Indian Journal of Chemistry Vol. 38B, June 1999, pp. 709-712

Structural studies on a polysaccharide from an edible mushroom, Termitomyces eurhizus

Amar Pramanik & Syed Sirajul Islam*

Department of Chemistr~ and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal, India

Received 20 May 1998; accepted (revised) 7 April 1999

A polysaccharide (PS), isolated from the hot aqueous extract of the mushroom, Temlitomyces eurhiZlIs, is found to contain, D-glucose, D-galactose, D-mannose and D-galacturonic acid in a mole ratio of about 0.9:0.9: 1.0:0.9. From the peiiodate oxidation and methylation analysis of the PS, the linkages among the constituent sugar residues are identified as, D-Galp (1,6 linked), D­Manp (1 ,2,3 linked), D-G1cp (1,3 linked) and D-galacturonic acid. The anomeric configuration of the constituent sugar residues are detennined by the chromium trioxide oxidation studies of the acetylated polysaccharide. It is concluded that galactose and mannose have the f)-configuration while glucose and galacturonic acid have the a-configuration. Based on the above observations, the structure of the repeating unit of the polysaccharide was assigned as,

-6)-f)-o-Galp-( 1-3)-f)-o-Manp-( 1-3)-a-o-G1cp-( I-

I (2-1 )-a-o-GalpA

Five mushroom of the genus Termitomyces, namely Termitomyces eurhizus, T. microcarpus, T. robustus, T. straitus, and T. clypeatus have since been identified as edible mushroom with high nutritive value. T. robustus and T. clypeatus contained I 31 % protein, 32% carbohydrate and 10-14% ascorbic acid. Several enzymes of high therapeutic value have been reported in the species T. microcarpui and T. clypeatus3

.7

•

Nutrient content of T. robustuS8.9 was analyzed and

found to contain free amino acids, protein, glycogen, lipid, sugar and ascorbic acid and trace minerals in varying proportions.

T. eurhizus 10 obtained from liquid culture medium was found to contain protein, 14-27% (in dry wet basis), ten amino acids of which have been identified previously. This mus.hroom grows on the laterite soil of the forest region of Bankura and Midnapore district of West Bengal. It is abundantly available during the months of Sept.-Oct. and local people consume them as delicious and edible mushroom. No work has been reported on the polysaccharide isolated from the aqueous extract of this mushroom. Therefore a detailed investigation was carried out on the PS of this mushroom and the results are reported in this paper.

Results and discussion

The purified PS had [a.]~ + 110; Total sugars

(98%) were estimated by phenol-sulphuric acid

method I I and acid sugar was estimated as 24% by carbazole-sulphuric acid method 12

• The PS was hydrolyzed with 2M trifluoroacetic acid with inositol as the internal standard. The hydrolyzed PS on paper chromato graphic analysis, showed the presence of D­glucose, D-galactose, D-mannose and D-galacturonic acid. Alditol acetates of the hydrolysate on analysis with GLC showed D-glucose, D-galactose, and D­man nose in almost equimolar ratio. D-galacturonic acid was estimated as 24% by carbazole-sulphuric acid method (Table I). Carboxyl-reduced13 PS on hydrolysis and followed by GLC examination of the corresponding alditol acetates showed the presence of D-glucose, D-galactose and D-mannose in a mole ratio of almost 1 :2: 1. The increase in the mole ratio of galactose further indicated that, D-galcturonic acid

Table I-Sugar content of the PS of Termilomyces eurhizus.

PS RPS mole mole mole mole

% ratio % ratio Galactose 23 .5 0.9 48.0 1.9 Glucose 24.5 0.9 24.5 0.9 Mannose 26.0 1.0 25.5 0.9 Galacturonic-acid 24.0 0.9

PS: Polysaccharide isolated from aq. extract of the mushroom, Termilomyces eurhizus.

RPS: Carboxyl-reduced polysaccharide of this mushroom.

