Title of Invention

"NOVEL POLYSACCHARIDES WITH ANTIOXIDANT PROPERTY"

Abstract

The invention is specifically related to new molecules,which are useful as antioxidants. These molecules are polysaccharides designated as Agarinan A and Agarinan B. These polysaccharides are partially fucosylated galactoglucans. A polysaccharide designated as Agarinan A, having structural formula shown in figure 1 and comprising glucose, galactose and fucose in a ratio of 1:1:0.25. A polysaccharide designated as Agarinan B having the structural formula as shown in figure 2 and comprising glucose, galactose and fucose in a ratio of 3:1:0.25. This invention also relates to a process for isolating these compounds and compositions comprising these compounds.

Full Text

1. FIELD OF INVENTION The invention specifically deals with new molecules, which are useful as antioxidants. These molecules are polysaccharides designated as Agarinan A and Agarinan B. These polysaccharides are partially fucosylated galactoglucans. These polysaccharides contain glucose in the ß linkage and galactose and fucose in α linkage. The IUPAC representation of the polysaccharide is as follows: Agarinan A: (Formula Removed) Agarinan B: (Formula Removed) 2. DESCRIPTION OF THE PRIOR ART It is reported that chronic infections contribute to about one-third of the world's cancer (Ames. B, et al, Proc. Natl. Acad. Sci., U.S.A. 1995; 92: 5258-65). Hepatitis B and C viruses are a major cause of chronic inflammation leading to liver cancer, which is one of the most common cancers in Asia and Africa. Reactive oxygen species (ROS), formed during the combat of infection are indicated in etiology of several degenerative diseases such as cancer and arthritis. Leukocytes and other phagocytic cells combat bacteria, parasites, and virus-infected cells by destroying them with nitrogen oxide and superoxide, which react to form peroxy nitrite, a powerful mutagenic oxidizing and nitrating agent; hypochlorite, a mutagenic chlorinating and oxidizing agent. Although, these oxidants protect humans from immediate death due to infection, they also cause damage to sensitive biological structures such as DNA, lipids and proteins. Oxidative damage caused to DNA leads to mutation and chronic cell killing, thereby contributing to the carcinogenic process (Collins.A. BioEssays 1999;21: 238-46; Hagen. T et al., Proc. Natl. Acad. Sci., U.S.A. 1994; 91: 12808-12; Halliwell. B, Nutr. Rev.1994; 52: 253-65; Halliwell. B. Free Radic. Biol. Med. 2002; 32: 968-74). Several mushroom species have been studied for anti-inflammatory and antioxidant activities (Ukai, et al., 1983. Journal of Pharmacology Dynamics 6, 983-990.) Extracts of Ganoderma lucidum that apparently remove the hyperoxide radical are believed to be an important main factor in the human ageing process. Significant superoxide and hydroxyl radical scavenging activities have been demonstrated for several mushroom polysaccharides (Liu. F, et al, 1997. Life Sciences 64,1005-011). A Ganoderma lucidum polysaccharide GLB7 decreased the production of oxygen free radicals and antagonised the respiratory burst induced by PMA in murine peritoneal macrophages (Li, M. and Lei, L.S. 2000. Zhongguo Yadikxue Yu Dulixue Zazhi - Chinese Journal of Pharmacology and Toxicology 14, 65-68). Antioxidant activity of various polysaccharides has been reported to have been isolated from certain mushrooms. United States Patent 6,616,928 describes an active oxygen scavenger and cancer chemopreventer obtained from Grifola frondosa.. The invention of the said patent teaches the use of extract of Grifola frondosa as oxygen scavenging agent. Similarly, extract of Ganoderma lucidum has also been patented for wound healing vide US Patent No. 5,547,672. Also US Patent No. 6,783,771 discloses a biologically active substance labeled as EEM-S obtained from mushrooms such as lentinus edodes and it is claimed to exhibit anti-cancer and immunopotentiating activity. The exact nature of the substance is not disclosed. Thus, prior art workers have performed several studies on mushrooms available in their respective local areas. Agaricus bisporus is a widely cultivated and easily available mushroom in India. It is also called 'white button mushroom'. Hence, the inventors herein undertook a detailed study of this mushroom, and now provide novel polysaccharide compounds isolated from the said mushroom. 