Note Formation of an Inclusion Complex of Anthocyanin With
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Agric. Biol. Chem., 44 (6), 1411•`1413 , 1980 1411 Note extracted from the seeds of black soy bean (KUROMAME) (Glycine max Merill). Nasunin was ex tracted from the fruits of egg plant (Solanum melongena L .). Formation of an Inclusion Complex These three anthocyanins extracted were proved to be pure of Anthocyanin with Cyclodextrin by paper chromatography after some preparation procedure.6) ƒ¿-CD and ƒÀ-CD used were commercially available Tetsuya YAMADA,Takashi KOMIYA (Hayashibara Biochemical Research), and amylose was and Morio AKAKI prepared from a high amylose corn starch. Amylose was dissolved in dimethylsulfoxide (DMSO) and the solution Facultyof Agriculture,Mie University, was diluted with water, final concentration of DMSO being Tsu 514,Japan 33%. ReceivedJuly 24, 1979 Each anthocyanin was dissolved in water, further diluted properly with water and adjusted to pH 2.0 with HCl. To each of these anthocyanin solutions was added cyclodextrin solution or amylose solution at various concentrations. Recently, many studies concerning the inclusion complex Addition of ƒÀ-CD resulted in the fading of two an forming activity of cyclodextrin were reported .1•`3) We thocyanin solutions (callistephin and chrysanthemin), and were also interested in this activity because cyclodextrin is this fading effect was greater with higher concentrations of analogous to amylose and amylopectin. Since many natural -CD added (Table I) . On the other hand, addition of ƒ¿-CD ƒÀ pigments in food coexist with saccharides, it was wondered had almost no fading effect in chrysanthemin solution, if the stability of these pigments was affected with the although it showed a less but significant fading effect in presence of such saccharides. callistephin solution. Addition of amylose resulted in the A cyclodextrin molecule is said to have a tubular form fading only in callistephin solution. However because of with an inner diameter of 6 A in a-cyclodextrin (ƒ¿-CD) or immediate precipitation, the extent of this effect could not 7.5 A in ƒÀ-cyclodextrin (ƒÀ-CD). Both cyclodextrins are 7 A be estimated. There was no change in nasunin solution with long and the inner side of the tube is rather basic and addition of ƒ¿-CD, ƒÀ-CD and amylose solutions. hydrophobic. Amylose is known to have a helical form with It must be pointed out that the establishment of equilib a helix consisting of six glucose units in the aqueous rium of the fading reaction of anthocyanin with ƒÀ-CD solution in the presence of n-butanol or thymol resembling needed much time as shown in Fig. 1, whereas the -CD rather than ƒÀ-CD ƒ¿ . Amylose was also reported to have equilibrium of the formation of the inclusion complex of p a helical form in dimethylsulfoxide solution .4) nitro phenol with ƒÀ-CD is almost instantly established.1) Many phenolic substances can easily form their inclusion As this fading reaction of anthocyanin must be based on complexes with cyclodextrin if their molecular sizes fit the the change of an oxonium ion into a pseudobase in the hole.1,2) Since anthocyanins have phenolic moeities, we anthocyanidin moeity, the above result may be explained were interested in whether or not anthocyanin could form with a hypothesis that the pseudobase forms through two an inclusion complex with cyclodextrin. steps in this case. The first step is the formation of a Three anthocyanins were examined: callistephin inclusion complex of anthocyanin with ƒÀ-CD and the (pelargonidin-3-glucoside), chrysanthemin (cyanidin-3 second is change of the oxonium ion to the pseudobase by a glucosidc) and nasunin5) [delphindin-3-(4-(p-coumaroyl) - catalytic action of ƒÀ-CD. The former maybe rapid, and the L-rhamnosyl-(1,6)-glucosido)-5-glucoside]. Callistephin latter may be slow and the rate determining step of this was extracted from the fresh fruits of strawberry (Fragaria fading reaction. chiroensis Duch. var. annassa Baillon). Chrysanthemin was A small depression of pH (0.050.1) and a small TABLE I. THE DEGREE OF THE FADING OF ANTHOCYANIN BY CD ADDITION Cal., Callistephin; Chr., Chrysanthemin. The anthocyanin solution was adjusted to pH 2.0, and the absorbance was measured at 498nm for callistephin and at 512nm for chrysanthemin at 26•Ž, 60min after CD addition. 1412 T. YAMADA, T. KOMIYA and M. AKAKI FIG. 1. Change in Absorbance of Anthocyanin Solution by Cyclodextrin Addition. Call, callistephin; Chr, chrysanthemin. B2, relative absorbance of anthocyanin solution in the absence of ƒÀ-CD at pH 2.0; Bo, relative absorbance of anthocyanin solution in the absence of ƒÀ-CD at pH near zero. Condition, ƒÀ-CD solution was added to anthocyanin solution which had been adjusted to pH 2.0, the final concentration of ƒÀ-CD varied from zero to 1.0%. A part of each solution was strongly acidified to pH near zero with conc. HCl at regular time intervals. Absorbance of anthocyanin solution was measured at 498nm for callistephin, and at 512nm for chrysanthemin at 18•Ž. The absorbance of all anthocyanin solutions in the presence of ƒÀ-CD instantly recovered just to the same level as B0 by strong acidification every time. FIG. 2. Change in Absorbance of Anthocyanin Solution in the Presence of ƒÀ-CD by Strong Acidification. Condition, the pH of anthocyanin solution was adjusted with HC1; concentration of ƒÀ-CD, 1.0%. increment of absorbance at ƒÉmax in the ultraviolet region of shows that in spite of the presence of ƒÀ-CD the absorbance callistephin solution (pH 2.0) were observed with the of both anthocyanin solutions (callistephin and chrysan addition of ƒÀ-CD. This observation also supports the themin) instantly recovered to the initial value without formation of the inclusion complex. CD even after 24hr if the solution was ƒÀ-strongly By the addition of ƒÀ-CD the ƒÉmax positions in the visible acidified. This result suggests that the inclusion complex region of the spectra of the anthocyanin solutions (498nm may protect anthocyanin from decomposition in spite of its for callistephin, 512nm for chrysanthemin) were not labile pseudobase form. changed, but the extent of the absorbance at ƒÉmax was changed. Acknowledgment. This study was financially sup Figure 2 shows disappearance of this fading effect under ported by the Grant-in-Aid for Scientific Research. the extremely acidic condition near pH zero, and Fig. I Inclusion Comples of Anthocyanin 1413 4) M. Fujii, K. Honda and H. Fujita, Biopolymers, 12, REFERENCES 1177 (1973). 5) S. Watanabe, S. Sakamura and Y. Obata, Agric. Biol. 1) F. Cramer, W. Saenger and H. Spatz, J. Am. Chem. Chem., 30, 420 (1966). Soc., 89, 14 (1967). 6) T. Nakabayashi, S. Kimura and H. Kato, "Shokuhin 2) K. Harata, J. Jap. Soc. Starch Sci., 26, 198 (1979). no Henshoku to Sono Kagaku," Korin-shoin, Tokyo, 3) J. Szejtli, Stdrke, 30, 427 (1978). 1967, pp. 38•`45..