Structural studies of a polysaccharide isolated from an edible mushroom, Astraeus hygrometricus

Indian Journal of Chemistry Vol. 398, July 2000, pp. 525 - 529

Structural studies of a polysaccharide isolated from an edible mushroom, Astraeus hygrometricus

Amar Pramanik & Syed Sirajul Islam*

Department of Chemistry and Chemical Technology, Vidyasagar Uni versi ty, Midnapore 721102, Indi a.

Received 26 September 1997; accepted (revised) 15 No vember 1999

The polysaccharide (PS) isolated from the hot aqueous extract of the mushroom, Astraeus hygrometricus cor1tains D­galactose, o-glucose and o-galacturonic acid in a mole ratio of about 1.9:0.9: 1.0. From the periodate oxidation and methylation studies of PS , the linkages among the constituent sugar residues are identified as, D-Galr ( 1,6)-linked, D-Galr (1,2,6)-li nked, o-Gler (1,3)-linked and o-Galp UA is glycosidically linked. The linkages have been confirmed by partial acid hydrolysi s studies of PS fo llowed by GLC analysis. The anomeric configurations of the constituent sugar residues have been determined by chromium trioxide oxidation of the acetylated PS fo llowed by GLC analysis of the surviving sugar residues and it has been found that galactose and galacturonic acid have P-configuration and glucose has a-configuration.

Based on the above studies, the structure of the repeating unit of PS has been assigned as:

--6 )-p-o-Galp-( 1- 6)-P-o-Gal,-( 1-3 )-a-o-Gle,-( I­I (2-1)-P-o-Gal, UA

The mushroom, Astraeus hygrometricus grows on the laterite soil bed of the forest belt of Bankura and Midnapore districts , West Bengal. It is abundantly available during the months of Aug.-Sept. and local people consume it as edible mushroom. No work has been reported so far on this mushroom. A detailed investigation has been carried out on the polysaccharide (PS) isolated from the aqueous extract of this mushroom and reported in thi s paper.

Results and Discussion

The lyophilized PS, 120 mg, had [a]~, 0°. Total

sugars (98.7%) were estimated by phenol-sulphuric acid method 1 and acid sugar (25%) was estimated by carbazole-sulphuric acid mcthod2

. The polysaccharide was hydrolyzed by 2M, trifluoroacetic acid with inositol as internal standard. Paper chromatographic analys is of the hydrolysed PS showed the presence of D-glucose, D-galactose and D-galacturonic acid. Alditol acetates of the hydrolysate, on GLC analysis, showed 24.4% D-glucose, 49% D-galactose and D­galacturonic acid, 25%. Carboxyl-reduced PS, on hydrolysis followed by GLC analysis of the corresponding alditol acetates, showed the presence of D-glucose and D-galactose in the mole ratio of

almost 1:3. The increase in the ratio of galactose further indicates that D-galacturonic acid moiety is also present in PS.

For detailed structural investigation of PS, it was methylated by Ciucanu and Kerek3 method foll owed by Purdie method4 and then hydrolysed. The alditol acetates of the hydrolysate were prepared and examined by GLC using column (A) and column (B) and found to contain 2,3,4-tri-0-methyi-D-galactose, 2,4,6-tri-0-methyl-D-glucose and 3,4-di-0-methyi-D­galactose in a mole ratio of about 1.0:0.9:0.9 (Table 1). These results indicate that one D-galacto­pyranosyl moiety is (I ,6)-linked and D-glucopyra­nosyl moiety is (I ,3)-linked. The other D-galactopyra­nosyl moiety is (1,2,6)-linked. The methylated PS was reduced by lithium-aluminium-hydride and the alditol acetates of the reduced methylated sug9-rs were analyzed by GLC using columns (A) and (B); the peaks of 2,3,4-tri-0-methyi-D-galactose, 2,4,6-tri-0-methyl-D-glucose and 3,4-di-0-methyi-D-galactose were obtained. A significant observation is that , in this case, the peak of 2,3,4-tri-0-methyi-D-galactose has become almost double than that before reduction . It therefore reveals that D-galacturonic acid is glycosidicall y linked.

