Bot. Mag. Tokyo, 74: 186-189 (April 25, 1961)

Concerning the of Two Garden Varieties of Tulipa Gesneriana

by Mannen SHIBATA and Emi SAKAI*

Received October 1, 1960

In the preceding papers of this series, the structure and properties of two chlorides tulipanin and keracyanin isolated in crystalline state from tulip flowers have been described1'2), On the other hand, paper-chromatographic survey o f anthocyanins recently carriedd out in our laboratory on 107 garden varieties of tulip has disclosed that the flowers ranging from red to dark purple contain 2-4 anthocyanins in general, and that six kinds of anthocyanin may be enumerated throughout the varieties examined3,4). The present paper includes further examples for the crystallization of two antho cyanins from tulip varieties. The perianths of two garden varieties, " The Bishop (Darwin strain) and " Parrot Pierson " (Parrot strain), were used as material. The extraction and crystallization of the pigments were carried out as usual. The yieldd of crystalline chloride was exceedingly poor; namely, 0.012 0o for " The Bishop " and. 0.006 % for " Parrot Pierson " on a fresh weight basis. The sugar moieties in these anthocyanins were found to be glucose and rhamnose. The were identified as and , respectively. Papar-chromatographic as well as spectrophotometric examinations and elementary analyses have shown that of these two anthocyanins, the one was nothing but tulipanin (delphinidin-3-glucorhamnoside) and the other was keracyanin (cyanidin-3-glucorhamnoside).

Experimental

A. "The Bishop "-perianths Isolation of tulipanin as chloride. Fresh perianths (ca. 28 kg.) were immersed in cold methanol (10 l.) containing 1 % hydrochloric acid. After standing for 2 days, the filtered red extract (9 l.) was added with a saturated methanolic solution of basic lead acetate (ca. 2l.), under continuous agitation. The blue lead salt precipitated was collected by suction and washed with water and methanol. The dried lead salt. (ca. 370 g.) was finely powdered and converted into chloride by dissolution in 5 % methanolic hydrochloric acid (ca. 2l.). The dark red filtrate was concentrated to 2/5 volume in vacuo below 35°, and stored in a refrigerator to make further impurities separate out. After filtration, the anthocyanin in the filtrate was precipitated with ether (7 vol.). The dark red resinous precipitate obtained was dissolved in aa small amount of 1 % ethanolic hydrochloric acid and precipitated again with ether (5 vol.). Amorphous precipitate formed was then dissolved in a small quantity of ethanol containing 1 % hydrochloric acid and filtered, and 1/2 vol. of 5 % ethanolic hydrochloric acid was added to it. Soon, purplish red-brown needles commenced to separate in sea-urchin-like cluster. Yield 3.6838 g. Crude crystals were recrystallized from warm water containing 5 % ethanolic hydrochloric acid. The yield of pure crystals corresponded to about 0.012 % of the fresh weight of perianths used. * Laboratory of Plant Physiology , Biological Institute, Toyama University, Toyama, Japan.. April, 1961 SHIBATA, M., and SAKAI, E. 187

Anthocyanin chloride (tulipanin). The "Bishop "-anthocyanin crystallized in pur- plish red-brown needles, which decomposed at 178°, either alone or on admixture with authentic specimen of tulipanin. The distribution number between iso-amyl alcohol and 0.5 % hydrochloric acid was about 9.3. On irrigation with n-butanol/conc. hy- drochloric acid/water (7:2:5, v/v), one single spot was obtained on the chromatogram showing Rf = 0.38. This was also the case on co-chromatography. The light absorp- tion (Fig. 1), solubility in usual solvents and colour reactions were quite identical with those of tulipanin. Found : C 49.99; H 4.74. Calc. for C27 H31O16C1: C 50.12; H 4.83. Water of crystallization: Found : H2O 10.22. Calc. for C27H31016C1.4H20: TT !1 in nn,

Wave length (mn) Fig. 1. Absorption spectra of "The Bishop "-anthocyanin and tulipanin. "The Bishop "-anthocyanin , 4/10,000 mol in 60 % EtOH (HC1-conc. 0.1 %) ------Tulipanin (from "Queen of Night "), 4/10,000 mol in 60% EtOH (HC1-conc. 0.1

