J. Home Econ. Jpn.Vol.60No.9785～790 (2009)

New Metalloanthocyanin from Blue Petals of Salvia uliginosa

1 ,2 ,3 ,＊ １ １ ２ Takemitsu MIZUNAGA, Megumi SAWAMURA, Satomi YOSHIOKA, Yasuko SONE ２ and Yuzuru OTSUKA

１ Keisen College of Horticulture, Isehara, Kanagawa 259‒1103, Japan ２ Institute of Environmental Science for Human Life, Ochanomizu University, Bunkyo-ku, Tokyo 112‒8610, Japan ３ Faculty of Human and Social Studies, Keisen University, Tama, Tokyo 206‒8586, Japan

A blue pigment complex was isolated from the blue flowers of Salvia uliginosa. The absorption spec- trum of its aqueous solution had three maxima at 645, 589 and 314 nm. The blue pigment did not per- meate a cellulose membrane, and contained magnesium as the metallic component, but not manganese, aluminum, zinc, calcium or iron. The blue pigment was composed of acetylated malonyl awobanine (Ishikawa, T. et al., Phytochemistry, 52,517‒521 (1999)), apigenin 7-cellobioside-4′-glucoside, and magne- sium. This composition was confirmed by an in vitro synthesis of the blue color from its component parts. No blue color was formed without magnesium. These results show the blue color of the flower is manifested by a metalloanthocyanin containing magnesium as the chelating metal. There was no dif- ference in the blue pigment formation among apigenin 7-cellobioside-4′-glucose, apigenin 7-cellobioside, and apigenin 7-glucoside. This blue pigment has potential use as a food colorants. (Received December 24, 2008; Accepted in revised form June 6, 2009)

Keywords: new metalloanthocyanin, Salvia uliginosa, blue pigment, magnesium-containing compound.

and flower color variation have been carried out and INTRODUCTION several proposals have been presented,４)５) i.e. Blue, violet, red, orange colors of flower are mostly metalloanthocyanin,４) copigmentation of anthocyanin composed of anthocyanins, betalains and carotenoids. and flavone,６) and intracellular sandwich-type stack- Among them, the anthocyanins are widely used as ing.７) In nature, there are many blue flowers, and it is natural food colorants additives１) because the not necessarily clear that each pigment of the all blue anthocyanins are thought to be safe as food additives. flower colors is suited to which one of the above pro- Anthocyanin changes its color with pH change; in posals. Because of wide use of anthocyanin for food strongly acidic medium, it shows orange to red color, additives, the structure of each anthocyanin is very whereas reddish violet to violet color appears in important to understand the mechanism of coloring weakly acidic or neutral solutions. Blue color can be and to understand the stability of anthocyanins in produced only in alkaline pH.２) Willstatter, who deter- food. mined the basic structure of anthocyanin around We have been interested in the flower color of Sal- 1913, attributed the variety of flower colors to pH via uliginosa and found that its pigment contains variation in flower cell sap.３) However, most blue magnesium and clarified that for the blue color flower cell sup is weakly acidic and never basic, and expression, magnesium is essential. This paper de- the blue color could not be explained by the scribes about a new metalloanthocyan from blue pet- Willsatter's pH theory. Extensive studies on the cor- als of S. uliginosa. relation between the structure of the anthocyanins MATERIALS AND METHODS

＊ To whom correspondence should be addressed. Fax: Materials 042‒482‒1559, E-mail: [email protected] S. uliginosa was grown in the farm of Keisen College Abbreviation: acetylated malonyl awaobanin, campus (Isehara, Kanagawa, Japan) and the flower delphinidine 3-(6-p-coumaroylglucoside)-5-(4-acetyl-6- petals were harvested from the mid July to late malonyl-glucoside) August and were freeze-dried and stored at

()785 13 J. Home Econ. Jpn.Vol.60No.9(2009)