3. OBJECTS OF THE INVENTION The main object of the invention is to provide novel polysaccharide compounds useful as antioxidants and isolated from Agaricus bisporous. Another object is to provide a method for large scale production of the said polysaccharide compounds. 4. DESCRIPTION OF THE ACCOMPANYING DRAWINGS: Figure 1 shows the structure of Agarinan A. Figure 2 shows the structure of Agarinan B. Figure 3 shows respiratory oxy burst process 5. DETAILED DESCRIPTION OF THE INVENTION Definitions: Polysaccharides - Sugars that can be broken down into infinite number of monosaccharide units Monosaccharides- Simplest sugar molecules, that cannot be broken down further e.g. glucose, galactose, fructose, fucose etc. These molecules when linked together in large chains form polysaccharides. Glucans- polysaccharide that is composed of glucose monosaccharide units Galactoglucans- polysaccharide that is composed of galactose and glucose monosaccharide units Homoglycans- Composed of single type of monosaccharide unit Heteroglycans - Composed of more than one type of monosaccharide unit Accordingly, in one aspect, the present invention provides polysaccharides, which are useful powerful antioxidants. The novel polysaccharides of the invention are mainly fucose- containing galactoglucans, which compounds are designated as Agarinan A and Agarinan B. The novel polysaccharides are represented by the formula 1 and 2 and shown in Figures 1 and 2. As evident from the figures, Agarinan A is a polysaccharide composed of glucose, galactose and fucose in the ratio 1:1:0.25 whereas in Agarinan B is a polysaccharide wherein glucose, galactose and fucose moieties are present in a ratio of 3:1:0.25. The polysaccharides compounds of the present invention are unique in that they consist of three sugars viz. glucose, galactose and fucose, the moieties being linked in a specific ratio, in a specific manner and in two dimensional structure. While workers in prior art have obtained polysaccharides from different sources with varying bioactivity, this is the first time that such polysaccharides possessing antioxidant activity have been identified and reported. It is important to understand that Agaricus bisporous is a common mushroom that contains a number of active principles. Some of these principles are even carcinogenic. Infact, there is evidence and reports that extract of Agaricus bisporous could cause cancer. For example it is said that the uncooked mushroom caused tumors in bone, fore stomach, liver, and lungs when administered per-orally for 3 days (Anticancer Res. 1986; 6: 917-20). Similarly lyophilized mushroom in a range of 2.5, 5 and 10 % of the diet caused tumors in the lungs, fore-stomach, glandular stomach, and ovaries (In Vivo 1998; 12: 239-44). One of the major metabolites of Agaricus bisporus is Agaritine. The breakdown products of Agaritine are carcinogenic in nature. Hence, most of the compounds isolated are expected to be carcinogenic. This is also one reason why Agaricus bisporous is not preferred. [Chem. Biol. Interact, 1995; 94: 21-36]. Further, in medicinal preparations, extracts are not preferred since they are difficult to standardize (as they vary in each preparation) whereas, on the other hand, if a pure molecule or moiety is obtained, it can be directly and easily formulated into a drug. Thus, obtaining pure compounds is always advantageous. Yet another factor is that the moiety obtained should be as pure as possible without any contaminants since such contaminants could eventually cause side-effects or harm patients. So far, prior art workers have succeeded in obtaining extracts of mushrooms and used them for medicinal purposes and if at all pure compounds are isolated, they are obtained from mushrooms not found in India. Further, compounds isolated so far are only Beta glucan type compounds in that they contain glucose monosaccharide units. Polysaccharides