Astaxanthin And/Or Canthaxanthin-Actomyosin Complex in Salmon Muscle*1
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Nippon Suisan Gakkaishi 55(9), 1583-1589 (1989) Astaxanthin and/or Canthaxanthin-actomyosin Complex in Salmon Muscle*1 Hikaru Henmi,*2 Masahiro Hata,*2 and Mitsuo Hata*3(Received March 17, 1989) It was proposed that astaxanthin and/or canthaxanthin in salmon muscle binds with one ƒÀ- ionone ring to a hydrophobic binding site which exists on the surface of actomyosin . The binding of carotenoids to actomyosin is not specific. The hydroxyl and keto groups of ƒÀ-end group in tensify the binding strength of carotenoids to actomyosin . In a previous paper,1) we reported that asta and zeaxanthin (physalis). xanthin in salmon muscle existed in the water insoluble fraction, associated with actomyosin pre Measurement of the Absorption Spectrum of the pared from myofibrils, and solublized easily Muscle by Opal Glass Method with Triton X-100, suggesting that astaxanthin The absorption spectrum of the muscle was binds to protein with a weak hydrophobic bond. measured using a Hitachi 150-20 spectrophoto Astaxanthin, canthaxanthin, zeaxanthin, cyn meter according to the method of Shibata.6) Thin thiaxanthin, ƒÀ-doradexanthin and diatoxanthin freshly sliced muscle was used. were identified as major carotenoids of salmonid muscle.*4,2-5) Astaxanthin, canthaxanthin and Reduction of Astaxanthin and Canthaxanthin other carotenoid(s) were extracted from the The muscle astaxanthin and canthaxanthin actomyosin in cultured coho salmon muscle.1) were reduced by the method of Ako et al.7) The These results suggest that salmonid actomyosin water-insoluble fraction of the muscle was sus can combine with many carotenoids. pended in 2 volumes (v/v) of distilled water and In the present study, we investigated how carot solid NaBH4 was added to the suspension at enoids combine with salmon actomyosin and the a concentration of 100mM, and incubated at nature of the astaxanthin and/or canthaxanthin 5•Ž in darkness. The suspension was taken at actomvosin complex. various time intervals and acetone was added to stop the reaction. Authentic astaxanthin and Materials and Methods canthaxanthin were dissolved in 95% ethanol and solid NaBH4 was added to the solution to a con Materials centration of 100mM and incubated at 0•Ž in dark Sockeye salmon Oncorhynchus nerka and cul ness. Carotenoids were extracted with acetone tured coho salmon Oncorhynchus kisutch were petroleum ether (PE) or acetone-ethyl ether, and used. Coho salmon were cultured at Onagawa the reduction products were analyzed by thin layer Bay and fed canthaxanthin as a pigment source. chromatography (TLC) and high performance These specimens were kept at -80•Ž until use. liquid chromatography (HPLC). The following carotenoids were used: synthetic astaxanthin, echinenone, canthaxanthin (Hof Extraction of Carotenoids with Heptane fman-La Roche & Co.), ƒÀ-carotene (Wako Pure The method described by Goodman and Raz8 Chemical Industries LTD.), astaxanthin mono ) was used for the extraction of carotenoids with ester (shrimp), astaxanthin diester (Antarctic krill), heptane. *1 Studies on the carotenoids in the muscle of salmon-II. *2 Department of Fisheries, Tohoku University, Sendai, Miyagi 981, Japan (逸 見 光,秦 正 弘:東 北 大 学 農 学部). *3 Department of Fisheries, Nihon University, Shimouma, Setagaya, Tokyo 154, Japan (秦 満 夫:日 本 大 学 農 獣 医 学 部), *4 W. Miki, T. Mori, K. Yamaguch, and S. Konosu: Abstract of Papers, the Autumn Meeting of the Japanese Society of Scientfic Fisheries(Sendai), 1984, p. 114. 1584 Henmi, Hata, and Hata VIS detector (JASCO UVIDEC 100-VI) with the The water-insoluble fraction of the muscle was suspended in 20ml of distilled water and 20ml specially made columns (stainless steel, colum A; of n-heptane was added. The solution was LiChrosorb CN-5, Merck, 4.6•~250mm or col shaken at 5•Ž for 20h under nitrogen gas at a umn B; LiChrosorb SIL 100-5, Merck, 4.O •~ 250mm) were used. rate of 100 strokes/min in darkness. After shaking, Astaxanthin and its reduction products were the absorbance of the upper heptane phase was measured and carotenoid content was determined. analyzed on column A. The column was eluted with a solvent mixture of 29% n-hexane contain Combination of Actomyosin with Carotenoids ing 0.1%•@ N-ethyldiisopropylamine and 71% di Combination of actomyosin with carotenoid chloromethane containing 3% methanol at 2.0ml/ was carried out by the method originally de min. scribed by Ando and Yamamoto.9) Canthaxanthin and its reduction products were Carotenoid solution was prepared by the fol analyzed on column B. The column was eluted lowing method. Carotenoid (1.3mg) was dis with a solvent mixture of n-hexane: acetone: solved in chloroform. After addition of octa dichloromethane=90.08: 9.54: 0.38 at 2.0ml/min. ethyleneglycol mono n-dodecyl ether (50mg), The peaks were monitored by measuring the chloroform was removed under a stream of nitro absorption at 470nm and identified by comparing gen gas and one ml of distilled