Indian Journal of Chemistry Vol. 40B, December 2001, pp. 1287- 1 288

Note

Arabinoxylan from Cryptocarya wightiana Bark

Gudipati Muralikrishna & Rudrapatnam N . Tharanathan*

Department of Biochemistry And Nutrition, Central Food Technological Research Institute, Mysore 570 013 (India)

Received 22 November 1 999; accepted (revised) 8 December2000

A homogeneous arabinoxylan (Ara-Xyl in 1 .05: 1 ratio) has been obtained from the major water soluble fraction of Cryptocarya wightialla bark gum by employing gel filtration on Sephacryl S-400 and SE-HPLC techniques. Methylation of the pure polysaccharide followed by GLC-MS analysis reveales a highly substituted lA-linked xylan backbone.

Plant gumsj are useful in application in the food industry as thickeners and emulsion stabilizers, and in various non-food industries such as paper, pharmaceutical, textiles, oil drilling, etc l . They are complex carbohydrates containing a variety of monosaccharides (and some times their derivatives) in various linkages and structures3. The bark of the perennial tree Cryptocarya wightiana, widely grown in South Canara District of Kamataka, due to its excellent binding properties is used as a cementing material for mixing and smearing the various constituents such as saw dust or cowdung powder (serving as the cellulose base), chemicals (as perfumery compounds), and colour on to a thin wooden stick in the manufacture of agarbathis or incense sticks4• Chemical nature and preliminary fractionation data of a sticky gum from this bark have been reported5. In this paper the purification and structural elucidation of an arabinoxylan derived from the major water-soluble fraction of the gum are described.

Results and Discussion The water extracted gum from C. wightiana bark

was separated into water-soluble (- 83%) and water insoluble (- 1 6%) fractions. The major water-soluble fraction was fractionated by graded concentration of ethanol into four different fractions in 58(1), 1 2(11), 8(II!) and l (IV) % yields, respectively, each having different proportions of L-arabinose and D-xylose5• By GPC on Sephacryl S-400 fraction I was separated into one major peak (Ib, 80.6%) and three minor fractions (la, 5.3%; Ic., 6.8% and Id, 4.0%). Fraction

Ib was found to be pure by SE-HPLC and it contained arabinose and xylose in the ratio of 1 .05 : 1 , respectively.

Fraction Ib upon permethylation followed by GLC­MS analysis gave O-methyl ethers (Table1). Identification of mono-O-methyl xylose as well as free xylose is structurally significant and suggests that the polysaccharide is considerably branched. The high proportion of 2,3,5-tri-O-methyl-L-arabinose indicates that all the arabinose residues are essentially furanosidic, which is also corroborated by its easy cleavage upon mild acid hydrolysis. About two thirds of the total arabinose residues are involved in 1 ,3-linkages occupying either in the side chain or in the backbone, which is composed of doubly, singly and unsubstituted 1 ,4-linked D-xylose residues. The molar agreement in the proportion of non-reducing terminal and branching sugar residues leads additional support for the highly branched structure of this polysaccharide. Based on these results the major fraction of C. wightiana bark has a highly branched 1 ,4-linked xylan core substituted variously with both arabinosyl and xylosyl residues.

The high viscogenic nature of the C. wightiana bark is probably due to the branched arabinoxylan­type polysaccharide in it. Its easy water solubility and the build up of viscosity are attributable to its chemical nature. Pentosans, in general, are known to contribute significantly to the viscosity and consistency6. For example, arabinoxylans of wheat are known to affect significantly the dough properties, product (bread) making quality? and to affect the retrogradation characteristics of wheat starch.

Highly branched acidic arabinoxylans are reported in sapote8, brea9, bromellalO and cereals 1 I .

Arabinogalactan and xyloglucan-type polysaccharides occur in silk cotton tree bark 12. The arabinoxylan reported in the present study consisted more of arabinofuranosidic (-75%) residues compared to that of wheat6 and ryel3 flour arabinoxylans (- 35%) and it differed from linseed arabinoxylan in not possessing terminal galactose as well as pyranosidic arabinose residues 14. The purified and the fractionated polysaccharides were highly viscous in comparison with the crude water extracted gum. The native polysaccharide solution, at concentrations over 0.05%, showed excellent "pituity" characteristics. These properties are of value in food and non-food industries.

