Bot. Mag. Tokyo 76: 317-323(September 25, 1963)

Paper Chromatographic Survey of in Gladiolus-Flowers I

by Mannen SHIBATA* and Masayoshi NOZAKA*°**

ReceivedJuly 17, 1963

In the course of their analytical experiments on anthocyanins occurring in a variety of flowers, Robinson and Robinsonl' described that a salmon-pink variety of gladiolus (Gladiolus gandavensis) contains -3, 5-dimonoside, which is ac- companied in some cases by a small amount of or derivatives, as seen in "Flaming Sword ". Moreover, they stated that scarlet variety (e. g. " Scaretta ") produces pelargonidin -3-bioside, crimson variety cyanidin-3,5-dimonside (distribution number 0), and purple red variety (e.g. "Jacob von Bengeren ") forms -3,5-dimonoside. So far as we are aware, this is the one and only report that has appeared on components in gladiolus-flowers. The present study aims at a comprehensive survey of anthocyanins appearing in a great number of garden varieties of gladiolus chiefly by means of paper chromatographic analysis.

Materials and methods The flowers of ten garden varieties of gladiolus were examined in this experi- ment. They were obtained from Sakata Nursery Co., Ltd. in Yokohama, and grown in the garden of senior author (M. S.). Their names and flower colours are shown in Table 1. Analytical experiments were carried out according to the method that was pre- viously applied for the study of tulip flowers2'. Chromatographic separation of individual anthocyanins was effected by the one-dimensional ascending procedure on Toyo No. 52 filter paper (40X 40 cm) at 28±10 in an incubator, using the solvents as shown in Table 2, Table 1. Plant names and flower colours used for experiments. 318 Bot. Mag. Tokyo Vol. 76

Table 2. Designation and composition of the solvents used for chromatographic irrigation.

Collection of flower materials and extraction of anthocyanins : Early in July, 1962, two or three stalks bearing several flowers in each were collected and treated immediately in the following way: Fresh perianths were extracted with cold, 1 per cent methanolic hydrochloric acid (ca. 10 nil per perianth) overnight. The filtered solution was concentrated under reduced pressure at room temperature and stored in a refrigerator. Mass paper chromatographic separation and determination of Rf-values of com- ponent anthocyanins : Individual anthocyanins, which were separated into several colour-bands by means of mass paper chromatography (one-dimensional, ascending procedure), were subjected to colorimetric estimation of their amounts on the chro- matogram. Each of the colour bands was cut off and eluted in a separate manner with 1 per cent methanolic hydrochloric acid. With these eluates, Rf-values were measured as usual. In these cases, authentic specimens (, cyanin, chry- santhemin, keracyanin, delphin, , 3-monoglucoside (synthetic), etc.) were used for co-chromatographic purpose. Hydrolysis of anthocyanins and determination of the products were carried out in the same way as described in the previous report2' .

Results

A. Determination of the anthocyanins : The present survey has revealed that at least seven kinds of anthocynin are present in the flowers of ten gladiolus varie- ties, each containing 2-5 (mostly 2-3) kinds of anthocyanin. According to their Rf- values and colour shades on the chromatogram, the anthocyanin spots were designated tentatively as A1, A2, A3, A4, A5, A6 and A7, as shown in Table 3.

Table 3. The Rf-values and colours of anthocyanins obtained by irrigation with BuH.

i i i

B. Determination of the (aglycones) : The anthocyanidins obtained by acid hydrolysis of individual anthocyanins were chromatographed, and the Rf- September, 1963 SHIBATA, M, and NOZAKA, M. 319

Table 4. Comparison of RI-values of authentic specimens with those of gladiolus pigments.

values and colours on the chromatograms were carefully compared with those of authentic specimens run in parallel (Table 4). From Table 4, it is indicated that Al and A3 are glycosides of pelargonidin, As and A7 are glycosides of delphinidin, A2 belongs to peonidin glycoside, A4 to malvidin glycoside, and A5 to cyanidin gly- coside. In general, it appears that glycosides of pelargonidin occur most frequently, cyanidin and malvidin less frequently, and those of peonidin occur only rarely. C. Determination of the organic acids and sugars: a. Organic acids: All an- thocyanin solutions were saponified by potassium hydroxide in an atmosphere of hydrogen as usual. In every case saponified and unsaponified samples gave the same Rf-value. Moreover, it was also ascertained that ester combination with organic acid is not involved in any of the anthocyanins examined. b. Sugars involved in glycosidation : From the results of the present experi- ment, it was shown that sugar moieties in gladiolus-anthocyanins were glucose and rhamnose. In seven kinds of anthocyanins examined, two anthocyanins (A1 and A2) liberated both glucose and rhamnose, while the remaining five anthocyanins (A3- A7) gave glucose alone.