526 INDIAN J CHEM, SEC B, JULY 2000

Table !-Methylation analysis of PS, carboxyl-reduced PS 1md oligosaccharides

Methylated Retention time• Sugar Column A Column B

2,3 ,4-Tri-0-methyi-D-galactose 3.41 2.90

3,4-Di-0-methyl-o-galactose 6.93 5.50

2,4,6-Tri-0-methyi-D-glucose 1.95 1.82

3,4,6-Tri-0-methyl-o-galactose 2.50 2.15

2,3,4,6-Tetra-0-methyi-D-glucose 1.00 1.00

Mole Proportion PS RPS '0'-1

1.0 1.9 1.0

0.9 0.9

0.9 0.9

1.0

'0'-2

0.9

1.0

Mode of '0'-3 linkages

1.0 -6)-GalP-(1-GalP UA-(1-

-6)-Galp-(1-12

-3)-Gicp-(1-

1.0 -2)-GaiP-(1-

•Retention times relative to that of 1,5-di-0-acetyl-2,3,4,6-tetra-0-methyi-D-glucitol on column A (3% ECNSS-M) at 170°C and column B (I% OV -225) at 165°C; RPS=methylated reduced PS ; 0-2 and 0-3 are reduced oligosaccharides obtained from partial hydrolysis of PS.

The periodate-oxidized and then reduced PS followed by hydrolysis was found to contain only o-glucose residue on GLC analysis indicating that the two galactose moieties, present in the PS molecule has been destroyed during oxidation . A part of the periodate-oxidized PS, on hydrolysis showed the absence of D-galacturonic acid on PC examination, which supports that D-galacturonic acid present in the PS molecule has also been destroyed during oxidation. The periodate-oxidized material was subjected to Smith-degradation5

. The Smjth-degraded product on PC analysis and GLC examination showed the presence of only D-glucose and glycerol, further indicating the degradation of two D-galactose units and one D-galacturonic acid moiety, during oxidation.

Partial acid hydrolysis of PS by 0.5M CF3COOH gave three oligomers having Rr (GLC) 0.80, 0.66 and 0.46 along with D-galacturonic acid and D-galactose in solvent system A. Oligomer- 1 (0-1) on hydrolysis and methylation showed that o-galactose and o­glucose units are present in it in a mole ratio of 1.0:0.9 and it is 6-0-D-glucopyranosyl-D-galactose. Oligomer-2 (0-2) and Oligomer-3 (0-3) were purified by passing through a column of Sephadex G-50 and found to be homogeneous on PC examination. 0-2 on hydrolysis followed by PC examination showed the presence of o-galactose and o­galacturonic acid. Preparation of alditol acetates of the hydrolysate and its examination by GLC showed o-galactose as the only sugar present in it. 0-2 was reduced with diborane6 and the acid hydrolysis of the neutral oligomer showed only the presence of D­galactose, but the amount was double compared to

that obtained before reduction. This indicates that 0 -2 is an aldobiuronic acid. Methylation analysis of aldobiuronic acid followed by usual treatments showed a single peak of 3, 4, 6-tri-0-methyi-D­galactose. In another experiment, the methylated aldobiuronic acid was reduced with lithium­aluminium-hydride7. The product was hydrolysed and the hydrolysate was analyzed by GLC (as alditol acetates) . 3,4,6-Tri-0-methyl-o-galactose and 2,3,4-tri-0-methyl-o-galactose were obtained in the ratio of 1.0:0.9. All these observations lead to the conclusion that 0-2 is an aldobiuronic acid having the structure 2-0-(D-galactopyranosyl uronic acid)-D-galactose.

