Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-01-11
2002-11-19
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S127000, C514S053000, C514S054000
Reexamination Certificate
active
06482942
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the use of tannin related compounds for the selective extraction and purification of mucilaginous polysaccharides from biological materials such as plants, ground biological tissues, or fermented cultured broths from microorganisms. In particular, this invention relates to the precipitation of acetylated mannose polymers derived from the aloe plant and beta glucans from oats and fungi.
BACKGROUND OF THE INVENTION
Generally speaking, mucilaginous polysaccharides are defined as biopolymers characterized by hetero or polysaccharide chains, either linear or branched, having acetyl, nitrogen acetyl, or other nitrogen functional groups associated with the main polysaccharide chain, and containing protein chemically bound to one or more of the external OH groups of the main structure of the polysaccharide chains. Some of these mucilaginous polysaccharides are immunomodulators, and their biological and physical properties make them useful in a variety of applications as ingredients for cosmetics, beverages and pharmaceuticals and as viscosifiers in several multiple chemical production processes. Because of their complex native chemical structure, mucilaginous polysaccharides tend to form a colloidal network with other substances present in solution. It is difficult to separate or isolate these substances while at the same time retaining most or all of their native properties.
Aloe polysaccharides are known as acetylated hetero poly-mannose biopolymers having about one or more acetyl groups per saccharide. (Manna S., McAnalley B.H.; “Determination of the position of the )O-acetyl group in a beta- (1→4)-mannan (acemannan) from Aloe barbadensis miller”.
Carbohydrate Research
(1993) Mar 17; 241:317-319). Although the author takes for granted, without any previous carbohydrate analysis, that the sample that he was analyzing was 100% mannan in its composition, he concludes that the O-acetyl groups in Aloe polysaccharides are located at C-2/C-3 position and at C6 position in a 50:50 ratio.
The acetyl group : saccharide ratio in aloe polysaccharides can also vary with the age of the source plant and other environmental factors, but in general terms the plant in its natural state typically maintains the inner biopolymer with an acetyl group : per saccharide ratio of 1 or higher, wherein the polysaccharide is composed mainly, but not entirely, of mannose. It is believed that the biological and physical characteristics of aloe polysaccharides are attributable in large part to the presence of acetylated mannose residues. This biopolymer is different thanother poly-mannans such as locust bean gum or guar gum which have no reported immunological biological activity.
Mucilaginous polysaccharides have traditionally been isolated either by the use of organic solvents, the use of ammonium sulfate, quaternary ammonium salts and by the use of cationic detergents. However, some of these procedures tend to alter the initial chemical structure of the native biopolymer.
Polysaccharides and mucilaginous polysaccharides will generally form viscous solutions or dispersions exhibiting a typical non-newtonian viscosity profile in polar solvents due to hydrogen bonding (R. L. Whistler, “Industrial Gums”R. L. Whistler and J. N. B. Miller, eds. Academic Press Inc., New York, N.Y., 1959, p 1). Because of the general inability of polysaccharides to swell in organic liquids such as ethanol, methanol, or acetone, these organic solvents traditionally have been used to precipitate polysaccharides from their carrier solutions. However, aside from requiring large amounts of solvent, the solvent precipitation technique tends to provide, co-precipitation of other materials such as organic acids, certain salts, proteins, and other similar substances, giving low product yields and/a somewhat degraded biopolymer.
Ethanol is generally preferred for precipitating the mucilaginous polysaccharides network from aloe vera and other similar mucilages and polysaccharides. Typically, aqueous solutions or extracts of the mucilaginous polysaccharides are treated with five or more volumes of ethanol (U.S. Pat. Nos. 4,957,907, 4,917,890, 4,735,935) to precipitate the polysaccharide. This ethanol-based method of precipitating aloe polysaccharides tends to yield a final polysaccharide product having a significantly reduced acetyl group total saccharide ratio, which is different than the initial native chemical structure, and to denature the glycoproteins present in the hydroparenchima of aloe vera leaves. The reduced acetyl group: saccharide can be attributed to a variety of factors such as the time required for making the gel of aloe allowing enough time for hydrolytic enzymes present in the hydroparechima to act on the polysaccharide, and for the normal increase in temperature caused by the addition of ethanol to the aqueous extract. The second technique for isolating mucilaginous polysaccharides requires the use of large quantities of ammonium sulfate or quaternary ammonium salts to precipitate all the polysaccharides. Detergent cations, such as cetyltrimethylammonium (CTA) or cetylpyridium (CP) also have the ability to form insoluble salts with hydrophobic polyanions, and these insoluble salts then precipitate out from their aqueous solution (J. E. Scott, Chem and Industry (London) 1568 (1955), and A. S. Jones, Biochem. Biophys. Acta, 10, 607 (1953)). The use of CTA and other similar detergent cations for precipitating polysaccharides is another example of structural polysaccharide alteration. Dupont showed that different angiogenic biological activities were obtained from different samples of shark cartilage mucopolysacchardie recovered from initial water extracts, which were treated with different precipitation techniques. After in vivo and in vitro examination, only those shark cartilage mucopolysaccharides which were obtained using the technique of water extraction followed by molecular ultrafiltration, were able to show significant biological activity as compared with other shark cartilage polysaacharide samples obtained either by the classical solvent preciptation using ethanol or using detergent cations (Dupont,Eric et.al., U.S. Pat. No. 5,618,925: “Extracts of shark cartilage having an anti-angiogenic activity and an effect on tumor regression; process of making thereof.” Apr. 8,1997).
Another commonly used procedure for recovering polysaccharides is the use of ammonium salts. However this procedure works best when individual samples containing polysaccharides have similar ionic character. However, some biopolymers present in certain biological extracts often contain various varieties of polysaccharides, which can vary widely in ionic character. This variability makes the use of ammonium salts unsuitable for application in biological extracts containing heterogeneous types of biopolymers.
Tannins have been classified chemically either as (1) condensed tannins (known as proanthocyanidins), which are chemically defined as flavanoid-based polymers, or (2) hydrolyzable tannins (Haslam E., (1981). Vegetable tannins. In Conn, EE (ed.): “The biochemistry of plants Volume 7,” New York Academic Press, p 527-556. In the case of condensed tannins, the beta ring of the flava monomer is generally substituted with two or three ortho-hydroxyl groups. An example of a condensed tannin is the one found in the testa of the grain Sorghum bicolor. On the other hand, hydrolyzable tannins are characterized by a polyhydroxy alcohol esterified with gallic acid (3,4,5-trihydroxybenzoic acid). Hydrolyzable tannins include the family of substances known as ellagitannins and gallotannins which, upon acid hydrolysis, give rise to ellagic and gallic acid. The typical commercial form of hydrolyzable tannins is known as tannic acid. It is well known that hydrolyzable tannins tend to form insoluble complexes with proteins. These complexes are generally water insoluble, but they can be dissociated by various techniques including solvation with organic solvents. Both condensed and hydrolyzable tannins can form
Akin Gump Strauss Hauer & Feld L.L.P.
Biotechnology Services and Consulting, Inc.
Maier Leigh C.
Wilson James O.
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