710 IND IAN 1. CHEM. SEC B, JUNE 1999

Table II-Methylation analys is of PS and of RPS of Tennitomyces eurhiZlIs

Methylated sugars Retention time" Mole EroEortion Mode o f linkages Column A Column B PS *RPS +SDPS

2,3,4-tri-O-methyl-D- galactose 3.4 1 2.88 0.9 1.9 .LGaJI)-~-

--GaJpA-'-

4 ,6-di-O-methyl-D- man nose 3.29 2.93 1.0 0 .9 1.0 LManpJ.

II 2,4,6-tri-O-methyl-D-glucose 1.95 1.82 0 .9 0.9 1.0 .LGIcI'J.

"Retention time relative to that of 1 ,5-di -O-acetyl-2,3,4,6-tet ra-O-methyl-D-glucitol on column A (3% ECNSS-M) at 170°C and column B (3% of OV -225 ) at 165°C.

*RPS : methylated carboxyl-reduced PS .

+SDPS: Smith degraded PS.

galactose further indicated that, D-galc turonic acid moiety was present in the PS (Table I) .

The PS was methylated by Ciucanu and Kerek method '4 fo llowed by Purdie" method and then hydro lyzed. The ald itol acetates of the hydro lysate were prepared by GLC us ing column A and B (Table-II) and found to contain 2,3,4-tri -O-methyl-D­galac-tose, 4 ,6-di-O-methyl-D-mannose and 2,4,6-tri­O-methy l-D-glucose in a mo le ratio of 0.9: 1.0:0.9 . These resu lts indicated that, D-galactose and D­man nose residues are respectively ( 1,6) linked and (1,2,3) linked, whereas D-G lucose residue is ( 1,3) linked . The methy lated PS was reduced with lithium­alumi nium-hydride ' 6 and then hydrolyzed and the GLC examination of the corresponding alditol acetates showed the peaks of 2,3,4-tri-O-methy l-D­galactose, 4,6-di-O-methyl-D-mannose and 2,4,6-tri­O-methyl-D-glucose in a mole ratio of 1.9:0 .9:0 .9. The increase in the ratio of 2,3,4-tri-O-methyl-D­galactose revea ls that, D-galacturonic acid moi ety is glycosidica lly linked.

The periodate ox idized PS on hydrolysis, showed the absence of D-galactose and D-galacturonic acid moiet ies in the paper chromatographic analysi s, indicating that, both have been destroyed during ox idation . A part of the periodate oxidized and then reduced PS on hydrolysis and foll owed by GLC examination, was found to contain 4 ,6-di-O-methyl­D-mannose and 2,4,6-tri-O-methyl-D-glucose in the mole ratio of 1.0:0.9. This suggests that, both D­galactose res idue and D-ga lacturonic ac id residue have been destroyed during oxidation.

The periodate oxidized product was subjected to Smith-degradation l7

. T he Smith-degraded product on hydrolysis and fol lowed by paper chromatographic and GLC examinations showed the presence of on ly

Table I II-Survival of sugars" in the ox idati on (,j acetylated PS and carboxyl- redu ced PS with chromium trioxide.

Materials PS

Carboxyl­redu ced PS

Time (hr) 0

0.5 1.5 2.0

0 0.5 1.5 2.0

Galactose 18.20 0. 92 0. 5 1 0.1 3

35.8 1 15 .32 11. 21 10.1 4-

Glucose 20 .() 15 .X I J () 8.1

20. 5 ~ 5 .9 I ! 1. 14 lU 2

"Myoinositol was used as internal st::lIld:Jrd {[lc r (ellt)

Mannose 17.9 14.2 10.9 9.8

17 .64 13.87 I 1.1 0 10.0

D-glucose and D-mannose in a mo le rati o of about I: I . It further confirms the degradation of D­galacturonic acid moiety and D-ga lac tose moiety during the oxidation.

