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Three flavonol allosides from Glaucidium palmatum
0031-9422/90 $3.00 +O.OO
Phytochemistry, Vol. 29, No. 11, pp. 3639-3641, 1990 Printedin Great Britain.
THREE FLAVONOL
Pergamon Press plc
ALLOSIDES
FROM GLAUCIDIUM
TSUKASA IWASHINA
PALMATUM
and SHUNJI OOTANI*
Tsukuba Botanical Garden, National Science Museum, Tsukuba-shi, Ibaraki 305, Japan; *Faculty of Bio-industry, Tokyo University of Agriculture, Abashiri-shi, Hokkaido 099-24, Japan (Received 6 April 1990)
Key Word Index-Glaucidium
palmatum; Glaucidiaceae; kaempferol3-alloside;
quercetin 3-alloside; rhamnocitrin
3-alloside; FABMS; ‘H NMR.
Abstract-Three flavonol allosides were isolated from the leaves of Glaucidium palmatum endemic to Japan. Their structures were established as quercetin, kaempferol and rhamnocitrin 3-O-/I-D-allosides. The two latter glycosides were also isolated from the flowers.
INTRODUCTION
Glaucidium palmatum Sieb. et Zucc. is the only member of Glaucidiaceae, a family which is endemic to the subcold zone of Japan. Although this plant has been included in the Ranunculaceae for a long time, it has been recently separated by Tamura [l] into its own family on morphological grounds. The coumarin, glaupalol has been isolated from the roots of G. palmatum [2, 31, but no flavonoids have been reported. In the present study, one new and two rare flavonol allosides were isolated from a methanolic leaf extract and identified as the 3-allosides of quercetin, kaempferol and rhamnocitrin (l-3). Kaempferol 3-alloside (2) and rhamnocitrin 3-alloside (3) were also isolated from the flowers. Flavonoid allosides have been reported from three fern genera, Osmunda, Wagneriopteris and Davallia C&6], five angiosperm genera, Stachys, Sideritis, Teucrium (Labiatae) [7-lo], Veronica (Scrophulariaceae) [ 11) and Thalictrum (Ranunculaceae) [12], but are reported for the first time in Gluucidium (Glaucidiaceae). RESULTS AND
DISCUSSION
Extraction of the leaves of Glaucidium palmatum gave three flavonoid glycosides (l-3). The UV spectrum of 1 showed the presence of free 5,7,3’ and 4’-hydroxyl groups [13, 141. The aglycone, obtained by both acid hydrolysis and H,O, oxidation, was identified as quercetin by direct PC comparison with an authentic sample. Paper chromatographic properties of the glycosidic sugar, which was attached to 3-hydroxyl were very similar to those of glucose in solvent systems, BAW, BEW and BTPW. Also, the FAB mass spectrum exhibited [M + Na] + at m/z 487. This data appeared to indicate that 1 might be quercetin 3-glucoside (isoquercitrin). However, the R, on paper and retention time on HPLC of 1 were different from those of authentic isoquercitrin. ‘HNMR data of the original glycoside, moreover, indicated five aromatic protons and a sugar anomeric proton (6 5.67, J = 7.8 Hz), which appeared at a lower field than that of authentic quercetin 3glucoside (65.46) and was close to that of quercetin 3alloside [S]. Finally, the glycosidic hexose was identified
as allose by direct PC comparisons (six solvent systems) with authentic allose, glucose, galactose and mannose. From the results described above, 1 was identified as quercetin 3-0-fi-D-alloside. Acid hydrolysis and H,O, oxidation of 2 gave kaempferol and allose which were identified by direct PC comparisons with authentic specimens. UV spectra of the glycoside (2) showed the presence of free 57 and 4 hydroxyl groups [13,14]. FABMS exhibited [M +Na]+ at m/z 471. The ‘H NMR spectrum indicated six aromatic protons and an allosyl anomeric proton (65.67, J = 7.9 Hz), and agreed with ‘H NMR data of the corresponding kaempferol 3-O-/I-D-alloside [4]. The UV spectra of 3 indicated a 5,4’-dihydroxy-3,7substituted flavonol [13, 141. Both acid hydrolysis and H,O, oxidation of 3 produced a sugar and an aglycone, which was shown to have free 35 and 4’ hydroxyl groups by UV spectra analysis, and six aromatic protons and a methoxyl proton by ‘HNMR. Therefore, the aglycone was identified as rhamnocitrin (kaempferol 7-methyl ether). The glycosidic sugar attached to 3-hydroxyl was identified as allose by direct comparison (co-PC) in six solvent systems with four authentic hexoses. The ‘H NMR of the glycoside (3) showed the presence of six aromatic protons, a methoxyl proton and an allosyl H-l proton (65.70, J=7.9 Hz). The FAB mass spectrum exhibited [M + Na]+ at m/z 485. From the results described above, 3 was identified as rhamnocitrin 3-0-/I-D-alloside. Of the three flavonol 3-allosides found, quercetin 3alloside (1) and kaempferol 3-alloside (2) have been reported previously from the fern, Wagneriopteris formosa Loeve et Loeve [S] and 2 has also been found in Osmunda asiatica Ohwi [4]. This is the first report of 1 and 2 in angiosperms. Rhamnocitrin 3-alloside (3) was found in nature for the first time.
