l~\
`CYAN EXHIBIT 1020 JAE
`
`l~3
`
`,. .—~.\
`Physxol. Chem. Phys. & Med. NMR. (1990(2217—38
`’
`‘ \\,
`
`Inhibition of OXIdatlve Injury of
`Biologlcal Membranes by Astaxanthm
`
`Michi Kurashige, Eiji Okimasu, Masayasu Inoue] and Kozo Utsumi
`
`Department ofMedIcaI Biology, Kociu' Medical School, Nankoku-Shi. Kochl 718-51 and
`[Department of Biochemistry, Kumamoio University Medical School, Kumamoto 860, Japan
`Correspondence address-[(020 Utsumi, Department of Medical Biology, Kochi' Medical School,
`Nanko/Cu-Shf. Kochi 781-51,.lapan
`
`Abstract: The value of astaxanthin, a carotenoid pigment, in the treatment ofoxidative injury is as-
`sessed. Astaxanthin protects the mitochondria of vitamin E-deficient rats from damage by Fe2+-
`catalyzed lipid peroxidation both in vivo and in vitro. The inhibitory effect of astaxanthin on
`mitochondrial lipid peroxidation is stronger than that of a-tocopherol. Thin layer chromatographic
`analysis shows that the change in phospholipid components of erythrocytes from vitamin E-deficient
`rats induced by Fe2+ and Fe +-xanthine/xanthine oxidase system was significantly suppressed by as—
`taxanthin. Carrageenan-induced inflammation of the paw is also significantly inhibited by administra-
`tion of astaxanthin. These data indicate that astaxanthin functions as a potent antioxidant both in vivo
`and in vitro.
`
`REACTIVE OXYGENS are produced by various enzymatic and nonenzymatic processes
`in living organisms. Under pathological conditions they may induce peroxidation of
`polyunsaturated fatty acids in biological membranes leading to functional impairment (1,2).
`Such oxygen toxicity has been postulated to underlie the pathogenesis of several diseases
`such as postischemic reflow injury (3, 4), retinOpathy in premature infants (5), shock (6) and
`cerebral infarction (7). For example, paraquat (8) and adriamycin (9) also catalyze the for-
`mation of reactive oxygens and result in pulmonary fibrosis and cardiomyopathy, respec-
`tively. Furthermore, both activated neutrophils and macrophages produce active oxygen
`and significant fractions of these metabolites are released extracellularly, and induce
`
`various inflammatory reSponses (10, l 1).
`Superoxide has been assumed to be a primary source for other reactive oxygens, such as
`' H202 and ‘OH (1). Intracellular compartments are highly enriched with anti—oxidants such
`as superoxide dismutase, catalase, glutathion peroxidase, and glutathion. Thus, reactive
`oxygens are efficiently detoxicated (12). However, in extracellular space (l3, 14) such as
`
`'DOKHT
`
`vs. Aria
`
`5—223.
`3—26.
`
`31) /
`
`3, 4,6
`
`,New
`
`l
`
`.
`”m“
`
`1939‘
`l989
`
`
`
`

