J. A H C . FoodChem. 1001, 39, 1709-1714
1700
Composition and Molecular Species of Ceramide and Cerebroside in Scarlet Runner Beans (Phaseolus coccineus L.) and Kidney Beans (Phaseolus vulgaris L.) Michiyuki Kojima,' Masao Ohnishi,' and Seisuke Ito' Department of Bioresource Chemistry, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080, Japan
Ceramide and cerebroside were isolated from scarlet runner bean (Phaseolus coccineus L.) and kidney bean (Phaseolus vulgaris LJ, and their components and principal molecular species were studied. In the ceramide of the two beans, three types of normal, 2-hydroxy, and 2,3-dihydroxy fatty acids were observed and showed a ratio of about 25:74:1. The major component of the fatty acid was P-hydroxylignoceric acid, while the major component in the sphingoid was 4-hydroxy-trans-8-sphingenine.In cerebroside of the two beans, normal and 2-hydroxy fatty acids were observed, the latter showing more than about 90%. The major fatty acid of cerebroside was 2-hydroxypalmitic acid, while the major and trans-4,cis-8-sphingadieninea The sugars of both sphingoids were trans-4,trans-8-sphingadienine cerebrosides were almost glucose. From GC-MS and reversed-phase HPLC data, the major ceramide and species in the two beans was found to be N-2'-hydroxylignoceroyl-4-hydroxy-trans-8-sphingenine, the major cerebroside was found to be l-O-~-glucosyl-N-(2'-hydroxypalmitoyl)-trans-4,trans-8-sphingadienine. The major ceramide residues in the cerebroside from the two Phaseolus beans were similar to those of soybean and Adzuki bean but different from that of pea bean, which were mainly N-2'hydroxypalmitoyl-trans-8-sphingenine residues.
INTRODUCTION
It is well-known that glycolipids in animals, mostly sphingolipids, are membrane components and have important physiologicalactivities (Karlason, 1982). In higher plants, three series of glycolipids, namely glyceroglycolipids, sphingoglycolipids, and steryl glycosides, are widely known (Fujino, 19831, and we found not only the wellknown mono- and diglycosyl types but also the oligoglycosy1 types with up to five sugar chains, from rice (Fujino, 1983; Fujino et al., 1985a), wheat (Ohnishi et al., 1985; Fujino et al., 1985b), maize (Tanaka et al., 1984), and Adzuki bean (Kojima et al., 1989, 1990). Recently, plant glycolipids were also reported to have physiological effects (Okuyama and Yamazaki, 1983; Sakata and Ina, 1983), and we reported that certain molecular species of cerebrosides from wheat grain were active upon the fruiting of Schizophyllum commune (Kawai et al., 1986). The most remarkable characteristic in plant cerebroside was reported to be the structural variety of the ceramide residues according to plant species (Ohnishi and Fujino, 1981, 1982; Ito et al., 1985a). But the properties of ceramide and cerebroside species in the similar family have not yet been characterized. In this paper we describe the components and the molecular species of ceramides and cerebrosides in two near species of the Phaseolus genus, scarlet runner bean (Phaseolus coccineus L.) and kidney bean (Phaseolus vulgaris L.),in which phosphatidylcholine and phosphatidylethanolamine species resembled but triacylglycerol species differed from mutually (Sasaki et al., 1989). Moreover, the molecular species of cerebrosides were compared with those among several bean seeds. MATERIALS AND METHODS Isolation of Ceramides and Cerebrosides. Commercially available scarlet runner bean (P.coccineus L., 200 g) and kidney bean (P.vulgaris L., 100 g) seeds were ground to powders, immediately steamed to deactivate the enzymes under boiled water, and extractedthree times eachwith600mL of chloroform-
