Structural and Phytochemical Investigation of Water Soluble Polysaccharide Isolated From Seeds of Cassia Obtusifolia

World Health Organization (WHO) pleasing the importance of medicinal plants Cassia obtusifolia for human health care in developing nations. Cassia Obtusifolia (F. Leguminosae) is commonly known as known as Sickle pod, Takla, Chakunda and Coffee cassia, wildly grown throughout India and other tropical countries. The plant is widely distributed on wastelands road side in rainy season, which are used for skin diseases, ring worm and it antimicrobial activity, anthelmintic, antipyretic, diuretic, carminative, purgative, antidiabetic, antimicrobial, antioxidant and insecticidal effect, root and fruits portion has beneficial properties that can supply the mankind. The whole plant as well as specific parts such as roots, leaves and seeds have been widely used in traditional Indian and south Asian medicine. The plant and seeds are edible; the edible part of the plant varies from 30 to 40 percent. Young leaves can be cooked as a vegetable while the roasted are a good substitute for coffee. [1] The seeds are poisonous but when roasted, they are used as a substitute for coffee and they contain a gum of commercial interest in addition to protein, fat and nitrates. [2-3] The seeds are often roasted, then boiled in water to produce a tea. Roasted and ground, the seeds have also been used as a substitute for coffee (http://www.wikipedia.com).The pod is narrower (2–3.5 mm) in diameter and strongly curved. Seeds are rhomboidal, 4–5 mm long, shiny, and yellowish brown to dark red. In S. obtusifolia, the areole (marking on the seed coat) is very narrow (0.3–0.5 mm wide); in S. tora, it is large (1.5–2 mm wide) (Brenan, 1967).The Cassia obtusifolia seed is composed of hull, endosperm, and germ The plant‘s seeds are a source of cassia gum, a food additive usually used as a thickener. Vadivel and Janardhanan (2002) studied the chemical composition of C. obtusifolia seeds, collected from Western Ghats, South India; they found that Seeds of Cassia obtusifolia contain 18.56% to 22.93% of proteins, crude lipid was between 5.35% and 7.40%, crude fiber ranged from 6.83% to 9.45%, ash content ranged from 5.14% to 5.83%, and carbohydrate varied from 57.00% to 60.69%. The current investigation found that galactomannan consisting of D-galactose and D-mannose in the molar ratio 3 : 4 has been isolated from the seeds of Cassia obtusifolia Hydrolysis of the methylated polysaccharide resulted in three methylated sugars: (a) 2,4-di-O-methyl-D-mannose, (b) 2,4,6-tri-O-

Cassia obtustifolia pods were collected from the local area of Shirpur, Maharashtra region (India) in the month of October–December. The seeds were manually separated dried and kept in a cool, dry place until further use.

The seeds of Cassia obtustifolia were dry milled in a blade grinder to separate the endosperm from the seed coat. The endosperms of Cassia obtustifolia seeds (100 g) were soaked in distilled water and heated at 60 °C for one hrs. and kept aside at room temperature for 24 hrs. The result- tan viscous solution obtained was filtered through a muslin cloth. The filtrate was then kept in refrigerator until further use. Mucilage was precipitated after addition of equal volume of acetone with continuous stir- ring. The mucilage was then separated by filtration, and dried in an oven at about 50 °C. The dried mucilage was grinded by grinders and sieves and finally powdering to 100 mesh sizes.

Purification of galactomannans was done by following steps. 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 5 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-precipitation was repeated to obtained total materiel. The purified polysaccharide was a white, non-reducing amorphous material which was easily dispersed in water forming viscous solution at the room temperature. It was showed the optical rotation - 40.5˚ (water) and sulphated Ash 0.267% The methoxy and acetyl was negligible and found to be free from Halogen, nitrogen and sulphur.

3.0 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 Ist. The filtrate was again treated with 700 ml of 90% ethyl alcohol filtered washed and dried as above it was fraction IInd separated in to fractions with 2N sulphuric acid. By paper chromatography analysis of the fraction Ist and fraction IInd, gave D-galactose and D-mannose in molar ratio 2:4 and both the fraction retained the original specific rotation -30.6˚ (water) indicating homogeneous nature of polysaccharide.

