in the form of red flakes. After separation and
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`washing with 50% alcohol, the latter was mixed with a de-vitaminized vegetable oil equal to the
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`volume of saponified oil. The preparation was administered to vitamin A deficient rats: even at a dose
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`of 90 mg per
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`(1) Comptes rendus, 227, 1948, p. 568.
`(2) 5yn. : Penoeusfoliaeeus, Risso.
`(3) R. GRANGAUD, C. CHECHAN et Mlle R. MASSONET, C. R. Soc. Biol., 143, 1949, p. 1179.
`(4) R. GRANGAUD, C. CHECHAN et Mlle R. MASSONET, C. R. Soc. Biol. (Alger, seance du
`16 mars 1950).
`(5) P. KARRER et E. TUCKER, Carotino,','a’e, Verl. Birehauser (Basel, 1948), p. 244.
`(G) G. WALD, Vitamins and Hormones (Academie Press, New-York, 1, 1943 p. 213).
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`animal per day, with the exception of a slight, transitory improvement in the ocular infection in the
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`two subjects, there was no detectable action on the development of the signs of deficiency. This result
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`is in agreement with the traditional data: astacine does not present any vitamin A activity.
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`Under the assumption that natural carotenoid might behave differently, a new test was conducted by
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`administering separated pigment to the deficient animal not by oil saponification but by aluminum
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`oxide chromatography. 2 g of oil were dissolved in 200 ml of petrol ether. The solution was slowly
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`poured and ingested into an aluminum oxide column of 20 cm in height and 2 cm in diameter. The
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`pigment was kept in the upper part of the column. After development using petrol ether (200 ml),
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`elution was performed by agitation of the colored aluminum oxide with petrol ether diluted with 1%
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`methanol. The eluate was separated, then diluted with 1 ml of de-vitaminized oil containing 2 mg of
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`a-tocopherol then the solvent was flushed in an inert atmosphere under reduced pressure. The residual
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`oil (7) was administered under the same conditions as previous experiments (1) to vitamin A deficient
`white rats. At a daily dose of 40 mg, healing from xerophthalmic lesions was obtained in less than ten
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`days; the improvement was undisputable on the third day. The action on the weight gain was, at that
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`dose, practically null and the animals died within the three to four weeks after start of treatment.
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`Although chromatography was not able to obtain the pigment in its pure state, it is unlikely that the
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`antixerophthalmic factor was distinct: in fact, it must be admitted that it involved a colorless
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`substance present in the summer oil at the same time as the pigment, absent in that form in the winter
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`oil, absorbed in the aluminum oxide at the same level and eluated by the same solvent. On the other
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`hand, the disappearance of the activity after saponification indicates the nature of the active principle
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`by confirming that, in the Aristeomorpha oil, the pigment is not astacine but astaxanthin in the form of
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`esters (8). Our results are to be compared with those of Wald and Zussman (9) who observed the
`presence of these carotenoids in the retinas of certain birds, thus providing indication of its role in the
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`visual process (10).
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`Consideration of this data leads to the conclusion of astaxanthin as
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`(7) Pigment concentration is substantially equal to that of the initial oil.
`(8) The administration to deficient rats of Aristeomorpha eggs (where astaxanthin is protein-bound)
`crushed and suspended in devitaminized vegetable oil is followed by no attenuation of xerophthalmic
`lesions: the ease with which non-esterified astaxanthin is oxidized to astacin probably provides the
`explanation for this failure.
`(9).]. biol. Chem, 122, 1938, p. 449.
`(10) R. A. MORTON, The Application ofAbsorption Spectra to the Study of Vitamins, Hormones and
`Coenzymes, London, 1942.
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`a new Group A Vitamin, being distinguished from other factors in this group by the predominance of
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`the antixerophthalmic effect on the growth rate.
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