CYAN EXHIBIT 1014
`
`TITLE: Antixerophthalmic effect of the esters of astxanthin
`Authors: GRANGAUD, R.; MASSONET, R.
`
`Journal: Comptes rendus des seances de la Societe de biologie 1954 Vol. 148 pp. 1392-
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`While performing research on carotenoid pigments of penaeid shrimp (1), we specified the
`chromatographic behavior of astaxanthin and its esters: when filtered in an aluminum oxide column
`(2) of 20 cm height and 2 cm diameter, an etheropetrolic solution of a hepatopancreas oil or of a
`hypodermic extract ofAr1'ste0mp0rphaf0liacea which contains free and esterified astaxanthin in
`variable proportions (3), the pigment is adsorbed in the upper portion of the column. After petrol ether
`processing, a chromatogram, having five zones from top to bottom, is obtained: the pigment in the
`upper zone is pink with 1 cm of thickness and is composed of free astaxanthin as its hypophasic
`nature confirms:
`the pigment in the remaining four zones below it is a mix of epiphasic esters. In the
`extracts which were studied, we were never able to detect vitamin A nor carotenes (4). We have,
`however, recently verified that the chromatographic behavior of these factors is completely different
`from that of
`
`(1) R. Grangaud, “Astaxanthin Research, New Vitamin A Factor”. (Biochemical News, 15th Series,
`Editions Desoer, Liege, 1951).
`(2) Alumina for chromatography “Prolabo”.
`(3) In summer, the esterified form is more abundant than the free form. In winter, the proportion is
`inverse, the total concentration in the pigment is noticeably decreased.
`(4) R.Grangaud, C.Chéchan, R.Massonet and M.Odier. Bull. Soc. Chim. biol., 1950, 1, 32, p. 245.
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`astaxanthin and its esters (5): Free vitamin A is located on the aluminum oxide column below the
`astaxanthin esters, the vitamin A esters affix to the lower portion of the column, the carotenes slowly
`traverse the aluminum oxide and become part of the residue. These results, which are in agreement
`with Lederer (6) and those of K011 and colleagues (7), show that aluminum oxide chromatography
`alone separates the free and esterified astaxanthin from the free vitamin A, its esters and its carotenes
`if, despite our chemical and spectrophotometric controls, it is admitted that (8) the latter may be
`present in the extracts which we studied. On the other hand, since free vitamin A is hypophasic, as is
`free astaxanthin, by agitating their ethero-petrolic solution with ethanol at 85°, both vitamin and
`astaxanthin pass into the alcoholic phase.
`
`We used all of these properties to prepare an oily solution of astaxanthin esters which we
`administered per as of vitamin A deficient rats. The preparation technique described below is such
`that, in all objectivity, it would be hard to invoke the possible intervention of free or esterified vitamin
`A and its carotenes to explain the antixerophthalmic activity of the studied extracts.
`
`The l g of hypodermic ethero-petrolic solution (200 cm3) of the pigment obtained while monitoring
`the previously-described operational method (1) was agitated with an equal volume of ethanol at 85°.
`This treatment, repeated five times, separates the free astaxanthin (at the same time as it eliminates the
`free vitamin A if the latter is present). The separated epiphasic solution was washed with water to
`eliminate the alcohol which was dissolved in the petrol ether. It is dried on anhydrous sodium sulfate
`and then chromatographed on an aluminum oxide column of 20 cm in height and 2 cm in diameter.
`The chromatogram is developed with 200 cm3 of petrol ether and, after having separated the
`pigmented zone corresponding to the astaxanthin esters, it is then eluted by agitation with petrol ether
`diluted with 5 parts per 100 methanol. The eluate was washed with water, dried on anhydrous sodium
`sulfate then diluted with l g of devitaminized vegetable oil containing 27 mg of or-tocopherol as an
`anti-oxidant. While flushing the solvent with reduced-pressure distillation in an inert atmosphere, an
`oily solution of astaxanthin esters that can contain neither vitamin A (free or esterified) nor carotenes
`was obtained. It is this oily extract, which contains approximately 250 micrograms of astaxanthin per
`gram of oil, which, at the dose of 80 mg of such oil per animal per day, was administered to vitamin A
`deficient white rats.
`
`Eleven Wistar rats, which we raised, were deficient when they were weaned. The signs of deficiency
`are manifested within the usual period of time (between 40 and 45 days of deficiency) by weight-
`stabilization
`
`(5) R.Grangaud, R.Massonet and A.Sansae. C.R.Soc.Biol. 1954. v. 158, p. 533.
`(6) E.Lederer, Tchen Pau Kiun, H.Penau and G.Hagemann. Bull. Soc. Chim. biol. (works), 1944, v.
`26, p. 1032.
`(7) S.K.Don et al., in S.K.Kon, Bull. Soc. Chim. biol., 1954, v. 36, p. 239.
`(8) T.W.Goodwin, The Comparative Biochemistry of The Carotenoids, Chapman and Hall, ed.
`London. 1952, p. 173.
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`soon followed by the appearance of xerophthalmia. The weight curve being on a plateau for 15 days
`and the xerophthalmia being clearly exhibited, the animals were distributed into 2 lots: seven of them
`(2 males and 5 females), received per day in addition to the deficient regime, 80 mg of oily solution of
`astaxanthin esters; the other four served as controls (3 males, 1 female), received the deficient regime
`and 80 mg of devitaminized vegetable oil diluted with 28 mg of ct-tocopherol per gram.
`
`Among the control animals, the development of the deficiency was manifested by the growing
`intensity of xerophthalmia lesions, weight loss, and death between the 68th and 87th day of deficiency.
`At the same time, among the treated animals, the ocular lesions were regressing, the cornea were
`becoming perfectly healthy, healing was achieved between the 12”‘ and 15”‘ day of treatment. In
`parallel, among two of them, the weight curve was stable; in a third, which had an enormous abscess
`in the neck, a weight loss of 8 g was measured on the 23th day. Finally, among the other four, there
`was a slight gain in weight.
`
`In summary, in the vitamin A deficient white rat, with a stable weight curve and a fully-developing
`xerophthalmia, the administration of an oily solution of astaxanthin esters led, within a few days, to
`the complete healing of the ocular lesions, the weight gain was minimal or non-existent. These results
`are in agreement with those which we published previously (9). The preparation method for the
`administered extract does not allow invocation of the participation of the vitamin A or carotenes to
`explain the observed vitaminic activity.
`
`(9) R.Grangaud and R.Massonet. C.R.Acad.Sc. 1950. v. 230, p. 1319.
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