`

`28
`
`KURASHlGE et al.
`
`plasma, concentrations of these enzymes and scavengers are low. High levels of extracel-
`lularly released superoxide cannot therefore be dismutated efficiently enough to protect cell
`membranes from oxygen toxicity.
`Astaxanthin (AX) is a carotenoid pigment (Fig. 1) found in many animals and plants (15),
`and the compound can be purified in large quantity from yeast, Phafi‘ia rhodozyma Miller
`(16). This compound may serve as an efficient and safe antioxidant. However. relatively lit-
`tle attention has been brought to the action of this compound. During the course of our
`studies on the protective action of various drugs against the impediment of biological
`membrane by reactive oxygens, we have found by using vitamin E deficient rats that as-
`taxanthin protects biological membranes from hazardous oxygen species in vivo and in
`vitro.
`
`MW 596.82
`
`Astaxanthin (AX)
`structure of Astaxanthin.
`FIGURE 1. Chemical
`dihydroxy b,b-carotene~4,4’-dione), a carotenoid and a precursor of astacin.
`
`(3.3’-
`
`Materials and Methods
`
`Chemicals: Adenosine diphosphate (ADP), carrageenan (type IV), thiobarbituric acid
`(TBA), vitamin E (VE) and xanthine were purchased from Sigma Chemicals (St. Louis).
`Xanthine oxidase was obtained from Boehringer Mannheim Co. (West Germany). As—
`taxanthin was kindly donated by Suntory Co. (Osaka). Vitamin E deficient diet was ob-
`tained from Oriental Yeast Co. (Tokyo). Other chemicals were obtained from nacalai
`
`tesque Co. (Kyoto). For in vitro experiments, AX was dissolved in dimethyl sulfoxide. .
`Rats: Vitamin E deficient rats were prepared according to the method of Machilin et a1.
`(17). Three groups (10 rats for each group) of male Wistar rats (6 to 7 weeks of age) were
`fed for 2—4 months. Each group had a different diet, normal (group 1), vitamin E-free (group
`2) and vitamin E-free plus astaxanthin (containing 1 mg astaxanthin/lOO g) (group 3).
`Mitochondria and erythrocyte ghosts: Liver mitochondria were isolated from each
`group of animals according to a modification of Hogeboom and Schneider (18), and
`erythrocyte ghosts were prepared by the method of Dodge et al. (19).
`M itoclzondrialfunctions: Oxidative phosphorylation and respiratory control index (the
`ratio of phosphorylative accelerated respiration expressed by state 3 to the substrate level
`respiration expressed by state 4) were measured by 3 Clarktype oxygen electrode (20).
`Mitochondria were incubatedin a medium containing 0.15 M KCl 10 mM Tn's-l'lCl2 buff-
`er (pH 74) to eliminate the inhibitory effect of sucrose on lipid peroxidation by Fe2+and
`thiobarbituric acid reaction (21, 22).
`Lipid peroxidation and lipid analysis. Erythrocyte ghosts were prepared according to
`the method of Blight and Dyer (22). Fe2-induced lipid peroxidation of the ghosts was 3125-
`
`sayed by thiobarbituric acid reaction (21). Lipid peroxidationin vitro was induced by Fe2+
`
`

`

`INHIBITION OF OXIDATIVE INJURY OF BIOLOGICAL MEMBRANES BY ASTAXANIHIN
`
`29
`
`and xanthine/xanthine oxidase system. Membrane phospholipids were analyzed by thin
`layer chromatography.
`
`Protein content: Membrane protein was determined according to Lowry‘s method
`using bovine serum albnumin as a standard (23).
`
`RL Ml 2mg mot/ml
`
`lOOpM
`Fe“
`
`1\i
`Succ
`
`.. 150
`z
`Lu
`
`RC]
`
`L 18
`
`50
`
`Oil?
`OXYGENIpM)
`
`O>
`
`—
`
`X 0
`
`FIGURE 2. Protection by astaxanthin against Fe2+-induced dysfunction of rat liver mitochondria.
`Liver mitochondria were isolated from normal rats and animals fed a vitamin E-deficient diet for
`two months. The isolated samples were washed twice with a medium containing 0.15 M KCl and 10
`mM Tris-HCl buffer (pH 7.4). Respiratory activity of mitochondria was assayed with 21 Clark type
`oxygen electrode in a medium containing 0.15 M KC1, 3 mM MgC12 and 5 mM potassium phos-
`phate buffer (pH 7.4) at 25°C. Respiratory control index of untreated rat liver mitochondria was 4.18
`(trace 1
`in a), and that of vitamin E—deficient rat liver mitochondria was 4.0 under succinate as
`respiratory substrate (trace 1 in b . Dimethyl sulfoxide. a carrier for astaxanthin was added after ad-
`ding 4.2 uM astaxanthin, and Fe 4’-induced changes were observed (trace 3 in b). RL Mt, rat liver
`mitochondna: RCI, respiratory control
`index; DMSO. 5 LtI/ml dimethyl sulfoxide' Succ,
`1 mM
`sodium succinate: DNP. 25 uM dinitrophenol; ADP, 150 LLM ADP; Fe“, 100 pM Fe +.
`
`.SHIGE c! 0/.
`
`of extracel-
`)rotect cell
`
`tlants( 15).
`7770 Miller
`
`atively lit-
`rse of our
`
`biological
`[S that as-
`1-0 and in
`
`iric acid
`
`Louis).
`1y). As-
`was ob-
`nacalai
`ide.
`
`lin eta].
`
`e) were
`
`:(group
`3).
`4
`m . b
`8). and
`
`lex (the
`te level
`
`Ie (20).
`'1 buff-
`2+
`:
`and
`
`ding to
`was as-
`2+
`
`iy Fe
`
`