EXPERIMENTAL General. ‘H NMR spectra were recorded in DMSO-d, using TMS as an int. standard. FABMS: measured using NBA +NaCI. HPLC: A finepak SIL C,sS, 5 pm (i.d. 4.6 mm x 15 cm) column equipped with a finepak C,sT-P precolumn was used. The
3639
3640
T.
IWASHINA
flavonoids were dissolved in MeOH, filtered through Chromatodisc 13N (0.45 pm), and eluted with MeCN-H,O-H,PO, (110: 390: 1). R,s (min) were: rhamnocitrin 3-alloside 20.51; kaempferol 3-alloside 20.16; quercetin 3-alloside 15.00, quercetin 3glucoside 13.72 and quercetin 3-galactoside 11.72. Plant material. Glaucidium palmatum Sieb. et Zucc. was collected on Mt. Togakushi, Nagano Pref., Japan. Isolation ofjauonols. Fresh leaves (ca 230 g) were extracted x 2 with MeOH (2 I), filtered and coned and after washing with petrol, the concentrate was shaken with EtOAc (400ml). The organic layer was evapd to dryness, the residue dissolved in MeOH (30 ml) and stood at 5” for 2 days when 3 was obtained as pale yellow needles. The MeOH soln left after filtering off the crystals of 3 was coned to small vol. and fractionated on a polyamide column with MeOH. Fractions containing 1 and 2 were coned to small vol. and 1 and 2 were obtained as pale yellow or yellow needles after cooling. Fresh flowers (25 g) were extracted with MeOH (ca 200 ml) and the concn extract applied to multiple PC in BAW (nBuOH-HOAc-H,O, 4: 1:5, upper phase) and 15% HOAc, and the bands corresponding to 2 and 3 eluted with MeOH. Compounds 2 and 3 were obtained as crystals from MeOH. Identification offlaoonols. Quercetin 3-O-p-D-alloside (1). UV ni$“’ nm: 259,265sh, 30lsh, 363; +NaOMe 272,329,407 (inc.); +AlCl, 275, 304sh, 34Osh, 436; +AICI,-HCI 269, 296sh, 359, 400; + NaOAc 273,324,395; + NaOAc-H,BO, 262,295sh, 379. PC R, 0.72 (BAW), 0.68 (TBA), 0.65 (BEW), 0.13 (5% HOAc), 0.37 (15% HOAc); dark purple under UV, yellow under UV (NH,). ‘HNMR (2OOMHz, DMSO-d,) 67.59 (2H, d, J =7.6 Hz, H-2’ and H-6’); 6.83 (lH, d, J=8.1 Hz, H-5’); 6.4O(lH, d, J=2.0 Hz, H-8); 6.20 (lH, d, J=2.2 Hz, H-6); 5.67 (lH, d, J = 7.8 Hz, allosyl H-l”); 3.2-3.9 (6H, m, H-2”, H-3”, H-4”, H-5” and H,-6”). ‘HNMR (DMSO-d,+D,O) 67.59 (2H, d, J =8.3Hz,H-2’,andH-6’);6.86(1H,d,J=8.1Hz,H-5’);6.44(1H, d, J=2.0 Hz, H-8); 6.22 (lH, d, J=2.2 Hz, H-6); 5.66 (iH, d, J = 7.8 Hz, allosyl H-l”); 3.3-3.9 (6H, m, H-2”, H-3”, H-4”, H-S’ and H,-6”). FABMS (NBA + NaCl) Found: m/z 487 [M + Na]+ and 464 CM]’ calcd for C,,H,,O,,. Authentic quercetin 3-0-glucoside (isoquercitrin). UV A!