water was added, their retention times with those of the standard and then the solution was stirred. carotenoids. The actomyosin (4.0g) was suspended in 5ml of 0.1M phosphate buffer (pH 6.0), and 0.2ml Results of the carotenoid solution was added. The solu tion was then shaken gently at 5•Ž for 12 h under The Absorption Spectrum of the Muscle nitrogen gas. After shaking, the actomyosin The maximum of the absorption spectrum of was washed with water to remove the uncombined the muscle of sockeye salmon was 486nm with a carotenoid. shoulder at 420nm. The carotenoid extracted from the muscle showed an absorption maximum Preparation of Actomyosin at 468nm in hexane and showed one band on Actomyosin in the muscle of coho salmon was TLC. This carotenoid was identified as asta xanthin. Muscle astaxanthin showed a weak prepared by the method of Arai.10) bathochromic 2max (from 468nm in hexane to 486nm in the muscle) (Fig. 1). Treatment of Actomyosin with Triton X-100 Purified actomyosin was treated with Triton X-100 by the method of Ando11) to remove lipids Extraction of Carotenoids with Heptane associated to actomyosin. In the muscle of sockeye salmon, the ratio of extracted astaxanthin to total astaxanthin was 3.6%. In the muscle of coho salmon, the ratio Analytical methods Carotenoids were identified by the previously reported methods.1) The carotenoid content was determined from the absorbance at absorption maximum, using E1%1cm=2200. Lipids were extracted by the method of Bligh and Dyer.12) Protein was determined by the method of Lowry et al.13) Egg albumin was used as a standard. Thin Layer Chromatography (TLC) TLC was performed on silica gel plate (Wako gel B-5) using petroleum ether-acetone (7: 3) as solvent. Fig. 1. Absorption spectra of the muscle of sockeye salmon and astaxanthin. High Performance Liquid Chromatography (HPLC) -Muscle Nihon Bunko (JASCO BIP-1) pump and UV/ - - - Astaxanthin in hexane l. Astaxanthin, Canthaxanthin-actomyosin Complex 1585 Table 1. Combination of coho salmon actomyosin is similar to that of the naturally occurring asta with carotenoids xanthin-actomyosin complex. Carotenoids Carotenoid contents (ƒÊg/mg protein) Combination of Triton X-100 Treated Actomyosin with ketocarotenoids Astaxanthin 0.70-0.86 Astaxanthin monoester 0.20 After treatment with Triton X-100, the lipid Astaxanthin diester 0.00 content of the actomyosin changed from 0.35 to Canthaxanthin 0.49 0.14mg/mg protein. The contents of astaxanthin Echinenone 0.33 and canthaxanthin combined with Triton X-100 Zeaxanthin 0.47 treated actomyosin are given in Table 2. These ƒÀ-carotene 0.09 values were similar to those of untreated acto myosin complexes. The absorption maximum of Table 2. Combination of the Triton X-100 treated the astaxanthin-actomyosin complex was 486nm, actomyosin with astaxanthin and canthaxanthin. showing a weak bathochromic ƒÉmex (18nm), similar to that of the naturally occurring astaxanthin-acto Carotenoid Carotenoids myosin complex. The absorption maximum of contents (ƒÊg/mg protein) the canthaxanthin-actomyosin complex was 480 Astaxanthin 0.88 Canthaxanthin 0.41 nm, showing a weak bathochromic ƒÉmax (15nm), similar to that of the naturally occurring cantha xanthin-actomyosin complex. canthaxanthin of extracted canthaxanthin to total These results indicate that lipids in actomyosin was 38%. Combination of Actomyosin with Carotenoids Actomyosin combined with free-form asta xanthin, astaxanthin monoester, canthaxanthin, echinenone, zeaxanthin, and ƒÀ-carotene. The colors of astaxanthin-, astaxanthin monoester-, canthaxanthin-, zeaxanthin-, and ƒÀ-carotene-acto myosin complexes were red, red, orange, yellow, and yellow, respectively. However actomyosin did not combine with astaxanthin diester (Table 1). The absorption maximum of the astaxanthin actomyosin complex was 477nm, showing a weak bathochromic ƒÉmax (9nm). About 5.5% of astaxanthin was extracted with n-heptane from the astaxanthin-actomyosin complex. This value Fig. 2. Reduction of muscle astaxanthin. Fig. 3. Reduction of astaxanthin-actomyosin complex dissolved in 0.6 M KC 1586 Henmi, Hata, and Hata have no effect on carotenoid-protein binding. Reduction of Muscle Astaxanthin and/or Can thaxanthin After addition of NaBH4, the color of the muscle turned from orange to yellow. The reduction products of the carotenoids were extracted with acetone and separated by TLC and three bands was observed. These carotenoids were identified as astaxanthin, idoxanthin and crustaxanthin, respectively, from the absorption spectra and the behavior on TLC (data not shown). The changes of the existing ratio of these re duction products are shown in Fig. 2. Astaxanthin-actomyosin complex prepared from Fig. 4. Reduction of authentic astaxanthin. sockeye salmon muscle was dissolved in 0.6M KCl, and incubated at 5•Ž in the presence of Addition of NaBH4 caused a rapid decrease of 0.1M NaBH4. The reduction products of asta astaxanthin at first, followed by a slow decrease xanthin were extracted with acetone-ethyl ether in the ratio.