`

`30
`
`KURASHIGE e! a].
`
`Carrageenan-[nduced inflammation: Saline solutions (1 ml) containing either vitamin
`E or astaxanthin were injected intraperitoneally 30 min before carrageenan treatment. Ef—
`
`fect of astaxanthin on carrageenan-induced edema was examined by measuring the volume
`changes of rat paws after subcutaneous injection of carrageenan (24, 25). Anesthetized rats
`were given subcutaneous injections of 0.2 ml saline solution into the left paw and the same
`volume of saline solution containing 10 mg carrageenan into the right paw. Change in paw
`volume was measured hourly three times after the treatment.
`
`Results and Discussion
`
`Effect of astaxanthin on Fe2-induced changesIn mitochondrial functions of vitamin
`E deficient rats. Figure 2a shows the effect of Fe2+ on the respiratory activity of
`mitochondria from intact rats. Addition of 10-100 ttM Fe2+ to the reaction medium en-
`hanced oxygen consumption of mitochondria and slightly decreased respiratory control
`index (RC1; state 3/ state 4). The mitochondria from vitamin E deficient rats (group 2)
`revealed RC1 of 4.0 and ADP/O ratio 2.0; these values are within normal levels with suc-
`cinate as a respiratory substrate (trace 1 in Fig. 2b). However, when Fe2+ was added to the
`medium the rate of oxygen consumption increased and respiratory control index (RC1)
`decreased markedly (trace 2 in Fig 2b) When astaxanthin was added prior to the treat-
`ment the Fe2+-ir1duced increase in oxygen consumption and the decreasein respiratory
`Table I
`
`Effect of Fe2+ and astaxanthin on the oxidative phosphorylation of
`mitochondria from vitamin E deficient rats.
`
`
`
`ADP/O
`(%
`A
`Fe“
`(1.1M)
`(nM)
`RC1
`Decrease)
`Ratio
`(‘70)
`
`
`0
`
`10
`
`40
`
`O
`
`0
`
`O
`
`4.00
`
`3.37
`
`2.92
`
`(
`
`0)
`
`( 21)
`
`( 36)
`
`2.00
`
`2.04
`
`2.02
`
`(100)
`
`(102)
`
`(101)
`
`100
`O
`2.44
`( 52)
`1.98
`( 99)
`
`
`100
`
`100
`
`45
`
`420
`
`2.64
`
`3.21
`
`( 45)
`
`( 26)
`
`2.04
`
`1.85
`
`(102)
`
`( 93)
`
`( 89)
`1.77
`( 22)
`3.35
`4200
`100
`
`
`Experimental conditions were the same as in Figure 2. Activities of
`mitochondrial oxidative-phosphorylation were expressed in two
`characteristics, respiratory control index (RC1) and ADP/O ratio. Numbers in
`parentheses represent % ofthe control values obtained from mitochondria
`which were not treated with Fe2 and astaxanthin
`
`
`
`