－80℃. then 1% acetic acid, and eluted with 5% acetic acid Purification of the blue pigment －70% methanol. The pigment was again taken to The petals were immersed in water for 3 h and the dryness and further purified by paper chromatogra- filtered blue extract was fractionated with ethanol phy in the solvent BAW (n-butanol : acetic acid : wa- precipitation as shown diagrammatically in Fig. 1. ter, 4 : 1 : 5). For the reconstruction of blue complex, The residue was dissolved in water and the solution in vitro, anthocyanin was dissolved in 1.5% HCl, and was passed through a column of Sephadex LH 20 extracted with ethyl acetate for 4 times to remove (Pharmacia Fine Chemicals) (3×21 cm) using water as flavone.８) an eluant. A pure fraction of the blue pigment was Apigenin glucosides collected and the blue pigment was separated from Apigenin 7-glucoside was purchased from aqueous ethanol. Funakoshi Co. For the preparation of apigenin Purification of anthocyanin from S. uliginosa cellobiosides, 14 g of the blue petals of S. uliginosa Anthocyanin was extracted from dried petals of S. were extracted with 80% methanol. The extract was uliginosa with 5% acetic acid and adsorbed to Diaion then concentrated to about 2 ml. The concentrated HP20 (Mitsubishi Chem. Corp.) (2.5×24 cm) column. material was subjected to preparative paper chroma- After washing with 5% acetic acid, followed by 1% tography in 15% acetic acid, and the apigenin parts acetic acid, the pigment adsorbed was eluted with detected under UV light were isolated, then applied AMH (acetic acid : methanol : water, 5 : 70 : 25). The to the polyamide C (Wako Chem. Ind., Ltd.) column pigment was evaporated to dryness. The residue was (2×30 cm). Apigenin 7-cellobioside-4′-glucoside was dissolved in 5% acetic acid, and adsorbed to a eluted at about 40% of methanol in water‒methanol Sephadex LH 20 column, washed with 5% acetic acid, linear gradient elution, and apigenin-7-cellobioside was eluted at 70 ‒ 75% methanol. Each apigenin cellobioside was purified by means of two preparative paper chromatography steps using the solvents BAW and 15% aqueous acetic acid. Reconstruction of blue pigment The purified anthocyanin was dechlorinated with ammonium ion as was described in the literature.９) The anhydrobase of anthocyanin was mixed with an aqueous solution of apigenin derivatives and Mg ace- tate. The final concentration contained three compo- nentsinthemolarratioofapproximately,1:4:1. Dialysis Blue pigments and isolated anthocyanin from S. uliginosa was dissolved in water and 5% acetic acid re- spectively, and were dialysed through a cellophane membrane (Visking Cellulose Tubing) against 2 liter of water for 4 h with stirring at room temperature. RESULTS AND DISCUSSION

Purification and the absorption spectrum of the blue pigment from the petals of S. uliginosa The blue pigment complex, ploteodelphin from the petals of Salvia patens was composed of delphinidine 3-(6″-p-coumaroylglucoside)-5-(6″-malonylglucoside), i.e. malonyl awobanin, apigenin 7,4′-diglucoside and Mg.10) The sky blue petal of S. uliginosa, the same Fig. 1. Purification of the blue pigment from S. genus as S. patens, reportedly contains an acetylated uliginosa by ethanol precipitation and malonyl awobanin,11) as an anthocyanin responsible to Sephadex LH 20 column chromatography the blue color, and apigenin 7-cellobioside, apigenin 7-