A purified galactomannan was separated into two fractions by fractional precipitation with different volume of ethyl alcohol. The results obtained such

+60⁰ in water were identical to that of identical to that of original polysaccharides showing it to be homogeneous.

A part of polysaccharides was subjected to conventional zone electrophoresis [4] 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.

250g of pure polysaccharide dissolved in 25 ml of 2N sulphuric acid reflux for 40 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.

2.5g 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 (5:6:2) and water: 1-butanol: 2-propanol (4:11:7) 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, 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 pyridine: water: ethyl acetate (1:2:2) 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 bellow.

The first fraction was solid, it was recrystallized from 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 phenylhydrazone m.p.153°-155°C (literature:155°C ), so above result identified that fraction to be D- galactose.

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

Methylation of the pure polysaccharide done by Haworth‘s method by using sodium hydroxide and dimethyl sulphate and then by Purdie‘s 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 2:1 ratio also under stirring and maintaining the temperature 38 to 42°C. This process repeated and then the solution was concentrated with acetone under 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 9.7 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.

150 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. no.1 chromatography paper, by using mobile phase water : 1-butanol : ethanol (4:5:1) and water:1-butanol: 2-Propanol (3:11:6). The chromatography shows only three spot after spraying aniline phthalate and drying at 110°C C. The RTMG value were calculated in each case and compared those reported in giving in table no.-2

O H MeO H HO OMe H

HHOH

OH

O H MeO H HO OMe H

HHOH

OMe

O OMe H H MeO H OH OMeHH OMe

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

The identification of 2,4,6-tri-O-methyl-D-mannose as one of the hydrolysis product of the methylated polysaccharide indicates its appearance from monoterminal, unbranched D-mannopyranosyl

backbone of the galactomannan. Thus methylation analysis showed that the galactomannan consist of main chain of (1→4) linked D-mannopyranosyl units to which are attached at 6-position as side chain of (1→6) linked D-galactopyranosyl unit. The foregoing evidences indicate that the simplest repeating unit of the galactomannan consists of 7 sugar moieties, out of which 3 are galactose which are present as non reducing end groups while rest of them are 4 mannose units which form (1→4) linked main chain. Determination of terminal group by periodate oxidation [14,15] and subsequent titration of liberated formic acid corresponds to 0.225 mole of formic acid per 100 gram of the polysaccharides after composition of 1.135 mole of metaperiodate. On the basis of these results of terminal groups were found to be 45% per repeat ion unit which is in close resemblance with that of by methylation studies 45%. After oxidation polysaccharides examination in 4 hours showed the absence of D-galactose while a small amount D-mannose was detected. However prolonged oxidation (104 hours) destroyed both the hexodes. The considerable difference in rates of oxidation of galactose and mannose is probably attributable to a steric effect resolution from a ramified structure of the galactomannan. On the other hand, galactose is more accessible to the periodate regent since it is present as terminal unit only. The result may be explained on the basis of mannose residues linked throughC1, C4 and C6, a conclusion either drawn from methylation studies. Further information regarding anomeric nature of the linkage the and sequential arrangement of hexagon have been derived from studies of various oligosaccharides obtained by acid catalysed partial fragmentation of the galactomannan. The hydrolysate upon paper chromatographic separation on preparative scale afforded 4 oligosaccharides along with the building sugars, D-galactose and D-mannose. The following oligosaccharides were identified. Mannobiose; β-D-mannopyranosyl (1→4)-D-mannopyranose. Epimelibiose; α-D-galactopyranosyl (1→6)-D-mannopyranose. Mannotriose; β-D-mannopyranosyl (1→4) - β-D-mannopyranosyl (1→4)-D-mannopyranose. Galactosyl mannobiose; α-D-galactopyranosyl (1→6)- β-D-mannopyranosyl (1→4)-D-mannopyranose. mannopyranosyl (1→4)-D-mannopyranose: was obtained in crystalline form m.p.203-204°C, - 9°(water) and to be reduced sugar. Its complete hydrolysis gave only mannose. The equivalent weight 174.5 by hypo iodide method corresponded to a hexose disaccharide. Emulsion hydrolysis showed presence of β-glycosidic linkage and osazone derivative formed m.p.203°-205°C, Identical with Mannobiosazone. Periodate oxidation study showed the liberation of 2.03 moles of formic acid with consumption of 4.11 moles of sodium metaperiodate per mole of oligosaccharide indicating the presence of (1→4) linkage between mannose units. On the basis of above fact the oligosaccharide was identified as β-D-mannopyranosyl (1→4)-D-mannopyranose, (Structure No.-04)