Discussion

According to the present survey, at least seven kinds of anthocyanin are present in common garden varieties of gladiolus, as shown in Table 5. Here, a brief discussion will be made on the chemical nature of individual pigments. A 1-anthocyanin (Rf =0.61 with BuH, orange red in colour) is composed of pelar- gonidin, glucose and rhamnose. Probably, this is pelargonidin 3-rhamnoglucoside, which has been known to occur in bracts of poinsettia3' and in flowers of tulip2' . On irrigation with BuAA, it gives Rf-value, 0.36, which is identical with the value already obtained by Harborne4' . As yet, crystallization has not been achieved in our laboratory. A2-anthocyanin (RI =0.50 with BuH, reddish pink in colour) is made up of peo- nidin, glucose and rhamnose. RI-value with BuAA is 0.22. This agrees with the value, which has been given for peonidin-3-rhamnoglucosido-5-glucoside by Harborne4'. A~-anthocyanin (Rf =0.36 with BuH, orange yellow in colour) is composed of pelargonidin and glucose. Up to present, three kinds of pelargonidin have been described in literature, whose Rf-values are as follows: pelargonin (pelargonidin 3,5- diglucoside) 0.42 with BuH, (pelargonidin-3-glucoside) 0.384' (with n-butanol/ 320 Bot. Mag. Tokyo Vol. 76 September, 1963 SHIBATA, M., and NozAKA, M. 321

2N hydrochloric acid, 1:1, v/v, Whatman No. 1 filter paper, by one-dimensional des- cending method), and raphanusin (pelargonidin-3-diglucosido-5-glucoside) 0.395 with BuH. The Rf-values of A3-anthocyanin were 0.36 with BuH, 0.25 with BuAA and 0.67 with AAH, respectively. These values agree with those described by Harborne4~. So far as the present experiments are concerned, Aa-anthocyanin seems to be either pelargonidin-3-diglucosido-5-glucoside (raphanusin) or pelargonidin-3-triglucoside, chiefly as regards its colour, Rf -values, etc. A4-anthocyanin (Rf =0.32 with BuH, mauve in colour) is composed of malvidin Table 6. Identification of anthocyanins in gladiolus-flowers 322 Bot. Mag. Tokyo Vol. 76 and glucose. The Rf-value (0.32 with BuH) and colour of this pigment are identical with those of authentic (malvidin 3,5 diglucoside) run in parallel. A5-anthocyanin (RI =0.27 with BuH, magenta in colour) is composed of cyanidin and glucose. RI-value is 0.27 on irrigation with BuH, being completely in agreement with that of cyanin (cyanidin 3,5-diglucoside). A6-anthocyanin (RI =0.16 with BuH, purple in colour) is made up of delphinidin and glucose. In anthocyanins being composed of delphinidin and glucose only, -a71 (delphinidin-3-monoglucoside) and delphin (=hyacin)6~ (delphinidin 3,5- diglucoside) have been known, and the RI-values obtained with BuH are 0.27 for the former and 0.16 for the latter. The RI-values for A6-anthocyanins were determined as 0.16 with BuH, and 0.11 with BuAA. Delphin also showed quite identical values on parallel run. A7-anthocyanin (RI =0.12 with BuH, purple in colour) has not been described previously. This is shown to be composed of delphinidin and glucose alone, similar to the case of As-anthocyanin. However, both pigments may be distinguished by their absorption maxima (515-516 mp for A7 and 533-534 m~c for A6). A7 is a new delphinidin derivative, probably belonging to a class of delphinidin triglucoside. From these considerations seven anthocyanins occurring in a variety of gladiolus- flowers may be characterized as follows: A1: Pelargonidin-E-gluccse+rhamnose (unnamed, pelargonidin-3-rhamnoglucoside). A2: Peonidin + glucose + rhamnose (unnamed, peonidin-3-rhamnoglucosido-5-glu- coside). A3: Pelargonidin+glucose (raphanusin, pelargonidin-3-diglucosido-5-glucoside). A4: Malvidin + glucose (m alvin, malvidin-3, 5-diglucoside). A5: Cyanidin + glucose (cyanin, cyanidin-3,5-diglucoside). A6: Delphinidin+glucose (delphin, delphinidin-3,5-diglucoside). A7: Delphinidin+glucose (unnamed, delphinidin-triglucoside) The kinds and amounts of these pigment components which were found in flowers of common garden varieties of gladiolus were summarized in Table 6.