`

`\SHIGE era].
`
`1er vitamin
`
`1tment. Ef-
`
`the volume
`retized rats
`d the same
`
`1ge in paw
`
`Ifvitamin
`
`Ctivity of
`:dium en-
`
`y control
`
`(group 2)
`with suc-
`
`ghe
`led
`ex (KCI)
`the treat—
`
`spiratory
`
`.
`
`O
`
`.
`
`.
`
`O
`
`O
`
`.
`
`n t
`
`O
`
`.
`
`INHIBITION OF OXIDATIVE INJURY OF BIOLOGICAL MEMBRANES BY ASTAXANTHIN
`
`3l
`
`IO mM IOOLJM
`5 220 NdN3
`Fe"
`
`'LSZpMdxnnthin
`
`I
`
`0;CONSUMPTION(I1
`
`RL Mi 2 mg protrl ml
`
`
`180
`
`Control
`
`O
`
`A
`2
`TIME IN MINUTES
`
`6
`
`.
`7+
`.
`.
`.
`.
`.
`FIGURE 3. Enhancement of NaN3-insensitive oxygen consump-
`tion by Fe'
`and its suppress1on by astaxanthm. Mitochondria
`were isolated from normal
`rat
`liver (group 1). Astaxanthin (4.2
`uM) was added prior to each experiment. Other conditions were
`the same as in Figure 2.
`
`control index were inhibited significantly (Fig. 2b, trace 3). Both the hazardous effect of
`Fe2+ and the protective effect of astaxanthin depended on their concentrations (Table I).
`Figure 3 represents the enhancement of the rate of NaN3--independent oxygen consump-
`tion by Fe2+Since NaN3 inhibits electron transport of mitochondria, the Fe W—induced1n-
`crease in oxygen consumption would have occurred independently from the mitochondrial
`electron transport system.
`Since mitochondrial membranes are enriched 2with polyunsaturated fatty acids, the1n-
`creased oxygen consumption might reflect the Fe2+-induced lipid peroxidation Consistent
`with this notion is the fact that the Fe2-induced1ncrease in oxygen consumption was in-
`hibited by astaxanthin that has a polyunsaturated carbon chain.
`Mitochondrial function of the astaxanthin-administered vitamin E deficient rats. To
`
`test whether astaxanthin also protects mitochondrial function in vivo, the mitochondrial
`Fe-induced lipid peroxidation was investigated in vitamin E deficient2 rats with or
`without astaxanthin administration Figure 4 shows the effect of 100 ttM Fe2+ on the RC1
`values of mitochondria isolated from different groups of rats
`The respiratory control index of mitochondria from group 1 rats was slightly inhibited by
`Fe2-induced lipid peroxidation; no remarkable disorders of m1tochondnal function, such as
`oxygen consumption were induced Thus the defense mechanism against free radicals seems
`to function1n control group (group I). On the other hand mitochondria from vitamin 2E defi—
`cient rats (group 2) showed a remarkable decrease1n respiratory control index by Fe2+The
`deteriorationin respiratory control index was inhibited significantly by treating animals with
`
`

`

`32
`
`KURASHIGE er al.
`
`70
`
`60
`
`SO
`
`40
`
`30
`
`INHIBITION
`
`°/o 20
`
`\
`
`ooQ
`6
`
`0Q
`
`<9
`
`$2
`
`FIGURE 4. Effect of Fe2+ on the respiratory control index of rat
`liver mitochondria fed under different conditions. Animals were
`
`divided into three groups and fed different diets; group I fed nor—
`mally, group 2 fed vitamin E-deficient (Oriental Yeast Co.) and
`molecular distilled corn oil, and group 3 fed vitamin E-deficient
`plus lmg/IDOg astaxanthin. Rat liver mitochondria were prepared
`from each group of animals. Other conditions2were the same as in
`Figure 2. Data show the effect of 100 pM Fe2 on the respiratory
`control index of mitochondria from 3 groups. Each value is a mean
`+ SD of five experiments.
`
`-...:¢
`
`aStaxanthin (group 3). ADP/O ratio another indicator for mitochondrial function, was not af-
`fected remarkably by Fe2~induced lipid peroxidationin any animal groups.
`
`Lipid peroxidation by active oxygens of erythrocyte membranes from vitamin E
`deficient rats and its inhibition by astaxanthin administration. To confirm that the
`decrease in mitochondrial function of vitamin E deficient rats by Fe2might reflect the
`astaxanthin- inhibitable lipid peroxidation changes in TBA-reactive metabolites were
`determined in erythrocyte ghosts from three 3animal groups. Figure 5 shows the formation
`of thiobarbituric acid (TBA)-reactants by Fe3+and superoxide generated by xanthine/xan-
`
`”l
`
`
`
`