14() 786 A Blue Pigment from Salvia uliginosa

separated from the blue pigment was apigenin 7- cellobioside-4′-glucoside from the literature of Veitch et al.12) Evidence that the blue pigment is a metalloanthocyanin The solution of metalloanthocyanins hitherto re- ported showed maxima at around 640‒670 nm except 580‒590 nm.10)13)14) The absorption spectrum of the so- lution of purified blue pigment from S. uliginosa showed maxim at 645 nm, indicating that the pigment could contain metal as a component. We analyzed metals in the pigment with atomic absorption pho- Fig. 2. Absorption spectrum of the purified blue pig- tometer (Z-8000, Hitachi). The pigment contained Mg, ment from S. uliginosa but did not contain Fe, Mn, Al, Zn and Ca. The Blue pigment was dissolved in water. amount of Mg was estimated to be 1 moles of Mg per 8,000‒10,000 of molecular weight. Commelinin, a blue pigment from the flowers of Commelina communis con- cellobioside-4′-glucoside and apigenin 7,4′-diglucoside, tains 2 moles of Mg per 9,100 of molecular weight,15) as flavone glycosides.12) Therefore, the components of therefore the molecular structures of commelinin and the blue pigments from S. patens and S. uliginosa ap- that from S. uliginosa would differ with each other. pear to be very similar. The purified blue pigment Reconstruction of the pigment from the petals of S. uliginosa easily dissolved in An experiment to reconstruct the blue complex water. As shown in Fig. 2, the absorption spectrum of was carried out using the acetylated malonyl the aqueous solution showed maximum at 314, 589, awobanine from the petals of S. uliginosa, apigenin 7- 645 nm, and the pattern of the spectrum was similar cellobioside-4′-glucoside and Mg as in the case of to that of the purified proteodelphin (317, 590, 648 commelinin,９) and protodelphin.10) nm).10) The absorption spectrum of the blue pigment syn- In the literature of Takeda et al.10) the ratio of the thesized in vitro (Fig. 3A) was practically identical to absorbance at 317 nm to 590 nm is more than 1.0, that of the natural pigment (Fig. 2). The blue complex whereas that of the reconstituted proteodelphin from was not formed without Mg (Fig. 3B), and the color its component parts is less than 1.0.10) The ratio of the was purple. absorbance at 314 nm to 589 nm of the purified pig- An attempt to form the blue complex using other ment from S. uliginosa is less than 1.0, indicating that metals instead of Mg showed that similar blue com- our purified material does not contain impurities. plexes were formed with Mn and Cd, but not with Ni, The blue pigment from S. uliginosa was not Co and Ca (Table 1). Although the patterns of the ab- dialyzed against water sorption spectra of the metal complexes using Mn The anthocyanin from S. uliginosa was dialyzable. and Cd were similar to the blue pigment from S. However, the blue pigment was not dialyzable uliginosa, the amount of the blue pigment synthesized against water (data not shown), indicating that the using Mg was about 1.5 fold much more than that pigment forms high molecular complex. using Ni and Cd. Hence, we could clearly show that Flavone isolated from the blue complex is the blue bigment is a metalloanthocyanin composed apigenin 7-cellobioside-4′-glucoside of acetylated malonyl awaobanine, apigenin 7- The purified blue pigment was treated with 10% cellobioside-4′-glucoside and Mg. Hitherto, metalloan- trifluoroacetic acid, developed in the paper chroma- thocyanins have been shown in detail about only tography of BAW, and of 15% acetic acid, and the Rf three examples, i.e. commelinin from Commelina values were compared. The UV-absorbing substance communis,9) protodelphin from S. patens10) and protocya- is apigenin from the color under the UV lamp, and the nin from the blue flower of Centaurea cyanus.14) Now sample showed the Rf of 0.09 in BAW, and 0.48 in the we showed the forth example of metalloanthocyanin 15% HOAc. The developed substance was eluted with from S. uliginosa. 80% MeOH and the spectrum showed maxima at 318, Very recently, Mori et al.16) have also purified 268 nm. These results showed that the copigment cyanosalvianin, a metalloanthocyanin from S.

()787 15 J. Home Econ. Jpn.Vol.60No.9(2009)

Fig. 3. Absorption spectra of reconstituted blue pigments in the presence (A)orabsence(B)ofMg The pigments were dissolved in 4 M NaCl. Light path is 3 mm.

Table 1. Formation of blue pigment complexes in mixtures of acetylated malonyl awobanin, apigenin-7-glucoside and different kinds of metals

λmax of blue pigment Metal component Color of reaction fraction in the visible applied mixtures region (nm)＊ Mg Blue 641.0 591.8 Zn ― Mn (Ⅱ) Blue 646.0 592.4 Cd Blue 643.0 592.8 Ni ― Co ― Ca ― K Purple 580.0

＊ measured in 4 M NaCl.