The authors beg to acknowledge many thanks to Prof. K. Hayashi (Tokyo Uni- versity of Education) for supplying authentic specimens of peonidin and malvidin.

Summary

Anhhocyanins appearing in ten garden varieties of gladiolus (Gladiolus gandaven- sis) were paper chromatographically investigated. Individual pigments in flowers were separated from each other by mass paper chromatographic means in sufficient purity. Tests for hydrolysates, i.e. , sugar and organic acid were also carried out by the same procedure. In consequence, seven anthocyanins have been detected and identified as follows : pelargonidin-3-rhamnoglucoside, peonidin-3-rham- noglucosido-5-glucoside, delphinidin triglucoside (these three have never been isolated in crystals), pelargonidin-3-diglucosido-5-glucoside (raphanusin), malvidin-3,5-diglucoside (malvin), delphinidin-3,5-diglucoside (delphin) and cyanidin-3,5-diglucoside (cyanin). No acylation is involved in these pigments. Every varities contain 2-5 (mostly 2-3) kinds of anthocyanin, in general. Throughout the varieties examined, the glycosides of pelargonidin appeared most frequently, those of delphinidin, cyanidin and malvidin less frequently, and those of peonidin occurred least frequently. September, 1963 SHIBATA, M., and NOZAKA, M. 323

References

1) Robinson, G. M., and Robinson, R., Biochem. J. 25: 1687 (1931). 2) Shibata, M., and Ishikura, N., Naturwissenschaften 46: 601 (1959); Jap. Jour, Bot. 17: 230 (1960). 3) Asen, S., Plant Physiol. 33: 14 (1958). 4) Harborne, J. B., J. Chromat. 1: 473 (1958); Biochem. J. 70: 22 (1958). 5) Ishikura, N., and Hayashi, K., Bot. Mag. Tokyo 75: 28 (1962); ibid. 76: 6 (1963). 6) Saito, N., Mitsui, S., and Hayashi, K., Proc. Jap. Acad. 36: 340 (1960). 7) Reynolds, T M., and Robinson, R., J. Chem Soc. 1039 (1934).

摘 要

柴 田 萬 年*・ 野 坂 正 誼**:ペ ー パ ー ク ロ マ ト法 に よ る ・グ ラ ジ オ ラ ス 花 の ア ソ ト シ ア ン の 研 究

グ ラ ジ オ ラ ス(10品 種 に つ い て)花 の ア ソ トシ ア ソ 構 成 を ペ ー パ ー ク ロ マ ト法 に よ っ て 調 べ た.色 素 の 粗 抽 出 液 を マ ス ペ ー パ ー ク ロ マ ト法 に よ っ て 各 成 分 に 分 割 し,そ れ ぞ れ に つ い て 酸 水 解 お よ び ア ル カ リけ ん 化 を 行 な っ て ア グ リ コ ソ,糖 お よ び 有 機 酸 を ペ ー パ ー ク ロ マ ト法 で 調 べ た.そ の 結 果7種 類 の ア ソ トシ ア ニ ソ が 証 明 さ れ,そ れ ら はpelargonidin・3-rhamnoglucoside,peonidin・3-rhamnoglucosido-5・glucoside, delphinidin-triglucoside(以 上3種 は 未 だ 結 晶 と し て 得 ら れ た こ と が な い),raphanusinrpelargonidin- 3・diglucosldo・5・glucoside),malvin(Malvidin・3,5・diglucoside),cyanin(cyanidin-3,一5-diglucoside)お よ びdelPhin(de1Phinidin3,5・diglucoside)で,い ず れ も エ ス テ ル 型 の 有 機 酸 を 含 ん で い な い こ と が わ か っ た.各 品 種 に は 以 上 の 色 素 の う ち3-5種 類(多 くは2-3種 類)が 含 ま れ て い る.な おpelargonidin の 配 糖 体 が 最 も 多 く現 わ れde1Phinidin,cyanidinお よ びmalvidinの 配 糖 体 が これ に 次 ぎPeonidinの 配 糖 体 は 最 も ま れ で あ っ た.(*富 山 大 学 文 理 学 部 生 物 学 教 室,**静 岡 県 立 三 ケ 日高 等 学 校)