methanol (2:l v/v) and water-saturatedbutanol for 2 h of severe shaking, respectively. After the combined extracts were evaporated to dryness by use of a rotary evaporator, the residue was dissolved in 180mL of a chloroform-methanol solution (2:l v/v) and partitioned according to the method of Folch (Folch et al., 1957) to get total lipids. The total lipids were treated with 0.4 N KOH in methanol and then sonicated for 4 h to remove contaminating glycerolipids. The alkaline-stable lipids, which were prepared for a Folch's partition, were then applied by silicic acid column chromatography using the chloroform and methanol system. The crude ceramide and cerebroside fractions were eluted with chloroform-methanol from 982 and 95:5 (v/v) and chloroform-methanol from 90:lOto 80:20 (v/v), respectively. The individualsphingolipid fractions were further purified by silicic acid column chromatography and by acetylation followed by preparative thin-layer chromatography (TLC) with subsequent deacetylation (Fujino et el., 1985a; Ohnishi et al., 1985). Analyses of Fatty Acids. Ceramide (3mg) and cerebroside (3 mg) were heated at 100 O C under reflux with 1.5 mL of 5% methanolic HCl for 4 h. The solution was then cooled and extracted with hexane. The hexane phase was washed with an equivalent amount of water, evaporated to dryness, and then subjected to TLC on silica gel G in hexane-diethyletheracetic acid (80301 v/v) to separate normal and hydroxy fatty acid methyl esters. They were then analyzed by gas-liquid chromatography (GLC) as reported (Tanaka et al., 1984; Fujino et al., 1985b). Analyses of Sugars. The rest of the methanolic solution which extracted the fatty acid methyl ester as previously described was made alkaline(pH9-10) with 7 N NaOH solution and washed with diethyl ether to remove the sphingoids. The solution was deionized by passage through ion-exchangeresin (Dowex 50, H+ type; Amberlite IR-C, OH- type) columns. The eluate solutions were evaporated to dryness to yield the constituent sugar as methyl glycosides. Moreover, half of the methyl glycosides was hydrolyzed in 1 mL of 1 N HCl for 3 h at 100 "C. After the solution was cooled, it was deionized through an ion-exchange resin (Amberlite IR-C, OH- type) column. After the eluate solution was evaporated to dryness, NaBH4 (10 mg) and 1 mL of water were added for reduction and left for 1 h at room temperature. To finish the reduction, the excess material (NaBH4) was dissolved by using a few drops of acetic acid and then 0 1991 American Chemical Society
Kojlma et el.
17lQ J. Agrlc. FoodChem., Vol. 39,No. I O , I991
evaporated with methanol to remove the acetic acid. The residue (alditols) was heated a t 100 "C under reflux with 1mL of acetic anhydride for 2 h to be changed to alditol acetate. The methyl glycosides and alditol acetates were analyzed by GLC as reported (Fujino et al., 1985b; Ohnishi et al., 1985). Analyses of Sphingoids. Cerebrosides (5 mg) were hydrolyzed with 5 mL of 1 N aqueous HCl in methanol for 18 h at 70 "C. The ceramides (3 mg) were heated at 100 "C under reflux with 3 mL of 1 N KOH in methanol for 20 h. Each reaction solution was cooled and washed with hexane four times. The methanol phase of the remainder was made alkaline (pH 9-10) with 6 N KOH solution, and then the sphingoids were extracted with an equivalent volume of 3 mL of diethyl ether three times, washed, and evaporated to dryness. The sphingoids were solubilized in 1mL of methanol and then added to 0.1 mL of 0.2 M NaIO, solution. The mixture was then oxidized for 1h a t room