$“’ nm: 258,263sh, 3OOsh, 360; + NaOMe 272,330,408 (inc.); + AICI, 275, 303sh, 334sh, 436; + AlCl,/HCl 269, 296sh, 362, 402; + NaOAc 273,325,390; + NaOAc-H,BO, 262,294sh, 381. PC R, 0.63 (BAW), 0.62 (BEW), 0.38 (15% HOAc); dark purple under UV, yellow under UV (NH,). ‘HNMR (200 MHz, DMSO-d,) 67.58 (2H, d, J = 6.8 Hz, H-2’ and H-6’); 6.84 (lH, d, J =9.0Hz, H-5’); 6.40 (lH, d, J=1.7Hz, H-8); 6.20 (lH, d, J =2.0 Hz, H-6); 5.46 (IH, d, J = 7.3 Hz, glucosyl H-l”); 3.c3.7 (6H, m, H-2”. H-3”, H-4”, H-S’ and H,-6”). Kaempfirol 3-0-p-D-aUoside (2). IJV n!$‘” nm: 267, 3OOsh, 353; + NaOMe 275, 328,402 (inc.); + AlCI, 275, 306, 352, 399; + AlCI,-HCl 276, 304, 348, 396; +NaOAc 275, 309, 384; +NaOAc-H,BO, 268, 3OOsh, 354. PC R, 0.82 (BAW), 0.80 (TBA), 0.81 (BEW), 0.14 (5% HOAc), 0.36 (15% HOAc); dark purple under UV, dark greenish yellow under UV (NH,). ‘H NMR (400 MHz, DMSO-d,) 68.07 (2H, d, J=8.6 Hz, H-2 and H-6’); 6.88 (2H, d, 5=8.9 Hz, H-3’ and H-5’); 6.43 (lH, d, J=2.0Hz, H-8); 6.21 (lH, d, J=2.0Hz, H-6); 5.67 (1H. d, J =7.9 Hz, allosyl H-l”); 3.1-3.9 (6H, m, H-2”, H-3”, H-4”, H-S’ and H,-6”). FABMS (NBA+ NaCI) Found: m/z 471 [M +Na]+ and 448 [M]’ calcd for C,,HzoO,,. Rhamnocitrin (kaempferol7-methyl ether) 3-O-P-D-alloside (3). UV I f$‘” nm: 267,298sh, 352; + NaOMe 276,303sh, 350sh, 391 (inc); + AICI, 276,305,354,398; + AlCl,-HCI 276,303,348,396; + NaOAc 269,303sh, 359.406sh; + NaOAc-H,BO, 267,3OOsh, 352. PC R, 0.80 (BAW), 0.80 (TBA), 0.79 (BEW), 0.14 (5% HOAc), 0.41 (15% HOAc); dark purple under UV, dark greenish
and S. OOTANI
yellow under UV (NH,). ‘H NMR (400 MHz, DMSO-d,) 68.10 (2H, d, J = 8.9 Hz, H-2’ and H-6’); 6.89 (2H, d, J = 8.9 Hz, H-3 andH-5’);6.75(1H,d,J=2.3Hz,H-8);6.39(1H,d,J=2.0Hz,H6); 5.70 (lH, d, J = 7.9 Hz, allosyl H-l”); 3.87 (3H, s, OMe); 3.2-3.9 (6H, m, H-2”, H-3”, H-4”, H-5” and H,-6”). FABMS (NBA + NaCI) Found: m/z 485 [M + Na] + and 462 CM] ’ calcd for CZ~H,,OI,. Acid hydrolysis and H,O, oxidation of 3 gave rhamnocitrin and allose. rhamnocitrin. Yellow needles. UV A::$ nm: 255sh, 268,295sh, 328sh, 366; + NaOMe 273,33Osh, 423 (dec.); + AICI, 260sh, 271,305sh, 356,424; + AlCl,-HCI 260sh, 271,30Ssh, 353, 422; + NaOAc 270, 329sh, 378,408sh (dec.); + NaOAc-H,BO, 269, 329sh, 368. PC R, 0.91 (BAW), 0.84 (TBA), 0.05 (15% HOAc); yellow under UV, bright yellow under UV (NH,). ‘H NMR (400 MHz, DMSO-d,) 68.09 (2H, d, J = 8.9 Hz, H-2’ and H-6’); 6.95 (2H, d, J =8.9 Hz, H-3’ and H-5’); 6.75 (lH, d, J=2.0 Hz, H-8); 6.36 (lH, d, J=2.3 Hz, H-6); 3.87 (3H, s, OMe). Acid hydrolysis. Acid hydrolysis of the flavonol glycosides was performed in 12% HCI for 30 min at loo”. H,O, oxidation. To each flavonol. glycoside (ca 1 mg) was added 3 drops of 2 M NH,OH and 2-3 drops of H,O,. After standing for 2 hr the aglycone and sugar were obtained by evapn of excess H,Oz. Identijcation of sugars. Sugars were identified by direct PC comparison using 6 solvent systems, BAW (n-BuOH-HOAcH,O, 4: 1:5, upper phase), BEW (n-BuOH-EtOH-H,O, 4: 1:2.2), BTPW (n-BuOH-C,H,Me-pyridine-H,O, 5: 1:3:3), PhOH (PhOH satd with H,O), BPW (n-BuOH-pyridine-H,O, 6: 3: 1) and BBPW (n-BuOH-C,H,-pyridine-H,O, 5: 1: 3: 3) with authentic allose, glucose, galactose and mannose [14, 151. PC R,s of the glycosidic sugar of 1, 2 and 3: 0.15 (BAW), 0.13 (BEW), 0.23 (BTPW), 0.42 (PhOH), 0.23 (BPW) and 0.27 (BBPW); allose, 0.15 (BAW), 0.13 (BEW), 0.23 (BTPW), 0.42 (PhOH), 0.23 (BPW) and 0.27 (BBPW); glucose 0.13 (BAW), 0.13 (BEW), 0.23 (BTPW), 0.31 (PhOH), 0.26 (BPW) and 0.24 (BBPW); galactose 0.12 (BAW), 0.13 (BEW), 0.19 (BTPW), 0.38 (PhOH), 0.18 (BPW) and 0.20 (BBPW); mannose 0.17 (BAW), 0.18 (BEW), 0.28 (BTPW), 0.40 (PhOH), 0.29 (BPW) and 0.31 (BBPW). Acknowledgements-The authors express their sincere gratitude to Dr K&i, Hayashi (the Research Institute of Evolutionary Biology) for his interest and continuing support of this work. The Authors’ thanks are due also to Dr Fumiyuki Mitsumori (National Institute for Environmental Studies) and Ph.D. Shinyu Nunome (Tumura Lab.) for NMR measurements, and Miss Kazuko Akuzawa, Mr Kazutoshi Ozawa and Yahachiro Hori (Tsukuba Research Lab. Nippon Oil & Fats Co., Ltd) for FABMS measurements.
REFERENCES 1. Tamura, M. (1982) Wild Flowers of Japan. Herbaceous Plants-Choripetalue (Satake, Y., Ohwi, J., Kitamura, S., Watari, S. and Tominari, T., eds), p. 112. Heibonsya, Tokyo. 2. Irie, H., Kinoshita, K., Mizutani, H., Takahashi, T., Uyeo, S. and Yamamoto, K. (1968) Yakugaku Zasshi 88, 627. 3. Yamamoto, K., Irie, H. and Uyeo. S. (1971) Yakugaku Zasshi 91, 257. 4. Okuyama, T., Hosoyama, K., Hiraga, Y., Kurono, G. and Takemoto, T. (1978) Chem. Pharm. Bull. 26, 3071. 5. Murakami, T., Satake, T., Hirasawa, C., Ikeno, Y., Saiki, Y. and Chen, C.-M. (1984) Yakugaku Zasshi 104, 142. 6. Hwang, T.-H., Kashiwada, K., Nonaka, G.-I. and Nishioka, I. (1989) Phytochemistry 28, 891.
