Stero-Bile Acids and Bile Sterols* XLVI. Isolation of a New Bile Sterol, 3ƒ, 7ƒ, 12ƒ -Trihydroxy-26, 27-Epoxycholestane from Carp Bile

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1 The Journal of Biochemistry, Vol. 52, No. 2,1962 Stero-Bile Acids and Bile Sterols* XLVI. Isolation of a New Bile Sterol, 3ƒ, 7ƒ, 12ƒ -Trihydroxy-26, 27-Epoxycholestane from Carp Bile By TAKAHIKO HOSHITA (From the Department of Biochemistry, Hiroshima University School of Medicine, Hiroshima) (Received for publication, April 7, 1962) Our recent investigations have verified that bile sterols in bile of bull frog and toad are all derived from cholesterol (1, 2). These substances which consist of polyhydroxy steroid with cholane nucleus are considered as intermediates in the conversion of chole sterol to normal bile acid. In 1955, Has1e wood isolated a new bile sterol, cyprinol, from bile of the family Cyprinidae, which may be bile steroid with allocholane nucleus, but the chemical constitution of cyprinol is not clear (3, 4). In this communication, the isolation of a new bile sterol, 3ƒ, 7ƒ, 12ƒ -trihydroxy-26, 27-epoxycholestane from carp bile is reported. MATERIALS AND METHODS Carp bile was extracted with 95%o ethanol and the ethanol extract was evaporated to dryness under a reduced pressure. The residue was hydrolyzed with 2.5 N sodium hydroxide by heating in a sealed metal container for 8 hours at 160 C. The hydrolyzate was poured into a large amount of water to separate bile sterol mixture and then filtered. The residue was subjected to reversed phase partition chromatography according to the same method as previously described (5). Hostalen was used as supporting material for stationary phase and the chromatography was carried out at constant temperature of 23 C. The effluents were collected in test tubes by using an automatic fraction collector. Concentrated sulfuric acid was added to suitable aliquots of the effluents and after an hour standing at room temperature the concentra tion of the sterol was measured by extinction at 380 mp (6). The method of S j ova 11 was used for paper chromatography of bile sterol (7). * XLV Enomoto, S., J. Biochem., 52, 1 (1962) 3). 125 RESULTS 1) Isolation of the New Bile Sterol and Its Chemical Constitution One gram of the hydrolyzed bile sterol mixture was chromatographed with phase system A, and four peaks appeared as shown in Fig. 1. The effluents of the third curve (from 600 ml. to 1000 ml.) were combined and evaporated to dryness. The resulting gelati nous residue was crystallized from ethanol to give semicrystals which indicated a positive Hammarsten test and did not decolor bromine in glacial acetic acid. This material was acetylated with acetic anhydride and pyridine at room temperature to give crystals with m.p. 158 C. The analytical data of the pro duct were in accord with a diacetylated sterol. Analysis Calcd. for C31H50O6 C H 9.72 Found C H 9.52 The acetate was hydrolyzed with 2% methanolic sodium hydroxide by the usual method to give the starting material as semi crystalline form. Infrared spectrum of the new sterol indi cated the presence of allocholic acid nucleus (4) and a characteristic peak at 971cm-1 for the four-membered oxide ring in the molecule (Fig. 2A). Paper chromatogram of this mate rial showed the lower Rf value than that of 3ƒ, 7ƒ, 12ƒ, 26-tetrahydroxycoprostane (5) in the phase system consisting of 70% acetic acid as stationary phase and ethylene chlo ride : heptane (60: 40) as moving phase (Fig.

2 126 T. HOSHITA EFFLUENT (ml.) FIG. 1. Chromatography of hydrolyzed bile sterol of carp. Column, 90 g. of Hostalen ; phase system A. FIG. 3. Paper chromatogram of the new bile sterol obtained from the third curve in Fig. 1 (A), the tetra hydroxy compound (B) (m.p. 232 Ž) obtained by litium aluminum hydride from the new bile sterol, authentic 3ƒ, 7ƒ, 12ƒ, 26- (C) and 3ƒ, 7ƒ, 12ƒ, 24- tetrahydroxycoprostanes (D). Moving phase, isopropyl ether : heptane 60; 40; stationary phase 70% acetic acid. sterol in 10 ml. of glacial acetic acid, were added dropwise a solution of 100 mg. of chromic anhydride in one ml. of water and 10 ml. of glacial acetic acid. After being allowed to stand at room temperature for an hour, the reaction mixtur was diluted with a large amount of water to obtain a precipitate. Recrystallization of the preci pitate from methanol-water gave crystals with m.p. 187 Ž which was insoluble in aqueous WAVE NUMBER (Cm-1) FIG. 2. Infrared spectra of the new bile sterol obtained from the third curve in Fig. 1 ; A, the oxidation product (m.p. 187 C); B and the tetrahydroxy compound (232 C); C. Chromic Anhydride Oxidation of the New Bile Sterol-To a solution of 100 mg. of the new alkaline solution. Infrared spectrum of this dehydrogenated compound exhibited no hydroxyl group and the presence of the oxide ring (Fig. 2B). Analysis Calcd. for C27H40O4 C H 9.41 Found C H 9.35 To a solution of 50 mg. of the above dehydrogenated compound in absolute etha-