containing glucose in combination with other saccharide moieties are not reported so far, and certainly not from Agaricus bisporous. The inventors during their investigations on Agaricus bisporous casually isolated a few compounds and tested their activity, as a matter of routine on normal cells and expected as usual reactions leading to cancer. To their utter surprise and shock, it was found that these compounds are not carcinogenic as expected and instead act as free radical scavengers. When the tests were repeated, identical results were obtained and it was confirmed that the inventors' novel compounds are useful as anti-oxidants. These compounds were then designated as Agarinan A and Agarinan B. Based on these results, a series of tests were performed to ascertain and understand the nature, characteristics and other properties of the novel isolated compounds as compared to other compounds isolated from Agaricus bisporous. It was found that these compounds Agarinan A and Agarinan B contain glucose, galactose and fucose in a specific ratio and this combination of saccharide units is responsible for their activity. It is important to understand that in case of polysaccharides any change in the ratio of saccharide units or absence of certain moieties or any change in the linkage would lead to different molecules, each having completely different activity. As an example, several other polysaccharide compounds were isolated alongwith Agarinan A and B. However, each of these compounds had varying ratios of glucose-galactose and fucose. In one compound, where the ratio was 1:1:0.25, no anti-oxidant activity was observed. In another aspect, the invention provides a method for production of the novel compounds with >98% purity, from a readily available biosource i.e. the button mushroom. The said process comprises the steps of: i) preparing a dry powder of Agaricus bisporus ii) subjecting the powder to treatment with an organic solvent followed by treatment with an alcohol to remove lipids and polar compounds and obtaining a residue, iii) treating the residue with hot water at 70-80°C to obtain a hot-water extract, iv) precipitating the hot water extract with acetone to obtain a crude extract containing polysaccharides, v) dialyzing the crude extract against distilled water and obtaining polysaccharides of size greater than 15 kD and subjecting it to ion-exchange chromatography and eluting with water and sodium chloride to obtain at least two fractions, vi) isolating polysaccharide compound agarinan A (crude) from a first fraction, agarinan B (pure) from a second fraction, vii) optionally subjecting each fraction to gel chromatography, and eluting with water to obtain purified agarinan A and B, and freeze-drying the purified compounds and storing at room temperature. As in case of all biological materials, the amount of final desired product obtainable from the starting material is less and this is true in the case of isolation of polysaccharides from Agaricus species. Prior art workers have typically employed processes wherein the Agaricus powder is repeatedly subjected to steps of filtration, dialysis and precipitation to obtain the desired polysaccharide. Even after such tedious procedures, the polysaccharide obtained is very less, is not pure and is almost always contaminated. Such prior art procedures recommend use of acids at the end, which if left on polysaccharides are harmful to humans. Further, use of many columnar materials leads to wastage of raw materials. In order to avoid the problems of the prior art, the inventors have developed a process whereby polysaccharides may be obtained in less than 48 hours (compared to several weeks of prior art). The present process employs very few steps, and uses harmless materials like NaCl for dialysis. The dialysate can be directly used to obtain the final compound in purity of >98%. In fact, from lKg of Agaricus material, 100 mg and 160 mg of Agarinan A & B respectively were obtained. Yet another aspect of the invention relates food and beverage composition comprising the polysaccharide compounds of the present invention. Examples of such compositions