Some species of plants which belong to Asterales, Poales (Graminales), Liliales, Asparagas, and others contain fructan as a reserve carbohydrate (1). Three types of fructosylsucroses, isokestose (l-kestose, IF-.8-fructosylsucrose), kestose (6-kestose, 6F •.8-fructosylsucrose), and neokestose (6G-.8-fructosylsucrose) are key intermediates for the related highly polymerized oIigofructans (1, 2). Inulin is a higher homologous oligosaccharide of isokestose whose fructofuranosyl residues are (2-+1)-.8-linked, and phlein (or levan) is a higher homologue based on the kestose whose fructosyl residues are joined by a (2-+6)-, e-linkage. Furthermore, there are also highly polymerized oligofructans related to neokestose. In the members of Liliales, all, three types of fructans have been found (1). As for the tulip bulb, HAMMER investigated the trisaccharide from two species of tulips (Tulipa silvestris L. and Tulipa c/usiana), and the results showed that the tulip bulb appeared to contain mainly isokestose together with traces of neokestose based on the RP value obtained by thin-layer chromatography (TLC) (3). In this report, trisaccharide and tetrasaccharide fractions were extracted from the tulip bulb and their structure was determined by l3C-nuclear magnetic resonance spectroscopy (NMR) and methylation analysis whereby oligosaccharide is reduced by NaBD, (deuterium labeled NaBH,) in order to distinguish mass fragments of fructose derivatives originating from the three types of oligofructans. About 3 kg of bulb scales of tulip (Tulipa gesneriana L.) were extracted by 80% ethanol. The extract was concentrated to a syrup in vacuum, and diluted to 100 ml with water. The aliquot of sample was separated by siIicagel TLC with triple development using acetonitrile: water (83: 17, v/v) as a'solvent (Fig. lA). The standard solution which contained isokestose and its related oligofructans was spotted at the same time (Fig. 1 B). Diphenylamine-aniline-phosphate reagent (4) was sprayed and the col or of each spot of the bulb extract was the same as the corresponding standard fructan. Therefore, it was estimated that b, c, d, e shown in Fig. lA might be sucrose (Glucose-Fructose, GF), GFa, GFa, and GF, respectively, Thus the isolation of c and d was 'attempted by charcoal column chromatography. Charcoal (granular KLH, Takeda Chemical Industries, Ltd.) was preliminarily washed with 1 N HCI and packed in a column (3 x 40 cm). Tulip extract was eluted by two step linear gradient of ethanol (from 0 to 10%, and 10 to 30%) and every 16 ml of eluent was collected and its sugar content was measured by the phenol sui fate method. In the first elution, the separation of each saccharide was not complete, therefore, the eluents containing GF2, GFa, and GF, were condensed together, and then re-eluted by the same method. Figure 2 shows the results of the second elution of the bulb extract. The peaks-I, 2, and 3 were developed by TLC (Fig. IC, D, E), and they showed the same color and RFvalue as b', c', d' of Fig. IB, respectively. An aliquot of peak-I, 2, 3 was measured by HPLC (Waters 730) equipped with DEXTROPAK (Waters) column which was eluted with water, and the RTs (retention time) of peak-I, 2, 3 were exactly the same as those of standard sucrose, isokestose and nystose.