Allosides from Glaucidium palmatum 7. Lenherr, A., Lahloub, M. and Sticher, 0. (1984) Phytochemistry 23, 2343. 8. Rabanal, R. M., Valverde, S., Martin-Lomas, M., Rodriguez, B. and Chari, V. M. (1982) Phytochemistry 21, 1830. 9. Barberan, F. A. T., Tomas, F. and Ferreres, F. (1985) .I. Nat. Prod. 48,28.
10. Harborne, J. B. and Williams, C. A. (1988) Flauone and Flavonol Glycosides in The Flavonoids Advances in Research Since 1980. (Harborne, J. B., ed.), pp. 303-328. Chapman & Hall, London. 11. Chari, V. M., Grayer-Barkmeijer. R. J., Harborne, J. B. and
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bsterdahl, B. G. (1981) Phytochemistry 20, 1977. 12. Shimizu, E., Tomimatsu, T. and Nohara, T. (1984) Chem. Pharm. Bull. 32, 5023. 13. Mabry, T. J., Markham, K. R. and Thomas, M. B. (1970) 7’he Systematic Identification of Flavonoids. Springer, Berlin. 14. Markham, K. R. (1982) Techniques of Flavonoid Identijication. Academic Press, London. 15. Harborne, J. B. (1984) Phytochemical Method. A Guide to Modern Techniques of Plant Analysis 2nd Edn, p. 225. Chapman & Hall, London.
Phytochemistry, Vol. 29, No. 11, pp. 3639-3641, 1990 Printedin Great Britain.
THREE FLAVONOL
Pergamon Press plc
ALLOSIDES
FROM GLAUCIDIUM
TSUKASA IWASHINA
PALMATUM
and SHUNJI OOTANI*
Tsukuba Botanical Garden, National Science Museum, Tsukuba-shi, Ibaraki 305, Japan; *Faculty of Bio-industry, Tokyo University of Agriculture, Abashiri-shi, Hokkaido 099-24, Japan (Received 6 April 1990)
Key Word Index-Glaucidium
palmatum; Glaucidiaceae; kaempferol3-alloside;
quercetin 3-alloside; rhamnocitrin
3-alloside; FABMS; ‘H NMR.
Abstract-Three flavonol allosides were isolated from the leaves of Glaucidium palmatum endemic to Japan. Their structures were established as quercetin, kaempferol and rhamnocitrin 3-O-/I-D-allosides. The two latter glycosides were also isolated from the flowers.
INTRODUCTION
Glaucidium palmatum Sieb. et Zucc. is the only member of Glaucidiaceae, a family which is endemic to the subcold zone of Japan. Although this plant has been included in the Ranunculaceae for a long time, it has been recently separated by Tamura [l] into its own family on morphological grounds. The coumarin, glaupalol has been isolated from the roots of G. palmatum [2, 31, but no flavonoids have been reported. In the present study, one new and two rare flavonol allosides were isolated from a methanolic leaf extract and identified as the 3-allosides of quercetin, kaempferol and rhamnocitrin (l-3). Kaempferol 3-alloside (2) and rhamnocitrin 3-alloside (3) were also isolated from the flowers. Flavonoid allosides have been reported from three fern genera, Osmunda, Wagneriopteris and Davallia C&6], five angiosperm genera, Stachys, Sideritis, Teucrium (Labiatae) [7-lo], Veronica (Scrophulariaceae) [ 11) and Thalictrum (Ranunculaceae) [12], but are reported for the first time in Gluucidium (Glaucidiaceae). RESULTS AND
DISCUSSION