are biscuits, health drinks and so on. Preferably, the compounds of the invention may be used as active ingredients or supplements in the composition. Most preferably, the amount of the compounds in the composition may be 2 to 20 gm. Optionally, the compounds of the present invention may be used in personal care products such as skin care lotions, creams etc. The invention is now illustrated by the following examples, which are merely illustrative and not meant to limit the scope of the invention in any manner. Example 1: Isolation of Polysaccharides Agarinan A and Agarinan B. The isolation of the polysaccharides is given in the scheme below. After collection, the mushrooms were washed with tap water, dried at 37° C for 48 hrs and extracted sequentially with hexane and methanol. The residue left after extraction with methanol was dried for 12-15 hrs. Dried powder 1 Kg was soaked in 5 liter of distilled water and kept overnight at 10 °C. The material was stirred and filtered through muslin cloth. The material was heated with intermittent stirring for 4 hrs, with a further change of water at 4 hrs. The extract was filtered and concentrated on a rotavapour under reduced pressure to give the hot water extract. The concentrate was precipitated with acetone (2.5 v/v) and the precipitate was washed, dissolved in a small quantity of water and freeze-dried. The freeze-dried extract was dissolved in a minimum volume of distilled water and dialyzed (sigma dialysis tubing, molecular weight cut off 12,000 Dalton against de-mineralized water at 4° C for 24 hrs. The dialysate was centrifuged at 8000 rpm for 15 min to remove insoluble water matters and lyophilized to yield the crude polysaccharides (CP). Crude polysaccharides were loaded in a DEAE cellulose column and eluted successively with H2O and NaCl (1M) to yield a fraction 1C and pure polysaccharides 2. The fraction 1C was further purified by gel column chromatography to yield the pure polysaccharide 1. The isolated and purified polysaccharides 1 and 2 are named as 'Agarinan A' and 'Agarinan B'. A scheme for isolation of Agarinan A & B is shown here below: (Formula Removed) Example 2: Characterization of the isolated compounds. a) Spectral data of Agarinan A and Agarinan B NMR data of Agarinan A (1) (Table Removed) Underlined bold numbers indicate site of glycosylation * Unassignable NMR data of Agarinan B (2) (Table Removed) Underlined bold numbers indicate site of glycosylation * Unassignable b) Chemical characterization of polysaccharides 1. Estimation of carbohydrates (Dubois. M, et al Anal. Chem. 1956; 28: 350-6). Sample aliquots were taken and volume of each aliquot was adjusted to 2 mL with distilled water. Phenol solution (5%, 1 mL) was added to each aliquot followed by rapid addition of H2SO4 (5 mL), in ice bath. In blank, the sample aliquot was replaced by water (2 mL). All sample and blank solutions were incubated at room temperature for 30 min. UV absorbance was measured at 485 run. A standard curve was used for a set of reagents. 2. Estimation of proteins (Bradford. M, Anal. Biochem. 1976; 72: 248-54). Protein concentration was determined by a modified procedure of the Sigma dye-binding assay (Bradford reagent). The method was adapted for microtitre plate measurements. Protein standards (BSA) and sample solutions were diluted in phosphate buffer (0.01M) and transferred into a 96-well flat bottom microtitre plate (100 µL). Bradford reagent (100 µL) was added to the samples and incubated for 10 min. Plates were measured at a wavelength of 595 nm in micro titre plate reader. Each sample was measured four times and protein concentration was calculated by linear regression method. 