`

`\SHIGE er a!
`
`INHIBITION OF OXIDATIVE INJURY OF BIOLOGICAL MEMBRANES BY ASTAXANTHIN
`
`33
`
`
`
`._,.'
`
`-not af~
`
`min E
`hat the
`
`ect the
`s were
`
`mation
`
`1e/xan—
`
`
`
`TBA-REACTIVEMETABOLITESInmole/mgprot.)
`
`
`
`
`
`OJ
`
`I\)
`
`._a
`
`C)
`
`x
`
`"L
`
`rs
`
`09
`(3&0
`
`0Q
`(3&0
`
`0Q
`(5‘0
`
`FIGURE 5. Lipid peroxidation of erythrocyte membranes from
`animals fed under different conditions. Erythrocyte ghosts were
`prepared from 3 animal groups following the method of Dodge e:
`al.(19). Ghosts were suspended in a medium containing 20 mM
`Tn's~I-ICI buffer (pl-I 7.5) 1 mg protein/ml. Lipid peroxidation of
`the membranes was induced by 50 uM FeCI3 and the xanthine
`(200 uM)/xanthine oxidase (0.01 IU/ml) system for the production
`of ‘OH. After incubation at 37°C, thiobarbituric acid (TBA)-reac-
`tive metabolites were determined by spectrophotometer. Each
`value is a mean + SD of five experiments.
`
`thine oxidase system. Control group represented a low level of thiobarbituric acid—reac—
`tants even after incubation for 210 min under oxidative stress. On the other hand, the
`
`amount of thiobarbituric acid—reactants increased markedly in group 2. In erythrocyte
`membranes from astaxanthin-administrated group the formation of thiobarbituric acid-
`reactants was inhibited significantly. Thus the administration of astaxanthin in viva might
`inhibit the lipid peroxidation of erythrocyte membranes from VEDR under oxidative
`stress in i'irro.
`
`

`

`34
`
`KURASHIGE er al.
`
`Effect of astaxanthin and vitamin E on the Fe2+-induced lipid peroxidation of
`mitochondria obtained from vitamin E deficient rats. As described above, the ad-
`
`ministrated astaxanthin protected the mitochondrial function of vitamin E deficient rats
`from being impaired by reactive oxygens. Since vitamin E has been used as a potent an-
`tioxidant, the effect of astaxanthin on lipid peroxidation of mitochondrial membrane was
`compared with that of vitamin E. Figure 6 represents the inhibitory action of astaxanthin
`and vitamin E on the formation of thiobarbituric acid-reactants. This inhibitory effect of
`astaxanthin being 100 to 500 times stronger than that of vitamin E.
`To get further insight into the mechanism by which astaxanthin inhibited the Fe2+
`catalyzed lipid peroxidation, the change in lipid components of erythrocyte membrane was
`determined in three groups. Figure 7 shows thin layer chromatographic profiles of
`erythrocyte membrane phospholipids. After incubation of erythrocyte membranes with
`Fe3+ and xanthine/xanthine oxidase system for 210 min, the formation of lipid peroxides
`markedly increased in the vitamin E—deficient group (group 2) but not in control group when
`detected when smears between each spot for the identified lipids in thin layer chromatog—
`raphy, suggesting the promotion of lipid peroxidation. Under identical conditions, addition
`
`
`
`O
`
`/
`
`80
`
`Z O E
`
`so-
`
`99.
`E 40
`
`°
`
`20-
`
`O
`
`/
`o oooors
`
`L3
`
`1300
`
`CONCENTRAHON(pM)
`
`FIGURE 6. Effect of astaxanthin and vitamin E on Fe2+—induced lipid
`peroxidation.
`Intact mitochondria (2 mg protein/ml) prepared under the
`same conditions as in Figure 2, and washed three times with 0.15 M KCl
`and 10 mM Tris-HCl buffer solution (pH 7.4) at 0°C. The washed
`mitochondria were preincubated for one min with various concentrations
`of astaxanthin or vitamin E at 37°C. After incubation with 100 uM Fe2+
`(Mohr’s salt) at 37°C for one hour, level of thiobarbituric acid—reactants
`was measured. Data were expressed as % inhibition of thiobarbituric
`acid-reactant formation.
`
`AX, astaxanthin; V.E, vitamin E.
`
`v.41
`
`
`
`