Extraction of the leaves of Glaucidium palmatum gave three flavonoid glycosides (l-3). The UV spectrum of 1 showed the presence of free 5,7,3’ and 4’-hydroxyl groups [13, 141. The aglycone, obtained by both acid hydrolysis and H,O, oxidation, was identified as quercetin by direct PC comparison with an authentic sample. Paper chromatographic properties of the glycosidic sugar, which was attached to 3-hydroxyl were very similar to those of glucose in solvent systems, BAW, BEW and BTPW. Also, the FAB mass spectrum exhibited [M + Na] + at m/z 487. This data appeared to indicate that 1 might be quercetin 3-glucoside (isoquercitrin). However, the R, on paper and retention time on HPLC of 1 were different from those of authentic isoquercitrin. ‘HNMR data of the original glycoside, moreover, indicated five aromatic protons and a sugar anomeric proton (6 5.67, J = 7.8 Hz), which appeared at a lower field than that of authentic quercetin 3glucoside (65.46) and was close to that of quercetin 3alloside [S]. Finally, the glycosidic hexose was identified
as allose by direct PC comparisons (six solvent systems) with authentic allose, glucose, galactose and mannose. From the results described above, 1 was identified as quercetin 3-0-fi-D-alloside. Acid hydrolysis and H,O, oxidation of 2 gave kaempferol and allose which were identified by direct PC comparisons with authentic specimens. UV spectra of the glycoside (2) showed the presence of free 57 and 4 hydroxyl groups [13,14]. FABMS exhibited [M +Na]+ at m/z 471. The ‘H NMR spectrum indicated six aromatic protons and an allosyl anomeric proton (65.67, J = 7.9 Hz), and agreed with ‘H NMR data of the corresponding kaempferol 3-O-/I-D-alloside [4]. The UV spectra of 3 indicated a 5,4’-dihydroxy-3,7substituted flavonol [13, 141. Both acid hydrolysis and H,O, oxidation of 3 produced a sugar and an aglycone, which was shown to have free 35 and 4’ hydroxyl groups by UV spectra analysis, and six aromatic protons and a methoxyl proton by ‘HNMR. Therefore, the aglycone was identified as rhamnocitrin (kaempferol 7-methyl ether). The glycosidic sugar attached to 3-hydroxyl was identified as allose by direct comparison (co-PC) in six solvent systems with four authentic hexoses. The ‘H NMR of the glycoside (3) showed the presence of six aromatic protons, a methoxyl proton and an allosyl H-l proton (65.70, J=7.9 Hz). The FAB mass spectrum exhibited [M + Na]+ at m/z 485. From the results described above, 3 was identified as rhamnocitrin 3-0-/I-D-alloside. Of the three flavonol 3-allosides found, quercetin 3alloside (1) and kaempferol 3-alloside (2) have been reported previously from the fern, Wagneriopteris formosa Loeve et Loeve [S] and 2 has also been found in Osmunda asiatica Ohwi [4]. This is the first report of 1 and 2 in angiosperms. Rhamnocitrin 3-alloside (3) was found in nature for the first time.
EXPERIMENTAL General. ‘H NMR spectra were recorded in DMSO-d, using TMS as an int. standard. FABMS: measured using NBA +NaCI. HPLC: A finepak SIL C,sS, 5 pm (i.d. 4.6 mm x 15 cm) column equipped with a finepak C,sT-P precolumn was used. The
3639
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T.
IWASHINA