Table 2. Formation of blue pigment complexes in mixtures of acylated malonyl awobanin, Mg and different kind of apigenin glucosides

λmax of blue pigment Color of reaction Apigenin glucosides fraction in the visible mixtures region (nm)＊ Apigenin-7-cellobioside-4′-glucoside Blue 640.4, 588.2 Apigeni-7-cellobioside Blue 638.0, 593.0 Apigenin-glucoside Blue 641.0, 589.0

＊ measured in 4 M NaCl.

16() 788 A Blue Pigment from Salvia uliginosa uliginosa. The structure proposed by Mori et al. con- Carotenoids, Plant J., 54,733‒749 (2008) sisted of 3-O-(6-O-p-coumaroylglucopyranosyl)-5-O-(4- 2) Brouillard, R.: Chemical Structure of Anthocyanins, in O-acetyl-6-O-malonylglucopyranosyl) delphinidine, Anthocyanins as Food Colors (ed. by Markakis, P.), Aca- 7,4′-di-O-glucopyranosylapigenin and magnesium. demic Press, New York, 1‒40 (1982)   The structure of anthocyanin moiety is the same as 3) Willstatter, R., and Everest, A. E.: Uber den the structure reported by Ishikawa et al.11) The Farbstoff der kornblume. Liebigs, Ann. der Chem., 401, 189‒232 (1913) flavone moity is different from our results. Our pro- 4) Goto, T., Tamura, H., Kawai, T., Hoshino, T., Harada, posed flavone moity is apigenin 7-cellobioside-4- N., and Kondo, T.: Chemistry of Metalloanthocyanins, glucoside described above. We looked for apigenin 7, Ann. NY Acad. Sci., 471,155‒173 (1986) S. uliginosa 4′-di-O-glucoside in our petals of , but could 5) Goto, T., and Kondo, T.: Structure and Molecular not find it. The difference of the results may depend Stacking of Anthocyanins―Flower Color Variation, on varietal difference. Angew. Chem. Int. Ed. Engl., 30,17‒33 (1991) In order to examine the role of glucose moiety 6) Robinson, G. M., and Robinson, R.: A Survey of covalently bonded to apigenin, effects of apigenin 7- Anthocyanins I, Biochem. J., 25, 1687‒1705 (1931) glucoside, apigenin 7-cellobioside and apigenin 7- 7) Saito, N., Osawa, Y., and Hayashi, K.: Platyconin, a New cellobioside-4′-glucoside on the in vitro synthesis of Acylated Anthocyanin in Chinese Bell-Flower, the blue complex were compared (Table 2). The re- Platycodon gradiforum, Phytochemistry, 10,445‒ 447 sults show that no differences of the blue pigment for- (1971) mation were observed among these apigenin gluco- 8) Takeda, K., and Hayashi, K.: Anthocyanin, in sides, indicating that glucose moiety in the apigenin Shokubutsushikiso (ed. by Hayashi, K.), Yokendo, Tokyo, 7-cellobioside-4′-glucoside have no special role for the 151‒153 (1988) 9) Takeda, K., and Hayashi, K.: Metalloanthocyanins. I. in vitro synthesis of the blue pigment. Probably the Reconstruction of Commelinin from Its Components, hydrophobic interaction between apigenin and the Awobanin, Flavocommelinin and Magnesium, Proc. acetylated anthocyanin could play an important role Japan Acad., 53B,1‒5 (1977) in the blue color formation together with Mg. 10) Takeda, K., Yanagisawa, M., Kifune, T., Kinoshita, T., Stability of the reconstructed blue pigment and Timberlake, C. F.: A Blue Pigment Complex in Metalloanthocyanins have been known to be very Flowers of Salvia patens, Phytochemistry, 35, 1167‒1169 stable even in acidic conditions. Therefore the recon- (1994) structed blue pigment from acetylated anthocyanin, 11) Ishikawa, T., Kondo, T., Kinoshita, T., Haruyama, H., apigenin 7-glucoside and Mg was exposed to pH 5.2, Inaba, S., Takeda, K., Grayer, R. J., and Veitch, N. C.: 4.1, 3.1, and 2.1 in the 50 mM citrate-phosphate buffer An Acylated Anthocyanin from the Blue Petals of Sal- for 20 min and the resultant absorption spectrum at via uliginosa, Phytochemistry, 52, 517‒521 (1999) 641, 589 nm were examined. From the absorption 12) Veitch, N. C., Grayer, R. J., Irwin, J. L., and Takeda, K.: spectrum of the blue pigments, the remaining blue Flavonoid Cellobiosides from Salvia uliginosa, pigment were 97% at pH 5.2 and pH 4.1, 93% at pH Phytochemistry, 48,389‒393 (1998) 3.1, and 86% at pH 2.1. These results indicate the re- 13) Takeda, K., Metalloanthocyanins. Ⅱ. Further Experi- ments of Synthesizing Crystalline Blue Metal- constituted pigment is very stable even in the acidic loanthocyanins Using Various Kinds of Bivalent conditions like other matalloanthocyanins.９)10) There- Metals, Proc. Japan Acad., 53B,257‒261 (1977) fore, this anthocyanin from S. uliginosa will be useful 14) Takeda, K., Osakabe, A., Saito, S., Furuya, D., Tomita, for natural food colorants additives. And it is neces- A., Kojima, Y., Yamada, M., and Sakuta, M.: Compo- sary to investigate the safety evaluation to use as food nents of Protocyanin, a Blue Pigment from the Blue additives. Flowers of Centaurea cyanus, Phytochemistry, 66, 1607‒ 1613 (2005) The authors would like to thank Prof. Kosaku 15) Tamura, H., Kondo, T., and Goto, T.: The composition Kakeda (Tokyo Gakugei Univ., Tokyo) and Prof. Norio of Commelinin, a Highly Associated Metal- Saito (Meijigakuin Univ., Kanagawa) for many helpful loanthocyanin Present in the Blue Flower Petals of suggestions. Commelina communis, Tetrahedron Lett., 27, 1801‒1804 (1986) REFERENCES 16) Mori, M., Kondo, T., and Yoshida, K.: Cyanosalvianin, a 1) Tanaka, Y., Sasaki, N., and Ohyama, A.: Biosynthesis of Supramolecular Blue Metalloanthocyanin, from Petals Plant Pigments: Anthosyanins, Betalains and of Salvia uliginosa, Phytochemistry, 69, 3151‒3158 (2008)