Oligomer-3 (0-3), Rr (GLC) 0.46, on hydrolysis followed by PC analysis showed the presence of D­glucose, D-galactose and o-galacturonic acid. The hydrolysate on GLC examination showed 0-glucose and D-galactose in the mole ratio of l: l. 0-3 on reduction with diborane followed by GLC examination showed the ratio of D-galactose to D­glucose as 1.8: 1.0. The increase in the amount of o­galactose indicates that 0-3 contains a o-galacturonic acid moiety and also it is an aldotriuronic acid. GLC examination of methylated 0-3 showed the presence of 2,4,6-tri-0-methyi-D-glucose and 3,4,6-tri-0-methyl-o-galactose in almost equimolar ratio. The methylated carboxyl-reduced product of 0-3, on GLC examination showed the above peaks along with the appearance of a new peak due to 2,3 ,4-tri-0-methyi­D-galactose which is coming from the glycosidically linked galacturonic acid moiety. From these observations, the structure of 0-3 may be given as:

D-Galr UA-( l-2)-D-Galp-( 1-3)-D-Gic".

PRAMANIK eta/.: STUDIES ON POLYSACCHARIDE FROM ASTRAEUS HYGROMETRICUS 527

Table 11-Survival of sugarsb in the oxidation of acetylated PS and carboxyl reduced PS (from Astraeus hygrometric us) with

chromium trioxide

Material Oxidation Galactose Glucose period (hr)

PS 0 49.6 24.9 I 20.9 21.5 2 13.2 19.1

2.5 6.1 15.0

Carboxyl-reduced PS 0 72.8 24.8 I 30.4 21.7 2 18.1 18.9

2.5 10.8 15.1

b (per cent) myo-inositol was used as internal standard (per cent).

From the results of chromium trioxide oxidation" of acetylated PS (Table II) , it was found that galactose and ga!acturonic acid have the ~-configurations and glucose has a-configuration.

Thus from the methylation, periodate oxidation, partial acid hydrolysis and chromium trioxide oxidation studies of PS, isolated from the mushroom Astraeus hygrometricus, the tentative structure of the repeating unit may be assigned as:

OH ""' H CHz

H H ==-60 0

H H ~0H OH~H HO~C~O~H OHH O"' u= ...

H OH

H H --6)-~-o-Galp-( 1-6)-~-o-Galp-( 1-3)-a-o-Gicp-( I­

I (2-1)-~-o-Ga!P UA

Experimental Section General. All evaporations were conducted at 40 °C

unless stated otherwise. Paper partition chromato­graphy was performed on Whatman Nos.1 and 3 mrn sheets. Solvent systems (v/v) used were: (A) 1-butanol-acetic acid-water (4: 1 :5) (upper phase) and

(B) ethyl acetate-pyridine-water (8:2: 1); the spray reagent used was alkaline silver nitrate solution6

• All solvents were distilled before use. Optical rotations were measured on a Perkin-Elmer Model 241 MC spectropolarimeter. Colorimetric estimations were conducted on a Systronic UV-VIS spectrophotometer, Model-108.

Gas-liquid chromatography (GLC) was performed on a Hewlett-Packard Model 5730A gas chromato­graph having a flame ionization detector and glass columns ( 1.83x6 mrn) with 3% of ECNSS-M on Gaschrom Q ( 100-120 mesh) and 1% OV -225 on Gaschrom Q (100-120 mesh). All GLC analyses were conducted at 170°C for neutral sugars and 165°C for methylated sugars by converting the sugars into their alditol acetates. Retention times of partially methylated alditol acetates were measured with respected to that of 1 ,5-di-0-acetyl-2, 3, 4, 6-tetra-0-methyl-o-glucitol as unity.

Preparation of aqueous extract. The wet uncoated mushroom (350 g) was swollen in 150 ml water and heated at 60 °C for 30 min. The mixture was centrifuged at 10,000 rpm for I hr. The aqueous extract was collected and lyophilized, yield 190 mg. The material (160 mg) was extensively dialyzed to remove monosaccharides and other low molecular weight materials. The polysaccharide of this mushroom was collected from inside of the dialysis bag. The volume was reduced to 20 mL and treated with ethanol in 1:10 (v/v) ratio. The precipitate was collected and lyophilized, yield 130 mg.