`

`.
`
`O
`
`iSHlGE eral
`
`idafion of
`
`the ad-
`'e.
`icient rats
`potent an-
`brane was
`staxanthin
`' effect of
`
`the Fe2+
`Jrane was
`
`rofiles of
`mes with
`
`.
`
`aeroxides
`
`Jup when
`romatog-
`,addition
`
`_)
`
`-
`
`.
`
`I
`
`a
`
`'
`
`l
`
`O
`
`.
`
`INHIBITION OF OXIDATlVE INJURY OF BIOLOGICAL MEMBRANES BY ASTAXANTHIN
`
`35
`
`of 4.2 LLM astaxanthin or 420 uM vitamin E to the incubation mixtures inhibited the
`peroxidative changes considerably; the thin layer chromatographic profiles of the astaxan—
`thin- and vitamin E-treated groups were almost the same as those for the control membrane.
`In the experiment with astaxanthin-administered vitamin E deficient rats (group 3, lane 10),
`the changes in the thin layer chromatographic profile induced by Fe2+ was not remarkable
`when compared with that from group 2.
`
`Effect of astaxanthin on carrageenan-induced paw edema in the rat. As described
`above, astaxanthin showed a protective effect against the membrane impediments by reac-
`
`
`
`. .
`I I
`
`PE
`
`PS
`/Pl
`
`Pc
`‘
`SM
`n Origin
`
`mm
`
`0
`
`210
`
`O
`
`210 210 210 210
`
`Feml X-XOD —
`In Vitro
`
`+
`
`-
`—
`+
`+
`+
`
`+AX ¢V.E
`
`O
`
`—
`
`210 210
`
`—
`
`+
`
`Group 1
`
`Group 2
`
`Group 3
`
`EffeCt of reacrive oxygens on the phospholipid in erythrocyte membranes.
`FIGURE 7.
`After incubation for 210 minutes under identical conditions as in Figure 6, membrane lipids
`were extracted following the method of Blight and Dyer (22). and subjected to thin layer
`chromatographic analysis using a developer composed of chloroform/methanol/acetic
`acid/distilled water (100/60/16/8: v/v) and colored with 2% phosphorus molybdate. In the
`pattern of the vitamin E deficient ghosts (lanes 3. 4 and 5), some unknown lipid complexes
`appeared with the progress of peroxidation. The formation of these unknown lipid complexes
`was reduced in the ghost of group 3 (lanes 8, 9 and 10). Conversely, no such changes were
`seen with the membrane treated with 4.2 pM astaxanthin or 420 HM vitamin E in vitro (lanes
`6 and 7). Added concentrations of Fe“, xanthine, and xanthine oxidase were 50 MM, 0.2 mM,
`and 0.025 unit/ml, respectively.
`
`PE‘ phosphatidylethanolamine; PS. phosphatidylserine; SM, sphingomyelin; Pl, phosphatidylinositol;
`PC. ph03phatidylcholine; X-XOD, xanthine-xanlhine oxidase system; AX. astaxanthin; V.E, vitamin E;
`in vrtro. added in \‘iII'O: Group 1, normal diet; Group 2, vitamin E—free diet; Group 3, vitamin E-free and
`astaxanthin containing diet.
`
`

