Investigation and Chemical Study of Water-Soluble Polysaccharides Isolated from Seeds of Cassia Alata

1. INTRODUCTION:

Cassia alata Linn. Plant [1] belongs to the family-Caesalpiniaceae and commonly known as Dadmurdan and occurs in Garhwal region of Northern Himalayas. Plant is a large shrub and grown as an ornamental purpose. Cassia species have been well known for their laxative and purgative properties and for the treatment of skin diseases [2] there is now an increasing body of scientific evidence demonstrating that the plants possess many other beneficial properties [3]. Seeds are used in Ayurvedic system as medicine and leaves are used for the treatment of ringworm[4], skin diseases[5] and also for snakebite treatment. Cassia alata is also known as the Candle Bush and it is an important medicinal tree[6,7]. Present manuscript mainly deals with the isolation, purification, preliminary analysis and nature of the constituent of water-soluble sugars from seeds of Cassia alata. The current investigation found that galactomannan consisting of D-galactose and D-mannose in the molar ratio 1:3 has been isolated from the seeds of Cassia alata. Hydrolysis of the methylated polysaccharide resulted in three methylated sugars: (a) 2,3-di-O-methyl-D-mannose, (b) 2,3,6-tri-O-methyl-D-mannose, and (c) 2,3,4,6-tetra-O-methyl-D-galactose in the molar ratio 1:4:2.

Cassia alata pods were collected from the local area of Thane, Maharashtra (India) in the month of October–December. The seeds were manually separated dried and kept in a cool and dry place until further use. B. Isolation of Polysaccharide Polysaccharide was isolated from the crushed seeds (300 gm) in water (1000 ml) [8] for 24 hrs. at room temperature then stirred for 48 hrs. with the help of mechanical stirrer. Viscous solution was squeezed through muslin cloth to remove the insoluble matter. Filtrate was then centrifuged through a Sharple‘s super centrifuge [9] to remove finely suspended matter. Centrifugate was precipitated with ethanol (3 litre), by mechanical stirrer to precipitated out the whole polysaccharide in light brown form. Precipitate of polysaccharide was further filtered through a sintered funnel (G-3) under suction then dried in vacuo after washing with acetone and pet. Ether. Polysaccharide was obtained as a brownish crude powder and isolated polysaccharide was found with sulphated ash [10] (0.74%).

Purification of galactomannans was done by following steps.

In 1% acetic acid solution dissolved the crude polysaccharide and precipitated by adding the solution slow rate to the excess of ethyl alcohol under continues stirring dilution and re-precipitation process was repeated five times to obtain the total material. The precipitated polysaccharide was filtered, washed with ethanol and dried in air. White fibrous material was obtained

After the repeated precipitation of the polysaccharide was deproteinated by shaking its aqueous solution with the chloroform up till to formed a milky gel at the water - chloroform interface. This process was repeated six times for obtain the total rid of the proteins.

The deproteinized aqueous solution of the polysaccharides was added Fehling‘s solution, it was formed the blue copper complex and washed again with water. The complex was destroyed with 1.5 N hydrochloric acid solution. The polysaccharide was regenerated by adding slowly excess of ethyl alcohol under continues stirring. Dissolution and re- water forming viscous solution at the room temperature. It was showed the optical rotation - 40.5˚(water) and sulphated Ash 0.74% The methoxy and acetyl was negligible and found to be free from Halogen, nitrogen and sulphur.

5.0 gm. of purified polysaccharide was dissolved in 500 ml of distilled water and by Adding 700 m1 of 90% of ethyl alcohol precipitate was obtained. The precipitated polysaccharide was filtered, washed with ethanol followed by ether and dried in vacuum oven it was fraction first. The filtrate was again treated with 700 ml of 90% ethyl alcohol filtered washed and dried as above it was fraction second separated in to fractions with 2N sulphuric acid. By paper chromatography analysis of the fraction first and fraction second, gave D-galactose and D-mannose in molar ratio 1:3 and both the fraction retained the original specific rotation -40.6˚ (water) indicating homogeneous nature of polysaccharide.

The acetylation of the polysaccharide was done by acetic anhydride and sodium acetate. The deacetylated polysaccharide had the same optical rotation as original polysaccharide, which further indicated that the polysaccharide is homogeneous.

A part of polysaccharides was subjected to conventional zone electrophoresis [11] on Whatman no.-1 paper in borate buffer pH 9.2. The intensity of the characteristic yellow orange colour developed into the phenol-sulphuric acid regent, was measured in Klett-Summerson photoelectric colorimeter. A single sharp peak indicating the homogeneous nature of galactomannan by plot of absorbance against segment number.

900 mg of pure polysaccharide dissolved in 30 ml of 2N sulphuric acid reflux for 55 hrs. on water bath. The following mobile phase used for identification of the hydrolysate polysaccharide.