3 Stero-Bile Acids' and Bile Sterols. XLVI 127 nol, the alcohol solution of hydroxylamine hydrochloride and sodium acetate was added. The mixture was refluxed on a water bath for 5 hours, diluted with water to obtain white crystalline precipitate. Recrystalliza tion of this precipitate from ethanol-water yielded fine crystals with m.p. 235 C. Analysis Calcd. for C27H4304N3 N 8.65 Found N 8.23 Litium Aluminum Hydride Reduction of the New Sterol-To a solution of 500 mg. of the new sterol in dry tetrahydrofuran, 500 mg. of litium aluminum hydride suspended in the same solvent were added and then the reac tion mixture was refluxed on a water bath for 8 hours, The reaction mixture was de composed with ice cold dilute sulfuric acid, and the resulting precipitate was filtered and washed with water. Recrystallizatian of the precipitate from methanol gave 350 mg. of fine rods with m.p. 232 C. Infrared spectrum of this compound exhibited loss of the absorp tion at 971 cm-1 (Fig. 2C). This material and 3ƒ, 7ƒ, 12ƒ, 26-tetrahydroxycoprostane ran at the same rate on paper in the phase system as described Above (Fig. 3). Analysis Calcd. for C27H48O4 C H Found C H Chromic Anhydride Oxidation of the Material with m.p. 232 C-To a solution of 200 mg. of the above sterol with m.p. 232 C in 3 ml. of glacial acetic acid, a solution of 300 mg. of chromic anhydride in 3 ml. of acetic acid was added at room temperature. The oxida tion product thus obtained was dissolved in an aqueous solution of sodium carbonate and then acidified with dilute hydrochloric acid to obtain a precipitate. Recrystallization of the precipitate from aqueous methanol gave crystals with m.p. 207 C. This acid was proved to be a monocarboxylic acid by titra tion of 0.02 N methanolic potassium hydroxide, and assumed to be triketocholestanic acid- Analysis Calcd. for C27H40O5 C H 9.09 Found C H 9.06 Molecular weight Calcd, for C27H40O5 465 Found 446 The methyl ester obtained by esterifica tion of the above acid by esterified with ethereal diazomethane solution was treated with hydroxylamine by the analogous as des cribed above. The oxim obtained was recry stallized from ethanol-water to give crystals with m.p Ž. Analysis Calcd. for C28H45O5N3 N 8.34 Found N 8.30 Chromic Anhydride Oxidation of the Acetylated New Sterol-One gram of the new sterol was acetylated with acetic anhydride and sodium acetate by heating on a water bath as usual. To the acetylated material dissolved in 2(~ ml. of glacial acetic acid, one gram of chromic anhydride in acetic acid were added and heated on a water bath at 70 C for an hour. The reaction mixture was then poured into. a large amount of water, filtered and extract ed with ether. The ether extract was shaken with an aqueous sodium carbonate solution- The alkaline solution was acidified with dilute hydrochloric acid and extracted with ethyl acetate. The ethyl acetate extract was. washed with water and evaporated to dry ness. The residue was hydrolyzed with 1.5 N sodium hydroxide, solution. The paper chromatogram of the acid mixture thus ob tained gave two spots showing the same R?? value as cholic and homocholic acids respec tively (Fig. 4). The acid mixture were sub jected to column chromatography by usingphase system C. The chromatogram showedtwo curves as shown in Fig. 5. The effluentṣ from 400 ml. to 600 ml. were combined and evaporated to dryness. The residue was recrystallized from ethyl acetate to give crystalswith m.p C. This crystals showed a positive Hammarsten test and ran on paper chromatogram at the same rate as cholic acid. Infrared spectrum of the acid methyl ester was identical with that of methyl allo cholate synthesized by Anderson and. Haslewood (8). Analysis Calcd. for C24H4005 C H Found C H The effluents from 700 ml. to 1000 ml. of-

