(+)-Pinitol as a major soluble carbohydrate in soybean plant

A large number of investigations have been carried out on the carbohydrates of legumes (1, 2), but only a few on the functions of individual carbohydrates. For studies on the symbiotic nitrogen fixation system of leguminous plants, it would be very important to determine the role of carbohydrates as energy suppliers in the nodules. We analyzed the soluble carbohydrates in soybean plants with gas-liquid chromatography (GLC) and found that the stem contained an unknown compound as abundant as sucrose. It was thought that unknown material could possibly be (+)-pinitol reported by Phillips and Smith (3) and Streeter et al. (4, 5). However the method applied by these investigators did not enable to identify this compound as (+)-pinitol due to the lack of data on IH-NMR and optical activity. We identified the compound by using gas-liquid chromatography, mass spectrometry and other physicochemica1 methods. 1. Gas-liquid chromatography (GLC). Soybean plants (Glycine max. cv. Norin- 2) were inoculated with Rhizobium japonicum (NIAS J-501) and grown in water culture. After harvest, the stems were separated and dried at 80°C. Ten grams of dried stems were extracted with 80% ethanol. The crude extract was evaporated in vacuum and filled up to 50 ml with distilled water. An aliquot of the solution was dried up completely in a small vial, trimethylsilylated with 0.1 ml N-TMS-imidazole pyridine solution, and analyzed by GLC. TMS-derivatives dissolved in pyridine were separated by the column (2 m) filled with 3% OV-17 on Chromosorb W(HP) 80/100 mesh, at 120°C with graded temperatures up to 280°C at 5°C/min and identified with a hydrogen ionization detector Figure 1 shows a chromatogram of the TMS-derivatives of the crude extract. Several organic acids, fructose, an unknown compound (Unknown 1), glucose, minor unknown compound (Unknown 2), myo-inositol, and sucrose were detected. A portion of the crude extract (17 ml) was passed through a column of Oowex 1-X8 (OH-) (6 cm × 8 mm 1.0.). The eluted fraction (17 ml) was collected. trimethy-lsilylated and analyzed by GLC. As shown in Fig. 2 the eluted fraction contained the Unknown compound 1, tbe Unknown compound 2 as tbe minor component, and myo-inositol. With this column, glucose, fructose, and sucrose were absorbed and not eluted.