Phytochemistry, Vol. 29, No. 3, pp. 853-855, 1990 Printed in Great Britain.

0

A GALACTOMANNAN

FROM

ASHOK

MEDICAGINEA

CROTALARIA

K. GUPTA*~

0031 9422/90 s3.00t0.00 1990 Pergamon Press plc

SEEDS

JAMES N. BEMILLER

and

The Whistler Center for Carbohydrate Research, Purdue University, West

Lafayette, IN 47907, U.S.A.

(Received in revised form 27 July 1989)

medicaginea; Leguminosae; polysaccharide; galactomannan.

Key Word Index-Crotalaria

polysaccharide isolated from the seeds of Crotalaria medicaginea is composed of D-galactose and Dmannose in the molar ratio of 10:31. Structural studies were performed by methylation analysis, partial acid hydrolysis, chromic oxide oxidation, mild hydrolysis with dilute oxalic acid and 13C NMR analysis of the polymer.

Abstract-A

galactose and D-mannose in the molar ratio of 10: 31. The polysaccharide was fully methylated by the Haworth method [4], followed by the Hakomori method [S]. Hydrolysis of the permethylated polymer gave six partially methylated sugars (Table 1) and suggested a branched galactomannan structure having an average of 16 non-reducing end units per 41 main chain units. Partial, acid hydrolysis of the polysaccharide yielded: OS-I, fi-D-Man,-( l-+4)-D-Maq,; OS-II, a-D-Gal,-( l--+3)D-Man,; OS-III, cr-D-Gal,-( 1 +6)-D-Manp; OS-IV, a-DGal,-(1 -+6)-/&D-Man,-(1 -+4)-D-Manp and OS-V, B-DMan,-(1 +4)-B-D-Man,-( l-+4)-D-Man, (see also Table 1). Chromic oxide oxidation [6, 71 of the polymer suggested the presence of D-mannose in /?-(oxidized more rapidly) and D-galactose in cr-configuration. Mild hydrolysis with 0.02 M oxalic acid for 6.5 hr, released galactose only, suggesting that some galactose residues are in the furanose ring form [S]. After this treatment, the resulting polysaccharide had a galactose and mannose ratio of 10:30.8. In the 13CNMR spectrum of the polymer, the three anomeric signals at 6 100.9, 100.7 and 99.5 suggested the presence of cc-D-Gal,, D-D-Man, and tl-DGal, residues, respectively, and were in agreement with literature data [9, lo].

INTRODUCTION

Seeds of C. medicaginea are a rich source of a mucilaginous polysaccharide. In view of the medicinal value and the industrial use of similar plant gums, it was subjected to structural characterization.

RESULTS AND DISCUSSION

Extraction of the seeds of C. medicaginea with water furnished a crude polysaccharide which was purified by dialysis followed by ion-exchange resin treatment arid fractionated via its copper complex [ 11. The purified and homogeneous polymer gave a single peak on movingboundary electrophoresis [2] and gel-filtration chromatography [3], had [a]k”+23.6”, was water soluble, netural and free of nitrogen, sulphur, halogens, methoxyl groups, pentoses and uranic acids, and contained D-

*Author to whom correspondence should be addressed. tPresent address: Department of Chemistry, Shivaji University, Kolhapur 416004, India,

Table 1. Methylation analysis of the native polysaccharide (A) and the oligosaccharides (B) Mol proportion B

A

R&t Methylated sugar*

(i)

(ii)

2,3,4,6-Me,-Man 2,3,5,6-Me,-Gal 2,3,4,6_Me,-Gal 2,3,6-Me,-Man 2,4,6-Me,-Man 2,3,4-Me,-Man 2,6-Me,-Man

1.02 1.07 1.22 1.86 1.89 2.01 2.86

0.97 1.02 1.13 1.79 1.68 1.90 2.53

1.00 6.00 10.20 30.06 6.06

2,3-Me,-Man

3.61

3.21

9.96

I

II

III

IV

v

Mode of linkage

1 1 -l_--

1 -

1 -

1 1

1 2

_

-ll_.

_

-

-

-

-

-

-

-

Man,-( 1 + Gal&l+ Gal,-(1 + -+4)-Man,+ -+ -+3)-Man,-(l-+ +6)-Man,-(l+ +3,4)-Man,-(I-+ +4,6)-Man,-(l +

*The methylated sugars were identified as the corresponding alditol acetates. tR,s of the corresponding alditol acetates relative to that of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-o-glucitol, on a column of(i) SP-1000 at 190”and (ii) 3% of OV-225 at 190”;the values are identical with those of standard sugars. 553

A. K. GUPTA and J. N. BEMILLER

854

1 The structure 1 proposed for C. medicaginea galactomannan based upon the above findings is unique due to the presence of (l-3)-linked x-D-Gal, units and differs from the classical structure of most galactomannans of leguminous seeds [ 111. EXPERIMENTAL