4 128 T. HOSHITA the curve in Fig. 5 were combined and evaporated to dryness. The residue was esterified with ethereal diazomethane solution. The resulting methyl ester was recrystallized from methanol to give crystals with m.p C. Paper chromatogram of this materi al and methyl homocholate showed the same R?? value. Its infrared spectrum showed the presence of allocholic acid nucleus. (2) Isolation of the other Sterols from Carp Bile The effluents of the first curve in Fig. 1 were combined and evaporated to dryness. The residue was crystallized from acetone, then methanol-ethyl acetate and finally from methanol to give rods melting at 242 C. This substance was identical with cyprinol previously isolated by Has 1ewood (3). The effluents of the second curve in Fig. I were combined and evaporated to dryness. The residue was recrystallized from methanol ethyl acetate to give crystals of the new sterol with m.p. 227 C. The other new sterol with m.p C was isolated from the effluents of the last curve in Fig. 1. DISCUSSION Four kinds of bile sterols were isolated from carp bile by using revesed phase column chromatography. Among these the sterol FIG. 4. Paper chromatogram of acidic substances obtained by chromic anhydride oxidation of the acetylated new bile sterol (X), authentic cholic (E) and homocholic acids (F). Moving phase, ethylene chloride : heptane 60: 40; stationary phase 70% acetic acid. (m.p. 242 C) obtained from the first peak in Fig. 1 was identical with cyprinol (3). Two of the new sterols isolated from the second and last curves in Fig. I melted at 227 and C respectively. All of these showed the presence of allocholic acid nucleus in the molecule by infrared spectrum. The other new bile sterol obtained from the third curve in Fig. 1 was characterized as 3ƒ, 7ƒ, 12ƒ -trihydroxy-26, 27-epoxy-cholestane (II), and gave a positive Hammarsten test and did not decolore bromine in glacial acetic acid solution. Infrared spectrum of this material showed the presence of allocho lic acid nucleus and a characteristic peak at 971 cm-1 for the four-membered oxide ring in the molecule. Analytical data of the di acetylated material (158 C) showed that the EFFLUENT (ml.) FIG. 5. Chromatography of acidic substances obtained by chromic anhydride oxidation of the acetylated new bile sterol. Column, 45 g. of Hostalen ; phase syftem C. new bile sterol has four oxygen atoms. De hydrogenated compound (187 C) (VI) derived from the new sterol by chromic anhydride oxidation possesses three ketone groups (tri oxime melting at 235 C) and the oxide ring in the molecule as proved by infrared spect rum.

5 Stero-bile Acids and Bile Sterols. XLVI 129 R'-COOH + R'-CH2-000H (N) (V) Litium aluminum hydride reduction of this new sterol gave a saturated neutral product melting at 232 C (III) in which the oxide ring has been lost as indicated by in frared spectrum. It showed the same R?? value as that of 3ƒ, 7ƒ, 12ƒ, 26-tetrahydroxycopro stane on paper chromatography and convert ed to the triketo monocarboxylic acid (m.p. 207 C) ( Z) by chromic anhydride oxidation. The above described data indicated that the oxide ring in the molecule of the new sterol was opened to a primary hydroxyl group by litium aluminum hydride to yield the tetra hydroxy compound. On the other hand the acetylated new bile sterol was oxidized with chromic anhy dride at 70 C to give both allocholic ( W) and allohomocholic acids (V). All these facts mentioned above clearly indicate that the new sterol consists of 3ƒ, 7ƒ, 12ƒ -trihydroxy-5ƒ -cholane nucleus and a four-membered oxide ring of a structure 6H2-O-CH,-G= in the end of the side chain, and may be 3ƒ, 7ƒ, 12ƒ -trihydroxy-26, 27-epoxycholestane (II) as shown in the follow ing formula. It was reported that on alkaline hydrolysis the natural scymnol sulfate will give anhy droscymnol which possesses four-membered oxide ring in the molecule (9, 10). This indeed suggested that the natural form of this new bile sterol might be 3ƒ, 7ƒ, 12ƒ, 26, 27-pentahydroxycholestane-sulfate-(26) (I). Chemical constitution of the other new bile sterols are now under further investiga tion. SUMMARY 1. Four kinds of bile sterols were isolated from carp bile by use of reversed phase partition chromatography. 2. It was verified that chemical constitution of one of the new bile sterols corresponds to 3ƒ, 7ƒ, 12ƒ -trihydroxy-26, 27-epoxy-cholestane. The author wishes to express his deep gratitude to Prof. T. Kazuno for his kind advice throughout this work. He also thanks Prof. G.A.D. Haslewood of Guy's Hospital Medical School, London for his generous help in identification of allocholic acid.

6 130 T. HOSHITA REFERENCE (1) Masui, T., J. Biochem., 51, 112 (1962) (2) Kazuno, T., and Okuda, K., J. Biochem., 50, 354 (1961) (3) Haslewood, G.A.D., Biochem. J., 59, xi (1955) (4) Briggs, T., and Haslewood, G.A.D., Biochem.,J., 82, 26p (1962) (5) Kazuno, T., Masui, T., and Hoshita, T., J. Biochem., 50, 12 (1961) (6) Kazuno, T., Seno, H., Goto, T., and Fujiwara, K.. J. Japan. Biochem. Soc. (in Japanese), 28, 1 (1956) (7) Sjovall, J., Acta Chem. Scand., 8, 339 (1954) (8) Anderson, I.G., and Haslewood, G.A.D., Bioch em. J., 81, 15p (1961) (9) Briggs, T., and Haslewood, G.A.D., Biochem. J., 79, 5p (1961) (10) Bridgewater, R.J., Briggs, T., and Haslewood, G.A.D., Biochem. J., 82, 285 (1962)

ON TEA TANNIN ISOLATED FROM GREEN TEA.

70 [Vol. 6 ON TEA TANNIN ISOLATED FROM GREEN TEA. By MICHIYO TSUJIMIIRA. (Received September 8th., 1930). The author(1) has recently isolated Tea catechin from green tea and pro posed the following formula