In all above three mobile phases the result was essentially the same showing homogeneity of the polysaccharide.

2.5 g of pure polysaccharides dissolved in 2N sulphuric acid and reflux for 40 hours on water bath and hydrolysate substance neutralized with barium carbonate, filtered, diluted and analyzed with chromatographically.

Prepared two sheets of Whatman no. -1 paper and a small quantity of hydrolysate polysaccharide was dissolved in water and this solution spots on the sheets. The paper was developed in mobile phase water: 1-butanol: ethanol (6:5:1) and water: 1-butanol: 2-propanol (3:9:5) Chromatogram were dried in air and sprayed with aniline hydrogen phthalate. Two spots were observed on the chromatogram after heating at 125°C. The RF and RG value of two spots corresponded to. D-galactose and D-mannose. Result as table no: 1.

The identity of D-galactose and D-mannose was confirmed by co-chromatography with authentic samples of D-galactose and D-mannose when the chromatograms were developed in the mobile phase no.-3

The hydrolysate substance was fractionated by elution from cellulose column [13], small sample of hydrolysate dissolved in methanol –water mix solution and examine and on the column 2x25 cm. The column was left overnight for separation with mobile phase water: 1-butanol: ethanol (6:5:1) and fraction of elute were collected in tubes. Fractions were examined by paper chromatography with standard samples of D-galactose and D-mannose. The fractions containing same sugars combined together, concentrated and recrystallized, so obtained two fractions examined as below. aqueous methanol had m.p.131°-132°C, +12.6°(water), it formed the derivative D-mannose Phenylhydrazone and m.p. 195°-196°C (literature:199°-201°C), so this fraction was identified D-mannose.

After recrystallization of second fraction from aqueous methanol found m.p. 165°- 166°C, +78.6°(water) and derivative D-galactose phenyl hydrazone [14] m.p.168°-170°C (lit.m.p.170-171°C [12] ), so above result identified that fraction to be D- galactose.

Hydrolysate was resolved into its components by paper chromatography on Whatman No. 3 MM filter paper sheet in solvent mixture water: 1-butanol: ethanol (6:5:1) and used aniline hydrogen phthalate [12] as spray reagent. Areas of individual sugar components were cut out with the help of guide spots and eluted with water according to Dent‘s method [15]. The eluted sugars were estimated by periodate oxidation method [16] with sodium metaperiodate solution (0.3 M) for necessary corrections the blank reading was also made. Molar ratio of D-galactose and D-mannose in purified seeds polysaccharide was found to be 1:3 molar ratio

400 mg of polysaccharide was dissolved in 20ml of 0.1N sulphuric acid hydrolysate was carried out at water bath for 8 hrs. The hydrolysate was taken out at the various intervals of time and examined chromatographically used mobile phase no.-3 At the time hydrolysis of the polysaccharide D-galactose obtained first and then D-mannose. From above it is clear that D-galactose units are present at the periphery as end group and D-mannose forms basic chain of the polysaccharide and it is attached to main chain by weaker bond.

Methylation of the pure polysaccharide done by Haworth‘s [17] method by using sodium hydroxide and dimethyl sulphate and then by Purdie‘s [18] method using silver oxide and methyl iodide. 10 g of pure polysaccharide taken in 500ml of round bottom flask and filtered with ground glass joint dissolved it in 150 ml of 10% sodium hydroxide solution under stirring. The mixed solution of (50% solution of NaOH) sodium hydroxide and dimethyl sulphate in the reduce pressure and extracted for the removing of sodium sulphate, this extraction repeated four times for the complete methylation of the process. Final extraction done by chloroform and dried over anhydrous sodium sulphate, The partly methylated product was 8.3 g and colour was whitish brown. This product was further methylated by the Purdie‘s method. The partly methylated polysaccharide was dissolved in moisture free methanol in round bottom flask. The temperature of the reaction mixture maintaining at the 38 to 42°C C on the water bath. A calcium chloride tube was placed at the top of the condenser to prevent the entry of the moisture at the time of reaction. Add silver oxide 10 g and methyl iodide 12 ml by the addition in 10 hrs. Vise-versa of about similar quantity of methyl iodide and silver oxide. The contents were stirred continuously during the reaction after the completion of addition; reaction mixture was heated on a water bath under stirring for reflux using the calcium chloride guard tube and nitrogen gas. The total filtrate and extracts were evaporated under reduced pressure and resulting thick material was methylated two times under the same condition. The methylated product was obtained as brownish masses.

200 mg of methylated polysaccharide was dissolved in 30 ml of 85% of formic acid and the solution was reflux for 8 hours on the water bath. The solution was cooled and concentrated under reduced pressure to a thick from which acid was removed under vacuum and it dissolved in 20 ml of 2N sulphuric acid and hydrolyzed for 15 hours on the water bath, cooled and neutralized with barium carbonate and filtered with filter paper. The precipitate washed with distilled water. The total filtrates concentrated under reduced pressure yellowish brown colour thick material obtained.