P urification of the polysaccharide. The poly­saccharide (110 mg) was purified by passing through a column of Sephadex G-50 using water as eluent; sixty fractions were collected and Moli' s8 test was performed and monitored spectrophotometrically at 490 nm using phenol sulphuric acid method. Fractions 11 to 30 were collected and lyophilized, yield 90 mg.

Homogeneity of the polysaccharide was established by high voltage electrophoresis in borate­buffer in a Shandon Model L-24 apparatus.

Acid hydrolysis of the polysaccharide. The polysaccharide (2.1 mg) was hydrolyzed with 2M CF3COOH for 18 hr at 100 °C. The acid was completely removed by co-distillation with water and the hydrolysate divided into two parts. One part was examined by PC in solvent systems A and B; spots for D-galactose, D-glucose and D-galacturonic acid were detected. The other part of the filtrate was

528 INDIAN J CHEM, SEC 8, JULY 2000

reduced by NaBH4 (40%) and the alditol acetates were prepared in the usual way and analyzed by GLC (colunms A and B) (Table I).

Preparation of carboxyl-reduced PS. To a solution (7 mL) of PS (7 mg) in water, was added 1-cyclohexyl-3-(2-morpholinoethyl) - carbo-di-imide-p­toluene sulphonate (CMC) (188 mg) with stirring and the pH kept at -4.75 during reaction by adding 0.01M HCI. After 2 hr, aq. 2M NaBH4 (4 mL) was added dropwi se during 45 min, and the pH was kept at 7 by simultaneous addition of 4M HCI. After I hr, the solution was dialyzed against distilled water and lyophilized, yield 5.3 mg. The procedure was repeated once again to ensure complete reduction . The carboxyl-reduced PS (2 mg) was hydrolyzed with 2M CF3COOH for 18 hr at I 00°C and after usual treatments, the sugars were es timated by GLC (Table I).

Methylation analysis. PS ( 12.0 mg) was methylated according to Ciucanu and Kerek method and the product isolated by part ition between CHC13

and H20. It was remethylated twice by Purdie method, yield 8.7 mg. The product showed no band at 3600-3300 cm·1 in IR spectrum. A portion (2.0 mg) of the methylated product was hydrolysed with 90% HCOOH at I 00°C for 1 hr. HCOOH was evaporated off and the res idue again hydrolyzed with 1M CF3COOH fo r 18 hr. The hydrolysate was reduced with NaBH4, acety lated in pyridine and analyzed by GLC usi ng colunms (A) and (B). Another port ion of the methylated product (4.6 mg) was dissolved in dry THF (5 mL) and refluxed with lithium-aluminium­hydride 7 ( I 00 mg) fo r 5 hr and kept overnight at room temperature. The excess of reductant was decomposed by dropwise addition of ethyl acetate and aqueous THF. The inorganic materials were filtered off. The filtra~e was evaporated to dryness g1vmg carboxyl-reduced permethyiated product (3.2 mg). The product was hydrolyzed with formic acid followed by IM CF3COOH as before and the alditol acetates of the reduced methylated sugars were prepared in the usual way and analyzed by GLC. CMC-reduced PS was also methylated following the same procedure and analyzed.

Periodate oxidation. The PS (I 0 mg) in water (5 mL) was treated with 0.1 M sodium-meta-periodate (6 mL) and the mixture was kept for 48 hr in the dark at 4°C. The excess periodate was destroyed by ethylene glycol (3 mL) and the solution dialyzed against distilled water and dried, yield 8 mg. A

portion (2 mg) of it was reduced with NaB~ and after usual treatments, the product was hydrolysed by 2M CF3COOH for 18 hr at 1 00°C. The sugars in the hydrolysate was identified as D-glucose only by GLC.