flavonoids were dissolved in MeOH, filtered through Chromatodisc 13N (0.45 pm), and eluted with MeCN-H,O-H,PO, (110: 390: 1). R,s (min) were: rhamnocitrin 3-alloside 20.51; kaempferol 3-alloside 20.16; quercetin 3-alloside 15.00, quercetin 3glucoside 13.72 and quercetin 3-galactoside 11.72. Plant material. Glaucidium palmatum Sieb. et Zucc. was collected on Mt. Togakushi, Nagano Pref., Japan. Isolation ofjauonols. Fresh leaves (ca 230 g) were extracted x 2 with MeOH (2 I), filtered and coned and after washing with petrol, the concentrate was shaken with EtOAc (400ml). The organic layer was evapd to dryness, the residue dissolved in MeOH (30 ml) and stood at 5” for 2 days when 3 was obtained as pale yellow needles. The MeOH soln left after filtering off the crystals of 3 was coned to small vol. and fractionated on a polyamide column with MeOH. Fractions containing 1 and 2 were coned to small vol. and 1 and 2 were obtained as pale yellow or yellow needles after cooling. Fresh flowers (25 g) were extracted with MeOH (ca 200 ml) and the concn extract applied to multiple PC in BAW (nBuOH-HOAc-H,O, 4: 1:5, upper phase) and 15% HOAc, and the bands corresponding to 2 and 3 eluted with MeOH. Compounds 2 and 3 were obtained as crystals from MeOH. Identification offlaoonols. Quercetin 3-O-p-D-alloside (1). UV ni$“’ nm: 259,265sh, 30lsh, 363; +NaOMe 272,329,407 (inc.); +AlCl, 275, 304sh, 34Osh, 436; +AICI,-HCI 269, 296sh, 359, 400; + NaOAc 273,324,395; + NaOAc-H,BO, 262,295sh, 379. PC R, 0.72 (BAW), 0.68 (TBA), 0.65 (BEW), 0.13 (5% HOAc), 0.37 (15% HOAc); dark purple under UV, yellow under UV (NH,). ‘HNMR (2OOMHz, DMSO-d,) 67.59 (2H, d, J =7.6 Hz, H-2’ and H-6’); 6.83 (lH, d, J=8.1 Hz, H-5’); 6.4O(lH, d, J=2.0 Hz, H-8); 6.20 (lH, d, J=2.2 Hz, H-6); 5.67 (lH, d, J = 7.8 Hz, allosyl H-l”); 3.2-3.9 (6H, m, H-2”, H-3”, H-4”, H-5” and H,-6”). ‘HNMR (DMSO-d,+D,O) 67.59 (2H, d, J =8.3Hz,H-2’,andH-6’);6.86(1H,d,J=8.1Hz,H-5’);6.44(1H, d, J=2.0 Hz, H-8); 6.22 (lH, d, J=2.2 Hz, H-6); 5.66 (iH, d, J = 7.8 Hz, allosyl H-l”); 3.3-3.9 (6H, m, H-2”, H-3”, H-4”, H-S’ and H,-6”). FABMS (NBA + NaCl) Found: m/z 487 [M + Na]+ and 464 CM]’ calcd for C,,H,,O,,. Authentic quercetin 3-0-glucoside (isoquercitrin). UV A!$“’ nm: 258,263sh, 3OOsh, 360; + NaOMe 272,330,408 (inc.); + AICI, 275, 303sh, 334sh, 436; + AlCl,/HCl 269, 296sh, 362, 402; + NaOAc 273,325,390; + NaOAc-H,BO, 262,294sh, 381. PC R, 0.63 (BAW), 0.62 (BEW), 0.38 (15% HOAc); dark purple under UV, yellow under UV (NH,). ‘HNMR (200 MHz, DMSO-d,) 67.58 (2H, d, J = 6.8 Hz, H-2’ and H-6’); 6.84 (lH, d, J =9.0Hz, H-5’); 6.40 (lH, d, J=1.7Hz, H-8); 6.20 (lH, d, J =2.0 Hz, H-6); 5.46 (IH, d, J = 7.3 Hz, glucosyl H-l”); 3.c3.7 (6H, m, H-2”. H-3”, H-4”, H-S’ and H,-6”). Kaempfirol 3-0-p-D-aUoside (2). IJV n!$‘” nm: 267, 3OOsh, 353; + NaOMe 275, 328,402 (inc.); + AlCI, 275, 306, 352, 399; + AlCI,-HCl 276, 304, 348, 396; +NaOAc 275, 309, 384; +NaOAc-H,BO, 268, 3OOsh, 354. PC R, 0.82 (BAW), 0.80 (TBA), 0.81 (BEW), 0.14 (5% HOAc), 0.36 (15% HOAc); dark purple under UV, dark greenish yellow under UV (NH,). ‘H NMR (400 MHz, DMSO-d,) 68.07 (2H, d, J=8.6 Hz, H-2 and H-6’); 6.88 (2H, d, 5=8.9 Hz, H-3’ and H-5’); 6.43 (lH, d, J=2.0Hz, H-8); 6.21 (lH, d, J=2.0Hz, H-6); 5.67 (1H. d, J =7.9 Hz, allosyl H-l”); 3.1-3.9 (6H, m, H-2”, H-3”, H-4”, H-S’ and H,-6”). FABMS (NBA+ NaCI) Found: m/z 471 [M +Na]+ and 448 [M]’ calcd for C,,HzoO,,. Rhamnocitrin (kaempferol7-methyl ether) 3-O-P-D-alloside (3). UV I f$‘” nm: 267,298sh, 352; + NaOMe 276,303sh, 350sh, 391 (inc); + AICI, 276,305,354,398; + AlCl,-HCI 276,303,348,396; + NaOAc 269,303sh, 359.406sh; + NaOAc-H,BO, 267,3OOsh, 352. PC R, 0.80 (BAW), 0.80 (TBA), 0.79 (BEW), 0.14 (5% HOAc), 0.41 (15% HOAc); dark purple under UV, dark greenish