Hydrolysis. The purified glycoside (1 g.) was dissolved in water (80 ml.), and after addition of conc. hydrochloric acid (50 ml., d 1.18), boiled for 3 min. and stood in a refrigerator for 24 hrs. The aglycone separated completely in blackish mass having greenish luster (0.47 g.). The acidic mother liquor was shaken with ether, in which no trace of organic acids could be detected. The acidic solution was then shaken with iso-amyl alcohol to remove a last trace of aglycone, and subjected to the ex- amination of sugar components. Finally, the sugars were identified as glucose and rhamnose, respectively, by colour reactions, paper-chromatographic test as well as by osazone formation. Aglycone (delphinidin chloride). The sugar-free pigment obtained above was recrystallized from a mixture of ethanol and 5 % ethanolic hydrochloric acid. All of its properties were quite similar to those of delphinidin described by Willstatter et al.5). No methoxyl group was present in the molecule. Rf value was 0.23 on irriga- tion with acetic acid/conc. hydrochloric acid/water (5:1:5, v/v) at 28±1°, using Toyo No. 52 filter paper. Found on anhydrous specimen: C 53.29; H 3.24. Calc. for C15H11O7C1: C 53.18, H 3.27. Water of crystallization. Found : H2O 10.09. Calc. for C15H11O7C1.2H20: HBO 9.61. B. "Parrot Pierson "-perianths Isolation of anthocyanin (keracyanin) as chloride. Fresh perianths (7.1 kg.) were immersed in 1 % methanolic hydrochloric acid (51.) for 2 days. The dark red ex- tract was decanted and the residue was extracted again with methanol (1.5 1.) for 3 hrs. A saturated methanolic solution of basic lead acetate was slowly added to the combined filtrate (61. ), and the bluish green precipitate formed was collected and 188 Bot. Mag. Tokyo Vol. 74 washed thoroughly with water and methanol. This was immediately converted into chloride by dissolving in 5 % methanolic hydrochloric acid, and the anthocyanin was precipitated with ether (10 vol.). The amorphous red precipitate was dissolved in 1 % methanolic hydrochloric acid and precipitated with ether (5 vol.). The same process was repeated further four times. Finally, the product was dissolved in 1 % ethanolic hydrochloric acid and, after addition of 5 % ethanolic hydrochloric acid (1/10 vol.), the mixture was allowed to stand for 2-5 days, whereby the anthocyanin commenced to crystallize. Recrystallization was effected by dissolution in 1 % ethanolic hy- drochloric acid or warm water and addition of 5 % ethanolic hydrochloric acid. The yield of the purest specimen was 0.45 g., corresponding to 0.006 °o of fresh weight of the perianths. Properties of "Parrot Pierson "-ant hocyanin. The anthocyanin recrystallized thrice consisted of red-brown needles, and decomposed at 177°. Distribution number 6.9. Rf value found on irrigation with acetic acid/conc. hydrochloric acid/water (3:1:3, v/v) at 28±1° using Toyo No. 52 filter paper was 0.47. The solubility, ab- sorption spectrum (cf. Fig. 2) and colour reactions showed a good agreement with those of keracyanin previously isolated from the tulip-flower (" Eclipse ")6). Anal. Found: C 51.11, H 4.83. Calc. for C27H31O15C1: C 51.33; H 4.92. Water of crystal- lization. Found: H2O 7.56. Calc. for C27H31O15C1.3H2O:H2O 7.98.