`

`36
`
`KURASHZGE et al.
`
`tive oxygens and its effective concentration was significantly lower than that of vitamin
`E. To test whether such a potent protective action can also be seen in inflammatory tis-
`sues, we tested the effect of astaxanthin and vitamin E on carrageenan-induced paw
`edema in which reactive oxygens play a phlogogenic role (27). As shown in Figure 8, car-
`rageenan markedly increased the paw volume by an astaxanthiminhibitable mechanism.
`Under identical conditions, vitamin E failed to show such anti-inflammatory action. The
`mechanism by which astaxanthin inhibited the carrageenan-induced paw edema is not
`clear at present. In this context, the carrageenan-induced inflammation is highly sensitive
`
`sofi—~———~——5—l
`/
`
`
`
`0..
`
`o E
`
`m
`2
`LU 10
`o:
`C.)
`
`1;
`L
`l Control
`3 40
`-/t
`s
`LL. /
`0 3m
`V.E
`L“
`l
`:23
`
`/
`
`o
`
`1
`
`1
`
`
`
`l
`
`A/l
`[3/1
`AX
`l
`
`E
`
`O
`
`O
`
`60
`
`120
`
`l80
`
`TIME IN HOURS
`
`FIGURE 8. Effect of antioxidants on carrageenan-induced in-
`flammation. Under
`light ether anesthesis, animals were sub-
`cutaneously injected with 0.2 ml saline solution in the left paw.
`The same volume of carrageenan solution (10 mgj0.2 ml) was ad-
`ministrated in the right paw, and the'change in paw volume was
`measured at the indicated times. The control group was treated
`with an intraperitoneal injection of 1 ml of saline solution (Con-
`trol), and the experimental group was administered the same
`volume of saline solution containing 1 mg vitamin E (V. E) or as-
`taxanthin (AX) 30 min prior to the experiment [26]. Each point is a
`mean ‘1; SD of five experiments.
`
`

`

`SINCE el al.
`
`INHIBITION OF OXIDATIVE INJURY OF BIOLOGICAL MEMBRANES BY ASTAXANTHIN
`
`37
`
`of vitamin
`
`natory tis-
`
`luced paw
`ure 8. car-
`
`echanism.
`:tion. The
`
`ma is not
`' sensitive
`
`0
`
`to inhibitors for phospholipase A2 (25). Thus, the results do not rule out a possible con-
`tribution of the inhibitory activity of astaxanthin on phospholipase A2. However, the phar-
`macological effects of astaxanthin make this antioxidant a valuable model
`for
`the
`development of novel anti~inflammatory agents for therapeutic use.
`At present. the mechanism of protective action of astaxanthin against active oxygen in-
`duced membrane damage has not been clarified. It is known that carotene, having a polyun-
`saturated carbon chain like that of astaxanthin, has a higher reactivity with singlet oxygen
`(28-31) than does vitamin E (32). Therefore, it is possible that astaxanthin can also quench
`the active oxygen. However, further work is needed to fully understand the mechanism of
`antioxidant and anti-inflammatory actions of astaxanthin.
`
`We are grateful to Dr. W. Miki, Dr. T. Ando and Dr. M. Mori for their help in these experiments. We
`thank Miss Y. Okazoe and Y. Kitaoka for their excellent technical assistance in the preparation of this
`manuscript. We would also like to thank Dr. W. Miki, Research Center, Suntory Ltd. Osaka, Japan,
`for kindly donating the astaxanthin for these experiments.
`
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