and S. OOTANI
yellow under UV (NH,). ‘H NMR (400 MHz, DMSO-d,) 68.10 (2H, d, J = 8.9 Hz, H-2’ and H-6’); 6.89 (2H, d, J = 8.9 Hz, H-3 andH-5’);6.75(1H,d,J=2.3Hz,H-8);6.39(1H,d,J=2.0Hz,H6); 5.70 (lH, d, J = 7.9 Hz, allosyl H-l”); 3.87 (3H, s, OMe); 3.2-3.9 (6H, m, H-2”, H-3”, H-4”, H-5” and H,-6”). FABMS (NBA + NaCI) Found: m/z 485 [M + Na] + and 462 CM] ’ calcd for CZ~H,,OI,. Acid hydrolysis and H,O, oxidation of 3 gave rhamnocitrin and allose. rhamnocitrin. Yellow needles. UV A::$ nm: 255sh, 268,295sh, 328sh, 366; + NaOMe 273,33Osh, 423 (dec.); + AICI, 260sh, 271,305sh, 356,424; + AlCl,-HCI 260sh, 271,30Ssh, 353, 422; + NaOAc 270, 329sh, 378,408sh (dec.); + NaOAc-H,BO, 269, 329sh, 368. PC R, 0.91 (BAW), 0.84 (TBA), 0.05 (15% HOAc); yellow under UV, bright yellow under UV (NH,). ‘H NMR (400 MHz, DMSO-d,) 68.09 (2H, d, J = 8.9 Hz, H-2’ and H-6’); 6.95 (2H, d, J =8.9 Hz, H-3’ and H-5’); 6.75 (lH, d, J=2.0 Hz, H-8); 6.36 (lH, d, J=2.3 Hz, H-6); 3.87 (3H, s, OMe). Acid hydrolysis. Acid hydrolysis of the flavonol glycosides was performed in 12% HCI for 30 min at loo”. H,O, oxidation. To each flavonol. glycoside (ca 1 mg) was added 3 drops of 2 M NH,OH and 2-3 drops of H,O,. After standing for 2 hr the aglycone and sugar were obtained by evapn of excess H,Oz. Identijcation of sugars. Sugars were identified by direct PC comparison using 6 solvent systems, BAW (n-BuOH-HOAcH,O, 4: 1:5, upper phase), BEW (n-BuOH-EtOH-H,O, 4: 1:2.2), BTPW (n-BuOH-C,H,Me-pyridine-H,O, 5: 1:3:3), PhOH (PhOH satd with H,O), BPW (n-BuOH-pyridine-H,O, 6: 3: 1) and BBPW (n-BuOH-C,H,-pyridine-H,O, 5: 1: 3: 3) with authentic allose, glucose, galactose and mannose [14, 151. PC R,s of the glycosidic sugar of 1, 2 and 3: 0.15 (BAW), 0.13 (BEW), 0.23 (BTPW), 0.42 (PhOH), 0.23 (BPW) and 0.27 (BBPW); allose, 0.15 (BAW), 0.13 (BEW), 0.23 (BTPW), 0.42 (PhOH), 0.23 (BPW) and 0.27 (BBPW); glucose 0.13 (BAW), 0.13 (BEW), 0.23 (BTPW), 0.31 (PhOH), 0.26 (BPW) and 0.24 (BBPW); galactose 0.12 (BAW), 0.13 (BEW), 0.19 (BTPW), 0.38 (PhOH), 0.18 (BPW) and 0.20 (BBPW); mannose 0.17 (BAW), 0.18 (BEW), 0.28 (BTPW), 0.40 (PhOH), 0.29 (BPW) and 0.31 (BBPW). Acknowledgements-The authors express their sincere gratitude to Dr K&i, Hayashi (the Research Institute of Evolutionary Biology) for his interest and continuing support of this work. The Authors’ thanks are due also to Dr Fumiyuki Mitsumori (National Institute for Environmental Studies) and Ph.D. Shinyu Nunome (Tumura Lab.) for NMR measurements, and Miss Kazuko Akuzawa, Mr Kazutoshi Ozawa and Yahachiro Hori (Tsukuba Research Lab. Nippon Oil & Fats Co., Ltd) for FABMS measurements.
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