Wave length (mµ) Fig. 2. Absorption spectra of "Parrot Pierson "-anthocyanin and keracyanin. "Parrot Pierson " -anthocyanin , 5/10,000 mol in 60 % EtOH (HC1-conc. 0.1 %) ------Keracyanin (from "Eclipse ") , 5/10,000 mol in 60 % EtOH (HC1-conc. 0.13 %) Hydrolysis. Anhydrous glycoside chloride (85 mg.) was dissolved in warm water (10 ml.), and the solution was added with an equal volume of 36 °o hydrochloric acid and boiled for 3 min. After standing in a refrigerator overnight, dark chocolate- brown crystals were collected. Yield 43.4 mg (51.02 %). The mother liquor was shaken with iso-amyl alcohol to remove any trace of aglycone, and was used for the determination of sugars. The qualitative examinations by means of colour reaction, osazone formation and Rf value have demonstrated the presence of glucose and rhamose. Aglycone (cyanidin chloride). The sugar-free pigment obtained above was recry- stallized five times from ethanol containing about 5 % hydrochloric acid. It was ob- tained in characteristic, long red-brown needles. On co-chromatography with cyanidin chloride obtained from Pennisetum6) and Tulipa2), only one spot was obtained on the chromatogram, giving Rf value 0.34 by April, 1961 SHIBATA,M., and SAKAI,E. 189 means of acetic acid/conc. hydrochloric acid/water (5:1:5, v/v) using Toyo No. 52 filter paper. The solubility and colour reactions also proved the identity with cya- nidin. Micro-Zeisel estimation proved the absence of methoxyl group. Anal. Found : C 55.66; H 3.41. Calc. for C15H11O6C1: C 55.82; H 3.43. Water of crystallization. Found : H2O 7.29. Caic. for C15H11O5C1.11/2H2O: H2O 7.72. Finally, it may be noted that according to the paper chromatographic and spectrophotometric studies, "The Bishop "-flower contained four anthocyanins approx- imately in the following proportion: delphin (1 part), tulipanin (7 parts), keracyanin (2 parts) and glucorhamnoside (trace), and "Parrot Pierson "-flower three anthocyanins, i.e., keracyanin (4 parts), pelargonidin glucorhamnoside (4.5 parts) and tulipanin (1.5 parts) (M. Shibata and N. Ishikura, unpublished results).

Summary

Two anthocyanins in the flower of garden varieties of tulip were isolated in crystalline state and their properties and structure have been described. The one from the perianth of the garden variety, "The Bishop" (strain of Darwin, bright violet, large flower) was tulipanin (delphinidin-3-glucorhamoside), which was previously isolated by one of us (M. S .) from the dark purple perianth of a variety of tulip (" Queen of Night "), and the other from the perianth of the garden variety, "Parrot Pierson " (strain of Parrot, dark red flower), was nothing but keracyanin (cyanidin-3- glucorhamnoside), which had been isolated by us from the blood-red perianth of a variety of tulip (" Eclipse ").

The authors are indebted to Prof. K. Hayashi (Tokyo University of Education) for elementary analysis, and also to Mr. I. Demura in Tonami city and Mr. N. Kobaya- shi in Nyuzen town for their donation of flowers used in this experiment.

References

1) Shibata, M., Bot. Mag. Tokyo 69: 462 (1956). 2) , and Sakai, E., ibid. 71: 6 (1958). 3) and Ishikura, N., Naturwissenschaften 46: 602 (1959). 4) , and , Jap. Jour. Bot. 17: 230 (1960). 5) Willstatter, R. and Mieg, W., Ann. Chem. 468: 61 (1914). 6) Shibata, M., and Sakai, E., Bot. Mag. Tokyo 71: 193 (1958).

摘 要

柴 田 万 年 ・堺 恵 美;チ ュ ー リ ッ プ の2品 種 の ア ン トシ ア ニ ン に つ い て

さ き に チ ュー リップ の 花 の ア ソ トシ アニ ソ を結 晶 状 に 単 離 し,そ の性 質,構 造 な ど を報 告 した が,本 報 で は 更 に他 の 品種 ザ ・ビ シ ョ ップ(ダ ー ウ ィ ソ系,紫 色 大 花)と パ ロ ッ ト ・ピア ス ソ(パ ロ ッ ト系,濃 赤 色 花)一 か らそ れ ぞ れ 色素 を結 晶状 に単 離 す る こ とが で きた ・ 前 者 か らは チ ュ リパ ニ ソ(デ ル フ ィ ニ ジ ソの ラ ム ノグ ル コ シ ド),後 者 か らは ケ ラ シア ニ ソ(シ ア ニ ジソ の ラム ノグ ル コ シ ド)が 得 られ た.な お, ペ ー パ ー一ク ロマ トグ ラ フ ィー お よび 分 光光 度 法 的 測 定 に よれ ば 前 者 の 花 に は4種 類,後 者 の 花 には3種 類 の ア ソ トシア ニ ソが 含 まれ て い る こ とが わ か った.(富 山 大 学 文 理 学部 生 物 学 教 室)