O H HO H HO OH H HH OH O H H HO OH H

HHOH

OH O

The oligosaccharides {2} Epimelibiose; α-D-galactopyranosyl (1→6)-D-mannopyranose. A crystalline sugar, m.p.-200°-202°C +120.5°(water). Its reduced Fehling‘s solutions. Paper chromatography in mobile phase {2}, {5} and {7} proved to be a single identity. Complete hydrolysis and subsequent paper chromatographic examination of hydrolysate indicated the presence of D-galactose and D-mannose in oligosaccharides. Quantitative estimation of Hirst & Jones method showed molar ratio of two as 1:1. The equivalent weight 171.1 corresponded to hexose disaccharides and periodate oxidation showed that one mole of oligosaccharide consumed 5.5 moles of sodium metaperiodate and yielded 3.2 moles of formic acid. Emulsion failed to hydrolyse the oligosaccharide, indicating the presence of (1→6)-α-linkage. It gives osazone derivatives which was identified with epimelliboisazone and the oligosaccharide was confirmed to be epimelibiose; α-D-galactopyranosyl (1→6)-D-mannopyranose (Structure No-05).

H H

HOOHHH

O H HO H HO OH H

HHOH

O

Oligosaccharides {3} Mannotriose; β-D-mannopyranosyl (1→4) - β-D-mannopyranosyl (1→4)-D-mannopyranose was obtained as solid, m.p. 165-167°C C. -19°(water) was found chromatographically pure in mobile phase (2), (3) and (7). Equivalent weight 265 determined by hypoiodite method showed it to be monohydrate of trisaccharide. Emulsion hydrolysis gives the indication the presence of β-anomeric linkage among the mannose unit. By periodate study indicates the consumption of 5.21 moles of metaperiodate with liberation of 3.03 moles of formic acid per mole of sugar. Partial hydrolysis gave the mannose and mannobiose. Thus the above result showed the presence of (1→4) linkage among mannose units. So the oligosaccharide identified as mannotriose; β-D-mannopyranosyl (1→4) - β-D-mannopyranosyl (1→4)-D-mannopyranose (Structure No-06).

O H HO H HO OH

HHH

OH O H H HO OH

HHHOH

OH O H H HO OH

HHH

OH OO

Structure No.-06 Oligosaccharides(4) Galactosyl mannobiose; α-D-galactopyranosyl (1→6)- β-D-mannopyranosyl (1→4)-D-mannopyranose also obtained in crystalline form m.p.226°-228°C +92°(water) and was found pure in mobile phase {2} and {7}. The complete hydrolysis of sugar yielded galactose and mannose in molar ratio 1:2. Its equivalent weight 265.7 corresponded to a monohydrate of trisaccharide. Acid catalysed partial hydrolysis produced epimelibiose, mannobiose, galactose and mannose. By the periodate oxidation it liberated 3.04 mole of formic acid with the consumption of 6.12 mole of metaperiodate. When emulsion hydrolysis for 6 days gave epimelibiose and mannose only. This sugar was identified as α-D-galactopyranosyl (1→6) -

O OH H H HO H

OHHH

O H HO H HO OH

HHH

O O H O H HO OH

HHHOH

OH OH

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 structural study of the polysaccharides obtained from the seeds of Cassia obtusifolia. The current investigation found that galactomannan consisting of D-galactose and D-mannose in the molar ratio 3 : 4 has been isolated from the seeds of Cassia obtusifolia .Hydrolysis of the methylated polysaccharide resulted in three methylated sugars: (a) 2,4-di-O-methyl-D-mannose, (b) 2,4,6-tri-O-methyl-D-mannose, and (c) 2,3,4,6-tetra-O-methyl-D-galactose in the molar ratio 2: 3: 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, and Gujarat for providing facilities and encouragement throughout the work.

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