Oligosaccharides structure by partial acid hydrolysis from Rauwolfia serpentina Benth . seeds polysaccharide

Upon partial acid hydrolysis of water soluble Rauwolfia serpentina Benth. seeds polysaccharide gave a mixture of three disaccharides and one trisaccharide as : 6-Oα-D-mannopyranosyl-(1→6)-Oα-D-mannopyranose (I); 6-O-α-D-glucopyranosyl-(1→6)-O-α-D-glucopyranose (II); 4-O-β-Dglucopyranosyl-(1→4)-O-β-D-mannopyranose (III) and 4-O-β-D-mannopyranosyl-(1→4)-O-β-Dmannopyanosyl-(1→4)-O-β-mannopyranose (IV).

paper afforded three disaccharides and one trisaccharide.These oligosaccharides were characterised by its optical rotation, formation of crystalline derivatives (disaccharides only), degree of polymerization 6 , reduction with sodium borohydride, complete acid hydrolysis and periodate oxidation studies 7

EXPERIMENTAL
Separation of oligosaccharides from the hydrolysed compound of Rauwolfia serpentina Benth.water soluble seeds polysaccharide was carried out by descending technique on Whatman No. 3 MM filter paper sheet by paper chromatography 8 .The upper phase of following solvent mixture (v/v) were used for the detection of monosaccharide and oligosaccharides as : (A) nbutanol-acetic acid-water (4:1:5) 9 , (B) ethyl acetateacetic acid-water (9:2:2) 10 and (C) ethyl acetatepyridine water (10:4:3) 11 .The spray reagent (R) panisidine phosphate was used for the detection of monosaccharide and oligosaccharides from the hydrolysed compound of polysaccharide.All evaporation of oligosacchrides was carried out under reduced pressure at 40-50 0 C. The optical rotation values were recoded after equilibrium and melting points are uncontrolled.The Rgal and Rglu refer to the rate of movement of sugars releative to D-galactose and D-glucose respectively.Oligosaccharides mixtures were separated on charcoal-celite column (1:1, w/w) using water followed by 2.5, 5.0, 7.5 and 10.0% (v/v) with aqueous ethanol as eluants.These eluants were further separated by paper chromatography on Whatman No. 3 MM filter paper sheet.The degree of polymerization (DP) 13 was determined by Timell's method and Deionisation was done with Amberlite ion-exchange resins 14 , IR-45 (OH -) and IR-120 (H + ).

Partial acid hydrolysis of oligosaccharides
After a series of trial experiments the following method was carried out for the partial acid hydrolysis 15 to obtain the oligosaccharides.Polysaccharide of Rauwolfia serpentina Benth.seeds (18 gm) was hydrolysed with sulphuric acid (1.5 N, 500 ml) for 24 hrs at 4.5°C in refrigerator then the content was heated for 50 min.over boiling water bath.The obtained hydrolysate was cooled, filtered and neutralized with barium carbonate slurry.It was again filtered and filtrate concentrated to a small volume about 30 ml.Ethanol about 450 ml was added with the help of mechanical stirrer when the degraded polysaccharides was precipitated out as white coarse power after filtration then dried.The paper chromatographic analysis 5 of the hydrolysate syrup on Whatman No. 3 MM filter paper sheet in solvent mixture (A) obtained after concentration of ethanolic extract showed the presence of D-glucose and Dmannose and a member of oligosaccharides.The degraded polysaccharide was again hydrolysed by keeping it in sulphuric acid (1.5 N, 600 ml) at room temperature for 72 hrs.It was heated on boiling water bath for 1 hr followed by cooling it in the same bath for 30 min then concentrated to a syrup.The syrup was deionised with Amberlite ion exchange resin, IR-45 (OH -) and IR-120 (H -).

Separation of oligosaccharides
The oligosaccharides were seprated 16 by chromatographic adsorption method on charcoalceilte (1:1, w/w) glass column (60 x 2.5 cm) using the graded elution method 4 .The Column was eluted with water (2 liters) under 7 lbs/sq.inch pressure to remove the monosaccharides then successively with 3 liters each of 2.5, 5.0, 7.5 and 10.0 % aqueous ethanol (v/v) as eluant.Oligosaccharides fraction (100 ml) was concentrated and then examined by paper chromatographic analysis on Whantman No. 3 MM filter paper sheet in solvent mixture (A) and used (R) as spray reagent.The corresponding sugar strips of oligosaccharides 17 were cut out with the help of guide spots and eluted with water according to the Dent's method then finally concentrated to a thin syrup.It is observed that each fraction was not a single component but a mixture of 4 oligosaccharides.It led to the isolation of oligosaccharides were identified as 3 disaccharides 17 and one trisaccharide 17 19 .Degree of polymerization was found to be 2.28 indicating that this oligosaccharide was a disaccharide.Acid hydrolysis of disaccharide with sulphuric acid (1N) showed the presence of D-mannose sugars only which was identified by paper chromatographic analysis.Derivatives of disaccharide (60 mg) was prepared by usual manner as 6-O-α-D-mannopyranosyl-Dmannopyranose-octa-acetate, had m.p. 151-152°C, Lit.m.p. 152-153°C 19 .
Periodate oxidation of disaccharide (60 mg) was carried out with sodium metperiodate at 4-8 0 C for 55 hrs.It consumed 5.86 moles of periodate oxidant and liberated 3.28 moles of formic acid per mole of disaccharide and results are given in table 1. α -D -g l u c o py r a n o s y l -( 20 and having m.p. 182-184°C.Acid hydrolysis with sulphuric acid (1N) by usual manner to obtained the hydrolysate which was paper chromatographically examined to show the presence of D-glucose only as determined by phenol sulphuric acid method 21 .Degree of polymerization was formed to be 1.86 indicating that this oligosaccharide was a disaccharide.Derivatives of oligosaccharide was prepared by usual manner as phenyl osazone having m.p. 174-176°C 22 .Peridate oxidation 23

-O -β -D -g l u c o p y r a n o s y l -( 1 → ) -O -β -Dmannopyranose
Sugar syrup (280 mg) had Rgal 0.64 in solvent (B) and Rglu 0.53 in solvent (C), optical rotation [α] D 24 + 15.8°C (H 2 O) and had m.p. 193-195°C.The DP was found to be 1.86 which indicating that the oligosaccharide was a disaccharide.Oligosaccharide was hydrolysed with sulphuric acid (1N) and subsequent paper chromatography of the hydrolyzate indicated that the presence of monosaccharide, D-glucose and D-mannose are in equal amount 21 .The phenyl hydrazone derivative 22 of disaccharide (50 mg) was prepared by usual manner having m.p. 192-194°C 19 .The periodate oxidation of oligosaccharide (60 mg) by usual manner consumed 5.98 moles of periodate and liberated 3.60 moles of formic acid per mole of disaccharide after 55 hrs and results are shown in table 3.  24 .Degree of polymerization was found to be 3.14 indicating that the oligosaccharide was a trisaccharide.Methylation studies of trisaccharide by Hakomari's method on paper chromatography gave presence of 2,3,4,6-tetra-O-methyl-D-mannose and 2,3,6-tri-O-methyl-D-mannose which showed (1→4)-β-type linkages by emulsin.On periodate oxidation studies of the trisaccharide by usual manner consumed 6.18 moles of periodate and liberated 3.56 moles of formic acid per moles of trisaccharide after 55 hrs and results are shown table 4.