3. Estimation of sulfate (Dodgson. K and Price. R. Biochem. J. 1962; 84: 106-10). Aliquots of K2SO4 working solution were taken and volume of each aliquot was adjusted with distilled water to 0.2 mL. Tri-chloro-acetic acid (TCA) (3.8 mL) was added to each aliquot followed by BaCl2- gelatin solution (1 mL). Blank was prepared in the same way by replacing K2SO4 solution with distilled water. After incubation for 15-20 min at room temperature, UV absorbance was measured at 360 nm. In the first set of experiments (A), each experimental solution was prepared with a particular aliquot of sample and volume was made up to 0.2 mL with HC1 (IN) and TCA (3.8 mL) was added followed by gelatin-BaCl2 solution (1 mL). The UV absorbance was measured (A) at 360 nm against a blank solution containing HC1 (IN, 0.2 mL), TCA (3.88 mL) and gelatin- BaCl2; (1 mL). In the second set of the experiment (B), each experimental solution was prepared with same sample aliquots volume which was used for set A experiment and the volume was made up to 0.2 mL with IN HC1 and addition of 3.8 mL TCA followed by 1 mL of gelatin solution. The UV absorbance was measured (B) at 360 nm against a blank solution containing HC1 (IN, 0.2 mL), TCA (3.8 mL) and gelatin solution (1 mL). Absorption due to sulfate is equal to (A-B). 4. Estimation of uronic acid (Knutson. C and Jeanes. A. Anal. Biochem. 1968; 24: 470-81). Sample aliquots were made up to 0.7 mL in H2SO4-borate reagent (6 mL) was added in ice bath and volume was adjusted with distilled water to 6.7 mL, shaken well and incubated in ice bath for 10 min. Each aliquot has its own blank; both experiment and blank had the same volume of sample aliquots but in the blank 0.2 mL of EtOH were added, instead of carbazole. Standard curve was obtained by using the aliquots, for which blank was prepared with distilled water (0.7 mL) and mixed with carbazole (0.2 mL). The UV absorbance was measured at 530 nm. 5. Paper chromatography (Trevelyan. W, et al., Nature 1950; 166: 444-5). Samples were run on Whattman filter paper # 42, solvent system was (n-BuOH:Pyridine:H2O, 6:4:3), permanent chromatograms were obtained by detection with silver nitrate- sodium hydroxide method (Trevelyan, et al, 1950). After the papers were dried, they were dipped in a solution made by diluting saturated aqueous AgNO3 solution (0.1 mL) with Me2CO (20 niL) and adding water drop-wise until the AgNO3 which separates get re-dissolved. After 3-4 min, the papers were sprayed with NaOH (0.5N in 40% aqueous EtOH). The sugars gave brown to black spots. After 5-10 min, the papers were soaked in NH4OH (6N) followed by dilute Na2S2O3. 6. Molecular weight determination a. Seralose-CL-4B column (45 x 1 cm) was eluted with H2O containing NaN3 (0.02%) at 25 °C at a flow rate of 6 mL/h. Molecular weight of the samples were determined from standard graphic curve on logarithmic scale against elution volume. Blue dextran was used for void volume determination. A standard curve was prepared with polymeric dextrans having different molecular weights (9,500 Da; 39,500 Da; 66,900 Da; 5,00,000 Da). Blue dextrans and the standards were used in a concentration of 1 mg/mL. b. Waters HPLC, column: Shodex protein KW-804 column, dim 8 x 300 mm, mobile phase: H2O (isocratic), flow rate of 1.0 mL/min at room temperature, exclusion limit 6 x 105 Da, Waters RI-410 detector. 7. Preparation of alditol acetates and partially methylated alditol acetates (Ciucanu. I and Kerek. F. Carb. Res. 1984; 131: 209-17). a. Hydrolysis : Aq. TFA (2M, 2.5 mL) was added to the sample (5 mg) and the sample was hydrolyzed for 2 h at 110-120 °C in a fan forced oven. The tubes were cooled and evaporated to dryness with a stream of nitrogen. The residue was oily in nature. b. Reduction :The residue from hydrolysis was dissolved in NH4OH (2M, 500 µl) and freshly prepared solution of NaBIL; (1M) in NH4OH (2M, 50 µl) was added to it. The mixture was sonicated for 1 min maintained at 60 °C in an oil bath for 1 h. The excess reductant was destroyed with glacial AcOH (50 µl). The sample was dried with a stream of nitrogen, washed with MeOH twice and dried again. c. Acetylation: Samples were refluxed with (CH3CO)2O and pyridine (1:1, 250 µl) for 20 min; excess reagents were removed by evaporating repeatedly using toluene. d. Per-methylation : The samples (10 mg) were dried by repeatedly adding and evaporating MeOH, and further dried by passing a stream of N2. The samples were suspended in dry DMSO (500 µl), sonicated for 20 min and left overnight. Slurry of NaOH (approximately 120 mg/mL) in DMSO was prepared by grinding NaOH (3 pellets) in DMSO (1 mL) in a glass pestle and mortar. Immediately DMSO/NaOH (500 µl) slurry was added to each sample using a glass pipette and sonicated for 30 min. CH3I (100 µl) was added and further sonicated for 20 min. This step was repeated twice. Freshly prepared Na2S2O3 (100 mg/mL, 500 µl) in H2O and CHCl3 (500 µl) were added successively to the sample. The upper aqueous phase was discarded; the lower CHCl3 layer was washed with water (500 ul, 4 times) and dried by a stream of N2. The samples were methylated two more times to ensure complete methylation. IR further confirmed complete methylation. The permethylated samples were then hydrolyzed, reduced and acetylated by procedures described above (G, a-c). e. Analysis of alditol acetates and partially methylated alditol acetates The samples were analyzed by GC-MS. TABLE The results of chemical analysis obtained for the isolated compounds are given below: (Table Removed) Example 3: Description of the method of in vitro antioxidant activity Antioxidant activity can be ascertained by a set of three assays, namely the DPPH, NBT reduction and luminol-based chemiluminescence (CL) assay. Accordingly, the antioxidant property of Agarinan A and B were evaluated using luminol-based chemiluminescence assay in polymorpho-nuclear (PMN) cells (differentiated lymphocytes, that are responsible for protecting humans from infection) cells (Pawar, R., et al., J. Nat. Prod. 2004; 67: 668-71; Lilius, et al, Analytical applications of bioluminescence and chemiluminescence, 1984). To the PMN cell suspension, luminol stock solution was added to attain the concentration of 0.5 mM. The cell suspension containing luminol was added to the microtitre plates containing test compounds in triplicate. After 5 min PMA (150 µL) was added to trigger the reaction. The microtitre plate was measured in kinetic mode for 90 min. during which each well was read for 740 ms. A curve of light intensity (RLU) was plotted against time and the area under curve (AUC) was calculated as total luminescence. The percent inhibition of luminescence was calculated as % Inhibition of chemiluminescence = [(Control - sample)\ Control]× 100. The luminol (5-amino-2,3-dihydro-l,4-phthalazinedione) enhanced chemiluminescence assay determines the production of hypochlorous acid by the myeloperoxidase-H2O2 system (see Figure 3). A compound that is active in luminol-dependent chemiluminescence assay indicates that the compound acts on the myeloperoxidase-H2O2 system. This indicates that the compound has higher reproducibility when tested invivo. TABLE 1 Antioxidant activity in luminol-based chemiluminescence assay (Table Removed) The IC50 value of Agarinan A is less than quercetin, showing that it is more active than quercetin (indicating that lesser amount of Agarinan A is required to inhibit 50% of free radicals produced in comparison to that of quercetin). Thus, the polysaccharides 1 and 2 were found to contain luminol-based antioxidant activity in PMN cells. Further the activity of 1 was found to be more potent than quercetin, which is a known as an antioxidant and is commercially available. Example 4. i) Health drink:- Polysaccharide lemonade Ingredients One and a half cups water One and a half cups sugar One and a half cups lemon juice Grated rind of one lemon Two table spoons of polysaccharides (approx 3-4g) Ice cubes Cold water or club soda Preparation Combine the water and sugar in a saucepan and bring the mixture to a boil over high heat. Boil the liquid three minutes. Remove the pan from the heat and stir in the lemon juice, lemon rind. Dissolve the polysaccharides in a small quantity of water and add to pan. Refrigerate at least one hour or until very cold. Strain the mixture into a storage container. When you want some lemonade, place 5 to 6 ice cubes in a drinking glass. Fill the glass about a third full with the lemon syrup, add water or club soda to the top of the glass, stir and drink. Makes about 3 cups. ii) Polysaccharide Punch Ingredients: Two table spoons of polysaccharides (approx 3-4g) 2 cups water 1 liter ginger ale, chilled 12 oz frozen orange pineapple juice Preparation Dissolve the polysaccharides