Partial add hydrolysis. Partial hydrolysis of PS (55 mg) was carried out with 0.5M CF3COOH at I 00°C for 5 hr. After removal of the acid, the hydrolysate was chromatographed on Whatman No. 3mm sheets . The spots corresponding to monosac­charides andl slow moving spots of oligosaccharides were obtained. The zones containing the oligosaccha­rides fractions were cut out and eluted with ethanolic water and then dried. Three ol igosaccharides were obtained and purified by pass ing th rough Sephadex G-50 colunm using water as eluent. The oligosac­charides, '0'- I (6 mg), '0 ' -2 (9 mg) and '0 ' -3 (10 mg) were collected and they were found chro­matographically homogeneous (PC) .

Methylation analysis of aldobiuronic acid. The aldobiuronic ac id (3.0 mg) was methylated by the method already described3

.4. GLC analysis of the alditol acetates prepared from a portion of the methylated product, showed the presence of 3,4,6-tri-0-methyi-D-galactose. Another portion (3 .5 mg) was reduced wi th lithium-aluminium-hydride and the product hydrolysed. The alditol acetates were prepared, analyzed by GLC (Table I).

Hydrolysis of aldotriuronic acid. The aldotri­uronic ac id (0.2 mg), [a]0

23+8°, was hydrolysed wi th 2M trifluoroacetic acid for 18 hr at I 00°C and the alditol acetates were prepared in the usual manner and analyzed by GLC.

Methylation analysis of aldotriuronic acid. MethylationH of aldotriuronic acid t2 mg), prepara­tion of alditol acetates were carried out as before. A portion of the permethylated aldotriuronic acid was reduced with lithium-aluminium-hydride and the alditol acetates were analyzed by GLC (Table 1).

Chromium trioxide oxidation. To a mixture containing PS (6 mg) and myo-inositol (5 mg) in (DMF, I mL) were added acetic anhydride and pyridi ne (2 mL), with stirring. After 16 hr at room temperature, the mixture was dissolved in chloroform (I 0 mL) and the solution washed thrice with water, dried over anhydrous sod ium sulphate and evaporated to dryness. The material was reacetylated in the same way. The acetylation product was dissolved in glacial acetic acid and the solution treated wi th chromium trioxide at 50°C. Aliquots were removed at different time intervals and immediately diluted with water to

PRAMANIK eta!. : STUDIES ON POLYSACCHARIDE FROM ASTRAEUS HYGROMETRICUS 529

stop oxidation. The mixture was extracted with chloroform and the extract dried and evaporated to dryness. The resulting material was hydrolyzed with O.SM trifluoroacetic acid for 18 hr at l 00°C. The sugars in the hydrolysate were converted into their alditol acetates, and estimated by GLC. Carboxyl­reduced PS was also acetylated in the same way and then oxidized and hydrolysed. The hydrolysate in the form of alditol acetate was estimated by GLC.

Acknowledgement The authors are grateful to Prof. N Roy, Prof. B P

Chatterjee and (late) DrS Basu of the Department of Biological Chemistry, lACS, Jadavpur, Calcutta for providing facilities and constant encouragement during the work. The authors are also thankful to Dr. Nirmal Samajpati of the Department of Botany,

Calcutta University, Calcutta for identification of the mushroom. One of the authors (A P) is grateful to UGC, New Delhi for the award of a research fellowship.

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Anal Chern, 28, 1952, 350. 2 Dische Z, 1 Bioi Chem,167,1947, 189. 3 Ciucanu I & Kerek F, Cari?ohydrate Res, 131, 1984, 209. 4 Purdie T & Irvine J C, 1 Chern Soc, 85,1964, 1049. 5 Akher M A, Hamilton J K, Montogomery R & Smith F, 1 Am

ChemSoc, 74,1952,4970. 6 Hoffman J, Lindbergh B & Svensson S, Acta Chern Scand,

26, 1972, 661. 7 Lindberg B, Longren J & Nimmich W, Carbohydrate

Research, 23, 1972,47. 8 Sadasivam S & Manikam A, Biochemical methods for

agricultural science (Wiley Eastern Ltd.) 1992, p. 179. 9 Angyal S J & James K, Aust 1 Chern, 23, 1970, 1209.