()789 17 J. Home Econ. Jpn.Vol.60No.9(2009)

サルビア・ウリギノーサの青色花弁の新規メタロアントシアニン

水永武光1,2,3,沢村恵１, 吉岡里美１, 曽根保子２,大塚譲２ (１ 恵泉園芸短期大学, ２ お茶の水女子大学生活環境教育研究センター, ３ 恵泉女学園大学人間社会学部) 原稿受付平成 20 年 12 月 24 日；原稿受理平成 21 年６月６日

サルビア・ウリギノーサの青色花弁から青色色素複合体を精製した. 精製青色色素は 645, 589, 314 nm に吸収極大を示した. この青色色素はセロハン透析チューブを通さなかった. 原 子吸光分析の結果 Mg が検出され, Fe, Mn, Al, Ca, Zn は検出されなかった. アントシア ニンはアセチル化マロニルアオバニンであり, フラボン部分はアピゲニン７‒セロビオシド‒ 4′‒グルコシドであった. この複合体に Mg が必須であることを再構成により示した. また, Mg 以外にも Mn, Cd も青色形成をしたが, Mg が最も効果があった. アピゲニン配糖体はそ の分子中に糖が１個でも２個でも３個でも青色形成に差がなかった. この色素は食品添加物と して利用の可能性が考えられた.

キーワード：新規メタロアントシアニン, サルビア・ウリギノーサ, 青色色素, Mg 含有化合 物.

18() 790