Concn was performed under red. pres. at 40-50”. [a]p were recorded at 589 nm. Moving-boundary electrophoresis: 1% polysaccharide soln, 0.2 M Tris-HC1 buffer (pH 8.5), 5 hr, and at 12 V and 5 mA. Gel-filtration chromatography: Sephadex G100, polysaccharide soln. in H,O (2.5 mg/0.5 ml), flow rate I5 ml/hr, eluant H,O. Elutions were monitored by the PhOH-H,SO, method 1121. PC (descending): Whatman No. 1 and 3 MM papers in solvent systems (A) n-BuOH-EtOH-H,O (4: 1:5, upper layer), (B) n-BuOH-pyridine-H,O (6:4:3). Spray reagent: (a) alkaline AgNO,. IR: CHCI,. GC-FID: glass column (1.80 m x 2.0 mm) containing: (i) SP-loo0 at 200-220” (for neutral sugars) and at 190” (for partially methylated sugars); and (ii) 3% OV-225 at 190”. MS: 70 eV. Paper electrophoresis: 0.05 M borate buffer (pH 9.2) and at 400 V. “C NMR: 100.6 MHz, polysaccharide in D,O (50 mg/ml), 75, TMS as int. standard. Isolation and purification of the polysaccharide. The powdered seeds (135 g) were defatted, extracted with H,O at 50” for 10 hr. The isolation, purification and fractionation of the polysaccharide was carried out using the procedure of ref. [13], and gave 9.18 g; ash 0.11%. Characterization and estimation of sugars. Based on the results of pilot expts, the polysaccharide (30 mg) was hydrolysed with TFA (2.5 ml, 2 M) in a sealed tube for 14 hr at 100”. After removal of TFA, the components of the hydrolysate were resolved and analysed by PC using solvent (A) and spray reagent (a) and by GC and GC-MS of their alditol acetates 1133. DMannose, [a] i” + 13.9” (H,O; c 0.4); R, 24.89. D-Galactose, R, 27.63 was shown to be D by virtue of its oxidation with Dgalaclose oxidase (Sigma Co.) [14]. Mass fragments of the released sugars were in agreement with literature data [13]. DGalactose and D-Mannose were present in the molar ratio of 10: 31, as estimated by CC. Methylation of the polysaccharide. A dried sample of the polysaccharide (2OOmg) was methylated by the Haworth method [4] followed by the Hakomori method [5] to yield fully methylated polymer (no OH bands in the IR), yield 78 mg. Hydrolysis of the methylated polymer with H$O, [lS] gave a mixture of partially methylated sugars. Conversion of these into their alditol acetates, followed by CC (Table 1) and GC-MS indicated the presence of six partially methylated sugars. GC-MS: 2,3,4,6-Me,mannose: m/z (rel. int.): 205 (4) 161 (21) 145 (14), 129 (21), 117 (24) 101 (37), 87 (16) 71 (14), 45 (33), 43 (100). 2,3,5,6-Me,galactose: m/z(rel. int.): 205(19), 117(33), 101 (51), 89(34), 59 (21), 45 (25), 43 (100). 2,3,4,6-Me,-galactose: m/z(rel. int.): 205 (lo), 161 (22) 145(28), 129(24),117(40),101(40),87(16),71(12),45(38),43 (100). 2,3,6-Me,-mannose: m/z (rel. int.): 233 (15) 117 (48) I I3 (18) 101 (20), 99 (17). 87 (17) 45 (20) 43 (100). 2.6-Me,-mannose:

m/z (rel. int.): 129 (16) 117 (63),87 (8),45 (13) 43 (100). 2,3-Me,mannose: m/z (rel. int.): 261 (6). 117 (43). 101 (IS), 43 (100). Partial hydrolysis. Based on the results of pilot expts, the polysaccharide (2.0 g) was hydrolysed with HISO, (60 ml, 0.025 M) at 85 + 2” for 13 hr. PC using solvent (B) indicated the presence of o-galactose, o-mannose and five oligosaccharides. The isolation of the oligosaccharides (in pure form) and their characterization were done using the procedures of ref. 1131. Methylation analysis of each oligosaccharide is shown in Table 1. OS-I, R,,, 0.83; [r]p-8.07 (HZ; c 0.8): DP 1.98. On acid hydrolysis, it gave b-mannose only. OS-II. R,,, 0.76: [x]; +98.6’ (H,O; c 0.6); DP 2.01. Acid hydrolysis gave t,-galactose and b-mannose (I : I); NaBH, reduction and subsequent hydrolysis afforded galactose only. OS-III, R,,, 0.65; [r]p + 121.3> (H,O; c 0.6); DP 2.03. Acid hydrolysis yielded D-galactose and Dmannose (1 : I); NaBH, reduction, followed by hydrolysis gave galactose only. OS-IV. R,,, 0.41: [r]:h+36.6“ (H,O; c 1.0); DP 3.0. Acid hydrolysis gave o-galactose and p-mannose (1: 2). NaBH, reduction, followed by partial hydrolysis gave a disaccharide identical to OS-III. indicating presence of a galactose moiety at the non-reducing end of the trisaccharide. OS-V, R,,, 0.15; [c(]k -21.7 (H,O; c 0.9); DP 3.02. Acid hydrolysis gave Dmannose only. CrO, oxidation of the polysaccharide. The polysaccharide (20 mg) was dispersed in formamide-pyridine (2: 1, 14 ml) by vigorous stirring for 2 hr. Ac,O (5 ml) was added and stirred for 25 hr at room temp. The reaction mixture was added to H,O (40 ml), dialysed and freeze-dried. CrO, oxidation was carried out using the method of ref. [6] with myoinositol as int. standard and analysed by GC (as the alditol acetates). The ratios of myoinositol, mannose, and galactose in the control and oxidized material were 120: 87: 28 and 1000: 8:89. respectively. Mild hydrolysis of the polysaccharide with oxalic acid. The polysaccharide (600 mg) was hydrolysed with oxalic acid (60 ml, 0.02 M) at 100” for 6.5 hr, followed by complete hydrolysis with 2 M TFA in the usual manner. After work-up using the method of ref. 1161, the neutral sugars (as their alditol acetates) were analysed and estimated by GC, in hourly collected aliquots.

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