]n order to ascertain the anomeric confi gurati on of the different sugar res idues, the acety lated derivat ive of PS was subjected to oxidati on w ith chromium trioxide in acetic acid fo r different time interval at 60°C, using myo-i nos ito l as inte rnal standard. The oxidized materials were hydrol yzed and a ldito l acetates of the different sugars were es timated by GLC. It showed that , the amount o f D-galactose and D-mannose decreases very rap id ly with time compared to D-gluc.ose (Table-III). This is due to abstraction of an axially oriented C- l proton to yi e ld a 5-hexu losonic ac id IS, th'us lead ing to the dis­

appearance of the sugar residues having ~-gl ycosidic linkages . A part of the carboxyl-reduced PS was al so oxidized w ith chromium trioxide and fo ll owed by usual treatments, showed that the a mount of D­ga lac tose decreases less rap idl y compared to that obtained from the unreduced PS , thus indica ting the

a-configuration of the D-galacturoni c ac id res idue. Thus from the periodate oxida tion and methylation

PRAMANIK e/ of.: STRUcrURAL STUDIES ON A POLYSACCHARIDE 71 J

studies of PS and chromium trioxide oxidation of the acetylated PS, isolated from the mushroom, Termitomyces eurhizus, among four possible structures of the repeating unit of the PS, one of which may be assigned as,

-6)-~-D-Galp-( l-3)-~-D-Manp-( 1-3)-a-D-Glcp-( I-

I (2-1 )-a-D-GalpA

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

unless stated otherwise. Paper partition chromato­graphy was performed on Whatman Nos.1 MM and 3 MM papers. Solvent systems (v/v) used were, (A) 1-butanol-acetic acid-water (4: I :5) (upper phase) and (B) ethyl acetate-pyridine-water (8:2 : I); the spray reagent used was alkaline silver nitrate solution l9

. All solvents were distilled before use. Optical rotations were measured with a Perkin-Elmer Model 241 MC spectropolarimeter. Colorimetric estimations were conducted with Systronic UV -VIS spectro­photometer, mode l-I 08 .

Gas-liguid chromatography (GLC) was performed using Hewlett-Packard Model 5730A gas chromato­graph having a flame ionization detec tor and glass

columns (1.83x6 mm) with ( I) 3% of ECNSS-M on Gaschrom Q (100-120 mesh). All GLC analysis were conducted at 170°C for neutral sugars and 165°C for methyl ated sugars by converting the sugars into the ir alditol acetates . Retenti on time of partially methylated alditol acetates were measured with respected to that of 1,5-di-O-acetyl-2,3,4 ,6-tetra-O­methyl-D-glucitol as unity .

Preparation of aqueous extract. Mushroom (500 gms) were swollen in 200 mL water and heated at 70°C for 30 min . The mixture was centrifuged at 10,000 rpm for I hr. The ago ex tract was collected and lyophilized, yield 350 mg. The materi al (200 mg) was extensively dialyzed to remove monosaccharides and other low molecular we ight materi als. The poly­saccharide of the mushroom was collected from inside of the dia lysi s bag. The vo lume was reduced to 20 mL and treated with ethanol in I: IO (v/v). The precipitate was collected and lyophilized, yield 160 mg.

Purification of the polysaccharide. The poly­saccharide ( 130 mg) was puri fied by pass ing through a column of Sephadex G-50 us ing water as e luent ; sixty fractions were collected and monitored

spectrophotometrically at 490 nmZD

using phenol sulphuric acid method. Fractions (19 to 39) were collected and lyophilized , yield 100 mg.

The homogeneity of the polysaccharide was established by means of high voltage electrophoresis in borate-buffer in a Shandon Model L-24 apparatus.

Acid hydrolysis of the polysaccharide. The polysaccharide (2.1 mg) was hydrolyzed with 2 M CF,COOH for 18 hr at 100De. The ac id was completely removed by co-distillation with water and the hydrolysate was 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 hydrolysate was reduced with NaBH4 (40%) and the alditol acetates were prepared in the usual way and analyzed by GLC (column A and B) Table II .