The methylated sugar was separated on Whatman no.1 chromatography paper, by using mobile phase water: 1-butanol: ethanol (6:5:1) and water: 1-butanol: 2-propanol (3:9:5). The chromatography shows only three spots after spraying aniline phthalate and drying at 110°C C. The RTMG value were calculated in each case and compared those reported in given in table no.-2

O OH H MeO OMe H HH OH

O H OH H MeO OMe H HH OH OMe

O OMe H H MeO H OH OMeH H OMe

The methylated monosaccharide quantitative estimation done by the method of Hirst, Hough, and Jones indicated that the methylated sugars 2,3-di-O-methyl-D-mannose [structure No.-01], 2,3,6-tri-O-methyl-D-mannose [structure No.-02] and 2,3,4,6-tetra-O-methyl-D-galactose [structure No.-3] were present in molar ratio 1:4:2 respectively in the methylated galactomannan

The polysaccharide (100 mg) was dissolved in water (25 ml) and the solution was cooled to 0 °C. A cold solution of sodium metaperiodate (0.20 mole, 45 ml) was added to the solution and volume was made up to 100 ml. The reaction was conducted at 6 °C and the amounts of periodate consumed and formic acid

The polysaccharide was subjected to partial hydrolysis with 0.1 N sulphuric acids. After the paper chromatographic examination of hydrolysate, it was showed the separation on preparative scale given four oligosaccharides, which identification as below. a. Epimelibiose: 6-O-α-D-galactosyl-D- mannose, m. p. 200° C, : + 122° (water), Lit.[19] m.p. 201-202° C, : 121-124° (water). Acid hydrolysis gave galactose and mannose in equal proportion. Methylation and subsequent hydrolysis gave 2, 3, 4, 6-tetra-Omethyl-D-galactose and 2, 3, 6-tri-O-methyl-D-mannose. It was not hydrolysed by emulsin showing the absence of β-linkage b. Galactosylmannose: 6-O-α-D-galactosyl (1→6)-O-α-D-galactosyl-D-mannose, m.p. 123°C, :+ 120° (water), lit. [20] m. p. 124° C, : + 119° (water). Acid hydrolysis gave D-galactose and D-mannose in the molar ratio 2: 1. Methylation and its hydrolysis gave 2, 3, 6-tri-O-methyl-D-mannose, 2, 3, 6-tri-O-methyl-D-galactose, and 2, 3, 4, 6-tetra-O-methyl-Dgalactose. It was not cleaved by emulsin indicating the absence of β-linkage c. Mannobiose, 4-O-β-D-mannosyl-D-mannose, m. p. 204° C, : -10.1° (water), lit. [21] m. p. 202- 203° C, : -5.2° to 8.2° (water). Its phenyl hydrazone had m. p. 203° C, lit. m. p. 203°-206°C. Acid hydrolysis gave D-mannose only. Methylation and its hydrolysis yielded 2, 3, 4, 6-tetra-O-methyl-D-mannose and 2, 3, 6-tri-O-methyl-D-mannose. It was cleaved by emulsin showing the presence of β-linkage. d. Mannotriose : 4-O-β-D-mannosyl-(1→4)-O-β- D-mannosyl-D-mannose, m.p.211°-213°C, : - 14°(water), lit. [22] m. p. 214°-215°C, : -15° to -26° (water). Acid hydrolysis indicated the presence of D-mannose only whereas partial acid fission afforded mannose and mannobiose. It was hydrolysed by emulsin showing the presence of β-linkage. Methylation and subsequent hydrolysis afforded 2, 3, 4, 6-tetra-O-methyl-D-mannose and 2, 3, 6-tri-O-methyl-D-mannose.

The plants of the genera cassia generally possess considerable medicinal value and are also a good source of mucilages. Owing to the high medicinal value and increasing industrial demand of plant mucilages, we were prompted to undertake a and D-mannose in the molar ratio 1:3 has been isolated from the seeds of Cassia alata. Hydrolysis of the methylated polysaccharide resulted in three methylated sugars: (a) 2,3-di-O-methyl-D-mannose, (b) 2,3,6-tri-O-methyl-D-mannose, and (c) 2,3,4,6-tetra-O-methyl-D-galactose in the molar ratio 1:4:2.

The authors are thankful to research guide Dr. D.S. Singh and Director of Dexo Chem Laboratories, Ankleshwar Gujarat for providing facilities and helpful discussions and direction of research work. The authors are also thankful to the director of Indian Management Academy, Ahmedabad, Gujarat for providing facilities and encouragement throughout the work.

5. REFERENCES:

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Research Scholar of University of Entrepreneurship & Technology, USA