1 288 INDIAN J CHEM, SEC B, DECEMBER 2001

Table I-GLC-MS analysis of partially methylated alditol ( l -2H) acetates derived from C. wightiana bark arabinoxylan

Peak R/ Relative molar ratiob Diagnostic Ions, mJz Mode of l inkage Araf - ( I

-.. 2,3.5-MeJ-Ara 0.41 5.3 45, 1 18 , 16 1 , 162 2,3,4-MerAra 0.54 5.5 10 1 , 1 18 , 16 1 , 1 62 Xylp - ( 1 -..

2,5-MerAra 0.84 10.0 45,87, 1 18 , 173,233 � 3) Araf - ( l -..

2,3-MerXyl I . l 9 5.3 43,87,102, 1 1 8, 1 29 , 189 � 4) - Xylp - ( l -..

2-Me-Xyl 2. 1 5 1 .0 43,85, 1 18, 127,201 � 3,4) - Xylp - (1 -..

Xylose 2.98 3.0 128, 1 29,145,146,2 17,218,289,290 � 2,3,4) - Xylp - ( 1 -..

a. With respect to 1 ,5 - Di-O-acetyl 2,3,4,6 - tetra - 0 - methyl glucitol, b. With respect to 2-0-methyl-xylose.

Experimental Section Methods. Some of the general methods have been

previously reported5. 15. 16. Gel permeation chromatography (GPC) on Sephacryl S-400 column ( 1 .7x92 cm) was carried out using 0.05M NaCl as the eluent ( 1 6 mL hr-I ) . Size exclusion-high performance liquid chromatography (SE-HPLC) was performed on a Shimadzu HIC-6A ion chromatographl5 using E­linear and E- 1 000 Il-Bondagel columns (SS, 30cmx3.9mm i .d. Water Associates, Milford, USA) connected in series with a guard column. Elution was done with water (0.2 mL min-I ) at 40°C. The Yo was measured using Sesbanium mosaic virus of MW, -6x l 06 Da.

Isolation and purification of the bark gum. The bark pieces were finely macerated and sieved to get 60-80 mesh powder, which was subsequently defatted by Soxhlet extraction with hexane and chloroform (2: I , v/v) for 10- 1 2 hr. The defatted material (50 g) was stirred in water (4 L) for 12 hr at room temperature and subjected for a brief centrifugation (4000 rpm, 5 min) to remove the insoluble residue. The clear supernatant was concentrated, precipitated with alcohol (3 vol.) and the crude polysaccharide gum was purified by reprecipitation from aqueous solution with acetone, dialyzed and lyophilized (yield, 5.6 g).

Alcohol fractionation of the polysaccharide gum. The polysaccharide gum ( 1 g) was solubilized in water (500 mL) followed by centrifugation to remove any insoluble material (- 1 80 mg). To the soluble fraction (- 800 mg) ethanol was added slowly to incipient turbidity and subsequent precipitation, which was collected by centrifugation. To the supernatant addition of alcohol was continued and the various fractions thus obtained were recovered by lyophilization.

The major fraction (no. l , total sugar 90%, pentose 89%, protein 8%) was purified by GPC on a precalibrated Sephacryl S-4OO column into a single

peak (Fra. Ib), which was found to be homogenous by SE-HPLC method.

Permethylation analysis. Fraction Ib ( 1 0 mg ) was permethylated by the method of Hakomori 1 7, as modified by Lindberg et al 1 8, acid hydrolyzed and the derived neutral O-methyl sugars were reduced (NaB2� in 2H20) and O-acetylated. The products were analyzed by GLC and GLC-MS. The individual O-methyl ethers were identified by (a) RT with respect to 1 ,5-di-O-acetyl-2,3.4,6-tetra O-methyl glucitol and (b) the presence of characteristic mass fragments in the mass spectruml8 .

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