Preparation of carboxyl-reduced PS. To a solution of the PS (9 mg, 9 mL) in water, was added I-cyclohex y 1-3-(2-morphol ino-ehty I )-carbo-d i-i mide­p-toluene sulphonate ( 188 mg) with stirring and the pH was kept at 4 .75 during the reacti on by adding 0.01 M HC!. After 2 hr, ago 2 M, NaBH4 (4 mL) was added dropwise during 45 min . and the pH was kept as 7 by simultaneous addition of 4M , HC!. After I hr the solution was di alyzed again st di stilled water and lyophilized, yield 6.0 mg. The procedure was repeated once again to ensure complete reducti on. Carboxyl-reduced PS (2 mg) was hydrolyzed with 2M, CF,COOH for 18 hrs at J OODC and after usual treatments the sugars were estimated by GLC (Table II) .

Methylation analysis. PS ( 12.0 mg) was methy­lated according to Ciucanu and Kerek method and the product was isolated by partition between CHCI1 and HzO. It was remethylated twice by Purdie method , yield 8.2 mg. The product showed no band at 3600-3300 cm- I in IR spec trum. A porti on (2.0 mg) of the methylated product was hydrol yzed with 90% HCOOH at 100DC ("or I hr. The HCOO H was evaporated off and it was again hydrolyzed with I M, CF.,COOH for 18 hrs. The hydro lysate was reduced with NaBH4, acetylated in pyridine and analyzed by GLC, column (A) and (B). Another porti on of the methylated product (4.6 mg) was disso lved ill dry THF (5 mL) and reflu xed with lithium-aluminium­hydride 15 (100 mg) for 5 hrs and kept overni ght at room temperature. The excess of the reductant was decomposed by dropwise addition of ethyl acetate and ago THF. The inorganic mate rial s were filt ered

712 INDIAN J. CHEM. SEC B, JUNE 1999

off. The filtrate was evaporated to dryness glVlng carboxyl-reduced permethylated product (3.2 mg). The product was hydrolyzed by I M, CFJCOOH as before and the alditol acetates of the reduced methylated sugars were prepared in the usual way and analyzed by GLe. CMC reduced PS was also methylated following the same procedure and analyzed.

Periodate oxidation. The PS (10 mg) in water (5 mL) was treated with 0.1 M, sodium metaperiodate (6 mL) and the mixture was kept at 48 hrs in the dark at 4°e. The excess periodate was destroyed by ethylene glycol (3 mL) and the solution was dialyzed against distilled water and dried, yield 8 mg. A portion of it (2 mg) was reduced with NaBH4 and after usual treatments, the product was hydrolyzed by 2 M , CFJCOOH for 18 hrs at IOO°e. The sugars in the hydrolysate were identified by GLC as usual manner.

Chromium trioxide oxidation21 . To a mixture containing PS (6 mg) and myoi nositol (5 mg) in DMF ( I mL) were added acetic anhydride and pyridine (2 mL) with stirring. After 16 hrs at room temp the mixture was dissolved in CHCIJ ( 10 mL) and the solution was washed thrice with H20, dried with anhyd. Na2S04 at'ld evaporated to dryness. The material was reacetylated in the same way. The acetylation product was dissolved in gl. acetic acid and the solution was treated with chromium trioxide at 50°C. Aliquots were removed at different time intervals and immediate ly diluted with H20 (to stop oxidation). The mixture was extracted with CHClJ and the extract was dried and evaporated to dryness . The resulting material was hydrolyzed with 0.5 M, CF,COOH for 18 hrs at 100°e. The sugars in the hydrolysate were converted into their alditol acetates and estimated by GLe. Carboxyl-reduced PS was also acetylated in the same way and then oxidized and hydrolyzed . The hydrolysate in the form of alditol acetates were estimated by GLe.

Acknowledgement The authors are grateful to Prof B P Chatteljee and

Dr S Basu of IACS, Jadavpur, Calcutta fo r providing facilities and constant encourabement during the work. The authors are also thankful to Prof B R De, and Dr · S Chattopadhyaya of the Depart ment. of Chemistry and Chemical Technology, Vidyasagar University., Midnapore, for their co-operation. One of them (AP) is grateful to UGC, New Delhi, for the award of a research fellowship .

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