in a small quantity of water. Add the rest of water in a saucepan and bring to a boil, stirring. Simmer for 5 minutes. Cover and allow cooling and strain. Combine with the orange pineapple juice and stir well. Refrigerate until ready to use. Then combine with the gingerale. iii) Biscuits : Bran containing biscuits Ingredients ½ cup (10 - 15 g) Polysaccharides ½ cup bran 1½ cups flour 5 teaspoons baking powder ¾ teaspoon salt ½ cup water 2 tablespoons melted shortening Preparation Mix thoroughly polysaccharides, bran, flour, baking powder, and salt, then the shortening, and sufficient water to make soft dough. Roll out on floured pastry board. Cut round, and bake on greased tin in a moderate oven for about 20 minutes. iv) Basic biscuits Ingredients 2 cups flour ½ cup (10 - 15 g) Polysaccharides 4 teaspoons baking powder 1 teaspoon salt 2 tablespoons shortening ¾ cup liquid (all milk or half milk and half water) Preparation Mix dry ingredients including the polysaccharides and sift twice. Work in fat with tips of the fingers, or cut in with two knives. Add the liquid gradually, mixing with a knife in a soft dough. Owing to differences in flours, it is impossible to determine the exact amount of liquid. Toss on a floured board, pat and roll lightly to one-half inch in thickness. Shape with a biscuit cutter. Bake in hot oven (450-460 degrees F.) twelve to fifteen minutes. Materials: The inventors employs Agaricus bisporous which is a commonly occurring mushroom. It may be grown or purchased from the market. We claim: 1. A polysaccharide designated as Agarinan A, having formula (Formula Removed) and the structural formula as shown in figure 1 and comprising glucose, galactose and fucose in a ratio of 1:1:0.25. 2. A polysaccharide designated as Agarinan B, having the formula (Formula Removed) and the structural formula as shown in figure 2 and comprising glucose, galactose and fucose in a ratio of 3:1:0.25. 3. A food composition comprising an effective amount of the compound as claimed in any of claims 1 to 2. 4. The composition as claimed in claim 3 wherein the amount of Agarinan A is 2 to 20 gm. 5. The composition as claimed in claim 3 wherein the amount of Agarinan B is 2 to 20 gm. 6. A beverage composition comprising an effective amount of a compound as claimed in claims 1 & 2. 7. The composition as claimed in claim 6, wherein the amount of Agarinan A or Agarinan B is 2 to 20 gm. 8. A method for isolation of a polysaccharide as claimed in any of claims 1 and 2 comprising the steps of: i. preparing a dry powder of Agaricus bisporus; ii. subjecting the powder to treatment with an organic solvent followed by treatment with an alcohol to remove lipids and polar compounds and obtaining a residue; iii. treating the residue with hot water at 70-80°C to obtain a hot-water extract; iv. precipitating the hot water extract with acetone to obtain a crude extract containing polysaccharides; v. dialyzing the crude extract against distilled water and obtaining polysaccharides of size greater than 15 kD and subjecting it to ion-exchange chromatography and eluting with water and sodium chloride to obtain at least two fractions; vi. isolating polysaccharide compound agarinan A (crude) from a first fraction, agarinan B (pure) from a second fraction; and vii. optionally subjecting each to gel chromatography, and eluting with water to obtain purified agarinan A and B, and freeze-drying the purified compounds and storing at room temperature.

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668-del-2005-Abstract-(14-09-2012).pdf

668-del-2005-Claims-(14-09-2012).pdf

668-del-2005-Claims.pdf

668-del-2005-Correspondence Others-(07-06-2012).pdf

668-del-2005-Correspondence-Others-(14-09-2012).pdf

668-del-2005-Correspondence-others.pdf

668-del-2005-Description (Complete).pdf

668-del-2005-Drawings-(14-09-2012).pdf

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668-del-2005-Form-1.pdf

668-del-2005-Form-18.pdf

668-del-2005-Form-2.pdf

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668-del-2005-Form-3.pdf

668-del-2005-Form-5.pdf

668-del-2005-GPA-(07-06-2012).pdf

668-del-2005-GPA-(14-09-2012).pdf