temperature. The fatty aldehyde induced from the sphingoids was extracted with hexane, washed, and evaporated to dryness. Moreover, half of the aldehyde was dissolved with 2 mL of chloroform-methanol (1:3 v/v) and then added to 2 mL of 0.1 M NaOH solution which included 1mg of NaBH,. The mixture was then left for 2 h. After the reaction, Folch's partition (Folch et al., 1957) was done and the lower phase was evaporated to dryness to yield the fatty alcohol. Analyses of Molecular Species of Ceramides and Cerebrosides. To examine the molecular species of the ceramides, the ceramides and cerebrosides were changed to trimethylsilyl ether derivatives, which was performed on a gas chromatographmass spectrometer (GC-MS) (Hitachi RMU-6MG instrument) (Ohnishi and Fujino, 1982;Fujino et al., 1985b). Moreover, the underived cerebrosides were analyzed by reversed-phase highperformance liquid chromatography to separate the geometric isomer. Infrared Spectrometer. Infrared spectra were measured on an infrared spectrometer (IR-3Atype, Nippon Bunko-KogyoCo., Ltd., Tokyo) using KBr pellets with 1 mg of lipid to 100 mg of KBr. Gas-Liquid Chromatography (GLC). GLC analyses were performed on Hitachi 063 and 163 gas chromatographs (Hitachi Seisakusho Co., Ltd., Tokyo), fitted with flame ionization detectors, and the carrier gas was high-purity nitrogen used at a flow rate of 30-40 mL/min. The normal fatty acid methyl esters and fatty aldehydes were analyzed on a 3 mm X 2 m glass column of 5% DEGS on 80-100-mesh ChromosorbW-AW-DMCS (Gaskuro Kogyo Inc., Tokyo) at 180 and 140 "C, respectively. The hydroxy fatty acid methyl esters (from 180 to 290 "C a t 2 "C/min), methyl glycosides (at 160 "C), sphingoids (at 190 "C), and fatty alcohols (at 175 "C) were analyzed as trimethylsilyl ether derivatives on a 3 mm x 2 m glass column of 3% silicone SE30 on 80-100-mesh Chromosorb W-AW-DMCS(Gaskuro Kogyo). The hydroxy fatty acid methyl esters were identified by comparison with authentic esters. The alditol acetates were then analyzed on a 3 mm X 2 m glass column of 3% ECNSS-M on 80-100 mesh Chromosorb W-AW-DMCS (Nihonchromato Inc., Tokyo) at 190 "C. Trimethylsilyl ether derivatives of ceramides and cerebrosides were analyzed on a 3 mm x 1 m glass column of Diasolid ZT (180-100 mesh, Nihonchromato). Column temperatures were from 200 to 260 "C at 3 "C/min for the ceramides and from 250 to 300 "C a t 5 "C/min for the cerebrosides. Gas-Liquid Chromatography-Mass Spectrometry (GCMS). GC-MS analyses were performed on the Hitachi RMU6MG instrument and interfaced with the Hitachi 0002B-8DK computer data system. The trimethylsilyl ether derivatives of the ceramides and cerebrosides were analyzed on a Diasolid ZT column (3 mm X 1m) at 300 and 330 "C, respectively. All mass spectra were taken under the same conditions as described in a previous paper (Ohnishi and Fujino, 1982). High-Performance Liquid Chromatography (HPLC). HPLC analyses were done with a Shimadzu Model 6A instrument (Shimadzu Co., Kyoto). Reversed-phase HPLC was performed by using an Inertail ODs-2 column (250 X 4.6 mm, Gaskuro Kogyo). A variable-wavelength spectromonitor Model SPD-6Awas used a t 220 nm for the underivatized cerebroside at 40 "C. Methanol-water (25:l v/v) was used as eluent at a flow rate of 1 mL/min.
Table I. Composition (Percent) of Fatty Acids of Ceramides and Cerebrosides in Scarlet Runner Beans and Kidney Bean Seeds ceramide cerebroside kidney scarlet kidney fatty scarlet runner bean bean runnerbean bean acid 160 2.7 8.6 3.6 4.6 161 0.2 0.3 0.1 0.7 0.6 3.0 0.3 0.7 180 0.6 1.4 0.2 0.4 18:l 0.7 0.4 0.3 0.2 182 0.5 0.9 0.2
